U.S. patent application number 17/255304 was filed with the patent office on 2022-04-28 for guided microbial remodeling, a platform for the rational improvement of microbial species for agriculture.
The applicant listed for this patent is Pivot Bio, Inc.. Invention is credited to Sarah BLOCH, Rosemary CLARK, Austin DAVIS-RICHARDSON, Shayin GOTTLIEB, Douglas HIGGINS, Alvin TAMSIR, Karsten TEMME.
Application Number | 20220127627 17/255304 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-28 |
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United States Patent
Application |
20220127627 |
Kind Code |
A1 |
BLOCH; Sarah ; et
al. |
April 28, 2022 |
GUIDED MICROBIAL REMODELING, A PLATFORM FOR THE RATIONAL
IMPROVEMENT OF MICROBIAL SPECIES FOR AGRICULTURE
Abstract
The present disclosure provides guided microbial remodeling
(GMR) methods for the rational improvement of plant-associated
microbes to perform plant-beneficial functions. The GMR methods
described herein allow for non-intergeneric genetic optimization of
key regulatory networks within the microbes, which improve
plant-beneficial functions over wild-type microbes but don't have
the risks associated with transgenic approaches (e.g.,
unpredictable gene function, public and regulatory concerns, etc.).
The present disclosure also provides remodeled microbes and
compositions thereof. The utilization of remodeled microbes and
compositions thereof will enable farmers to realize more productive
and predictable crop yields without the nutrient degradation,
leaching, or toxic runoff associated with traditional synthetically
derived fertilizers.
Inventors: |
BLOCH; Sarah; (Berkeley,
CA) ; TEMME; Karsten; (Berkeley, CA) ; TAMSIR;
Alvin; (Berkeley, CA) ; HIGGINS; Douglas;
(Berkeley, CA) ; DAVIS-RICHARDSON; Austin;
(Berkeley, CA) ; CLARK; Rosemary; (Berkeley,
CA) ; GOTTLIEB; Shayin; (Berkeley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pivot Bio, Inc. |
Berkeley |
CA |
US |
|
|
Appl. No.: |
17/255304 |
Filed: |
June 27, 2019 |
PCT Filed: |
June 27, 2019 |
PCT NO: |
PCT/US2019/039528 |
371 Date: |
December 22, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62690619 |
Jun 27, 2018 |
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International
Class: |
C12N 15/74 20060101
C12N015/74; C12Q 1/6869 20060101 C12Q001/6869; C12N 1/20 20060101
C12N001/20 |
Claims
1. A guided microbial remodeling method for the rational
improvement of plant-associated microbes to perform
plant-beneficial functions, comprising: a. providing a plurality of
microbial species that are associated with a target plant of
interest; b. assaying the plurality of microbial species for:
colonization metrics, and ability to perform a plant-beneficial
function of interest; c. selecting a candidate microbial species
from the plurality of assayed microbial species; d. introducing one
or more targeted non-intergeneric genetic variations into the
candidate microbial species; e. confirming integration of the
non-intergeneric genetic variation at the target genomic locus and
absence of any transgenetic sequence; and f. repeating steps d) and
e) one or more times, until the candidate microbial species has
acquired an improved ability to perform the plant-beneficial
function of interest.
2. The guided microbial remodeling method according to claim 1,
wherein the step b) comprises assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions.
3. The guided microbial remodeling method according to claim 1,
comprising a step of sequencing the genomes of the plurality of
microbial species obtained in step a) and characterizing one or
more genomic pathways, or sets of genes, that are associated with a
plant-beneficial function of interest.
4. The guided microbial remodeling method according to claim 1,
wherein the step d) of introducing one or more targeted
non-intergeneric genetic variations into the candidate microbial
species comprises: a. transforming the candidate microbial species
with a transformation plasmid comprising (i) a selection marker,
(ii) a counterselection marker, (iii) a DNA fragment comprising: a
non-intergeneric genetic variation to be introduced into the
candidate microbial species at a target genomic locus in one or
more genomic pathways, or sets of genes, that are associated with a
plant-beneficial function of interest, and homology arms to the
target genomic locus flanking the non-intergeneric genetic
variation, and (iv) plasmid backbone; b. selecting for a candidate
microbial species that has undergone an initial homologous
recombination and has the non-intergeneric genetic variation
integrated into the target genomic locus based on the presence of
the selection marker in the genome; and c. selecting for a
candidate microbial species that has the non-intergeneric genetic
variation integrated into the target genomic locus, but has
undergone an additional homologous recombination that loops-out the
plasmid backbone, based on the absence of the counterselection
marker.
5. The guided microbial remodeling method according to claim 1,
wherein the step e) of confirming integration of the
non-intergeneric genetic variation at the target genomic locus and
absence of any transgenetic sequence comprises sequencing the
genome of the transformed candidate microbial species.
6. (canceled)
7. The guided microbial remodeling method according to claim 1,
wherein the plant-beneficial function of interest is nitrogen
fixation, phosphate solubilizatoin, or microbial colonization.
8. (canceled)
9. (canceled)
10. The guided microbial remodeling method according to claim 3,
wherein the step of sequencing comprises whole genome
sequencing.
11. The guided microbial remodeling method according to claim 3,
comprising characterizing the nitrogen fixation pathway.
12. (canceled)
13. The guided microbial remodeling method according to claim 3,
comprising characterizing one or more genes involved in a pathway
selected from the group consisting of: exopolysaccharide
production, endo-polygalaturonase production, trehalose production,
and glutamine conversion.
14. (canceled)
15. The guided microbial remodeling method according to claim 3,
comprising characterizing one or more genes selected from the group
consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding
glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide
encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY,
nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a
gene associated with biosynthesis of a nitrogenase enzyme, bcsii,
bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, and combinations
thereof.
16. (canceled)
17. (canceled)
18. The guided microbial remodeling method according to claim 1,
wherein step b) comprises assaying the plurality of microbial
species for colonization metrics under greenhouse conditions, lab
based conditions, and/or under field conditions.
19. The guided microbial remodeling method according to claim 1,
wherein a colonization metric assayed in step b) comprises at least
one of the following: spatial colonization patterns, temporal
colonization dynamics, density of colonization, or combinations
thereof.
20. (canceled)
21. The guided microbial remodeling method according to claim 2,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs in vitro, under greenhouse, or lab
based, or field conditions and comprises measuring the
transcriptomic profile of the microbial species.
22. The guided microbial remodeling method according to claim 2,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs in vitro, under greenhouse, or lab
based, or field conditions and comprises measuring the
transcriptomic activity of genes associated with the microbial
species ability to perform a plant-beneficial function of
interest.
23. The guided microbial remodeling method according to claim 2,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs under greenhouse or lab based, or
field conditions and comprises at least one of: measuring the
transcriptomic activity of regulatory gene sequences, measuring the
transcriptomic activity of promotor sequences; measuring the
transcriptomic activity of promotor sequences in the presence of
exogenous nitrogen, and measuring the transcriptomic activity of
promoter sequences in the presence of exogenous nitrogen, wherein
said transcriptomic activity of the promoter sequences is measured
by quantifying the expression of a regulated gene.
24.-36. (canceled)
37. The guided microbial remodeling method according to claim 2,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs in vitro and comprises at least one
of: measuring the transcriptomic activity of regulatory gene
sequences, measuring the transcriptomic activity of promotor
sequences; measuring the transcriptomic activity of promotor
sequences in N-depleted and N-replete conditions, and measuring the
transcriptomic activity of promoter sequences in N-depleted and
N-replete conditions, wherein said transcriptomic activity of the
promoter sequences is measured by quantifying the expression of a
regulated gene.
38.-41. (canceled)
42. The guided microbial remodeling method according to claim 1,
wherein assaying the plurality of microbial species for
colonization metrics, comprises: growing said plurality of
microbial species in intimate association with a target plant under
greenhouse conditions, or lab based conditions, and/or field
conditions.
43. (canceled)
44. (canceled)
45. The guided microbial remodeling method according to claim 2,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions, comprises: growing said plurality of
microbial species in intimate association with a target plant under
greenhouse conditions, lab based conditions, or field
conditions.
46. (canceled)
47. (canceled)
48. The guided microbial remodeling method according to claim 1,
wherein step b) comprises assaying the plurality of microbial
species for ability to perform a plant-beneficial function of
interest under greenhouse or lab based conditions.
49. The guided microbial remodeling method according to claim 1,
wherein step b) comprises assaying the plurality of microbial
species for nitrogen fixation activity.
50. The guided microbial remodeling method according to claim 1,
wherein step b) comprises assaying the plurality of microbial
species for nitrogen fixation activity in an acetylene reduction
assay or ammonium excretion assay.
51. (canceled)
52. The guided microbial remodeling method according to claim 5,
wherein the sequencing comprises whole genome sequencing.
53. (canceled)
54. (canceled)
55. The guided microbial remodeling method according to claim 1,
wherein in step d), said non-intergeneric genetic variations are
selected from the group consisting of: full gene deletions, partial
gene deletions, promoter insertions, single base pair changes, and
combinations thereof.
56. (canceled)
57. (canceled)
58. (canceled)
59. (canceled)
60. (canceled)
61. A computationally guided microbial remodeling method for the
rational improvement of plant-associated microbes to perform
plant-beneficial functions, comprising: a. accessing a plurality of
microbial whole genome sequences; b. identifying a plurality of
regulatory gene sequences that actively regulate the transcription
of a gene under a metabolically relevant environmental condition;
c. identifying a plurality of genes associated with a
plant-beneficial function; d. selecting a regulatory gene sequence
and a gene associated with a plant-beneficial function from said
pluralities; wherein steps a)-d) occur in silico; and e.
manufacturing, in vivo, a remodeled microbial cell that has the
selected regulatory gene sequence operably linked to the selected
gene associated with a plant-beneficial function, thereby improving
the expression of the gene associated with a plant-beneficial
function.
62. A computationally guided microbial remodeling system for the
rational improvement of plant-associated microbes to perform
plant-beneficial functions, comprising: a. one or more processors;
and b. one or more memories operatively coupled to the one or more
processors and having instructions stored thereon, that when
executed by the one or more processors, cause the system to: i.
access a plurality of microbial whole genome sequences; ii.
identify a plurality of regulatory gene sequences that actively
regulate the transcription of a gene under a metabolically relevant
environmental condition; iii. identify a plurality of genes
associated with a plant-beneficial function; and iv. select a
regulatory gene sequence and a gene associated with a
plant-beneficial function from said pluralities.
63. A computationally guided microbial remodeling method for the
rational improvement of plant-associated microbes to perform
plant-beneficial functions, comprising: a. activating a computer
system having: one or more processors and one or more memories
operatively coupled to the one or more processors and having
instructions stored thereon, thereby causing the one or more
processors to execute the instructions, and cause the system to: i.
access a plurality of microbial whole genome sequences; ii.
identify a plurality of regulatory gene sequences that actively
regulate the transcription of a gene under a metabolically relevant
environmental condition; iii. identify a plurality of genes
associated with a plant-beneficial function; iv. select a
regulatory gene sequence and a gene associated with a
plant-beneficial function from said pluralities; and b.
manufacturing, in vivo, a remodeled microbial cell that has the
selected regulatory gene sequence operably linked to the selected
gene associated with a plant-beneficial function, thereby improving
the expression of the gene associated with a plant-beneficial
function.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of International
PCT Application No. PCT/US2019/039528, filed Jun. 27, 2019, which
claims the benefit of priority to U.S. Provisional Application No.
62/690,619, filed on Jun. 27, 2018, which is incorporated by
reference herein in its entirety.
INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING
[0002] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing filename:
PIVO_007_01WO_SeqList_ST25.txt, date created, Jun. 26, 2019, file
size 602 kilobytes.
BACKGROUND OF THE DISCLOSURE
[0003] By 2050 the United Nations' Food and Agriculture
Organization projects that total food production must increase by
70% to meet the needs of a growing population, a challenge that is
exacerbated by numerous factors, including: diminishing freshwater
resources, increasing competition for arable land, rising energy
prices, increasing input costs, and the likely need for crops to
adapt to the pressures of a drier, hotter, and more extreme global
climate.
[0004] Current agricultural practices are not well equipped to meet
this growing demand for food production, while simultaneously
balancing the environmental impacts that result from increased
agricultural intensity.
[0005] One of the major agricultural inputs needed to satisfy
global food demand is nitrogen fertilizer. However, the current
industrial standard utilized to produce nitrogen fertilizer, is an
artificial nitrogen fixation method called the Haber-Bosch process,
which converts atmospheric nitrogen (N.sub.2) to ammonia (NH.sub.3)
by a reaction with hydrogen (H.sub.2) using a metal catalyst under
high temperatures and pressures. This process is resource intensive
and deleterious to the environment.
[0006] In contrast to the synthetic Haber-Bosch process, certain
biological systems have evolved to fix atmospheric nitrogen. These
systems utilize an enzyme called nitrogenase that catalyzes the
reaction between N.sub.2 and H.sub.2, and results in nitrogen
fixation. For example, rhizobia are diazotrophic bacteria that fix
nitrogen after becoming established inside root nodules of legumes.
An important goal of nitrogen fixation research is the extension of
this phenotype to non-leguminous plants, particularly to important
agronomic grasses such as wheat, rice, and corn. However, despite
the significant progress made in understanding the development of
the nitrogen-fixing symbiosis between rhizobia and legumes, the
path to use that knowledge to induce nitrogen-fixing nodules on
non-leguminous crops is still not clear.
[0007] Consequently, the vast majority of modern row crop
agriculture utilizes nitrogen fertilizer that is produced via the
resource intensive and environmentally deleterious Haber-Bosch
process. For instance, the USDA indicates that the average U.S.
corn farmer typically applies between 130 and 200 lb. of nitrogen
per acre (146 to 224 kg/ha). This nitrogen is not only produced in
a resource intensive synthetic process, but is applied by heavy
machinery crossing/impacting the field's soil, burning petroleum,
and requiring hours of human labor.
[0008] Furthermore, the nitrogen fertilizer produced by the
industrial Haber-Bosch process is not well utilized by the target
crop. Rain, runoff, heat, volatilization, and the soil microbiome
degrade the applied chemical fertilizer. This equates to not only
wasted money, but also adds to increased pollution instead of
harvested yield. To this end, the United Nations has calculated
that nearly 80% of fertilizer is lost before a crop can utilize it.
Consequently, modern agricultural fertilizer production and
delivery is not only deleterious to the environment, but it is
extremely inefficient.
[0009] In order to meet the world's growing food supply
needs--while also balancing resource utilization and providing
minimal impacts upon environmental systems--a better approach to
nitrogen fixation and delivery to plants is urgently needed.
SUMMARY OF THE DISCLOSURE
[0010] Provided herein are guided microbial remodeling (GMR)
methods for the rational improvement of plant-associated microbes
to perform plant-beneficial functions.
[0011] In one aspect, the GMR method comprises: (a) providing a
plurality of microbial species that are associated with a target
plant of interest; (b) assaying the plurality of microbial species
for: colonization metrics, and ability to perform a
plant-beneficial function of interest; (c) selecting a candidate
microbial species from the plurality of assayed microbial species;
(d) introducing one or more targeted non-intergeneric genetic
variations into the candidate microbial species; (e) confirming
integration of the non-intergeneric genetic variation at the target
genomic locus and absence of any transgenetic sequence; and (f)
repeating steps d) and e) one or more times, until the candidate
microbial species has acquired an improved ability to perform the
plant-beneficial function of interest.
[0012] In one aspect, the step b) of assaying the plurality of
microbial species comprises assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions.
[0013] In one aspect, the GMR method comprises a step of sequencing
the genomes of the plurality of microbial species obtained in step
a) and characterizing one or more genomic pathways, or sets of
genes, that are associated with a plant-beneficial function of
interest.
[0014] In one aspect, the step d) of introducing one or more
targeted non-intergeneric genetic variations into the candidate
microbial species comprises: (a) transforming the candidate
microbial species with a transformation plasmid comprising (i) a
selection marker, (ii) a counterselection marker, (iii) a DNA
fragment comprising: a non-intergeneric genetic variation to be
introduced into the candidate microbial species at a target genomic
locus in one or more genomic pathways, or sets of genes, that are
associated with a plant-beneficial function of interest, and
homology arms to the target genomic locus flanking the
non-intergeneric genetic variation, and (iv) plasmid backbone; (b)
selecting for a candidate microbial species that has undergone an
initial homologous recombination and has the non-intergeneric
genetic variation integrated into the target genomic locus based on
the presence of the selection marker in the genome; and (c)
selecting for a candidate microbial species that has the
non-intergeneric genetic variation integrated into the target
genomic locus, but has undergone an additional homologous
recombination that loops-out the plasmid backbone, based on the
absence of the counterselection marker.
[0015] In one aspect, the step e) of confirming integration of the
non-intergeneric genetic variation at the target genomic locus and
absence of any transgenetic sequence comprises sequencing the
genome of the transformed candidate microbial species and
confirming absence of any transgenetic sequence from the genome of
the transformed candidate microbial species. In one aspect, the
step e) comprises confirming absence of any transgenetic sequence
from the transformation plasmid.
[0016] In one aspect, the GMR method comprises: (a) providing a
plurality of microbial species that are associated with a target
plant of interest; (b) sequencing the genomes of the plurality of
microbial species and characterizing one or more genomic pathways,
or sets of genes, that are associated with a plant-beneficial
function of interest; (c) assaying the plurality of microbial
species for: colonization metrics, transcriptionally active genes
under metabolically relevant environmental conditions, and ability
to perform a plant-beneficial function of interest; (d) selecting a
candidate microbial species from the plurality of assayed microbial
species; (e) transforming the candidate microbial species with a
transformation plasmid comprising: (i) a selection marker, (ii) a
counterselection marker, (iii) a DNA fragment comprising: a
non-intergeneric genetic variation to be introduced into the
candidate microbial species at a target genomic locus in one or
more genomic pathways, or sets of genes, that are associated with a
plant-beneficial function of interest, and homology arms to the
target genomic locus flanking the non-intergeneric genetic
variation; and (iv) plasmid backbone; (f) selecting for a candidate
microbial species that has undergone an initial homologous
recombination and has the non-intergeneric genetic variation
integrated into the target genomic locus based on the presence of
the selection marker in the genome; (g) selecting for a candidate
microbial species that has the non-intergeneric genetic variation
integrated into the target genomic locus, but has undergone an
additional homologous recombination that loops-out the plasmid
backbone, based on the absence of the counterselection marker; (h)
sequencing the genome of the transformed candidate microbial
species and confirming integration of the non-intergeneric genetic
variation at the target genomic locus and absence of any genetic
sequence from the transformation plasmid; and (i) repeating steps
e)-h) one or more times, until a candidate microbial species has
acquired an improved ability to perform the plant-beneficial
function of interest.
[0017] In one aspect, the plant-beneficial functions of interest in
the methods described herein can be nitrogen fixation, phosphate
solubilization, microbial colonization, or combinations
thereof.
[0018] In one aspect, the step of sequencing the genomes of the
plurality of microbial species and characterizing one or more
genomic pathways, or sets of genes, that are associated with a
plant-beneficial function of interest (e.g. step b of the method
described above) comprises whole genome sequencing and/or
characterizing the nitrogen fixation pathway.
[0019] In one aspect, characterizing the nitrogen fixation pathway
comprises characterizing a set of genes selected from the group
consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding
glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide
encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY,
nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a
gene associated with biosynthesis of a nitrogenase enzyme, and
combinations thereof.
[0020] In one aspect, characterizing the microbial colonization
pathway comprises characterizing one or more genes involved in a
pathway selected from the group consisting of: exopolysaccharide
production, endo-polygalaturonase production, trehalose production,
and glutamine conversion.
[0021] In one aspect, characterizing the microbial colonization
pathway comprises characterizing one or more genes selected from
the group consisting of: bcsii, bcsiii, yjbE, fhaB, pehA, otsB,
treZ, glsA2, and combinations thereof.
[0022] In one aspect, characterizing the microbial colonization
pathway comprises characterizing one or more genes selected from
the group consisting of: nifA, nijL, ntrB, ntrC, polynucleotide
encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB,
polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD,
nijK, nifY, nijE, nijN, nifU, nijS, nifV, nifW, nijZ, nifM, nijF,
nijB, nifQ, a gene associated with biosynthesis of a nitrogenase
enzyme, bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, and
combinations thereof.
[0023] In one aspect, the step of assaying the plurality of
microbial species for colonization metrics (e.g. in step c of the
method described above) comprises assaying the plurality of
microbial species for colonization metrics under greenhouse or lab
based conditions and/or under field conditions.
[0024] In one aspect, the colonization metric comprises at least
one of the following: spatial colonization patterns, temporal
colonization dynamics, density of colonization, or combinations
thereof.
[0025] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the method described above) occurs under greenhouse or lab based
conditions.
[0026] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the method described above) occurs under field conditions.
[0027] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the method described above) occurs in vitro.
[0028] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the method described above) occurs under greenhouse or lab based
conditions; under field conditions; and/or in vitro.
[0029] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the method described above) occurs under greenhouse or lab based
conditions and comprises (i) measuring the transcriptomic profile
of the microbial species; (ii) measuring the transcriptomic
activity of genes associated with the microbial species' ability to
perform a plant-beneficial function of interest; (iii) measuring
the transcriptomic activity of regulatory gene sequences; (iv)
measuring the transcriptomic activity of promoter sequences; (v)
measuring the transcriptomic activity of promoter sequences in the
presence of exogenous nitrogen; and/or (vi) measuring the
transcriptomic activity of promoter sequences in the presence of
exogenous nitrogen, wherein said transcriptomic activity of the
promoter sequences is measured by quantifying the expression of a
regulated gene.
[0030] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the method described above) occurs under field conditions and
comprises (i) measuring the transcriptomic profile of the microbial
species; (ii) measuring the transcriptomic activity of genes
associated with the microbial species' ability to perform a
plant-beneficial function of interest; (iii) measuring the
transcriptomic activity of regulatory gene sequences; (iv)
measuring the transcriptomic activity of promoter sequences; (v)
measuring the transcriptomic activity of promoter sequences in the
presence of exogenous nitrogen; and/or (vi) measuring the
transcriptomic activity of promoter sequences in the presence of
exogenous nitrogen, wherein said transcriptomic activity of the
promoter sequences is measured by quantifying the expression of a
regulated gene.
[0031] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the method described above) occurs in vitro and comprises (i)
measuring the transcriptomic profile of the microbial species; (ii)
measuring the transcriptomic activity of genes associated with the
microbial species' ability to perform a plant-beneficial function
of interest; (iii) measuring the transcriptomic activity of
regulatory gene sequences; (iv) measuring the transcriptomic
activity of promoter sequences; (v) measuring the transcriptomic
activity of promoter sequences in nitrogen-depleted and
nitrogen-replete conditions; and/or (vi) measuring the
transcriptomic activity of promoter sequences in nitrogen-depleted
and nitrogen-replete conditions, wherein said transcriptomic
activity of the promoter sequences is measured by quantifying the
expression of a regulated gene.
[0032] In one aspect, the step of assaying the plurality of
microbial species for colonization metrics (e.g. in step c of the
guided microbial remodeling method described above) comprises:
growing said plurality of microbial species in intimate association
with a target plant.
[0033] In one aspect, the step of assaying the plurality of
microbial species for colonization metrics (e.g. in step c of the
guided microbial remodeling method described above) comprises:
growing said plurality of microbial species in intimate association
with a target plant under greenhouse or lab based conditions.
[0034] In one aspect, the step of assaying the plurality of
microbial species for colonization metrics (e.g. in step c of the
guided microbial remodeling method described above) comprises:
growing said plurality of microbial species in intimate association
with a target plant under field conditions.
[0035] In one aspect, the step of assaying the plurality of
microbial species for colonization metrics (e.g. in step c of the
guided microbial remodeling method described above) comprises:
growing said plurality of microbial species in intimate association
with a target plant under greenhouse or lab based conditions and
under field conditions.
[0036] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the guided microbial remodeling method described above) comprises:
growing said plurality of microbial species in intimate association
with a target plant.
[0037] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the guided microbial remodeling method described above) comprises:
growing said plurality of microbial species in intimate association
with a target plant under greenhouse or lab based conditions.
[0038] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the guided microbial remodeling method described above) comprises:
growing said plurality of microbial species in intimate association
with a target plant under field conditions.
[0039] In one aspect, the step of assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions (e.g. in step c of
the guided microbial remodeling method described above) comprises:
growing said plurality of microbial species in intimate association
with a target plant under greenhouse or lab based conditions and
under field conditions.
[0040] In one aspect, the step of assaying the plurality of
microbial species for colonization metrics and transcriptionally
active genes under metabolically relevant environmental conditions
(e.g. in step c of the guided microbial remodeling method described
above) comprises: growing said plurality of microbial species in
intimate association with a target plant under field
conditions.
[0041] In one aspect, the step of assaying the plurality of
microbial species in step c of the guided microbial remodeling
method described above comprises assaying the plurality of
microbial species for ability to perform a plant-beneficial
function of interest under greenhouse or lab based conditions.
[0042] In one aspect, the step of assaying the plurality of
microbial species in step c of the guided microbial remodeling
method described above comprises assaying the plurality of
microbial species for nitrogen fixation activity. In one aspect,
the nitrogen fixation activity is assayed in an acetylene reduction
assay or ammonium excretion assay.
[0043] In one aspect, the transformation plasmid used in step e of
the guided microbial remodeling method described above is a suicide
plasmid.
[0044] In one aspect, sequencing the genome of the transformed
candidate microbial species and confirming integration of the
non-intergeneric genetic variation at the target genomic locus and
absence of any genetic sequence from the transformation plasmid
(step h of the guided microbial remodeling method described above)
comprises whole genome sequencing.
[0045] In one aspect, provided herein is a guided microbial
remodeling method for the rational improvement of plant-associated
microbes to perform plant-beneficial functions, comprising: (a)
providing a plurality of microbial species; (b) assaying the
plurality of microbial species for: colonization metrics, and
ability to perform a plant-beneficial function of interest; (c)
selecting a candidate microbial species from the plurality of
assayed microbial species; (d) introducing one or more targeted
non-intergeneric genetic variations into the candidate microbial
species at a target genomic locus in one or more genomic pathways,
or sets of genes, that are associated with a plant-beneficial
function of interest; (e) confirming integration of the
non-intergeneric genetic variation at the target genomic locus and
absence of any transgenic genetic sequence; and (0 repeating steps
d)-e) one or more times, until a candidate microbial species has
acquired an improved ability to perform the plant-beneficial
function of interest. In a further aspect of this embodiment, the
step b) comprises assaying transcriptionally active genes under
metabolically relevant environmental conditions. In a further
aspect of this embodiment, in step d), said non-intergeneric
genetic variations are selected from the group consisting of: full
gene deletions, partial gene deletions, promoter insertions, single
base pair changes, and combinations thereof. In yet further aspect
of this embodiment, step e) comprises sequencing the genome of the
candidate microbial species.
[0046] In one aspect, provided herein is A guided microbial
remodeling method for the rational improvement of plant-associated
microbes to perform plant-beneficial functions, comprising: (a)
providing a plurality of microbial species; (b) assaying the
plurality of microbial species for: colonization metrics, and
ability to perform a plant-beneficial function of interest; (c)
selecting a candidate microbial species from the plurality of
assayed microbial species; (d) introducing two or more targeted
non-intergeneric genetic variations into the candidate microbial
species at two or more target genomic loci, in one or more genomic
pathways, or sets of genes, that are associated with a
plant-beneficial function of interest; and (e) confirming
introduction of the non-intergeneric genetic variations at the
target genomic loci and absence of any transgenic genetic sequence.
In a further aspect of this embodiment, step b) comprises assaying
transcriptionally active genes under metabolically relevant
environmental conditions. In a further aspect of this embodiment,
in step d), said non-intergeneric genetic variations are selected
from the group consisting of: full gene deletions, partial gene
deletions, promoter insertions, single base pair changes, and
combinations thereof. In a further aspect of this embodiment, step
e) comprises sequencing the genome of the candidate microbial
species.
[0047] In one aspect, provided herein is a guided microbial
remodeling method for the rational improvement of plant-associated
microbes to perform plant-beneficial functions, comprising: (a)
providing a plurality of microbial species; (b) assaying the
plurality of microbial species for: colonization metrics,
transcriptionally active genes under metabolically relevant
environmental conditions, and ability to perform a plant-beneficial
function of interest; (c) selecting a candidate microbial species
from the plurality of assayed microbial species; (d) introducing
one or more targeted non-intergeneric genetic variations into the
candidate microbial species at a target genomic locus in one or
more genomic pathways, or sets of genes, that are associated with a
plant-beneficial function of interest, said non-intergeneric
genetic variations selected from the group consisting of: full gene
deletions, partial gene deletions, promoter insertions, single base
pair changes, and combinations thereof (e) sequencing the genome of
the candidate microbial species and confirming integration of the
non-intergeneric genetic variation at the target genomic locus and
absence of any transgenic genetic sequence; and (0 repeating steps
d)-e) one or more times, until a candidate microbial species has
acquired an improved ability to perform the plant-beneficial
function of interest.
[0048] In one aspect, provided herein is a guided microbial
remodeling method for the rational improvement of plant-associated
microbes to perform plant-beneficial functions, comprising: (a)
providing a plurality of microbial species; (b) assaying the
plurality of microbial species for: colonization metrics,
transcriptionally active genes under metabolically relevant
environmental conditions, and ability to perform a plant-beneficial
function of interest; (c) selecting a candidate microbial species
from the plurality of assayed microbial species; (d) introducing
two or more targeted non-intergeneric genetic variations into the
candidate microbial species at two or more target genomic loci, in
one or more genomic pathways, or sets of genes, that are associated
with a plant-beneficial function of interest, said non-intergeneric
genetic variations selected from the group consisting of: full gene
deletions, partial gene deletions, promoter insertions, single base
pair changes, and combinations thereof; and (e) sequencing the
genome of the candidate microbial species and confirming
introduction of the non-intergeneric genetic variations at the
target genomic loci and absence of any transgenic genetic
sequence.
[0049] In one aspect, provided is a computationally guided
microbial remodeling method for the rational improvement of
plant-associated microbes to perform plant-beneficial functions,
comprising: (a) accessing a plurality of microbial whole genome
sequences; (b) identifying a plurality of regulatory gene sequences
that actively regulate the transcription of a gene under a
metabolically relevant environmental condition; (c) identifying a
plurality of genes associated with a plant-beneficial function; (d)
selecting a regulatory gene sequence and a gene associated with a
plant-beneficial function from said pluralities; wherein steps
a)-d) occur in silico; and (e) manufacturing, in vivo, a remodeled
microbial cell that has the selected regulatory gene sequence
operably linked to the selected gene associated with a
plant-beneficial function, thereby improving the expression of the
gene associated with a plant-beneficial function.
[0050] In one aspect, provided is a computationally guided
microbial remodeling system for the rational improvement of
plant-associated microbes to perform plant-beneficial functions,
comprising: (a) one or more processors; and (b) one or more
memories operatively coupled to the one or more processors and
having instructions stored thereon, that when executed by the one
or more processors, cause the system to: (i) access a plurality of
microbial whole genome sequences; (ii) identify a plurality of
regulatory gene sequences that actively regulate the transcription
of a gene under a metabolically relevant environmental condition;
(iii) identify a plurality of genes associated with a
plant-beneficial function; and (iv) select a regulatory gene
sequence and a gene associated with a plant-beneficial function
from said pluralities.
[0051] In one aspect, provided is a computationally guided
microbial remodeling method for the rational improvement of
plant-associated microbes to perform plant-beneficial functions,
comprising: (a) activating a computer system having: one or more
processors and one or more memories operatively coupled to the one
or more processors and having instructions stored thereon, thereby
causing the one or more processors to execute the instructions, and
cause the system to: (i) access a plurality of microbial whole
genome sequences; (ii) identify a plurality of regulatory gene
sequences that actively regulate the transcription of a gene under
a metabolically relevant environmental condition; (iii) identify a
plurality of genes associated with a plant-beneficial function;
(iv) select a regulatory gene sequence and a gene associated with a
plant-beneficial function from said pluralities; and (b)
manufacturing, in vivo, a remodeled microbial cell that has the
selected regulatory gene sequence operably linked to the selected
gene associated with a plant-beneficial function, thereby improving
the expression of the gene associated with a plant-beneficial
function.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1A depicts an overview of the guided microbial
remodeling process, in accordance with embodiments.
[0053] FIG. 1B depicts an expanded view of the measurement of
microbiome composition as shown in FIG. 1A.
[0054] FIG. 1C depicts a problematic "traditional bioprospecting"
approach, which has several drawbacks compared to the taught guided
microbial remodeling (GMR) platform.
[0055] FIG. 1D depicts a problematic "field-first approach to
bioprospecting" system, which has several drawbacks compared to the
taught guided microbial remodeling (GMR) platform.
[0056] FIG. 1E depicts the time period in the corn growth cycle, at
which nitrogen is needed most by the plant.
[0057] FIG. 1F depicts an overview of a field development process
for a remodeled microbe.
[0058] FIG. 1G depicts an overview of a guided microbial remodeling
platform embodiment.
[0059] FIG. 1H depicts an overview of a computationally-guided
microbial remodeling platform.
[0060] FIG. 1I depicts the use of field data combined with modeling
in aspects of the guided microbial remodeling platform.
[0061] FIG. 1J depicts 5 properties that can be possessed by
remodeled microbes of the present disclosure.
[0062] FIG. 1K depicts a schematic of a remodeling approach for a
microbe, PBC6.1.
[0063] FIG. 1L depicts decoupled nifA expression from endogenous
nitrogen regulation in remodeled microbes.
[0064] FIG. 1M depicts improved assimilation and excretion of fixed
nitrogen by remodeled microbes.
[0065] FIG. 1N depicts corn yield improvement attributable to
remodeled microbes.
[0066] FIG. 1O illustrates the inefficiency of current nitrogen
delivery systems, which result in underfertilized fields, over
fertilized fields, and environmentally deleterious nitrogen
runoff
[0067] FIG. 2 illustrates PBC6.1 colonization to nearly 21%
abundance of the root-associated microbiota in corn roots.
Abundance data is based on 16S amplicon sequencing of the
rhizosphere and endosphere of corn plants inoculated with PBC6.1
and grown in greenhouse conditions.
[0068] FIGS. 3A-3E illustrate derivative microbes that fix and
excrete nitrogen in vitro under conditions similar to high nitrate
agricultural soils. FIG. 3A illustrates the regulatory network
controlling nitrogen fixation and assimilation in PBC6.1 is shown,
including the key nodes NifL, NifA, GS, GlnE depicted as the
two-domain ATase-AR enzyme, and AmtB. FIG. 3B illustrates the
genome of Kosakonia sacchari isolate PBC6.1. The three tracks
circumscribing the genome convey transcription data from PBC6.1,
PBC6.38, and the differential expression between the strains
respectively. FIG. 3C illustrates the nitrogen fixation gene
cluster and transcription data is expanded for finer detail. FIG.
3D illustrates nitrogenase activity under varying concentrations of
exogenous nitrogen is measured with the acetylene reduction assay.
The wild type strain exhibits repression of nitrogenase activity as
glutamine concentrations increase, while derivative strains show
varying degrees of robustness. In the line graph, triangles
represent strain PBC6.22; circles represent strain PBC6.1; squares
represent strain PBC6.15; and diamonds represent strain PBC6.14.
Error bars represent standard error of the mean of at least three
biological replicates. FIG. 3E illustrates temporal excretion of
ammonia by derivative strains is observed at mM concentrations.
Wild type strains are not observed to excrete fixed nitrogen, and
negligible ammonia accumulates in the media. Error bars represent
standard error of the mean.
[0069] FIG. 4 illustrates transcriptional rates of nifA in
derivative strains of PBC6.1 correlated with acetylene reduction
rates. An ARA assay was performed as described in the Methods,
after which cultures were sampled and subjected to qPCR analysis to
determine nifA transcript levels. Error bars show standard error of
the mean of at least three biological replicates in each
measure.
[0070] FIGS. 5A-5C illustrate greenhouse experiments that
demonstrate microbial nitrogen fixation in corn. FIG. 5A
illustrates microbe colonization six weeks after inoculation of
corn plants by PBC6.1 derivative strains. Error bars show standard
error of the mean of at least eight biological replicates. FIG. 5B
illustrates in planta transcription of nifH measured by extraction
of total RNA from roots and subsequent Nanostring analysis. Only
derivative strains show nifH transcription in the root environment.
Error bars show standard error of the mean of at least 3 biological
replicates. FIG. 5C illustrates microbial nitrogen fixation
measured by the dilution of isotopic tracer in plant tissues.
Derivative microbes exhibit substantial transfer of fixed nitrogen
to the plant. Error bars show standard error of the mean of at
least ten biological replicates.
[0071] FIG. 6 depicts the lineage of modified strains that were
derived from strain C1006.
[0072] FIG. 7 depicts the lineage of modified strains that were
derived from strain C1019.
[0073] FIG. 8 depicts a heatmap of the pounds of nitrogen delivered
per acre-season by microbes of the present disclosure recorded as a
function of microbes per g-fresh weight by mmol of
nitrogen/microbe-hr. Below the thin line that transects the larger
image are the microbes that deliver less than one pound of nitrogen
per acre-season, and above the line are the microbes that deliver
greater than one pound of nitrogen per acre-season. The table below
the heatmap gives the precise value of mmol N produced per microbe
per hour (mmol N/Microbe hr) along with the precise CFU per gram of
fresh weight (CFU/g fw) for each microbe shown in the heatmap. The
microbes utilized in the heatmap were assayed for N production in
corn. For the WT strains C1006 and C1019, corn root colonization
data was taken from a single field site. For the remaining strains,
colonization was assumed to be the same as the WT field level.
N-fixation activity was determined using an in vitro ARA assay at 5
mM glutamine.
[0074] FIG. 9 depicts the plant yield of plants having been exposed
to strain C1006. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0075] FIG. 10 depicts the plant yield of plants having been
exposed to strain CM029. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0076] FIG. 11 depicts the plant yield of plants having been
exposed to strain CM038. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0077] FIG. 12 depicts the plant yield of plants having been
exposed to strain CI019. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0078] FIG. 13 depicts the plant yield of plants having been
exposed to strain CM081. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0079] FIG. 14 depicts the plant yield of plants having been
exposed to strains CM029 and CM081. The area of the circles
corresponds to the relative yield, while the shading corresponds to
the particular MRTN treatment. The x-axis is the p value and the
y-axis is the win rate.
[0080] FIG. 15 depicts the plant yield of plants as the aggregated
bushel gain/loss. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0081] FIG. 16 illustrates results from a summer 2017 field testing
experiment. The yield results obtained demonstrate that the
microbes of the disclosure can serve as a potential fertilizer
replacement. For instance, the utilization of a microbe of the
disclosure (i.e. 6-403) resulted in a higher yield than the wild
type strain (WT) and a higher yield than the untreated control
(UTC). The "-25 lbs N" treatment utilizes 25 lbs less N per acre
than standard agricultural practices of the region. The "100% N"
UTC treatment is meant to depict standard agricultural practices of
the region, in which 100% of the standard utilization of N is
deployed by the farmer. The microbe "6-403" was deposited as NCMA
201708004 and can be found in Table 1. This is a mutant Kosakonia
sacchari (also called CM037) and is a progeny mutant strain from
CI006 WT.
[0082] FIG. 17 illustrates results from a summer 2017 field testing
experiment. The yield results obtained demonstrate that the
microbes of the disclosure perform consistently across locations.
Furthermore, the yield results demonstrate that the microbes of the
disclosure perform well in both a nitrogen stressed environment, as
well as an environment that has sufficient supplies of nitrogen.
The microbe "6-881" (also known as CM094, PBC6.94), and which is a
progeny mutant Kosakonia sacchari strain from CI006 WT, was
deposited as NCMA 201708002 and can be found in Table 1. The
microbe "137-1034," which is a progeny mutant Klebsiella variicola
strain from CI137 WT, was deposited as NCMA 201712001 and can be
found in Table 1. The microbe "137-1036," which is a progeny mutant
Klebsiella variicola strain from CI137 WT, was deposited as NCMA
201712002 and can be found in Table 1. The microbe "6-404" (also
known as CM38, PBC6.38), and which is a progeny mutant Kosakonia
sacchari strain from CI006 WT, was deposited as NCMA 201708003 and
can be found in Table 1. The "Nutrient Stress" condition
corresponds to the 0% nitrogen regime. The "Sufficient Fertilizer"
condition corresponds to the 100% nitrogen regime.
[0083] FIG. 18 depicts the lineage of modified strains that were
derived from strain CI006 (also termed "6", Kosakonia sacchari
WT).
[0084] FIG. 19 depicts the lineage of modified strains that were
derived from strain CI019 (also termed "19", Rahnella aquatilis
WT).
[0085] FIG. 20 depicts the lineage of modified strains that were
derived from strain CI137 (also termed ("137", Klebsiella variicola
WT).
[0086] FIG. 21 depicts the lineage of modified strains that were
derived from strain 1021 (Kosakonia pseudosacchari WT).
[0087] FIG. 22 depicts the lineage of modified strains that were
derived from strain 910 (Kluyvera intermedia WT).
[0088] FIG. 23 depicts the lineage of modified strains that were
derived from strain 63 (Rahnella aquatilis WT).
[0089] FIG. 24 depicts a heatmap of the pounds of nitrogen
delivered per acre-season by microbes of the present disclosure
recorded as a function of microbes per g-fresh weight by mmol of
nitrogen/microbe-hr. Below the thin line that transects the larger
image are the microbes that deliver less than one pound of nitrogen
per acre-season, and above the line are the microbes that deliver
greater than one pound of nitrogen per acre-season. The Table 28 in
Example 5 gives the precise value of mmol N produced per microbe
per hour (mmol N/Microbe hr) along with the precise CFU per gram of
fresh weight (CFU/g fw) for each microbe shown in the heatmap. The
data in FIG. 24 is derived from microbial strains assayed for N
production in corn in field conditions. Each point represents lb
N/acre produced by a microbe using corn root colonization data from
a single field site. N-fixation activity was determined using in
vitro ARA assay at 5 mM N in the form of glutamine or ammonium
phosphate.
[0090] FIG. 25 depicts a heatmap of the pounds of nitrogen
delivered per acre-season by microbes of the present disclosure
recorded as a function of microbes per g-fresh weight by mmol of
nitrogen/microbe-hr. Below the thin line that transects the larger
image are the microbes that deliver less than one pound of nitrogen
per acre-season, and above the line are the microbes that deliver
greater than one pound of nitrogen per acre-season. The Table 29 in
Example 5 gives the precise value of mmol N produced per microbe
per hour (mmol N/Microbe hr) along with the precise CFU per gram of
fresh weight (CFU/g fw) for each microbe shown in the heatmap. The
data in FIG. 25 is derived from microbial strains assayed for N
production in corn in laboratory and greenhouse conditions. Each
point represents lb N/acre produced by a single strain. White
points represent strains in which corn root colonization data was
gathered in greenhouse conditions. Black points represent mutant
strains for which corn root colonization levels are derived from
average field corn root colonization levels of the wild-type parent
strain. Hatched points represent the wild type parent strains at
their average field corn root colonization levels. In all cases,
N-fixation activity was determined by in vitro ARA assay at 5 mM N
in the form of glutamine or ammonium phosphate.
[0091] FIG. 26 illustrates excretion of ammonia excretion by WT
CI137 and mutant strains described in Example 7. The WT strain was
observed not to excrete ammonia, and negligible ammonia accumulates
in the media.
[0092] FIG. 27 illustrates microbial colonization in corn three
weeks after planting. The corn seed was inoculated at planting by
CI137 WT and derivative strains described in Example 7.
[0093] FIG. 28 illustrates the same data from FIG. 27 but in a
slightly different way. FIG. 28 illustrates average microbial
colonization in corn three weeks after planting. The corn seed was
inoculated at planting by CI137 WT and derivative strains described
in Example 7.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0094] While various embodiments of the disclosure have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions may occur to those
skilled in the art without departing from the disclosure. It should
be understood that various alternatives to the embodiments of the
disclosure described herein may be employed.
[0095] Increased fertilizer utilization brings with it
environmental concerns and is also likely not possible for many
economically stressed regions of the globe. Furthermore, many
industry players in the microbial arena are focused on creating
intergeneric microbes. However, there is a heavy regulatory burden
placed on engineered microbes that are characterized/classified as
intergeneric. These intergeneric microbes face not only a higher
regulatory burden, which makes widespread adoption and
implementation difficult, but they also face a great deal of public
perception scrutiny.
[0096] Currently, there are no engineered microbes on the market
that are non-intergeneric and that are capable of increasing
nitrogen fixation in non-leguminous crops. This dearth of such a
microbe is a missing element in helping to usher in a truly
environmentally friendly and more sustainable 21.sup.st century
agricultural system.
[0097] The present disclosure solves the aforementioned problems
and provides a non-intergeneric microbe that has been engineered to
readily fix nitrogen in crops. These microbes are not
characterized/classified as intergeneric microbes and thus will not
face the steep regulatory burdens of such. Further, the taught
non-intergeneric microbes will serve to help 21.sup.st century
farmers become less dependent upon utilizing ever increasing
amounts of exogenous nitrogen fertilizer.
Definitions
[0098] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the disclosure (especially
in the context of the following claims) are to be construed to
cover both the singular and the plural, unless otherwise indicated
herein or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. For example, if the range 10-15 is disclosed, then
11, 12, 13, and 14 are also disclosed. All methods described herein
can be performed in any suitable order unless otherwise indicated
herein or otherwise clearly contradicted by context. The use of any
and all examples, or exemplary language (e.g., "such as") provided
herein, is intended merely to better illuminate the disclosure and
does not pose a limitation on the scope of the disclosure unless
otherwise claimed. No language in the specification should be
construed as indicating any non-claimed element as essential to the
practice of the disclosure.
[0099] The terms "polynucleotide", "nucleotide", "nucleotide
sequence", "nucleic acid" and "oligonucleotide" are used
interchangeably. They refer to a polymeric form of nucleotides of
any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Polynucleotides may have any three-dimensional
structure, and may perform any function, known or unknown. The
following are non-limiting examples of polynucleotides: coding or
non-coding regions of a gene or gene fragment, loci (locus) defined
from linkage analysis, exons, introns, messenger RNA (mRNA),
transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA
(siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes,
cDNA, recombinant polynucleotides, branched polynucleotides,
plasmids, vectors, isolated DNA of any sequence, isolated RNA of
any sequence, nucleic acid probes, and primers. A polynucleotide
may comprise one or more modified nucleotides, such as methylated
nucleotides and nucleotide analogs. If present, modifications to
the nucleotide structure may be imparted before or after assembly
of the polymer. The sequence of nucleotides may be interrupted by
non-nucleotide components. A polynucleotide may be further modified
after polymerization, such as by conjugation with a labeling
component.
[0100] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson Crick base pairing, Hoogstein
binding, or in any other sequence specific manner according to base
complementarity. The complex may comprise two strands forming a
duplex structure, three or more strands forming a multi stranded
complex, a single self-hybridizing strand, or any combination of
these. A hybridization reaction may constitute a step in a more
extensive process, such as the initiation of PCR, or the enzymatic
cleavage of a polynucleotide by an endonuclease. A second sequence
that is complementary to a first sequence is referred to as the
"complement" of the first sequence. The term "hybridizable" as
applied to a polynucleotide refers to the ability of the
polynucleotide to form a complex that is stabilized via hydrogen
bonding between the bases of the nucleotide residues in a
hybridization reaction.
[0101] "Complementarity" refers to the ability of a nucleic acid to
form hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick or other non-traditional types. A percent
complementarity indicates the percentage of residues in a nucleic
acid molecule which can form hydrogen bonds (e.g., Watson-Crick
base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7,
8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%
complementary, respectively). "Perfectly complementary" means that
all the contiguous residues of a nucleic acid sequence will
hydrogen bond with the same number of contiguous residues in a
second nucleic acid sequence. "Substantially complementary" as used
herein refers to a degree of complementarity that is at least 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a
region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to
two nucleic acids that hybridize under stringent conditions.
Sequence identity, such as for the purpose of assessing percent
complementarity, may be measured by any suitable alignment
algorithm, including but not limited to the Needleman-Wunsch
algorithm (see e.g. the EMBOSS Needle aligner available at
www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally
with default settings), the BLAST algorithm (see e.g. the BLAST
alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi,
optionally with default settings), or the Smith-Waterman algorithm
(see e.g. the EMBOSS Water aligner available at
www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optionally
with default settings). Optimal alignment may be assessed using any
suitable parameters of a chosen algorithm, including default
parameters.
[0102] In general, "stringent conditions" for hybridization refer
to conditions under which a nucleic acid having complementarity to
a target sequence predominantly hybridizes with a target sequence,
and substantially does not hybridize to non-target sequences.
Stringent conditions are generally sequence-dependent and vary
depending on a number of factors. In general, the longer the
sequence, the higher the temperature at which the sequence
specifically hybridizes to its target sequence. Non-limiting
examples of stringent conditions are described in detail in Tijssen
(1993), Laboratory Techniques In Biochemistry And Molecular
Biology-Hybridization With Nucleic Acid Probes Part I, Second
Chapter "Overview of principles of hybridization and the strategy
of nucleic acid probe assay", Elsevier, N.Y.
[0103] As used herein, "expression" refers to the process by which
a polynucleotide is transcribed from a DNA template (such as into
and mRNA or other RNA transcript) and/or the process by which a
transcribed mRNA is subsequently translated into peptides,
polypeptides, or proteins. Transcripts and encoded polypeptides may
be collectively referred to as "gene product." If the
polynucleotide is derived from genomic DNA, expression may include
splicing of the mRNA in a eukaryotic cell.
[0104] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component. As
used herein the term "amino acid" includes natural and/or unnatural
or synthetic amino acids, including glycine and both the D or L
optical isomers, and amino acid analogs and peptidomimetics.
[0105] As used herein, the term "about" is used synonymously with
the term "approximately." Illustratively, the use of the term
"about" with regard to an amount indicates that values slightly
outside the cited values, e.g., plus or minus 0.1% to 10%.
[0106] The term "biologically pure culture" or "substantially pure
culture" refers to a culture of a bacterial species described
herein containing no other bacterial species in quantities
sufficient to interfere with the replication of the culture or be
detected by normal bacteriological techniques.
[0107] "Plant productivity" refers generally to any aspect of
growth or development of a plant that is a reason for which the
plant is grown. For food crops, such as grains or vegetables,
"plant productivity" can refer to the yield of grain or fruit
harvested from a particular crop. As used herein, improved plant
productivity refers broadly to improvements in yield of grain,
fruit, flowers, or other plant parts harvested for various
purposes, improvements in growth of plant parts, including stems,
leaves and roots, promotion of plant growth, maintenance of high
chlorophyll content in leaves, increasing fruit or seed numbers,
increasing fruit or seed unit weight, reducing NO.sub.2 emission
due to reduced nitrogen fertilizer usage and similar improvements
of the growth and development of plants.
[0108] Microbes in and around food crops can influence the traits
of those crops. Plant traits that may be influenced by microbes
include: yield (e.g., grain production, biomass generation, fruit
development, flower set); nutrition (e.g., nitrogen, phosphorus,
potassium, iron, micronutrient acquisition); abiotic stress
management (e.g., drought tolerance, salt tolerance, heat
tolerance); and biotic stress management (e.g., pest, weeds,
insects, fungi, and bacteria). Strategies for altering crop traits
include: increasing key metabolite concentrations; changing
temporal dynamics of microbe influence on key metabolites; linking
microbial metabolite production/degradation to new environmental
cues; reducing negative metabolites; and improving the balance of
metabolites or underlying proteins.
[0109] As used herein, a "control sequence" refers to an operator,
promoter, silencer, or terminator.
[0110] As used herein, "in planta" may refer to in the plant, on
the plant, or intimately associated with the plant, depending upon
context of usage (e.g. endophytic, epiphytic, or rhizospheric
associations). The plant may comprise plant parts, tissue, leaves,
roots, root hairs, rhizomes, stems, seed, ovules, pollen, flowers,
fruit, etc.
[0111] In some embodiments, native or endogenous control sequences
of genes of the present disclosure are replaced with one or more
intrageneric control sequences.
[0112] As used herein, "introduced" refers to the introduction by
means of modern biotechnology, and not a naturally occurring
introduction.
[0113] In some embodiments, the bacteria of the present disclosure
have been modified such that they are not naturally occurring
bacteria.
[0114] In some embodiments, the bacteria of the present disclosure
are present in the plant in an amount of at least 10.sup.3 cfu,
10.sup.4 cfu, 10.sup.5 cfu, 10.sup.6 cfu, 10.sup.7 cfu, 10.sup.8
cfu, 10.sup.9 cfu, 10.sup.10 cfu, 10.sup.11 cfu, or 10.sup.12 cfu
per gram of fresh or dry weight of the plant. In some embodiments,
the bacteria of the present disclosure are present in the plant in
an amount of at least about 10.sup.3 cfu, about 10.sup.4 cfu, about
10.sup.5 cfu, about 10.sup.6 cfu, about 10.sup.7 cfu, about
10.sup.8 cfu, about 10.sup.9 cfu, about 10.sup.10 cfu, about
10.sup.11 cfu, or about 10.sup.12 cfu per gram of fresh or dry
weight of the plant. In some embodiments, the bacteria of the
present disclosure are present in the plant in an amount of at
least 10.sup.3 to 10.sup.9, 10.sup.3 to 10.sup.7, 10.sup.3 to
10.sup.5, 10.sup.5 to 10.sup.9, 10.sup.5 to 10.sup.7, 10.sup.6 to
10.sup.10, 10.sup.6 to 10.sup.7 cfu per gram of fresh or dry weight
of the plant.
[0115] Fertilizers and exogenous nitrogen of the present disclosure
may comprise the following nitrogen-containing molecules: ammonium,
nitrate, nitrite, ammonia, glutamine, etc. Nitrogen sources of the
present disclosure may include anhydrous ammonia, ammonia sulfate,
urea, diammonium phosphate, urea-form, monoammonium phosphate,
ammonium nitrate, nitrogen solutions, calcium nitrate, potassium
nitrate, sodium nitrate, etc.
[0116] As used herein, "exogenous nitrogen" refers to
non-atmospheric nitrogen readily available in the soil, field, or
growth medium that is present under non-nitrogen limiting
conditions, including ammonia, ammonium, nitrate, nitrite, urea,
uric acid, ammonium acids, etc.
[0117] As used herein, "non-nitrogen limiting conditions" refers to
non-atmospheric nitrogen available in the soil, field, media at
concentrations greater than about 4 mM nitrogen, as disclosed by
Kant et al. (2010. J. Exp. Biol. 62(4):1499-1509), which is
incorporated herein by reference.
[0118] As used herein, an "intergeneric microorganism" is a
microorganism that is formed by the deliberate combination of
genetic material originally isolated from organisms of different
taxonomic genera. An "intergeneric mutant" can be used
interchangeably with "intergeneric microorganism". An exemplary
"intergeneric microorganism" includes a microorganism containing a
mobile genetic element that was first identified in a microorganism
in a genus different from the recipient microorganism. Further
explanation can be found, inter alia, in 40 C.F.R. .sctn.
725.3.
[0119] In aspects, microbes taught herein are "non-intergeneric,"
which means that the microbes are not intergeneric.
[0120] As used herein, an "intrageneric microorganism" is a
microorganism that is formed by the deliberate combination of
genetic material originally isolated from organisms of the same
taxonomic genera. An "intrageneric mutant" can be used
interchangeably with "intrageneric microorganism".
[0121] As used herein, "introduced genetic material" means genetic
material that is added to, and remains as a component of, the
genome of the recipient.
[0122] As used herein, in the context of non-intergeneric
microorganisms, the term "remodeled" is used synonymously with the
term "engineered". Consequently, a "non-intergeneric remodeled
microorganism" has a synonymous meaning to "non-intergeneric
engineered microorganism," and will be utilized interchangeably.
Further, the disclosure may refer to an "engineered strain" or
"engineered derivative" or "engineered non-intergeneric microbe,"
these terms are used synonymously with "remodeled strain" or
"remodeled derivative" or "remodeled non-intergeneric microbe."
[0123] In some embodiments, the nitrogen fixation and assimilation
genetic regulatory network comprises polynucleotides encoding genes
and non-coding sequences that direct, modulate, and/or regulate
microbial nitrogen fixation and/or assimilation and can comprise
polynucleotide sequences of the nif cluster (e.g., nifA, nijB,
nifC, . . . nifZ), polynucleotides encoding nitrogen regulatory
protein C, polynucleotides encoding nitrogen regulatory protein B,
polynucleotide sequences of the gln cluster (e.g. glnA and glnD),
draT, and ammonia transporters/permeases. In some cases, the Nif
cluster may comprise NifB, NifH, NifD, NifK, NifE, NifN, NifX,
hesa, and NifV. In some cases, the Nif cluster may comprise a
subset of NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and
NifV.
[0124] In some embodiments, fertilizer of the present disclosure
comprises at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,
28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,
41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,
54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% nitrogen by weight.
[0125] In some embodiments, fertilizer of the present disclosure
comprises at least about 5%, about 6%, about 7%, about 8%, about
9%, about 10%, about 11%, about 12%, about 13%, about 14%, about
15%, about 16%, about 17%, about 18%, about 19%, about 20%, about
21%, about 22%, about 23%, about 24%, about 25%, about 26%, about
27%, about 28%, about 29%, about 30%, about 31%, about 32%, about
33%, about 34%, about 35%, about 36%, about 37%, about 38%, about
39%, about 40%, about 41%, about 42%, about 43%, about 44%, about
45%, about 46%, about 47%, about 48%, about 49%, about 50%, about
51%, about 52%, about 53%, about 54%, about 55%, about 56%, about
57%, about 58%, about 59%, about 60%, about 61%, about 62%, about
63%, about 64%, about 65%, about 66%, about 67%, about 68%, about
69%, about 70%, about 71%, about 72%, about 73%, about 74%, about
75%, about 76%, about 77%, about 78%, about 79%, about 80%, about
81%, about 82%, about 83%, about 84%, about 85%, about 86%, about
87%, about 88%, about 89%, about 90%, about 91%, about 92%, about
93%, about 94%, about 95%, about 96%, about 97%, about 98%, or
about 99% nitrogen by weight.
[0126] In some embodiments, fertilizer of the present disclosure
comprises about 5% to 50%, about 5% to 75%, about 10% to 50%, about
10% to 75%, about 15% to 50%, about 15% to 75%, about 20% to 50%,
about 20% to 75%, about 25% to 50%, about 25% to 75%, about 30% to
50%, about 30% to 75%, about 35% to 50%, about 35% to 75%, about
40% to 50%, about 40% to 75%, about 45% to 50%, about 45% to 75%,
or about 50% to 75% nitrogen by weight.
[0127] In some embodiments, the increase of nitrogen fixation
and/or the production of 1% or more of the nitrogen in the plant
are measured relative to control plants, which have not been
exposed to the bacteria of the present disclosure. All increases or
decreases in bacteria are measured relative to control bacteria.
All increases or decreases in plants are measured relative to
control plants.
[0128] As used herein, a "constitutive promoter" is a promoter,
which is active under most conditions and/or during most
development stages. There are several advantages to using
constitutive promoters in expression vectors used in biotechnology,
such as: high level of production of proteins used to select
transgenic cells or organisms; high level of expression of reporter
proteins or scorable markers, allowing easy detection and
quantification; high level of production of a transcription factor
that is part of a regulatory transcription system; production of
compounds that requires ubiquitous activity in the organism; and
production of compounds that are required during all stages of
development. Non-limiting exemplary constitutive promoters include,
CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol
dehydrogenase promoter, etc.
[0129] As used herein, a "non-constitutive promoter" is a promoter
which is active under certain conditions, in certain types of
cells, and/or during certain development stages. For example,
tissue specific, tissue preferred, cell type specific, cell type
preferred, inducible promoters, and promoters under development
control are non-constitutive promoters. Examples of promoters under
developmental control include promoters that preferentially
initiate transcription in certain tissues.
[0130] As used herein, "inducible" or "repressible" promoter is a
promoter that is under chemical or environmental factor's control.
Examples of environmental conditions that may affect transcription
by inducible promoters include anaerobic conditions, certain
chemicals, the presence of light, acidic or basic conditions,
etc.
[0131] As used herein, a "tissue specific" promoter is a promoter
that initiates transcription only in certain tissues. Unlike
constitutive expression of genes, tissue-specific expression is the
result of several interacting levels of gene regulation. As such,
in the art sometimes it is preferable to use promoters from
homologous or closely related species to achieve efficient and
reliable expression of transgenes in particular tissues. This is
one of the main reasons for the large amount of tissue-specific
promoters isolated from particular tissues found in both scientific
and patent literature.
[0132] As used herein, the term "operably linked" refers to the
association of nucleic acid sequences on a single nucleic acid
fragment so that the function of one is regulated by the other. For
example, a promoter is operably linked with a coding sequence when
it is capable of regulating the expression of that coding sequence
(i.e., that the coding sequence is under the transcriptional
control of the promoter). Coding sequences can be operably linked
to regulatory sequences in a sense or antisense orientation. In
another example, the complementary RNA regions of the disclosure
can be operably linked, either directly or indirectly, 5' to the
target mRNA, or 3' to the target mRNA, or within the target mRNA,
or a first complementary region is 5' and its complement is 3' to
the target mRNA.
[0133] In aspects, "applying to the plant a plurality of
non-intergeneric bacteria," includes any means by which the plant
(including plant parts such as a seed, root, stem, tissue, etc.) is
made to come into contact (i.e. exposed) with said bacteria at any
stage of the plant's life cycle. Consequently, "applying to the
plant a plurality of non-intergeneric bacteria," includes any of
the following means of exposing the plant (including plant parts
such as a seed, root, stem, tissue, etc.) to said bacteria:
spraying onto plant, dripping onto plant, applying as a seed coat,
applying to a field that will then be planted with seed, applying
to a field already planted with seed, applying to a field with
adult plants, etc.
[0134] As used herein "MRTN" is an acronym for maximum return to
nitrogen and is utilized as an experimental treatment in the
Examples. MRTN was developed by Iowa State University and
information can be found at: cnrc.agron.iastate.edu. The MRTN is
the nitrogen rate where the economic net return to nitrogen
application is maximized. The approach to calculating the MRTN is a
regional approach for developing corn nitrogen rate guidelines in
individual states. The nitrogen rate trial data was evaluated for
Illinois, Iowa, Michigan, Minnesota, Ohio, and Wisconsin where an
adequate number of research trials were available for corn
plantings following soybean and corn plantings following corn. The
trials were conducted with spring, sidedress, or split
preplant/sidedress applied nitrogen, and sites were not irrigated
except for those that were indicated for irrigated sands in
Wisconsin. MRTN was developed by Iowa State University due to
apparent differences in methods for determining suggested nitrogen
rates required for corn production, misperceptions pertaining to
nitrogen rate guidelines, and concerns about application rates. By
calculating the MRTN, practitioners can determine the following:
(1) the nitrogen rate where the economic net return to nitrogen
application is maximized, (2) the economic optimum nitrogen rate,
which is the point where the last increment of nitrogen returns a
yield increase large enough to pay for the additional nitrogen, (3)
the value of corn grain increase attributed to nitrogen
application, and the maximum yield, which is the yield where
application of more nitrogen does not result in a corn yield
increase. Thus, the MRTN calculations provide practitioners with
the means to maximize corn crops in different regions while
maximizing financial gains from nitrogen applications.
[0135] The term mmol is an abbreviation for millimole, which is a
thousandth (10.sup.-s) of a mole, abbreviated herein as mol.
[0136] As used herein the terms "microorganism" or "microbe" should
be taken broadly. These terms, used interchangeably, include but
are not limited to, the two prokaryotic domains, Bacteria and
Archaea. The term may also encompass eukaryotic fungi and
protists.
[0137] The term "microbial consortia" or "microbial consortium"
refers to a subset of a microbial community of individual microbial
species, or strains of a species, which can be described as
carrying out a common function, or can be described as
participating in, or leading to, or correlating with, a
recognizable parameter, such as a phenotypic trait of interest.
[0138] The term "microbial community" means a group of microbes
comprising two or more species or strains. Unlike microbial
consortia, a microbial community does not have to be carrying out a
common function, or does not have to be participating in, or
leading to, or correlating with, a recognizable parameter, such as
a phenotypic trait of interest.
[0139] As used herein, "isolate," "isolated," "isolated microbe,"
and like terms, are intended to mean that the one or more
microorganisms has been separated from at least one of the
materials with which it is associated in a particular environment
(for example soil, water, plant tissue, etc.). Thus, an "isolated
microbe" does not exist in its naturally occurring environment;
rather, it is through the various techniques described herein that
the microbe has been removed from its natural setting and placed
into a non-naturally occurring state of existence. Thus, the
isolated strain or isolated microbe may exist as, for example, a
biologically pure culture, or as spores (or other forms of the
strain). In aspects, the isolated microbe may be in association
with an acceptable carrier, which may be an agriculturally
acceptable carrier.
[0140] In certain aspects of the disclosure, the isolated microbes
exist as "isolated and biologically pure cultures." It will be
appreciated by one of skill in the art that an isolated and
biologically pure culture of a particular microbe denotes that said
culture is substantially free of other living organisms and
contains only the individual microbe in question. The culture can
contain varying concentrations of said microbe. The present
disclosure notes that isolated and biologically pure microbes often
"necessarily differ from less pure or impure materials." See, e.g.
In re Bergstrom, 427 F.2d 1394, (CCPA 1970)(discussing purified
prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA
1979)(discussing purified microbes), see also, Parke-Davis &
Co. v. H. K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned
Hand discussing purified adrenaline), aff'd in part, rev'd in part,
196 F. 496 (2d Cir. 1912), each of which are incorporated herein by
reference. Furthermore, in some aspects, the disclosure provides
for certain quantitative measures of the concentration, or purity
limitations, that must be found within an isolated and biologically
pure microbial culture. The presence of these purity values, in
certain embodiments, is a further attribute that distinguishes the
presently disclosed microbes from those microbes existing in a
natural state. See, e.g., Merck & Co. v. Olin Mathieson
Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity
limitations for vitamin B12 produced by microbes), incorporated
herein by reference.
[0141] As used herein, "individual isolates" should be taken to
mean a composition, or culture, comprising a predominance of a
single genera, species, or strain, of microorganism, following
separation from one or more other microorganisms.
[0142] Microbes of the present disclosure may include spores and/or
vegetative cells. In some embodiments, microbes of the present
disclosure include microbes in a viable but non-culturable (VBNC)
state. As used herein, "spore" or "spores" refer to structures
produced by bacteria and fungi that are adapted for survival and
dispersal. Spores are generally characterized as dormant
structures; however, spores are capable of differentiation through
the process of germination. Germination is the differentiation of
spores into vegetative cells that are capable of metabolic
activity, growth, and reproduction. The germination of a single
spore results in a single fungal or bacterial vegetative cell.
Fungal spores are units of asexual reproduction, and in some cases
are necessary structures in fungal life cycles. Bacterial spores
are structures for surviving conditions that may ordinarily be
nonconducive to the survival or growth of vegetative cells.
[0143] As used herein, "microbial composition" refers to a
composition comprising one or more microbes of the present
disclosure. In some embodiments, a microbial composition is
administered to plants (including various plant parts) and/or in
agricultural fields.
[0144] As used herein, "carrier," "acceptable carrier," or
"agriculturally acceptable carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the microbe can be administered,
which does not detrimentally effect the microbe.
Regulation of Nitrogen Fixation
[0145] In some cases, nitrogen fixation pathway may act as a target
for genetic engineering and optimization. One trait that may be
targeted for regulation by the methods described herein is nitrogen
fixation. Nitrogen fertilizer is the largest operational expense on
a farm and the biggest driver of higher yields in row crops like
corn and wheat. Described herein are microbial products that can
deliver renewable forms of nitrogen in non-leguminous crops. While
some endophytes have the genetics necessary for fixing nitrogen in
pure culture, the fundamental technical challenge is that wild-type
endophytes of cereals and grasses stop fixing nitrogen in
fertilized fields. The application of chemical fertilizers and
residual nitrogen levels in field soils signal the microbe to shut
down the biochemical pathway for nitrogen fixation.
[0146] Changes to the transcriptional and post-translational levels
of components of the nitrogen fixation regulatory network may be
beneficial to the development of a microbe capable of fixing and
transferring nitrogen to corn in the presence of fertilizer. To
that end, described herein is Host-Microbe Evolution (HoME)
technology to precisely evolve regulatory networks and elicit novel
phenotypes. Also described herein are unique, proprietary libraries
of nitrogen-fixing endophytes isolated from corn, paired with
extensive omics data surrounding the interaction of microbes and
host plant under different environmental conditions like nitrogen
stress and excess. In some embodiments, this technology enables
precision evolution of the genetic regulatory network of endophytes
to produce microbes that actively fix nitrogen even in the presence
of fertilizer in the field. Also described herein are evaluations
of the technical potential of evolving microbes that colonize corn
root tissues and produce nitrogen for fertilized plants and
evaluations of the compatibility of endophytes with standard
formulation practices and diverse soils to determine feasibility of
integrating the microbes into modern nitrogen management
strategies.
[0147] In order to utilize elemental nitrogen (N) for chemical
synthesis, life forms combine nitrogen gas (N.sub.2) available in
the atmosphere with hydrogen in a process known as nitrogen
fixation. Because of the energy-intensive nature of biological
nitrogen fixation, diazotrophs (bacteria and archaea that fix
atmospheric nitrogen gas) have evolved sophisticated and tight
regulation of the nif gene cluster in response to environmental
oxygen and available nitrogen. Nif genes encode enzymes involved in
nitrogen fixation (such as the nitrogenase complex) and proteins
that regulate nitrogen fixation. Shamseldin (2013. Global J.
Biotechnol. Biochem. 8(4):84-94) discloses detailed descriptions of
nif genes and their products, and is incorporated herein by
reference. Described herein are methods of producing a plant with
an improved trait comprising isolating bacteria from a first plant,
introducing a genetic variation into a gene of the isolated
bacteria to increase nitrogen fixation, exposing a second plant to
the variant bacteria, isolating bacteria from the second plant
having an improved trait relative to the first plant, and repeating
the steps with bacteria isolated from the second plant.
[0148] In Proteobacteria, regulation of nitrogen fixation centers
around the .sigma..sub.54-dependent enhancer-binding protein NifA,
the positive transcriptional regulator of the nif cluster.
Intracellular levels of active NifA are controlled by two key
factors: transcription of the nifLA operon, and inhibition of NifA
activity by protein-protein interaction with NifL. Both of these
processes are responsive to intracellular glutamine levels via the
PII protein signaling cascade. This cascade is mediated by GlnD,
which directly senses glutamine and catalyzes the uridylylation or
deuridylylation of two PII regulatory proteins--GlnB and GlnK--in
response the absence or presence, respectively, of bound glutamine.
Under conditions of nitrogen excess, unmodified GlnB signals the
deactivation of the nifLA promoter. However, under conditions of
nitrogen limitation, GlnB is post-translationally modified, which
inhibits its activity and leads to transcription of the nifLA
operon. In this way, nifLA transcription is tightly controlled in
response to environmental nitrogen via the PII protein signaling
cascade. On the post-translational level of NifA regulation, GlnK
inhibits the NifL/NifA interaction in a matter dependent on the
overall level of free GlnK within the cell.
[0149] NifA is transcribed from the nifLA operon, whose promoter is
activated by phosphorylated NtrC, another .sigma..sub.54-dependent
regulator. The phosphorylation state of NtrC is mediated by the
histidine kinase NtrB, which interacts with deuridylylated GlnB but
not uridylylated GlnB. Under conditions of nitrogen excess, a high
intracellular level of glutamine leads to deuridylylation of GlnB,
which then interacts with NtrB to deactivate its phosphorylation
activity and activate its phosphatase activity, resulting in
dephosphorylation of NtrC and the deactivation of the nifLA
promoter. However, under conditions of nitrogen limitation, a low
level of intracellular glutamine results in uridylylation of GlnB,
which inhibits its interaction with NtrB and allows the
phosphorylation of NtrC and transcription of the nifLA operon. In
this way, nifLA expression is tightly controlled in response to
environmental nitrogen via the PII protein signaling cascade. nifA,
ntrB, ntrC, and glnB, are all genes that can be mutated in the
methods described herein. These processes may also be responsive to
intracellular or extracellular levels of ammonia, urea or
nitrates.
[0150] The activity of NifA is also regulated post-translationally
in response to environmental nitrogen, most typically through
NifL-mediated inhibition of NifA activity. In general, the
interaction of NifL and NifA is influenced by the PII protein
signaling cascade via GlnK, although the nature of the interactions
between GlnK and NifL/NifA varies significantly between
diazotrophs. In Klebsiella pneumoniae, both forms of GlnK inhibit
the NifL/NifA interaction, and the interaction between GlnK and
NifL/NifA is determined by the overall level of free GlnK within
the cell. Under nitrogen-excess conditions, deuridylylated GlnK
interacts with the ammonium transporter AmtB, which serves to both
block ammonium uptake by AmtB and sequester GlnK to the membrane,
allowing inhibition of NifA by NifL. On the other hand, in
Azotobacter vinelandii, interaction with deuridylylated GlnK is
required for the NifL/NifA interaction and NifA inhibition, while
uridylylation of GlnK inhibits its interaction with NifL. In
diazotrophs lacking the nifL gene, there is evidence that NifA
activity is inhibited directly by interaction with the
deuridylylated forms of both GlnK and GlnB under nitrogen-excess
conditions. In some bacteria the Nif cluster may be regulated by
glnR, and further in some cases this may comprise negative
regulation. Regardless of the mechanism, post-translational
inhibition of NifA is an important regulator of the nif cluster in
most known diazotrophs. Additionally, nifL, amtB, glnK, and glnR
are genes that can be mutated in the methods described herein.
[0151] In addition to regulating the transcription of the nif gene
cluster, many diazotrophs have evolved a mechanism for the direct
post-translational modification and inhibition of the nitrogenase
enzyme itself, known as nitrogenase shutoff. This is mediated by
ADP-ribosylation of the Fe protein (NifH) under nitrogen-excess
conditions, which disrupts its interaction with the MoFe protein
complex (NifDK) and abolishes nitrogenase activity. DraT catalyzes
the ADP-ribosylation of the Fe protein and shutoff of nitrogenase,
while DraG catalyzes the removal of ADP-ribose and reactivation of
nitrogenase. As with nifLA transcription and NifA inhibition,
nitrogenase shutoff is also regulated via the PII protein signaling
cascade. Under nitrogen-excess conditions, deuridylylated GlnB
interacts with and activates DraT, while deuridylylated GlnK
interacts with both DraG and AmtB to form a complex, sequestering
DraG to the membrane. Under nitrogen-limiting conditions, the
uridylylated forms of GlnB and GlnK do not interact with DraT and
DraG, respectively, leading to the inactivation of DraT and the
diffusion of DraG to the Fe protein, where it removes the
ADP-ribose and activates nitrogenase. The methods described herein
also contemplate introducing genetic variation into the nifH, nifD,
nifK, and draT genes.
[0152] Although some endophytes have the ability to fix nitrogen in
vitro, often the genetics are silenced in the field by high levels
of exogenous chemical fertilizers. One can decouple the sensing of
exogenous nitrogen from expression of the nitrogenase enzyme to
facilitate field-based nitrogen fixation. Improving the integral of
nitrogenase activity across time further serves to augment the
production of nitrogen for utilization by the crop. Specific
targets for genetic variation to facilitate field-based nitrogen
fixation using the methods described herein include one or more
genes selected from the group consisting of nifA, nifL, ntrB, ntrC,
glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK,
nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB,
and nifQ.
[0153] An additional target for genetic variation to facilitate
field-based nitrogen fixation using the methods described herein is
the NifA protein. The NifA protein is typically the activator for
expression of nitrogen fixation genes. Increasing the production of
NifA (either constitutively or during high ammonia condition)
circumvents the native ammonia-sensing pathway. In addition,
reducing the production of NifL proteins, a known inhibitor of
NifA, also leads to an increased level of freely active NifA. In
addition, increasing the transcription level of the nifAL operon
(either constitutively or during high ammonia condition) also leads
to an overall higher level of NifA proteins. Elevated level of
nifAL expression is achieved by altering the promoter itself or by
reducing the expression of NtrB (part of ntrB and ntrC signaling
cascade that originally would result in the shutoff of nifAL operon
during high nitrogen condition). High level of NifA achieved by
these or any other methods described herein increases the nitrogen
fixation activity of the endophytes.
[0154] Another target for genetic variation to facilitate
field-based nitrogen fixation using the methods described herein is
the GlnD/GlnB/GlnK PII signaling cascade. The intracellular
glutamine level is sensed through the GlnD/GlnB/GlnK PII signaling
cascade. Active site mutations in GlnD that abolish the
uridylyl-removing activity of GlnD disrupt the nitrogen-sensing
cascade. In addition, reduction of the GlnB concentration short
circuits the glutamine-sensing cascade. These mutations "trick" the
cells into perceiving a nitrogen-limited state, thereby increasing
the nitrogen fixation level activity. These processes may also be
responsive to intracellular or extracellular levels of ammonia,
urea or nitrates.
[0155] The amtB protein is also a target for genetic variation to
facilitate field-based nitrogen fixation using the methods
described herein. Ammonia uptake from the environment can be
reduced by decreasing the expression level of amtB protein. Without
intracellular ammonia, the endophyte is not able to sense the high
level of ammonia, preventing the down-regulation of nitrogen
fixation genes. Any ammonia that manages to get into the
intracellular compartment is converted into glutamine.
Intracellular glutamine level is the major currency of nitrogen
sensing. Decreasing the intracellular glutamine level prevents the
cells from sensing high ammonium levels in the environment. This
effect can be achieved by increasing the expression level of
glutaminase, an enzyme that converts glutamine into glutamate. In
addition, intracellular glutamine can also be reduced by decreasing
glutamine synthase (an enzyme that converts ammonia into
glutamine). In diazotrophs, fixed ammonia is quickly assimilated
into glutamine and glutamate to be used for cellular processes.
Disruptions to ammonia assimilation may enable diversion of fixed
nitrogen to be exported from the cell as ammonia. The fixed ammonia
is predominantly assimilated into glutamine by glutamine synthetase
(GS), encoded by glnA, and subsequently into glutamine by glutamine
oxoglutarate aminotransferase (GOGAT). In some examples, glnS
encodes a glutamine synthetase. GS is regulated
post-translationally by GS adenylyl transferase (GlnE), a
bi-functional enzyme encoded by glnE that catalyzes both the
adenylylation and de-adenylylation of GS through activity of its
adenylyl-transferase (AT) and adenylyl-removing (AR) domains,
respectively. Under nitrogen limiting conditions, glnA is
expressed, and GlnE's AR domain de-adynylylates GS, allowing it to
be active. Under conditions of nitrogen excess, glnA expression is
turned off, and GlnE's AT domain is activated allosterically by
glutamine, causing the adenylylation and deactivation of GS.
[0156] Furthermore, the draT gene may also be a target for genetic
variation to facilitate field-based nitrogen fixation using the
methods described herein. Once nitrogen fixing enzymes are produced
by the cell, nitrogenase shut-off represents another level in which
cell downregulates fixation activity in high nitrogen condition.
This shut-off could be removed by decreasing the expression level
of DraT.
[0157] Methods for imparting new microbial phenotypes can be
performed at the transcriptional, translational, and
post-translational levels. The transcriptional level includes
changes at the promoter (such as changing sigma factor affinity or
binding sites for transcription factors, including deletion of all
or a portion of the promoter) or changing transcription terminators
and attenuators. The translational level includes changes at the
ribosome binding sites and changing mRNA degradation signals. The
post-translational level includes mutating an enzyme's active site
and changing protein-protein interactions. These changes can be
achieved in a multitude of ways. Reduction of expression level (or
complete abolishment) can be achieved by swapping the native
ribosome binding site (RBS) or promoter with another with lower
strength/efficiency. ATG start sites can be swapped to a GTG, TTG,
or CTG start codon, which results in reduction in translational
activity of the coding region. Complete abolishment of expression
can be done by knocking out (deleting) the coding region of a gene.
Frameshifting the open reading frame (ORF) likely will result in a
premature stop codon along the ORF, thereby creating a
non-functional truncated product. Insertion of in-frame stop codons
will also similarly create a non-functional truncated product.
Addition of a degradation tag at the N or C terminal can also be
done to reduce the effective concentration of a particular
gene.
[0158] Conversely, expression level of the genes described herein
can be achieved by using a stronger promoter. To ensure high
promoter activity during high nitrogen level condition (or any
other condition), a transcription profile of the whole genome in a
high nitrogen level condition could be obtained and active
promoters with a desired transcription level can be chosen from
that dataset to replace the weak promoter. Weak start codons can be
swapped out with an ATG start codon for better translation
initiation efficiency. Weak ribosomal binding sites (RBS) can also
be swapped out with a different RBS with higher translation
initiation efficiency. In addition, site specific mutagenesis can
also be performed to alter the activity of an enzyme.
[0159] Increasing the level of nitrogen fixation that occurs in a
plant can lead to a reduction in the amount of chemical fertilizer
needed for crop production and reduce greenhouse gas emissions
(e.g., nitrous oxide).
Regulation of Colonization Potential
[0160] One trait that may be targeted for regulation by the methods
described herein is colonization potential. Accordingly, in some
embodiments, pathways and genes involved in colonization may act as
a target for genetic engineering and optimization.
[0161] In some cases, exopolysaccharides may be involved in
bacterial colonization of plants. In some cases, plant colonizing
microbes may produce a biofilm. In some cases, plant colonizing
microbes secrete molecules which may assist in adhesion to the
plant, or in evading a plant immune response. In some cases, plant
colonizing microbes may excrete signaling molecules which alter the
plants response to the microbes. In some cases, plant colonizing
microbes may secrete molecules which alter the local
microenvironment. In some cases, a plant colonizing microbe may
alter expression of genes to adapt to a plant said microbe is in
proximity to. In some cases, a plant colonizing microbe may detect
the presence of a plant in the local environment and may change
expression of genes in response.
[0162] In some embodiments, to improve colonization, a gene
involved in a pathway selected from the group consisting of:
exopolysaccharide production, endo-polygalaturonase production,
trehalose production, and glutamine conversion may be targeted for
genetic engineering and optimization.
[0163] In some embodiments, an enzyme or pathway involved in
production of exopolysaccharides may be genetically modified to
improve colonization. Exemplary genes encoding an exopolysaccharide
producing enzyme that may be targeted to improve colonization
include, but are not limited to, bcsii, bcsiii, and yjbE.
[0164] In some embodiments, an enzyme or pathway involved in
production of a filamentous hemagglutinin may be genetically
modified to improve colonization. For example, a fhaB gene encoding
a filamentous hemagglutinin may be targeted to improve
colonization.
[0165] In some embodiments, an enzyme or pathway involved in
production of an endo-polygalaturonase may be genetically modified
to improve colonization. For example, a pehA gene encoding an
endo-polygalaturonase precursor may be targeted to improve
colonization.
[0166] In some embodiments, an enzyme or pathway involved in
production of trehalose may be genetically modified to improve
colonization. Exemplary genes encoding a trehalose producing enzyme
that may be targeted to improve colonization include, but are not
limited to, otsB and treZ.
[0167] In some embodiments, an enzyme or pathway involved in
conversion of glutamine may be genetically modified to improve
colonization. For example, the glsA2 gene encodes a glutaminase
which converts glutamine into ammonium and glutamate. Upregulating
glsA2 improves fitness by increasing the cell's glutamate pool,
thereby increasing available N to the cells. Accordingly, in some
embodiments, the glsA2 gene may be targeted to improve
colonization.
[0168] In some embodiments, colonization genes selected from the
group consisting of: bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ,
glsA2, and combinations thereof, may be genetically modified to
improve colonization.
[0169] Colonization genes that may be targeted to improve the
colonization potential are also described in a PCT publication,
WO/2019/032926, which is incorporated by reference herein in its
entirety.
Generation of Bacterial Populations
Isolation of Bacteria
[0170] Microbes useful in methods and compositions disclosed herein
can be obtained by extracting microbes from surfaces or tissues of
native plants. Microbes can be obtained by grinding seeds to
isolate microbes. Microbes can be obtained by planting seeds in
diverse soil samples and recovering microbes from tissues.
Additionally, microbes can be obtained by inoculating plants with
exogenous microbes and determining which microbes appear in plant
tissues. Non-limiting examples of plant tissues may include a seed,
seedling, leaf, cutting, plant, bulb, or tuber.
[0171] A method of obtaining microbes may be through the isolation
of bacteria from soils. Bacteria may be collected from various soil
types. In some example, the soil can be characterized by traits
such as high or low fertility, levels of moisture, levels of
minerals, and various cropping practices. For example, the soil may
be involved in a crop rotation where different crops are planted in
the same soil in successive planting seasons. The sequential growth
of different crops on the same soil may prevent disproportionate
depletion of certain minerals. The bacteria can be isolated from
the plants growing in the selected soils. The seedling plants can
be harvested at 2-6 weeks of growth. For example, at least 400
isolates can be collected in a round of harvest. Soil and plant
types reveal the plant phenotype as well as the conditions, which
allow for the downstream enrichment of certain phenotypes.
[0172] Microbes can be isolated from plant tissues to assess
microbial traits. The parameters for processing tissue samples may
be varied to isolate different types of associative microbes, such
as rhizopheric bacteria, epiphytes, or endophytes. The isolates can
be cultured in nitrogen-free media to enrich for bacteria that
perform nitrogen fixation. Alternatively, microbes can be obtained
from global strain banks.
[0173] In planta analytics are performed to assess microbial
traits. In some embodiments, the plant tissue can be processed for
screening by high throughput processing for DNA and RNA.
Additionally, non-invasive measurements can be used to assess plant
characteristics, such as colonization. Measurements on wild
microbes can be obtained on a plant-by-plant basis. Measurements on
wild microbes can also be obtained in the field using medium
throughput methods. Measurements can be done successively over
time. Model plant system can be used including, but not limited to,
Setaria.
[0174] Microbes in a plant system can be screened via
transcriptional profiling of a microbe in a plant system. Examples
of screening through transcriptional profiling are using methods of
quantitative polymerase chain reaction (qPCR), molecular barcodes
for transcript detection, Next Generation Sequencing, and microbe
tagging with fluorescent markers. Impact factors can be measured to
assess colonization in the greenhouse including, but not limited
to, microbiome, abiotic factors, soil conditions, oxygen, moisture,
temperature, inoculum conditions, and root localization. Nitrogen
fixation can be assessed in bacteria by measuring 15N
gas/fertilizer (dilution) with IRMS or NanoSIMS as described herein
NanoSIMS is high-resolution secondary ion mass spectrometry. The
NanoSIMS technique is a way to investigate chemical activity from
biological samples. The catalysis of reduction of oxidation
reactions that drive the metabolism of microorganisms can be
investigated at the cellular, subcellular, molecular and elemental
level. NanoSIMS can provide high spatial resolution of greater than
0.1 .mu.m. NanoSIMS can detect the use of isotope tracers such as
.sup.13C, .sup.15N, and .sup.18O. Therefore, NanoSIMS can be used
to the chemical activity nitrogen in the cell.
[0175] Automated greenhouses can be used for in planta analytics.
Plant metrics in response to microbial exposure include, but are
not limited to, biomass, chloroplast analysis, CCD camera,
volumetric tomography measurements.
[0176] One way of enriching a microbe population is according to
genotype. For example, a polymerase chain reaction (PCR) assay with
a targeted primer or specific primer. Primers designed for the nifH
gene can be used to identity diazotrophs because diazotrophs
express the nifH gene in the process of nitrogen fixation. A
microbial population can also be enriched via single-cell
culture-independent approaches and chemotaxis-guided isolation
approaches. Alternatively, targeted isolation of microbes can be
performed by culturing the microbes on selection media.
Premeditated approaches to enriching microbial populations for
desired traits can be guided by bioinformatics data and are
described herein.
Enriching for Microbes with Nitrogen Fixation Capabilities Using
Bioinformatics
[0177] Bioinformatics tools can be used to identify and isolate
plant growth promoting rhizobacteria (PGPRs), which are selected
based on their ability to perform nitrogen fixation. Microbes with
high nitrogen fixing ability can promote favorable traits in
plants. Bioinformatic modes of analysis for the identification of
PGPRs include, but are not limited to, genomics, metagenomics,
targeted isolation, gene sequencing, transcriptome sequencing, and
modeling.
[0178] Genomics analysis can be used to identify PGPRs and confirm
the presence of mutations with methods of Next Generation
Sequencing as described herein and microbe version control.
[0179] Metagenomics can be used to identify and isolate PGPR using
a prediction algorithm for colonization. Metadata can also be used
to identify the presence of an engineered strain in environmental
and greenhouse samples.
[0180] Transcriptomic sequencing can be used to predict genotypes
leading to PGPR phenotypes. Additionally, transcriptomic data is
used to identify promoters for altering gene expression.
Transcriptomic data can be analyzed in conjunction with the Whole
Genome Sequence (WGS) to generate models of metabolism and gene
regulatory networks.
Domestication of Microbes
[0181] Microbes isolated from nature can undergo a domestication
process wherein the microbes are converted to a form that is
genetically trackable and identifiable. One way to domesticate a
microbe is to engineer it with antibiotic resistance. The process
of engineering antibiotic resistance can begin by determining the
antibiotic sensitivity in the wild type microbial strain. If the
bacteria are sensitive to the antibiotic, then the antibiotic can
be a good candidate for antibiotic resistance engineering.
Subsequently, an antibiotic resistant gene or a counterselectable
suicide vector can be incorporated into the genome of a microbe
using recombineering methods. A counterselectable suicide vector
may consist of a deletion of the gene of interest, a selectable
marker, and the counterselectable marker sacB. Counterselection can
be used to exchange native microbial DNA sequences with antibiotic
resistant genes. A medium throughput method can be used to evaluate
multiple microbes simultaneously allowing for parallel
domestication. Alternative methods of domestication include the use
of homing nucleases to prevent the suicide vector sequences from
looping out or from obtaining intervening vector sequences.
[0182] DNA vectors can be introduced into bacteria via several
methods including electroporation and chemical transformations. A
standard library of vectors can be used for transformations. An
example of a method of gene editing is CRISPR preceded by Cas9
testing to ensure activity of Cas9 in the microbes.
Non-Transgenic Engineering of Microbes
[0183] A microbial population with favorable traits can be obtained
via directed evolution. Direct evolution is an approach wherein the
process of natural selection is mimicked to evolve proteins or
nucleic acids towards a user-defined goal. An example of direct
evolution is when random mutations are introduced into a microbial
population, the microbes with the most favorable traits are
selected, and the growth of the selected microbes is continued. The
most favorable traits in growth promoting rhizobacteria (PGPRs) may
be in nitrogen fixation. The method of directed evolution may be
iterative and adaptive based on the selection process after each
iteration.
[0184] Plant growth promoting rhizobacteria (PGPRs) with high
capability of nitrogen fixation can be generated. The evolution of
PGPRs can be carried out via the introduction of genetic variation.
Genetic variation can be introduced via polymerase chain reaction
mutagenesis, oligonucleotide-directed mutagenesis, saturation
mutagenesis, fragment shuffling mutagenesis, homologous
recombination, CRISPR/Cas9 systems, chemical mutagenesis, and
combinations thereof. These approaches can introduce random
mutations into the microbial population. For example, mutants can
be generated using synthetic DNA or RNA via
oligonucleotide-directed mutagenesis. Mutants can be generated
using tools contained on plasmids, which are later cured. Genes of
interest can be identified using libraries from other species with
improved traits including, but not limited to, improved PGPR
properties, improved colonization of cereals, increased oxygen
sensitivity, increased nitrogen fixation, and increased ammonia
excretion. Intrageneric genes can be designed based on these
libraries using software such as Geneious or Platypus design
software. Mutations can be designed with the aid of machine
learning. Mutations can be designed with the aid of a metabolic
model. Automated design of the mutation can be done using a la
Platypus and will guide RNAs for Cas-directed mutagenesis.
[0185] The intra-generic genes can be transferred into the host
microbe. Additionally, reporter systems can also be transferred to
the microbe. The reporter systems characterize promoters, determine
the transformation success, screen mutants, and act as negative
screening tools.
[0186] The microbes carrying the mutation can be cultured via
serial passaging. A microbial colony contains a single variant of
the microbe. Microbial colonies are screened with the aid of an
automated colony picker and liquid handler. Mutants with gene
duplication and increased copy number express a higher genotype of
the desired trait.
Selection of Plant Growth Promoting Microbes Based on Nitrogen
Fixation
[0187] The microbial colonies can be screened using various assays
to assess nitrogen fixation. One way to measure nitrogen fixation
is via a single fermentative assay, which measures nitrogen
excretion. An alternative method is the acetylene reduction assay
(ARA) with in-line sampling over time. ARA can be performed in high
throughput plates of microtube arrays. ARA can be performed with
live plants and plant tissues. The media formulation and media
oxygen concentration can be varied in ARA assays. Another method of
screening microbial variants is by using biosensors. The use of
NanoSIMS and Raman microspectroscopy can be used to investigate the
activity of the microbes. In some cases, bacteria can also be
cultured and expanded using methods of fermentation in bioreactors.
The bioreactors are designed to improve robustness of bacteria
growth and to decrease the sensitivity of bacteria to oxygen.
Medium to high TP plate-based microfermentors are used to evaluate
oxygen sensitivity, nutritional needs, nitrogen fixation, and
nitrogen excretion. The bacteria can also be co-cultured with
competitive or beneficial microbes to elucidate cryptic pathways.
Flow cytometry can be used to screen for bacteria that produce high
levels of nitrogen using chemical, colorimetric, or fluorescent
indicators. The bacteria may be cultured in the presence or absence
of a nitrogen source. For example, the bacteria may be cultured
with glutamine, ammonia, urea or nitrates.
Guided Microbial Remodeling--An Overview
[0188] Guided microbial remodeling is a method to systematically
identify and improve the role of species within the crop
microbiome. In some aspects, and according to a particular
methodology of grouping/categorization, the method comprises three
steps: 1) selection of candidate species by mapping plant-microbe
interactions and predicting regulatory networks linked to a
particular phenotype, 2) pragmatic and predictable improvement of
microbial phenotypes through intra-species crossing of regulatory
networks and gene clusters within a microbe's genome, and 3)
screening and selection of new microbial genotypes that produce
desired crop phenotypes.
[0189] To systematically assess the improvement of strains, a model
is created that links colonization dynamics of the microbial
community to genetic activity by key species. The model is used to
predict genetic targets for non-intergeneric genetic remodeling
(i.e. engineering the genetic architecture of the microbe in a
non-transgenic fashion). See, FIG. 1A for a graphical
representation of an embodiment of the process.
[0190] As illustrated in FIG. 1A, rational improvement of the crop
microbiome may be used to increase soil biodiversity, tune impact
of keystone species, and/or alter timing and expression of
important metabolic pathways.
[0191] To this end, the inventors have developed a platform to
identify and improve the role of strains within the crop
microbiome. In some aspects, the inventors call this process
microbial breeding.
[0192] The aforementioned "Guided Microbial Remodeling" process
will be further elaborated upon in the Examples, for instance in
Example 1, entitled: "Guided Microbial Remodeling--A Platform for
the Rational Improvement of Microbial Species for Agriculture."
Serial Passage
[0193] Production of bacteria to improve plant traits (e.g.,
nitrogen fixation) can be achieved through serial passage. The
production of these bacteria can be done by selecting plants, which
have a particular improved trait that is influenced by the
microbial flora, in addition to identifying bacteria and/or
compositions that are capable of imparting one or more improved
traits to one or more plants. One method of producing a bacteria to
improve a plant trait includes the steps of: (a) isolating bacteria
from tissue or soil of a first plant; (b) introducing a genetic
variation into one or more of the bacteria to produce one or more
variant bacteria; (c) exposing a plurality of plants to the variant
bacteria; (d) isolating bacteria from tissue or soil of one of the
plurality of plants, wherein the plant from which the bacteria is
isolated has an improved trait relative to other plants in the
plurality of plants; and (e) repeating steps (b) to (d) with
bacteria isolated from the plant with an improved trait (step (d)).
Steps (b) to (d) can be repeated any number of times (e.g., once,
twice, three times, four times, five times, ten times, or more)
until the improved trait in a plant reaches a desired level.
Further, the plurality of plants can be more than two plants, such
as 10 to 20 plants, or 20 or more, 50 or more, 100 or more, 300 or
more, 500 or more, or 1000 or more plants.
[0194] In addition to obtaining a plant with an improved trait, a
bacterial population comprising bacteria comprising one or more
genetic variations introduced into one or more genes (e.g., genes
regulating nitrogen fixation) is obtained. By repeating the steps
described above, a population of bacteria can be obtained that
include the most appropriate members of the population that
correlate with a plant trait of interest. The bacteria in this
population can be identified and their beneficial properties
determined, such as by genetic and/or phenotypic analysis. Genetic
analysis may occur of isolated bacteria in step (a). Phenotypic
and/or genotypic information may be obtained using techniques
including: high through-put screening of chemical components of
plant origin, sequencing techniques including high throughput
sequencing of genetic material, differential display techniques
(including DDRT-PCR, and DD-PCR), nucleic acid microarray
techniques, RNA-sequencing (Whole Transcriptome Shotgun
Sequencing), and qRT-PCR (quantitative real time PCR). Information
gained can be used to obtain community profiling information on the
identity and activity of bacteria present, such as phylogenetic
analysis or microarray-based screening of nucleic acids coding for
components of rRNA operons or other taxonomically informative loci.
Examples of taxonomically informative loci include 16S rRNA gene,
23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S
rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA
gene, coxl gene, nifD gene. Example processes of taxonomic
profiling to determine taxa present in a population are described
in US20140155283. Bacterial identification may comprise
characterizing activity of one or more genes or one or more
signaling pathways, such as genes associated with the nitrogen
fixation pathway. Synergistic interactions (where two components,
by virtue of their combination, increase a desired effect by more
than an additive amount) between different bacterial species may
also be present in the bacterial populations.
Genetic Variation--Locations and Sources of Genomic Alteration
[0195] The genetic variation may be a variation in a gene selected
from the group consisting of: nifA, nifL, ntrB, ntrC, glnA, glnB,
glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE,
nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. The
genetic variation may be a variation in a gene encoding a protein
with functionality selected from the group consisting of: glutamine
synthetase, glutaminase, glutamine synthetase adenylyltransferase,
transcriptional activator, anti-transcriptional activator, pyruvate
flavodoxin oxidoreductase, flavodoxin, or NAD+-dinitrogen-reductase
aDP-D-ribosyltransferase. The genetic variation may be a mutation
that results in one or more of: increased expression or activity of
NifA or glutaminase; decreased expression or activity of NifL,
NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased
adenylyl-removing activity of GlnE; or decreased uridylyl-removing
activity of GlnD. The genetic variation may be a variation in a
gene selected from the group consisting of: bcsii, bcsiii, yjbE,
fhaB, pehA, otsB, treZ, glsA2, and combinations thereof. In some
embodiments, a genetic variation may be a variation in any of the
genes described throughout this disclosure.
[0196] Introducing a genetic variation may comprise insertion
and/or deletion of one or more nucleotides at a target site, such
as 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or more nucleotides.
The genetic variation introduced into one or more bacteria of the
methods disclosed herein may be a knock-out mutation (e.g. deletion
of a promoter, insertion or deletion to produce a premature stop
codon, deletion of an entire gene), or it may be elimination or
abolishment of activity of a protein domain (e.g. point mutation
affecting an active site, or deletion of a portion of a gene
encoding the relevant portion of the protein product), or it may
alter or abolish a regulatory sequence of a target gene. One or
more regulatory sequences may also be inserted, including
heterologous regulatory sequences and regulatory sequences found
within a genome of a bacterial species or genus corresponding to
the bacteria into which the genetic variation is introduced.
Moreover, regulatory sequences may be selected based on the
expression level of a gene in a bacterial culture or within a plant
tissue. The genetic variation may be a pre-determined genetic
variation that is specifically introduced to a target site. The
genetic variation may be a random mutation within the target site.
The genetic variation may be an insertion or deletion of one or
more nucleotides. In some cases, a plurality of different genetic
variations (e.g. 2, 3, 4, 5, 10, or more) are introduced into one
or more of the isolated bacteria before exposing the bacteria to
plants for assessing trait improvement. The plurality of genetic
variations can be any of the above types, the same or different
types, and in any combination. In some cases, a plurality of
different genetic variations are introduced serially, introducing a
first genetic variation after a first isolation step, a second
genetic variation after a second isolation step, and so forth so as
to accumulate a plurality of genetic variations in bacteria
imparting progressively improved traits on the associated
plants.
Genetic Variation--Methods of Introducing Genomic Alteration
[0197] In general, the term "genetic variation" refers to any
change introduced into a polynucleotide sequence relative to a
reference polynucleotide, such as a reference genome or portion
thereof, or reference gene or portion thereof. A genetic variation
may be referred to as a "mutation," and a sequence or organism
comprising a genetic variation may be referred to as a "genetic
variant" or "mutant". Genetic variations can have any number of
effects, such as the increase or decrease of some biological
activity, including gene expression, metabolism, and cell
signaling. Genetic variations can be specifically introduced to a
target site, or introduced randomly. A variety of molecular tools
and methods are available for introducing genetic variation. For
example, genetic variation can be introduced via polymerase chain
reaction mutagenesis, oligonucleotide-directed mutagenesis,
saturation mutagenesis, fragment shuffling mutagenesis, homologous
recombination, recombineering, lambda red mediated recombination,
CRISPR/Cas9 systems, chemical mutagenesis, and combinations
thereof. Chemical methods of introducing genetic variation include
exposure of DNA to a chemical mutagen, e.g., ethyl methanesulfonate
(EMS), methyl methanesulfonate (MMS), N-nitrosourea (EN U),
N-methyl-N-nitro-N'-nitrosoguanidine, 4-nitroquinoline N-oxide,
diethylsulfate, benzopyrene, cyclophosphamide, bleomycin,
triethylmelamine, acrylamide monomer, nitrogen mustard,
vincristine, diepoxyalkanes (for example, diepoxybutane), ICR-170,
formaldehyde, procarbazine hydrochloride, ethylene oxide,
dimethylnitrosamine, 7,12 dimethylbenz(a)anthracene, chlorambucil,
hexamethylphosphoramide, bisulfan, and the like. Radiation
mutation-inducing agents include ultraviolet radiation,
.gamma.-irradiation, X-rays, and fast neutron bombardment. Genetic
variation can also be introduced into a nucleic acid using, e.g.,
trimethylpsoralen with ultraviolet light. Random or targeted
insertion of a mobile DNA element, e.g., a transposable element, is
another suitable method for generating genetic variation. Genetic
variations can be introduced into a nucleic acid during
amplification in a cell-free in vitro system, e.g., using a
polymerase chain reaction (PCR) technique such as error-prone PCR.
Genetic variations can be introduced into a nucleic acid in vitro
using DNA shuffling techniques (e.g., exon shuffling, domain
swapping, and the like). Genetic variations can also be introduced
into a nucleic acid as a result of a deficiency in a DNA repair
enzyme in a cell, e.g., the presence in a cell of a mutant gene
encoding a mutant DNA repair enzyme is expected to generate a high
frequency of mutations (i.e., about 1 mutation/100 genes-1
mutation/10,000 genes) in the genome of the cell. Examples of genes
encoding DNA repair enzymes include but are not limited to Mut H,
Mut S, Mut L, and Mut U, and the homologs thereof in other species
(e.g., MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1, and the like).
Example descriptions of various methods for introducing genetic
variations are provided in e.g., Stemple (2004) Nature 5:1-7;
Chiang et al. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994)
Proc. Natl. Acad. Sci. USA 91:10747-10751; and U.S. Pat. Nos.
6,033,861, and 6,773,900.
[0198] Genetic variations introduced into microbes may be
classified as transgenic, cisgenic, intragenomic, intrageneric,
intergeneric, synthetic, evolved, rearranged, or SNPs.
[0199] Genetic variation may be introduced into numerous metabolic
pathways within microbes to elicit improvements in the traits
described above. Representative pathways include sulfur uptake
pathways, glycogen biosynthesis, the glutamine regulation pathway,
the molybdenum uptake pathway, the nitrogen fixation pathway,
ammonia assimilation, ammonia excretion or secretion, Nitrogen
uptake, glutamine biosynthesis, annamox, phosphate solubilization,
organic acid transport, organic acid production, agglutinins
production, reactive oxygen radical scavenging genes, Indole Acetic
Acid biosynthesis, trehalose biosynthesis, plant cell wall
degrading enzymes or pathways, root attachment genes,
exopolysaccharide secretion, glutamate synthase pathway, iron
uptake pathways, siderophore pathway, chitinase pathway, ACC
deaminase, glutathione biosynthesis, phosphorous signaling genes,
quorum quenching pathway, cytochrome pathways, hemoglobin pathway,
bacterial hemoglobin-like pathway, small RNA rsmZ, rhizobitoxine
biosynthesis, lapA adhesion protein, AHL quorum sensing pathway,
phenazine biosynthesis, cyclic lipopeptide biosynthesis, and
antibiotic production.
[0200] CRISPR/Cas9 (Clustered regularly interspaced short
palindromic repeats)/CRISPR-associated (Cas) systems can be used to
introduce desired mutations. CRISPR/Cas9 provide bacteria and
archaea with adaptive immunity against viruses and plasmids by
using CRISPR RNAs (crRNAs) to guide the silencing of invading
nucleic acids. The Cas9 protein (or functional equivalent and/or
variant thereof, i.e., Cas9-like protein) naturally contains DNA
endonuclease activity that depends on the association of the
protein with two naturally occurring or synthetic RNA molecules
called crRNA and tracrRNA (also called guide RNAs). In some cases,
the two molecules are covalently link to form a single molecule
(also called a single guide RNA ("sgRNA"). Thus, the Cas9 or
Cas9-like protein associates with a DNA-targeting RNA (which term
encompasses both the two-molecule guide RNA configuration and the
single-molecule guide RNA configuration), which activates the Cas9
or Cas9-like protein and guides the protein to a target nucleic
acid sequence. If the Cas9 or Cas9-like protein retains its natural
enzymatic function, it will cleave target DNA to create a
double-stranded break, which can lead to genome alteration (i.e.,
editing, deletion, insertion (when a donor polynucleotide is
present), replacement, etc.), thereby altering gene expression.
Some variants of Cas9 (which variants are encompassed by the term
Cas9-like) have been altered such that they have a decreased DNA
cleaving activity (in some cases, they cleave a single strand
instead of both strands of the target DNA, while in other cases,
they have severely reduced to no DNA cleavage activity). Further
exemplary descriptions of CRISPR systems for introducing genetic
variation can be found in, e.g. U.S. Pat. No. 8,795,965.
[0201] As a cyclic amplification technique, polymerase chain
reaction (PCR) mutagenesis uses mutagenic primers to introduce
desired mutations. PCR is performed by cycles of denaturation,
annealing, and extension. After amplification by PCR, selection of
mutated DNA and removal of parental plasmid DNA can be accomplished
by: 1) replacement of dCTP by hydroxymethylated-dCTP during PCR,
followed by digestion with restriction enzymes to remove
non-hydroxymethylated parent DNA only; 2) simultaneous mutagenesis
of both an antibiotic resistance gene and the studied gene changing
the plasmid to a different antibiotic resistance, the new
antibiotic resistance facilitating the selection of the desired
mutation thereafter; 3) after introducing a desired mutation,
digestion of the parent methylated template DNA by restriction
enzyme Dpn1 which cleaves only methylated DNA, by which the
mutagenized unmethylated chains are recovered; or 4)
circularization of the mutated PCR products in an additional
ligation reaction to increase the transformation efficiency of
mutated DNA. Further description of exemplary methods can be found
in e.g. U.S. Pat. Nos. 7,132,265, 6,713,285, 6,673,610, 6,391,548,
5,789,166, 5,780,270, 5,354,670, 5,071,743, and US20100267147.
[0202] Oligonucleotide-directed mutagenesis, also called
site-directed mutagenesis, typically utilizes a synthetic DNA
primer. This synthetic primer contains the desired mutation and is
complementary to the template DNA around the mutation site so that
it can hybridize with the DNA in the gene of interest. The mutation
may be a single base change (a point mutation), multiple base
changes, deletion, or insertion, or a combination of these. The
single-strand primer is then extended using a DNA polymerase, which
copies the rest of the gene. The gene thus copied contains the
mutated site, and may then be introduced into a host cell as a
vector and cloned. Finally, mutants can be selected by DNA
sequencing to check that they contain the desired mutation.
[0203] Genetic variations can be introduced using error-prone PCR.
In this technique, the gene of interest is amplified using a DNA
polymerase under conditions that are deficient in the fidelity of
replication of sequence. The result is that the amplification
products contain at least one error in the sequence. When a gene is
amplified and the resulting product(s) of the reaction contain one
or more alterations in sequence when compared to the template
molecule, the resulting products are mutagenized as compared to the
template. Another means of introducing random mutations is exposing
cells to a chemical mutagen, such as nitrosoguanidine or ethyl
methanesulfonate (Nestmann, Mutat Res 1975 June; 28(3):323-30), and
the vector containing the gene is then isolated from the host.
[0204] Saturation mutagenesis is another form of random
mutagenesis, in which one tries to generate all or nearly all
possible mutations at a specific site, or narrow region of a gene.
In a general sense, saturation mutagenesis is comprised of
mutagenizing a complete set of mutagenic cassettes (wherein each
cassette is, for example, 1-500 bases in length) in defined
polynucleotide sequence to be mutagenized (wherein the sequence to
be mutagenized is, for example, from 15 to 100,000 bases in
length). Therefore, a group of mutations (e.g. ranging from 1 to
100 mutations) is introduced into each cassette to be mutagenized.
A grouping of mutations to be introduced into one cassette can be
different or the same from a second grouping of mutations to be
introduced into a second cassette during the application of one
round of saturation mutagenesis. Such groupings are exemplified by
deletions, additions, groupings of particular codons, and groupings
of particular nucleotide cassettes.
[0205] Fragment shuffling mutagenesis, also called DNA shuffling,
is a way to rapidly propagate beneficial mutations. In an example
of a shuffling process, DNAse is used to fragment a set of parent
genes into pieces of e.g. about 50-100 bp in length. This is then
followed by a polymerase chain reaction (PCR) without primers--DNA
fragments with sufficient overlapping homologous sequence will
anneal to each other and are then be extended by DNA polymerase.
Several rounds of this PCR extension are allowed to occur, after
some of the DNA molecules reach the size of the parental genes.
These genes can then be amplified with another PCR, this time with
the addition of primers that are designed to complement the ends of
the strands. The primers may have additional sequences added to
their 5' ends, such as sequences for restriction enzyme recognition
sites needed for ligation into a cloning vector. Further examples
of shuffling techniques are provided in US20050266541.
[0206] Homologous recombination mutagenesis involves recombination
between an exogenous DNA fragment and the targeted polynucleotide
sequence. After a double-stranded break occurs, sections of DNA
around the 5' ends of the break are cut away in a process called
resection. In the strand invasion step that follows, an overhanging
3' end of the broken DNA molecule then "invades" a similar or
identical DNA molecule that is not broken. The method can be used
to delete a gene, remove exons, add a gene, and introduce point
mutations. Homologous recombination mutagenesis can be permanent or
conditional. Typically, a recombination template is also provided.
A recombination template may be a component of another vector,
contained in a separate vector, or provided as a separate
polynucleotide. In some embodiments, a recombination template is
designed to serve as a template in homologous recombination, such
as within or near a target sequence nicked or cleaved by a
site-specific nuclease. A template polynucleotide may be of any
suitable length, such as about or more than about 10, 15, 20, 25,
50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In
some embodiments, the template polynucleotide is complementary to a
portion of a polynucleotide comprising the target sequence. When
optimally aligned, a template polynucleotide might overlap with one
or more nucleotides of a target sequences (e.g. about or more than
about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100
or more nucleotides). In some embodiments, when a template sequence
and a polynucleotide comprising a target sequence are optimally
aligned, the nearest nucleotide of the template polynucleotide is
within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500,
1000, 5000, 10000, or more nucleotides from the target sequence.
Non-limiting examples of site-directed nucleases useful in methods
of homologous recombination include zinc finger nucleases, CRISPR
nucleases, TALE nucleases, and meganuclease. For a further
description of the use of such nucleases, see e.g. U.S. Pat. No.
8,795,965 and US20140301990.
[0207] Mutagens that create primarily point mutations and short
deletions, insertions, transversions, and/or transitions, including
chemical mutagens or radiation, may be used to create genetic
variations. Mutagens include, but are not limited to, ethyl
methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosourea,
triethylmelamine, N-methyl-N-nitrosourea, procarbazine,
chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide
monomer, melphalan, nitrogen mustard, vincristine,
dimethylnitrosamine, N-methyl-N'-nitro-Nitrosoguanidine,
nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene,
ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes
(diepoxyoctane, diepoxybutane, and the like),
2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine
dihydrochloride and formaldehyde.
[0208] Introducing genetic variation may be an incomplete process,
such that some bacteria in a treated population of bacteria carry a
desired mutation while others do not. In some cases, it is
desirable to apply a selection pressure so as to enrich for
bacteria carrying a desired genetic variation. Traditionally,
selection for successful genetic variants involved selection for or
against some functionality imparted or abolished by the genetic
variation, such as in the case of inserting antibiotic resistance
gene or abolishing a metabolic activity capable of converting a
non-lethal compound into a lethal metabolite. It is also possible
to apply a selection pressure based on a polynucleotide sequence
itself, such that only a desired genetic variation need be
introduced (e.g. without also requiring a selectable marker). In
this case, the selection pressure can comprise cleaving genomes
lacking the genetic variation introduced to a target site, such
that selection is effectively directed against the reference
sequence into which the genetic variation is sought to be
introduced. Typically, cleavage occurs within 100 nucleotides of
the target site (e.g. within 75, 50, 25, 10, or fewer nucleotides
from the target site, including cleavage at or within the target
site). Cleaving may be directed by a site-specific nuclease
selected from the group consisting of a Zinc Finger nuclease, a
CRISPR nuclease, a TALE nuclease (TALEN), and a meganuclease. Such
a process is similar to processes for enhancing homologous
recombination at a target site, except that no template for
homologous recombination is provided. As a result, bacteria lacking
the desired genetic variation are more likely to undergo cleavage
that, left unrepaired, results in cell death. Bacteria surviving
selection may then be isolated for use in exposing to plants for
assessing conferral of an improved trait.
[0209] A CRISPR nuclease may be used as the site-specific nuclease
to direct cleavage to a target site. An improved selection of
mutated microbes can be obtained by using Cas9 to kill non-mutated
cells. Plants are then inoculated with the mutated microbes to
re-confirm symbiosis and create evolutionary pressure to select for
efficient symbionts. Microbes can then be re-isolated from plant
tissues. CRISPR nuclease systems employed for selection against
non-variants can employ similar elements to those described above
with respect to introducing genetic variation, except that no
template for homologous recombination is provided. Cleavage
directed to the target site thus enhances death of affected
cells.
[0210] Other options for specifically inducing cleavage at a target
site are available, such as zinc finger nucleases, TALE nuclease
(TALEN) systems, and meganuclease. Zinc-finger nucleases (ZFNs) are
artificial DNA endonucleases generated by fusing a zinc finger DNA
binding domain to a DNA cleavage domain. ZFNs can be engineered to
target desired DNA sequences and this enables zinc-finger nucleases
to cleave unique target sequences. When introduced into a cell,
ZFNs can be used to edit target DNA in the cell (e.g., the cell's
genome) by inducing double stranded breaks. Transcription
activator-like effector nucleases (TALENs) are artificial DNA
endonucleases generated by fusing a TAL (Transcription
activator-like) effector DNA binding domain to a DNA cleavage
domain. TALENS can be quickly engineered to bind practically any
desired DNA sequence and when introduced into a cell, TALENs can be
used to edit target DNA in the cell (e.g., the cell's genome) by
inducing double strand breaks. Meganucleases (homing endonuclease)
are endodeoxyribonucleases characterized by a large recognition
site (double-stranded DNA sequences of 12 to 40 base pairs.
Meganucleases can be used to replace, eliminate or modify sequences
in a highly targeted way. By modifying their recognition sequence
through protein engineering, the targeted sequence can be changed.
Meganucleases can be used to modify all genome types, whether
bacterial, plant or animal and are commonly grouped into four
families: the LAGLIDADG family (SEQ ID NO: 1), the GIY-YIG family,
the His-Cyst box family and the HNH family. Exemplary homing
endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV,
I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII
and I-TevIII.
Genetic Variation--Methods of Identification
[0211] The microbes of the present disclosure may be identified by
one or more genetic modifications or alterations, which have been
introduced into said microbe. One method by which said genetic
modification or alteration can be identified is via reference to a
SEQ ID NO that contains a portion of the microbe's genomic sequence
that is sufficient to identify the genetic modification or
alteration.
[0212] Further, in the case of microbes that have not had a genetic
modification or alteration (e.g. a wild type, WT) introduced into
their genomes, the disclosure can utilize 16S nucleic acid
sequences to identify said microbes. A 16S nucleic acid sequence is
an example of a "molecular marker" or "genetic marker," which
refers to an indicator that is used in methods for visualizing
differences in characteristics of nucleic acid sequences. Examples
of other such indicators are restriction fragment length
polymorphism (RFLP) markers, amplified fragment length polymorphism
(AFLP) markers, single nucleotide polymorphisms (SNPs), insertion
mutations, microsatellite markers (SSRs), sequence-characterized
amplified regions (SCARs), cleaved amplified polymorphic sequence
(CAPS) markers or isozyme markers or combinations of the markers
described herein which defines a specific genetic and chromosomal
location. Markers further include polynucleotide sequences encoding
16S or 18S rRNA, and internal transcribed spacer (ITS) sequences,
which are sequences found between small-subunit and large-subunit
rRNA genes that have proven to be especially useful in elucidating
relationships or distinctions when compared against one another.
Furthermore, the disclosure utilizes unique sequences found in
genes of interest (e.g. nifH, D, K, L, A, glnE, amtB, etc.) to
identify microbes disclosed herein.
[0213] The primary structure of major rRNA subunit 16S comprise a
particular combination of conserved, variable, and hypervariable
regions that evolve at different rates and enable the resolution of
both very ancient lineages such as domains, and more modern
lineages such as genera. The secondary structure of the 16S subunit
include approximately 50 helices which result in base pairing of
about 67% of the residues. These highly conserved secondary
structural features are of great functional importance and can be
used to ensure positional homology in multiple sequence alignments
and phylogenetic analysis. Over the previous few decades, the 16S
rRNA gene has become the most sequenced taxonomic marker and is the
cornerstone for the current systematic classification of bacteria
and archaea (Yarza et al. 2014. Nature Rev. Micro. 12:635-45).
[0214] Thus, in certain aspects, the disclosure provides for a
sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to any sequence in Tables 23, 24, 30, 31, and
32.
[0215] Thus, in certain aspects, the disclosure provides for a
microbe that comprises a sequence, which shares at least about 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 62-303.
These sequences and their associated descriptions can be found in
Tables 31 and 32.
[0216] In some aspects, the disclosure provides for a microbe that
comprises a 16S nucleic acid sequence, which shares at least about
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 85,
96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269,
277-283. These sequences and their associated descriptions can be
found in Table 32.
[0217] In some aspects, the disclosure provides for a microbe that
comprises a nucleic acid sequence, which shares at least about 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 86-95,
97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176,
263-268, 270-274, 275, 276, 284-295. These sequences and their
associated descriptions can be found in Table 32.
[0218] In some aspects, the disclosure provides for a microbe that
comprises a nucleic acid sequence, which shares at least about 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 177-260,
296-303. These sequences and their associated descriptions can be
found in Table 32.
[0219] In some aspects, the disclosure provides for a microbe that
comprises, or primer that comprises, or probe that comprises, or
non-native junction sequence that comprises, a nucleic acid
sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to SEQ ID NOs: 304-424. These sequences and their
associated descriptions can be found in Table 30.
[0220] In some aspects, the disclosure provides for a microbe that
comprises a non-native junction sequence that shares at least about
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:
372-405. These sequences and their associated descriptions can be
found in Table 30.
[0221] In some aspects, the disclosure provides for a microbe that
comprises an amino acid sequence, which shares at least about 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 77, 78, 81,
82, or 83. These sequences and their associated descriptions can be
found in Table 31.
Genetic Variation--Methods of Detection: Primers, Probes, and
Assays
[0222] The present disclosure teaches primers, probes, and assays
that are useful for detecting the microbes taught herein. In some
aspects, the disclosure provides for methods of detecting the WT
parental strains. In other aspects, the disclosure provides for
methods of detecting the non-intergeneric engineered microbes
derived from the WT strains. In aspects, the present disclosure
provides methods of identifying non-intergeneric genetic
alterations in a microbe.
[0223] In aspects, the genomic engineering methods of the present
disclosure lead to the creation of non-natural nucleotide
"junction" sequences in the derived non-intergeneric microbes.
These non-naturally occurring nucleotide junctions can be used as a
type of diagnostic that is indicative of the presence of a
particular genetic alteration in a microbe taught herein.
[0224] The present techniques are able to detect these
non-naturally occurring nucleotide junctions via the utilization of
specialized quantitative PCR methods, including uniquely designed
primers and probes. In some aspects, the probes of the disclosure
bind to the non-naturally occurring nucleotide junction sequences.
In some aspects, traditional PCR is utilized. In other aspects,
real-time PCR is utilized. In some aspects, quantitative PCR (qPCR)
is utilized.
[0225] Thus, the disclosure can cover the utilization of two common
methods for the detection of PCR products in real-time: (1)
non-specific fluorescent dyes that intercalate with any
double-stranded DNA, and (2) sequence-specific DNA probes
consisting of oligonucleotides that are labelled with a fluorescent
reporter which permits detection only after hybridization of the
probe with its complementary sequence. In some aspects, only the
non-naturally occurring nucleotide junction will be amplified via
the taught primers, and consequently can be detected via either a
non-specific dye, or via the utilization of a specific
hybridization probe. In other aspects, the primers of the
disclosure are chosen such that the primers flank either side of a
junction sequence, such that if an amplification reaction occurs,
then said junction sequence is present.
[0226] Aspects of the disclosure involve non-naturally occurring
nucleotide junction sequence molecules per se, along with other
nucleotide molecules that are capable of binding to said
non-naturally occurring nucleotide junction sequences under mild to
stringent hybridization conditions. In some aspects, the nucleotide
molecules that are capable of binding to said non-naturally
occurring nucleotide junction sequences under mild to stringent
hybridization conditions are termed "nucleotide probes."
[0227] In aspects, genomic DNA can be extracted from samples and
used to quantify the presence of microbes of the disclosure by
using qPCR. The primers utilized in the qPCR reaction can be
primers designed by Primer Blast
(www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify unique
regions of the wild-type genome or unique regions of the engineered
non-intergeneric mutant strains. The qPCR reaction can be carried
out using the SYBR GreenER qPCR SuperMix Universal (Thermo Fisher
P/N 11762100) kit, using only forward and reverse amplification
primers; alternatively, the Kapa Probe Force kit (Kapa Biosystems
P/N KK4301) can be used with amplification primers and a TaqMan
probe containing a FAM dye label at the 5' end, an internal ZEN
quencher, and a minor groove binder and fluorescent quencher at the
3' end (Integrated DNA Technologies).
[0228] Certain primer, probe, and non-native junction sequences are
listed in Table 30. qPCR reaction efficiency can be measured using
a standard curve generated from a known quantity of gDNA from the
target genome. Data can be normalized to genome copies per g fresh
weight using the tissue weight and extraction volume.
[0229] Quantitative polymerase chain reaction (qPCR) is a method of
quantifying, in real time, the amplification of one or more nucleic
acid sequences. The real time quantification of the PCR assay
permits determination of the quantity of nucleic acids being
generated by the PCR amplification steps by comparing the
amplifying nucleic acids of interest and an appropriate control
nucleic acid sequence, which may act as a calibration standard.
[0230] TaqMan probes are often utilized in qPCR assays that require
an increased specificity for quantifying target nucleic acid
sequences. TaqMan probes comprise an oligonucleotide probe with a
fluorophore attached to the 5' end and a quencher attached to the
3' end of the probe. When the TaqMan probes remain as is with the
5' and 3' ends of the probe in close contact with each other, the
quencher prevents fluorescent signal transmission from the
fluorophore. TaqMan probes are designed to anneal within a nucleic
acid region amplified by a specific set of primers. As the Taq
polymerase extends the primer and synthesizes the nascent strand,
the 5' to 3' exonuclease activity of the Taq polymerase degrades
the probe that annealed to the template. This probe degradation
releases the fluorophore, thus breaking the close proximity to the
quencher and allowing fluorescence of the fluorophore. Fluorescence
detected in the qPCR assay is directly proportional to the
fluorophore released and the amount of DNA template present in the
reaction.
[0231] The features of qPCR allow the practitioner to eliminate the
labor-intensive post-amplification step of gel electrophoresis
preparation, which is generally required for observation of the
amplified products of traditional PCR assays. The benefits of qPCR
over conventional PCR are considerable, and include increased
speed, ease of use, reproducibility, and quantitative ability.
Improvement of Traits
[0232] Methods of the present disclosure may be employed to
introduce or improve one or more of a variety of desirable traits.
Examples of traits that may introduced or improved include: root
biomass, root length, height, shoot length, leaf number, water use
efficiency, overall biomass, yield, fruit size, grain size,
photosynthesis rate, tolerance to drought, heat tolerance, salt
tolerance, resistance to nematode stress, resistance to a fungal
pathogen, resistance to a bacterial pathogen, resistance to a viral
pathogen, level of a metabolite, and proteome expression. The
desirable traits, including height, overall biomass, root and/or
shoot biomass, seed germination, seedling survival, photosynthetic
efficiency, transpiration rate, seed/fruit number or mass, plant
grain or fruit yield, leaf chlorophyll content, photosynthetic
rate, root length, or any combination thereof, can be used to
measure growth, and compared with the growth rate of reference
agricultural plants (e.g., plants without the improved traits)
grown under identical conditions.
[0233] A preferred trait to be introduced or improved is nitrogen
fixation, as described herein. In some cases, a plant resulting
from the methods described herein exhibits a difference in the
trait that is at least about 5% greater, for example at least about
5%, at least about 8%, at least about 10%, at least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
75%, at least about 80%, at least about 80%, at least about 90%, or
at least 100%, at least about 200%, at least about 300%, at least
about 400% or greater than a reference agricultural plant grown
under the same conditions in the soil. In additional examples, a
plant resulting from the methods described herein exhibits a
difference in the trait that is at least about 5% greater, for
example at least about 5%, at least about 8%, at least about 10%,
at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 75%, at least about 80%, at least about
80%, at least about 90%, or at least 100%, at least about 200%, at
least about 300%, at least about 400% or greater than a reference
agricultural plant grown under similar conditions in the soil.
[0234] The trait to be improved may be assessed under conditions
including the application of one or more biotic or abiotic
stressors. Examples of stressors include abiotic stresses (such as
heat stress, salt stress, drought stress, cold stress, and low
nutrient stress) and biotic stresses (such as nematode stress,
insect herbivory stress, fungal pathogen stress, bacterial pathogen
stress, and viral pathogen stress).
[0235] The trait improved by methods and compositions of the
present disclosure may be nitrogen fixation, including in a plant
not previously capable of nitrogen fixation. In some cases,
bacteria isolated according to a method described herein produce 1%
or more (e.g. 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or
more) of a plant's nitrogen, which may represent an increase in
nitrogen fixation capability of at least 2-fold (e.g. 3-fold,
4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,
50-fold, 100-fold, 1000-fold, or more) as compared to bacteria
isolated from the first plant before introducing any genetic
variation. In some cases, the bacteria produce 5% or more of a
plant's nitrogen. The desired level of nitrogen fixation may be
achieved after repeating the steps of introducing genetic
variation, exposure to a plurality of plants, and isolating
bacteria from plants with an improved trait one or more times (e.g.
1, 2, 3, 4, 5, 10, 15, 25, or more times). In some cases, enhanced
levels of nitrogen fixation are achieved in the presence of
fertilizer supplemented with glutamine, ammonia, or other chemical
source of nitrogen. Methods for assessing degree of nitrogen
fixation are known, examples of which are described herein.
[0236] Microbe breeding is a method to systematically identify and
improve the role of species within the crop microbiome. The method
comprises three steps: 1) selection of candidate species by mapping
plant-microbe interactions and predicting regulatory networks
linked to a particular phenotype, 2) pragmatic and predictable
improvement of microbial phenotypes through intra-species crossing
of regulatory networks and gene clusters, and 3) screening and
selection of new microbial genotypes that produce desired crop
phenotypes. To systematically assess the improvement of strains, a
model is created that links colonization dynamics of the microbial
community to genetic activity by key species. The model is used to
predict genetic targets for breeding and improve the frequency of
selecting improvements in microbiome-encoded traits of agronomic
relevance.
Measuring Nitrogen Delivered in an Agriculturally Relevant Field
Context
[0237] In the field, the amount of nitrogen delivered can be
determined by the function of colonization multiplied by the
activity.
Nitrogen .times. .times. delivered = .intg. Time .times. &
.times. .times. Space .times. Colonization .times. Activity
##EQU00001##
[0238] The above equation requires (1) the average colonization per
unit of plant tissue, and (2) the activity as either the amount of
nitrogen fixed or the amount of ammonia excreted by each microbial
cell. To convert to pounds of nitrogen per acre, corn growth
physiology is tracked over time, e.g., size of the plant and
associated root system throughout the maturity stages.
[0239] The pounds of nitrogen delivered to a crop per acre-season
can be calculated by the following equation:
Nitrogen delivered=.intg.Plant
Tissue(t).times.Colonization(t).times.Activity(t)dt
[0240] The Plant Tissue(t) is the fresh weight of corn plant tissue
over the growing time (t). Values for reasonably making the
calculation are described in detail in the publication entitled
Roots, Growth and Nutrient Uptake (Mengel. Dept. of Agronomy Pub.
#AGRY-95-08 (Rev. May-95. p. 1-8).
[0241] The Colonization (t) is the amount of the microbes of
interest found within the plant tissue, per gram fresh weight of
plant tissue, at any particular time, t, during the growing season.
In the instance of only a single timepoint available, the single
timepoint is normalized as the peak colonization rate over the
season, and the colonization rate of the remaining timepoints are
adjusted accordingly.
[0242] Activity(t) is the rate of which N is fixed by the microbes
of interest per unit time, at any particular time, t, during the
growing season. In the embodiments disclosed herein, this activity
rate is approximated by in vitro acetylene reduction assay (ARA) in
ARA media in the presence of 5 mM glutamine or Ammonium excretion
assay in ARA media in the presence of 5 mM ammonium ions.
[0243] The Nitrogen delivered amount is then calculated by
numerically integrating the above function. In cases where the
values of the variables described above are discretely measured at
set timepoints, the values in between those timepoints are
approximated by performing linear interpolation.
Nitrogen Fixation
[0244] Described herein are methods of increasing nitrogen fixation
in a plant, comprising exposing the plant to bacteria comprising
one or more genetic variations introduced into one or more genes
regulating nitrogen fixation, wherein the bacteria produce 1% or
more of nitrogen in the plant (e.g. 2%, 5%, 10%, or more), which
may represent a nitrogen-fixation capability of at least 2-fold as
compared to the plant in the absence of the bacteria. The bacteria
may produce the nitrogen in the presence of fertilizer supplemented
with glutamine, urea, nitrates or ammonia. Genetic variations can
be any genetic variation described herein, including examples
provided above, in any number and any combination. The genetic
variation may be introduced into a gene selected from the group
consisting of nifA, nifL, ntrB, ntrC, glutamine synthetase, glnA,
glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifJ, nifH, nifD,
nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF,
nifB, and nifQ. The genetic variation may be a mutation that
results in one or more of: increased expression or activity of nifA
or glutaminase; decreased expression or activity of nifL, ntrB,
glutamine synthetase, glnB, glnK, draT, amtB; decreased
adenylyl-removing activity of GlnE; or decreased uridylyl-removing
activity of GlnD. The genetic variation introduced into one or more
bacteria of the methods disclosed herein may be a knock-out
mutation or it may abolish a regulatory sequence of a target gene,
or it may comprise insertion of a heterologous regulatory sequence,
for example, insertion of a regulatory sequence found within the
genome of the same bacterial species or genus. The regulatory
sequence can be chosen based on the expression level of a gene in a
bacterial culture or within plant tissue. The genetic variation may
be produced by chemical mutagenesis. The plants grown in step (c)
may be exposed to biotic or abiotic stressors.
[0245] The amount of nitrogen fixation that occurs in the plants
described herein may be measured in several ways, for example by an
acetylene-reduction (AR) assay. An acetylene-reduction assay can be
performed in vitro or in vivo. Evidence that a particular bacterium
is providing fixed nitrogen to a plant can include: 1) total plant
N significantly increases upon inoculation, preferably with a
concomitant increase in N concentration in the plant; 2) nitrogen
deficiency symptoms are relieved under N-limiting conditions upon
inoculation (which should include an increase in dry matter); 3)
N.sub.2 fixation is documented through the use of an .sup.15N
approach (which can be isotope dilution experiments, .sup.15N.sub.2
reduction assays, or .sup.15N natural abundance assays); 4) fixed N
is incorporated into a plant protein or metabolite; and 5) all of
these effects are not be seen in non-inoculated plants or in plants
inoculated with a mutant of the inoculum strain.
[0246] The wild-type nitrogen fixation regulatory cascade can be
represented as a digital logic circuit where the inputs O.sub.2 and
NH.sub.4.sup.+ pass through a NOR gate, the output of which enters
an AND gate in addition to ATP. In some embodiments, the methods
disclosed herein disrupt the influence of NH.sub.4.sup.+ on this
circuit, at multiple points in the regulatory cascade, so that
microbes can produce nitrogen even in fertilized fields. However,
the methods disclosed herein also envision altering the impact of
ATP or O.sub.2 on the circuitry, or replacing the circuitry with
other regulatory cascades in the cell, or altering genetic circuits
other than nitrogen fixation. Gene clusters can be re-engineered to
generate functional products under the control of a heterologous
regulatory system. By eliminating native regulatory elements
outside of, and within, coding sequences of gene clusters, and
replacing them with alternative regulatory systems, the functional
products of complex genetic operons and other gene clusters can be
controlled and/or moved to heterologous cells, including cells of
different species other than the species from which the native
genes were derived. Once re-engineered, the synthetic gene clusters
can be controlled by genetic circuits or other inducible regulatory
systems, thereby controlling the products' expression as desired.
The expression cassettes can be designed to act as logic gates,
pulse generators, oscillators, switches, or memory devices. The
controlling expression cassette can be linked to a promoter such
that the expression cassette functions as an environmental sensor,
such as an oxygen, temperature, touch, osmotic stress, membrane
stress, or redox sensor.
[0247] As an example, the nifL, nifA, nifT, and nifX genes can be
eliminated from the nif gene cluster. Synthetic genes can be
designed by codon randomizing the DNA encoding each amino acid
sequence. Codon selection is performed, specifying that codon usage
be as divergent as possible from the codon usage in the native
gene. Proposed sequences are scanned for any undesired features,
such as restriction enzyme recognition sites, transposon
recognition sites, repetitive sequences, sigma 54 and sigma 70
promoters, cryptic ribosome binding sites, and rho independent
terminators. Synthetic ribosome binding sites are chosen to match
the strength of each corresponding native ribosome binding site,
such as by constructing a fluorescent reporter plasmid in which the
150 bp surrounding a gene's start codon (from -60 to +90) is fused
to a fluorescent gene. This chimera can be expressed under control
of the Ptac promoter, and fluorescence measured via flow cytometry.
To generate synthetic ribosome binding sites, a library of reporter
plasmids using 150 bp (-60 to +90) of a synthetic expression
cassette is generated. Briefly, a synthetic expression cassette can
consist of a random DNA spacer, a degenerate sequence encoding an
RBS library, and the coding sequence for each synthetic gene.
Multiple clones are screened to identify the synthetic ribosome
binding site that best matched the native ribosome binding site.
Synthetic operons that consist of the same genes as the native
operons are thus constructed and tested for functional
complementation. A further exemplary description of synthetic
operons is provided in US20140329326.
Bacterial Species
[0248] Microbes useful in the methods and compositions disclosed
herein may be obtained from any source. In some cases, microbes may
be bacteria, archaea, protozoa or fungi. The microbes of this
disclosure may be nitrogen fixing microbes, for example a nitrogen
fixing bacteria, nitrogen fixing archaea, nitrogen fixing fungi,
nitrogen fixing yeast, or nitrogen fixing protozoa. Microbes useful
in the methods and compositions disclosed herein may be
spore-forming microbes, for example spore forming bacteria. In some
cases, bacteria useful in the methods and compositions disclosed
herein may be Gram positive bacteria or Gram negative bacteria. In
some cases, the bacteria may be an endospore forming bacteria of
the Firmicute phylum. In some cases, the bacteria may be a
diazotroph. In some cases, the bacteria may not be a
diazotroph.
[0249] The methods and compositions of this disclosure may be used
with an archaea, such as, for example, Methanothermobacter
thermoautotrophicus.
[0250] In some cases, bacteria which may be useful include, but are
not limited to, Agrobacterium radiobacter, Bacillus acidocaldarius,
Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus
albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus
aminoglucosidicus, Bacillus aminovorans, Bacillus amylolyticus
(also known as Paenibacillus amylolyticus) Bacillus
amyloliquefaciens, Bacillus aneurinolyticus, Bacillus atrophaeus,
Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms:
Bacillus endorhythmos, Bacillus medusa), Bacillus chitinosporus,
Bacillus circulars, Bacillus coagulans, Bacillus endoparasiticus
Bacillus fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacillus
lacticola, Bacillus lactimorbus, Bacillus lactis, Bacillus
laterosporus (also known as Brevibacillus laterosporus), Bacillus
lautus, Bacillus lentimorbus, Bacillus lentus, Bacillus
licheniformis, Bacillus maroccanus, Bacillus megaterium, Bacillus
metiens, Bacillus mycoides, Bacillus natto, Bacillus nematocida,
Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus,
Bacillus popillae, Bacillus psychrosaccharolyticus, Bacillus
pumilus, Bacillus siamensis, Bacillus smithii, Bacillus sphaericus,
Bacillus subtilis, Bacillus thuringiensis, Bacillus uniflagellatus,
Bradyrhizobium japonicum, Brevibacillus brevis Brevibacillus
laterosporus (formerly Bacillus laterosporus), Chromobacterium
subtsugae, Delftia acidovorans, Lactobacillus acidophilus,
Lysobacter antibioticus, Lysobacter enzymogenes, Paenibacillus
alvei, Paenibacillus polymyxa, Paenibacillus popilliae (formerly
Bacillus popilliae), Pantoea agglomerans, Pasteuria penetrans
(formerly Bacillus penetrans), Pasteuria usgae, Pectobacterium
carotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa,
Pseudomonas aureofaciens, Pseudomonas cepacia (formerly known as
Burkholderia cepacia), Pseudomonas chlororaphis, Pseudomonas
fluorescens, Pseudomonas proradix, Pseudomonas putida, Pseudomonas
syringae, Serratia entomophila, Serratia marcescens, Streptomyces
colombiensis, Streptomyces galbus, Streptomyces goshikiensis,
Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces
prasinus, Streptomyces saraceticus, Streptomyces venezuelae,
Xanthomonas campestris, Xenorhabdus luminescens, Xenorhabdus
nematophila, Rhodococcus globerulus AQ719 (NRRL Accession No.
B-21663), Bacillus sp. AQ175 (ATCC Accession No. 55608), Bacillus
sp. AQ 177 (ATCC Accession No. 55609), Bacillus sp. AQ178 (ATCC
Accession No. 53522), and Streptomyces sp. strain NRRL Accession
No. B-30145. In some cases the bacterium may be Azotobacter
chroococcum, Methanosarcina barkeri, Klebsiella pneumoniae,
Azotobacter vinelandii, Rhodobacter spharoides, Rhodobacter
capsulatus, Rhodobacter palustris, Rhodosporillum rubrum, Rhizobium
leguminosarum or Rhizobium etli.
[0251] In some cases, the bacterium may be a species of
Clostridium, for example Clostridium pasteurianum, Clostridium
beijerinckii, Clostridium perfringens, Clostridium tetani,
Clostridium acetobutylicum.
[0252] In some cases, bacteria used with the methods and
compositions of the present disclosure may be cyanobacteria.
Examples of cyanobacterial genuses include Anabaena (for example
Anagaena sp. PCC7120), Nostoc (for example Nostoc punctiforme), or
Synechocystis (for example Synechocystis sp. PCC6803).
[0253] In some cases, bacteria used with the methods and
compositions of the present disclosure may belong to the phylum
Chlorobi, for example Chlorobium tepidum.
[0254] In some cases, microbes used with the methods and
compositions of the present disclosure may comprise a gene
homologous to a known NifH gene. Sequences of known NifH genes may
be found in, for example, the Zehr lab NifH database,
(wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, Apr. 4, 2014), or the
Buckley lab NifH database
(www.css.cornell.edu/faculty/buckley/nifh, and Gaby, John
Christian, and Daniel H. Buckley. "A comprehensive aligned nifH
gene database: a multipurpose tool for studies of nitrogen-fixing
bacteria." Database 2014 (2014): bau001). In some cases, microbes
used with the methods and compositions of the present disclosure
may comprise a sequence which encodes a polypeptide with at least
60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99% or more than 99%
sequence identity to a sequence from the Zehr lab NifH database,
(wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, Apr. 4, 2014). In some
cases, microbes used with the methods and compositions of the
present disclosure may comprise a sequence which encodes a
polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%,
98%, 99% or more than 99% sequence identity to a sequence from the
Buckley lab NifH database, (Gaby, John Christian, and Daniel H.
Buckley. "A comprehensive aligned nifH gene database; a
multipurpose tool for studies of nitrogen-fixing bacteria."
Database 2014 (2014): bau00I).
[0255] Microbes useful in the methods and compositions disclosed
herein can be obtained by extracting microbes from surfaces or
tissues of native plants; grinding seeds to isolate microbes;
planting seeds in diverse soil samples and recovering microbes from
tissues; or inoculating plants with exogenous microbes and
determining which microbes appear in plant tissues. Non-limiting
examples of plant tissues include a seed, seedling, leaf, cutting,
plant, bulb, tuber, root, and rhizomes. In some cases, bacteria are
isolated from a seed. The parameters for processing samples may be
varied to isolate different types of associative microbes, such as
rhizospheric, epiphytes, or endophytes. Bacteria may also be
sourced from a repository, such as environmental strain
collections, instead of initially isolating from a first plant. The
microbes can be genotyped and phenotyped, via sequencing the
genomes of isolated microbes; profiling the composition of
communities in planta; characterizing the transcriptomic
functionality of communities or isolated microbes; or screening
microbial features using selective or phenotypic media (e.g.,
nitrogen fixation or phosphate solubilization phenotypes). Selected
candidate strains or populations can be obtained via sequence data;
phenotype data; plant data (e.g., genome, phenotype, and/or yield
data); soil data (e.g., pH, N/P/K content, and/or bulk soil biotic
communities); or any combination of these.
[0256] The bacteria and methods of producing bacteria described
herein may apply to bacteria able to self-propagate efficiently on
the leaf surface, root surface, or inside plant tissues without
inducing a damaging plant defense reaction, or bacteria that are
resistant to plant defense responses. The bacteria described herein
may be isolated by culturing a plant tissue extract or leaf surface
wash in a medium with no added nitrogen. However, the bacteria may
be unculturable, that is, not known to be culturable or difficult
to culture using standard methods known in the art. The bacteria
described herein may be an endophyte or an epiphyte or a bacterium
inhabiting the plant rhizosphere (rhizospheric bacteria). The
bacteria obtained after repeating the steps of introducing genetic
variation, exposure to a plurality of plants, and isolating
bacteria from plants with an improved trait one or more times (e.g.
1, 2, 3, 4, 5, 10, 15, 25, or more times) may be endophytic,
epiphytic, or rhizospheric. Endophytes are organisms that enter the
interior of plants without causing disease symptoms or eliciting
the formation of symbiotic structures, and are of agronomic
interest because they can enhance plant growth and improve the
nutrition of plants (e.g., through nitrogen fixation). The bacteria
can be a seed-borne endophyte. Seed-borne endophytes include
bacteria associated with or derived from the seed of a grass or
plant, such as a seed-borne bacterial endophyte found in mature,
dry, undamaged (e.g., no cracks, visible fungal infection, or
prematurely germinated) seeds. The seed-borne bacterial endophyte
can be associated with or derived from the surface of the seed;
alternatively, or in addition, it can be associated with or derived
from the interior seed compartment (e.g., of a surface-sterilized
seed). In some cases, a seed-borne bacterial endophyte is capable
of replicating within the plant tissue, for example, the interior
of the seed. Also, in some cases, the seed-borne bacterial
endophyte is capable of surviving desiccation.
[0257] The bacterial isolated according to methods of the
disclosure, or used in methods or compositions of the disclosure,
can comprise a plurality of different bacterial taxa in
combination. By way of example, the bacteria may include
Proteobacteria (such as Pseudomonas, Enterobacter,
Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea,
Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter,
Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas),
Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus,
Mycoplasma, and Acetabacterium), and Actinobacteria (such as
Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium). The
bacteria used in methods and compositions of this disclosure may
include nitrogen fixing bacterial consortia of two or more species.
In some cases, one or more bacterial species of the bacterial
consortia may be capable of fixing nitrogen. In some cases, one or
more species of the bacterial consortia may facilitate or enhance
the ability of other bacteria to fix nitrogen. The bacteria which
fix nitrogen and the bacteria which enhance the ability of other
bacteria to fix nitrogen may be the same or different. In some
examples, a bacterial strain may be able to fix nitrogen when in
combination with a different bacterial strain, or in a certain
bacterial consortia, but may be unable to fix nitrogen in a
monoculture. Examples of bacterial genuses which may be found in a
nitrogen fixing bacterial consortia include, but are not limited
to, Herbaspirillum, Azospirillum, Enterobacter, and Bacillus.
[0258] Bacteria that can be produced by the methods disclosed
herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp.,
and Sinorhizobium sp. In some cases, the bacteria may be selected
from the group consisting of: Azotobacter vinelandii,
Bradyrhizobium japonicum, Klebsiella pneumoniae, and Sinorhizobium
meliloti. In some cases, the bacteria may be of the genus
Enterobacter or Rahnella. In some cases, the bacteria may be of the
genus Frankia, or Clostridium. Examples of bacteria of the genus
Clostridium include, but are not limited to, Clostridium
acetobutilicum, Clostridium pasteurianum, Clostridium beijerinckii,
Clostridium perfringens, and Clostridium tetani. In some cases, the
bacteria may be of the genus Paenibacillus, for example
Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus
durus, Paenibacillus macerans, Paenibacillus polymyxa,
Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus
campinasensis, Paenibacillus chibensis, Paenibacillus
glucanolyticus, Paenibacillus illinoisensis, Paenibacillus larvae
subsp. Larvae, Paenibacillus larvae subsp. Pulvifaciens,
Paenibacillus lautus, Paenibacillus macerans, Paenibacillus
macquariensis, Paenibacillus macquariensis, Paenibacillus pabuli,
Paenibacillus peoriae, or Paenibacillus polymyxa.
[0259] In some examples, bacteria isolated according to methods of
the disclosure can be a member of one or more of the following
taxa: Achromobacter, Acidithiobacillus, Acidovorax, Acidovoraz,
Acinetobacter, Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas,
Afipia, Agromyces, Ancylobacter, Arthrobacter, Atopostipes,
Azospirillum, Bacillus, Bdellovibrio, Beijerinckia, Bosea,
Bradyrhizobium, Brevibacillus, Brevundimonas, Burkholderia,
Candidatus Haloredivivus, Caulobacter, Cellulomonas, Cellvibrio,
Chryseobacterium, Citrobacter, Clostridium, Coraliomargarita,
Corynebacterium, Cupriavidus, Curtobacterium, Curvibacter,
Deinococcus, Delftia, Desemzia, Devosia, Dokdonella, Dyella,
Enhydrobacter, Enterobacter, Enterococcus, Erwinia, Escherichia,
Escherichia/Shigella, Exiguobacterium, Ferroglobus, Filimonas,
Finegoldia, Flavisolibacter, Flavobacterium, Frigoribacterium,
Gluconacetobacter, Hafnia, Halobaculum, Halomonas, Halosimplex,
Herbaspirillum, Hymenobacter, Klebsiella, Kocuria, Kosakonia,
Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas,
Massilia, Mesorhizobium, Methylobacterium, Microbacterium,
Micrococcus, Microvirga, Mycobacterium, Neisseria, Nocardia,
Oceanibaculum, Ochrobactrum, Okibacterium, Oligotropha, Oryzihumus,
Oxalophagus, Paenibacillus, Panteoa, Pantoea, Pelomonas,
Perlucidibaca, Plantibacter, Polynucleobacter, Propionibacterium,
Propioniciclava, Pseudoclavibacter, Pseudomonas, Pseudonocardia,
Pseudoxanthomonas, Psychrobacter, Rahnella, Ralstonia,
Rheinheimera, Rhizobium, Rhodococcus, Rhodopseudomonas, Roseateles,
Ruminococcus, Sebaldella, Sediminibacillus, Sediminibacterium,
Serratia, Shigella, Shigella, Sinorhizobium, Sinosporangium,
Sphingobacterium, Sphingomonas, Sphingopyxis, Sphingosinicella,
Staphylococcus, 25 Stenotrophomonas, Strenotrophomonas,
Streptococcus, Streptomyces, Stygiolobus, Sulfurisphaera,
Tatumella, Tepidimonas, Thermomonas, Thiobacillus, Variovorax,
WPS-2 genera incertae sedis, Xanthomonas, and Zimmermannella.
[0260] In some cases, a bacterial species selected from at least
one of the following genera are utilized: Enterobacter, Klebsiella,
Kosakonia, and Rahnella. In some cases, a combination of bacterial
species from the following genera are utilized: Enterobacter,
Klebsiella, Kosakonia, and Rahnella. In some cases, the species
utilized can be one or more of: Enterobacter sacchari, Klebsiella
variicola, Kosakonia sacchari, and Rahnella aquatilis.
[0261] In some cases, a Gram positive microbe may have a
Molybdenum-Iron nitrogenase system comprising: nifH, nifD, nifK,
nifB, nifE, nifN, nifX, hesA, nifV, nifW, nifU, nifS, and MN. In
some cases, a Gram positive microbe may have a vanadium nitrogenase
system comprising: vnfDG, vnfK, vnfE, vnfN, vupC, vupB, vupA, vnfV,
vnfR1, vnfH, vnfR2, vnfA (transcriptional regulator). In some
cases, a Gram positive microbe may have an iron-only nitrogenase
system comprising: anfK, anfG, anfD, anfH, anfA (transcriptional
regulator). In some cases a Gram positive microbe may have a
nitrogenase system comprising glnB, and glnK (nitrogen signaling
proteins). Some examples of enzymes involved in nitrogen metabolism
in Gram positive microbes include glnA (glutamine synthetase), gdh
(glutamate dehydrogenase), bdh (3-hydroxybutyrate dehydrogenase),
glutaminase, gltAB/gltB/gltS (glutamate synthase), asnA/asnB
(aspartate-ammonia ligase/asparagine synthetase), and ansA/ansZ
(asparaginase). Some examples of proteins involved in nitrogen
transport in Gram positive microbes include amtB (ammonium
transporter), glnK (regulator of ammonium transport),
glnPHQ/glnQHMP (ATP-dependent glutamine/glutamate transporters),
glnT/alsT/yrbD/yflA (glutamine-like proton symport transporters),
and gltP/gltT/yhcl/nqt (glutamate-like proton symport
transporters).
[0262] Examples of Gram positive microbes which may be of
particular interest include Paenibacillus polymixa, Paenibacillus
riograndensis, Paenibacillus sp., Frankia sp., Heliobacterium sp.,
Heliobacterium chlorum, Heliobacillus sp., Heliophilum sp.,
Heliorestis sp., Clostridium acetobutylicum, Clostridium sp.,
Mycobacterium flaum, Mycobacterium sp., Arthrobacter sp., Agromyces
sp., Corynebacterium autitrophicum, Corynebacterium sp.,
Micromonspora sp., Propionibacteria sp., Streptomyces sp., and
Microbacterium sp.
[0263] Some examples of genetic alterations which may be made in
Gram positive microbes include: deleting glnR to remove negative
regulation of BNF in the presence of environmental nitrogen,
inserting different promoters directly upstream of the nif cluster
to eliminate regulation by GlnR in response to environmental
nitrogen, mutating glnA to reduce the rate of ammonium assimilation
by the GS-GOGAT pathway, deleting amtB to reduce uptake of ammonium
from the media, mutating glnA so it is constitutively in the
feedback-inhibited (FBI-GS) state, to reduce ammonium assimilation
by the GS-GOGAT pathway.
[0264] In some cases, glnR is the main regulator of N metabolism
and fixation in Paenibacillus species. In some cases, the genome of
a Paenibacillus species may not contain a gene to produce glnR. In
some cases, the genome of a Paenibacillus species may not contain a
gene to produce glnE or glnD. In some cases, the genome of a
Paenibacillus species may contain a gene to produce glnB or glnK.
For example, Paenibacillus sp. WLY78 doesn't contain a gene for
glnB, or its homologs found in the archaeon Methanococcus
maripaludis, nifI1 and nifI2. In some cases, the genomes of
Paenibacillus species may be variable. For example, Paenibacillus
polymixa E681 lacks glnK and gdh, has several nitrogen compound
transporters, but only amtB appears to be controlled by GlnR. In
another example, Paenibacillus sp. JDR2 has glnK, gdh and most
other central nitrogen metabolism genes, has many fewer nitrogen
compound transporters, but does have glnPHQ controlled by GlnR.
Paenibacillus riograndensis SBR5 contains a standard glnRA operon,
an fdx gene, a main nif operon, a secondary nif operon, and an anf
operon (encoding iron-only nitrogenase). Putative glnR/tnrA sites
were found upstream of each of these operons. GlnR may regulate all
of the above operons, except the anf operon. GlnR may bind to each
of these regulatory sequences as a dimer.
[0265] Paenibacillus N-fixing strains may fall into two subgroups:
Subgroup I, which contains only a minimal nif gene cluster and
subgroup II, which contains a minimal cluster, plus an
uncharacterized gene between nifX and hesA, and often other
clusters duplicating some of the nif genes, such as nifH, nifHDK,
nifBEN, or clusters encoding vanadium nitrogenase (vnf) or
iron-only nitrogenase (anf) genes.
[0266] In some cases, the genome of a Paenibacillus species may not
contain a gene to produce glnB or glnK. In some cases, the genome
of a Paenibacillus species may contain a minimal nif cluster with 9
genes transcribed from a sigma-70 promoter. In some cases, a
Paenibacillus nif cluster may be negatively regulated by nitrogen
or oxygen. In some cases, the genome of a Paenibacillus species may
not contain a gene to produce sigma-54. For example, Paenibacillus
sp. WLY78 does not contain a gene for sigma-54. In some cases, a
nif cluster may be regulated by glnR, and/or TnrA. In some cases,
activity of a nif cluster may be altered by altering activity of
glnR, and/or TnrA.
[0267] In Bacilli, glutamine synthetase (GS) is feedback-inhibited
by high concentrations of intracellular glutamine, causing a shift
in confirmation (referred to as FBI-GS). Nif clusters contain
distinct binding sites for the regulators GlnR and TnrA in several
Bacilli species. GlnR binds and represses gene expression in the
presence of excess intracellular glutamine and AMP. A role of GlnR
may be to prevent the influx and intracellular production of
glutamine and ammonium under conditions of high nitrogen
availability. TnrA may bind and/or activate (or repress) gene
expression in the presence of limiting intracellular glutamine,
and/or in the presence of FBI-GS. In some cases, the activity of a
Bacilli nif cluster may be altered by altering the activity of
GlnR.
[0268] Feedback-inhibited glutamine synthetase (FBI-GS) may bind
GlnR and stabilize binding of GlnR to recognition sequences.
Several bacterial species have a GlnR/TnrA binding site upstream of
the nif cluster. Altering the binding of FBI-GS and GlnR may alter
the activity of the nif pathway.
Sources of Microbes
[0269] The bacteria (or any microbe according to the disclosure)
may be obtained from any general terrestrial environment, including
its soils, plants, fungi, animals (including invertebrates) and
other biota, including the sediments, water and biota of lakes and
rivers; from the marine environment, its biota and sediments (for
example, sea water, marine muds, marine plants, marine
invertebrates (for example, sponges), marine vertebrates (for
example, fish)); the terrestrial and marine geosphere (regolith and
rock, for example, crushed subterranean rocks, sand and clays); the
cryosphere and its meltwater; the atmosphere (for example, filtered
aerial dusts, cloud and rain droplets); urban, industrial and other
man-made environments (for example, accumulated organic and mineral
matter on concrete, roadside gutters, roof surfaces, and road
surfaces).
[0270] The plants from which the bacteria (or any microbe according
to the disclosure) are obtained may be a plant having one or more
desirable traits, for example a plant that naturally grows in a
particular environment or under certain conditions of interest. By
way of example, a certain plant may naturally grow in sandy soil or
sand of high salinity, or under extreme temperatures, or with
little water, or it may be resistant to certain pests or disease
present in the environment, and it may be desirable for a
commercial crop to be grown in such conditions, particularly if
they are, for example, the only conditions available in a
particular geographic location. By way of further example, the
bacteria may be collected from commercial crops grown in such
environments, or more specifically from individual crop plants best
displaying a trait of interest amongst a crop grown in any specific
environment: for example the fastest-growing plants amongst a crop
grown in saline-limiting soils, or the least damaged plants in
crops exposed to severe insect damage or disease epidemic, or
plants having desired quantities of certain metabolites and other
compounds, including fiber content, oil content, and the like, or
plants displaying desirable colors, taste or smell. The bacteria
may be collected from a plant of interest or any material occurring
in the environment of interest, including fungi and other animal
and plant biota, soil, water, sediments, and other elements of the
environment as referred to previously.
[0271] The bacteria (or any microbe according to the disclosure)
may be isolated from plant tissue. This isolation can occur from
any appropriate tissue in the plant, including for example root,
stem and leaves, and plant reproductive tissues. By way of example,
conventional methods for isolation from plants typically include
the sterile excision of the plant material of interest (e.g. root
or stem lengths, leaves), surface sterilization with an appropriate
solution (e.g. 2% sodium hypochlorite), after which the plant
material is placed on nutrient medium for microbial growth.
Alternatively, the surface-sterilized plant material can be crushed
in a sterile liquid (usually water) and the liquid suspension,
including small pieces of the crushed plant material spread over
the surface of a suitable solid agar medium, or media, which may or
may not be selective (e.g. contain only phytic acid as a source of
phosphorus). This approach is especially useful for bacteria which
form isolated colonies and can be picked off individually to
separate plates of nutrient medium, and further purified to a
single species by well-known methods. Alternatively, the plant root
or foliage samples may not be surface sterilized but only washed
gently thus including surface-dwelling epiphytic microorganisms in
the isolation process, or the epiphytic microbes can be isolated
separately, by imprinting and lifting off pieces of plant roots,
stem or leaves onto the surface of an agar medium and then
isolating individual colonies as above. This approach is especially
useful for bacteria, for example. Alternatively, the roots may be
processed without washing off small quantities of soil attached to
the roots, thus including microbes that colonize the plant
rhizosphere. Otherwise, soil adhering to the roots can be removed,
diluted and spread out onto agar of suitable selective and
non-selective media to isolate individual colonies of rhizospheric
bacteria.
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedures
[0272] The microbial deposits of the present disclosure were made
under the provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purpose of
Patent Procedure (Budapest Treaty).
[0273] Applicants state that pursuant to 37 C.F.R. .sctn. 1.808
(a)(2) "all restrictions imposed by the depositor on the
availability to the public of the deposited material will be
irrevocably removed upon the granting of the patent." This
statement is subject to paragraph (b) of this section (i.e. 37
C.F.R. .sctn. 1.808 (b)).
[0274] The Enterobacter sacchari has now been reclassified as
Kosakonia sacchari, the name for the organism may be used
interchangeably throughout the manuscript.
[0275] Many microbes of the present disclosure are derived from two
wild-type strains, as depicted in FIG. 6 and FIG. 7. Strain CI006
is a bacterial species previously classified in the genus
Enterobacter (see aforementioned reclassification into Kosakonia),
and FIG. 6 identifies the lineage of the mutants that have been
derived from CI006. Strain CI019 is a bacterial species classified
in the genus Rahnella, and FIG. 7 identifies the lineage of the
mutants that have been derived from CI019. With regard to FIG. 6
and FIG. 7, it is noted that strains comprising CM in the name are
mutants of the strains depicted immediately to the left of said CM
strain. The deposit information for the CI006 Kosakonia wild type
(WT) and CI019 Rahnella WT are found in the below Table 1.
[0276] Some microorganisms described in this application were
deposited on Jan. 6, 2017 or Aug. 11, 2017 with the Bigelow
National Center for Marine Algae and Microbiota (NCMA), located at
60 Bigelow Drive, East Boothbay, Me. 04544, USA. As aforementioned,
all deposits were made under the terms of the Budapest Treaty on
the International Recognition of the Deposit of Microorganisms for
the Purposes of Patent Procedure. The Bigelow National Center for
Marine Algae and Microbiota accession numbers and dates of deposit
for the aforementioned Budapest Treaty deposits are provided in
Table 1.
[0277] Biologically pure cultures of Kosakonia sacchari (WT),
Rahnella aquatilis (WT), and a variant/remodeled Kosakonia sacchari
strain were deposited on Jan. 6, 2017 with the Bigelow National
Center for Marine Algae and Microbiota (NCMA), located at 60
Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA
Patent Deposit Designation numbers 201701001, 201701003, and
201701002, respectively. The applicable deposit information is
found below in Table 1.
[0278] Biologically pure cultures of variant/remodeled Kosakonia
sacchari strains were deposited on Aug. 11, 2017 with the Bigelow
National Center for Marine Algae and Microbiota (NCMA), located at
60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA
Patent Deposit Designation numbers 201708004, 201708003, and
201708002, respectively. The applicable deposit information is
found below in Table 1.
[0279] A biologically pure culture of Klebsiella variicola (WT) was
deposited on Aug. 11, 2017 with the Bigelow National Center for
Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive,
East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit
Designation number 201708001. Biologically pure cultures of two
Klebsiella variicola variants/remodeled strains were deposited on
Dec. 20, 2017 with the Bigelow National Center for Marine Algae and
Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me.
04544, USA, and assigned NCMA Patent Deposit Designation numbers
201712001 and 201712002, respectively. The applicable deposit
information is found below in Table 1.
TABLE-US-00001 TABLE 1 Microorganisms Deposited under the Budapest
Treaty Pivot Strain Designation (some strains have multiple
Accession Date of Depository designations) Taxonomy Number Deposit
NCMA CI006, Kosakonia sacchari (WT) 201701001 Jan. 6, 2017 PBC6.1,
6 NCMA CI019, 19 Rahnella aquatilis (WT) 201701003 Jan. 6, 2017
NCMA CM029, 6-412 Kosakonia sacchari 201701002 Jan. 6, 2017 NCMA
6-403 CM037 Kosakonia sacchari 201708004 Aug. 11, 2017 NCMA 6-404,
CM38, Kosakonia sacchari 201708003 Aug. 11, 2017 PBC6.38 NCMA
CM094, 6-881, Kosakonia sacchari 201708002 Aug. 11, 2017 PBC6.94
NCMA CI137, 137, Klebsiella variicola (WT) 201708001 Aug. 11, 2017
PB137 NCMA 137-1034 Klebsiella variicola 201712001 Dec. 20, 2017
NCMA 137-1036 Klebsiella variicola 201712002 Dec. 20, 2017
Isolated and Biologically Pure Microorganisms
[0280] The present disclosure, in certain embodiments, provides
isolated and biologically pure microorganisms that have
applications, inter alia, in agriculture. The disclosed
microorganisms can be utilized in their isolated and biologically
pure states, as well as being formulated into compositions (see
below section for exemplary composition descriptions). Furthermore,
the disclosure provides microbial compositions containing at least
two members of the disclosed isolated and biologically pure
microorganisms, as well as methods of utilizing said microbial
compositions. Furthermore, the disclosure provides for methods of
modulating nitrogen fixation in plants via the utilization of the
disclosed isolated and biologically pure microbes.
[0281] In some aspects, the isolated and biologically pure
microorganisms of the disclosure are those from Table 1. In other
aspects, the isolated and biologically pure microorganisms of the
disclosure are derived from a microorganism of Table 1. For
example, a strain, child, mutant, or derivative, of a microorganism
from Table 1 are provided herein. The disclosure contemplates all
possible combinations of microbes listed in Table 1, said
combinations sometimes forming a microbial consortia. The microbes
from Table 1, either individually or in any combination, can be
combined with any plant, active molecule (synthetic, organic,
etc.), adjuvant, carrier, supplement, or biological, mentioned in
the disclosure.
[0282] In some aspects, the disclosure provides microbial
compositions comprising species as grouped in Tables 2-8. In some
aspects, these compositions comprising various microbial species
are termed a microbial consortia or consortium.
[0283] With respect to Tables 2-8, the letters A through I
represent a non-limiting selection of microorganisms of the present
disclosure, defined as:
[0284] A=Microbe with accession number 201701001 identified in
Table 1;
[0285] B=Microbe with accession number 201701003 identified in
Table 1;
[0286] C=Microbe with accession number 201701002 identified in
Table 1;
[0287] D=Microbe with accession number 201708004 identified in
Table 1;
[0288] E=Microbe with accession number 201708003 identified in
Table 1;
[0289] F=Microbe with accession number 201708002 identified in
Table 1;
[0290] G=Microbe with accession number 201708001 identified in
Table 1;
[0291] H=Microbe with accession number 201712001 identified in
Table 1; and
[0292] I=Microbe with accession number 201712002 identified in
Table 1.
TABLE-US-00002 TABLE 2 Eight and Nine Strain Compositions A, B, C,
D, E, F, G, H A, B, C, D, E, F, G, I A, B, C, D, E, F, H, I A, B,
C, D, E, G, H, I A, B, C, D, F, G, H, I A, B, C, E, F, G, H, I A,
B, D, E, F, G, H, I A, C, D, E, F, G, H, I B, C, D, E, F, G, H, I
A, B, C, D, E, F, G, H, I
TABLE-US-00003 TABLE 3 Seven Strain Compositions A, B, C, D, E, F,
G A, B, C, D, E, F, H A, B, C, D, E, F, I A, B, C, D, E, G, H A, B,
C, D, E, G, I A, B, C, D, E, H, I A, B, C, D, F, G, H A, B, C, D,
F, G, I A, B, C, D, F, H, I A, B, C, D, G, H, I A, B, C, E, F, G, H
A, B, C, E, F, G, I A, B, C, E, F, H, I A, B, C, E, G, H, I A, B,
C, F, G, H, I A, B, D, E, F, G, H A, B, D, E, F, G, I A, B, D, E,
F, H, I A, B, D, E, G, H, I A, B, D, F, G, H, I A, B, E, F, G, H, I
A, C, D, E, F, G, H A, C, D, E, F, G, I A, C, D, E, F, H, I A, C,
D, E, G, H, I A, C, D, F, G, H, I A, C, E, F, G, H, I A, D, E, F,
G, H, I B, C, D, E, F, G, H B, C, D, E, F, G, I B, C, D, E, F, H, I
B, C, D, E, G, H, I B, C, D, F, G, H, I B, C, E, F, G, H, I B, D,
E, F, G, H, I C, D, E, F, G, H, I
TABLE-US-00004 TABLE 4 Six Strain Compositions A, B, C, D, E, F A,
B, C, D, E, G A, B, C, D, E, H A, B, C, D, E, I A, B, C, D, F, G A,
B, C, D, F, H A, B, C, D, F, I A, B, C, D, G, H A, B, C, D, G, I A,
B, C, D, H, I A, B, C, E, F, G A, B, C, E, F, H A, B, C, E, F, I A,
B, C, E, G, H A, B, C, E, G, I A, B, C, E, H, I A, B, C, F, G, H A,
B, C, F, G, I A, B, C, F, H, I A, B, C, G, H, I A, B, D, E, F, G A,
B, D, E, F, H A, B, D, E, F, I A, B, D, E, G, H A, B, D, E, G, I A,
B, D, E, H, I A, B, D, F, G, H A, B, D, F, G, I D, E, F, G, H, I C,
E, F, G, H, I A, B, D, F, H, I A, B, D, G, H, I A, B, E, F, G, H A,
B, E, F, G, I A, B, E, F, H, I A, B, E, G, H, I A, B, F, G, H, I A,
C, D, E, F, G A, C, D, E, F, H A, C, D, E, F, I A, C, D, E, G, H A,
C, D, E, G, I A, C, D, E, H, I A, C, D, F, G, H A, C, D, F, G, I A,
C, D, F, H, I A, C, D, G, H, I A, C, E, F, G, H A, C, E, F, G, I A,
C, E, F, H, I A, C, E, G, H, I A, C, F, G, H, I A, D, E, F, G, H A,
D, E, F, G, I A, D, E, F, H, I A, D, E, G, H, I A, D, F, G, H, I A,
E, F, G, H, I B, C, D, E, F, G B, C, D, E, F, H B, C, D, E, F, I B,
C, D, E, G, H B, C, D, E, G, I B, C, D, E, H, I B, C, D, F, G, H B,
C, D, F, G, I B, C, D, F, H, I B, C, D, G, H, I B, C, E, F, G, H B,
C, E, F, G, I B, C, E, F, H, I B, C, E, G, H, I B, C, F, G, H, I B,
D, E, F, G, H B, D, E, F, G, I B, D, E, F, H, I B, D, E, G, H, I B,
D, F, G, H, I B, E, F, G, H, I C, D, E, F, G, H C, D, E, F, G, I C,
D, E, F, H, I C, D, E, G, H, I C, D, F, G, H, I
TABLE-US-00005 TABLE 5 Five Strain Compositions A, B, C, D, E A, B,
C, D, F A, B, C, D, G A, B, C, D, H A, B, C, D, I A, B, C, E, F A,
B, C, E, G A, B, C, E, H A, B, C, F, H A, B, C, F, G A, B, C, F, I
A, B, C, G, H A, B, C, G, I A, B, C, H, I A, B, D, E, F A, B, D, E,
G A, B, D, E, I A, B, D, F, G A, B, D, F, H A, B, D, F, I A, B, D,
G, H A, B, D, G, I A, B, D, H, I A, B, E, F, G A, B, E, F, I A, B,
E, G, H A, B, E, G, I A, B, E, H, I A, B, F, G, H A, B, F, G, I A,
B, F, H, I A, B, G, H, I A, C, D, E, G A, C, D, E, H A, C, D, E, I
A, C, D, F, G A, C, D, F, H A, C, D, F, I A, C, D, G, H A, C, D, G,
I A, C, E, F, G A, C, E, F, H A, C, E, F, I A, C, E, G, H A, C, E,
G, I A, C, E, H, I A, C, F, G, H A, C, F, G, I A, C, G, H, I A, D,
E, F, G A, D, E, F, H A, D, E, F, I A, D, E, G, H A, D, E, G, I A,
D, E, H, I A, D, F, G, H A, D, F, H, I A, D, G, H, I A, E, F, G, H
A, E, F, G, I A, E, F, H, I A, E, G, H, I A, F, G, H, I B, C, D, E,
F B, C, D, E, H B, C, D, E, I B, C, D, F, G B, C, D, F, H B, C, D,
F, I B, C, D, G, H B, C, D, G, I B, C, D, H, I B, C, E, F, H B, C,
E, F, I B, C, E, G, H B, C, E, G, I B, C, E, H, I B, C, F, G, H B,
C, F, G, I B, C, F, H, I B, D, E, F, G B, D, E, F, H B, D, E, F, I
B, D, E, G, H B, D, E, G, I B, D, E, H, I B, D, F, G, H B, D, F, G,
I B, D, G, H, I B, E, F, G, H B, E, F, G, I B, E, F, H, I B, E, G,
H, I B, F, G, H, I C, D, E, F, G C, D, E, F, H C, D, E, G, H C, D,
E, G, I C, D, E, H, I C, D, F, G, H C, D, F, G, I C, D, F, H, I C,
D, G, H, I C, E, F, G, H C, E, F, H, I C, E, G, H, I C, F, G, H, I
D, E, F, G, H D, E, F, G, I D, E, F, H, I D, E, G, H, I D, F, G, H,
I A, B, C, E, I A, B, D, E, H A, B, E, F, H A, C, D, E, F A, C, D,
H, I A, C, F, H, I A, D, F, G, I B, C, D, E, G B, C, E, F, G B, C,
G, H, I B, D, F, H, I C, D, E, F, I C, E, F, G, I E, F, G, H, I
TABLE-US-00006 TABLE 6 Four Strain Compositions A, B, C, D A, B, C,
E A, B, C, F A, B, C, G A, B, C, H A, B, C, I A, B, D, E A, B, D, F
D, G, H, I A, B, D, G A, B, D, H A, B, D, I A, B, E, F A, B, E, G
A, B, E, H A, B, E, I A, B, F, G E, F, G, H A, B, F, H A, D, F, H
A, D, F, I A, D, G, H A, D, G, I A, D, H, I A, E, F, G A, E, F, H
E, F, G, I A, B, F, I A, B, G, H A, B, G, I A, B, H, I A, C, D, E
A, C, D, F A, C, D, G A, C, D, H E, F, H, I A, C, D, I A, C, E, F
A, C, E, G A, C, E, H A, C, E, I A, C, F, G A, C, F, H A, C, F, I
E, G, H, I A, C, G, H A, C, G, I A, C, H, I A, D, E, F A, D, E, G
A, D, E, H A, D, E, I A, D, F, G F, G, H, I A, E, F, I A, E, G, H
A, E, G, I A, E, H, I A, F, G, H A, F, G, I A, F, H, I A, G, H, I
D, E, F, H B, C, D, E B, C, D, F B, C, D, G B, C, D, H B, C, D, I
B, C, E, F B, C, E, G B, C, E, H D, E, F, I B, C, E, I B, C, F, G
B, C, F, H B, C, F, I B, C, G, H B, C, G, I B, C, H, I B, D, E, F
D, E, G, H B, D, E, G B, D, E, H B, D, E, I B, D, F, G B, D, F, H
B, D, F, I B, D, G, H B, D, G, I D, E, G, I B, D, H, I B, E, F, G
B, E, F, H B, E, F, I B, E, G, H B, E, G, I B, E, H, I B, F, G, H
D, E, H, I B, F, G, I B, F, H, I B, G, H, I C, D, E, F C, D, E, G
C, D, E, H C, D, E, I C, D, F, G D, F, G, H C, D, F, H C, D, F, I
C, D, G, H C, D, G, I C, D, H, I C, E, F, G C, E, F, H C, E, F, I
D, F, G, I C, E, G, H C, E, G, I C, E, H, I C, F, G, H C, F, G, I
C, F, H, I C, G, H, I D, E, F, G D, F, H, I
TABLE-US-00007 TABLE 7 Three Strain Compositions A, B, C A, B, D A,
B, E A, B, F A, B, G A, B, H A, B, I A, C, D A, C, E G, H, I E, F,
H A, C, F A, C, G A, C, H A, C, I A, D, E A, D, F A, D, G A, D, H
A, D, I F, H, I E, F, G A, E, F A, E, G A, E, H A, E, I A, F, G A,
F, H A, F, I A, G, H A, G, I F, G, I D, H, I A, H, I B, C, D B, C,
E B, C, F B, C, G B, C, H B, C, I B, D, E B, D, F F, G, H D, G, I
B, D, G B, D, H B, D, I B, E, F B, E, G B, E, H B, E, I B, F, G B,
F, H E, H, I E, F, I B, F, I B, G, H B, G, I B, H, I C, D, E C, D,
F C, D, G C, D, H C, D, I E, G, I D, G, H C, E, F C, E, G C, E, H
C, E, I C, F, G C, F, H C, F, I C, G, H C, G, I E, G, H D, F, I C,
H, I D, E, F D, E, G D, E, H D, E, I D, F, G D, F, H
TABLE-US-00008 TABLE 8 Two Strain Compositions A, B A, C A, D A, E
A, F A, G A, H A, I B, C B, D B, E B, F B, G B, H B, I C, D C, E C,
F C, G C, H C, I D, E D, F D, G D, H D, I E, F E, G E, H E, I F, G
F, H F, I G, H G, I H, I
[0293] In some embodiments, microbial compositions may be selected
from any member group from Tables 2-8.
Agricultural Compositions
[0294] Compositions comprising bacteria or bacterial populations
produced according to methods described herein and/or having
characteristics as described herein can be in the form of a liquid,
a foam, or a dry product. Compositions comprising bacteria or
bacterial populations produced according to methods described
herein and/or having characteristics as described herein may also
be used to improve plant traits. In some examples, a composition
comprising bacterial populations may be in the form of a dry
powder, a slurry of powder and water, or a flowable seed treatment.
The compositions comprising bacterial populations may be coated on
a surface of a seed, and may be in liquid form.
[0295] The composition can be fabricated in bioreactors such as
continuous stirred tank reactors, batch reactors, and on the farm.
In some examples, compositions can be stored in a container, such
as a jug or in mini bulk. In some examples, compositions may be
stored within an object selected from the group consisting of a
bottle, jar, ampule, package, vessel, bag, box, bin, envelope,
carton, container, silo, shipping container, truck bed, and/or
case.
[0296] Compositions may also be used to improve plant traits. In
some examples, one or more compositions may be coated onto a seed.
In some examples, one or more compositions may be coated onto a
seedling. In some examples, one or more compositions may be coated
onto a surface of a seed. In some examples, one or more
compositions may be coated as a layer above a surface of a seed. In
some examples, a composition that is coated onto a seed may be in
liquid form, in dry product form, in foam form, in a form of a
slurry of powder and water, or in a flowable seed treatment. In
some examples, one or more compositions may be applied to a seed
and/or seedling by spraying, immersing, coating, encapsulating,
and/or dusting the seed and/or seedling with the one or more
compositions. In some examples, multiple bacteria or bacterial
populations can be coated onto a seed and/or a seedling of the
plant. In some examples, at least two, at least three, at least
four, at least five, at least six, at least seven, at least eight,
at least nine, at least ten, or more than ten bacteria of a
bacterial combination can be selected from one of the following
genera: Acidovorax, Agrobacterium, Bacillus, Burkholderia,
Chryseobacterium, Curtobacterium, Enterobacter, Escherichia,
Methylobacterium, Paenibacillus, Pantoea, Pseudomonas, Ralstonia,
Saccharibacillus, Sphingomonas, and Stenotrophomonas.
[0297] In some examples, at least two, at least three, at least
four, at least five, at least six, at least seven, at least eight,
at least nine, at least ten, or more than ten bacteria and
bacterial populations of an endophytic combination are selected
from one of the following families: Bacillaceae, Burkholderiaceae,
Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae,
Methylobacteriaceae, Microbacteriaceae, Paenibacillileae,
Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae,
Cladosporiaceae, Gnomoniaceae, Incertae sedis, Lasiosphaeriaceae,
Netriaceae, and Pleosporaceae.
[0298] In some examples, at least two, at least three, at least
four, at least five, at least six, at least seven, at least eight,
at least night, at least ten, or more than ten bacteria and
bacterial populations of an endophytic combination are selected
from one of the following families: Bacillaceae, Burkholderiaceae,
Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae,
Methylobacteriaceae, Microbacteriaceae, Paenibacillileae,
Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae,
Cladosporiaceae, Gnomoniaceae, Incertae sedis, Lasiosphaeriaceae,
Netriaceae, Pleosporaceae.
[0299] Examples of compositions may include seed coatings for
commercially important agricultural crops, for example, sorghum,
canola, tomato, strawberry, barley, rice, maize, and wheat.
Examples of compositions can also include seed coatings for corn,
soybean, canola, sorghum, potato, rice, vegetables, cereals, and
oilseeds. Seeds as provided herein can be genetically modified
organisms (GMO), non-GMO, organic, or conventional. In some
examples, compositions may be sprayed on the plant aerial parts, or
applied to the roots by inserting into furrows in which the plant
seeds are planted, watering to the soil, or dipping the roots in a
suspension of the composition. In some examples, compositions may
be dehydrated in a suitable manner that maintains cell viability
and the ability to artificially inoculate and colonize host plants.
The bacterial species may be present in compositions at a
concentration of between 10.sup.8 to 10.sup.10 CFU/ml. In some
examples, compositions may be supplemented with trace metal ions,
such as molybdenum ions, iron ions, manganese ions, or combinations
of these ions. The concentration of ions in examples of
compositions as described herein may between about 0.1 mM and about
50 mM. Some examples of compositions may also be formulated with a
carrier, such as beta-glucan, carboxylmethyl cellulose (CMC),
bacterial extracellular polymeric substance (EPS), sugar, animal
milk, or other suitable carriers. In some examples, peat or
planting materials can be used as a carrier, or biopolymers in
which a composition is entrapped in the biopolymer can be used as a
carrier. The compositions comprising the bacterial populations
described herein can improve plant traits, such as promoting plant
growth, maintaining high chlorophyll content in leaves, increasing
fruit or seed numbers, and increasing fruit or seed unit
weight.
[0300] The compositions comprising the bacterial populations
described herein may be coated onto the surface of a seed. As such,
compositions comprising a seed coated with one or more bacteria
described herein are also contemplated. The seed coating can be
formed by mixing the bacterial population with a porous, chemically
inert granular carrier. Alternatively, the compositions may be
inserted directly into the furrows into which the seed is planted
or sprayed onto the plant leaves or applied by dipping the roots
into a suspension of the composition. An effective amount of the
composition can be used to populate the sub-soil region adjacent to
the roots of the plant with viable bacterial growth, or populate
the leaves of the plant with viable bacterial growth. In general,
an effective amount is an amount sufficient to result in plants
with improved traits (e.g. a desired level of nitrogen
fixation).
[0301] Bacterial compositions described herein can be formulated
using an agriculturally acceptable carrier. The formulation useful
for these embodiments may include at least one member selected from
the group consisting of a tackifier, a microbial stabilizer, a
fungicide, an antibacterial agent, a preservative, a stabilizer, a
surfactant, an anti-complex agent, an herbicide, a nematicide, an
insecticide, a plant growth regulator, a fertilizer, a rodenticide,
a desiccant, a bactericide, a nutrient, and any combination
thereof. In some examples, compositions may be shelf-stable. For
example, any of the compositions described herein can include an
agriculturally acceptable carrier (e.g., one or more of a
fertilizer such as a non-naturally occurring fertilizer, an
adhesion agent such as a non-naturally occurring adhesion agent,
and a pesticide such as a non-naturally occurring pesticide). A
non-naturally occurring adhesion agent can be, for example, a
polymer, copolymer, or synthetic wax. For example, any of the
coated seeds, seedlings, or plants described herein can contain
such an agriculturally acceptable carrier in the seed coating. In
any of the compositions or methods described herein, an
agriculturally acceptable carrier can be or can include a
non-naturally occurring compound (e.g., a non-naturally occurring
fertilizer, a non-naturally occurring adhesion agent such as a
polymer, copolymer, or synthetic wax, or a non-naturally occurring
pesticide). Non-limiting examples of agriculturally acceptable
carriers are described below. Additional examples of agriculturally
acceptable carriers are known in the art.
[0302] In some cases, bacteria are mixed with an agriculturally
acceptable carrier. The carrier can be a solid carrier or liquid
carrier, and in various forms including microspheres, powders,
emulsions and the like. The carrier may be any one or more of a
number of carriers that confer a variety of properties, such as
increased stability, wettability, or dispersability. Wetting agents
such as natural or synthetic surfactants, which can be nonionic or
ionic surfactants, or a combination thereof can be included in the
composition. Water-in-oil emulsions can also be used to formulate a
composition that includes the isolated bacteria (see, for example,
U.S. Pat. No. 7,485,451). Suitable formulations that may be
prepared include wettable powders, granules, gels, agar strips or
pellets, thickeners, and the like, microencapsulated particles, and
the like, liquids such as aqueous flowables, aqueous suspensions,
water-in-oil emulsions, etc. The formulation may include grain or
legume products, for example, ground grain or beans, broth or flour
derived from grain or beans, starch, sugar, or oil.
[0303] In some embodiments, the agricultural carrier may be soil or
a plant growth medium. Other agricultural carriers that may be used
include water, fertilizers, plant-based oils, humectants, or
combinations thereof. Alternatively, the agricultural carrier may
be a solid, such as diatomaceous earth, loam, silica, alginate,
clay, bentonite, vermiculite, seed cases, other plant and animal
products, or combinations, including granules, pellets, or
suspensions. Mixtures of any of the aforementioned ingredients are
also contemplated as carriers, such as but not limited to, pesta
(flour and kaolin clay), agar or flour-based pellets in loam, sand,
or clay, etc. Formulations may include food sources for the
bacteria, such as barley, rice, or other biological materials such
as seed, plant parts, sugar cane bagasse, hulls or stalks from
grain processing, ground plant material or wood from building site
refuse, sawdust or small fibers from recycling of paper, fabric, or
wood.
[0304] For example, a fertilizer can be used to help promote the
growth or provide nutrients to a seed, seedling, or plant.
Non-limiting examples of fertilizers include nitrogen, phosphorous,
potassium, calcium, sulfur, magnesium, boron, chloride, manganese,
iron, zinc, copper, molybdenum, and selenium (or a salt thereof).
Additional examples of fertilizers include one or more amino acids,
salts, carbohydrates, vitamins, glucose, NaCl, yeast extract,
NH.sub.4H.sub.2PO.sub.4, (NH.sub.4).sub.2SO.sub.4, glycerol,
valine, L-leucine, lactic acid, propionic acid, succinic acid,
malic acid, citric acid, KH tartrate, xylose, lyxose, and lecithin.
In one embodiment, the formulation can include a tackifier or
adherent (referred to as an adhesive agent) to help bind other
active agents to a substance (e.g., a surface of a seed). Such
agents are useful for combining bacteria with carriers that can
contain other compounds (e.g., control agents that are not
biologic), to yield a coating composition. Such compositions help
create coatings around the plant or seed to maintain contact
between the microbe and other agents with the plant or plant part.
In one embodiment, adhesives are selected from the group consisting
of: alginate, gums, starches, lecithins, formononetin, polyvinyl
alcohol, alkali formononetinate, hesperetin, polyvinyl acetate,
cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, Polyethylene
Glycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, Methyl
Cellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate,
Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate,
Gellan Gum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum
Ghatti, and polyoxyethylene-polyoxybutylene block copolymers.
[0305] In some embodiments, the adhesives can be, e.g. a wax such
as carnauba wax, beeswax, Chinese wax, shellac wax, spermaceti wax,
candelilla wax, castor wax, ouricury wax, and rice bran wax, a
polysaccharide (e.g., starch, dextrins, maltodextrins, alginate,
and chitosans), a fat, oil, a protein (e.g., gelatin and zeins),
gum arables, and shellacs. Adhesive agents can be non-naturally
occurring compounds, e.g., polymers, copolymers, and waxes. For
example, non-limiting examples of polymers that can be used as an
adhesive agent include: polyvinyl acetates, polyvinyl acetate
copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl
alcohols, polyvinyl alcohol copolymers, celluloses (e.g.,
ethylcelluloses, methylcelluloses, hydroxymethylcelluloses,
hydroxypropylcelluloses, and carboxymethylcelluloses),
polyvinylpyrolidones, vinyl chloride, vinylidene chloride
copolymers, calcium lignosulfonates, acrylic copolymers,
polyvinylacrylates, polyethylene oxide, acylamide polymers and
copolymers, polyhydroxyethyl acrylate, methylacrylamide monomers,
and polychloroprene.
[0306] In some examples, one or more of the adhesion agents,
anti-fungal agents, growth regulation agents, and pesticides (e.g.,
insecticide) are non-naturally occurring compounds (e.g., in any
combination). Additional examples of agriculturally acceptable
carriers include dispersants (e.g., polyvinylpyrrolidone/vinyl
acetate PVPIVA S-630), surfactants, binders, and filler agents.
[0307] The formulation can also contain a surfactant. Non-limiting
examples of surfactants include nitrogen-surfactant blends such as
Prefer 28 (Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfarm) and
Patrol (Helena); esterified seed oils include Sun-It II (AmCy), MSO
(UAP), Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); and
organo-silicone surfactants include Silwet L77 (UAP), Silikin
(Terra), Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309
(Wilbur-Ellis) and Century (Precision). In one embodiment, the
surfactant is present at a concentration of between 0.01% v/v to
10% v/v. In another embodiment, the surfactant is present at a
concentration of between 0.1% v/v to 1% v/v.
[0308] In certain cases, the formulation includes a microbial
stabilizer. Such an agent can include a desiccant, which can
include any compound or mixture of compounds that can be classified
as a desiccant regardless of whether the compound or compounds are
used in such concentrations that they in fact have a desiccating
effect on a liquid inoculant. Such desiccants are ideally
compatible with the bacterial population used, and should promote
the ability of the microbial population to survive application on
the seeds and to survive desiccation. Examples of suitable
desiccants include one or more of trehalose, sucrose, glycerol, and
Methylene glycol. Other suitable desiccants include, but are not
limited to, non-reducing sugars and sugar alcohols (e.g., mannitol
or sorbitol). The amount of desiccant introduced into the
formulation can range from about 5% to about 50% by weight/volume,
for example, between about 10% to about 40%, between about 15% to
about 35%, or between about 20% to about 30%. In some cases, it is
advantageous for the formulation to contain agents such as a
fungicide, an antibacterial agent, an herbicide, a nematicide, an
insecticide, a plant growth regulator, a rodenticide, bactericide,
or a nutrient. In some examples, agents may include protectants
that provide protection against seed surface-borne pathogens. In
some examples, protectants may provide some level of control of
soil-borne pathogens. In some examples, protectants may be
effective predominantly on a seed surface.
[0309] In some examples, a fungicide may include a compound or
agent, whether chemical or biological, that can inhibit the growth
of a fungus or kill a fungus. In some examples, a fungicide may
include compounds that may be fungistatic or fungicidal. In some
examples, fungicide can be a protectant, or agents that are
effective predominantly on the seed surface, providing protection
against seed surface-borne pathogens and providing some level of
control of soil-borne pathogens. Non-limiting examples of
protectant fungicides include captan, maneb, thiram, or
fludioxonil.
[0310] In some examples, fungicide can be a systemic fungicide,
which can be absorbed into the emerging seedling and inhibit or
kill the fungus inside host plant tissues. Systemic fungicides used
for seed treatment include, but are not limited to the following:
azoxystrobin, carboxin, mefenoxam, metalaxyl, thiabendazole,
trifloxystrobin, and various triazole fungicides, including
difenoconazole, ipconazole, tebuconazole, and triticonazole.
Mefenoxam and metalaxyl are primarily used to target the water mold
fungi Pythium and Phytophthora. Some fungicides are preferred over
others, depending on the plant species, either because of subtle
differences in sensitivity of the pathogenic fungal species, or
because of the differences in the fungicide distribution or
sensitivity of the plants. In some examples, fungicide can be a
biological control agent, such as a bacterium or fungus. Such
organisms may be parasitic to the pathogenic fungi, or secrete
toxins or other substances which can kill or otherwise prevent the
growth of fungi. Any type of fungicide, particularly ones that are
commonly used on plants, can be used as a control agent in a seed
composition.
[0311] In some examples, the seed coating composition comprises a
control agent that has antibacterial properties. In one embodiment,
the control agent with antibacterial properties is selected from
the compounds described herein elsewhere. In another embodiment,
the compound is Streptomycin, oxytetracycline, oxolinic acid, or
gentamicin. Other examples of antibacterial compounds which can be
used as part of a seed coating composition include those based on
dichlorophene and benzylalcohol hemi formal (Proxel.RTM. from ICI
or Acticide.RTM. RS from Thor Chemie and Kathon.RTM. MK 25 from
Rohm & Haas) and isothiazolinone derivatives such as
alkylisothiazolinones and benzisothiazolinones (Acticide.RTM. MBS
from Thor Chemie).
[0312] In some examples, growth regulator is selected from the
group consisting of: Abscisic acid, amidochlor, ancymidol,
6-benzylaminopurine, brassinolide, butralin, chlormequat
(chlormequat chloride), choline chloride, cyclanilide, daminozide,
dikegulac, dimethipin, 2,6-dimethylpuridine, ethephon, flumetralin,
flurprimidol, fluthiacet, forchlorfenuron, gibberellic acid,
inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide,
mepiquat (mepiquat chloride), naphthaleneacetic acid,
N-6-benzyladenine, paclobutrazol, prohexadione phosphorotrithioate,
2,3,5-tri-iodobenzoic acid, trinexapac-ethyl and uniconazole.
Additional non-limiting examples of growth regulators include
brassinosteroids, cytokinins (e.g., kinetin and zeatin), auxins
(e.g., indolylacetic acid and indolylacetyl aspartate), flavonoids
and isoflavanoids (e.g., formononetin and diosmetin), phytoaixins
(e.g., glyceolline), and phytoalexin-inducing oligosaccharides
(e.g., pectin, chitin, chitosan, polygalacuronic acid, and
oligogalacturonic acid), and gibellerins. Such agents are ideally
compatible with the agricultural seed or seedling onto which the
formulation is applied (e.g., it should not be deleterious to the
growth or health of the plant). Furthermore, the agent is ideally
one which does not cause safety concerns for human, animal or
industrial use (e.g., no safety issues, or the compound is
sufficiently labile that the commodity plant product derived from
the plant contains negligible amounts of the compound).
[0313] Some examples of nematode-antagonistic biocontrol agents
include ARF18; 30 Arthrobotrys spp.; Chaetomium spp.;
Cylindrocarpon spp.; Exophilia spp.; Fusarium spp.; Gliocladium
spp.; Hirsutella spp.; Lecanicillium spp.; Monacrosporium spp.;
Myrothecium spp.; Neocosmospora spp.; Paecilomyces spp.; Pochonia
spp.; Stagonospora spp.; vesicular-arbuscular mycorrhizal fungi,
Burkholderia spp.; Pasteuria spp., Brevibacillus spp.; Pseudomonas
spp.; and Rhizobacteria. Particularly preferred
nematode-antagonistic biocontrol agents include ARF18, Arthrobotrys
oligospora, Arthrobotrys dactyloides, Chaetomium globosum,
Cylindrocarpon heteronema, Exophilia jeanselmei, Exophilia
pisciphila, Fusarium aspergilus, Fusarium solani, Gliocladium
catenulatum, Gliocladium roseum, Gliocladium vixens, Hirsutella
rhossiliensis, Hirsutella minnesotensis, Lecanicillium lecanii,
Monacrosporium drechsleri, Monacrosporium gephyropagum, Myrotehcium
verrucaria, Neocosmospora vasinfecta, Paecilomyces lilacinus,
Pochonia chlamydosporia, Stagonospora heteroderae, Stagonospora
phaseoli, vesicular-arbuscular mycorrhizal fungi, Burkholderia
cepacia, Pasteuria penetrans, Pasteuria thornei, Pasteuria
nishizawae, Pasteuria ramosa, Pasteuria usage, Brevibacillus
laterosporus strain G4, Pseudomonas fluorescens and
Rhizobacteria.
[0314] Some examples of nutrients can be selected from the group
consisting of a nitrogen fertilizer including, but not limited to
Urea, Ammonium nitrate, Ammonium sulfate, Non-pressure nitrogen
solutions, Aqua ammonia, Anhydrous ammonia, Ammonium thiosulfate,
Sulfur-coated urea, Urea-formaldehydes, IBDU, Polymer-coated urea,
Calcium nitrate, Ureaform, and Methylene urea, phosphorous
fertilizers such as Diammonium phosphate, Monoammonium phosphate,
Ammonium polyphosphate, Concentrated superphosphate and Triple
superphosphate, and potassium fertilizers such as Potassium
chloride, Potassium sulfate, Potassium-magnesium sulfate, Potassium
nitrate. Such compositions can exist as free salts or ions within
the seed coat composition. Alternatively, nutrients/fertilizers can
be complexed or chelated to provide sustained release over
time.
[0315] Some examples of rodenticides may include selected from the
group of substances consisting of 2-isovalerylindan-1,3-dione,
4-(quinoxalin-2-ylamino) benzenesulfonamide, alpha-chlorohydrin,
aluminum phosphide, antu, arsenous oxide, barium carbonate,
bisthiosemi, brodifacoum, bromadiolone, bromethalin, calcium
cyanide, chloralose, chlorophacinone, cholecalciferol, coumachlor,
coumafuryl, coumatetralyl, crimidine, difenacoum, difethialone,
diphacinone, ergocalciferol, flocoumafen, fluoroacetamide,
flupropadine, flupropadine hydrochloride, hydrogen cyanide,
iodomethane, lindane, magnesium phosphide, methyl bromide,
norbormide, phosacetim, phosphine, phosphorus, pindone, potassium
arsenite, pyrinuron, scilliroside, sodium arsenite, sodium cyanide,
sodium fluoroacetate, strychnine, thallium sulfate, warfarin and
zinc phosphide.
[0316] In the liquid form, for example, solutions or suspensions,
bacterial populations can be mixed or suspended in water or in
aqueous solutions. Suitable liquid diluents or carriers include
water, aqueous solutions, petroleum distillates, or other liquid
carriers.
[0317] Solid compositions can be prepared by dispersing the
bacterial populations in and on an appropriately divided solid
carrier, such as peat, wheat, bran, vermiculite, clay, talc,
bentonite, diatomaceous earth, fuller's earth, pasteurized soil,
and the like. When such formulations are used as wettable powders,
biologically compatible dispersing agents such as non-ionic,
anionic, amphoteric, or cationic dispersing and emulsifying agents
can be used.
[0318] The solid carriers used upon formulation include, for
example, mineral carriers such as kaolin clay, pyrophyllite,
bentonite, montmorillonite, diatomaceous earth, acid white soil,
vermiculite, and pearlite, and inorganic salts such as ammonium
sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium
chloride, and calcium carbonate. Also, organic fine powders such as
wheat flour, wheat bran, and rice bran may be used. The liquid
carriers include vegetable oils such as soybean oil and cottonseed
oil, glycerol, ethylene glycol, polyethylene glycol, propylene
glycol, polypropylene glycol, etc.
Pests
[0319] Agricultural compositions of the disclosure, which may
comprise any microbe taught herein, are sometimes combined with one
or more pesticides.
[0320] The pesticides that are combined with the microbes of the
disclosure may target any of the pests mentioned below.
[0321] "Pest" includes but is not limited to, insects, fungi,
bacteria, nematodes, mites, ticks and the like. Insect pests
include insects selected from the orders Coleoptera, Diptera,
Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera
Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura,
Siphonaptera, Trichoptera, etc., particularly Lepidoptera and
Coleoptera.
[0322] Those skilled in the art will recognize that not all
compounds are equally effective against all pests. Compounds that
may be combined with microbes of the disclosure may display
activity against insect pests, which may include economically
important agronomic, forest, greenhouse, nursery ornamentals, food
and fiber, public and animal health, domestic and commercial
structure, household and stored product pests.
[0323] As aforementioned, the agricultural compositions of the
disclosure (which may comprise any microbe taught herein) are in
embodiments combined with one or more pesticides. These pesticides
may be active against any of the following pests:
[0324] Larvae of the order Lepidoptera include, but are not limited
to, armyworms, cutworms, loopers and heliothines in the family
Noctuidae Spodoptera frugiperda J E Smith (fall armyworm); S.
exigua Hubner (beet armyworm); S. litura Fabricius (tobacco
cutworm, cluster caterpillar); Mamestra configurata Walker (bertha
armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis Ipsilon
Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm);
A. subterranea Fabricius (granulate cutworm); Alabama argillacea
Hubner (cotton leaf worm); Trichoplusia ni Hubner (cabbage looper);
Pseudoplusia includens Walker (soybean looper); Anticarsia
gemmatalis Hubner (velvet bean caterpillar); Hypena scabra
Fabricius (green clover worm); Heliothis virescens Fabricius
(tobacco budworm); Pseudaletia umpuncta Haworth (armyworm); Athetis
mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa
messoria Harris (darksided cutworm); Earias insulana Boisduval
(spiny bollworm); E. vittella Fabricius (spotted bollworm);
Helicoverpa armigera Hubner (American bollworm); H. zea Boddie
(corn earworm or cotton bollworm); Melanchra picta Harris (zebra
caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm);
borers, case bearers, webworms, coneworms, and skeletonizers from
the family Pyralidae Ostrinia nubilalis Hubner (European corn
borer); Amyelois transitella Walker (naval orangeworm); Anagasta
kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker
(almond moth); Chilo suppressalis Walker (rice stem borer); C.
partellus, (sorghum borer); Corcyra cephalonica Stainton (rice
moth); Crambus caliginosellus Clemens (corn root webworm); C.
teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis
Guenee (rice leaf roller); Desmia funeralis Hubner (grape
leaffolder); Diaphania hyalinata Linnaeus (melon worm); D.
nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar
(southwestern corn borer), D. saccharalis Fabricius (surgarcane
borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia
elutella Hubner (tobacco (cacao) moth); Galleria mellonella
Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod
webworm); Homoeosoma electellum Hulst (sunflower moth);
Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia
grisella Fabricius (lesser wax moth); Loxostege sticticalis
Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web
moth); Maruca testulalis Geyer (bean pod borer); Plodia
interpunctella Hubner (Indian meal moth); Scirpophaga incertulas
Walker (yellow stem borer); Udea rubigalis Guenee (celery
leaftier); and leafrollers, budworms, seed worms and fruit worms in
the family Tortricidae Acleris gloverana Walsingham (Western
blackheaded budworm); A. variana Fernald (Eastern blackheaded
budworm); Archips argyrospila Walker (fruit tree leaf roller); A.
rosana Linnaeus (European leaf roller); and other Archips species,
Adoxophyes orana Fischer von Rosslerstamm (summer fruit tortrix
moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia
latiferreana Walsingham (filbertworm); C. pomonella Linnaeus
(colding moth); Platynota flavedana Clemens (variegated
leafroller); P. stultana Walsingham (omnivorous leafroller);
Lobesia botrana Denis & Schiffermuller (European grape vine
moth); Spilonota ocellana Denis & Schiffermuller (eyespotted
bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia
ambiguella Hubner (vine moth); Bonagota salubricola Meyrick
(Brazilian apple leafroller); Grapholita molesta Busck (oriental
fruit moth); Suleima helianthana Riley (sunflower bud moth);
Argyrotaenia spp.; Choristoneura spp.
[0325] Selected other agronomic pests in the order Lepidoptera
include, but are not limited to, Alsophila pometaria Harris (fall
cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota
senatoria J. E. Smith (orange striped oakworm); Antheraea pernyi
Guerin-Meneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus
(Silkworm); Bucculatrix thurberiella Busck (cotton leaf
perforator); Collas eurytheme Boisduval (alfalfa caterpillar);
Datana integerrima Grote & Robinson (walnut caterpillar);
Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos
subsignaria Hubner (elm spanworm); Erannis tiliaria Harris (linden
looper); Euproctis chrysorrhoea Linnaeus (browntail moth);
Harrisina americana Guerin-Meneville (grapeleaf skeletonizer);
Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea
Drury (fall web-worm); Keiferia lycopersicella Walsingham (tomato
pinworm); Lambdina fiscellaria Hulst (Eastern hemlock looper); L.
fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma
salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy
moth); Manduca quinquemaculata Haworth (five spotted hawk moth,
tomato hornworm); M. sexta Haworth (tomato homworm, tobacco
hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita
vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant
swallowtail orange dog); Phryganidia californica Packard
(California oakworm); Phyllocnistis citrella Stainton (citrus
leafminer); Phyllonorycter blancardella Fabricius (spotted
tentiform leafminer); Pieris brassicae Linnaeus (large white
butterfly); P. rapae Linnaeus (small white butterfly); P. napi
Linnaeus (green veined white butterfly); Platyptilia carduidactyla
Riley (artichoke plume moth); Plutella xylostella Linnaeus
(diamondback moth); Pectinophora gossypiella Saunders (pink
bollworm); Pontia protodice Boisduval and Leconte (Southern
cabbage-worm); Sabulodes aegrotata Guenee (onmivorous looper);
Schizura concinna J. E. Smith (red humped caterpillar); Sitotroga
cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa
Schiffermuller (pine processionary caterpillar); Tineola
bisselliella Hummel (webbing clothes moth); Tuta absoluta Meyrick
(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth);
Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.;
Ostrinia nubilalis (European corn borer); seed corn maggot; Agrotis
ipsilon (black cutworm).
[0326] Larvae and adults of the order Coleoptera including weevils
from the families Anthribidae, Bruchidae and Curculionidae
(including, but not limited to: Anthonomus grandis Boheman (boll
weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);
Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus
(rice weevil); Hypera punctata Fabricius (clover leaf weevil);
Cylindrocopturus adspersus LeConte (sunflower stem weevil);
Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus
LeConte (gray sunflower seed weevil); Sphenophorus maidis
Chittenden (maize billbug)); flea beetles, cucumber beetles,
rootworms, leaf beetles, potato beetles and leafminers in the
family Chrysomelidae (including, but not limited to: Leptinotarsa
decemlineata Say (Colorado potato beetle); Diabrotica virgifera
LeConte (western corn rootworm); D. barberi Smith and Lawrence
(northern corn rootworm); D. undecimpunctata howardi Barber
(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn
flea beetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);
Phyllotreta striolata (stripped flea beetle); Colaspis brunnea
Fabricius (grape colaspis); Oulema melanopus Linnaeus (cereal leaf
beetle); Zygogramma exclamationis Fabricius (sunflower beetle));
beetles from the family Coccinellidae (including, but not limited
to: Epilachna varivestis Mulsant (Mexican bean beetle)); chafers
and other beetles from the family Scarabaeidae (including, but not
limited to: Popillia japonica Newman (Japanese beetle);
Cyclocephala borealis Arrow (northern masked chafer, white grub);
C. immaculata Olivier (southern masked chafer, white grub);
Rhizotrogus majalis Razoumowsky (European chafer); Phyllophaga
crinita Burmeister (white grub); Ligyrus gibbosus De Geer (carrot
beetle)); carpet beetles from the family Dermestidae; wireworms
from the family Elateridae, Eleodes spp., Melanotus spp.; Conoderus
spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.;
bark beetles from the family Scolytidae and beetles from the family
Tenebrionidae; Cerotoma trifurcate (bean leaf beetle); and
wireworm.
[0327] Adults and immatures of the order Diptera, including
leafminers Agromyza parvicornis Loew (corn blotch leafminer);
midges (including, but not limited to: Contarinia sorghicola
Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly);
Sitodiplosis mosellana Gehin (wheat midge); Neolasioptera
murtfeldtiana Felt, (sunflower seed midge)); fruit flies
(Tephritidae), Oscinella frit Linnaeus (fruit flies); maggots
(including, but not limited to: Delia platura Meigen (seedcorn
maggot); D. coarctata Fallen (wheat bulb fly) and other Delia spp.,
Meromyza americana Fitch (wheat stem maggot); Musca domestica
Linnaeus (house flies); Fannia canicularis Linnaeus, F. femoralis
Stein (lesser house flies); Stomoxys calcitrans Linnaeus (stable
flies)); face flies, horn flies, blow flies, Chrysomya spp.;
Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.;
bot flies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma
spp.; deer flies Chrysops spp.; Melophagus ovinus Linnaeus (keds)
and other Brachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex
spp.; black flies Prosimulium spp.; Simulium spp.; biting midges,
sand flies, sciarids, and other Nematocera.
[0328] Adults and nymphs of the orders Hemiptera and Homoptera such
as, but not limited to, adelgids from the family Adelgidae, plant
bugs from the family Miridae, cicadas from the family Cicadidae,
leafhoppers, Empoasca spp.; from the family Cicadellidae,
planthoppers from the families Cixiidae, Flatidae, Fulgoroidea,
Issidae and Delphacidae, treehoppers from the family Membracidae,
psyllids from the family Psyllidae, whiteflies from the family
Aleyrodidae, aphids from the family Aphididae, phylloxera from the
family Phylloxeridae, mealybugs from the family Pseudococcidae,
scales from the families Asterolecanidae, Coccidae, Dactylopiidae,
Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae and
Margarodidae, lace bugs from the family Tingidae, stink bugs from
the family Pentatomidae, cinch bugs, Blissus spp.; and other seed
bugs from the family Lygaeidae, spittlebugs from the family
Cercopidae squash bugs from the family Coreidae and red bugs and
cotton stainers from the family Pyrrhocoridae.
[0329] Agronomically important members from the order Homoptera
further include, but are not limited to: Acyrthisiphon pisum Harris
(pea aphid); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli
(black bean aphid); A. gossypii Glover (cotton aphid, melon aphid);
A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple
aphid); A. spiraecola Patch (spirea aphid); Aulacorthum solani
Kaltenbach (foxglove aphid); Chaetosiphon fragaefolii Cockerell
(strawberry aphid); Diuraphis noxia Kurdjumov/Mordvilko (Russian
wheat aphid); Dysaphis plantaginea Paaserini (rosy apple aphid);
Eriosoma lanigerum Hausmann (woolly apple aphid); Brevicoryne
brassicae Linnaeus (cabbage aphid); Hyalopterus pruni Geoffroy
(mealy plum aphid); Lipaphis erysimi Kaltenbach (turnip aphid);
Metopolophium dirrhodum Walker (cereal aphid); Macrosiphum
euphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach
potato aphid, green peach aphid); Nasonovia ribisnigri Mosley
(lettuce aphid); Pemphigus spp. (root aphids and gall aphids);
Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus
(bird cherry-oat aphid); Schizaphis graminum Rondani (greenbug);
Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae
Fabricius (English grain aphid); Therioaphis maculata Buckton
(spotted alfalfa aphid); Toxoptera aurantii Boyer de Fonscolombe
(black citrus aphid) and T. citricida Kirkaldy (brown citrus
aphid); Melanaphis sacchari (sugarcane aphid); Adelges spp.
(adelgids); Phylloxera devastatrix Pergande (pecan phylloxera);
Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly);
B. argentifolii Bellows & Perring (silverleaf whitefly);
Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes
abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood
(greenhouse whitefly); Empoasca fabae Harris (potato leafhopper);
Laodelphax striatellus Fallen (smaller brown planthopper);
Macrolestes quadrilineatus Forbes (aster leafhopper); Nephotettix
cinticeps Uhler (green leafhopper); N. nigropictus Stal (rice
leafhopper); Nilaparvata lugens Stal (brown planthopper);
Peregrinus maidis Ashmead (corn planthopper); Sogatella furcifera
Horvath (white backed planthopper); Sogatodes orizicola Muir (rice
delphacid); Typhlocyba pomaria McAtee (white apple leafhopper);
Erythroneoura spp. (grape leafhoppers); Magicicada septendecim
Linnaeus (periodical cicada); Icerya purchasi Maskell (cottony
cushion scale); Quadraspidiotus perniciosus Comstock (San Jose
scale); Planococcus citri Risso (citrus mealybug); Pseudococcus
spp. (other mealybug complex); Cacopsylla pyricola Foerster (pear
psylla); Trioza diospyri Ashmead (persimmon psylla).
[0330] Species from the order Hemiptera include, but are not
limited to: Acrosternum hilare Say (green stink bug); Anasa tristis
De Geer (squash bug); Blissus leucopterus Say (chinch bug);
Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta
Distant (tomato bug); Dysdercus suturellus Herrich-Schaffer (cotton
stainer); Euschistus servus Say (brown stink bug); E. variolarius
Palisot de Beauvais (one spotted stink bug); Graptostethus spp.
(complex of seed bugs); Leptoglossus corculus Say (leaf footed pine
seed bug); Lygus lineolaris Palisot de Beauvais (tarnished plant
bug); L. Hesperus Knight (Western tarnished plant bug); L.
pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius
(European tarnished plant bug); Lygocoris pabulinus Linnaeus
(common green capsid); Nezara viridula Linnaeus (southern green
stink bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus
fasciatus Dallas (large milk-weed bug); Pseudatomoscelis seriatus
Reuter (cotton flea hopper).
[0331] Hemiptera such as, Calocoris norvegicus Gmelin (strawberry
bug); Orthops campestris Linnaeus; Plesiocoris rugicollis Fallen
(apple capsid); Cyrtopeltis modestus Distant (tomato bug);
Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatus
Reuter (whitemarked fleahopper); Diaphnocoris chlorionis Say
(honeylocust plant bug); Labopidicola allii Knight (onion plant
bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);
Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatus
Fabricius (four lined plant bug); Nysius ericae Schilling (false
chinch bug); Nysius raphanus Howard (false chinch bug); Nezara
viridula Linnaeus (Southern green stink bug); Eurygaster spp.;
Coreidae spp.; Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae
spp.; Reduviidae spp. and Cimicidae spp.
[0332] Adults and larvae of the order Acari (mites) such as Aceria
tosichella Keifer (wheat curl mite); Petrobia latens Muller (brown
wheat mite); spider mites and red mites in the family
Tetranychidae, Panonychus ulmi Koch (European red mite);
Tetranychus urticae Koch (two spotted spider mite); (T. mcdanieli
McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider
mite); T. turkestani Ugarov & Nikolski (strawberry spider
mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisi
McGregor (citrus flat mite); rust and bud mites in the family
Eriophyidae and other foliar feeding mites and mites important in
human and animal health, i.e., dust mites in the family
Epidermoptidae, follicle mites in the family Demodicidae, grain
mites in the family Glycyphagidae, ticks in the order Ixodidae.
Ixodes scapularis Say (deer tick); I. holocyclus Neumann
(Australian paralysis tick); Dermacentor variabilis Say (American
dog tick); Amblyomma americanum Linnaeus (lone star tick) and scab
and itch mites in the families Psoroptidae, Pyemotidae and
Sarcoptidae.
[0333] Insect pests of the order Thysanura, such as Lepisma
saccharina Linnaeus (silverfish); Thermobia domestica Packard
(firebrat).
[0334] Additional arthropod pests include: spiders in the order
Araneae such as Loxosceles reclusa Gertsch and Mulaik (brown
recluse spider) and the Latrodectus mactans Fabricius (black widow
spider) and centipedes in the order Scutigeromorpha such as
Scutigera coleoptrata Linnaeus (house centipede).
[0335] Superfamily of stink bugs and other related insects
including but not limited to species belonging to the family
Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus
guildini, Euschistus servus, Acrosternum hilare, Euschistus heros,
Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus,
Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), the
family Plataspidae (Megacopta cribraria-Bean plataspid) and the
family Cydnidae (Scaptocoris castanea-Root stink bug) and
Lepidoptera species including but not limited to: diamond-back
moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g.,
Pseudoplusia includens Walker and velvet bean caterpillar e.g.,
Anticarsia gemmatalis Hubner.
[0336] Nematodes include parasitic nematodes such as root-knot,
cyst and lesion nematodes, including Heterodera spp., Meloidogyne
spp. and Globodera spp.; particularly members of the cyst
nematodes, including, but not limited to, Heterodera glycines
(soybean cyst nematode); Heterodera schachtii (beet cyst nematode);
Heterodera avenae (cereal cyst nematode) and Globodera
rostochiensis and Globodera pailida (potato cyst nematodes). Lesion
nematodes include Pratylenchus spp.
Pesticidal Compositions Comprising a Pesticide and Microbe of the
Disclosure
[0337] As aforementioned, agricultural compositions of the
disclosure, which may comprise any microbe taught herein, are
sometimes combined with one or more pesticides. Pesticides can
include herbicides, insecticides, fungicides, nematicides, etc.
[0338] In some embodiments, the pesticides/microbial combinations
can be applied in the form of compositions and can be applied to
the crop area or plant to be treated, simultaneously or in
succession, with other compounds. These compounds can be
fertilizers, weed killers, cryoprotectants, surfactants,
detergents, pesticidal soaps, dormant oils, polymers, and/or time
release or biodegradable carrier formulations that permit long term
dosing of a target area following a single application of the
formulation. They can also be selective herbicides, chemical
insecticides, virucides, microbicides, amoebicides, pesticides,
fungicides, bacteriocides, nematicides, molluscicides or mixtures
of several of these preparations, if desired, together with further
agriculturally acceptable carriers, surfactants or application
promoting adjuvants customarily employed in the art of formulation.
Suitable carriers (i.e. agriculturally acceptable carriers) and
adjuvants can be solid or liquid and correspond to the substances
ordinarily employed in formulation technology, e.g. natural or
regenerated mineral substances, solvents, dispersants, wetting
agents, sticking agents, tackifiers, binders or fertilizers.
Likewise, the formulations may be prepared into edible baits or
fashioned into pest traps to permit feeding or ingestion by a
target pest of the pesticidal formulation.
[0339] Exemplary chemical compositions, which may be combined with
the microbes of the disclosure, include:
[0340] Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron,
Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop,
Glufosinate, Halo sulfuron Gowan, Paraquat, Propyzamide,
Sethoxydim, Butafenacil, Halosulfuron, Indaziflam;
Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuringiensis,
Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin,
Diazinon, Malathion, Abamectin, Cyfluthrin/betacyfluthrin,
Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate,
Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid,
Dinotefuran, FluaCrypyrim, Tolfenpyrad, Clothianidin,
Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad,
Rynaxypyr, Cyazypyr, Spinoteram, Triflumuron, Spirotetramat,
Imidacloprid, Flubendiamide, Thiodicarb, Metaflumizone,
Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Imidacloprid,
Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb, Flonicamid,
Methiocarb, Emamectin benzoate, Indoxacarb, Forthiazate,
Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin oxide, Hexthiazox,
Methomyl,
4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;
Fruits Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,
Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,
Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,
Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin,
Fenhexamid, Oxpoconazole fumarate, Cyazofamid, Fenamidone,
Zoxamide, Picoxystrobin, Pyraclostrobin, Cyflufenamid,
Boscalid;
[0341] Cereals Herbicides: Isoproturon, Bromoxynil, Ioxynil,
Phenoxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican,
Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron, Triasulfuron,
Flucarbazone, Iodosulfuron, Propoxycarbazone, Picolin-afen,
Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron,
Thifensulfuron Methyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron,
Pyrasulfotole, Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon;
Cereals Fungicides: Carbendazim, Chlorothalonil, Azoxystrobin,
Cyproconazole, Cyprodinil, Fenpropimorph, Epoxiconazole,
Kresoxim-methyl, Quinoxyfen, Tebuconazole, Trifloxystrobin,
Simeconazole, Picoxystrobin, Pyraclostrobin, Dimoxystrobin,
Prothioconazole, Fluoxastrobin; Cereals Insecticides: Dimethoate,
Lambda-cyhalothrin, Deltamethrin, alpha-Cypermethrin,
.beta.-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin,
Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos,
Metamidophos, Oxidemethon methyl, Pirimicarb, Methiocarb;
[0342] Maize Herbicides: Atrazine, Alachlor, Bromoxynil,
Acetochlor, Dicamba, Clopyralid, S-Dimethenamid, Glufosinate,
Glyphosate, Isoxaflutole, S-Metolachlor, Mesotrione, Nicosulfuron,
Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron,
Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet,
Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos,
Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin,
Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide,
Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb,
.beta.-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron,
Triflumoron, Tefluthrin, Tebupirim-phos, Ethiprole, Cyazypyr,
Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb,
Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan, Thiram,
Prothioconazole, Tebuconazole, Trifloxystrobin;
[0343] Rice Herbicides: Butachlor, Propanil, Azimsulfuron,
Bensulfuron, Cyhalo-fop, Daimuron, Fentrazamide, Imazosulfuron,
Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb,
Quinclorac, Thiobencarb, Indanofan, Flufenacet, Fentrazamide,
Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid,
Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor,
Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan;
Rice Insecticides: Diazinon, Fenitro-thion, Fenobucarb,
Monocrotophos, Benfuracarb, Buprofezin, Dinotefuran, Fipronil,
Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid,
Dinotefuran, Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr,
Deltamethrin, Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad,
Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyriphos,
Cartap, Methamidophos, Etofen-prox, Triazophos,
4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,
Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,
Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,
Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,
Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil;
[0344] Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen,
Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl,
Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium,
Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin,
Thidiazuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos,
Cypermethrin, Deltamethrin, Malathion, Monocrotophos, Abamectin,
Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb,
Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin,
Spiromesifen, Pyridalyl, Flonicamid, Flubendiamide, Triflumuron,
Rynaxypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin,
Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr,
Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)
methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Thiodicarb,
Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor,
Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:
Etridiazole, Metalaxyl, Quintozene;
[0345] Soybean Herbicides: Alachlor, Bentazone, Trifluralin,
Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen,
Flu-azifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr,
(S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,
Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,
Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,
Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,
Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil,
Ethiprole, Deltamethrin, .beta.-Cyfluthrin, gamma and lambda
Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)
methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Spirotetramat,
Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,
beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,
Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,
Trifloxystrobin, Prothioconazole, Tetraconazole;
[0346] Sugarbeet Herbicides: Chloridazon, Desmedipham,
Ethofumesate, Phenmedipham, Triallate, Clopyralid, Fluazifop,
Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron,
Tepral-oxydim, Quizalofop; Sugarbeet Insecticides: Imidacloprid,
Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran,
Deltamethrin, .beta.-Cyfluthrin, gamma/lambda Cyhalothrin,
4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluor-ethyl)amino]furan-2(5H)-on,
Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran;
[0347] Canola Herbicides: Clopyralid, Diclofop, Fluazifop,
Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron,
Quinmerac, Quizalofop, Clethodim, Tepraloxydim; Canola Fungicides:
Azoxystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz,
Vinclozolin; Canola Insecticides: Carbofuran organophos-phates,
Pyrethroids, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin,
Thiamethoxam, Acetamiprid, Dineto-furan, .beta.-Cyfluthrin, gamma
and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad,
Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,
4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.
Insecticidal Compositions Comprising an Insecticide and Microbe of
the Disclosure
[0348] As aforementioned, agricultural compositions of the
disclosure, which may comprise any microbe taught herein, are
sometimes combined with one or more insecticides.
[0349] In some embodiments, insecticidal compositions may be
included in the compositions set forth herein, and can be applied
to a plant(s) or a part(s) thereof simultaneously or in succession,
with other compounds. Insecticides include ammonium carbonate,
aqueous potassium silicate, boric acid, copper sulfate, elemental
sulfur, lime sulfur, sucrose octanoate esters,
4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,
abamectin, notenone, fenazaquin, fenpyroximate, pyridaben,
pyrimedifen, tebufenpyrad, tolfenpyrad, acephate, emamectin
benzoate, lepimectin, milbemectin, hdroprene, kinoprene,
methoprene, fenoxycarb, pyriproxyfen, methryl bromide and other
alkyl halides, fulfuryl fluoride, chloropicrin, borax, disodium
octaborate, sodium borate, sodium metaborate, tartar emetic,
dazomet, metam, pymetrozine, pyrifluquinazon, flofentezine,
diflovidazin, hexythiazox, bifenazate, thiamethoxam, imidacloprid,
fenpyroximate, azadirachtin, permethrin, esfenvalerate,
acetamiprid, bifenthrin, indoxacarb, azadirachtin, pyrethrin,
imidacloprid, beta-cyfluthrin, sulfotep, tebupirimfos, temephos,
terbufos, tetrachlorvinphos, thiometon, triazophos, alanycarb,
aldicarb, bendiocarb, benfluracarb, butocarboxim, butoxycarboxim,
carbaryl, carbofuran, carbosulfan, ethiofencarb, fenobucarb,
formetanate, furathiocarb, isoprocarb, methiocarb, methymyl,
metolcarb, oxamyl, primicarb, propoxur, thiodicarb, thiofanox,
triazamate, trimethacarb, XMC, xylylcarb, acephate, azamethiphos,
azinphos-ethyl, azinphos-methyl, cadusafos, chlorethoxyfox,
trichlorfon, vamidothion, chlordane, endosulfan, ethiprole,
fipronil, acrinathrin, allethrin, bifenthrin, bioallethrin,
bioalletherin X-cyclopentenyl, bioresmethrin, cyclorothrin,
cyfluthrin, cyhalothrin, cypermethrin, cyphenothrin
[(1R)-trans-isomers], deltamethrin, empenthrin [(EZ)-(1R)-isomers],
esfenvalerate, etofenprox, fenpropathrin, fenvalerate,
flucythrinate, flumethrin, halfenprox, kadathrin, phenothrin
[(1R)-trans-isomer] prallethrin, pyrethrins (pyrethrum),
resmethrin, silafluofen, tefluthrin, tetramethrin, tetramethrin
[(1R)-isomers], tralomethrin, transfluthrin, alpha-cypermethrin,
beta-cyfluthrin, beta-cypermethrin, d-cis-trans allethrin, d-trans
allethrin, gamma-cyhalothrin, lamda-cyhalothrin, tau-fluvalinate,
theta-cypermethrin, zeta-cypermethrin, methoxychlor, nicotine,
sulfoxaflor, acetamiprid, clothianidin, dinotefuran, imidacloprid,
nitenpyram, thiacloprid, thiamethoxan, tebuprimphos,
beta-cyfluthrin, clothianidin, flonicamid, hydramethylnon, amitraz,
flubendiamide, blorantraniliprole, lambda cyhalothrin, spinosad,
gamma cyhalothrin, Beauveria bassiana, capsicum oleoresin extract,
garlic oil, carbaryl, chlorpyrifos, sulfoxaflor, lambda
cyhalothrin, Chlorfenvinphos, Chlormephos, Chlorpyrifos,
Chlorpyrifos-methyl, Coumaphos, Cyanophos, Demeton-S-methyl,
Diazinon, Dichlorvos/DDVP, Dicrotophos, Dimethoate,
Dimethylvinphos, Disulfoton, EPN, Ethion, Ethoprophos, Famphur,
Fenamiphos, Fenitrothion, Fenthion, Fosthiazate, Heptenophos,
Imicyafos, Isofenphos, Isopropyl O-(methoxyaminothio-phosphoryl)
salicylate, Isoxathion, Malathion, Mecarbam, Methamidophos,
Methidathion, Mevinphos, Monocrotophos, Naled, Omethoate,
Oxydemeton-methyl, Parathion, Parathion-methyl, Phenthoate,
Phorate, Phosalone, Phosmet, Phosphamidon, Phoxim,
Pirimiphos-methyl, Profenofos, Propetamphos, Prothiofos,
Pyraclofos, Pyridaphenthion, Quinalphosfluacrypyrim, tebufenozide,
chlorantraniliprole, Bacillus thuringiensis subs. Kurstaki,
terbufos, mineral oil, fenpropathrin, metaldehyde, deltamethrin,
diazinon, dimethoate, diflubenzuron, pyriproxyfen, reosemary oil,
peppermint oil, geraniol, azadirachtin, piperonyl butoxide,
cyantraniliprole, alpha cypermethrin, tefluthrin, pymetrozine,
malathion, Bacillus thuringiensis subsp. israelensis, dicofol,
bromopropylate, benzoximate, azadirachtin, flonicamid, soybean oil,
Chromobacterium subtsugae strain PRAA4-1, zeta cypermethrin,
phosmet, methoxyfenozide, paraffinic oil, spirotetramat, methomyl,
Metarhizium anisopliae strain F52, ethoprop, tetradifon,
propargite, fenbutatin oxide, azocyclotin, cyhexatin,
diafenthiuron, Bacillus sphaericus, etoxazole, flupyradifurone,
azadirachtin, Beauveria bassiana, cyflumetofen, azadirachtin,
chinomethionat, acephate, Isaria fumosorosea Apopka strain 97,
sodium tetraborohydrate decahydrate, emamectin benzoate, cryolite,
spinetoram, Chenopodium ambrosioides extract, novaluron,
dinotefuran, carbaryl, acequinocyl, flupyradifurone, iron
phosphate, kaolin, buprofezin, cyromazine, chromafenozide,
halofenozide, methoxyfenozide, tebufenozide, bistrifluron,
chlorfluazuron, diflubenzuron, flucycloxuron, flufenoxuron,
hexaflumuron, lufenuron, nocaluron, noviflumuron, teflubenzuron,
triflumuron, bensultap, cartap hydrochloride, thiocyclam,
thiosultap-sodium, DNOC, chlorfenapyr, sulfuramid, phorate,
tolfenpyrad, sulfoxaflor, neem oil, Bacillus thuringiensis subsp.
tenebrionis strain SA-10, cyromazine, heat-killed Burkholderia
spp., cyantraniliprole, cyenopyrafen, cyflumetofen, sodium cyanide,
potassium cyanide, calcium cyanide, aluminum phosphide, calcium
phosphide, phosphine, zinc phosphide, spriodiclofen, spiromesifen,
spirotetramat, metaflumizone, flubendiamide, pyflubumide, oxamyl,
Bacillus thuringiensis subsp. aizawai, etoxazole, and
esfenvalerate
TABLE-US-00009 TABLE 9 Exemplary insecticides associated with
various modes of action, which can be combined with microbes of the
disclosure Physiological Exemplary function(s) Mode of Action
Compound class insecticides affected acetylcholinesterase
carbamates Alanycarb, Aldicarb, Nerve and (AChE) inhibitors
Bendiocarb, Benfuracarb, muscle Butocarboxim, Butoxycarboxim,
Carbaryl, Carbofuran, Carbosulfan, Ethiofencarb, Fenobucarb,
Formetanate, Furathiocarb, Isoprocarb, Methiocarb, Methomyl,
Metolcarb, Oxamyl, Pirimicarb, Propoxur, Thiodicarb, Thiofanox,
Triazamate, Trimethacarb, XMC, Xylylcarb acetylcholinesterase
organophosphates Acephate, Azamethiphos, Nerve and (AChE)
inhibitors Azinphos-ethyl, Azinphos- muscle methyl, Cadusafos,
Chlorethoxyfos, Chlorfenvinphos, Chlormephos, Chlorpyrifos,
Chlorpyrifos-methyl, Coumaphos, Cyanophos, Demeton-S-methyl,
Diazinon, Dichlorvos/DDVP, Dicrotophos, Dimethoate,
Dimethylvinphos, Disulfoton, EPN, Ethion, Ethoprophos, Famphur,
Fenamiphos, Fenitrothion, Fenthion, Fosthiazate, Heptenophos,
Imicyafos, Isofenphos, Isopropyl O- (methoxyaminothio- phosphoryl)
salicylate, Isoxathion, Malathion, Mecarbam, Methamidophos,
Methidathion, Mevinphos, Monocrotophos, Naled, Omethoate,
Oxydemeton- methyl, Parathion, Parathion- methyl, Phenthoate,
Phorate, Phosalone, Phosmet, Phosphamidon, Phoxim,
Pirimiphos-methyl, Profenofos, Propetamphos, Prothiofos,
Pyraclofos, Pyridaphenthion, Quinalphos, Sulfotep, Tebupirimfos,
Temephos, Terbufos, Tetrachlorvinphos, Thiometon, Triazophos,
Trichlorfon, Vamidothion GABA-gated cyclodiene Chlordane,
Endosulfan Nerve and chloride channel organochlorines muscle
blockers GABA-gated phenylpyrazoles Ethiprole, Fipronil Nerve and
chloride channel (Fiproles) muscle blockers sodium channel
pyrethroids, Acrinathrin, Allethrin, Nerve and modulators
pyrethrins Bifenthrin, Bioallethrin, muscle Bioallethrin
S-cyclopentenyl, Bioresmethrin, Cycloprothrin, Cyfluthrin,
Cyhalothrin, Cypermethrin, Cyphenothrin [(1R)-trans-isomers],
Deltamethrin, Empenthrin [(EZ)-(1R)-isomers], Esfenvalerate,
Etofenprox, Fenpropathrin, Fenvalerate, Flucythrinate, Flumethrin,
Halfenprox, Kadathrin, Phenothrin [(1R)-trans- isomer],
Prallethrin, Pyrethrins (pyrethrum), Resmethrin, Silafluofen,
Tefluthrin, Tetramethrin, Tetramethrin [(1R)-isomers],
Tralomethrin, Transfluthrin, alpha- Cypermethrin, beta-Cyfluthrin,
beta-Cypermethrin, d-cis-trans Allethrin, d-trans Allethrin,
gamma-Cyhalothrin, lambda- Cvhalothrin, tau-Fluvalinate,
theta-Cypermethrin, zeta- Cypermethrin sodium channel DDT, DDT,
methoxychlor Nerve and modulators methoxychlor muscle nicotinic
neonicotinoids Acetamiprid, Clothianidin, Nerve and acetylcholine
Dinotefuran, Imidacloprid, muscle receptor (nAChR) Nitenpyram,
Thiacloprid, competitive Thiamethoxam modulators nicotinic nicotine
nicotine Nerve and acetylcholine muscle receptor (nAChR)
competitive modulators nicotinic sulfoximines sulfoxaflor Nerve and
acetylcholine muscle receptor (nAChR) competitive modulators
nicotinic butenolides Flupyradifurone Nerve and acetylcholine
muscle receptor (nAChR) competitive modulators nicotinic spinosyns
Spinetoram, Spinosad Nerve and acetylcholine muscle receptor
(nAChR) allosteric modulators Glutamate-gated avermectins,
Abamectin, Emamectin Nerve and chloride channel milbemycins
benzoate, Lepimectin, muscle (GluCl) allosteric Milbemectin
modulators juvenile hormone juvenile hormone Hydroprene, Kinoprene,
Growth mimics analogues Methoprene juvenile hormone Fenoxycarb
Fenoxycarb Growth mimics juvenile hormone Pyriproxyfen Pyriproxyfen
Growth mimics miscellaneous non- alkyl halides Methyl bromide and
other Unknown or specific (multi-site) alkyl halides non-specific
inhibitors miscellaneous non- Chloropicrin Chloropicrin Unknown or
specific (multi-site) non-specific inhibitors miscellaneous non-
fluorides Cryolite, sulfuryl fluoride Unknown or specific
(multi-site) non-specific inhibitors miscellaneous non- borates
Borax, Boric acid, Disodium Unknown or specific (multi-site)
octaborate, Sodium borate, non-specific inhibitors Sodium
metaborate miscellaneous non- tartar emetic tartar emetic Unknown
or specific (multi-site) non-specific inhibitors miscellaneous non-
methyl Dazomet, Metam Unknown or specific (multi-site)
isothiocyanate non-specific inhibitors generators modulators of
Pyridine Pymetrozine, Pyrifluquinazon Nerve and chordotonal organs
azomethine muscle derivatives mite growth Clofentezine,
Clofentezine, Diflovidazin, Growth inhibitors Diflovidazin,
Hexythiazox Hexythiazox mite growth Etoxazole Etoxazole Growth
inhibitors microbial Bacillus Bt var. aizawai, Bt var. Midgut
disruptors of insect thuringiensis and israelensis, Bt var.
kurstaki, Bt midgut membranes the insecticidal var. tenebrionensis
proteins they produce microbial Bacillus Bacillus sphaericus Midgut
disruptors of insect sphaericus midgut membranes inhibitors of
Diafenthiuron Diafenthiuron Respiration mitochondrial ATP synthase
inhibitors of organotin Azocyclotin, Cyhexatin, Respiration
mitochondrial ATP miticides Fenbutatin oxide synthase inhibitors of
Propargite Propargite Respiration mitochondrial ATP synthase
inhibitors of Tetradifon Tetradifon Respiration mitochondrial ATP
synthase uncouplers of Chlorfenapyr, Chlorfenapyr, DNOC,
Respiration oxidative DNOC, Sulfuramid phosphorylation via
Sulfuramid disruption of the proton gradient Nicotinic nereistoxin
Bensultap, Cartap Nerve and acetylcholine analogues hydrochloride,
Thiocyclam, muscle receptor (nAChR) Thiosultap-sodium channel
blockers inhibitors of chitin benzoylureas Bistrifluron,
Chlorfluazuron, Growth biosynthesis, type 0 Diflubenzuron,
Flucycloxuron, Flufenoxuron, Hexaflumuron, Lufenuron, Novaluron,
Noviflumuron, Teflubenzuron, Triflumuron inhibitors of chitin
Buprofezin Buprofezin Growth biosynthesis, type 1 moulting
disruptor, Cyromazine Cyromazine Growth Dipteran ecdysone receptor
diacylhydrazines Chromafenozide, Growth agonists Halofenozide,
Methoxyfenozide, Tebufenozide octopamine Amitraz Amitraz Nerve and
receptor agonists muscle mitochondrial Hydramethylnon
Hydramethylnon Respiration complex III electron transport
inhibitors mitochondrial Acequinocyl Acequinocyl Respiration
complex III electron transport inhibitors mitochondrial
Fluacrypyrim Fluacrypyrim Respiration complex III electron
transport inhibitors mitochondrial Bifenazate Bifenazate
Respiration complex III electron transport inhibitors mitochondrial
Meti acaricides Fenazaquin, Fenpyroximate, Respiration complex I
electron and insecticides Pyridaben, Pyrimidifen, transport
inhibitors Tebufenpyrad, Tolfenpyrad mitochondrial Rotenone
Rotenone Respiration complex I electron transport inhibitors
voltage-dependent oxadiazines Indoxacarb Nerve and sodium channel
muscle blockers voltage-dependent semicarbazones Metaflumizone
Nerve and sodium channel muscle blockers inhibitors of acetyl
tetronic and Spirodiclofen, Spiromesifen, Growth CoA carboxylase
tetramic acid Spirotetramat derivatives mitochondrial phosphides
Aluminium phosphide, Respiration complex IV Calcium phosphide,
electron transport Phosphine, Zinc phosphide inhibitors
mitochondrial cyanides Calcium cyanide, Respiration complex IV
Potassium cyanide, electron transport Sodium cyanide inhibitors
mitochondrial beta-ketonitrile Cyenopyrafen, Cyflumetofen
Respiration complex II electron derivatives transport inhibitors
mitochondrial carboxanilides Pyflubumide Respiration complex II
electron transport inhibitors ryanodine receptor diamides
Chlorantraniliprole, Nerve and modulators Cyantraniliprole, muscle
Flubendiamide Chordotonal organ Flonicamid Flonicamid Nerve and
modulators - muscle
undefined target site compounds of Azadirachtin Azadirachtin
Unknown unknown or uncertain mode of action compounds of
Benzoximate Benzoximate Unknown unknown or uncertain mode of action
compounds of Bromopropylate Bromopropylate Unknown unknown or
uncertain mode of action compounds of Chinomethionat Chinomethionat
Unknown unknown or uncertain mode of action compounds of Dicofol
Dicofol Unknown unknown or uncertain mode of action compounds of
lime sulfur lime sulfur Unknown unknown or uncertain mode of action
compounds of Pyridalyl Pyridalyl Unknown unknown or uncertain mode
of action compounds of sulfur sulfur Unknown unknown or uncertain
mode of action
TABLE-US-00010 TABLE 10 Exemplary list of pesticides, which can be
combined with microbes of the disclosure Category Compounds
INSECTICIDES arsenical insecticides calcium arsenate copper
acetoarsenite copper arsenate lead arsenate potassium arsenite
sodium arsenite botanical insecticides allicin anabasine
azadirachtin carvacrol d-limonene matrine nicotine nornicotine
oxymatrine pyrethrins cinerins cinerin I cinerin II jasmolin I
jasmolin II pyrethrin I pyrethrin II quassia rhodojaponin-III
rotenone ryania sabadilla sanguinarine triptolide carbamate
insecticides bendiocarb carbaryl benzofuranyl methylcarbamate
insecticides benfuracarb carbofuran carbosulfan decarbofuran
furathiocarb dimethylcarbamate insecticides dimetan dimetilan
hyquincarb isolan pirimicarb pyramat pyrolan oxime carbamate
insecticides alanycarb aldicarb aldoxycarb butocarboxim
butoxycarboxim methomyl nitrilacarb oxamyl tazimcarb thiocarboxime
thiodicarb thiofanox phenyl methylcarbamate insecticides allyxycarb
aminocarb bufencarb butacarb carbanolate cloethocarb CPMC dicresyl
dimethacarb dioxacarb EMPC ethiofencarb fenethacarb fenobucarb
isoprocarb methiocarb metolcarb mexacarbate promacyl promecarb
propoxur trimethacarb XMC xylylcarb diamide insecticides
broflanilide chlorantraniliprole cyantraniliprole cyclaniliprole
cyhalodiamide flubendiamide tetraniliprole dinitrophenol
insecticides dinex dinoprop dinosam DNOC fluorine insecticides
barium hexafluorosilicate cryolite flursulamid sodium fluoride
sodium hexafluorosilicate sulfluramid formamidine insecticides
amitraz chlordimeform formetanate formparanate medimeform
semiamitraz fumigant insecticides acrylonitrile carbon disulfide
carbon tetrachloride carbonyl sulfide chloroform chloropicrin
cyanogen para-dichlorobenzene 1,2-dichloropropane dithioether ethyl
formate ethylene dibromide ethylene dichloride ethylene oxide
hydrogen cyanide methyl bromide methyl iodide methylchloroform
methylene chloride naphthalene phosphine sodium tetrathiocarbonate
sulfuryl fluoride tetrachloroethane inorganic insecticides borax
boric acid calcium polysulfide copper oleate diatomaceous earth
mercurous chloride potassium thiocyanate silica gel sodium
thiocyanate insect growth regulators chitin synthesis inhibitors
buprofezin cyromazine benzoylphenylurea chitin synthesis
bistrifluron inhibitors chlorbenzuron chlorfluazuron
dichlorbenzuron diflubenzuron flucycloxuron flufenoxuron
hexaflumuron lufenuron novaluron noviflumuron penfluron
teflubenzuron triflumuron juvenile hormone mimics dayoutong
epofenonane fenoxycarb hydroprene kinoprene methoprene pyriproxyfen
triprene juvenile hormones juvenile hormone I juvenile hormone II
juvenile hormone III moulting hormone agonists chromafenozide furan
tebufenozide halofenozide methoxyfenozide tebufenozide yishijing
moulting hormones .alpha.-ecdysone ecdysterone moulting inhibitors
diofenolan precocenes precocene I precocene II precocene III
unclassified insect growth regulators dicyclanil macrocyclic
lactone insecticides avermectin insecticides abamectin doramectin
emamectin eprinomectin ivermectin selamectin milbemycin
insecticides lepimectin milbemectin milbemycin oxime moxidectin
spinosyn insecticides spinetoram spinosad neonicotinoid
insecticides nitroguanidine neonicotinoid insecticides clothianidin
dinotefuran imidacloprid imidaclothiz thiamethoxam nitromethylene
neonicotinoid insecticides nitenpyram nithiazine pyridylmethylamine
neonicotinoid acetamiprid insecticides imidacloprid nitenpyram
paichongding thiacloprid nereistoxin analogue insecticides
bensultap cartap polythialan thiocyclam thiosultap organochlorine
insecticides bromo-DDT camphechlor DDT pp'-DDT ethyl-DDD HCH
gamma-HCH lindane methoxychlor pentachlorophenol TDE cyclodiene
insecticides aldrin bromocyclen chlorbicyclen chlordane chlordecone
dieldrin dilor endosulfan alpha-endosulfan endrin HEOD heptachlor
HHDN isobenzan isodrin kelevan mirex organophosphorus insecticides
organophosphate insecticides bromfenvinfos calvinphos
chlorfenvinphos crotoxyphos dichlorvos dicrotophos
dimethylvinphos fospirate heptenophos methocrotophos mevinphos
monocrotophos naled naftalofos phosphamidon propaphos TEPP
tetrachlorvinphos organothiophosphate insecticides dioxabenzofos
fosmethilan phenthoate aliphatic organothiophosphate insecticides
acethion acetophos amiton cadusafos chlorethoxyfos chlormephos
demephion demephion-O demephion-S demeton demeton-O demeton-S
demeton-methyl demeton-O-methyl demeton-S-methyl
demeton-S-methylsulphon disulfoton ethion ethoprophos IPSP
isothioate malathion methacrifos methylacetophos oxydemeton-methyl
oxydeprofos oxydisulfoton phorate sulfotep terbufos thiometon
aliphatic amide organothiophosphate amidithion insecticides
cyanthoate dimethoate ethoate-methyl formothion mecarbam omethoate
prothoate sophamide vamidothion oxime organothiophosphate
insecticides chlorphoxim phoxim phoxim-methyl heterocyclic
organothiophosphate azamethiphos insecticides colophonate coumaphos
coumithoate dioxathion endothion menazon morphothion phosalone
pyraclofos pyrazothion pyridaphenthion quinothion benzothiopyran
organothiophosphate dithicrofos insecticides thicrofos
benzotriazine organothiophosphate azinphos-ethyl insecticides
azinphos-methyl isoindole organothiophosphate insecticides dialifos
phosmet isoxazole organothiophosphate insecticides isoxathion
zolaprofos pyrazolopyrimidine organothiophosphate chlorprazophos
insecticides pyrazophos pyridine organothiophosphate insecticides
chlorpyrifos chlorpyrifos-methyl pyrimidine organothiophosphate
butathiofos insecticides diazinon etrimfos lirimfos pirimioxyphos
pirimiphos-ethyl pirimiphos-methyl primidophos pyrimitate
tebupirimfos quinoxaline organothiophosphate quinalphos
insecticides quinalphos-methyl thiadiazole organothiophosphate
athidathion insecticides lythidathion methidathion prothidathion
triazole organothiophosphate insecticides isazofos triazophos
phenyl organothinphosphate insecticides azothoate bromophos
bromophos-ethyl carbophenothion chlorthiophos cyanophos cythioate
dicapthon dichlofenthion etaphos famphur fenchlorphos fenitrothion
fensulfothion fenthion fenthion-ethyl heterophos jodfenphos
mesulfenfos parathion parathion-methyl phenkapton phosnichlor
profenofos prothiofos sulprofos temephos trichlormetaphos-3
trifenofos xiaochongliulin phosphonate insecticides butonate
trichlorfon phosphonothioate insecticides mecarphon phenyl
ethylphosphonothioate insecticides fonofos trichloronat phenyl
phenylphosphonothioate cyanofenphos insecticides EPN leptophos
phosphoramidate insecticides crufomate fenamiphos fosthietan
mephosfolan phosfolan phosfolan-methyl pirimetaphos
phosphoramidothioate insecticides acephate chloramine phosphorus
isocarbophos isofenphos isofenphos-methyl methamidophos phosglycin
propetamphos phosphorodiamide insecticides dimefox mazidox mipafox
schradan oxadiazine insecticides indoxacarb oxadiazolone
insecticides metoxadiazone phthalimide insecticides dialifos
phosmet tetramethrin physical insecticides maltodextrin desiccant
insecticides boric acid diatomaceous earth silica gel pyrazole
insecticides chlorantraniliprole cyantraniliprole cyclaniliprole
dimetilan isolan tebufenpyrad tetraniliprole tolfenpyrad
phenylpyrazole insecticides acetoprole ethiprole fipronil
flufiprole pyraclofos pyrafluprole pyriprole pyrolan vaniliprole
pyrethroid insecticides pyrethroid ester insecticides acrinathrin
allethrin bioallethrin esdepallethrine barthrin bifenthrin
kappa-bifenthrin bioethanomethrin brofenvalerate brofluthrinate
bromethrin butethrin chlorempenthrin cyclethrin cycloprothrin
cyfluthrin beta-cyfluthrin cyhalothrin gamma-cyhalothrin
lambda-cyhalothrin cypermethrin alpha-cypermethrin
beta-cypermethrin theta-cypermethrin zeta-cypermethrin cyphenothrin
deltamethrin dimefluthrin dimethrin empenthrin
d-fanshiluquebingjuzhi chloroprallethrin fenfluthrin fenpirithrin
fenpropathrin fenvalerate esfenvalerate flucythrinate fluvalinate
tau-fluvalinate furamethrin furethrin heptafluthrin imiprothrin
japothrins kadethrin methothrin metofluthrin epsilon-metofluthrin
momfluorothrin epsilon-momfluorothrin pentmethrin permethrin
biopermethrin transpermethrin phenothrin prallethrin profluthrin
proparthrin pyresmethrin renofluthrin meperfluthrin resmethrin
bioresmethrin cismethrin
tefluthrin kappa-tefluthrin terallethrin tetramethrin
tetramethylfluthrin tralocythrin tralomethrin transfluthrin
valerate pyrethroid ether insecticides etofenprox flufenprox
halfenprox protrifenbute silafluofen pyrethroid oxime insecticides
sulfoxime thiofluoximate pyrimidinamine insecticides flufenerim
pyrimidifen pyrrole insecticides chlorfenapyr quaternary ammonium
insecticides sanguinarine sulfoximine insecticides sulfoxaflor
tetramic acid insecticides spirotetramat tetronic acid insecticides
spiromesifen thiazole insecticides clothianidin imidaclothiz
thiamethoxam thiapronil thiazolidine insecticides tazimcarb
thiacloprid thiourea insecticides diafenthiuron urea insecticides
flucofuron sulcofuron zwitterionic insecticides dicloromezotiaz
triflumezopyrim unclassified insecticides afidopyropen afoxolaner
allosamidin closantel copper naphthenate crotamiton EXD fenazaflor
fenoxacrim flometoquin flonicamid fluhexafon flupyradifurone
fluralaner fluxametamide hydramethylnon isoprothiolane
jiahuangchongzong malonoben metaflumizone nifluridide plifenate
pyridaben pyridalyl pyrifluquinazon rafoxanide thuringiensin
triarathene triazamate ACARICIDES botanical acaricides carvacrol
sanguinarine bridged diphenyl acaricides azobenzene benzoximate
benzyl benzoate bromopropylate chlorbenside chlorfenethol
chlorfenson chlorfensulphide chlorobenzilate chloropropylate
cyflumetofen DDT dicofol diphenyl sulfone dofenapyn fenson
fentrifanil fluorbenside genit hexachlorophene phenproxide
proclonol tetradifon tetrasul carbamate acaricides benomyl
carbanolate carbaryl carbofuran methiocarb metolcarb promacyl
propoxur oxime carbamate acaricides aldicarb butocarboxim oxamyl
thiocarboxime thiofanox carbazate acaricides bifenazate
dinitrophenol acaricides binapacryl dinex dinobuton dinocap
dinocap-4 dinocap-6 dinocton dinopenton dinosulfon dinoterbon DNOC
formamidine acaricides amitraz chlordimeform chloromebuform
formetanate formparanate medimeform semiamitraz macrocyclic lactone
acaricides tetranactin avermectin acaricides abamectin doramectin
eprinomectin ivermectin selamectin milbemycin acaricides
milbemectin milbemycin oxime moxidectin mite growth regulators
clofentezine cyromazine diflovidazin dofenapyn fluazuron
flubenzimine flucycloxuron flufenoxuron hexythiazox organochlorine
acaricides bromocyclen camphechlor DDT dienochlor endosulfan
lindane organophosphorus acaricides organophosphate acaricides
chlorfenvinphos crotoxyphos dichlorvos heptenophos mevinphos
monocrotophos naled TEPP tetrachlorvinphos organothiophosphate
acaricides amidithion amiton azinphos-ethyl azinphos-methyl
azothoate benoxafos bromophos bromophos-ethyl carbophenothion
chlorpyrifos chlorthiophos coumaphos cyanthoate demeton demeton-O
demeton-S demeton-methyl demeton-O-methyl demeton-S-methyl
demeton-S-methylsulphon dialifos diazinon dimethoate dioxathion
disulfoton endothion ethion ethoate-methyl formothion malathion
mecarbam methacrifos omethoate oxydeprofos oxydisulfoton parathion
phenkapton phorate phosalone phosmet phostin phoxim
pirimiphos-methyl prothidathion prothoate pyrimitate quinalphos
qumtiofos sophamide sulfotep thiometon triazophos trifenofos
vamidothion phosphonate acaricides trichlorfon phosphoramidothioate
acaricides isocarbophos methamidophos propetamphos phosphorodiamide
acaricides dimefox mipafox schradan organotin acaricides
azocyclotin cyhexatin fenbutatin oxide phostin phenylsulfamide
acaricides dichlofluanid phthalimide acaricides dialifos phosmet
pyrazole acaricides cyenopyrafen fenpyroximate pyflubumide
tebufenpyrad phenylpyrazole acaricides acetoprole fipronil
vaniliprole pyrethroid acaricides pyrethroid ester acaricides
acrinathrin bifenthrin brofluthrinate cyhalothrin cypermethrin
alpha-cypermethrin fenpropathrin fenvalerate flucythrinate
flumethrin fluvalinate tau-fluvalinate permethrin pyrethroid ether
acaricides halfenprox pyrimidinamine acaricides pyrimidifen pyrrole
acaricides chlorfenapyr quaternary ammonium acaricides sanguinarine
quinoxaline acaricides chinomethionat thioquinox
strobilurin acaricides methoxyacrylate strobilurin acaricides
bifujunzhi fluacrypyrim flufenoxystrobin pyriminostrobin sulfite
ester acaricides aramite propargite tetronic acid acaricides
spirodiclofen tetrazine acaricides clofentezine diflovidazin
thiazolidine acaricides flubenzimine hexythiazox thiocarbamate
acaricides fenothiocarb thiourea acaricides chloromethiuron
diafenthiuron unclassified acaricides acequinocyl afoxolaner
amidoflumet arsenous oxide clenpirin closantel crotamiton
cycloprate cymiazole disulfiram etoxazole fenazaflor fenazaquin
fluenetil fluralaner mesulfen MNAF nifluridide nikkomycins
pyridaben sulfiram sulfluramid sulfur thuringiensin triarathene
CHEMOSTERILANTS apholate bisazir busulfan diflubenzuron dimatif
hemel hempa metepa methiotepa methyl apholate morzid penfluron tepa
thiohempa thiotepa tretamine uredepa INSECT REPELLENTS acrep
butopyronoxyl camphor d-camphor carboxide dibutyl phthalate
diethyltoluamide dimethyl carbate dimethyl phthalate dibutyl
succinate ethohexadiol hexamide icaridin methoquin-butyl
methylneodecanamide 2-(octylthio)ethanol oxamate quwenzhi
quyingding rebemide zengxiaoan NEMATICIDES avermectin nematicides
abamectin botanical nematicides carvacrol carbamate nematicides
benomyl carbofuran carbosulfan cloethocarb oxime carbamate
nematicides alanycarb aldicarb aldoxycarb oxamyl tirpate fumigant
nematicides carbon disulfide cyanogen 1,2-dichloropropane
1,3-dichloropropene dithioether methyl bromide methyl iodide sodium
tetrathiocarbonate organophosphorus nematicides organophosphate
nematicides diamidafos fenamiphos fosthietan phosphamidon
organothiophosphate nematicides cadusafos chlorpyrifos
dichlofenthion dimethoate ethoprophos fensulfothion fosthiazate
heterophos isamidofos isazofos phorate phosphocarb terbufos
thionazin triazophos phosphonothioate nematicides imicyafos
mecarphon unclassified nematicides acetoprole benclothiaz
chloropicrin dazomet DBCP DCIP fluazaindolizine fluensulfone
furfural metam methyl isothiocyanate tioxazafen xylenols
[0350] Insecticides also include synergists or activators that are
not in themselves considered toxic or insecticidal, but are
materials used with insecticides to synergize or enhance the
activity of the insecticides. Synergists or activators include
piperonyl butoxide.
Biorational Pesticides
[0351] Insecticides can be biorational, or can also be known as
biopesticides or biological pesticides. Biorational refers to any
substance of natural origin (or man-made substances resembling
those of natural origin) that has a detrimental or lethal effect on
specific target pest(s), e.g., insects, weeds, plant diseases
(including nematodes), and vertebrate pests, possess a unique mode
of action, are non-toxic to man, domestic plants and animals, and
have little or no adverse effects on wildlife and the
environment.
[0352] Biorational insecticides (or biopesticides or biological
pesticides) can be grouped as: (1) biochemicals (hormones, enzymes,
pheromones and natural agents, such as insect and plant growth
regulators), (2) microbial (viruses, bacteria, fungi, protozoa, and
nematodes), or (3) Plant-Incorporated protectants (PIPs)--primarily
transgenic plants, e.g., Bt corn.
[0353] Biopesticides, or biological pesticides, can broadly include
agents manufactured from living microorganisms or a natural product
and sold for the control of plant pests. Biopesticides can be:
microorganisms, biochemicals, and semiochemicals. Biopesticides can
also include peptides, proteins and nucleic acids such as
double-stranded DNA, single-stranded DNA, double-stranded RNA,
single-stranded RNA and hairpin DNA or RNA.
[0354] Bacteria, fungi, oomycetes, viruses and protozoa are all
used for the biological control of insect pests. The most widely
used microbial biopesticide is the insect pathogenic bacteria
Bacillus thuringiensis (Bt), which produces a protein crystal (the
Bt .delta.-endotoxin) during bacterial spore formation that is
capable of causing lysis of gut cells when consumed by susceptible
insects. Microbial Bt biopesticides consist of bacterial spores and
.delta.-endotoxin crystals mass-produced in fermentation tanks and
formulated as a sprayable product. Bt does not harm vertebrates and
is safe to people, beneficial organisms and the environment. Thus,
Bt sprays are a growing tactic for pest management on fruit and
vegetable crops where their high level of selectivity and safety
are considered desirable, and where resistance to synthetic
chemical insecticides is a problem. Bt sprays have also been used
on commodity crops such as maize, soybean and cotton, but with the
advent of genetic modification of plants, farmers are increasingly
growing Bt transgenic crop varieties.
[0355] Other microbial insecticides include products based on
entomopathogenic baculoviruses. Baculoviruses that are pathogenic
to arthropods belong to the virus family and possess large
circular, covalently closed, and double-stranded DNA genomes that
are packaged into nucleocapsids. More than 700 baculoviruses have
been identified from insects of the orders Lepidoptera,
Hymenoptera, and Diptera. Baculoviruses are usually highly specific
to their host insects and thus, are safe to the environment,
humans, other plants, and beneficial organisms. Over 50 baculovirus
products have been used to control different insect pests
worldwide. In the US and Europe, the Cydia pomonella granulovirus
(CpGV) is used as an inundative biopesticide against codling moth
on apples. Washington State, as the biggest apple producer in the
US, uses CpGV on 13% of the apple crop. In Brazil, the
nucleopolyhedrovirus of the soybean caterpillar Anticarsia
gemmatalis was used on up to 4 million ha (approximately 35%) of
the soybean crop in the mid-1990s. Viruses such as Gemstar.RTM.
(Certis USA) are available to control larvae of Heliothis and
Helicoverpa species.
[0356] At least 170 different biopesticide products based on
entomopathogenic fungi have been developed for use against at least
five insect and acarine orders in glasshouse crops, fruit and field
vegetables as well as commodity crops. The majority of products are
based on the ascomycetes Beauveria bassiana or Metarhizium
anisopliae. M anisopliae has also been developed for the control of
locust and grasshopper pests in Africa and Australia and is
recommended by the Food and Agriculture Organization of the United
Nations (FAO) for locust management.
[0357] A number of microbial pesticides registered in the United
States are listed in Table 16 of Kabaluk et al. 2010 (Kabaluk, J.
T. et al. (ed.). 2010. The Use and Regulation of Microbial
Pesticides in Representative Jurisdictions Worldwide. IOBC Global.
99pp.) and microbial pesticides registered in selected countries
are listed in Annex 4 of Hoeschle-Zeledon et al. 2013
(Hoeschle-Zeledon, I., P. Neuenschwander and L. Kumar. (2013).
Regulatory Challenges for biological control. SP-IPM Secretariat,
International Institute of Tropical Agriculture (IITA), Ibadan,
Nigeria. 43 pp.), each of which is incorporated herein in its
entirety.
[0358] Plants produce a wide variety of secondary metabolites that
deter herbivores from feeding on them. Some of these can be used as
biopesticides. They include, for example, pyrethrins, which are
fast-acting insecticidal compounds produced by Chrysanthemum
cinerariaefolium. They have low mammalian toxicity but degrade
rapidly after application. This short persistence prompted the
development of synthetic pyrethrins (pyrethroids). The most widely
used botanical compound is neem oil, an insecticidal chemical
extracted from seeds of Azadirachta indica. Two highly active
pesticides are available based on secondary metabolites synthesized
by soil actinomycetes, but they have been evaluated by regulatory
authorities as if they were synthetic chemical pesticides. Spinosad
is a mixture of two macrolide compounds from Saccharopolyspora
spinosa. It has a very low mammalian toxicity and residues degrade
rapidly in the field. Farmers and growers used it widely following
its introduction in 1997 but resistance has already developed in
some important pests such as western flower thrips. Abamectin is a
macrocyclic lactone compound produced by Streptomyces avermitilis.
It is active against a range of pest species but resistance has
developed to it also, for example, in tetranychid mites.
[0359] Peptides and proteins from a number of organisms have been
found to possess pesticidal properties. Perhaps most prominent are
peptides from spider venom (King, G. F. and Hardy, M. C. (2013)
Spider-venom peptides: structure, pharmacology, and potential for
control of insect pests. Annu. Rev. Entomol. 58: 475-496). A unique
arrangement of disulfide bonds in spider venom peptides render them
extremely resistant to proteases. As a result, these peptides are
highly stable in the insect gut and hemolymph and many of them are
orally active. The peptides target a wide range of receptors and
ion channels in the insect nervous system. Other examples of
insecticidal peptides include: sea anemone venom that act on
voltage-gated Na+ channels (Bosmans, F. and Tytgat, J. (2007) Sea
anemone venom as a source of insecticidal peptides acting on
voltage-gated Na+ channels. Toxicon. 49(4): 550-560); the PA1b (Pea
Albumin 1, subunit b) peptide from Legume seeds with lethal
activity on several insect pests, such as mosquitoes, some aphids
and cereal weevils (Eyraud, V. et al. (2013) Expression and
Biological Activity of the Cystine Knot Bioinsecticide PA1b (Pea
Albumin 1 Subunit b). PLoS ONE 8(12): e81619); and an internal 10
kDa peptide generated by enzymatic hydrolysis of Canavalia
ensiformis (jack bean) urease within susceptible insects
(Martinelli, A. H. S., et al. (2014) Structure--function studies on
jaburetox, a recombinant insecticidal peptide derived from jack
bean (Canavalia ensiformis) urease. Biochimica et Biophysica Acta
1840: 935-944). Examples of commercially available peptide
insecticides include Spear.TM.--T for the treatment of thrips in
vegetables and ornamentals in greenhouses, Spear.TM.--P to control
the Colorado Potato Beetle, and Spear.TM.--C to protect crops from
lepidopteran pests (Vestaron Corporation, Kalamazoo, Mich.). A
novel insecticidal protein from Bacillus bombysepticus, called
parasporal crystal toxin (PC), shows oral pathogenic activity and
lethality towards silkworms and Cry1Ac-resistant Helicoverpa
armigera strains (Lin, P. et al. (2015) PC, a novel oral
insecticidal toxin from Bacillus bombysepticus involved in host
lethality via APN and BtR-175. Sci. Rep. 5: 11101).
[0360] A semiochemical is a chemical signal produced by one
organism that causes a behavioral change in an individual of the
same or a different species. The most widely used semiochemicals
for crop protection are insect sex pheromones, some of which can
now be synthesized and are used for monitoring or pest control by
mass trapping, lure-and-kill systems and mating disruption.
Worldwide, mating disruption is used on over 660,000 ha and has
been particularly useful in orchard crops.
[0361] As used herein, "transgenic insecticidal trait" refers to a
trait exhibited by a plant that has been genetically engineered to
express a nucleic acid or polypeptide that is detrimental to one or
more pests. In one embodiment, the plants of the present disclosure
are resistant to attach and/or infestation from any one or more of
the pests of the present disclosure. In one embodiment, the trait
comprises the expression of vegetative insecticidal proteins (VIPs)
from Bacillus thuringiensis, lectins and proteinase inhibitors from
plants, terpenoids, cholesterol oxidases from Streptomyces spp.,
insect chitinases and fungal chitinolytic enzymes, bacterial
insecticidal proteins and early recognition resistance genes. In
another embodiment, the trait comprises the expression of a
Bacillus thuringiensis protein that is toxic to a pest. In one
embodiment, the Bt protein is a Cry protein (crystal protein). Bt
crops include Bt corn, Bt cotton and Bt soy. Bt toxins can be from
the Cry family (see, for example, Crickmore et al., 1998,
Microbiol. Mol. Biol. Rev. 62: 807-812), which are particularly
effective against Lepidoptera, Coleoptera and Diptera.
[0362] Bt Cry and Cyt toxins belong to a class of bacterial toxins
known as pore-forming toxins (PFT) that are secreted as
water-soluble proteins undergoing conformational changes in order
to insert into, or to translocate across, cell membranes of their
host. There are two main groups of PFT: (i) the .alpha.-helical
toxins, in which .alpha.-helix regions form the trans-membrane
pore, and (ii) the .beta.-barrel toxins, that insert into the
membrane by forming a .beta.-barrel composed of .beta.sheet
hairpins from each monomer. See, Parker M W, Feil S C,
"Pore-forming protein toxins: from structure to function," Prog.
Biophys. Mol. Biol. 2005 May; 88(1):91-142. The first class of PFT
includes toxins such as the colicins, exotoxin A, diphtheria toxin
and also the Cry three-domain toxins. On the other hand, aerolysin,
.alpha.-hemolysin, anthrax protective antigen,
cholesterol-dependent toxins as the perfringolysin O and the Cyt
toxins belong to the .beta.-barrel toxins. Id. In general, PFT
producing-bacteria secrete their toxins and these toxins interact
with specific receptors located on the host cell surface. In most
cases, PFT are activated by host proteases after receptor binding
inducing the formation of an oligomeric structure that is insertion
competent. Finally, membrane insertion is triggered, in most cases,
by a decrease in pH that induces a molten globule state of the
protein. Id.
[0363] The development of transgenic crops that produce Bt Cry
proteins has allowed the substitution of chemical insecticides by
environmentally friendly alternatives. In transgenic plants the Cry
toxin is produced continuously, protecting the toxin from
degradation and making it reachable to chewing and boring insects.
Cry protein production in plants has been improved by engineering
cry genes with a plant biased codon usage, by removal of putative
splicing signal sequences and deletion of the carboxy-terminal
region of the protoxin. See, Schuler T H, et al., "Insect-resistant
transgenic plants," Trends Biotechnol. 1998; 16:168-175. The use of
insect resistant crops has diminished considerably the use of
chemical pesticides in areas where these transgenic crops are
planted. See, Qaim M, Zilberman D, "Yield effects of genetically
modified crops in developing countries," Science. 2003 Feb. 7;
299(5608):900-2.
[0364] Known Cry proteins include: .delta.-endotoxins including but
not limited to: the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8,
Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17,
Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26,
Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34, Cry35,
Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44,
Cry45, Cry 46, Cry47, Cry49, Cry 51, Cry52, Cry 53, Cry 54, Cry55,
Cry56, Cry57, Cry58, Cry59. Cry60, Cry61, Cry62, Cry63, Cry64,
Cry65, Cry66, Cry67, Cry68, Cry69, Cry70 and Cry71 classes of
.delta.-endotoxin genes and the B. thuringiensis cytolytic cyt1 and
cyt2 genes.
[0365] Members of these classes of B. thuringiensis insecticidal
proteins include, but are not limited to: Cry1Aa1 (Accession
#AAA22353); Cry1Aa2 (Accession #Accession #AAA22552); Cry1Aa3
(Accession #BAA00257); Cry1Aa4 (Accession #CAA31886); Cry1Aa5
(Accession #BAA04468); Cry1Aa6 (Accession #AAA86265); Cry1Aa7
(Accession #AAD46139); Cry1Aa8 (Accession #126149); Cry1Aa9
(Accession #BAA77213); Cry1Aa10 (Accession #AAD55382); Cry1Aa1 1
(Accession #CAA70856); Cry1Aa12 (Accession #AAP80146); Cry1Aa13
(Accession #AAM44305); Cry1Aa14 (Accession #AAP40639); Cry1Aa15
(Accession #AAY66993); Cry1Aa16 (Accession #HQ439776); Cry1Aa17
(Accession #HQ439788); Cry1Aa18 (Accession #HQ439790); Cry1Aa19
(Accession #HQ685121); Cry1Aa20 (Accession #JF340156); Cry1Aa21
(Accession #JN651496); Cry1Aa22 (Accession #KC158223); Cry1Ab1
(Accession #AAA22330); Cry1Ab2 (Accession #AAA22613); Cry1Ab3
(Accession #AAA22561); Cry1Ab4 (Accession #BAA00071); Cry1Ab5
(Accession #CAA28405); Cry1Ab6 (Accession #AAA22420); Cry1Ab7
(Accession #CAA31620); Cry1Ab8 (Accession #AAA22551); Cry1Ab9
(Accession #CAA38701); Cry1Ab10 (Accession #A29125); Cry1Ab11
(Accession #112419); Cry1Ab12 (Accession #AAC64003); Cry1Ab13
(Accession #AAN76494); Cry1Ab14 (Accession #AAG16877); Cry1Ab15
(Accession #AA013302); Cry1Ab16 (Accession #AAK55546); Cry1Ab17
(Accession #AAT46415); Cry1Ab18 (Accession #AAQ88259); Cry1Ab19
(Accession #AAW31761); Cry1Ab20 (Accession #ABB72460); Cry1Ab21
(Accession #ABS18384); Cry1Ab22 (Accession #ABW87320); Cry1Ab23
(Accession #HQ439777); Cry1Ab24 (Accession #HQ439778); Cry1Ab25
(Accession #HQ685122); Cry1Ab26 (Accession #HQ847729); Cry1Ab27
(Accession #JN135249); Cry1Ab28 (Accession #JN135250); Cry1Ab29
(Accession #JN135251); Cry1Ab30 (Accession #JN135252); Cry1Ab31
(Accession #JN135253); Cry1Ab32 (Accession #JN135254); Cry1Ab33
(Accession #AAS93798); Cry1Ab34 (Accession #KC156668); Cry1Ab-like
(Accession #AAK14336); Cry1Ab-like (Accession #AAK14337);
Cry1Ab-like (Accession #AAK14338); Cry1Ab-like (Accession
#ABG88858); Cry1Ac1 (Accession #AAA22331); Cry1Ac2 (Accession
#AAA22338); Cry1Ac3 (Accession #CAA38098); Cry1Ac4 (Accession
#AAA73077); Cry1Ac5 (Accession #AAA22339); Cry1Ac6 (Accession
#AAA86266); Cry1Ac7 (Accession #AAB46989); Cry1Ac8 (Accession
#AAC44841); Cry1Ac9 (Accession #AAB49768); Cry1Ac10 (Accession
#CAA05505); Cry1Ac11 (Accession #CAA10270); Cry1Ac12 (Accession
#112418); Cry1Ac13 (Accession #AAD38701); Cry1Ac14 (Accession
#AAQ06607); Cry1Ac15 (Accession #AAN07788); Cry1Ac16 (Accession
#AAU87037); Cry1Ac17 (Accession #AAX18704); Cry1Ac18 (Accession
#AAY88347); Cry1Ac19 (Accession #ABD37053); Cry1Ac20 (Accession
#ABB89046); Cry1Ac21 (Accession #AAY66992); Cry1Ac22 (Accession
#ABZ01836); Cry1Ac23 (Accession #CAQ30431); Cry1Ac24 (Accession
#ABL01535); Cry1Ac25 (Accession #FJ513324); Cry1Ac26 (Accession
#FJ617446); Cry1Ac27 (Accession #FJ617447); Cry1Ac28 (Accession
#ACM90319); Cry1Ac29 (Accession #DQ438941); Cry1Ac30 (Accession
#GQ227507); Cry1Ac31 (Accession #GU446674); Cry1Ac32 (Accession
#HM061081); Cry1Ac33 (Accession #GQ866913); Cry1Ac34 (Accession
#HQ230364); Cry1Ac35 (Accession #JF340157); Cry1Ac36 (Accession
#JN387137); Cry1Ac37 (Accession #JQ317685); Cry1Ad1 (Accession
#AAA22340); Cry1Ad2 (Accession #CAA01880); Cry1Ae1 (Accession
#AAA22410); Cry1Af1 (Accession #AAB82749); Cry1Ag1 (Accession
#AAD46137); Cry1Ah1 (Accession #AAQ14326); Cry1Ah2 (Accession
#ABB76664); Cry1Ah3 (Accession #HQ439779); Cry1Ai1 (Accession
#AA039719); Cry1Ai2 (Accession #HQ439780); Cry1A-like (Accession
#AAK14339); Cry1Ba1 (Accession #CAA29898); Cry1Ba2 (Accession
#CAA65003); Cry1Ba3 (Accession #AAK63251); Cry1Ba4 (Accession
#AAK51084); Cry1Ba5 (Accession #AB020894); Cry1Ba6 (Accession
#ABL60921); Cry1Ba7 (Accession #HQ439781); Cry1Bb1 (Accession
#AAA22344); Cry1Bb2 (Accession #HQ439782); Cry1Bc1 (Accession
#CAA86568); Cry1Bd1 (Accession #AAD10292); Cry1Bd2 (Accession
#AAM93496); Cry1Be1 (Accession #AAC32850); Cry1Be2 (Accession
#AAQ52387); Cry1Be3 (Accession #ACV96720); Cry1Be4 (Accession
#HM070026); Cry1Bf1 (Accession #CAC50778); Cry1Bf2 (Accession
#AAQ52380); Cry1Bg1 (Accession #AA039720); Cry1Bh1 (Accession
#HQ589331); Cry1Bi1 (Accession #KC156700); Cry1Ca1 (Accession
#CAA30396); Cry1Ca2 (Accession #CAA31951); Cry1Ca3 (Accession
#AAA22343); Cry1Ca4 (Accession #CAA01886); Cry1Ca5 (Accession
#CAA65457); Cry1Ca6 [1] (Accession #AAF37224); Cry1Ca7 (Accession
#AAG50438); Cry1Ca8 (Accession #AAM00264); Cry1Ca9 (Accession
#AAL79362); Cry1Ca10 (Accession #AAN16462); Cry1Ca11 (Accession
#AAX53094); Cry1Ca12 (Accession #HM070027); Cry1Ca13 (Accession
#HQ412621); Cry1Ca14 (Accession #JN651493); Cry1Cb1 (Accession
#M97880); Cry1Cb2 (Accession #AAG35409); Cry1Cb3 (Accession
#ACD50894); Cry1Cb-like (Accession #AAX63901); Cry1Da1 (Accession
#CAA38099); Cry1Da2 (Accession #176415); Cry1Da3 (Accession
#HQ439784); Cry1 Db1 (Accession #CAA80234); Cry1 Db2 (Accession
#AAK48937); Cry1 Dc1 (Accession #ABK35074); Cry1Ea1 (Accession
#CAA37933); Cry1Ea2 (Accession #CAA39609); Cry1Ea3 (Accession
#AAA22345); Cry1Ea4 (Accession #AAD04732); Cry1Ea5 (Accession
#A15535); Cry1Ea6 (Accession #AAL50330); Cry1Ea7 (Accession
#AAW72936); Cry1Ea8 (Accession #ABX11258); Cry1Ea9 (Accession
#HQ439785); Cry1Ea10 (Accession #ADR00398); Cry1Ea11 (Accession
#JQ652456); Cry1Eb1 (Accession #AAA22346); Cry1Fa1 (Accession
#AAA22348); Cry1Fa2 (Accession #AAA22347); Cry1Fa3 (Accession
#HM070028); Cry1Fa4 (Accession #HM439638); Cry1 Fb1 (Accession
#CAA80235); Cry1Fb2 (Accession #BAA25298); Cry1Fb3 (Accession
#AAF21767); Cry1Fb4 (Accession #AAC10641); Cry1Fb5 (Accession
#AA013295); Cry1Fb6 (Accession #ACD50892); Cry1Fb7 (Accession
#ACD50893); Cry1Ga1 (Accession #CAA80233); Cry1 Ga2 (Accession
#CAA70506); Cry1Gb1 (Accession #AAD10291); Cry1Gb2 (Accession
#AA013756); Cry1Gc1 (Accession #AAQ52381); Cry1Ha1 (Accession
#CAA80236); Cry1Hb1 (Accession #AAA79694); Cry1Hb2 (Accession
#HQ439786); Cry1H-like (Accession #AAF01213); Cry1Ia1 (Accession
#CAA44633); Cry1Ia2 (Accession #AAA22354); Cry1Ia3 (Accession
#AAC36999); Cry1Ia4 (Accession #AAB00958); Cry1Ia5 (Accession
#CAA70124); Cry1Ia6 (Accession #AAC26910); Cry1Ia7 (Accession
#AAM73516); Cry1Ia8 (Accession #AAK66742); Cry1Ia9 (Accession
#AAQ08616); Cry1Ia10 (Accession #AAP86782); Cry1Ia11 (Accession
#CAC85964); Cry1Ia12 (Accession #AAV53390); Cry1Ia13 (Accession
#ABF83202); Cry1Ia14 (Accession #ACG63871); Cry1Ia15 (Accession
#FJ617445); Cry1Ia16 (Accession #FJ617448); Cry1Ia17 (Accession
#GU989199); Cry1Ia18 (Accession #ADK23801); Cry1Ia19 (Accession
#HQ439787); Cry1Ia20 (Accession #JQ228426); Cry1Ia21 (Accession
#JQ228424); Cry1Ia22 (Accession #JQ228427); Cry1Ia23 (Accession
#JQ228428); Cry1Ia24 (Accession #JQ228429); Cry1Ia25 (Accession
#JQ228430); Cry1Ia26 (Accession #JQ228431); Cry1Ia27 (Accession
#JQ228432); Cry1Ia28 (Accession #JQ228433); Cry1Ia29 (Accession
#JQ228434); Cry1Ia30 (Accession #JQ317686); Cry1Ia31 (Accession
#JX944038); Cry1Ia32 (Accession #JX944039); Cry1Ia33 (Accession
#JX944040); Cry1Ib1 (Accession #AAA82114); Cry1Ib2 (Accession
#ABW88019); Cry1Ib3 (Accession #ACD75515); Cry1Ib4 (Accession
#HM051227); Cry1Ib5 (Accession #HM070028); Cry1Ib6 (Accession
#ADK38579); Cry1Ib7 (Accession #JN571740); Cry1Ib8 (Accession
#JN675714); Cry1Ib9 (Accession #JN675715); Cry1Ib10 (Accession
#JN675716); Cry1Ib11 (Accession #JQ228423); Cry1Ic1 (Accession
#AAC62933); Cry1Ic2 (Accession #AAE71691); Cry1Id1 (Accession
#AAD44366); Cry1Id2 (Accession #JQ228422); Cry1Ie1 (Accession
#AAG43526); Cry1Ie2 (Accession #HM439636); Cry1Ie3 (Accession
#KC156647); Cry1Ie4 (Accession #KC156681); Cry1If1 (Accession
#AAQ52382); Cry1Ig1 (Accession #KC156701); Cry1I-like (Accession
#AAC31094); Cry1I-like (Accession #ABG88859); Cry1Ja1 (Accession
#AAA22341); Cry1Ja2 (Accession #HM070030); Cry1Ja3 (Accession
#JQ228425); Cry1Jb1 (Accession #AAA98959); Cry1Jc1 (Accession
#AAC31092); Cry1Jc2 (Accession #AAQ52372); Cry1Jd1 (Accession
#CAC50779); Cry1Ka1 (Accession #AAB00376); Cry1Ka2 (Accession
#HQ439783); Cry1La1 (Accession #AAS60191); Cry1La2 (Accession
#HM070031); Cry1Ma1 (Accession #FJ884067); Cry1Ma2 (Accession
#KC156659); Cry1Na1 (Accession #KC156648); Cry1Nb1 (Accession
#KC156678); Cry1-like (Accession #AAC31091); Cry2Aa1 (Accession
#AAA22335); Cry2Aa2 (Accession #AAA83516); Cry2Aa3 (Accession
#D86064); Cry2Aa4 (Accession #AAC04867); Cry2Aa5 (Accession
#CAA10671); Cry2Aa6 (Accession #CAA10672); Cry2Aa7 (Accession
#CAA10670); Cry2Aa8 (Accession #AA013734); Cry2Aa9 (Accession
#AA013750); Cry2Aa1 O (Accession #AAQ04263); Cry2Aa1 1 (Accession
#AAQ52384); Cry2Aa12 (Accession #AB183671); Cry2Aa13 (Accession
#ABL01536); Cry2Aa14 (Accession #ACF04939); Cry2Aa15 (Accession
#JN426947); Cry2Ab1 (Accession #AAA22342); Cry2Ab2 (Accession
#CAA39075); Cry2Ab3 (Accession #AAG36762); Cry2Ab4 (Accession
#AA013296); Cry2Ab5 (Accession #AAQ04609); Cry2Ab6 (Accession
#AAP59457); Cry2Ab7 (Accession #AAZ66347); Cry2Ab8 (Accession
#ABC95996); Cry2Ab9 (Accession #ABC74968); Cry2Ab10 (Accession
#EF157306); Cry2Ab11 (Accession #CAM84575); Cry2Ab12 (Accession
#ABM21764); Cry2Ab13 (Accession #ACG76120); Cry2Ab14 (Accession
#ACG76121); Cry2Ab15 (Accession #HM037126); Cry2Ab16 (Accession
#GQ866914); Cry2Ab1 7 (Accession #HQ439789); Cry2Ab18 (Accession
#JN135255); Cry2Ab19 (Accession #JN135256); Cry2Ab20 (Accession
#JN135257); Cry2Ab21 (Accession #JN135258); Cry2Ab22 (Accession
#JN135259); Cry2Ab23 (Accession #JN135260); Cry2Ab24 (Accession
#JN135261); Cry2Ab25 (Accession #JN415485); Cry2Ab26 (Accession
#JN426946); Cry2Ab27 (Accession #JN415764); Cry2Ab28 (Accession
#JN651494); Cry2Ac1 (Accession #CAA40536); Cry2Ac2 (Accession
#AAG35410); Cry2Ac3 (Accession #AAQ52385); Cry2Ac4 (Accession
#ABC95997); Cry2Ac5 (Accession #ABC74969); Cry2Ac6 (Accession
#ABC74793); Cry2Ac7 (Accession #CAL18690); Cry2Ac8 (Accession
#CAM09325); Cry2Ac9 (Accession #CAM09326); Cry2Ac10 (Accession
#ABN15104); Cry2Ac11 (Accession #CAM83895); Cry2Ac12 (Accession
#CAM83896); Cry2Ad1 (Accession #AAF09583); Cry2Ad2 (Accession
#ABC86927); Cry2Ad3 (Accession #CAK29504); Cry2Ad4 (Accession
#CAM32331); Cry2Ad5 (Accession #CA078739); Cry2Ae1 (Accession
#AAQ52362); Cry2Af1 (Accession #AB030519); Cry2Af2 (Accession
#GQ866915); Cry2Ag1 (Accession #ACH91610); Cry2Ah1 (Accession
#EU939453); Cry2Ah2 (Accession #ACL80665); Cry2Ah3 (Accession
#GU073380); Cry2Ah4 (Accession #KC156702); Cry2Ai1 (Accession
#FJ788388); Cry2Aj (Accession #); Cry2Ak1 (Accession #KC156660);
Cry2Ba1 (Accession #KC156658); Cry3Aa1 (Accession #AAA22336);
Cry3Aa2 (Accession #AAA22541); Cry3Aa3 (Accession #CAA68482);
Cry3Aa4 (Accession #AAA22542); Cry3Aa5 (Accession #AAA50255);
Cry3Aa6 (Accession #AAC43266); Cry3Aa7 (Accession #CAB41411);
Cry3Aa8 (Accession #AAS79487); Cry3Aa9 (Accession #AAW05659);
Cry3Aa10 (Accession #AAU29411); Cry3Aa11 (Accession #AAW82872);
Cry3Aa12 (Accession #ABY49136); Cry3Ba1 (Accession #CAA34983);
Cry3Ba2 (Accession #CAA00645); Cry3Ba3 (Accession #JQ397327);
Cry3Bb1 (Accession #AAA22334); Cry3Bb2 (Accession #AAA74198);
Cry3Bb3 (Accession #115475); Cry3Ca1 (Accession #CAA42469); Cry4Aa1
(Accession #CAA68485); Cry4Aa2 (Accession #BAAOO1 79); Cry4Aa3
(Accession #CAD30148); Cry4Aa4 (Accession #AFB18317); Cry4A-like
(Accession #AAY96321); Cry4Ba1 (Accession #CAA30312); Cry4Ba2
(Accession #CAA30114); Cry4Ba3 (Accession #AAA22337); Cry4Ba4
(Accession #BAAOO1 78); Cry4Ba5 (Accession #CAD30095); Cry4Ba-like
(Accession #ABC47686); Cry4Ca1 (Accession #EU646202); Cry4Cb1
(Accession #FJ403208); Cry4Cb2 (Accession #FJ597622); Cry4Cc1
(Accession #FJ403207); Cry5Aa1 (Accession #AAA67694); Cry5Ab1
(Accession #AAA67693); Cry5Ac1 (Accession #134543); Cry5Ad1
(Accession #ABQ82087); Cry5Ba1 (Accession #AAA68598); Cry5Ba2
(Accession #ABW88931); Cry5Ba3 (Accession #AFJ04417); Cry5Ca1
(Accession #HM461869); Cry5Ca2 (Accession #ZP_04123426); Cry5Da1
(Accession #HM461870); Cry5Da2 (Accession #ZP_04123980); Cry5Ea1
(Accession #HM485580); Cry5Ea2 (Accession #ZP_04124038); Cry6Aa1
(Accession #AAA22357); Cry6Aa2 (Accession #AAM46849); Cry6Aa3
(Accession #ABH03377); Cry6Ba1 (Accession #AAA22358); Cry7 Aa1
(Accession #AAA22351); Cry7Ab1 (Accession #AAA21120); Cry7Ab2
(Accession #AAA21121); Cry7Ab3 (Accession #ABX24522); Cry7 Ab4
(Accession #EU380678); Cry7 Ab5 (Accession #ABX79555); Cry7 Ab6
(Accession #ACI44005); Cry7 Ab7 (Accession #ADB89216); Cry7 Ab8
(Accession #GU145299); Cry7Ab9 (Accession #ADD92572); Cry7Ba1
(Accession #ABB70817); Cry7Bb1 (Accession #KC156653); Cry7Ca1
(Accession #ABR67863); Cry7Cb1 (Accession #KC156698); Cry7Da1
(Accession #ACQ99547); Cry7Da2 (Accession #HM572236); Cry7Da3
(Accession #KC156679); Cry7Ea1 (Accession #HM035086); Cry7Ea2
(Accession #HM132124); Cry7Ea3 (Accession #EEM19403); Cry7Fa1
(Accession #HM035088); Cry7Fa2 (Accession #EEM19090); Cry7Fb1
(Accession #HM572235); Cry7Fb2 (Accession #KC156682); Cry7Ga1
(Accession #HM572237); Cry7Ga2 (Accession #KC156669); Cry7Gb1
(Accession #KC156650); Cry7Gc1 (Accession #KC156654); Cry7Gd1
(Accession #KC156697); Cry7Ha1 (Accession #KC156651); Cry7Ia1
(Accession #KC156665); Cry7Ja1 (Accession #KC156671); Cry7Ka1
(Accession #KC156680); Cry7Kb1 (Accession #BAM99306); Cry7La1
(Accession #BAM99307); Cry8Aa1 (Accession #AAA21117); Cry8Ab1
(Accession #EU044830); Cry8Ac1 (Accession #KC156662); Cry8Ad1
(Accession #KC156684); Cry8Ba1 (Accession #AAA21118); Cry8Bb1
(Accession #CAD57542); Cry8Bc1 (Accession #CAD57543); Cry8Ca1
(Accession #AAA21119); Cry8Ca2 (Accession #AAR98783); Cry8Ca3
(Accession #EU625349); Cry8Ca4 (Accession #ADB54826); Cry8Da1
(Accession #BAC07226); Cry8Da2 (Accession #BD133574); Cry8Da3
(Accession #BD133575); Cry8Db1 (Accession #BAF93483); Cry8Ea1
(Accession #AAQ73470); Cry8Ea2 (Accession #EU047597); Cry8Ea3
(Accession #KC855216); Cry8Fa1 (Accession #AAT48690); Cry8Fa2
(Accession #HQ174208); Cry8Fa3 (Accession #AFH78109); Cry8Ga1
(Accession #AAT46073); Cry8Ga2 (Accession #ABC42043); Cry8Ga3
(Accession #FJ198072); Cry8Ha1 (Accession #AAW81032); Cry8Ia1
(Accession #EU381044); Cry8Ia2 (Accession #GU073381); Cry8Ia3
(Accession #HM044664); Cry8Ia4 (Accession #KC156674); Cry8Ib1
(Accession #GU325772); Cry8Ib2 (Accession #KC156677); Cry8Ja1
(Accession #EU625348); Cry8Ka1 (Accession #FJ422558); Cry8Ka2
(Accession #ACN87262); Cry8Kb1 (Accession #HM123758); Cry8Kb2
(Accession #KC156675); Cry8La1 (Accession #GU325771); Cry8Ma1
(Accession #HM044665); Cry8Ma2 (Accession #EEM86551); Cry8Ma3
(Accession #HM210574); Cry8Na1 (Accession #HM640939); Cry8Pa1
(Accession #HQ388415); Cry8Qa1 (Accession #HQ441166); Cry8Qa2
(Accession #KC152468); Cry8Ra1 (Accession #AFP87548); Cry8Sa1
(Accession #JQ740599); Cry8Ta1 (Accession #KC156673); Cry8-like
(Accession #FJ770571); Cry8-like (Accession #ABS53003); Cry9Aa1
(Accession #CAA41122); Cry9Aa2 (Accession #CAA41425); Cry9Aa3
(Accession #GQ249293); Cry9Aa4 (Accession #GQ249294); Cry9Aa5
(Accession #JX1 74110); Cry9Aa like (Accession #AAQ52376); Cry9Ba1
(Accession #CAA52927); Cry9Ba2 (Accession #GU299522); Cry9Bb1
(Accession #AAV28716); Cry9Ca1 (Accession #CAA85764); Cry9Ca2
(Accession #AAQ52375); Cry9Da1 (Accession #BAA1 9948); Cry9Da2
(Accession #AAB97923); Cry9Da3 (Accession #GQ249293); Cry9Da4
(Accession #GQ249297); Cry9Db1 (Accession #AAX78439); Cry9Dc1
(Accession #KCl 56683); Cry9Ea1 (Accession #BAA34908); Cry9Ea2
(Accession #AA012908); Cry9Ea3 (Accession #ABM21765); Cry9Ea4
(Accession #ACE88267); Cry9Ea5 (Accession #ACF04743); Cry9Ea6
(Accession #ACG63872); Cry9Ea7 (Accession #FJ380927); Cry9Ea8
(Accession #GQ249292); Cry9Ea9 (Accession #JN651495); Cry9Eb1
(Accession #CAC50780); Cry9Eb2 (Accession #GQ249298); Cry9Eb3
(Accession #KC156646); Cry9Ec1 (Accession #AAC63366); Cry9Ed1
(Accession #AAX78440); Cry9Ee1 (Accession #GQ249296); Cry9Ee2
(Accession #KC156664); Cry9Fa1 (Accession #KC156692); Cry9Ga1
(Accession #KC156699); Cry9-like (Accession #AAC63366); Cry1OAa1
(Accession #AAA22614); Cry10Aa2 (Accession #E00614); Cry10Aa3
(Accession #CAD30098); Cry10Aa4 (Accession #AFB18318); Cry1OA-like
(Accession #DQ167578); Cry1 1Aa1 (Accession #AAA22352); Cry1 1Aa2
(Accession #AAA22611); Cry1 1Aa3 (Accession #CAD30081); Cry1 1Aa4
(Accession #AFB18319); Cry11Aa-like (Accession #DQ166531); Cry11Ba1
(Accession #CAA60504); Cry11Bb1 (Accession #AAC97162); Cry1 1Bb2
(Accession #HM068615); Cry12Aa1 (Accession #AAA22355); Cry13Aa1
(Accession #AAA22356); Cry14Aa1 (Accession #AAA21516); Cry14Ab1
(Accession #KC156652); Cry15Aa1 (Accession #AAA22333); Cry16Aa1
(Accession #CAA63860); Cry17Aa1 (Accession #CAA67841); Cry18Aa1
(Accession #CAA67506); Cry18Ba1 (Accession #AAF89667); Cry18Ca1
(Accession #AAF89668); Cry19Aa1 (Accession #CAA68875); Cry19Ba1
(Accession #BAA32397); Cry19Ca1 (Accession #AFM37572); Cry20Aa1
(Accession #AAB93476); Cry20Ba1 (Accession #ACS93601); Cry20Ba2
(Accession #KC156694); Cry20-like (Accession #GQ144333); Cry21Aa1
(Accession #132932); Cry21Aa2 (Accession #166477); Cry21Ba1
(Accession #BAC06484); Cry21Ca1 (Accession #JF521577); Cry21Ca2
(Accession #KC156687); Cry21Da1 (Accession #JF521578); Cry22Aa1
(Accession #134547); Cry22Aa2 (Accession #CAD43579); Cry22Aa3
(Accession #ACD93211); Cry22Ab1 (Accession #AAK50456); Cry22Ab2
(Accession #CAD43577); Cry22Ba1 (Accession #CAD43578); Cry22Bb1
(Accession #KC156672); Cry23Aa1 (Accession #AAF76375); Cry24Aa1
(Accession #AAC61891); Cry24Ba1 (Accession #BAD32657); Cry24Ca1
(Accession #CAJ43600); Cry25Aa1 (Accession #AAC61892); Cry26Aa1
(Accession #AAD25075); Cry27Aa1 (Accession #BAA82796); Cry28Aa1
(Accession
#AAD24189); Cry28Aa2 (Accession #AAG00235); Cry29Aa1 (Accession
#CAC80985); Cry30Aa1 (Accession #CAC80986); Cry30Ba1 (Accession
#BAD00052); Cry30Ca1 (Accession #BAD67157); Cry30Ca2 (Accession
#ACU24781); Cry30Da1 (Accession #EF095955); Cry30Db1 (Accession
#BAE80088); Cry30Ea1 (Accession #ACC95445); Cry30Ea2 (Accession
#FJ499389); Cry30Fa1 (Accession #ACI22625); Cry30Ga1 (Accession
#ACG60020); Cry30Ga2 (Accession #HQ638217); Cry31Aa1 (Accession
#BAB11 757); Cry31Aa2 (Accession #AAL87458); Cry31Aa3 (Accession
#BAE79808); Cry31Aa4 (Accession #BAF32571); Cry31Aa5 (Accession
#BAF32572); Cry31Aa6 (Accession #BA144026); Cry31Ab1 (Accession
#BAE79809); Cry31Ab2 (Accession #BAF32570); Cry31Ac1 (Accession
#BAF34368); Cry31Ac2 (Accession #AB731600); Cry31Ad1 (Accession
#BA144022); Cry32Aa1 (Accession #AAG36711); Cry32Aa2 (Accession
#GU063849); Cry32Ab1 (Accession #GU063850); Cry32Ba1 (Accession
#BAB78601); Cry32Ca1 (Accession #BAB78602); Cry32Cb1 (Accession
#KC156708); Cry32Da1 (Accession #BAB78603); Cry32Ea1 (Accession
#GU324274); Cry32Ea2 (Accession #KC156686); Cry32Eb1 (Accession
#KC156663); Cry32Fa1 (Accession #KC156656); Cry32Ga1 (Accession
#KC156657); Cry32Ha1 (Accession #KC156661); Cry32Hb1 (Accession
#KC156666); Cry32Ia1 (Accession #KCl 56667); Cry32Ja1 (Accession
#KCl 56685); Cry32Ka1 (Accession #KCl 56688); Cry32La1 (Accession
#KC156689); Cry32Ma1 (Accession #KC156690); Cry32Mb1 (Accession
#KC156704); Cry32Na1 (Accession #KC156691); Cry32Oa1 (Accession
#KC156703); Cry32Pa1 (Accession #KC156705); Cry32Qa1 (Accession
#KC156706); Cry32Ra1 (Accession #KC156707); Cry32Sa1 (Accession
#KC156709); Cry32Ta1 (Accession #KC156710); Cry32Ua1 (Accession
#KC156655); Cry33Aa1 (Accession #AAL26871); Cry34Aa1 (Accession
#AAG50341); Cry34Aa2 (Accession #AAK64560); Cry34Aa3 (Accession
#AAT29032); Cry34Aa4 (Accession #AAT29030); Cry34Ab1 (Accession
#AAG41671); Cry34Ac1 (Accession #AAG50118); Cry34Ac2 (Accession
#AAK64562); Cry34Ac3 (Accession #AAT29029); Cry34Ba1 (Accession
#AAK64565); Cry34Ba2 (Accession #AAT29033); Cry34Ba3 (Accession
#AAT29031); Cry35Aa1 (Accession #AAG50342); Cry35Aa2 (Accession
#AAK64561); Cry35Aa3 (Accession #AAT29028); Cry35Aa4 (Accession
#AAT29025); Cry35Ab1 (Accession #AAG41672); Cry35Ab2 (Accession
#AAK64563); Cry35Ab3 (Accession #AY536891); Cry35Ac1 (Accession
#AAG50117); Cry35Ba1 (Accession #AAK64566); Cry35Ba2 (Accession
#AAT29027); Cry35Ba3 (Accession #AAT29026); Cry36Aa1 (Accession
#AAK64558); Cry37 Aa1 (Accession #AAF76376); Cry38Aa1 (Accession
#AAK64559); Cry39Aa1 (Accession #BAB72016); Cry40Aa1 (Accession
#BAB72018); Cry40Ba1 (Accession #BAC77648); Cry40Ca1 (Accession
#EU381045); Cry40Da1 (Accession #ACF15199); Cry41Aa1 (Accession
#BAD35157); Cry41Ab1 (Accession #BAD35163); Cry41Ba1 (Accession
#HM461871); Cry41Ba2 (Accession #ZP_04099652); Cry42Aa1 (Accession
#BAD35166); Cry43Aa1 (Accession #BAD15301); Cry43Aa2 (Accession
#BAD95474); Cry43Ba1 (Accession #BAD15303); Cry43Ca1 (Accession
#KC156676); Cry43Cb1 (Accession #KC156695); Cry43Cc1 (Accession
#KC156696); Cry43-like (Accession #BAD15305); Cry44Aa (Accession
#BAD08532); Cry45Aa (Accession #BAD22577); Cry46Aa (Accession
#BAC79010); Cry46Aa2 (Accession #BAG68906); Cry46Ab (Accession
#BAD35170); Cry47 Aa (Accession #AAY24695); Cry48Aa (Accession
#CAJ18351); Cry48Aa2 (Accession #CAJ86545); Cry48Aa3 (Accession
#CAJ86546); Cry48Ab (Accession #CAJ86548); Cry48Ab2 (Accession
#CAJ86549); Cry49Aa (Accession #CAH56541); Cry49Aa2 (Accession
#CAJ86541); Cry49Aa3 (Accession #CAJ86543); Cry49Aa4 (Accession
#CAJ86544); Cry49Ab1 (Accession #CAJ86542); Cry50Aa1 (Accession
#BAE86999); Cry50Ba1 (Accession #GU446675); Cry50Ba2 (Accession
#GU446676); Cry51Aa1 (Accession #AB114444); Cry51Aa2 (Accession
#GU570697); Cry52Aa1 (Accession #EF613489); Cry52Ba1 (Accession
#FJ361760); Cry53Aa1 (Accession #EF633476); Cry53Ab1 (Accession
#FJ361759); Cry54Aa1 (Accession #ACA52194); Cry54Aa2 (Accession
#GQ140349); Cry54Ba1 (Accession #GU446677); Cry55Aa1 (Accession
#ABW88932); Cry54Ab1 (Accession #JQ916908); Cry55Aa2 (Accession
#AAE33526); Cry56Aa1 (Accession #ACU57499); Cry56Aa2 (Accession
#GQ483512); Cry56Aa3 (Accession #JX025567); Cry57Aa1 (Accession
#ANC87261); Cry58Aa1 (Accession #ANC87260); Cry59Ba1 (Accession
#JN790647); Cry59Aa1 (Accession #ACR43758); Cry60Aa1 (Accession
#ACU24782); Cry60Aa2 (Accession #EA057254); Cry60Aa3 (Accession
#EEM99278); Cry60Ba1 (Accession #GU810818); Cry60Ba2 (Accession
#EA057253); Cry60Ba3 (Accession #EEM99279); Cry61Aa1 (Accession
#HM035087); Cry61Aa2 (Accession #HM132125); Cry61Aa3 (Accession
#EEM19308); Cry62Aa1 (Accession #HM054509); Cry63Aa1 (Accession
#BA144028); Cry64Aa1 (Accession #BAJ05397); Cry65Aa1 (Accession
#HM461868); Cry65Aa2 (Accession #ZP_04123838); Cry66Aa1 (Accession
#HM485581); Cry66Aa2 (Accession #ZP_04099945); Cry67Aa1 (Accession
#HM485582); Cry67Aa2 (Accession #ZP_04148882); Cry68Aa1 (Accession
#HQ113114); Cry69Aa1 (Accession #HQ401006); Cry69Aa2 (Accession
#JQ821388); Cry69Ab1 (Accession #JN209957); Cry70Aa1 (Accession
#JN646781); Cry70Ba1 (Accession #AD051070); Cry70Bb1 (Accession
#EEL67276); Cry71Aa1 (Accession #JX025568); Cry72Aa1 (Accession
#JX025569); Cyt1Aa (GenBank Accession Number X03182); Cyt1Ab
(GenBank Accession Number X98793); Cyt1B (GenBank Accession Number
U37196); Cyt2A (GenBank Accession Number Z14147); and Cyt2B
(GenBank Accession Number U52043).
[0366] Examples of .delta.-endotoxins also include but are not
limited to Cry1A proteins of U.S. Pat. Nos. 5,880,275, 7,858,849
8,530,411, 8,575,433, and 8,686,233; a DIG-3 or DIG-11 toxin
(N-terminal deletion of a-helix 1 and/or a-helix 2 variants of cry
proteins such as Cry1A, Cry3A) of U.S. Pat. Nos. 8,304,604,
8,304,605 and 8,476,226; Cry1B of U.S. patent application Ser. No.
10/525,318; Cry1C of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat.
Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras of U.S. Pat. Nos.
7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Ab
protein of U.S. Pat. No. 7,064,249); a Cry3A protein including but
not limited to an engineered hybrid insecticidal protein (eHIP)
created by fusing unique combinations of variable regions and
conserved blocks of at least two different Cry proteins (US Patent
Application Publication Number 2010/0017914); a Cry4 protein; a
Cry5 protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos.
7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499
and 7,462,760; a Cry9 protein such as such as members of the Cry9A,
Cry9B, Cry9C, Cry9D, Cry9E and Cry9F families, including but not
limited to the Cry9D protein of U.S. Pat. No. 8,802,933 and the
Cry9B protein of U.S. Pat. No. 8,802,934; a Cry15 protein of
Naimov, et al., (2008), "Applied and Environmental Microbiology,"
74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat. Nos.
6,127,180, 6,624,145 and 6,340,593; a CryET33 and cryET34 protein
of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626,
7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US
Patent Publication Number 2006/0191034, 2012/0278954, and PCT
Publication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat.
Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51
protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of
US Patent Application Publication Number 2008/0295207; ET29, ET37,
TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006/033867;
TIC853 toxins of U.S. Pat. No. 8,513,494, AXMI-027, AXMI-036, and
AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039, AXMI-040,
AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018, AXMI-020 and
AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of
WO 2005/021585; AXMI-008 of US Patent Application Publication
Number 2004/0250311; AXMI-006 of US Patent Application Publication
Number 2004/0216186; AXMI-007 of US Patent Applica-tion Publication
Number 2004/0210965; AXMI-009 of US Patent Application Number
2004/0210964; AXMI-014 of US Patent Application Publication Number
2004/0197917; AXMI-004 of US Patent Application Publication Number
2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007,
AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO
2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US
Patent Application Publication Number 2011/0023184; AXMI-011,
AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037,
AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041,
AXMI-063 and AXMI-064 of US Patent Application Publication Number
2011/0263488; AXMI-R1 and related proteins of US Patent Application
Publication Number 2010/0197592; AXMI221Z, AXMI222z, AXMI223z,
AXMI224z and AXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220,
AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO
2011/103247 and U.S. Pat. No. 8,759,619; AXMI-115, AXMI-113,
AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;
AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US Patent
Application Publication Number 2010/0298211; AXMI-066 and AXMI-076
of US Patent Application Publication Number 2009/0144852; AXMI128,
AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143,
AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154,
AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166,
AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173,
AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180,
AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of
U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082,
AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100,
AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109,
AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118,
AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257,
AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183,
AXMI132, AXMI138, AXMI137 of US Patent Application Publication
Number 2010/0005543, AXMI270 of US Patent Application Publication
US20140223598, AXMI279 of US Patent Application Publication
US20140223599, cry proteins such as Cry1A and Cry3A having modified
proteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aa and
Cry1 Ca toxin protein from Bacillus thuringiensis strain VBTS 2528
of US Patent Application Publication Number 2011/0064710. Other Cry
proteins are well known to one skilled in the art. See, N.
Crickmore, et al., "Revision of the Nomenclature for the Bacillus
thuringiensis Pesticidal Crystal Proteins," Microbiology and
Molecular Biology Reviews," (1998) Vol 62: 807-813; see also, N.
Crickmore, et al., "Bacillus thuringiensis toxin nomenclature"
(2016), at www.btnomenclature.info/.
[0367] The use of Cry proteins as transgenic plant traits is well
known to one skilled in the art and Cry-transgenic plants including
but not limited to plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab,
Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A,
Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have
received regulatory approval. See, Sanahuja et al., "Bacillus
thuringiensis: a century of research, development and commercial
applications," (2011) Plant Biotech Journal, April 9(3):283-300 and
the CERA (2010) GM Crop Database Center for Environmental Risk
Assessment (CERA), ILSI Research Foundation, Washington D.C. at
cera-gmc.org/index.php?action=gm_crop_database, which can be
accessed on the world-wide web using the "www" prefix). More than
one pesticidal proteins well known to one skilled in the art can
also be expressed in plants such as Vip3Ab & Cry1Fa
(US2012/0317682); Cry1BE & Cry1F (US2012/0311746); Cry1CA &
Cry1AB (US2012/0311745); Cry1F & CryCa (US2012/0317681);
Cry1DA& Cry1BE (US2012/0331590); Cry1DA & Cry1Fa
(US2012/0331589); Cry1AB & Cry1BE (US2012/0324606); Cry1Fa
& Cry2Aa and Cry11 & Cry1E (US2012/0324605); Cry34Ab/35Ab
and Cry6Aa (US20130167269); Cry34Ab/VCry35Ab & Cry3Aa
(US20130167268); Cry1Ab & Cry1F (US20140182018); and Cry3A and
Cry1Ab or Vip3Aa (US20130116170). Pesticidal proteins also include
insecticidal lipases including lipid acyl hydrolases of U.S. Pat.
No. 7,491,869, and cholesterol oxidases such as from Streptomyces
(Purcell et al. (1993) Biochem Biophys Res Commun
15:1406-1413).
[0368] Pesticidal proteins also include VIP (vegetative
insecticidal proteins) toxins. Entomopathogenic bacteria produce
insecticidal proteins that accumulate in inclusion bodies or
parasporal crystals (such as the aforementioned Cry and Cyt
proteins), as well as insecticidal proteins that are secreted into
the culture medium. Among the latter are the Vip proteins, which
are divided into four families according to their amino acid
identity. The Vip1 and Vip2 proteins act as binary toxins and are
toxic to some members of the Coleoptera and Hemiptera. The Vip1
component is thought to bind to receptors in the membrane of the
insect midgut, and the Vip2 component enters the cell, where it
displays its ADP-ribosyltransferase activity against actin,
preventing microfilament formation. Vip3 has no sequence similarity
to Vip1 or Vip2 and is toxic to a wide variety of members of the
Lepidoptera. Its mode of action has been shown to resemble that of
the Cry proteins in terms of proteolytic activation, binding to the
midgut epithelial membrane, and pore formation, although Vip3A
proteins do not share binding sites with Cry proteins. The latter
property makes them good candidates to be combined with Cry
proteins in transgenic plants (Bacillus thuringiensis-treated crops
[Bt crops]) to prevent or delay insect resistance and to broaden
the insecticidal spectrum. There are commercially grown varieties
of Bt cotton and Bt maize that express the Vip3Aa protein in
combination with Cry proteins. For the most recently reported Vip4
family, no target insects have been found yet. See, Chakroun et
al., "Bacterial Vegetative Insecticidal Proteins (Vip) from
Entomopathogenic Bacteria," Microbiol Mol Biol Rev. 2016 Mar. 2;
80(2):329-50. VIPs can be found in U.S. Pat. Nos. 5,877,012,
6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020 and the
like. Other VIP proteins are well known to one skilled in the art
(see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html, which
can be accessed on the world-wide web using the "www" prefix).
[0369] Pesticidal proteins also include toxin complex (TC)
proteins, obtainable from organisms such as Xenorhabdus,
Photorhabdus and Paenibacillus (see, U.S. Pat. Nos. 7,491,698 and
8,084,418). Some TC proteins have "stand alone" insecticidal
activity and other TC proteins enhance the activity of the
stand-alone toxins produced by the same given organism. The
toxicity of a "stand-alone" TC protein (from Photorhabdus,
Xenorhabdus or Paenibacillus, for example) can be enhanced by one
or more TC protein "potentiators" derived from a source organism of
a different genus. There are three main types of TC proteins. As
referred to herein, Class A proteins ("Protein A") are stand-alone
toxins. Class B proteins ("Protein B") and Class C proteins
("Protein C") enhance the toxicity of Class A proteins. Examples of
Class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of Class
B proteins are TcaC, TcdB, XptB1Xb and XptC1 Wi. Examples of Class
C proteins are TccC, XptC1Xb and XptB1 Wi. Pesticidal proteins also
include spider, snake and scorpion venom proteins. Examples of
spider venom peptides include, but are not limited to lycotoxin-1
peptides and mutants thereof (U.S. Pat. No. 8,334,366).
[0370] Some currently registered PIPs are listed in Table 11.
Transgenic plants have also been engineered to express dsRNA
directed against insect genes (Baum, J. A. et al. (2007) Control of
coleopteran insect pests through RNA interference. Nature
Biotechnology 25: 1322-1326; Mao, Y. B. et al. (2007) Silencing a
cotton bollworm P450 monooxygenase gene by plant-mediated RNAi
impairs larval tolerance of gossypol. Nature Biotechnology 25:
1307-1313). RNA interference can be triggered in the pest by
feeding of the pest on the transgenic plant. Pest feeding thus
causes injury or death to the pest.
TABLE-US-00011 TABLE 11 List of exemplary Plant-incorporated
Protectants, which can be combined with microbes of the disclosure
Plant-Incorporated Company and Trade Pesticide Registration
Protectants (PIPs) Names Numbers Potato Potato Cry3A Potato PC Code
006432 Naturemark 524-474 New Leaf Monsanto Cry3A & PLRV Potato
Monsanto 524-498 PC Codes 006432, 006469 New Leaf Plus Corn Cry1Ab
Corn Event 176 PC Code 006458 Mycogen Seeds/Dow 68467-1 Agro
66736-1 Syngenta Seeds Cry1Ab Corn Event Bt11 EPA PC Code Agrisure
CB (with 67979-1 006444 OECD Unique Identifier SYN- Yieldgard)
65268-1 BTO11-1, Attribute Insect Protected Sweet Corn Syngenta
Seeds Cry1Ab Corn Event MON 801 Monsanto 524-492 Cry1Ab corn Event
MON 810 PC Code Monsanto 524-489 006430 OECD Unique Identifier MON-
OO81O-6 Cry1Ac Corn PC Code 006463 Dekalb Genetics c/o 69575-2
Monsanto BT-XTRA Cry1F corn Event TC1507 PC Code Mycogen Seeds/Dow
68467-2 006481 OECD Unique Identifier DAS- Agro 29964-3 O15O7-1
Pioneer Hi- Bred/Dupont moCry1F corn Event DAS-O6275-8 PC Mycogen
Seeds/Dow 68467-4 Code 006491 OECD Unique Identifier Agro
DAS-O6275-8 Cry9C Corn Aventis 264-669 StarLink Cry3Bb1 corn Event
MON863 PC Code Monsanto 524-528 006484 YielGard RW OECD Unique
Identifier MON-OO863-5 Cry3Bb1 corn Event MON 88017 PC Code
Monsanto 524-551 006498 YieldGrad VT OECD Unique Identifier
MON-88O17-3 Rootworm Cry34Ab1/Cry35Ab1 corn Event DAS- Mycogen
Seeds/Dow 68467-5 591227-7 Agro 29964-4 PC Code 006490 Pioneer Hi-
OECD Unique Identifier DAS-59122-7 Bred/Dupont Herculex Rootworm
Cry34Ab1/Cry35Ab1 and Cry1F corn Pioneer Hi- 29964-17 Event 4114
Bred/Dupont PC Codes 006555, 006556 mCry3A corn Event MIR 604
Syngenta Seeds 67979-5 PC Code 006509 OECD Unique Identifier
Agrisure RW SYN-IR604-8 Cry1A.105 and Cry2Ab2 corn Event MON
Monsanto 524-575 89034 PC Codes 006515 and 006514 Genuity VT Double
Pro Vip3Aa20 corn Event MIR 162 Syngenta Seeds 67979-14 PC Code
006599 OECD Unique Identifier Agrisure Viptera SYN-IR162-4
eCry3.1Ab corn in Event 5307 PC Code Syngenta 67979-22 016483 OECD
Unique Identifier SYN- 53 7-1 Stacked Events and Seed Blend Corn
MON863 .times. MON810 with Cry3Bb1 + Monsanto YieldGard 524-545
Cry1Ab Plus DAS-59122-7 .times. TC1507 with Mycogen Seeds/Dow
68467-6 Cry34Ab1/Cry35Ab1 + Cry1F Agro Pioneer Hi- 29964-5
Bred/Dupont Herculex Xtra MON 88017 .times. MON 810 with Cry1AB +
Monsanto 524-552 Cry3Bb YieldGard VT Triple YieldGard VT Plus MIR
604 .times. Bt11 with mCry3A + Cry1Ab Syngenta 67979-8 Agrisure
CB/RW Agrisure 3000GT Mon 89034 .times. Mon 88017 with Cry1A.105 +
Monsanto 524-576 Cry2Ab2 + Cry3Bb1 Genuity VT Triple PRO Bt11
.times. MIR 162 with Cry1Ab + Vip3Aa 20 Syngenta Seeds 67979-12
Agrisure 2100 Bt11 .times. MIR 162 .times. MIR 604 with Cry1Ab +
Syngenta Seeds 67979-13 Vip3Aa20 + mCry3A Agrisure 3100 MON 89034
.times. TC1507 .times. MON 88017 .times. Monsanto Company 524-581
DAS-59122-7 with Cry1A.105 + Cry2Ab2 + Mycogen Seeds/Dow 68467-7
Cry1F + Cry3Bb1 + Agro Cry34Ab1/Cry35Ab1 Genuity SmartStax
SmartStax MON 89034 .times. TC1507 x MON 88017 .times. Monsanto
Company 524-595 DAS-59122-7 Seed Blend Mycogen Seeds/Dow 68467-16
Agro Genuity SmartStax RIB Complete SmartStax Refuge Advanced;
Refuge Advanced Powered by SmartStax Seed Blend of Herculex Xtra +
Herculex I Pioneer Hi- 29964-6 Bred/Dupont Optimum AcreMax1 Insect
Protection Seed Blend of Herculex RW + Non-Bt corn Pioneer Hi-
29964-10 Bred/Dupont Optimum AcreMax RW (Cry1F .times. Cry34/35
.times. Cry1Ab) - seed blend Pioneer Hi- 29964-11 Bred/Dupont
Optimum AcreMax Xtra (Cry1F .times. Cry1Ab) - seed blend Pioneer
Hi- 29964-12 Bred/Dupont Optimum AcreMax Insect Protection (Cry1F
.times. mCry3A) Pioneer Hi- 29964-13 Bred/Dupont Optimum Trisect
(Cry1F .times. Cry34/35 .times. Cry1Ab .times. mCry3A) Pioneer Hi-
29964-14 Bred/Dupont Optimum Intrasect Xtreme 59122 .times. MON 810
.times. MIR 604 (Cry34/35 .times. Pioneer Hi- 29964-15 Cry1Ab
.times. mCry3A) Bred/Dupont Optimum AcreMax Xtreme (Cry1F .times.
Pioneer Hi- 29964-16 Cry34/35 .times. Cry1Ab .times. mCry3A) - seed
Bred/Dupont blend Optimum AcreMax Xtreme (seed blend) MON 810
.times. MIR 604 (Cry1Ab .times. mCry3A) Pioneer Hi- 29964-18
Bred/Dupont 1507 .times. MON810 .times. MIR 162 (Cry1F .times.
Pioneer Hi- 29964-19 Cry1Ab .times. Vip 3Aa20) Bred/Dupont Optimum
Intrasect Leptra 1507 .times. MIR 162 (Cry1F .times. Vip30Aa20)
Pioneer Hi- 29964-20 Bred/Dupont 4114 .times. MON 810 .times. MIR
604 (Cry34/35 .times. Pioneer Hi- 29964-21 Cry1F .times. Cry1Ab
.times. mCry3A) - seed blend Bred/Dupont 4114 .times. MON 810
.times. MIR 604 (Cry34/35 .times. Pioneer Hi- 29964-22 Cry1F
.times. Cry1Ab .times. mCry3A) Bred/Dupont 1507 .times. MON810
.times. MIR 604 (Cry1F .times. Pioneer Hi- 29964-23 Cry1Ab .times.
mCry3A) - seed blend Bred/Dupont Optimum AcreMax Trisect 1507
.times. MON810 .times. MIR 604 (Cry1F .times. Pioneer Hi- 29964-24
Cry1Ab .times. mCry3A) Bred/Dupont Optimum Intrasect Trisect 4114
.times. MON 810 (Cry34/35 .times. Cry1F .times. Pioneer Hi-
29964-25 Cry1Ab) Bred/Dupont 1507 .times. MON810 .times. MIR 162
(Cry1F .times. Pioneer Hi- 29964-26 Cry1Ab .times. Vip 3Aa20) -
seed blend Bred/Dupont Optimum AcreMax Leptra SmartStax
Intermediates (8 products) Monsanto 524-583, 524-584, 524- 586,
524-587, 524- 588, 524-589, 524-590 MON 89034 .times. 1507
(Cry1A.105 .times. Monsanto 524-585 Cry2Ab2 .times. Cry1F) Genuity
Power-Core MON 89034 (Cry1A.105 .times. Cry2Ab2) - Monsanto 524-597
seed blend Genuity VT Double PRO RIB Complete MON 89034 .times.
88017 RIB Complete Monsanto 524-606 (Cry1A.105 .times. Cry2Ab2
.times. Cry3Bb1) - seed Genuity VT Triple PRO blend RIB Complete
MON 89034 .times. 1507 (Cry1A.105 .times. Monsanto 524-612 Cry2Ab2
.times. Cry1F) - seed blend Genuity PowerCore RIB Complete Bt11
.times. MIR162 .times. 1507 (Cry1Ab .times. Syngenta Seeds 67979-15
Vip3Aa20 .times. Cry1F) Agrisure Viptera 3220 Refuge Renew Bt11
.times. 59122-7 .times. MIR 604 .times. 1507 (Cry1Ab .times.
Syngenta Seeds 67979-17 Cry34/35 .times. mCry3A .times. Cry1F)
Agrisure 3122 Bt11 .times. MIR162 .times. TC1507 (Cry1Ab .times.
Syngenta Seeds 67979-19 Vip3Aa20 .times. Cry1F) - seed blend
Agisure Viptera 3220 (E-Z Refuge) (Refuge Advanced) Bt11 .times.
DAS 59122-7 .times. MIR604 .times. TC1507 Syngenta Seeds 67979-20
(Cry1Ab .times. Cry34/35 .times. mCry3A .times. Cry1F) - Agisure
Viptera 3122 seed blend (E-Z Refuge) (Refuge Advanced) Bt11 .times.
MIR 162 .times. MIR 604 .times. TC1507 .times. Syngenta Seeds
67979-23 5307 (Cry1Ab .times. Vip3Aa20 .times. mCry3A .times.
Agrisure Duracade Cry1F .times. eCry3.1Ab) (Refuge Renew) 5222 Bt11
.times. MIR 604 .times. TC1507 .times. 5307 (Cry1Ab .times.
Syngenta Seeds 67979-24 mCry3A .times. Cry1F .times. eCry3.1Ab)
Agrisure Duracade (Refuge Renew) 5122 Bt11 .times. MIR 604 .times.
TC1507 .times. 5307 (Cry1Ab .times. Syngenta Seeds 67979-25 mCry3A
.times. Cry1F .times. eCry3.1Ab) - seed Agisure Duracade 5122 blend
E-Z Refuge Bt11 .times. MIR 162 .times. MIR 604 .times. TC1507
.times. Syngenta Seeds 67979-26 5307 (Cry1Ab .times. Vip3Aa20
.times. mCry3A .times. Agisure Duracade 5222 Cry1F .times.
eCry3.1Ab) - seed blend E-Z Refuge Bt11 .times. MIR 162 .times. MIR
604 .times. TC1507 .times. Syngenta Seeds 67979-27 5307 (Cry1Ab
.times. Vip3Aa20 x mCry3A .times. Agrisure Duracade Cry1F .times.
eCry3.1Ab) (Refuge Renew) 5022 MIR604 .times. DAS-59122-7 .times.
TC1507 Syngenta Seeds 67979-29 (mCry3A .times. Cry34/35 x Cry1F)
SmartStax Intermediates (8 products) Mycogen Seeds/Dow 68467-8,
68467-9, Agro 68467-10, 68467-11, 68467-13, 68467-14, 68467-15 MON
89034 .times. 1507 (Cry1A.105 .times. Mycogen Seeds/Dow 68467-12
Cry2Ab2 .times. Cry1F) Agro PowerCore; PowerCore Enlist MON 89034
.times. 1507 (Cry1A.105 .times. Mycogen Seeds/Dow 68467-21 Cry2Ab2
.times. Cry1F) - seed blend Agro PowerCore Refuge Advanced; Refuge
Advanced Powered by PowerCore 1507 .times. MON 810 Pioneer Hi-
29964-7 Bred/Dupont Optimum Intrasect 59122 .times. 1507 .times.
MON 810 Pioneer Hi- 29964-8 Bred/Dupont 59122 .times. MON 810
Pioneer Hi- 29964-9 Bred/Dupont Cotton Cry1Ac Cotton Monsanto
524-478 BollGard Cry1Ac and Cry2Ab2 in Event 15985 Monsanto 524-522
Cotton PC Codes 006445, 006487 BollGard II Bt cotton Event MON531
with Cry1Ac Monsanto 524-555 (breeding nursery use only) Bt cotton
Event MON15947 with Cry2Ab2 Monsanto 524-556 (breeding nursery use
only) COT102 .times. MON 15985 (Vip3Aa19 .times. Monsanto 524-613
Cry1Ac .times. Cry2Ab2) Bollgard III Cry1F and Cry1Ac (Events
DAS-21023-5 .times. Mycogen Seeds/Dow 68467-3 DAS-24236-5) Cotton
PC Codes 006512, Agro 006513 Widestrike Event 3006-210-23 (Cry1Ac)
Mycogen Seeds/Dow 68467-17 Agro Event 281-24-236 (Cry1F) Mycogen
Seeds/Dow 68467-18 Agro WideStrike .times. COT102 (Cry1F .times.
Cry1Ac .times. Mycogen Seeds/Dow 68467-19
Vip3Aa19) Agro WideStrike 3 Vip3Aa19 and FLCry1Ab (Events Syngenta
Seeds 67979-9 Cot102 .times. Cot67B) Cotton PC Codes 016484,
(Formally VipCot) 016486 OECD Unique Identifier SYN- IR102-7 X
SYN-IR67B-1 COT102 (Vip3Aa19) Syngenta Seeds 67979-18 COT67B
(FLCry1Ab) Syngenta Seeds 67979-21 T304-40 (Cry1Ab) Bayer
CropScience 264-1094 GHB119 (Cry2Ae) Bayer CropScience 264-1095
T304-40 .times. GHB119 (Cry1Ab .times. Cry2Ae) Bayer CropScience
264-1096 OECD Unique Identifier: BCS-GHOO4-7 .times. TwinLink
BCS-GHOO5-8 Soybean Cry1Ac in Event 87701 Soybean PC Code Monsanto
524-594 006532 OECD Unique Identifier Inacta Cry1A.105 and Cry2Ab2
in Event 87751 Monsanto 524-619 Soybean PC Codes 006614, 006615
OECD Unique Identifier MON-87751-7 Cry1Ac .times. Cry1F in Event
DAS 81419 Mycogen Seeds/Dow 68467-20 Soybean PC Codes 006527,
006528 OECD Agro Unique Identifier DAS 81419 (Cry1Ac .times.
Cry1F)
[0371] In some embodiments, any one or more of the pesticides set
forth herein may be utilized with any one or more of the microbes
of the disclosure and can be applied to plants or parts thereof,
including seeds.
Herbicides
[0372] As aforementioned, agricultural compositions of the
disclosure, which may comprise any microbe taught herein, are
sometimes combined with one or more herbicides.
[0373] Compositions comprising bacteria or bacterial populations
produced according to methods described herein and/or having
characteristics as described herein may further include one or more
herbicides. In some embodiments, herbicidal compositions are
applied to the plants and/or plant parts. In some embodiments,
herbicidal compositions may be included in the compositions set
forth herein, and can be applied to a plant(s) or a part(s) thereof
simultaneously or in succession, with other compounds.
[0374] Herbicides include 2,4-D, 2,4-DB, acetochlor, acifluorfen,
alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron,
bensulide, bentazon, bicyclopyrone, bromacil, bromoxynil, butylate,
carfentrazone, chlorimuron, chlorsulfuron, clethodim, clomazone,
clopyralid, cloransulam, cycloate, DCPA, desmedipham, dicamba,
dichlobenil, diclofop, diclosulam, diflufenzopyr, dimethenamid,
diquat, diuron, DSMA, endothall, EPTC, ethalfluralin, ethofumesate,
fenoxaprop, fluazifop-P, flucarbzone, flufenacet, flumetsulam,
flumiclorac, flumioxazin, fluometuron, fluroxypyr, fomesafen,
foramsulfuron, glufosinate, glyphosate, halosulfuron, hexazinone,
imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr,
isoxaflutole, lactofen, linuron, MCPA, MCPB, mesotrione,
metolachlor-s, metribuzin, indaziflam, metsulfuron, molinate, MSMA,
napropamide, naptalam, nicosulfuron, norflurazon, oryzalin,
oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin,
phenmedipham, picloram, primisulfuron, prodiamine, prometryn,
pronamide, propanil, prosulfuron, pyrazon, pyrithioac, quinclorac,
quizalofop, rimsulfuron, S-metolachlor, sethoxydim, siduron,
simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron,
tembotrione, terbacil, thiazopyr, thifensulfuron, thiobencarb,
topramezone, tralkoxydim, triallate, triasulfuron, tribenuron,
triclopyr, trifluralin, and triflusulfuron.
[0375] In some embodiments, any one or more of the herbicides set
forth herein may be utilized with any one or more of the plants or
parts thereof set forth herein.
[0376] Herbicidal products may include CORVUS, BALANCE FLEXX,
CAPRENO, DIFLEXX, LIBERTY, LAUDIS, AUTUMN SUPER, and DIFLEXX
DUO.
[0377] In some embodiments, any one or more of the herbicides set
forth in the below Table 12 may be utilized with any one or more of
the microbes taught herein, and can be applied to any one or more
of the plants or parts thereof set forth herein.
TABLE-US-00012 TABLE 12 List of exemplary herbicides, which can be
combined with microbes of the disclosure Herbicide Site of Action
Group Number Chemical Family Herbicide ACCase 1 Cyclohexanediones
Sethoxydim (Poast, inhibitors Poast Plus) Clethodim (Select, Select
Max, Arrow) Aryloxyphenoxypropionates Fluazifop (Fusilade DX,
component in Fusion) Fenoxaprop (Puma, component in Fusion)
Quizalofop (Assure II, Targa) Phenylpyrazolins Pinoxaden (Axial XL)
ALS inhibitors 2 Imidazolinones Imazethapyr (Pursuit) Imazamox
(Raptor) Sulfonylureas Chlorimuron (Classic) Halosulfuron (Permit,
Sandea) Iodosulfuron (component in Autumn Super) Mesosulfuron
(Osprey) Nicosulfuron (Accent Q) Primisulfuron (Beacon) Prosulfuron
(Peak) Rimsulfuron (Matrix, Resolve) Thifensulfuron (Harmony)
Tribenuron (Express) Triflusulfuron (UpBeet) Triazolopyrimidine
Flumetsulam (Python) Cloransulam-methyl (FirstRate) Pyroxsulam
(PowerFlex HL) Florasulam (component in Quelex)
Sulfonylaminocarbonyltriazolinones Propoxycarbazone (Olympus)
Thiencarbazone-methyl (component in Capreno) Microtubule 3
Dinitroanilines Trifluralin (many inhibitors (root names)
inhibitors) Ethalfluralin (Sonalan) Pendimethalin (Prowl/Prowl
H.sub.2O) Benzamide Pronamide (Kerb) Synthetic auxins 4
Arylpicolinate Halauxifen (Elevore, component in Quelex) Phenoxy
acetic acids 2,4-D (Enlist One, others) 2,4-DB (Butyrac 200,
Butoxone 200) MCPA Benzoic acids Dicamba (Banvel, Clarity, DiFlexx,
Eugenia, XtendiMax; component in Status) Pyridines Clopyralid
(Stinger) Fluroxypyr (Starane Ultra) Photosystem II 5 Triazines
Atrazine inhibitors Simazine (Princep, Sim- Trol) Triazinone
Metribuzin (Metribuzin, others) Hexazinone (Velpar)
Phenyl-carbamates Desmedipham (Betenex) Phenmedipham (component in
Betamix) Uracils Terbacil (Sinbar) 6 Benzothiadiazoles Bentazon
(Basagran, others) Nitriles Bromoxynil (Buctril, Moxy, others) 7
Phenylureas Linuron (Lorox, Linex) Lipid synthesis 8 Thiocarbamates
EPTC (Eptam) inhibitor EPSPS inhibitor 9 Organophosphorus
Glyphosate Glutamine 10 Organophosphorus Glufosinate (Liberty,
synthetase Rely) inhibitor Diterpene 13 Isoxazolidinone Clomazone
(Command) biosynthesis inhibitor (bleaching) Protoporphyrinogen 14
Diphenylether Acifluorfen (Ultra oxidase Blazer) inhibitors (PPO)
Fomesafen (Flexstar, Reflex) Lactofen (Cobra, Phoenix)
N-phenylphthalimide Flumiclorac (Resource) Flumioxazin (Valor,
Valor EZ, Rowel) Aryl triazolinone Sulfentrazone (Authority,
Spartan) Carfentrazone (Aim) Fluthiacet-methyl (Cadet) Pyrazoles
Pyraflufen-ethyl (Vida) Pyrimidinedione Saflufenacil (Sharpen)
Long-chain fatty 15 Acetamides Acetochlor (Harness, acid inhibitors
Surpass NXT, Breakfree NXT, Warrant) Dimethenamid-P (Outlook)
Metolachlor (Parallel) Pyroxasulfone (Zidua, Zidua SC)
s-metolachlor (Dual Magnum, Dual II Magnum, Cinch) Flufenacet
(Define) Specific site 16 Benzofuranes Ethofumesate (Nortron)
unknown Auxin transport 19 Semicarbazone diflufenzopyr inhibitor
(component in Status) Photosystem I 22 Bipyridiliums Paraquat
(Gramoxone, inhibitors Parazone) Diquat (Reglone) 4-HPPD 27
Isoxazole Isoxaflutole (Balance inhibitors Pyrazole Flexx)
(bleaching) Pyrazolone Pyrasulfotole Triketone (component in
Huskie) Topramezone (Armezon/Impact) Bicyclopyrone (component in
Acuron) Mesotrione (Callisto) Tembotrione (Laudis)
Fungicides
[0378] As aforementioned, agricultural compositions of the
disclosure, which may comprise any microbe taught herein, are
sometimes combined with one or more fungicides.
[0379] Compositions comprising bacteria or bacterial populations
produced according to methods described herein and/or having
characteristics as described herein may further include one or more
fungicides. In some embodiments, fungicidal compositions may be
included in the compositions set forth herein, and can be applied
to a plant(s) or a part(s) thereof simultaneously or in succession,
with other compounds. The fungicides include azoxystrobin, captan,
carboxin, ethaboxam, fludioxonil, mefenoxam, fludioxonil,
thiabendazole, thiabendaz, ipconazole, mancozeb, cyazofamid,
zoxamide, metalaxyl, PCNB, metaconazole, pyraclostrobin, Bacillus
subtilis strain QST 713, sedaxane, thiamethoxam, fludioxonil,
thiram, tolclofos-methyl, trifloxystrobin, Bacillus subtilis strain
MBI 600, pyraclostrobin, fluoxastrobin, Bacillus pumilus strain QST
2808, chlorothalonil, copper, flutriafol, fluxapyroxad, mancozeb,
gludioxonil, penthiopyrad, triazole, propiconaozole,
prothioconazole, tebuconazole, fluoxastrobin, pyraclostrobin,
picoxystrobin, qols, tetraconazole, trifloxystrobin, cyproconazole,
flutriafol, SDHI, EBDCs, sedaxane, MAXIM QUATTRO (gludioxonil,
mefenoxam, azoxystrobin, and thiabendaz), RAXIL (tebuconazole,
prothioconazole, metalaxyl, and ethoxylated tallow alkyl amines),
and benzovindiflupyr.
[0380] In some embodiments, any one or more of the fungicides set
forth herein may be utilized with any one or more of the plants or
parts thereof set forth herein.
Nematicides
[0381] As aforementioned, agricultural compositions of the
disclosure, which may comprise any microbe taught herein, are
sometimes combined with one or more nematicides.
[0382] Compositions comprising bacteria or bacterial populations
produced according to methods described herein and/or having
characteristics as described herein may further include one or more
nematicide. In some embodiments, nematicidal compositions may be
included in the compositions set forth herein, and can be applied
to a plant(s) or a part(s) thereof simultaneously or in succession,
with other compounds. The nematicides may be selected from D-D,
1,3-dichloropropene, ethylene dibromide,
1,2-dibromo-3-chloropropane, methyl bromide, chloropicrin, metam
sodium, dazomet, methylisothiocyanate, sodium tetrathiocarbonate,
aldicarb, aldoxycarb, carbofuran, oxamyl, ethoprop, fenamiphos,
cadusafos, fosthiazate, terbufos, fensulfothion, phorate, DiTera,
clandosan, sincocin, methyl iodide, propargyl bromide,
2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP), any one or
more of the avermectins, sodium azide, furfural, Bacillus firmus,
abamectrin, thiamethoxam, fludioxonil, clothiandin, salicylic acid,
and benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl
ester.
[0383] In some embodiments, any one or more of the nematicides set
forth herein may be utilized with any one or more of the plants or
parts thereof set forth herein.
[0384] In some embodiments, any one or more of the nematicides,
fungicides, herbicides, insecticides, and/or pesticides set forth
herein may be utilized with any one or more of the plants or parts
thereof set forth herein.
Fertilizers, Nitrogen Stabilizers, and Urease Inhibitors
[0385] As aforementioned, agricultural compositions of the
disclosure, which may comprise any microbe taught herein, are
sometimes combined with one or more of a: fertilizer, nitrogen
stabilizer, or urease inhibitor.
[0386] In some embodiments, fertilizers are used in combination
with the methods and bacteria of the present disclosure.
Fertilizers include anhydrous ammonia, urea, ammonium nitrate, and
urea-ammonium nitrate (UAN) compositions, among many others. In
some embodiments, pop-up fertilization and/or starter fertilization
is used in combination with the methods and bacteria of the present
disclosure.
[0387] In some embodiments, nitrogen stabilizers are used in
combination with the methods and bacteria of the present
disclosure. Nitrogen stabilizers include nitrapyrin,
2-chloro-6-(trichloromethyl) pyridine, N-SERVE 24, INSTINCT,
dicyandiamide (DCD).
[0388] In some embodiments, urease inhibitors are used in
combination with the methods and bacteria of the present
disclosure. Urease inhibitors include N-(n-butyl)-thiophosphoric
triamide (NBPT), AGROTAIN, AGROTAIN PLUS, and AGROTAIN PLUS SC.
Further, the disclosure contemplates utilization of AGROTAIN
ADVANCED 1.0, AGROTAIN DRI-MAXX, and AGROTAIN ULTRA.
[0389] Further, stabilized forms of fertilizer can be used. For
example, a stabilized form of fertilizer is SUPER U, containing 46%
nitrogen in a stabilized, urea-based granule, SUPERU contains
urease and nitrification inhibitors to guard from denitrification,
leaching, and volatilization. Stabilized and targeted foliar
fertilizer such as NITAMIN may also be used herein.
[0390] Pop-up fertilizers are commonly used in corn fields. Pop-up
fertilization comprises applying a few pounds of nutrients with the
seed at planting. Pop-up fertilization is used to increase seedling
vigor.
[0391] Slow- or controlled-release fertilizer that may be used
herein entails: A fertilizer containing a plant nutrient in a form
which delays its availability for plant uptake and use after
application, or which extends its availability to the plant
significantly longer than a reference `rapidly available nutrient
fertilizer` such as ammonium nitrate or urea, ammonium phosphate or
potassium chloride. Such delay of initial availability or extended
time of continued availability may occur by a variety of
mechanisms. These include controlled water solubility of the
material by semi-permeable coatings, occlusion, protein materials,
or other chemical forms, by slow hydrolysis of water-soluble low
molecular weight compounds, or by other unknown means.
[0392] Stabilized nitrogen fertilizer that may be used herein
entails: A fertilizer to which a nitrogen stabilizer has been
added. A nitrogen stabilizer is a substance added to a fertilizer
which extends the time the nitrogen component of the fertilizer
remains in the soil in the urea-N or ammoniacal-N form.
[0393] Nitrification inhibitor that may be used herein entails: A
substance that inhibits the biological oxidation of ammoniacal-N to
nitrate-N. Some examples include: (1)
2-chloro-6-(trichloromethyl-pyridine), common name Nitrapyrin,
manufactured by Dow Chemical; (2) 4-amino-1,2,4-6-triazole-HCl,
common name ATC, manufactured by Ishihada Industries; (3)
2,4-diamino-6-trichloro-methyltriazine, common name CI-1580,
manufactured by American Cyanamid; (4) Dicyandiamide, common name
DCD, manufactured by Showa Denko; (5) Thiourea, common name TU,
manufactured by Nitto Ryuso; (6) 1-mercapto-1,2,4-triazole, common
name MT, manufactured by Nippon; (7)
2-amino-4-chloro-6-methyl-pyramidine, common name AM, manufactured
by Mitsui Toatsu; (8) 3,4-dimethylpyrazole phosphate (DMPP), from
BASF; (9) 1-amide-2-thiourea (ASU), from Nitto Chemical Ind.; (10)
Ammoniumthiosulphate (ATS); (11) 1H-1,2,4-triazole (HPLC); (12)
5-ethylene oxide-3-trichloro-methlyl 1,2,4-thiodiazole (Terrazole),
from Olin Mathieson; (13) 3-methylpyrazole (3-MP); (14)
1-carbamoyle-3-methyl-pyrazole (CMP); (15) Neem; and (16) DMPP.
[0394] Urease inhibitor that may be used herein entails: A
substance that inhibits hydrolytic action on urea by the enzyme
urease. Thousands of chemicals have been evaluated as soil urease
inhibitors (Kiss and Simihaian, 2002). However, only a few of the
many compounds tested meet the necessary requirements of being non
toxic, effective at low concentration, stable, and compatible with
urea (solid and solutions), degradable in the soil and inexpensive.
They can be classified according to their structures and their
assumed interaction with the enzyme urease (Watson, 2000, 2005).
Four main classes of urease inhibitors have been proposed: (a)
reagents which interact with the sulphydryl groups (sulphydryl
reagents), (b) hydroxamates, (c) agricultural crop protection
chemicals, and (d) structural analogues of urea and related
compounds. N-(n-Butyl) thiophosphoric triamide (NBPT),
phenylphosphorodiamidate (PPD/PPDA), and hydroquinone are probably
the most thoroughly studied urease inhibitors (Kiss and Simihaian,
2002). Research and practical testing has also been carried out
with N-(2-nitrophenyl) phosphoric acid triamide (2-NPT) and
ammonium thiosulphate (ATS). The organo-phosphorus compounds are
structural analogues of urea and are some of the most effective
inhibitors of urease activity, blocking the active site of the
enzyme (Watson, 2005).
Insecticidal Seed Treatments (ISTs) for Corn
[0395] Corn seed treatments normally target three spectrums of
pests: nematodes, fungal seedling diseases, and insects.
[0396] Insecticide seed treatments are usually the main component
of a seed treatment package. Most corn seed available today comes
with a base package that includes a fungicide and insecticide. In
some aspects, the insecticide options for seed treatments include
PONCHO (clothianidin), CRUISER/CRUISER EXTREME (thiamethoxam) and
GAUCHO (Imidacloprid). All three of these products are
neonicotinoid chemistries. CRUISER and PONCHO at the 250 (0.25 mg
AI/seed) rate are some of the most common base options available
for corn. In some aspects, the insecticide options for treatments
include CRUISER 250 thiamethoxam, CRUISER 250 (thiamethoxam) plus
LUMIVIA (chlorantraniliprole), CRUISER 500 (thiamethoxam), and
PONCHO VOTIVO 1250 (Clothianidin & Bacillus firmus 1-1582).
[0397] Pioneer's base insecticide seed treatment package consists
of CRUISER 250 with PONCHO/VOTIVO 1250 also available. VOTIVO is a
biological agent that protects against nematodes.
[0398] Monsanto's products including corn, soybeans, and cotton
fall under the ACCELERON treatment umbrella. Dekalb corn seed comes
standard with PONCHO 250. Producers also have the option to upgrade
to PONCHO/VOTIVO, with PONCHO applied at the 500 rate.
[0399] Agrisure, Golden Harvest and Garst have a base package with
a fungicide and CRUISER 250. AVICTA complete corn is also
available; this includes CRUISER 500, fungicide, and nematode
protection. CRUISER EXTREME is another option available as a seed
treatment package, however; the amounts of CRUISER are the same as
the conventional CRUISER seed treatment, i.e. 250, 500, or
1250.
[0400] Another option is to buy the minimum insecticide treatment
available, and have a dealer treat the seed downstream.
[0401] Commercially available ISTs for corn are listed in the below
Table 13 and can be combined with one or more of the microbes
taught herein.
TABLE-US-00013 TABLE 13 List of exemplary seed treatments,
including ISTs, which can be combined with microbes of the
disclosure Treatment Type Active Ingredient(s) Product Trade Name
Crop F azoxystrobin DYNASTY Corn, Soybean PROTEGE FL Corn F
Bacillus pumilus YIELD SHIELD Corn, Soybean F Bacillus subtilis
HISTICK N/T Soybean VAULT HP Corn, Soybean F Captan CAPTAN 400
Corn, Soybean CAPTAN 400-C Corny Soybean F Fludioxonil MAXIM 4FS
Corn, Soybean F Hydrogen peroxide OXIDATE Soybean STOROX Soybean F
ipconazole ACCELERON DC-509 Corn RANCONA 3.8 FS Corn, Soybean
VORTEX Corn F mancozeb BONIDE MANCOZEB w/Zinc Corn Concentrate
DITHANE 75DF RAINSHIELD Corn DITHANE DF RAINSHIELD Corn DITHANE F45
RAINSHIELD Corn DITHANE M45 Corn LESCO 4 FLOWABLE Corn MANCOZEB
PENNCOZEB 4FL FLOWABLE Corn PENNCOZEB 75DF DRY Corn FLOWABLE
PENNCOZEB 80WP Corn F mefenoxam APRON XL Corn, Soybean F metalaxyl
ACCELERON DC-309 Corn ACCELERON DX-309 Corn, Soybean ACQUIRE Corn,
Soybean AGRI STAR METALAXYL 265 Corn, Soybean ST ALLEGIANCE DRY
Corn, Soybean ALLEGIANCE FL Corn, Soybean BELMONT 2.7 FS Corn,
Soybean DYNA-SHIELD METALAXYL Corn, Soybean SEBRING 2.65 ST Corn,
Soybean SEBRING 318 FS Corn, Soybean SEBRING 480 FS Corn, Soybean
VIREO MEC Soybean F pyraclostrobin ACCELERON DX-109 Soybean STAMINA
Corn F Streptomyces MYCOSTOP Corn, Soybean griseoviridis F
Streptomyces lydicus ACTINOGROW ST Corn, Soybean F tebuconazole
AMTIDE TEBU 3.6F Corn SATIVA 309 FS Corn SATIVA 318 FS Corn TEBUSHA
3.6FL Corn TEBUZOL 3.6F Corn F thiabendazole MERTECT 340-F Soybean
F thiram 42-S THIRAM Corn, Soybean FLOWSAN Corn, Soybean SIGNET 480
FS Corn, Soybean F Trichoderma T-22 HC Corn, Soybean harzianum
Rifai F trifloxystrobin ACCELERON DX-709 Corn TRILEX FLOWABLE Corn,
soybean I chlorpyrifos LORSBAN 50W in water soluble Corn packets I
clothianidin ACCELERON IC-609 Corn NIPSIT INSIDE Corn, Soybean
PONCHO 600 Corn I imidacloprid ACCELERON IX-409 Corn AGRI STAR
MACHO 600 ST Corn, Soybean AGRISOLUTIONS NITRO Corn, Soybean SHIELD
ATTENDANT 600 Corn, Soybean AXCESS Corn, Soybean COURAZE 2F Soybean
DYNA-SHIELD Corn, Soybean IMIDACLOPRID 5 GAUCHO 480 FLOWABLE Corn,
Soybean GAUCHO 600 FLOWABLE Corn, Soybean GAUCHO SB FLOWABLE Corn,
Soybean NUPRID 4.6F PRO Soybean SENATOR 600 FS Corn, Soybean I
thiamethoxam CRUISER 5FS Corn, Soybean N abamectin AVICTA 500 FS
Corn, Soybean N Bacillus firmus VOTIVO FS Soybean P cytokinin SOIL
X-CYTO Soybean X-CYTE Soybean P harpin alpha beta ACCELERON HX-209
Corn, Soybean protein N-HIBIT GOLD CST Corn, Soybean N-HIBIT HX-209
Corn, Soybean P indole butyric acid KICKSTAND PGR Corn, Soybean I,
N thiamethoxam, AVICTA DUO CORN Corn abamectin AVICTA DUO 250 I, F
clothianidin, PONCHO VOTIVO Corn, Soybean Bacillus firmus F, F
carboxin, captan ENHANCE Soybean I, F permethrin, carboxin KERNEL
GUARD SUPREME Corn, Soybean F, F carboxin, thiram VITAFLO 280 Corn,
Soybean F, F mefenoxam, fludioxonil MAXIM XL Corn, Soybean WARDEN
RTA Soybean APRON MAXX RFC APRON MAXX RTA + MOLY APRON MAXX RTA I,
F imidacloprid, metalaxyl AGRISOLUTIONS CONCUR Corn F, F metalaxyl,
ipconazole RANCONA SUMMIT Soybean RANCONA XXTRA F, F thiram,
metalaxyl PROTECTOR-L-ALLEGIANCE Soybean F, F trifloxystrobin,
TRILEX AL Soybean metalaxyl TRILEX 2000 P, P, P cytokinin,
gibberellic STIMULATE YIELD Corn, Soybean acid, indole butyric acid
ENHANCER ASCEND F, F, I mefenoxam, CRUISERMAXX PLUS Soybean
fludioxonil, thiamethoxam F, F, F captan, carboxin, BEAN
GUARD/ALLEGIANCE Soybean metalaxyl F, F, I captan, carboxin,
ENHANCE AW Soybean imidacloprid F, F, I carboxin, LATITUDE Corn,
Soybean metalaxyl, imidacloprid F, F, F metalaxyl, STAMINA F3 HL
Corn pyraclostrobin, triticonazole F, F, F, I azoxystrobin, CRUISER
EXTREME Corn fludioxonil, mefenoxam, thiamethoxam F, F, F, F, F
azoxystrobin, MAXIM QUATTRO Corn fludioxonil, mefenoxam,
thiabendazole I Chlorantraniliprole LUMIVIA Corn F = Fungicide; I =
Insecticide; N = Nematicide; P = Plant Growth Regulator
Application of Bacterial Populations on Crops
[0402] The composition of the bacteria or bacterial population
described herein can be applied in furrow, in talc, or as seed
treatment. The composition can be applied to a seed package in
bulk, mini bulk, in a bag, or in talc.
[0403] The planter can plant the treated seed and grows the crop
according to conventional ways, twin row, or ways that do not
require tilling. The seeds can be distributed using a control
hopper or an individual hopper. Seeds can also be distributed using
pressurized air or manually. Seed placement can be performed using
variable rate technologies. Additionally, application of the
bacteria or bacterial population described herein may be applied
using variable rate technologies. In some examples, the bacteria
can be applied to seeds of corn, soybean, canola, sorghum, potato,
rice, vegetables, cereals, pseudocereals, and oilseeds. Examples of
cereals may include barley, fonio, oats, palmer's grass, rye, pearl
millet, sorghum, spelt, teff, triticale, and wheat. Examples of
pseudocereals may include breadnut, buckwheat, cattail, chia, flax,
grain amaranth, hanza, quinoa, and sesame. In some examples, seeds
can be genetically modified organisms (GMO), non-GMO, organic or
conventional.
[0404] Additives such as micro-fertilizer, PGR, herbicide,
insecticide, and fungicide can be used additionally to treat the
crops. Examples of additives include crop protectants such as
insecticides, nematicides, fungicide, enhancement agents such as
colorants, polymers, pelleting, priming, and disinfectants, and
other agents such as inoculant, PGR, softener, and micronutrients.
PGRs can be natural or synthetic plant hormones that affect root
growth, flowering, or stem elongation. PGRs can include auxins,
gibberellins, cytokinins, ethylene, and abscisic acid (ABA).
[0405] The composition can be applied in furrow in combination with
liquid fertilizer. In some examples, the liquid fertilizer may be
held in tanks. NPK fertilizers contain macronutrients of sodium,
phosphorous, and potassium.
[0406] The composition may improve plant traits, such as promoting
plant growth, maintaining high chlorophyll content in leaves,
increasing fruit or seed numbers, and increasing fruit or seed unit
weight. Methods of the present disclosure may be employed to
introduce or improve one or more of a variety of desirable traits.
Examples of traits that may introduced or improved include: root
biomass, root length, height, shoot length, leaf number, water use
efficiency, overall biomass, yield, fruit size, grain size,
photosynthesis rate, tolerance to drought, heat tolerance, salt
tolerance, tolerance to low nitrogen stress, nitrogen use
efficiency, resistance to nematode stress, resistance to a fungal
pathogen, resistance to a bacterial pathogen, resistance to a viral
pathogen, level of a metabolite, modulation in level of a
metabolite, proteome expression. The desirable traits, including
height, overall biomass, root and/or shoot biomass, seed
germination, seedling survival, photosynthetic efficiency,
transpiration rate, seed/fruit number or mass, plant grain or fruit
yield, leaf chlorophyll content, photosynthetic rate, root length,
or any combination thereof, can be used to measure growth, and
compared with the growth rate of reference agricultural plants
(e.g., plants without the introduced and/or improved traits) grown
under identical conditions. In some examples, the desirable traits,
including height, overall biomass, root and/or shoot biomass, seed
germination, seedling survival, photosynthetic efficiency,
transpiration rate, seed/fruit number or mass, plant grain or fruit
yield, leaf chlorophyll content, photosynthetic rate, root length,
or any combination thereof, can be used to measure growth, and
compared with the growth rate of reference agricultural plants
(e.g., plants without the introduced and/or improved traits) grown
under similar conditions.
[0407] An agronomic trait to a host plant may include, but is not
limited to, the following: altered oil content, altered protein
content, altered seed carbohydrate composition, altered seed oil
composition, and altered seed protein composition, chemical
tolerance, cold tolerance, delayed senescence, disease resistance,
drought tolerance, ear weight, growth improvement, health
e4nhancement, heat tolerance, herbicide tolerance, herbivore
resistance improved nitrogen fixation, improved nitrogen
utilization, improved root architecture, improved water use
efficiency, increased biomass, increased root length, increased
seed weight, increased shoot length, increased yield, increased
yield under water-limited conditions, kernel mass, kernel moisture
content, metal tolerance, number of ears, number of kernels per
ear, number of pods, nutrition enhancement, pathogen resistance,
pest resistance, photosynthetic capability improvement, salinity
tolerance, stay-green, vigor improvement, increased dry weight of
mature seeds, increased fresh weight of mature seeds, increased
number of mature seeds per plant, increased chlorophyll content,
increased number of pods per plant, increased length of pods per
plant, reduced number of wilted leaves per plant, reduced number of
severely wilted leaves per plant, and increased number of
non-wilted leaves per plant, a detectable modulation in the level
of a metabolite, a detectable modulation in the level of a
transcript, and a detectable modulation in the proteome, compared
to an isoline plant grown from a seed without said seed treatment
formulation.
[0408] In some cases, plants are inoculated with bacteria or
bacterial populations that are isolated from the same species of
plant as the plant element of the inoculated plant. For example, an
bacteria or bacterial population that is normally found in one
variety of Zea mays (corn) is associated with a plant element of a
plant of another variety of Zea mays that in its natural state
lacks said bacteria and bacterial populations. In one embodiment,
the bacteria and bacterial populations is derived from a plant of a
related species of plant as the plant element of the inoculated
plant. For example, an bacteria and bacterial populations that is
normally found in Zea diploperennis Iltis et al., (diploperennial
teosinte) is applied to a Zea mays (corn), or vice versa. In some
cases, plants are inoculated with bacteria and bacterial
populations that are heterologous to the plant element of the
inoculated plant. In one embodiment, the bacteria and bacterial
populations is derived from a plant of another species. For
example, an bacteria and bacterial populations that is normally
found in dicots is applied to a monocot plant (e.g., inoculating
corn with a soybean-derived bacteria and bacterial populations), or
vice versa. In other cases, the bacteria and bacterial populations
to be inoculated onto a plant is derived from a related species of
the plant that is being inoculated. In one embodiment, the bacteria
and bacterial populations is derived from a related taxon, for
example, from a related species. The plant of another species can
be an agricultural plant. In another embodiment, the bacteria and
bacterial populations is part of a designed composition inoculated
into any host plant element.
[0409] In some examples, the bacteria or bacterial population is
exogenous wherein the bacteria and bacterial population is isolated
from a different plant than the inoculated plant. For example, in
one embodiment, the bacteria or bacterial population can be
isolated from a different plant of the same species as the
inoculated plant. In some cases, the bacteria or bacterial
population can be isolated from a species related to the inoculated
plant.
[0410] In some examples, the bacteria and bacterial populations
described herein are capable of moving from one tissue type to
another. For example, the present disclosure's detection and
isolation of bacteria and bacterial populations within the mature
tissues of plants after coating on the exterior of a seed
demonstrates their ability to move from seed exterior into the
vegetative tissues of a maturing plant. Therefore, in one
embodiment, the population of bacteria and bacterial populations is
capable of moving from the seed exterior into the vegetative
tissues of a plant. In one embodiment, the bacteria and bacterial
populations that is coated onto the seed of a plant is capable,
upon germination of the seed into a vegetative state, of localizing
to a different tissue of the plant. For example, bacteria and
bacterial populations can be capable of localizing to any one of
the tissues in the plant, including: the root, adventitious root,
seminal 5 root, root hair, shoot, leaf, flower, bud, tassel,
meristem, pollen, pistil, ovaries, stamen, fruit, stolon, rhizome,
nodule, tuber, trichome, guard cells, hydathode, petal, sepal,
glume, rachis, vascular cambium, phloem, and xylem. In one
embodiment, the bacteria and bacterial populations is capable of
localizing to the root and/or the root hair of the plant. In
another embodiment, the bacteria and bacterial populations is
capable of localizing to the photosynthetic tissues, for example,
leaves and shoots of the plant. In other cases, the bacteria and
bacterial populations is localized to the vascular tissues of the
plant, for example, in the xylem and phloem. In still another
embodiment, the bacteria and bacterial populations is capable of
localizing to the reproductive tissues (flower, pollen, pistil,
ovaries, stamen, fruit) of the plant. In another embodiment, the
bacteria and bacterial populations is capable of localizing to the
root, shoots, leaves and reproductive tissues of the plant. In
still another embodiment, the bacteria and bacterial populations
colonizes a fruit or seed tissue of the plant. In still another
embodiment, the bacteria and bacterial populations is able to
colonize the plant such that it is present in the surface of the
plant (i.e., its presence is detectably present on the plant
exterior, or the episphere of the plant). In still other
embodiments, the bacteria and bacterial populations is capable of
localizing to substantially all, or all, tissues of the plant. In
certain embodiments, the bacteria and bacterial populations is not
localized to the root of a plant. In other cases, the bacteria and
bacterial populations is not localized to the photosynthetic
tissues of the plant.
[0411] The effectiveness of the compositions can also be assessed
by measuring the relative maturity of the crop or the crop heating
unit (CHU). For example, the bacterial population can be applied to
corn, and corn growth can be assessed according to the relative
maturity of the corn kernel or the time at which the corn kernel is
at maximum weight. The crop heating unit (CHU) can also be used to
predict the maturation of the corn crop. The CHU determines the
amount of heat accumulation by measuring the daily maximum
temperatures on crop growth.
[0412] In examples, bacterial may localize to any one of the
tissues in the plant, including: the root, adventitious root,
seminal root, root hair, shoot, leaf, flower, bud tassel, meristem,
pollen, pistil, ovaries, stamen, fruit, stolon, rhizome, nodule,
tuber, trichome, guard cells, hydathode, petal, sepal, glume,
rachis, vascular cambium, phloem, and xylem. In another embodiment,
the bacteria or bacterial population is capable of localizing to
the photosynthetic tissues, for example, leaves and shoots of the
plant. In other cases, the bacteria and bacterial populations is
localized to the vascular tissues of the plant, for example, in the
xylem and phloem. In another embodiment, the bacteria or bacterial
population is capable of localizing to reproductive tissues
(flower, pollen, pistil, ovaries, stamen, or fruit) of the plant.
In another embodiment, the bacteria and bacterial populations is
capable of localizing to the root, shoots, leaves and reproductive
tissues of the plant. In another embodiment, the bacteria or
bacterial population colonizes a fruit or seed tissue of the plant.
In still another embodiment, the bacteria or bacterial population
is able to colonize the plant such that it is present in the
surface of the plant. In another embodiment, the bacteria or
bacterial population is capable of localizing to substantially all,
or all, tissues of the plant. In certain embodiments, the bacteria
or bacterial population is not localized to the root of a plant. In
other cases, the bacteria and bacterial populations is not
localized to the photosynthetic tissues of the plant.
[0413] The effectiveness of the bacterial compositions applied to
crops can be assessed by measuring various features of crop growth
including, but not limited to, planting rate, seeding vigor, root
strength, drought tolerance, plant height, dry down, and test
weight.
Plant Species
[0414] The methods and bacteria described herein are suitable for
any of a variety of plants, such as plants in the genera Hordeum,
Oryza, Zea, and Triticeae. Other non-limiting examples of suitable
plants include mosses, lichens, and algae. In some cases, the
plants have economic, social and/or environmental value, such as
food crops, fiber crops, oil crops, plants in the forestry or pulp
and paper industries, feedstock for biofuel production and/or
ornamental plants. In some examples, plants may be used to produce
economically valuable products such as a grain, a flour, a starch,
a syrup, a meal, an oil, a film, a packaging, a nutraceutical
product, a pulp, an animal feed, a fish fodder, a bulk material for
industrial chemicals, a cereal product, a processed human-food
product, a sugar, an alcohol, and/or a protein. Non-limiting
examples of crop plants include maize, rice, wheat, barley,
sorghum, millet, oats, rye triticale, buckwheat, sweet corn, sugar
cane, onions, tomatoes, strawberries, and asparagus. In some
embodiments, the methods and bacteria described herein are suitable
for any of a variety of transgenic plants, non-transgenic plants,
and hybrid plants thereof.
[0415] In some examples, plants that may be obtained or improved
using the methods and composition disclosed herein may include
plants that are important or interesting for agriculture,
horticulture, biomass for the production of biofuel molecules and
other chemicals, and/or forestry. Some examples of these plants may
include pineapple, banana, coconut, lily, grasspeas and grass; and
dicotyledonous plants, such as, for example, peas, alfalfa,
tomatillo, melon, chickpea, chicory, clover, kale, lentil, soybean,
tobacco, potato, sweet potato, radish, cabbage, rape, apple trees,
grape, cotton, sunflower, thale cress, canola, citrus (including
orange, mandarin, kumquat, lemon, lime, grapefruit, tangerine,
tangelo, citron, and pomelo), pepper, bean, lettuce, Panicum
virgatum (switch), Sorghum bicolor (sorghum, sudan), Miscanthus
giganteus (miscanthus), Saccharum sp. (energycane), Populus
balsamifera (poplar), Zea mays (corn), Glycine max (soybean),
Brassica napus (canola), Triticum aestivum (wheat), Gossypium
hirsutum (cotton), Oryza sativa (rice), Helianthus annuus
(sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet),
Pennisetum glaucum (pearl millet), Panicum spp. Sorghum spp.,
Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp.,
Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.
(eucalyptus), Triticosecale spp. (triticum-25 wheat X rye), Bamboo,
Carthamus tinctorius (safflower), Jatropha curcas (Jatropha),
Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenix
dactylifera (date palm), Archontophoenix cunninghamiana (king
palm), Syagrus romanzoffiana (queen palm), Linum usitatissimum
(flax), Brassica juncea, Manihot esculenta (cassaya), Lycopersicon
esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca
(banana), Solanum tuberosum (potato), Brassica oleracea (broccoli,
cauliflower, brussel sprouts), Camellia sinensis (tea), Fragaria
ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica
(coffee), Vitis vinifera (grape), Ananas comosus (pineapple),
Capsicum annum (hot & sweet pepper), Allium cepa (onion),
Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima
(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),
Citrullus lanatus (watermelon), Abelmoschus esculentus (okra),
Solanum melongena (eggplant), Papaver somniferum (opium poppy),
Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia
annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus,
Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrum
californica, Digitalis lanata, Digitalis purpurea, Dioscorea 5
spp., Andrographis paniculata, Atropa belladonna, Datura stomonium,
Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp.,
Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium
serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina,
Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula
officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum
parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber),
Mentha spicata (mint), Mentha piperita (mint), Bixa orellana,
Alstroemeria spp., Rosa spp. (rose), Dianthus caryophyllus
(carnation), Petunia spp. (petunia), Poinsettia pulcherrima
(poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin),
Uniola paniculata (oats), Hordeum vulgare (barley), and Lolium spp.
(rye).
[0416] In some examples, a monocotyledonous plant may be used.
Monocotyledonous plants belong to the orders of the Alismatales,
Arales, Arecales, Bromeliales, Commelinales, Cyclanthales,
Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales,
Najadales, Orchidales, Pandanales, Poales, Restionales,
Triuridales, Typhales, and Zingiberales. Plants belonging to the
class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and
Pinales. In some examples, the monocotyledonous plant can be
selected from the group consisting of a maize, rice, wheat, barley,
and sugarcane.
[0417] In some examples, a dicotyledonous plant may be used,
including those belonging to the orders of the Aristochiales,
Asterales, Batales, Campanulales, Capparales, Caryophyllales,
Casuarinales, Celastrales, Comales, Diapensales, Dilleniales,
Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales,
Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales,
Middles, Juglandales, Lamiales, Laurales, Lecythidales,
Leitneriales, Magniolales, Malvales, Myricales, Myrtales,
Nymphaeales, Papeverales, Piperales, Plantaginales, Plumb aginales,
Podostemales, Polemoniales, Polygalales, Polygonales, Primulales,
Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales,
Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae,
Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales,
and Violates. In some examples, the dicotyledonous plant can be
selected from the group consisting of cotton, soybean, pepper, and
tomato.
[0418] In some cases, the plant to be improved is not readily
amenable to experimental conditions. For example, a crop plant may
take too long to grow enough to practically assess an improved
trait serially over multiple iterations. Accordingly, a first plant
from which bacteria are initially isolated, and/or the plurality of
plants to which genetically manipulated bacteria are applied may be
a model plant, such as a plant more amenable to evaluation under
desired conditions. Non-limiting examples of model plants include
Setaria, Brachypodium, and Arabidopsis. Ability of bacteria
isolated according to a method of the disclosure using a model
plant may then be applied to a plant of another type (e.g. a crop
plant) to confirm conferral of the improved trait.
[0419] Traits that may be improved by the methods disclosed herein
include any observable characteristic of the plant, including, for
example, growth rate, height, weight, color, taste, smell, changes
in the production of one or more compounds by the plant (including
for example, metabolites, proteins, drugs, carbohydrates, oils, and
any other compounds). Selecting plants based on genotypic
information is also envisaged (for example, including the pattern
of plant gene expression in response to the bacteria, or
identifying the presence of genetic markers, such as those
associated with increased nitrogen fixation). Plants may also be
selected based on the absence, suppression or inhibition of a
certain feature or trait (such as an undesirable feature or trait)
as opposed to the presence of a certain feature or trait (such as a
desirable feature or trait).
Non-Genetically Modified Maize
[0420] The methods and bacteria described herein are suitable for
any of a variety of non-genetically modified maize plants or part
thereof. And in some aspects the corn is organic. Furthermore, the
methods and bacteria described herein are suitable for any of the
following non-genetically modified hybrids, varieties, lineages,
etc. In some embodiments, corn varieties generally fall under six
categories: sweet corn, flint corn, popcorn, dent corn, pod corn,
and flour corn.
Sweet Corn
[0421] Yellow su varieties include Earlivee, Early Sunglow,
Sundance, Early Golden Bantam, Iochief, Merit, Jubilee, and Golden
Cross Bantam. White su varieties include True Platinum, Country
Gentleman, Silver Queen, and Stowell's Evergreen. Bicolor su
varieties include Sugar & Gold, Quickie, Double Standard,
Butter & Sugar, Sugar Dots, Honey & Cream. Multicolor su
varieties include Hookers, Triple Play, Painted Hill, Black
Mexican/Aztec.
[0422] Yellow se varieties include Buttergold, Precocious, Spring
Treat, Sugar Buns, Colorow, Kandy King, Bodacious R/M, Tuxedo,
Incredible, Merlin, Miracle, and Kandy Korn EH. White se varieties
include Spring Snow, Sugar Pearl, Whiteout, Cloud Nine, Alpine,
Silver King, and Argent. Bicolor se varieties include Sugar Baby,
Fleet, Bon Jour, Trinity, Bi-Licious, Temptation, Luscious,
Ambrosia, Accord, Brocade, Lancelot, Precious Gem, Peaches and
Cream Mid EH, and Delectable R/M. Multicolor se varieties include
Ruby Queen.
[0423] Yellow sh2 varieties include Extra Early Super Sweet,
Takeoff, Early Xtra Sweet, Raveline, Summer Sweet Yellow, Krispy
King, Garrison, Illini Gold, Challenger, Passion, Excel, Jubilee
SuperSweet, Illini Xtra Sweet, and Crisp 'N Sweet. White sh2
varieties include Summer Sweet White, Tahoe, Aspen, Treasure, How
Sweet It Is, and Camelot. Bicolor sh2 varieties include Summer
Sweet Bicolor, Radiance, Honey 'N Pearl, Aloha, Dazzle, Hudson, and
Phenomenal.
[0424] Yellow sy varieties include Applause, Inferno, Honeytreat,
and Honey Select. White sy varieties include Silver Duchess,
Cinderella, Mattapoisett, Avalon, and Captivate. Bicolor sy
varieties include Pay Dirt, Revelation, Renaissance, Charisma,
Synergy, Montauk, Kristine, Serendipity/Providence, and Cameo.
[0425] Yellow augmented supersweet varieties include Xtra-Tender
1ddA, Xtra-Tender 11dd, Mirai 131Y, Mirai 130Y, Vision, and Mirai
002. White augmented supersweet varieties include Xtra-Tender 3dda,
Xtra-Tender 31dd, Mirai 421W, XTH 3673, and Devotion. Bicolor
augmented supersweet varieties include Xtra-Tender 2dda,
Xtra-Tender 21dd, Kickoff XR, Mirai 308BC, Anthem XR, Mirai 336BC,
Fantastic XR, Triumph, Mirth 301BC, Stellar, American Dream, Mirai
350BC, and Obsession.
Flint Corn
[0426] Flint corn varieties include Bronze-Orange, Candy Red Flint,
Floriani Red Flint, Glass Gem, Indian Ornamental (Rainbow), Mandan
Red Flour, Painted Mountain, Petmecky, Cherokee White Flour,
PopCorn
[0427] Pop corn varieties include Monarch Butterfly, Yellow
Butterfly, Midnight Blue, Ruby Red, Mixed Baby Rice, Queen Mauve,
Mushroom Flake, Japanese Hull-less, Strawberry, Blue Shaman,
Miniature Colored, Miniature Pink, Pennsylvania Dutch Butter
Flavor, and Red Strawberry.
Dent Corn
[0428] Dent corn varieties include Bloody Butcher, Blue Clarage,
Ohio Blue Clarage, Cherokee White Eagle, Hickory Cane, Hickory
King, Jellicorse Twin, Kentucky Rainbow, Daymon Morgan's Knt.
Butcher, Leaming, Leaming's Yellow, McCormack's Blue Giant, Neal
Paymaster, Pungo Creek Butcher, Reid's Yellow Dent, Rotten Clarage,
and Tennessee Red Cob.
[0429] In some embodiments, corn varieties include P1618W, P1306W,
P1345, P1151, P1197, P0574, P0589, and P0157. W=white corn.
[0430] In some embodiments, the methods and bacteria described
herein are suitable for any hybrid of the maize varieties setforth
herein.
Genetically Modified Maize
[0431] The methods and bacteria described herein are suitable for
any of a hybrid, variety, lineage, etc. of genetically modified
maize plants or part thereof.
[0432] Furthermore, the methods and bacteria described herein are
suitable for any of the following genetically modified maize
events, which have been approved in one or more countries: 32138
(32138 SPT Maintainer), 3272 (ENOGEN), 3272 x Bt11, 3272 x bt11 x
GA21, 3272 x Bt11 x MIR604, 3272 x Bt11 x MIR604 x GA21, 3272 x
Bt11 x MIR604 x TC1507 x 5307 x GA21, 3272 x GA21, 3272 x MIR604,
3272 x MIR604 x GA21, 4114, 5307 (AGRISURE Duracade), 5307 x GA21,
5307 x MIR604 x Bt11 x TC1507 x GA21 (AGRISURE Duracade 5122), 5307
x MIR604 x Bt11 x TC1507 x GA21 x MIR162 (AGRISURE Duracade 5222),
59122 (HERCULEX RW), 59122 x DAS40278, 59122 x GA21, 59122 x
MIR604, 59122 x MIR604 x GA21, 59122 x MIR604 x TC1507, 59122 x
MIR604 x TC1507 x GA21, 59122 x MON810, 59122 x MON810 x MIR604,
59122 x MON810 x NK603, 59122 x MON810 x NK603 x MIR604, 59122 x
MON88017, 59122 x MON88017 x DAS40278, 59122 x NK603 (Herculex RW
ROUNDUP READY 2), 59122 x NK603 x MIR604, 59122 x TC1507 x GA21,
676, 678, 680, 3751 IR, 98140, 98140 x 59122, 98140 x TC1507, 98140
x TC1507 x 59122, Bt10 (Bt10), Bt11 [X4334CBR, X4734CBR] (AGRISURE
CB/LL), Bt11 x 5307, Bt11 x 5307 x GA21, Bt11 x 59122 x MIR604,
Br11 x 59122 x MIR604 x GA21, Bt11 x 59122 x MIR604 x TC1507, M53,
M56, DAS-59122-7, Bt11 x 59122 x MIR604 x TC1507 x GA21, Bt11 x
59122 x TC1507, TC1507 x DAS-59122-7, Bt11 x 59122 x TC1507 x GA21,
Bt11 x GA21 (AGRISURE GT/CB/LL), Bt11 x MIR162 (AGRISURE Viptera
2100), BT11 x MIR162 x 5307, Bt11 x MIR162 x 5307 x GA21, Bt11 x
MIR162 x GA21 (AGRISURE Viptera 3110), Bt11 x MIR162 x MIR604
(AGRISURE Viptera 3100), Bt11 x MIR162 x MIR604 x 5307, Bt11 x
MIR162 x MIR604 x 5307 x GA21, Bt11 x MIR162 x MIR604 x GA21
(AGRISURE Viptera 3111/AGRISURE Viptera 4), Bt11, MIR162 x MIR604 x
MON89034 x 5307 x GA21, Bt11 x MIR162 x MIR604 x TC1507, Bt11 x
MIR162 x MIR604 x TC1507 x 5307, Bt11 x MIR162 x MIR604 x TC1507 x
GA21, Bt11 x MIR162 x MON89034, Bt11 x MIR162 x MON89034 x GA21,
Bt11 x MIR162 x TC1507, Bt11 x MIR162 x TC1507 x 5307, Bt11 x
MIR162 x TC1507 x 5307 x GA21, Bt11 x MR162 x TC1507 x GA21
(AGRISURE Viptera 3220), BT11 x MIR604 (Agrisure BC/LL/RW), Bt11 x
MIR604 x 5307, Bt11 x MIR604 x 5307 x GA21, Bt11 x MIR604 x GA21,
Bt11 x MIR604 x TC1507, Bt11 x MIR604 x TC1507 x 5307, Bt11 x
MIR604 x TC1507 x GA21, Bt11 x MON89034 x GA21, Bt11 x TC1507, Bt11
x TC1507 x 5307, Bt11 x TC1507 x GA21, Bt176 (NaturGard
KnockOut/Maximizer), BVLA430101, CBH-351 (STARLINK Maize), DAS40278
(ENLIST Maize), DAS40278 x NK603, DBT418 (Bt Xtra Maize), DLL25
[B16], GA21 (ROUNDUP READY Maize/AGRISURE GT), GA21 x MON810
(ROUNDUP READY Yieldgard Maize), GA21 x T25, HCEM485, LY038 (MAVERA
Maize), LY038 x MON810 (MAVERA Yieldgard Maize), MIR162 (AGRISURE
Viptera), MIR162 x 5307, MIR162 x 5307 x GA21, MIR162 x GA21,
MIR162 x MIR604, MIR162 x MIR604 x 5307, MIR162 x MIR604 x 5307 x
GA21, MIR162 x MIR604 x GA21, MIR162 x MIR604 x TC1507 x 5307,
MIR162 x MIR604 x TC1507 x 5307 x GA21, MIR162 x MIR604 x TC1507 x
GA21, MIR162 x MON89O34, MIR162 x NK603, MIR162 x TC1507, MIR162 x
TC1507 x 5307, MIR162 x TC1507 x 5307 x GA21, MIR162 x TC1507 x
GA21, MIR604 (AGRISURE RW), MIR604 x 5307, MIR604 x 5307 x GA21,
MIR604 x GA21 (AGRISURE GT/RW), MIR604 x NK603, MIR604 x TC1507,
MIR604 x TC1507 x 5307, MIR604 x TC1507 x 5307 x GA21, MIR604 x
TC1507 x GA21, MON801 [MON80100], MON802, MON809, MON810
(YIELDGARD, MAIZEGARD), MON810 x MIR162, MON810 x MIR162 x NK603,
MON810 x MIR604, MON810 x MON88017 (YIELDGARD VT Triple), MON810 x
NK603 x MIR604, MON832 (ROUNDUP READY Maize), MON863 (YIELDGARD
Rootworm RW, MAXGARD), MON863 x MON810 (YIELDGARD Plus), MON863 x
MON810 x NK603 (YIELDGARD Plus with RR), MON863 x NK603 (YIELDGARD
RW+RR), MON87403, MON87411, MON87419, MON87427 (ROUNDUP READY
Maize), MON87427 x 59122, MON87427 x MON88017, MON87427 x MON88017
x 59122, MON87427 x MON89034, MON87427 x MON89034 x 59122, MON87427
x MON89034 x MIR162 x MON87411, MON87427 x MON89034 x MON88017,
MON87427 x MON89034 x MON88017 x 59122, MON87427 x MON89034 x
NK603, MON87427 x MON89034 x TC1507, MON87427 x MON89034 x TC1507 x
59122, MON87427 x MON89034 x TC1507 x MON87411 x 59122, MON87427 x
MON89034 x TC1507 x MON87411 x 59122 x DAS40278, MON87427 x
MON89034 x TC1507 x MON88017, MON87427 x MON89034 x MIR162 x NK603,
MON87427 x MON89034 x TC15O7 x MON88017 x 59122, MON87427 x TC1507,
MON87427 x TC1507 x 59122, MON87427 x TC1507 x MON88017, MON87427 x
TC1507 x MON88017 x 59122, MON87460 (GENUITY DROUGHTGARD), MON87460
x MON88017, MON87460 x MON89034 x MON88017, MON87460 x MON89034 x
NK603, MON87460 x NK603, MON88017, MON88017 x DAS40278, MON89034,
MON89034 x 59122, MON89034 x 59122 x DAS40278, MON89034 x 59122 x
MON88017, MON89034 x 59122 x MON88017 x DAS40278, MON89034 x
DAS40278, MON89034 x MON87460, MON89034 x MON88017 (GENUITY VT
Triple Pro), MON89034 x MON88017 x DAS40278, MON89034 x NK603
(GENUITY VT Double Pro), MON89034 x NK603 x DAS40278, MON89034 x
TC1507, MON89034 x TC1507 x 59122, MON89034 x TC1507 x 59122 x
DAS40278, MON89034 x TC1507 x DAS40278, MON89034 x TC1507 x
MON88017, MON89034 x TC1507 x MON88017 x 59122 (GENUITY SMARTSTAX),
MON89034 x TC1507 x MON88017 x 59122 x DAS40278, MON89034 x TC1507
x MON88017 x DAS40278, MON89034 x TC1507 x NK603 (POWER CORE),
MON89034 x TC1507 x NK603 x DAS40278, MON89034 x TC1507 x NK603 x
MIR162, MON89034 x TC1507 x NK603 x MIR162 x DAS40278, MON89034 x
GA21, MS3 (INVIGOR Maize), MS6 (INVIGOR Maize), MZHG0JG, MZIR098,
NK603 (ROUNDUP READY 2 Maize), NK603 x MON810 x 4114 x MIR604,
NK603 x MON810 (YIELDGARD CB+RR), NK603 x T25 (ROUNDUP READY
LIBERTY LINK Maize), T14 (LIBERTY LINK Maize), T25 (LIBERTY LINK
Maize), T25 x MON810 (LIBERTY LINK YIELDGARD Maize), TC1507
(HERCULEX I, HERCULEX CB), TC1507 x 59122 x MON810 x MIR604 x NK603
(OPTIMUM INTRASECT XTREME), TC1507 x MON810 x MIR604 x NK603,
TC1507 x 5307, TC1507 x 5307 x GA21, TC1507 x 59122 (HERCULEX
XTRA), TC1507 x 59122 x DAS40278, TC1507 x 59122 x MON810, TC1507 x
59122 x MON810 x MIR604, TC1507 x 59122 x MON810 x NK603 (OPTIMUM
INTRASECT XTRA), TC1507 x 59122 x MON88017, TC1507 x 59122 x
MON88017 x DAS40278, TC1507 x 59122 x NK603 (HERCULEX XTRA RR),
TC1507 x 59122 x NK603 x MIR604, TC1507 x DAS40278, TC1507 x GA21,
TC1507 x MIR162 x NK603, TC1507 x MIR604 x NK603 (OPTIMUM TRISECT),
TC1507 x MON810, TC1507 x MON810 x MIR162, TC1507 x MON810 x MIR162
x NK603, TC1507 x MON810 x MIR604, TC1507 x MON810 x NK603 (OPTIMUM
INTRASECT), TC1507 x MON810 x NK603 x MIR604, TC1507 x MON88017,
TC1507 x MON88017 x DAS40278, TC1507 x NK603 (HERCULEX I RR),
TC1507 x NK603 x DAS40278, TC6275, and VCO-01981-5.
Additional Genetically Modified Plants
[0433] The methods and bacteria described herein are suitable for
any of a variety of genetically modified plants or part
thereof.
[0434] Furthermore, the methods and bacteria described herein are
suitable for any of the following genetically modified plant events
which have been approved in one or more countries.
TABLE-US-00014 TABLE 14 Rice Traits, which can be combined with
microbes of the disclosure Oryza sativa Rice Event Company
Description CL121, CL141, CFX51 BASF Inc. Tolerance to the
imidazolinone herbicide, imazethapyr, induced by chemical
mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl
methanesulfonate (EMS). IMINTA-1, IMINTA-4 BASF Inc. Tolerance to
imidazolinone herbicides induced by chemical mutagenesis of the
acetolactate synthase (ALS) enzyme using sodium azide. LLRICE06,
LLRICE62 Aventis CropScience Glufosinate ammonium herbicide
tolerant rice produced by inserting a modified phosphinothricin
acetyltransferase (PAT) encoding gene from the soil bacterium
Streptomyces hygroscopicus). LLRICE601 Bayer CropScience (Aventis
Glufosinate ammonium herbicide CropScience(AgrEvo)) tolerant rice
produced by inserting a modified phosphinothricin acetyltransferase
(PAT) encoding gene from the soil bacterium Streptomyces
hygroscopicus). PWC16 BASF Inc. Tolerance to the imidazolinone
herbicide, imazethapyr, induced by chemical mutagenesis of the
acetolactate synthase (ALS) enzyme using ethyl methanesulfonate
(EMS).
TABLE-US-00015 TABLE 15 Alfalfa Traits, which can be combined with
microbes of the disclosure Medicago sativa Alfalfa Event Company
Description J101, J163 Monsanto Company and Glyphosate herbicide
tolerant Forage Genetics alfalfa (lucerne) produced by
International inserting a gene encoding the enzyme
5-enolypyruvylshikimate- 3-phosphate synthase (EPSPS) from the CP4
strain of Agrobacterium tumefaciens.
TABLE-US-00016 TABLE 16 Wheat Traits, which can be combined with
microbes of the disclosure Triticum aestivum Wheat Event Company
Description AP205CL BASF Inc. Selection for a mutagenized version
of the enzyme acetohydroxyacid synthase (AHAS), also known as
acetolactate synthase (ALS) or acetolactate pyruvate-lyase. AP602CL
BASF Inc. Selection for a mutagenized version of the enzyme
acetohydroxyacid synthase (AHAS), also known as acetolactate
synthase (ALS) or acetolactate pyruvate-lyase. BW255-2, BW238-3
BASF Inc. Selection for a mutagenized version of the enzyme
acetohydroxyacid synthase (AHAS), also known as acetolactate
synthase (ALS) or acetolactate pyruvate-lyase. BW7 BASF Inc.
Tolerance to imidazolinone herbicides induced by chemical
mutagenesis of the acetohydroxyacid synthase (AHAS) gene using
sodium azide. MON71800 Monsanto Company Glyphosate tolerant wheat
variety produced by inserting a modified 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene
from the soil bacterium Agrobacterium tumefaciens, strain CP4.
SWP965001 Cyanamid Crop Selection for a mutagenized version
Protection of the enzyme acetohydroxyacid synthase (AHAS), also
known as acetolactate synthase (ALS) or acetolactate
pyruvate-lyase. Teal 11A BASF Inc. Selection for a mutagenized
version of the enzyme acetohydroxyacid synthase (AHAS), also known
as acetolactate synthase (ALS) or acetolactate pyruvate-lyase.
TABLE-US-00017 TABLE 17 Sunflower Traits, which can be combined
with microbes of the disclosure Helianthus annuus Sunflower Event
Company Description X81359 BASF Inc. Tolerance to imidazolinone
herbicides by selection of a naturally occurring mutant.
TABLE-US-00018 TABLE 18 Soybean Traits, which can be combined with
microbes of the disclosure Glycine max L. Soybean Event Company
Description A2704-12, A2704-21, Bayer CropScience Glufosinate
ammonium herbicide A5547-35 (Aventis CropScience tolerant soybean
produced by (AgrEvo)) inserting a modified phosphinothricin
acetyltransferase (PAT) encoding gene from the soil bacterium
Streptomyces viridochromogenes. A5547-127 Bayer CropScience
Glufosinate ammonium herbicide (Aventis CropScience tolerant
soybean produced by (AgrEvo)) inserting a modified phosphinothricin
acetyltransferase (PAT) encoding gene from the soil bacterium
Streptomyces viridochromogenes. BPS-CV127-9 BASF Inc. The
introduced csr1-2 gene from Arabidopsis thaliana encodes an
acetohydroxyacid synthase protein that confers tolerance to
imidazolinone herbicides due to a point mutation that results in a
single amino acid substitution in which the serine residue at
position 653 is replaced by asparagine (S653N). DP-305423 Pioneer
Hi-Bred High oleic acid soybean produced International Inc. by
inserting additional copies of a portion of the omega 6 desaturase
encoding gene, gm-fad2-1 resulting in silencing of the endogenous
omega-6 desaturase gene (FAD2-1). DP356043 Pioneer Hi-Bred Soybean
event with two herbicide International Inc. tolerance genes:
glyphosate N- acetlytransferase, which detoxifies glyphosate, and a
modified acetolactate synthase (ALS) gene which is tolerant to
ALS-inhibiting herbicides. G94-1, G94-19, G168 DuPont Canada High
oleic acid soybean produced Agricultural Products by inserting a
second copy of the fatty acid desaturase (Gm Fad2-1) encoding gene
from soybean, which resulted in "silencing" of the endogenous host
gene. GTS 40-3-2 Monsanto Company Glyphosate tolerant soybean
variety produced by inserting a modified 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene
from the soil bacterium Agrobacterium tumefaciens. GU262 Bayer
CropScience Glufosinate ammonium herbicide (Aventis tolerant
soybean produced by CropScience(AgrEvo)) inserting a modified
phosphinothricin acetyltransferase (PAT) encoding gene from the
soil bacterium Streptomyces viridochromogenes. MON87701 Monsanto
Company Resistance to Lepidopteran pests of soybean including
velvetbean caterpillar (Anticarsia gemmatalis) and soybean looper
(Pseudoplusia includens). MON87701 .times. Monsanto Company
Glyphosate herbicide tolerance MON89788 through expression of the
EPSPS encoding gene from A. tumefaciens strain CP4, and resistance
to Lepidopteran pests of soybean including velvetbean caterpillar
(Anticarsia gemmatalis) and soybean looper (Pseudoplusia includens)
via expression of the Cry1Ac encoding gene from B. thuringiensis.
MON89788 Monsanto Company Glyphosate-tolerant soybean produced by
inserting a modified 5- enolpyruvylshikimate-3-phosphate synthase
(EPSPS) encoding aroA (epsps) gene from Agrobacterium tumefaciens
CP4. OT96-15 Agriculture & Agri-Food Low linolenic acid soybean
Canada produced through traditional cross- breeding to incorporate
the novel trait from a naturally occurring fan1 gene mutant that
was selected for low linolenic acid. W62, W98 Bayer CropScience
Glufosinate ammonium herbicide (Aventis tolerant soybean produced
by CropScience(AgrEvo)) inserting a modified phosphinothricin
acetyltransferase (PAT) encoding gene from the soil bacterium
Streptomyces hygroscopicus.
TABLE-US-00019 TABLE 19 Corn Traits, which can be combined with
microbes of the disclosure Zea mays L. Maize Event Company
Description 176 Syngenta Seeds, Inc. Insect-resistant maize
produced by inserting the Cry1Ab gene from Bacillus thuringiensis
subsp. kurstaki. The genetic modification affords resistance to
attack by the European corn borer (ECB). 3751 IR Pioneer Hi-Bred
Selection of somaclonal variants 676, 678, 680 International Inc.
by culture of embryos on Pioneer Hi-Bred imidazolinone containing
media. International Inc. Male-sterile and glufosinate ammonium
herbicide tolerant maize produced by inserting genes encoding DNA
adenine methylase and phosphinothricin acetyltransferase (PAT) from
Escherichia coli and Streptomyces viridochromogenes, respectively.
B16 (DLL25) Dekalb Genetics Glufosinate ammonium herbicide
Corporation tolerant maize produced by inserting the gene encoding
phosphinothricin acetyltransferase (PAT) from Streptomyces
hygroscopicus. BT11 (X4334CBR, Syngenta Seeds, Inc.
Insect-resistant and herbicide X4734CBR) tolerant maize produced by
inserting the Cry1Ab gene from Bacillus thuringiensis subsp.
kurstaki, and the phosphinothricin N-acetyltransferase (PAT)
encoding gene from S. viridochromogenes. BT11 .times. GA21 Syngenta
Seeds, Inc. Stacked insect resistant and herbicide tolerant maize
produced by conventional cross breeding of parental lines BT11
(OECD unique identifier: SYN-BTO11-1) and GA21 (OECD unique
identifier: MON-OOO21-9). BT11 .times. MIR162 .times. Syngenta
Seeds, Inc. Resistance to Coleopteran pests, MIR604 .times. GA21
particularly corn rootworm pests (Diabrotica spp.) and several
Lepidopteran pests of corn, including European corn borer (ECB,
Ostrinia nubilalis), corn earworm (CEW, Helicoverpa zea), fall army
worm (FAW, Spodoptera frugiperda), and black cutworm (BCW, Agrotis
ipsilon); tolerance to glyphosate and glufosinate- ammonium
containing herbicides. BT11 .times. MIR162 Syngenta Seeds, Inc.
Stacked insect resistant and herbicide tolerant maize produced by
conventional cross breeding of parental lines BT11 (OECD unique
identifier: SYN-BTO11-1) and MIR162 (OECD unique identifier:
SYN-1R162-4). Resistance to the European Corn Borer and tolerance
to the herbicide glufosinate ammonium (Liberty) is derived from
BT11, which contains the Cry1Ab gene from Bacillus thuringiensis
subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT)
encoding gene from S. viridochromogenes. Resistance to other
Lepidopteran pests, including H. zea, S. frugiperda, A. ipsilon,
and S. albicosta, is derived from MIR162, which contains the vip3Aa
gene from Bacillus thuringiensis strain AB88. BT11 .times. MIR162
.times. Syngenta Seeds, Inc. Bacillus thuringiensis Cry1Ab MIR604
delta-endotoxin protein and the genetic material necessary for its
production (via elements of vector pZO1502) in Event Bt11 corn
(OECD Unique Identifier: SYNBTO11-1) .times. Bacillus thuringiensis
Vip3Aa20 insecticidal protein and the genetic material necessary
for its production (via elements of vector pNOV1300) in Event
MIR162 maize (OECD Unique Identifier: SYN-IR162-4) .times. modified
Cry3A protein and the genetic material necessary for its production
(via elements of vector pZM26) in Event MIR604 corn (OECD Unique
Identifier: SYN-1R604-5). CBH-351 Aventis CropScience
Insect-resistant and glufosinate ammonium herbicide tolerant maize
developed by inserting genes encoding Cry9C protein from Bacillus
thuringiensis subsp tolworthi and phosphinothricin
acetyltransferase (PAT) from Streptomyces hygroscopicus.
DAS-06275-8 DOW AgroSciences LLC Lepidopteran insect resistant and
glufosinate ammonium herbicide- tolerant maize variety produced by
inserting the Cry1F gene from Bacillus thuringiensis var aizawai
and the phosphinothricin acetyltransferase (PAT) from Streptomyces
hygroscopicus. BT11 .times. MIR604 Syngenta Seeds, Inc. Stacked
insect resistant and herbicide tolerant maize produced by
conventional cross breeding of parental lines BT11 (OECD unique
identifier: SYN-BTO11-1) and MIR604 (OECD unique identifier:
SYN-1R6O5-5). Resistance to the European Corn Borer and tolerance
to the herbicide glufosinate ammonium (Liberty) is derived from
BT11, which contains the Cry1Ab gene from Bacillus thuringiensis
subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT)
encoding gene from S. viridochromogenes. Corn rootworm -resistance
is derived from MIR604 which contains the mCry3A gene from Bacillus
thuringiensis. BT11 .times. MIR604 .times. Syngenta Seeds, Inc.
Stacked insect resistant and GA21 herbicide tolerant maize produced
by conventional cross breeding of parental lines BT11 (OECD unique
identifier: SYN-BTO11-1), MIR604 (OECD unique identifier:
SYN-1R6O5-5) and GA21 (OECD unique identifier: MON- OOO21-9).
Resistance to the European Corn Borer and tolerance to the
herbicide glufosinate ammonium (Liberty) is derived from BT11,
which contains the Cry1Ab gene from Bacillus thuringiensis subsp.
kurstaki, and the phosphinothricin N-acetyltransferase (PAT)
encoding gene from S. viridochromogenes. Corn rootworm-resistance
is derived from MIR604 which contains the mCry3A gene from Bacillus
thuringiensis. Tolerance to glyphosate herbicide is derived from
GA21 which contains a a modified EPSPS gene from maize. DAS-59122-7
DOW AgroSciences LLC Corn rootworm-resistant maize and Pioneer
Hi-Bred produced by inserting the International Inc. Cry34Ab1 and
Cry35Ab1 genes from Bacillus thuringiensis strain PS149B1. The PAT
encoding gene from Streptomyces viridochromogenes was introduced as
a selectable marker. DAS-59122-7 .times. DOW AgroSciences LLC
Stacked insect resistant and TC1507 .times. NK603 and Pioneer
Hi-Bred herbicide tolerant maize produced International Inc. by
conventional cross breeding of parental lines DAS-59122-7 (OECD
unique identifier: DAS- 59122-7) and TC1507 (OECD unique
identifier: DAS-01507-1) with NK603 (OECD unique identifier:
MON-00603-6). Corn rootworm-resistance is derived from DAS-59122- 7
which contains the Cry34Abl and Cry35Abl genes from Bacillus
thuringiensis strain P5149B1. Lepidopteran resistance and tolerance
to glufosinate ammonium herbicide is derived from TC1507. Tolerance
to glyphosate herbicide is derived from NK603. DBT418 Dekalb
Genetics Insect-resistant and glufosinate Corporation ammonium
herbicide tolerant maize developed by inserting genes encoding
Cry1AC protein from Bacillus thuringiensis subsp kurstaki and
phosphinothricin acetyltransferase (PAT) from Streptomyces
hygroscopicus. MIR604 .times. GA21 Syngenta Seeds, Inc. Stacked
insect resistant and herbicide tolerant maize produced by
conventional cross breeding of parental lines MIR604 (OECD unique
identifier: SYN-1R605-5) and GA21 (OECD unique identifier:
MON-00021-9). Corn rootworm-resistance is derived from MIR604 which
contains the mCry3A gene from Bacillus thuringiensis. Tolerance to
glyphosate herbicide is derived from GA21. MON80100 Monsanto
Company Insect-resistant maize produced by inserting the Cry1Ab
gene from Bacillus thuringiensis subsp. kurstaki. The genetic
modification affords resistance to attack by the European corn
borer (ECB). MON802 Monsanto Company Insect-resistant and
glyphosate herbicide tolerant maize produced by inserting the genes
encoding the Cry1Ab protein from Bacillus thuringiensis and the 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS) from A.
tumefaciens strain CP4. MON809 Pioneer Hi-Bred Resistance to
European corn borer International Inc. (Ostrinia nubilalis) by
introduction of a synthetic Cry1Ab gene. Glyphosate resistance via
introduction of the bacterial version of a plant enzyme,
5-enolpynivyl shikimate-3- phosphate synthase (EPSPS). MON810
Monsanto Company Insect-resistant maize produced by inserting a
truncated form of the Cry1Ab gene from Bacillus thuringiensis
subsp. kurstaki HD-1. The genetic modification affords resistance
to attack by the European corn borer (ECB). MON810 .times. LY038
Monsanto Company Stacked insect resistant and enhanced lysine
content maize derived from conventional crossbreeding of the
parental lines MON810 (OECD identifier: MON-OO81O-6) and LY038
(OECD identifier: REN-OOO38-3). MON810 .times. MON88017 Monsanto
Company Stacked insect resistant and glyphosate tolerant maize
derived from conventional cross-breeding of the parental lines
MON810 (OECD identifier: MON-OO81O- 6) and MON88017 (OECD
identifier: MON-88017-3). European corn borer (ECB) resistance is
derived from a truncated form of the Cry1Ab gene from Bacillus
thuringiensis subsp. kurstaki HD-1 present in MON810. Corn rootworm
resistance is derived from the Cry3Bbl gene from Bacillus
thuringiensis subspecies kumamotoensis strain EG4691 present in
MON88017. Glyphosate tolerance is derived from a 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene
from Agrobacterium tumefaciens strain CP4 present in MON88017.
MON832 Monsanto Company Introduction, by particle bombardment, of
glyphosate oxidase (GOX) and a modified 5- enolpyruvyl
shikimate-3-phosphate synthase (EPSPS), an enzyme involved in the
shikimate biochemical pathway for the production of the aromatic
amino acids. MON863 Monsanto Company Corn rootworm resistant maize
produced by inserting the Cry3Bbl gene from Bacillus thuringiensis
subsp. kumamotoensis. MON863 .times. MON810 Monsanto Company
Stacked insect resistant corn hybrid derived from conventional
cross-breeding of the parental lines MON863 (OECD identifier:
MON-00863-5) and MON810 (OECD identifier: MON-00810-6) MON863
.times. MON810 .times. Monsanto Company Stacked insect resistant
and Monsanto NK603 herbicide tolerant corn hybrid derived from
conventional crossbreeding of the stacked hybrid MON-00863-5
.times. MON- 00810-6 and NK603 (OECD identifier: MON-00603-6).
MON863 .times. NK603 Monsanto Company Stacked insect resistant and
herbicide tolerant corn hybrid derived from conventional
crossbreeding of the parental lines MON863 (OECD identifier:
MON-OO863-5) and NK603 (OECD identifier: MON-OO6O3-6). MON87460
Monsanto Company MON 87460 was developed to provide reduced yield
loss under water-limited conditions compared to conventional maize.
Efficacy in MON 87460 is derived by expression of the inserted
Bacillus subtilis cold shock protein B (CspB). MON88017 Monsanto
Company Corn rootworm-resistant maize produced by inserting the
Cry3Bbl gene from Bacillus thuringiensis subspecies kumamotoensis
strain EG4691. Glyphosate tolerance derived by inserting a 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene
from Agrobacterium tumefaciens strain CP4. MON89034 Monsanto
Company Maize event expressing two different insecticidal proteins
from Bacillus thuringiensis providing resistance to number of
Lepidopteran pests. MON89034 .times. Monsanto Company Stacked
insect resistant and MON88017 glyphosate tolerant maize derived
from conventional cross-breeding of the parental lines MON89034
(OECD identifier: MON-89O34-3) and MON88017 (OECD identifier:
MON-88O17-3). Resistance to Lepidopteran insects is derived from
two Cry genes present in MON89043. Corn rootworm resistance is
derived from a single Cry genes and glyphosate tolerance is derived
from the 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS)
encoding gene from Agrobacterium tumefaciens present in MON88017.
MON89034 .times. NK603 Monsanto Company Stacked insect resistant
and herbicide tolerant maize produced by conventional cross
breeding of parental lines MON89034 (OECD identifier: MON-89034-3)
with NK603 (OECD unique identifier: MON-00603-6). Resistance to
Lepidopteran insects is derived from two Cry genes present in
MON89043. Tolerance to glyphosate herbicide is derived from NK603.
NK603 .times. MON810 Monsanto Company Stacked insect resistant and
herbicide tolerant corn hybrid derived from conventional
crossbreeding of the parental lines NK603 (OECD identifier: MON-
00603-6) and MON810 (OECD identifier: MON-00810-6). MON89034
.times. TC1507 .times. Monsanto Company and Stacked insect
resistant and MON88017 .times. DAS- Mycogen Seeds c/o Dow herbicide
tolerant maize produced 59122-7 AgroSciences LLC by conventional
cross breeding of parental lines: MON89034, TC1507, MON88017, and
DAS-59 122. Resistance to the above- ground and below-ground insect
pests and tolerance to glyphosate and glufosinate-ammonium
containing herbicides. M53 Bayer CropScience Male sterility caused
by expression (Aventis of the barnase ribonuclease gene
CropScience(AgrEvo)) from Bacillus amyloliquefaciens; PPT
resistance was via PPT- acetyltransferase (PAT). M56 Bayer
CropScience Male sterility caused by expression (Aventis of the
barnase ribonuclease gene CropScience(AgrEvo) from Bacillus
amyloliquefaciens; PPT resistance was via PPT- acetyltransferase
(PAT). NK603 Monsanto Company Introduction, by particle
bombardment, of a modified 5- enolpyruvyl shikimate-3-phosphate
synthase (EPSPS), an enzyme involved in the shikimate biochemical
pathway for the production of the aromatic amino acids. NK603
.times. T25 Monsanto Company Stacked glufosinate ammonium and
glyphosate herbicide tolerant maize hybrid derived from
conventional cross-breeding of the parental lines NK603 (OECD
identifier: MON-00603-6) and T25 (OECD identifier: ACS-ZM003-2).
T25 .times. MON810 Bayer CropScience Stacked insect resistant and
(Aventis herbicide tolerant corn hybrid CropScience(AgrEvo))
derived from conventional crossbreeding of the parental lines T25
(OECD identifier: ACS- ZMOO3-2) and MON810 (OECD identifier:
MON-OO81O-6). TC1507 Mycogen (c/o Dow Insect-resistant and
glufosinate AgroSciences); Pioneer ammonium herbicide tolerant (c/o
DuPont) maize produced by inserting the Cry1F gene from Bacillus
thuringiensis var. aizawai and the phosphinothricin
N-acetyltransferase encoding gene from Streptomyces
viridochromogenes. TC1507 .times. NK603 DOW AgroSciences LLC
Stacked insect resistant and herbicide tolerant corn hybrid derived
from conventional crossbreeding of the parental lines 1507 (OECD
identifier: DAS- O15O7-1) and NK603 (OECD identifier: MON-OO6O3-6).
TC1507 .times. DAS-59122-7 DOW AgroSciences LLC Stacked insect
resistant and and Pioneer Hi-Bred herbicide tolerant maize produced
International Inc. by conventional cross breeding of parental lines
TC1507 (OECD unique identifier: DAS-O15O7-1) with DAS-59122-7 (OECD
unique identifier: DAS-59122-7). Resistance to Lepidopteran insects
is derived from TC1507 due the presence of the Cry1F gene from
Bacillus thuringiensis var. aizawai. Corn rootworm-resistance is
derived from DAS-59122-7 which contains the Cry34Ab1 and Cry35Ab1
genes from Bacillus thuringiensis strain P5149B1. Tolerance to
glufosinate ammonium herbicide is derived from TC1507 from the
phosphinothricin N-acetyltransferase encoding gene from
Streptomyces viridochromogenes. Event Company Description Hybrid
Family P0157 Dupont Pioneer P0157 P0157AM Dupont Pioneer AM LL RR2
P0157 P0157AMXT Dupont Pioneer AMXT LL RR2 P0157 P0157R Dupont
Pioneer RR2 P0157 P0339AM Dupont Pioneer AM LL RR2 P0339 P0339AMXT
Dupont Pioneer AMXT LL RR2 P0339 P0306AM Dupont Pioneer AM LL RR2
P0306 P0589 Dupont Pioneer P0589 P0589AM Dupont Pioneer AM LL RR2
P0589 P0589AMXT Dupont Pioneer AMXT LL RR2 P0589 P0589R Dupont
Pioneer RR2 P0589 P0574 Dupont Pioneer P0574 P0574AM Dupont Pioneer
AM LL RR2 P0574 P0574AMXT Dupont Pioneer AMXT LL RR2 P0574 P0533EXR
Dupont Pioneer HXX LL RR2 P0533 P0506AM Dupont Pioneer AM LL RR2
P0566 P0760AMXT Dupont Pioneer AMXT LL RR2 P0760 P0707AM Dupont
Pioneer AM LL RR2 P0707 P0707AMXT Dupont Pioneer AMXT LL RR2 P0707
P0825AM Dupont Pioneer AM LL RR2 P0825 P0825AMXT Dupont Pioneer
AMXT LL RR2 P0825 P0969AM Dupont Pioneer AM LL RR2 P0969 P0969AMXT
Dupont Pioneer AMXT LL RR2 P0969 P0937AM Dupont Pioneer AM LL RR2
P0937 P0919AM Dupont Pioneer AM LL RR2 P0919 P0905EXR Dupont
Pioneer HXX LL RR2 P0905 P1197 Dupont Pioneer P1197 P1197AM Dupont
Pioneer AM LL RR2 P1197 P1197AMXT Dupont Pioneer AMXT LL RR2 P1197
P1197R Dupont Pioneer RR2 P1197 P1151 Dupont Pioneer P1151 P1151AM
Dupont Pioneer AM LL RR2 P1151 P1151R Dupont Pioneer RR2 P1151
P1138AM Dupont Pioneer AM LL RR2 P1138 P1366AM Dupont Pioneer AM LL
RR2 P1366 P1366AMXT Dupont Pioneer AMXT LL RR2 P1366 P1365AMX
Dupont Pioneer AMX LL RR2 P1365 P1353AM Dupont Pioneer AM LL RR2
P1353 P1345 Dupont Pioneer P1345 P1311AMXT Dupont Pioneer AMXT LL
RR2 P1311 P1498EHR Dupont Pioneer HX1 LL RR2 P1498 P1498R Dupont
Pioneer RR2 P1498 P1443AM Dupont Pioneer AM LL RR2 P1443 P1555CHR
Dupont Pioneer RW HX1 LL RR2 P1555 P1751AMT Dupont Pioneer AMT LL
RR2 P1751 P2089AM Dupont Pioneer AM LL RR2 P2089 QROME Dupont
Pioneer Q LL RR2
[0435] The following are the definitions for the shorthand
occurring in Table 19. AM--OPTIMUM ACREMAX Insect Protection system
with YGCB, HX1, LL, AMT--OPTIMUM ACREMAX TRISECT insect Protection
System with RW, YGCB, HX1, LL, RR2. AMXT--(OPTIMUM ACREMAX XTreme).
HXX--HERCULEX XTRA contains the Herculex I and Herculex RW genes.
HX--Contains the HERCULEX 1 Insect Protection gene which provides
protection against European corn borer, southwestern corn borer,
black cutworm, fill armyworm, western bean cutworm, lesser corn
stalk borer, southern corn stalk borer, and sugarcane borer; and
suppresses corn earworm. LL--Contains the LIBERTYLINK gene for
resistance to LIBERTY herbicide. RR2--Contains the ROUNDUP READY
Corn 2 trait that provides crop safety for over-the-top
applications of labeled glyphosate herbicides when applied
according to label directions. YGCB--contains the YIELDGARD Corn
Borer gene offers a high level of resistance to European corn
borer, southwestern corn borer, and southern cornstalk borer;
moderate resistance to corn earworm and common stalk borer; and
above average resistance to fall armyworm. RW--contains the
AGRISURE rootworm resistance trait. Q--provides protection or
suppression against susceptible European corn borer, southwestern
corn borer, black cutworm, fall armyworm, lesser corn stalk borer,
southern corn stalk borer, stalk borer, sugarcane borer, and corn
earworm; and also provides protection from larval injury caused by
susceptible western corn rootworm, northern corn rootworm, and
Mexican corn rootworm; contains (1) HERCULEX XTRA insect Protection
genes that produce Cry1F and Cry34ab1 and Cry35ab1 proteins, (2)
AGRISURE RW trait that includes a gene that produces mCry3A
protein, and (3) YIELDGARD Corn Borer gene which produces Cry1 Ab
protein.
Concentrations and Rates of Application of Agricultural
Compositions
[0436] As aforementioned, the agricultural compositions of the
present disclosure, which comprise a taught microbe, can be applied
to plants in a multitude of ways. In two particular aspects, the
disclosure contemplates an in-furrow treatment or a seed
treatment
[0437] For seed treatment embodiments, the microbes of the
disclosure can be present on the seed in a variety of
concentrations. For example, the microbes can be found in a seed
treatment at a cfu concentration, per seed of: 1.times.10.sup.1,
1.times.10.sup.2, 1.times.10.sup.3, 1.times.10.sup.4,
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10, or more. In
particular aspects, the seed treatment compositions comprise about
1.times.10.sup.4 to about 1.times.10.sup.8 cfu per seed. In other
particular aspects, the seed treatment compositions comprise about
1.times.10.sup.5 to about 1.times.10.sup.7 cfu per seed. In other
aspects, the seed treatment compositions comprise about
1.times.10.sup.6 cfu per seed.
[0438] In the United States, about 10% of corn acreage is planted
at a seed density of above about 36,000 seeds per acre; 1/3 of the
corn acreage is planted at a seed density of between about 33,000
to 36,000 seeds per acre; 1/3 of the corn acreage is planted at a
seed density of between about 30,000 to 33,000 seeds per acre, and
the remainder of the acreage is variable. See, "Corn Seeding Rate
Considerations," written by Steve Butzen, available at:
www.pioneer.com/home/site/us/agronomy/library/corn-seeding-rate-considera-
tions/.
[0439] Table 20 below utilizes various cfu concentrations per seed
in a contemplated seed treatment embodiment (rows across) and
various seed acreage planting densities (1.sup.st column: 15K-41K)
to calculate the total amount of cfu per acre, which would be
utilized in various agricultural scenarios (i.e. seed treatment
concentration per seed.times.seed density planted per acre). Thus,
if one were to utilize a seed treatment with 1.times.10.sup.6 cfu
per seed and plant 30,000 seeds per acre, then the total cfu
content per acre would be 3.times.10.sup.10 (i.e.
30K*1.times.10.sup.6).
TABLE-US-00020 TABLE 20 Total CFU Per Acre Calculation for Seed
Treatment Embodiments Corn Population (i.e. seeds per acre)
1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08
1.00E+09 15,000 1.50E+06 1.50E+07 1.50E+08 1.50E+09 1.50E+10
1.50E+11 1.50E+12 1.50E+13 16,000 1.60E+06 1.60E+07 1.60E+08
1.60E+09 1.60E+10 1.60E+11 1.60E+12 1.60E+13 17,000 1.70E+06
1.70E+07 1.70E+08 1.70E+09 1.70E+10 1.70E+11 1.70E+12 1.70E+13
18,000 1.80E+06 1.80E+07 1.80E+08 1.80E+09 1.80E+10 1.80E+11
1.80E+12 1.80E+13 19,000 1.90E+06 1.90E+07 1.90E+08 1.90E+09
1.90E+10 1.90E+11 1.90E+12 1.90E+13 20,000 2.00E+06 2.00E+07
2.00E+08 2.00E+09 2.00E+10 2.00E+11 2.00E+12 2.00E+13 21,000
2.10E+06 2.10E+07 2.10E+08 2.10E+09 2.10E+10 2.10E+11 2.10E+12
2.10E+13 22,000 2.20E+06 2.20E+07 2.20E+08 2.20E+09 2.20E+10
2.20E+11 2.20E+12 2.20E+13 23,000 2.30E+06 2.30E+07 2.30E+08
2.30E+09 2.30E+10 2.30E+11 2.30E+12 2.30E+13 24,000 2.40E+06
2.40E+07 2.40E+08 2.40E+09 2.40E+10 2.40E+11 2.40E+12 2.40E+13
25,000 2.50E+06 2.50E+07 2.50E+08 2.50E+09 2.50E+10 2.50E+11
2.50E+12 2.50E+13 26,000 2.60E+06 2.60E+07 2.60E+08 2.60E+09
2.60E+10 2.60E+11 2.60E+12 2.60E+13 27,000 2.70E+06 2.70E+07
2.70E+08 2.70E+09 2.70E+10 2.70E+11 2.70E+12 2.70E+13 28,000
2.80E+06 2.80E+07 2.80E+08 2.80E+09 2.80E+10 2.80E+11 2.80E+12
2.80E+13 29,000 2.90E+06 2.90E+07 2.90E+08 2.90E+09 2.90E+10
2.90E+11 2.90E+12 2.90E+13 30,000 3.00E+06 3.00E+07 3.00E+08
3.00E+09 3.00E+10 3.00E+11 3.00E+12 3.00E+13 31,000 3.10E+06
3.10E+07 3.10E+08 3.10E+09 3.10E+10 3.10E+11 3.10E+12 3.10E+13
32,000 3.20E+06 3.20E+07 3.20E+08 3.20E+09 3.20E+10 3.20E+11
3.20E+12 3.20E+13 33,000 3.30E+06 3.30E+07 3.30E+08 3.30E+09
3.30E+10 3.30E+11 3.30E+12 3.30E+13 34,000 3.40E+06 3.40E+07
3.40E+08 3.40E+09 3.40E+10 3.40E+11 3.40E+12 3.40E+13 35,000
3.50E+06 3.50E+07 3.50E+08 3.50E+09 3.50E+10 3.50E+11 3.50E+12
3.50E+13 36,000 3.60E+06 3.60E+07 3.60E+08 3.60E+09 3.60E+10
3.60E+11 3.60E+12 3.60E+13 37,000 3.70E+06 3.70E+07 3.70E+08
3.70E+09 3.70E+10 3.70E+11 3.70E+12 3.70E+13 38,000 3.80E+06
3.80E+07 3.80E+08 3.80E+09 3.80E+10 3.80E+11 3.80E+12 3.80E+13
39,000 3.90E+06 3.90E+07 3.90E+08 3.90E+09 3.90E+10 3.90E+11
3.90E+12 3.90E+13 40,000 4.00E+06 4.00E+07 4.00E+08 4.00E+09
4.00E+10 4.00E+11 4.00E+12 4.00E+13 41,000 4.10E+06 4.10E+07
4.10E+08 4.10E+09 4.10E+10 4.10E+11 4.10E+12 4.10E+13
[0440] For in-furrow embodiments, the microbes of the disclosure
can be applied at a cfu concentration per acre of:
1.times.10.sup.6, 3.20.times.10.sup.10, 1.60.times.10.sup.11,
3.20.times.10.sup.11, 8.0.times.10.sup.11, 1.6.times.10.sup.12,
3.20.times.10.sup.12, or more. Therefore, in aspects, the liquid
in-furrow compositions can be applied at a concentration of between
about 1.times.10.sup.6 to about 3.times.10.sup.12 cfu per acre.
[0441] In some aspects, the in-furrow compositions are contained in
a liquid formulation. In the liquid in-furrow embodiments, the
microbes can be present at a cfu concentration per milliliter of:
1.times.10.sup.1, 1.times.10.sup.2, 1.times.10.sup.3,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.10, 1.times.10.sup.11, 1.times.10.sup.12,
1.times.10.sup.13, or more. In certain aspects, the liquid
in-furrow compositions comprise microbes at a concentration of
about 1.times.10.sup.6 to about 1.times.10.sup.11 cfu per
milliliter. In other aspects, the liquid in-furrow compositions
comprise microbes at a concentration of about 1.times.10.sup.7 to
about 1.times.10.sup.10 cfu per milliliter. In other aspects, the
liquid in-furrow compositions comprise microbes at a concentration
of about 1.times.10.sup.8 to about 1.times.10.sup.9 cfu per
milliliter. In other aspects, the liquid in-furrow compositions
comprise microbes at a concentration of up to about
1.times.10.sup.13 cfu per milliliter.
Transcriptomic Profiling of Candidate Microbes
[0442] Previous work by the inventors entailed transcriptomic
profiling of strain CI010 to identify promoters that are active in
the presence of environmental nitrogen. Strain CI010 was cultured
in a defined, nitrogen-free media supplemented with 10 mM
glutamine. Total RNA was extracted from these cultures (QIAGEN
RNeasy kit) and subjected to RNAseq sequencing via Illumina HiSeq
(SeqMatic, Fremont Calif.). Sequencing reads were mapped to the
CI010 genome data using Geneious, and highly expressed genes under
control of proximal transcriptional promoters were identified.
[0443] Tables 21-23 lists genes and their relative expression level
as measured through RNASeq sequencing of total RNA. Sequences of
the proximal promoters were recorded for use in mutagenesis of nif
pathways, nitrogen utilization related pathways, or other genes
with a desired expression level.
TABLE-US-00021 TABLE 21 Name Minimum Maximum Length Direction
murein lipoprotein CDS 2,929,898 2,930,134 237 forward membrane
protein CDS 5,217,517 5,217,843 327 forward zinc/cadmium-binding
3,479,979 3,480,626 648 forward protein CDS acyl carrier protein
CDS 4,563,344 4,563,580 237 reverse ompX CDS 4,251,002 4,251,514
513 forward DNA-binding protein 375,156 375,428 273 forward HU-beta
CDS sspA CDS 629,998 630,636 639 reverse taffi CDS 3,199,435
3,199,638 204 reverse LexA repressor CDS 1,850,457 1,851,065 609
forward hisS CDS <3999979 4,001,223 >1245 forward
TABLE-US-00022 TABLE 22 Differential Expression Differential
RNASeq_nifL - RNASeq_nifL - RNASeq_WT - RNASeq_WT - Absolute
Expression Raw Read Raw Transcript Raw Read Raw Transcript Name
Confidence Ratio Count Count Count Count murein 1000 -1.8 12950.5
10078.9 5151.5 4106.8 lipoprotein CDS membrane 1000 -1.3 9522.5
5371.3 5400 3120 protein CDS zinc/cadmium- 3.3 1.1 6461 1839.1 5318
1550.6 binding protein CDS acyl carrier 25.6 1.6 1230.5 957.6
1473.5 1174.7 protein CDS ompX CDS 1.7 1.1 2042 734.2 1687.5 621.5
DNA-binding 6.9 -1.3 1305 881.7 725 501.8 protein HU- beta CDS sspA
CDS 0.2 1 654 188.8 504.5 149.2 tatE CDS 1.4 1.3 131 118.4 125
115.8 LexA 0.1 -1.1 248 75.1 164 50.9 repressor CDS hisS CDS 0 -1.1
467 69.2 325 49.3
TABLE-US-00023 TABLE 23 Prm (In Forward direction, -250 Expressed
Neighbor to +10 region) Sequence Sequence Name SEQ ID NO: SEQ ID
NO: SEQ ID NO: murein lipoprotein CDS SEQ ID NO: 3 SEQ ID NO: 13
SEQ ID NO: 23 membrane protein CDS SEQ ID NO: 4 SEQ ID NO: 14 SEQ
ID NO: 24 zinc/cadmium-binding SEQ ID NO: 5 SEQ ID NO: 15 SEQ ID
NO: 25 protein CDS acyl carrier protein CDS SEQ ID NO: 6 SEQ ID NO:
16 SEQ ID NO: 26 ompX CDS SEQ ID NO: 7 SEQ ID NO: 17 SEQ ID NO. 27
DNA-binding protein SEQ ID NO: 8 SEQ ID NO: 18 SEQ ID NO: 28
HU-beta CDS sspA CDS SEQ ID NO: 9 SEQ ID NO: 19 SEQ ID NO: 29 tatE
CDS SEQ ID NO: 10 SEQ ID NO: 20 SEQ ID NO: 30 LexA repressor CDS
SEQ ID NO: 11 SEQ ID NO: 21 SEQ ID NO: 31 hisS CDS SEQ ID NO: 12
SEQ ID NO: 22 SEQ ID NO: 32
TABLE-US-00024 TABLE 24 Table of Strains Mutagenic DNA Gene 1 Gene
2 Name Lineage Description Genotype mutation mutation CI006
Isolated strain from None WT Enterobacter (now Kosakonia) genera
CI008 Isolated strain from None WT Burkholderia (now
Paraburkholderia) genera CI010 Isolated strain from None WT
Klebsiella genera CI019 Isolated strain from None WT Rahnella
genera CI028 Isolated strain from None WT Enterobacter genera CI050
Isolated strain from None WT Klebsiella genera CM002 Mutant of
CI050 Disruption of nifL gene .DELTA.nifL::KanR SEQ ID with a
kanamycin resistance NO: 33 expression cassette (KanR) encoding the
aminoglycoside O- phosphotransferase gene aph1 inserted. CM011
Mutant of CI019 Disruption of nifL gene .DELTA.nifL::SpecR SEQ ID
with a spectinomycin NO: 34 resistance expression cassette (SpecR)
encoding the streptomycin 3''-O- adenylyltransferase gene aadA
inserted. CM013 Mutant of CI006 Disruption of nifL gene
.DELTA.nifL::KanR SEQ ID with a kanamycin resistance NO: 35
expression cassette (KanR) encoding the aminoglycoside O-
phosphotransferase gene aph1 inserted. CM004 Mutant of CI010
Disruption of amtB gene .DELTA.amtB::KanR SEQ ID with a kanamycin
resistance NO: 36 expression cassette (KanR) encoding the
aminoglycoside O- phosphotransferase gene aph1 inserted. CM005
Mutant of CI010 Disruption of nifL gene .DELTA.nifL::KanR SEQ ID
with a kanamycin resistance NO: 37 expression cassette (KanR)
encoding the aminoglycoside O- phosphotransferase gene aph1
inserted. CM015 Mutant of CI006 Disruption of nifL gene
.DELTA.nifL::Prm5 SEQ ID with a fragment of the NO: 38 region
upstream of the ompX gene inserted (Prm5). CM021 Mutant of CI006
Disruption of nifL gene .DELTA.nifL::Prm2 SEQ ID with a fragment of
the NO: 39 region upstream of an unannotated gene and the first 73
bp of that gene inserted (Prm2). CM023 Mutant of CI006 Disruption
of nifL gene .DELTA.nifL::Prm4 SEQ ID with a fragm ent of the NO:
40 region upstream of the acpP gene and the first 121 bp of the
acpP gene inserted (Prm4). CM014 Mutant of CI006 Disruption of nifL
gene .DELTA.nifL::Prm1 SEQ ID with a fragment of the NO: 41 region
upstream of the lpp gene and the first 29 bp of the lpp gene
inserted (Prm1). CM016 Mutant of CI006 Disruption of nifL gene
.DELTA.nifL::Prm9 SEQ ID with a fragment of the NO: 42 region
upstream of the lexA 3 gene and the first 21 bp of the lexA 3 gene
inserted (Prm9). CM022 Mutant of CI006 Disruption of nifL gene
.DELTA.nifL::Prm3 SEQ ID with a fragment of the NO: 43 region
upstream of the mntP 1 gene and the first 53 bp of the mntP 1 gene
inserted (Prm3). CM024 Mutant of CI006 Disruption of nifL gene
.DELTA.nifL::Prm7 SEQ ID with a fragment of the NO: 44 region
upstream of the sspA gene inserted (Prm7). CM025 Mutant of CI006
Disruption of nifL gene .DELTA.nifL::Prm10 SEQ ID with a fragment
of the NO: 45 region upstream of the hisS gene and the first 52 bp
of the hisS gene inserted (Prm10). CM006 Mutant of CI010 Disruption
of glnB gene .DELTA.glnB::KanR SEQ ID with a kanamycin resistance
NO: 46 expression cassette (KanR) encoding the aminoglycoside O-
phosphotransferase gene aph1 inserted. CM017 Mutant of CI028
Disruption of nifL gene .DELTA.nifL::KanR SEQ ID with a kanamycin
resistance NO: 47 expression cassette (KanR) encoding the
aminoglycoside O- phosphotransferase gene aph1 inserted. CM011
Mutant of CI019 Disruption of nifL gene .DELTA.nifL::SpecR SEQ ID
with a spectinomycin NO: 48 resistance expression cassette (SpecR)
encoding the streptomycin 3''-O- adenylyltransferase gene aadA
inserted. CM013 Mutant of CI006 Disruption of nifL gene
.DELTA.nifL::KanR SEQ ID with a kanamycin resistance NO: 49
expression cassette (KanR) encoding the aminoglycoside O-
phosphotransferase gene aph1 inserted. CM005 Mutant of CI010
Disruption of nifL gene .DELTA.nifL::KanR SEQ ID with a kanamycin
resistance NO: 50 expression cassette (KanR) encoding the
aminoglycoside O- phosphotransferase gene aph1 inserted. CM014
Mutant of CI006 Disruption of nifL gene .DELTA.nifL::Prm1 SEQ ID
with a fragment of the NO: 51 region upstream of the lpp gene and
the first 29 bp of the lpp gene inserted (Prm1). CM015 Mutant of
CI006 Disruption of nifL gene .DELTA.nifL::Prm5 SEQ ID with a
fragment of the NO: 52 region upstream of the ompX gene inserted
(Pnn5). CM023 Mutant of CI006 Disruption of nifL gene
.DELTA.nifL::Prm4 SEQ ID with a fragment of the NO: 53 region
upstream of the acpP gene and the first 121 bp of the acpP gene
inserted (Prm4). CM029 Mutant of CI006 Disruption of nifL gene
.DELTA.nifL::Prm5 SEQ ID SEQ ID with a fragment of the .DELTA.glnE-
NO: 54 NO: 61 region upstream of the AR_KO1 ompX gene inserted
(Prm5) and deletion of the 1287 bp after the start codon of the
glnE gene containing the adenylyl-removing domain of
glutamate-ammonia- ligase adenylyltransferase (.DELTA.glnE-AR_KO1).
CM014 Mutant of CI006 Disruption of nifL gene .DELTA.nifL::Prm1 SEQ
ID with a fragment of the NO: 55 region upstream of the lpp gene
and the first 29 bp of the lpp gene inserted (Prm1). CM011 Mutant
of CI019 Disruption of nifL gene .DELTA.nifL::SpecR SEQ ID with a
spectinomycin NO: 56 resistance expression cassette (SpecR)
encoding the streptomycin 3''-O- adenylyltransferase gene aadA
inserted. CM011 Mutant of CI019 Disruption of nifL gene
.DELTA.nifL::SpecR SEQ ID with a spectinomycin NO: 57 resistance
expression cassette (SpecR) encoding the streptomycin 3''-O-
adenylyltransferase gene aadA inserted. CM013 Mutant of CI006
Disruption of nifL gene .DELTA.nifL::KanR SEQ ID with a kanamycin
resistance NO: 58 expression cassette (KanR) encoding the
aminoglycoside O- phosphotransferase gene aph1 inserted. CM011
Mutant of CI019 Disruption of nifL gene .DELTA.nifL::SpecR SEQ ID
with a spectinomycin NO: 59 resistance expression cassette (SpecR)
encoding the streptomycin 3''-O- adenylyltransferase gene aadA
inserted. CM011 Mutant of CI019 Disruption of nifL gene
.DELTA.nifL::SpecR SEQ ID with a spectinomycin NO: 60 resistance
expression cassette (SpecR) encoding the streptomycin 3''-O-
adenylyltransferase gene aadA inserted.
EXAMPLES
[0444] The following examples are given for the purpose of
illustrating various embodiments of the disclosure and are not
meant to limit the present disclosure in any fashion. Changes
therein and other uses which are encompassed within the spirit of
the disclosure, as defined by the scope of the claims, will be
recognized by those skilled in the art.
Example 1: Guided Microbial Remodeling--a Platform for the Rational
Improvement of Microbial Species for Agriculture
[0445] An example overview of an embodiment of the Guided Microbial
Remodeling (GMR) platform can be summarized in the schematic of
FIG. 1A.
[0446] FIG. 1A illustrates that the composition of the microbiome
can first be characterized and a species of interest is identified
(e.g. to find a microbe with the appropriate colonization
characteristics).
[0447] The metabolism of the species of interest can be mapped and
linked to genetics. For example, the nitrogen fixation pathway of
the microbe can be characterized. The pathway that is being
characterized can be examined under a range of environmental
conditions. For example, the microbe's ability to fix atmospheric
nitrogen in the presence of various levels of exogenous nitrogen in
its environment can be examined. The metabolism of nitrogen can
involve the entrance of ammonia (NH.sub.4.sup.+) from the
rhizosphere into the cytosol of the bacteria via the AmtB
transporter. Ammonia and L-glutamate (L-Glu) are catalyzed by
glutamine synthetase and ATP into glutamine. Glutamine can lead to
the formation of bacterial biomass and it can also inhibit
expression of the nif operon, i.e. it can be a competing force when
one desires the microbe to fix atmospheric nitrogen and excrete
ammonia. The nitrogen fixation pathway is characterized in great
detail in earlier sections of the specification.
[0448] Afterwards, a targeted non-intergeneric genomic alteration
can be introduced to the microbe's genome, using methods including,
but not limited to: conjugation and recombination, chemical
mutagenesis, adaptive evolution, and gene editing. The targeted
non-intergeneric genomic alteration can include an insertion,
disruption, deletion, alteration, perturbation, modification, etc.
of the genome.
[0449] Derivative remodeled microbes, which comprise the desired
phenotype resulting from the remodeled underlying genotype, are
then used to inoculate crops.
[0450] The present disclosure provides, in certain embodiments,
non-intergeneric remodeled microbes that are able to fix
atmospheric nitrogen and supply such nitrogen to a plant. In
aspects, these non-intergeneric remodeled microbes are able to fix
atmospheric nitrogen, even in the presence of exogenous
nitrogen.
[0451] FIG. 1B depicts an expanded view of the measurement of the
microbiome step. In some embodiments, the present disclosure finds
microbial species that have desired colonization characteristics,
and then utilizes those species in the subsequent remodeling
process.
[0452] The aforementioned Guided Microbial Remodeling (GMR)
platform will now be described with more specificity.
[0453] In aspects, the GMR platform comprises the following steps:
[0454] A. Isolation--Obtain microbes from the soil, rhizosphere,
surface, etc. of a crop plant of interest; [0455] B.
Characterization--Involves characterizing the isolated microbes for
genotype/phenotypes of interest (e.g. genome sequence, colonization
ability, nitrogen fixation activity, solubilization of P ability,
excretion of a metabolite of interest, excretion of a plant
promoting compound, etc.) [0456] C. Domestication--Development of a
molecular protocol for non-intergeneric genetic modification of the
microbe; [0457] D. Non-Intergeneric Engineering Campaign and
Optimization--Generation of derivative non-intergeneric microbial
strains with genetic modifications in key pathways (e.g.
colonization associated genes, nitrogen fixation/assimilation
genes, P solubilization genes); [0458] E. Analytics--Evaluation of
derived non-intergeneric strains for phenotypes of interest both in
vitro (e.g., ARA assays) and in planta (e.g. colonization assays).
[0459] F. Iterate Engineering Campaign/Analytics--Iteration of
steps D and E for further improvement of microbial strain.
[0460] Each of the GMR platform process steps will now be
elaborated upon below.
A. Isolation of Microbes
[0461] 1. Obtain a Soil Sample
[0462] Microbes will be isolated from soil and/or roots of a plant.
In one example, plants will be grown in a laboratory or a
greenhouse in small pots. Soil samples will be obtained from
various agricultural areas. For example, soils with diverse texture
characteristics can be collected, including loam (e.g. peaty clay
loam, sandy loam), clay soil (e.g. heavy clay, silty clay), sandy
soil, silty soil, peaty soil, chalky soil, and the like.
[0463] 2. Grow Bait Plants
[0464] Seeds of a bait plant (a plant of interest) (e.g. corn,
wheat, rice, sorghum, millet, soybean, vegetables, fruits, etc.)
will be planted into each soil type. In one example, different
varieties of a bait plant will be planted in various soil types.
For example, if the plant of interest is corn, seeds of different
varieties of corn such as field corn, sweet corn, heritage corn,
etc. will be planted in various soil types described above.
[0465] 3. Harvest Soil and/or Root Samples and Plate on Appropriate
Medium
[0466] Plants will be harvested by uprooting them after a few weeks
(e.g. 2-4 weeks) of growth. Alternative to growing plants in a
laboratory/greenhouse, soil and/or roots of the plant of interest
can be collected directly from the fields with different soil
types.
[0467] To isolate rhizosphere microbes and epiphytes, plants will
be removed gently by saturating the soil with distilled water or
gently loosening the soil by hand to avoid damage to the roots. If
larger soil particles are present, these particles will be removed
by submerging the roots in a still pool of distilled water and/or
by gently shaking the roots. The root will be cut and a slurry of
the soil sticking to the root will be prepared by placing the root
in a plate or tube with small amount of distilled water and gently
shaking the plate/tube on a shaker or centrifuging the tube at low
speed. This slurry will be processed as described below.
[0468] To isolate endophytes, excess soil on root surfaces will be
removed with deionized water. Following soil removal, plants will
be surface sterilized and rinsed vigorously in sterile water. A
cleaned, 1 cm section of root will be excised from the plant and
placed in a phosphate buffered saline solution containing 3 mm
steel beads. A slurry will be generated by vigorous shaking of the
solution with a Qiagen TissueLyser II.
[0469] The soil and/or root slurry can be processed in various ways
depending on the desired plant-beneficial trait of microbes to be
isolated. For example, the soil and root slurry can be diluted and
inoculated onto various types of screening media to isolate
rhizospheric, endophytic, epiphytic, and other plant-associated
microbes. For example, if the desired plant-beneficial trait is
nitrogen fixation, then the soil/root slurry will be plated on a
nitrogen free media (e.g. Nfb agar media) to isolate nitrogen
fixing microbes. Similarly, to isolate phosphate solubilizing
bacteria (PSB), media containing calcium phosphate as the sole
source of phosphorus can be used. PSB can solubilize calcium
phosphate and assimilate and release phosphorus in higher amounts.
This reaction is manifested as a halo or a clear zone on the plate
and can be used as an initial step for isolating PSB.
[0470] 4. Pick Colonies, Purify Cultures, and Screen for the
Presence of Genes of Interest
[0471] Populations of microbes obtained in step A3 are streaked to
obtain single colonies (pure cultures). A part of the pure culture
is resuspended in a suitable medium (e.g. a mixture of R2A and
glycerol) and subjected to PCR analysis to screen for the presence
of one or more genes of interest. For example, to identify nitrogen
fixing bacteria (diazotrophs), purified cultures of isolated
microbes can be subjected to a PCR analysis to detect the presence
of nif genes that encode enzymes involved in the fixation of
atmospheric nitrogen into a form of nitrogen available to living
organisms.
[0472] 5. Bank a Purified Culture
[0473] Purified cultures of isolated strains will be stored, for
example at -80.degree. C., for future reference and analysis.
B. Characterization of Isolated Microbes
[0474] 1. Phylogenetic Characterization and Whole Genome
Sequencing
[0475] Isolated microbes will be analyzed for phylogenetic
characterization (assignment of genus and species) and the whole
genome of the microbes will be sequenced.
[0476] For phylogenetic characterization, 16S rDNA of the isolated
microbe will be sequenced using degenerate 16S rDNA primers to
generate phylogenetic identity. The 16S rDNA sequence reads will be
mapped to a database to initially assign the genus, species and
strain name for isolated microbes. Whole genome sequencing is used
as the final step to assign phylogenetic genus/species to the
microbes.
[0477] The whole genome of the isolated microbes will be sequenced
to identify key pathways. For the whole genome sequencing, the
genomic DNA will be isolated using a genomic DNA isolation kit
(e.g. QIAmp DNA mini kit from QIAGEN) and a total DNA library will
be prepared using the methods known in the art. The whole genome
will be sequenced using high throughput sequencing (also called
Next Generation Sequencing) methods known in the art. For example,
Illumina, Inc., Roche, and Pacific Biosciences provide whole genome
sequencing tools that can be used to prepare total DNA libraries
and perform whole genome sequencing.
[0478] The whole genome sequence for each isolated strain will be
assembled; genes of interest will be identified; annotated; and
noted as potential targets for remodeling. The whole genome
sequences will be stored in a database.
[0479] 2. Assay the Microbe for Colonization of a Host Plant in a
Greenhouse
[0480] Isolated microbes will be characterized for the colonization
of host plants in a greenhouse. For this, seeds of the desired host
plant (e.g., corn, wheat, rice, sorghum, and soybean) will be
inoculated with cultures of isolated microbes individually or in
combination and planted into soil. Alternatively, cultures of
isolated microbes, individually or in combination, can be applied
to the roots of the host plant by inoculating the soil directly
over the roots. The colonization potential of the microbes will be
assayed, for example, using a quantitative PCR (qPCR) method
described in a greater detail below.
[0481] 3. Assay the Microbe for Colonization of the Host Plant in
Small-Scale Field Trials and Isolate RNA from Colonized Root
Samples (CAT Trials)
[0482] Isolated microbes will be assessed for colonization of the
desired host plant in small-scale field trials. Additionally, RNA
will be isolated from colonized root samples to obtain
transcriptome data for the strain in a field environment. These
small-scale field trials are referred to herein as CAT
(Colonization and Transcript) trials, as these trials provide
Colonization and Transcript data for the strain in a field
environment.
[0483] For these trials, seeds of the host plant (e.g., corn,
wheat, rice, sorghum, and soybean) will be inoculated using
cultures of isolated microbes individually or in combination and
planted into soil. Alternatively, cultures of isolated microbes,
individually or in combination, can be applied to the roots of the
host plant by inoculating the soil directly over the roots. The CAT
trials can be conducted in a variety of soils and/or under various
temperature and/or moisture conditions to assess the colonization
potential and obtain transcriptome profile of the microbe in
various soil types and environmental conditions.
[0484] Colonization of roots of the host plant by the inoculated
microbe(s) will be assessed, for example, using a qPCR method as
described below.
[0485] In one protocol, the colonization potential of isolated
microbes was assessed as follows. One day after planting of corn
seeds, 1 ml of microbial overnight culture (SOB media) was drenched
right at the spot of where the seed was located. 1 mL of this
overnight culture was roughly equivalent to about 10{circumflex
over ( )}9 cfu, varying within 3-fold of each other, depending on
which strain is being used. Each seedling was fertilized 3.times.
weekly with 50 mL modified Hoagland's solution supplemented with
either 2.5 mM or 0.25 mM ammonium nitrate. At four weeks after
planting, root samples were collected for DNA extraction. Soil
debris were washed away using pressurized water spray. These tissue
samples were then homogenized using QIAGEN Tissuelyzer and the DNA
was then extracted using QIAmp DNA Mini Kit (QIAGEN) according to
the recommended protocol. qPCR assay was performed using Stratagene
Mx3005P RT-PCR on these DNA extracts using primers that were
designed (using NCBI's Primer BLAST) to be specific to a loci in
each of the microbe's genome.
[0486] The presence of the genome copies of the microbe was
quantified, which reflected the colonization potential of the
microbe. Identity of the microbial species was confirmed by
sequencing the PCR amplification products.
[0487] Additionally, RNA will be isolated from colonized root
and/or soil samples and sequenced.
[0488] Unlike the DNA profile, an RNA profile varies depending on
the environmental conditions. Therefore, sequencing of RNA isolated
from colonized roots and/or soil will reflect the transcriptional
activity of genes in planta in the rhizosphere.
[0489] RNA can be isolated from colonized root and/or soil samples
at different time points to analyze the changes in the RNA profile
of the colonized microbe at these time points.
[0490] For example, RNA can be isolated from colonized root and/or
soil samples right after fertilization of the field and a few weeks
after fertilization of the field and sequenced to generate
corresponding transcriptional profile.
[0491] Similarly, RNA sequencing can be carried out under high
phosphate and low phosphate conditions to understand which genes
are transcriptionally active or repressed under these
conditions.
[0492] Methods for transcriptomic/RNA sequencing are known in the
art. Briefly, total RNA will be isolated from the purified culture
of the isolated microbe; cDNA will be prepared using reverse
transcriptase; and the cDNA will be sequenced using high throughput
sequencing tools described above.
[0493] Sequencing reads from the transcriptome analysis can be
mapped to the genomic sequence and transcriptional promoters for
the genes of interest can be identified.
[0494] 4. Assay the Plant-Beneficial Activity of Isolated
Microbes
[0495] The plant-beneficial activity of isolated microbes will be
assessed.
[0496] For example, nitrogen fixing microbes will be assayed for
nitrogen fixation activity using an acetylene reduction assay (ARA)
or phosphate solubilizing microbes will be assayed for phosphate
solubilization. Any parameter of interest can be utilized and an
appropriate assay developed for such. For instance, assays could
include growth curves for colonization metrics and assays for
production of phytohormones like indole acetic acid (IAA) or
gibberellins. An assay for any plant-beneficial activity that is of
interest can be developed.
[0497] This step will confirm the phenotype of interest and
eliminate any false positives.
[0498] 5. Selection of Potential Candidates from Isolated
Microbes
[0499] The data generated in the above steps will be used to select
microbes for further development. For example, microbes showing a
desired combination of colonization potential, plant-beneficial
activity, and/or relevant DNA and RNA profile will be selected for
domestication and remodeling.
C. Domestication of Selected Microbes
[0500] The selected microbes will be domesticated; wherein, the
microbes will be converted to a form that is genetically tractable
and identifiable.
[0501] 1. Test for Antibiotic Sensitivity
[0502] One way to domesticate the microbes is to engineer them with
antibiotic resistance. For this, the wild type microbial strain
will be tested for sensitivity to various antibiotics. If the
strain is sensitive to the antibiotic, then the antibiotic can be a
good candidate for use in genetic tools/vectors for remodeling the
strain.
[0503] 2. Design and Build a Vector
[0504] Vectors that are conditional for their replication (e.g. a
suicide plasmid) will be constructed to domesticate the selected
microbes (host microbes). For example, a suicide plasmid containing
an appropriate antibiotic resistance marker, a counter selectable
marker, an origin of replication for maintenance in a donor microbe
(e.g. E. coli), a gene encoding a fluorescent protein (GFP, RFP,
YFP, CFP, and the like) to screen for insertion through
fluorescence, an origin of transfer for conjugation into the host
microbe, and a polynucleotide sequence comprising homology arms to
the host genome with a desired genetic variation will be
constructed. The vector may comprise a SceI site and other
additional elements.
[0505] Exemplary antibiotic resistance markers include ampicillin
resistance marker, kanamycin resistance marker, tetracycline
resistance marker, chloramphenicol resistance marker, erythromycin
resistance marker, streptomycin resistance marker, spectinomycin
resistance marker, etc. Exemplary counter selectable markers
include sacB, rpsL, tetAR, pheS, thyA, lacY, gata-1, ccdB, etc.
[0506] 3. Generation of Donor Microbes
[0507] In one protocol, a suicide plasmid containing an appropriate
antibiotic resistance marker, a counter selectable marker, the
.lamda.pir origin of replication for maintenance in E. coli ST18
containing the pir replication initiator gene, a gene encoding
green fluorescent protein (GFP) to screen for insertion through
fluorescence, an origin of transfer for conjugation into the host
microbe, and a polynucleotide sequence comprising homology arms to
the host genome with a desired genetic variation (e.g. a promoter
from within the microbe's own genome for insertion into a
heterologous location) will be transformed into E. coli ST18 (an
auxotroph for aminolevulinic acid, ALA) to generate donor
microbes.
[0508] 4. Mix Donor Microbes with Host Microbes
[0509] Donor microbes will be mixed with host microbes (selected
candidate microbes from step B5) to allow conjugative integration
of the plasmid into the host genome. The mixture of donor and host
microbes will be plated on a medium containing the antibiotic and
not containing ALA. The suicide plasmid is able to replicate in
donor microbes (E. coli ST18), but not in the host. Therefore, when
the mixture containing donor and host microbes is plated on a
medium containing the antibiotic and not containing ALA, only host
cells that integrated the plasmid into its genome will be able to
grow and form colonies on the medium. The donor microbes will not
grow due to the absence of ALA.
[0510] 5. Confirm Integration of the Vector
[0511] A proper integration of the suicide plasmid containing the
fluorescent protein marker, the antibiotic resistance marker, the
counter selectable marker, etc. at the intended locus of the host
microbe will be confirmed through fluorescence of colonies on the
plate and using colony PCR.
[0512] 6. Streak Confirm Integration Colony
[0513] A second round of homologous recombination in the host
microbes will loop out (remove) the plasmid backbone leaving the
desired genetic variation (e.g. a promoter from within the
microbe's own genome for insertion into a heterologous location)
integrated into the host genome of a certain percentage of host
microbes, while reverting a certain percentage back to wild
type.
[0514] Colonies of host microbes that have looped out the plasmid
backbone (and therefore, looped out the counter selectable marker)
can be selected by growing them on an appropriate medium.
[0515] For example, if sacB is used as a counter selectable marker,
loss of this marker due to the loss of the plasmid backbone will be
tested by growing the colonies on a medium containing sucrose (sacB
confers sensitivity to sucrose). Colonies that grow on this medium
would have lost the sacB marker and the plasmid backbone and would
either contain the desired genetic variation or be reverted to wild
type. Also, these colonies will not fluoresce on the plate due to
the loss of the fluorescent protein marker.
[0516] In some isolates, the sacB or other counterselectable
markers do not confer full sensitivity to sucrose or other
counterselection mechanisms, which necessitates screening large
numbers of colonies to isolate a successful loop-out. In those
cases, loop-out may be aided by use of a "helper plasmid" that
replicates independently in the host cell and expresses a
restriction endonuclease, e.g. SceI, which recognizes a site in the
integrated suicide plasmid backbone. The strain with the integrated
suicide plasmid is transformed with the helper plasmid containing
an antibiotic resistance marker, an origin of replication
compatible with the host strain, and a gene encoding a restriction
endonuclease controlled by a constitutive or inducible promoter.
The double-strand break induced in the integrated plasmid backbone
by the restriction endonuclease promotes homologous recombination
to loop-out the suicide plasmid. This increases the number of
looped-out colonies on the counterselection plate and decreases the
number of colonies that need to be screened to find a colony
containing the desired mutation. The helper plasmid is then removed
from the strain by culture and serial passaging in the absence of
antibiotic selection for the plasmid. The passaged cultures are
streaked for single colonies, colonies are picked and screened for
sensitivity to the antibiotic used for selection of the helper
plasmid, as well as absence of the plasmid confirmed by colony PCR.
Finally, the genome is sequenced and the absence of helper plasmid
DNA is confirmed as described in D6.
[0517] 7. Confirm Integration of the Genetic Variation Through
Colony PCR
[0518] The colonies that grew better on the sucrose-containing
medium (or other appropriate media depending on the counter
selectable marked used) will be picked and the presence of the
genetic variation at the intended locus will be confirmed by
screening the colonies using colony PCR.
[0519] Although this example describes one protocol for
domesticating the microbe and introducing genetic variation into
the microbe, one of ordinary skill in the art would understand that
the genetic variation can be introduced into the selected microbes
using a variety of other techniques known in the art such as:
polymerase chain reaction mutagenesis, oligonucleotide-directed
mutagenesis, saturation mutagenesis, fragment shuffling
mutagenesis, homologous recombination, ZFN, TALENS, CRISPR systems
(Cas9, Cpf1, etc.), chemical mutagenesis, and combinations
thereof.
[0520] 8. Iterate Upon Steps C2-C7
[0521] If any of the steps C2-C7 fail to provide the intended
outcome, the steps will be repeated to design an alternative vector
that may comprise different elements for facilitating incorporation
of desired genetic variations and markers into the host
microbe.
[0522] 9. Develop a Standard Operating Procedure (SOP)
[0523] Once the steps C2-C7 can be reproduced consistently for a
given strain, the steps will be used to develop a standard
operating procedure (SOP) for that strain and vector. This SOP can
be used to improve other plant-beneficial traits of the
microbe.
D. Non-Intergeneric Engineering Campaign and Optimization
[0524] 1. Identify Gene Targets for Optimization
[0525] Selected microbes will be engineered/remodeled to improve
performance of the plant-beneficial activity. For this, gene
targets for improving the plant-beneficial activity will be
identified.
[0526] Gene targets can be identified in various ways. For example,
genes of interest can be identified while annotating the genes from
the whole genome sequencing of isolated microbes. They can be
identified through a literature search. For example, genes involved
in nitrogen fixation are known in the literature. These known genes
can be used as targets for introducing genetic variations. Gene
targets can also be identified based on the RNA sequencing data
obtained in the step B3 (small-scale field trials for colonization)
or by performing RNA sequencing described in the step below.
[0527] 2. Select Promoters for Promoter Swaps
[0528] A desired genetic variation for improving the
plant-beneficial activity can comprise promoter swapping, in which
the native promoter for a target gene is replaced with a stronger
or weaker promoter (when compared to the native promoter) from
within the microbe's genome, or differently regulated promoter
(e.g. a N-independent). If the expression of a target gene
increases the plant-beneficial activity (e.g., nifA, the expression
of which enhances nitrogen fixation in microbes), the desired
promoter for promoter swapping is a stronger promoter (compared to
the native promoter of the target gene) that would further increase
the expression level of the target gene compared to the native
promoter. If the expression of a target gene decreases the
plant-beneficial activity (e.g., nifL that downregulates nitrogen
fixation), the desired promoter for promoter swapping is a weak
promoter (compared to the native promoter of the target gene) that
would substantially decrease the expression level of the target
gene compared to the native promoter. Promoters can be inserted
into genes to "knock-out" a gene's expression, while at the same
time upregulating the expression of a downstream gene.
[0529] Promoters for promoter swapping can be selected based on the
RNA sequencing data. For example, the RNA sequencing data can be
used to identify strong and weak promoters, or constitutively
active vs. inducible promoters.
[0530] For example, to identify strong and weak promoters, or
constitutively active vs. inducible promoters, in the nitrogen
fixation pathway, selected microbes will be cultured in vitro under
nitrogen-depleted and nitrogen-replete conditions; RNA of the
microbe will be isolated from these cultures; and sequenced.
[0531] In one protocol, the RNA profile of the microbe under
nitrogen-depleted and nitrogen-replete conditions will be compared
and active promoters with a desired transcription level will be
identified. These promoters can be selected to swap a weak
promoter.
[0532] Promoters can also be selected using the RNA sequencing data
obtained in the step B3 that reflects the RNA profile of the
microbe in planta in the host plant rhizosphere.
[0533] RNA sequencing under various conditions allows for selection
of promoters that: a) are active in the rhizosphere during the host
plant growth cycle in fertilized field conditions, and b) are also
active in relevant in vitro conditions so they can be rapidly
screened.
[0534] In an exemplary protocol, in planta RNA sequencing data from
colonization assays (e.g. step B3) is used to measure the
expression levels of genes in isolated microbes. In one embodiment,
the level of gene expression is calculated as reads per kilobase
per million mapped reads (RPKM). The expression level of various
genes is compared to the expression level of a target gene and at
least the top 10, 20, 30, 40, 50, 60, or 70 promoters, associated
with the various genes, that show the highest or lowest level of
expression compared to the target gene are selected as possible
candidates for promoter swapping. Thus, one looks at expression
levels of various genes relative to a target gene and then selects
genes that demonstrate increased expression relative to a target
(or standard) gene and then find the promoters associated with said
genes.
[0535] For example, if the target gene is upregulation of nifA, the
first 10, 20, 30, 40, 50, or 60 promoters for genes that show the
highest level of expression compared to nifA are selected as
possible candidates for promoter swapping.
[0536] These candidates can be further short-listed based on in
vitro RNA sequencing data. For example, for nifA as the target
gene, possible promoter candidates selected based on the in planta
RNA sequencing data are further selected by choosing promoters with
similar or increased gene expression levels compared to nifA under
in vitro nitrogen-deplete vs. nitrogen-replete conditions.
[0537] The set of promoters selected in this step are used to swap
the native promoter of the target gene (e.g. nifA). Remodeled
strains with swapped promoters are tested in in vitro assays;
strains with lower than expected activity are eliminated; and
strains with expected or higher than expected activity are tested
in field. The cycle of promoter selection may be repeated on
remodeled strains to further improve their plant-beneficial
activity.
[0538] Described here is an exemplary promoter swap experiment that
was carried out based on in planta and in vitro RNA sequencing data
from Klebsiella variicola strain, CI137 to improve the nitrogen
fixation trait. CI137 was analyzed in ARA assays at 0 mM and 5 mM
glutamine concentration and RNA was extracted from these ARA
samples. The RNA was sequenced via NextSeq and a subset of reads
from one sample was mapped to the CI137 genome (in vitro RNA
sequencing data). RNA was extracted from the roots of corn plants
at V5 stage in the colonization and activity assay (e.g. step B3)
for CI137. Samples from 6 plants were pooled; the RNA from the
pooled sample was sequenced using NextSeq, and reads were mapped to
the CI137 genome (in planta RNA sequencing data). Out of
2.times.10.sup.8 total reads, 7.times.10.sup.4 reads mapped to
CI137. In planta RNA sequencing data was used to rank genes in
order of in planta expression levels and the expression levels were
compared to the native nifA expression level. The first 40
promoters that showed the highest expression level (based on gene
expression) compared to the native nifA expression level were
selected. These 40 promoters were further short-listed based on the
in vitro RNA sequencing data, where promoters with increased or
similar in vitro expression levels compared to nifA were selected.
The final list of promoters included 17 promoters and 2 versions of
most promoters were used to generate promoter swap mutants; thus a
total of 30 promoters were tested. Generation of a suite of CI137
mutants where nifL was deleted partially or completely and the 30
promoters inserted (.DELTA.nifL::Prm) was attempted. 28 out of 30
mutants were generated successfully. The .DELTA.nifL::Prm mutants
were analyzed in ARA assays at 0 mM and 5 mM glutamine
concentration and RNA was extracted from these ARA samples. Several
mutants showed lower than expected or decreased ARA activity
compared to the WT CI137 strain. A few mutants showed higher than
expected ARA activity.
[0539] A person of ordinary skill in the art would appreciate from
the above example that while in planta and/or in vitro RNA
sequencing data can be used to select promoters for promoter
swapping, the step of promoter selection is highly unpredictable
and involves many challenges.
[0540] For example, in planta RNA sequencing mainly reveals the
genes that are highly expressed; however, it is difficult to detect
fine differences in gene expression and/or genes with low
expression levels. For instance, in some in planta RNA sequencing
experiments, only about 40 out of about 5000 genes from a microbial
genome were detected. Thus, in planta RNA sequencing technique is
useful to identify abundantly expressed genes and their
corresponding promoters; however, the technique has difficulty in
identifying low expression genes and corresponding promoters and
small differences between gene expression.
[0541] Furthermore, in planta RNA profile reflects the status of
the genes at the time the microbes were isolated; however, a slight
change in the field conditions can substantially change the RNA
profile of rhizosphere/epiphytic/endophytic microbes. Therefore, it
is difficult to predict in advance whether the promoters selected
based on one field trial RNA sequencing data would provide
desirable expression levels of the target gene when remodeled
strains are tested in vitro and in field.
[0542] Additionally, in planta evaluation is time and
resource-consuming; therefore, in planta experiments cannot be
conducted often and/or repeated quickly or easily. On the other
hand, while in vitro RNA sequencing can be conducted relatively
quickly and easily, the in vitro conditions do not mimic the field
conditions and promoters that may show high activity in vitro may
not show comparable activity in planta.
[0543] Moreover, promoters often don't behave as predicted in a new
context. Therefore, in planta and in vitro RNA sequencing data can
at best serve as a starting point in the step of promoter
selection; however, arriving at any particular promoter that would
provide desirable expression levels of the target gene in the field
is, in some instances, unpredictable.
[0544] Another limitation in the step of promoter selection is the
number of available promoters. Because one of the goals of the
present invention is to provide non-transgenic microbes; promoters
for promoter swapping need to be selected from within the microbe's
genome, or genus. Thus, unlike a transgenic approach, the present
process cannot merely go out into the literature and find/use a
well-characterized transgenic promoter from a different host
organism.
[0545] Another constraint is that the promoter must be active in
planta during a desired growth phase. For example, the highest
requirement for nitrogen in plants is generally late in the growing
season, e.g. late vegetative and early reproductive phases. For
example, in corn, nitrogen uptake is the highest during V6 (6
leaves) through R1 (reproductive stage 1) stages. Therefore, to
increase the availability of nitrogen during V6 through R1 stages
of corn, remodeled microbes must show highest nitrogen fixation
activity during these stages of the corn lifecycle. Accordingly,
promoters that are active in planta during the late vegetative and
early reproductive stages of corn need to be selected. This
constraint not only reduces the number of promoters that may be
tested in promoter swapping, but also make the step of promoter
selection unpredictable. As discussed above, unpredictability
arises, in part, because although the RNA sequencing data from
small scale field trials (e.g. step B3) may be used to identify
promoters that are active in planta during a desired growth stage,
the RNA data is based on the field conditions (e.g., type of soil,
level of water in the soil, level of available nitrogen, etc.) at
the time of sample collection. As one of ordinary skill in the art
would understand, the field conditions may change over the period
of time within the same field and also change substantially across
various fields. Thus, the promoters selected under one field
condition may not behave as expected under other field conditions.
Similarly, selected promoters may not behave as expected after
swapping. Therefore, it is difficult to anticipate in advance
whether the selected promoters would be active in planta during a
desired growth phase of a plant of interest.
[0546] 3. Design Non-Intergeneric Genetic Variations
[0547] Based on steps D1 (identification of gene targets) and D2
(identification of promoters for promoter swaps), non-intergeneric
genetic variations will be designed.
[0548] The term "non-intergeneric" indicates that the genetic
variation to be introduced into the host does not contain a nucleic
acid sequence from outside the host genus (i.e., no transgenic
DNA). Although vectors and/or other genetic tools will be used to
introduce the genetic variation into the host microbe, the methods
of the present disclosure include steps to loop-out (remove) the
backbone vector sequences or other genetic tools introduced into
the host microbe leaving only the desired genetic variation into
the host genome. Thus, the resulting microbe is non-transgenic.
[0549] Exemplary non-intergeneric genetic variations include a
mutation in the gene of interest that may improve the function of
the protein encoded by the gene; a constitutionally active promoter
that can replace the endogenous promoter of the gene of interest to
increase the expression of the gene; a mutation that will
inactivate the gene of interest; the insertion of a promoter from
within the host's genome into a heterologous location, e.g.
insertion of the promoter into a gene that results in inactivation
of said gene and upregulation of a downstream gene; and the like.
The mutations can be point mutations, insertions, and/or deletions
(full or partial deletion of the gene). For example, in one
protocol, to improve the nitrogen fixation activity of the host
microbe, a desired genetic variation may comprise an inactivating
mutation of the nifL gene (negative regulator of nitrogen fixation
pathway) and/or comprise replacing the endogenous promoter of the
nifH gene (nitrogenase iron protein that catalyzes a key reaction
to fix atmospheric nitrogen) with a constitutionally active
promoter that will drive the expression of the nifH gene
constitutively.
[0550] 4. Generate Non-Intergeneric Derivative Strains
[0551] After designing the non-intergeneric genetic variations,
steps C2-C7 will be carried out to generate non-intergeneric
derivative strains (i.e. remodeled microbes).
[0552] 5. Bank a Purified Culture of the Remodeled Microbe
[0553] A purified culture of the remodeled microbe will be
preserved in a bank, so that gDNA can be extracted for whole genome
sequencing described below.
[0554] 6. Confirm Presence of the Desired Genetic Variation
[0555] The genomic DNA of the remodeled microbe will be extracted
and the whole genome sequencing will be performed on the genomic
DNA using methods described previously. The resulting reads will be
mapped to the reads previously stored in LIMS to confirm: a)
presence of the desired genetic variation, and b) complete absence
of reads mapping to vector sequences (e.g. plasmid backbone or
helper plasmid sequence) that were used to generate the remodeled
microbe.
[0556] This step allows sensitive detection of non-host genus DNA
(transgenic DNA) that may remain in the strain after looping out of
the vector backbone (e.g. suicide plasmid) method and could provide
a control for accidental off-target insertion of the genetic
variation, etc.
E. Analytics Upon Remodeled Microbes
[0557] 1. Analysis of the Plant-Beneficial Activity
[0558] The plant-beneficial activity and growth kinetics of the
remodeled microbes will be assessed in vitro.
[0559] For example, strains remodeled for improving nitrogen
fixation function will be assessed for nitrogen fixation activity
and fitness through acetylene reduction assays, ammonium excretion
assays, etc.
[0560] Strains remodeled for improved phosphate solubilization will
be assessed for the phosphate solubilization activity.
[0561] This step allows rapid, medium to high throughput screening
of remodeled strains for the phenotypes of interest.
[0562] 2. Analysis of Colonization and Transcription of the Altered
Genes
[0563] Remodeled strains will be assessed for colonization of the
host plant either in the greenhouse or in the field using the steps
described in B3. Additionally, RNA will be isolated from colonized
root and/or soil samples and sequenced to analyze the
transcriptional activity of target genes. Target genes comprise the
genes containing the genetic variation introduced and may also
comprise other genes that play a role in the plant-beneficial trait
of the microbe.
[0564] For example, a cluster of genes, the nif genes, controls the
nitrogen fixation activity of microbes. Using the protocol
described above, a genetic variation may be introduced into one of
the nif genes (e.g. a promoter insertion), whereas the other genes
in the nif cluster are in their endogenous form (i.e. their gene
sequence and/or the promoter region is not altered). The RNA
sequencing data will be analyzed for the transcriptional activity
of the nif gene containing the genetic variation and may also be
analyzed for other nif genes that are not altered directly, by the
inserted genetic change, but nonetheless may be influenced by the
introduced genetic change.
[0565] This step allows determination of the fitness of top in
vitro performing strains in the rhizosphere and allows measurement
of the transcriptional activity of altered genes in planta.
F. Iterate Engineering Campaign/Analytics
[0566] The data from in vitro and in planta analytics (steps E1 and
E2) will be used to iteratively stack beneficial mutations.
[0567] Furthermore, steps A-E described above may be repeated to
fine-tune the plant-beneficial traits of the microbes. For example,
plants will be inoculated using microbial strains remodeled in the
first round; harvested after a few weeks of growth; and microbes
from the soil and/or roots of the plants will be isolated. The
functional activity (plant-beneficial trait and colonization
potential) and the DNA and RNA profile of isolated microbes will be
characterized, in order to select microbes with improved
plant-beneficial activity and colonization potential. The selected
microbes will be remodeled to further improve the plant-beneficial
activity. Remodeled microbes will be screened for the functional
activity (plant-beneficial trait and colonization potential) and
RNA profile in vitro and in planta and the top performing strains
will be selected. If desired, steps A-E can be repeated to further
improve the plant-beneficial activity of the remodeled microbes
from the second round. The process can be repeated for 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, or more rounds.
[0568] The exemplary steps described above are summarized in Table
A below.
TABLE-US-00025 TABLE A An Overview of an Embodiment of the Guided
Microbial Remodeling Platform Steps Contribution Alternate Forms A
Isolation 1 Obtain a soil sample Provides WT soil microbes to be
isolated 2 Grow corn "bait Allows selection of plant- Wheat,
sorghum, rice, millet, plants" in soil sample beneficial microbes
by soybean, etc. rhizosphere 3 Harvest, clean and Down-select soil
microbes Other nitrogen-free media, other extract root sample to
those that a) colonize the selective or screening media (eg. and
plate on nitrogen- root and b) fix atmospheric for phosphate
solubilization) free (specifically nitrogen NfB) media 4 Pick
colonies, purify Down-select microbes to Degenerate primers for
other cultures and screen those containing the nifH genes of
interest, e.g. ipdC for presence of nifH gene (eliminate false-
(phytohormone biosynthesis) using degenerate positive from media
primers screen) 5 Bank a purified culture of the strain B
Characterization 1 Sequence and Characterize genome for assemble
the genome key pathways of the strain using Illumina and/or PacBio
platform 2 Assay the microbe for Down-select for microbes Wheat,
sorghum, rice, millet, colonization of corn that colonize the plant
well soybean, etc., other methods for roots in the assaying
colonization (e.g. greenhouse (qPCR- plating) based method) 3 Assay
the microbe for Known internally as "CAT" Larger field trials,
other crops, colonization of com trials, these provide other
methods for assaying roots in a small-scale Colonization And
colonization (e.g. plating) field trials (qPCR- Transcript data for
the based method) and strain in a field isolate RNA from
environment colonized root samples 4 Assay the microbe for Confirm
N-fixation nitrogen fixation phenotype of strain activity in an
acetylene reduction assay (ARA) 5 Use the above data to Allows
selection of select candidate greatest-potential microbe for
further candidates domestication and optimization C Domestication 1
Test microbes for Determine which antibiotic sensitivity to various
selection markers can be antibiotics used to transform genetic
tools 2 Design and build a These are the "parts" Plasmid could
contain a SceI site suicide plasmid necessary to maintain the or
other counter-selectable containing an plasmid and carry out
marker, alternate fluorescent appropriate antibiotic conjugation,
insertion and reporters, additional elements resistance marker,
"loop-out" of the host sacB counter- genome selectable marker,
origin of replication for maintenance in E. coli, GFP to screen for
insertion through fluorescence, origin of transfer for conjugation
into the host, homology arms to the host genome, and the desired
mutation. 3 Transform suicide Preparation for conjugation Could use
a different donor strain plasmid into E. coli into host; plasmid of
E. coli or other microbe; ST18 (an auxotroph maintenance different
auxotrophic marker for aminolevulinic acid, ALA) to generate donor
cells 4 Mix donor cells with The suicide plasmid is able Could use
a different donor strain recipient host cells to to replicate in E.
coli but of E. coli or other microbe; conjugate, and plate not in
the host Therefore different auxotrophic marker on media selecting
for plating of the mixture on the antibiotic such plates means that
only resistance marker and host cells that received the NOT
containing ALA plasmid and experience plasmid integration into the
chromosome will be able to grow and form colonies. The E coli ST18
is unable to grow due to the absence of ALA 5 Confirm integration
Confirms proper integration of the plasmid of the suicide plasmid
through GFP backbone containing GFP, fluorescence, and the
antibiotic resistance integration at the cassette, the sacB marker,
intended locus etc. through colony PCR 6 Streak confirmed The sacB
marker confers Different counter selectable integration colony on
sensitivity to sucrose; marker, SceI-mediated loop-out, a plate
containing colonies which have etc. sucrose and screen for
undergone a second round non-fluorescent of homologous colonies
recombination and "looped- out" the plasmid will grow better and
not fluoresce on the plate. 7 Screen looped-out Upon the second
colonies for the homologous recombination intended mutation event
only 50% of looped using colony PCR out colonies should contain the
mutation, the other 50% will be WT 8 If any of the steps 2-7 Allows
iterative fail, go back to step 2 troubleshooting of suicide and
re-design with plasmid to develop a alternate plasmid working
protocol parts 9 Once steps 2-7 can be reliably performed, develop
an SOP for that strain/plasmid to be used for Optimization D
Non-Intergeneric Engineering Campaign and Optimization 1 Identify
gene targets for optimizing a pathway, eg. nif genes through
literature search 2 Select promoters for Allows for selection of
Alternate crops; alternate RNAseq promoter swaps using promoters
that a) are active data conditions (greenhouse, field, RNAseq data
in the rhizosphere during in vitro, whatever's relevant for
collected both in vitro the corn growth cycle in the phenotype
targeted) in N-depleted and N- fertilized field conditions b)
replete conditions, are also active in in vitro N- and in planta
from replete conditions so they the corn rhizosphere can be rapidly
screened. (Collected in step B3) 3 Design non- No DNA from outside
the Alter regulatory sequences (e.g. intergeneric host chromosome
is added, RBS), non-coding RNAs, etc. mutations in key therefore
the resulting genes: deletions (full microbe is non-transgenic or
partial gene), promoter swaps, or single base pair changes; store
these designs in our LIMS 4 Using the established We perform this
in higher protocol, carry out throughput than the steps C2-7 to
generate domestication step - up to non-intergeneric 20 or so
strains at once per derivative strains person. (mutants) 5 Bank a
purified culture of the strain, extract gDNA and conduct WGS via
Illumina 6 Map the resulting Allows very sensitive Suicide plasmid
removal is fairly reads to the designs detection of non- reliable;
however use of other stored in LIMS to intergeneric DNA that may
stable plasmids in alternate confirm a) presence remain in the
strain after methods necessitates this extra of the desire mutation
the suicide plasmid method; step to ensure with complete and b)
complete confirm absence of confidence that no transgenic absence
of reads transgenic DNA, controls DNA that was previously mapping
to any for accidental off-target transformed in remains in the
suicide plasmid or insertion of the suicide strain. other plasmid
plasmid, etc. sequences used to generate the strains E Analytics 1
Analyze the strains Allow rapid, med- to high- Any other in vitro
assay, e.g. for in vitro nitrogen throughput screening of phosphate
solubilization, qPCR fixation activity and mutants for phenotypes
of for transcription of specific genes, fitness through ARA,
interest etc. ammonium excretion assays, and growth curves 2
Analyze the strains Measure fitness of top in for colonization
vitro performing strains in (qPCR) and the rhizosphere; measure
transcription of target transcription of promoter- and promoter-
swapped genes in planta swapped genes (Nanostring) in the plant
(greenhouse or field) F Iterate Engineering Campaign/Analytics 1
Use data from in vitro and in planta analytics to iteratively stack
beneficial mutations.
[0569] Traditional Approaches to Creating Biologicals for
Agriculture Suffer from Drawbacks Inherent in their Methodology
[0570] Unlike pure bioprospecting of wild type (WT) microbes or
transgenic approaches, GMR allows for non-intergeneric genetic
optimization of key regulatory networks within the microbe, which
improves plant-beneficial phenotypes over WT microbes, but doesn't
have the risks associated with transgenic approaches (e.g.
unpredictable gene function, public and regulatory concerns). See,
FIG. 1C for a depiction of a problematic "traditional
bioprospecting" approach, which has several drawbacks compared to
the taught GMR platform.
[0571] Other methods for developing microbials for agriculture are
focused on either extensive lab development, which often fails at
the field scale, or extensive greenhouse or "field-first" testing
without an understanding of the underlying mechanisms/plant-microbe
interactions. See, FIG. 1D for a depiction of a problematic
"field-first approach to bioprospecting" system, which has several
drawbacks compared to the taught GMR platform.
[0572] The GMR Platform Solves these Problems in Numerous Ways
[0573] One strength of the GMR platform is the identification of
active promoters, which are active at key physiologically important
times for a target crop, and which are also active under
particular, agriculturally relevant, environmental conditions.
[0574] As has been explained, within the context of nitrogen
fixation, the GMR platform is able to identify microbial promoter
sequences, which are active under environmental conditions of
elevated exogenous nitrogen, which thereby allows the remodeled
microbe to fix atmospheric nitrogen and deliver it to a target crop
plant, under modern agricultural row crop conditions, and at a time
when a plant needs the fixed nitrogen the most. See, FIG. 1E for a
depiction of the time period in the corn growth cycle, at which
nitrogen is needed most by the plant. The taught GMR platform is
able to create remodeled microbes that supply nitrogen to a corn
plant at the time period in which the nitrogen is needed, and also
deliver such nitrogen even in the presence of exogenous nitrogen in
the soil environment.
[0575] These promoters can be identified by rhizosphere RNA
sequencing and read mapping to the microbe's genome sequence, and
key pathways can be "reprogrammed" to be turned on or off during
key stages of the plant growth cycle. Additionally, through whole
genome sequencing of optimized microbes and mapping to
previously-transformed sequences, the method has the ability to
ensure that no transgenic sequences are accidentally released into
the field through off-target insertion of plasmid DNA, low-level
retention of plasmids not detected through PCR or antibiotic
resistance, etc.
[0576] The GMR platform combines these approaches by evaluating
microbes iteratively in the lab and plant environment, leading to
microbes that are robust in greenhouse and field conditions rather
than just in lab conditions.
[0577] Various aspects and embodiments of the taught GMR platform
can be found in FIGS. 1F-1I. The GMR platform culminates in the
derivation/creation/production of remodeled microbes that possess a
plant-beneficial property, e.g. nitrogen fixation.
[0578] The traditional bioprospecting methods are not able to
produce microbes having the aforementioned properties.
[0579] Properties of a Microbe Remodeled for Nitrogen Fixation
[0580] In the context of remodeling microbes for nitrogen fixation,
there are several properties that the remodeled microbe may
possess. For instance, FIG. 1J depicts 5 properties that can be
possessed by remodeled microbes of the present disclosure.
[0581] Furthermore, as can be seen in Example 2, the present
inventors have utilized the GMR platform to produce remodeled
non-intergeneric bacteria (i.e. Kosakonia sacchari) capable of
fixing atmospheric nitrogen and delivering said nitrogen to a corn
plant, even under conditions in which exogenous nitrogen is present
in the environment. See, FIG. 1K-M, which illustrate that the
remodeling process successfully: (1) decoupled nifA expression from
endogenous nitrogen regulation; and (2) improved the assimilation
and excretion of fixed nitrogen.
[0582] These remodeled microbes ultimately result in corn yield
improvement, when applied to corn crops. See, FIG. 1N.
[0583] The GMR Platform Provides an Approach to Nitrogen Fixation
and Delivery that Solves Pressing Environmental Concerns
[0584] As explained previously, the nitrogen fertilizer produced by
the industrial Haber-Bosch process is not well utilized by the
target crop. Rain, runoff, heat, volatilization, and the soil
microbiome degrade the applied chemical fertilizer. This equates to
not only wasted money, but also adds to increased pollution instead
of harvested yield. To this end, the United Nations has calculated
that nearly 80% of fertilizer is lost before a crop can utilize it.
Consequently, modern agricultural fertilizer production and
delivery is not only deleterious to the environment, but it is
extremely inefficient. See, FIG. 1O, illustrating the inefficiency
of current nitrogen delivery systems, which result in
underfertilized fields, over fertilized fields, and environmentally
deleterious nitrogen runoff.
[0585] The current GMR platform, and resulting remodeled microbes,
provide a better approach to nitrogen fixation and delivery to
plants. As will be seen in the below Examples, the non-intergeneric
remodeled microbes of the disclosure are able to colonize the roots
of a corn plant and spoon feed said corn plants with fixed
atmospheric nitrogen, even in the presence of exogenous nitrogen.
This system of nitrogen fixation and delivery--enabled by the
taught GMR platform--will help transform modern agricultural to a
more environmentally sustainable system.
Example 2: Guided Microbial Remodeling--an Example Embodiment for
the Rational Improvement of Nitrogen Fixation
[0586] A diversity of nitrogen fixing bacteria can be found in
nature, including in agricultural soils. However, the potential of
a microbe to provide sufficient nitrogen to crops to allow
decreased fertilizer use may be limited by repression of
nitrogenase genes in fertilized soils as well as low abundance in
close association with crop roots. Identification, isolation and
breeding of microbes that closely associate with key commercial
crops might disrupt and improve the regulatory networks linking
nitrogen sensing and nitrogen fixation and unlock significant
nitrogen contributions by crop-associated microbes. To this end,
nitrogen fixing microbes that associate with and colonize the root
system of corn were identified. This step corresponds to the
"Measure the Microbiome Composition" depicted in FIG. 1A and FIG.
1B.
[0587] Root samples from corn plants grown in agronomically
relevant soils were collected, and microbial populations extracted
from the rhizosphere and endosphere. Genomic DNA from these samples
was extracted, followed by 16S amplicon sequencing to profile the
community composition.
[0588] A Kosakonia sacchari microbe (strain PBC6.1) was isolated
and classified through 16S rRNA and whole genome sequencing. This
is a particularly interesting nitrogen fixer capable of colonizing
to nearly 21% abundance of the root-associated microbiota (FIG. 2).
To assess strain sensitivity to exogenous nitrogen, nitrogen
fixation rates in pure culture were measured with the classical
acetylene reduction assay (ARA) and varying levels of glutamine
supplementation. The species exhibited a high level of nitrogen
fixing activity in nitrogen-free media, yet exogenous fixed
nitrogen repressed nif gene expression and nitrogenase activity
(Strain PBC6.1, FIG. 3C, FIG. 3D). Additionally, when released
ammonia was measured in the supernatant of PBC6.1 grown in
nitrogen-fixing conditions, very little release of fixed nitrogen
could be detected (FIG. 3E).
[0589] We hypothesized that PBC6.1 could be a significant
contributor of fixed nitrogen in fertilized fields if regulatory
networks controlling nitrogen metabolism were remodeled to allow
optimal nitrogenase expression and ammonia release in the presence
of fixed nitrogen.
[0590] Sufficient genetic diversity should exist within the PBC6.1
genome to enable broad phenotypic remodeling (as a result of
remodeling the underlying genetic architecture in a
non-intergeneric manner) without the insertion of transgenes or
synthetic regulatory elements. The isolated strain has a genome of
at least 5.4 Mbp and a canonical nitrogen fixation gene cluster.
Related nitrogen metabolism pathways in PBC6.1 are similar to those
of the model organism for nitrogen fixation, Klebsiella oxytoca
m5al.
[0591] Several gene regulatory network nodes were identified which
may augment nitrogen fixation and subsequent transfer to a host
plant, particularly in high exogenous concentrations of fixed
nitrogen (FIG. 3A). The nifLA operon directly regulates the rest of
the nif cluster through transcriptional activation by NifA and
nitrogen- and oxygen-dependent repression of NifA by NifL.
Disruption of nifL can abolish inhibition of NifA and improve nif
expression in the presence of both oxygen and exogenous fixed
nitrogen. Furthermore, expressing nifA under the control of a
nitrogen-independent promoter may decouple nitrogenase biosynthesis
from regulation by the NtrB/NtrC nitrogen sensing complex.
[0592] The assimilation of fixed nitrogen by the microbe to
glutamine by glutamine synthetase (GS) is reversibly regulated by
the two-domain adenylyltransferase (ATase) enzyme GlnE through the
adenylylation and deadenylation of GS to attenuate and restore
activity, respectively. Truncation of the GlnE protein to delete
its adenylyl-removing (AR) domain may lead to constitutively
adenylated glutamine synthetase, limiting ammonia assimilation by
the microbe and increasing intra- and extracellular ammonia.
[0593] Finally, reducing expression of AmtB, the transporter
responsible for uptake of ammonia, could lead to greater
extracellular ammonia.
[0594] To generate rationally designed microbial phenotypes without
the use of transgenes, two approaches were employed to remodel the
underlying genetic architecture of the microbe: (1) creating
markerless deletions of genomic sequences encoding protein domains
or whole genes, and (2) rewiring regulatory networks by
intragenomic promoter rearrangement.
[0595] Through an iterative remodeling process, several
non-transgenic derivative strains of PBC6.1 were generated (Table
25).
TABLE-US-00026 TABLE 25 List of isolated and derivative K. sacchari
strains used in this work. Prm, promoter sequence derived from the
PBC6.1 genome; .DELTA.glnE.sub.AR1 and .DELTA.glnE.sub.AR2,
different truncated versions of glnE gene removing the
adenylyl-removing domain sequence. Strain ID Genotype PBC6.1 WT
PBC6.14 .DELTA.nifL::Prm1 PBC6.15 .DELTA.nifL::Prm5 PBC6.22
.DELTA.nifL::Prm3 PBC6.37 .DELTA.nifL::Prm1 .DELTA.glnE.sub.AR2
PBC6.38 .DELTA.nifL::Prm1 .DELTA.glnE.sub.AR1 PBC6.93
.DELTA.nifL::Prm1 .DELTA.glnE.sub.AR2 .DELTA.amtB PBC6.94
.DELTA.nifL::Prm1 .DELTA.glnE.sub.AR1 .DELTA.amtB
[0596] Several in vitro assays were performed to characterize
specific phenotypes of the derivative strains. The ARA was used to
assess strain sensitivity to exogenous nitrogen, in which PBC6.1
exhibited repression of nitrogenase activity at high glutamine
concentrations (FIG. 3D). In contrast, most derivative strains
showed a derepressed phenotype with varying levels of acetylene
reduction observed at high glutamine concentrations.
Transcriptional rates of nifA in samples analyzed by qPCR
correlated well with acetylene reduction rates (FIG. 4), supporting
the hypothesis that nifL disruption and insertion of a
nitrogen-independent promoter to drive nifA can lead to nif cluster
derepression.
[0597] Strains with altered GlnE or AmtB activity showed markedly
increased ammonium excretion rates compared to wild type or
derivative strains without these mutations (FIG. 3E), illustrating
the effect of these genotypes on ammonia assimilation and
reuptake.
[0598] Two experiments were performed to study the interaction of
PBC6.1 derivatives (remodeled microbes) with corn plants and
quantify incorporation of fixed nitrogen into plant tissues. First,
rates of microbial nitrogen fixation were quantified in a
greenhouse study using isotopic tracers. Briefly, plants are grown
with 15N labeled fertilizer, and diluted concentrations of 15N in
plant tissues indicate contributions of fixed nitrogen from
microbes. Corn seedlings were inoculated with selected microbial
strains, and plants were grown to the V6 growth stage. Plants were
subsequently deconstructed to enable measurement of microbial
colonization and gene expression as well as measurement of 15N/14N
ratios in plant tissues by isotope ratio mass spectrometry (IRMS).
Analysis of the aerial tissue showed a small, nonsignificant
contribution by PBC6.38 to plant nitrogen levels, and a significant
contribution by PBC6.94 (p=0.011). Approximately 20% of the
nitrogen found in above-ground corn leaves was produced by PBC6.94,
with the remainder coming from the seed, potting mix, or
"background" fixation by other soilborne microbes (FIG. 5C). This
illustrates that our microbial breeding and remodeling pipeline can
generate remodeled strains capable of making significant nitrogen
contributions to plants in the presence of nitrogen fertilizer.
Microbial transcription within plant tissues was measured, and
expression of the nif gene cluster was observed in derivative
remodeled strains, but not the wild type strain (FIG. 5B), showing
the importance of nif derepression for contribution of BNF to crops
in fertilized conditions. Root colonization measured by qPCR
demonstrated that colonization density is different for each of the
strains tested (FIG. 5A). A 50-fold difference in colonization was
observed between PBC6.38 and PBC6.94. This difference could be an
indication that PBC6.94 has reduced fitness in the rhizosphere
relative to PBC6.38 as a result of high levels of fixation and
excretion.
Methods
Media
[0599] Minimal medium contains (per liter) 25 g Na2HPO.sub.4, 0.1 g
CaCL.sub.2-2H.sub.2O, 3 g KH.sub.2PO.sub.4, 0.25 g
MgSO.sub.4.7H.sub.2O, 1 g NaCl, 2.9 mg FeCl.sub.3, 0.25 mg
Na.sub.2MoO.sub.4.2H.sub.2O, and 20 g sucrose. Growth medium is
defined as minimal medium supplemented with 50 ml of 200 mM
glutamine per liter.
Isolation of Diazotrophs
[0600] Corn seedlings were grown from seed (DKC 66-40, DeKalb,
Ill.) for two weeks in a greenhouse environment controlled from
22.degree. C. (night) to 26.degree. C. (day) and exposed to 16 hour
light cycles in soil collected from San Joaquin County, CA. Roots
were harvested and washed with sterile deionized water to remove
bulk soil. Root tissues were homogenized with 2 mm stainless steel
beads in a tissue lyser (TissueLyser II, Qiagen P/N 85300) for
three minutes at setting 30, and the samples were centrifuged for 1
minute at 13,000 rpm to separate tissue from root-associated
bacteria. Supernatants were split into two fractions, and one was
used to characterize the microbiome through 16S rRNA amplicon
sequencing and the remaining fraction was diluted and plated on
Nitrogen-free Broth (NfB) media supplemented with 1.5% agar. Plates
were incubated at 30.degree. C. for 5-7 days. Colonies that emerged
were tested for the presence of the nifH gene by colony PCR with
primers Ueda19f and Ueda406r. Genomic DNA from strains with a
positive nifH colony PCR was isolated (QIAamp DNA Mini Kit, Cat No.
51306, QIAGEN, Germany) and sequenced (Illumina MiSeq v3, SeqMatic,
Fremont, Calif.). Following sequence assembly and annotation, the
isolates containing nitrogen fixation gene clusters were utilized
in downstream research.
Microbiome Profiling of Isolation Seedlings
[0601] Genomic DNA was isolated from root-associated bacteria using
the ZR-96 Genomic DNA I Kit (Zymo Research P/N D3011), and 16S rRNA
amplicons were generated using nextera-barcoded primers targeting
799f and 1114r. The amplicon libraries were purified and sequenced
with the Illumina MiSeq v3 platform (SeqMatic, Fremont, Calif.).
Reads were taxonomically classified using Kraken using the
minikraken database (FIG. 2).
Acetylene Reduction Assay (ARA)
[0602] A modified version of the Acetylene Reduction Assay was used
to measure nitrogenase activity in pure culture conditions. Strains
were propagated from single colony in SOB (RPI, P/N S25040-1000) at
30.degree. C. with shaking at 200 RPM for 24 hours and then
subcultured 1:25 into growth medium and grown aerobically for 24
hours (30.degree. C., 200 RPM). 1 ml of the minimal media culture
was then added to 4 ml of minimal media supplemented with 0 to 10
mM glutamine in air-tight Hungate tubes and grown anaerobically for
4 hours (30.degree. C., 200 RPM). 10% headspace was removed then
replaced by an equal volume of acetylene by injection, and
incubation continued for 1 hr. Subsequently, 2 ml of headspace was
removed via gas tight syringe for quantification of ethylene
production using an Agilent 6850 gas chromatograph equipped with a
flame ionization detector (FID).
Ammonium Excretion Assay
[0603] Excretion of fixed nitrogen in the form of ammonia was
measured using batch fermentation in anaerobic bioreactors. Strains
were propagated from single colony in 1 ml/well of SOB in a 96 well
DeepWell plate. The plate was incubated at 30.degree. C. with
shaking at 200 RPM for 24 hours and then diluted 1:25 into a fresh
plate containing 1 ml/well of growth medium. Cells were incubated
for 24 hours (30.degree. C., 200 RPM) and then diluted 1:10 into a
fresh plate containing minimal medium. The plate was transferred to
an anaerobic chamber with a gas mixture of >98.5% nitrogen,
1.2-1.5% hydrogen and <30 ppM oxygen and incubated at 1350 RPM,
room temperature for 66-70 hrs. Initial culture biomass was
compared to ending biomass by measuring optical density at 590 nm.
Cells were then separated by centrifugation, and supernatant from
the reactor broth was assayed for free ammonia using the Megazyme
Ammonia Assay kit (P/N K-AMIAR) normalized to biomass at each
timepoint.
Extraction of Root-Associated Microbiome
[0604] Roots were shaken gently to remove loose particles, and root
systems were separated and soaked in a RNA stabilization solution
(Thermo Fisher P/N AM7021) for 30 minutes. The roots were then
briefly rinsed with sterile deionized water. Samples were
homogenized using bead beating with 1/2-inch stainless steel ball
bearings in a tissue lyser (TissueLyser II, Qiagen P/N 85300) in 2
ml of lysis buffer (Qiagen P/N 79216). Genomic DNA extraction was
performed with ZR-96 Quick-gDNA kit (Zymo Research P/N D3010), and
RNA extraction using the RNeasy kit (Qiagen P/N 74104).
Root Colonization Assay
[0605] Four days after planting, 1 ml of a bacterial overnight
culture (approximately 10.sup.9 cfu) was applied to the soil above
the planted seed. Seedlings were fertilized three times weekly with
25 ml modified Hoagland's solution supplemented with 0.5 mM
ammonium nitrate. Four weeks after planting, root samples were
collected and the total genomic DNA (gDNA) was extracted. Root
colonization was quantified using qPCR with primers designed to
amplify unique regions of either the wild type or derivative strain
genome. QPCR reaction efficiency was measured using a standard
curve generated from a known quantity of gDNA from the target
genome. Data was normalized to genome copies per g fresh weight
using the tissue weight and extraction volume. For each experiment,
the colonization numbers were compared to untreated control
seedlings.
In Planta Transcriptomics
[0606] Transcriptional profiling of root-associated microbes was
measured in seedlings grown and processed as described in the Root
Colonization Assay. Purified RNA was sequenced using the Illumina
NextSeq platform (SeqMatic, Fremont, Calif.). Reads were mapped to
the genome of the inoculated strain using bowtie2 using
`--very-sensitive-local` parameters and a minimum alignment score
of 30. Coverage across the genome was calculated using samtools.
Differential coverage was normalized to housekeeping gene
expression and visualized across the genome using Circos and across
the nif gene cluster using DNAplotlib. Additionally, the in planta
transcriptional profile was quantified via targeted Nanostring
analysis. Purified RNA was processed on an nCounter Sprint (Core
Diagnostics, Hayward, Calif.).
15N Dilution Greenhouse Study
[0607] A 15N fertilizer dilution experiment was performed to assess
optimized strain activity in planta. A planting medium containing
minimal background N was prepared using a mixture of vermiculite
and washed sand (5 rinses in DI H.sub.2O). The sand mixture was
autoclaved for 1 hour at 122.degree. C. and approximately 600 g
measured out into 40 cubic inch (656 mL) pots, which were saturated
with sterile DI H.sub.2O and allowed to drain 24 hours before
planting. Corn seeds (DKC 66-40) were surface sterilized in 0.625%
sodium hypochlorite for 10 minutes, then rinsed five times in
sterile distilled water and planted 1 cm deep. The plants were
maintained under fluorescent lamps for four weeks with 16-hour day
length at room temperatures averaging 22.degree. C. (night) to
26.degree. C. (day).
[0608] Five days after planting, seedlings were inoculated with a 1
ml suspension of cells drenched directly over the emerging
coleoptile. Inoculum was prepared from 5 ml overnight cultures in
SOB, which were spun down and resuspended twice in 5 ml PBS to
remove residual SOB before final dilution to OD of 1.0
(approximately 10.sup.9 CFU/ml). Control plants were treated with
sterile PBS, and each treatment was applied to ten replicate
plants.
[0609] Plants were fertilized with 25 ml fertilizer solution
containing 2% 15N-enriched 2 mM KNO.sub.3 on 5, 9, 14, and 19 days
after planting, and the same solution without KNO.sub.3 on 7, 12,
16, and 18 days after planting. The fertilizer solution contained
(per liter) 3 mmol CaCl.sub.2, 0.5 mmol KH.sub.2PO.sub.4, 2 mmol
MgSO.sub.4, 17.9 .mu.mol FeSO.sub.4, 2.86 mg H.sub.3BO.sub.3, 1.81
mg MnCl.sub.2.4H.sub.2O, 0.22 mg ZnSO.sub.4.7H.sub.2O, 51 .mu.g
CuSO.sub.4.5H.sub.2O, 0.12 mg Na.sub.2MoO.sub.4.2H.sub.2O, and 0.14
nmol NiCl.sub.2. All pots were watered with sterile DI H.sub.2O as
needed to maintain consistent soil moisture without runoff.
[0610] At four weeks, plants were harvested and separated at the
lowest node into samples for root gDNA and RNA extraction and
aerial tissue for IRMS. Aerial tissues were wiped as needed to
remove sand, placed whole into paper bags and dried for at least 72
hours at 60.degree. C. Once completely dry, total aerial tissue was
homogenized by bead beating and 5-7 mg samples were analyzed by
isotope ratio mass spectrometry (IRMS) for .delta.15N by the MBL
Stable Isotope Laboratory (The Ecosystems Center, Woods Hole,
Mass.). Percent NDFA was calculated using the following formula: %
NDFA=(.delta.15N of UTC average-.delta.15N of sample)/(.delta.15N
of UTC average).times.100.
Example 3: Field Trials with Remodeled Microbes of the
Disclosure--Summer 2016
[0611] In order to evaluate the efficacy of remodeled strains of
the present disclosure on corn growth and productivity under
varying nitrogen regimes, field trials were conducted.
[0612] Trials were conducted with (1) seven subplot treatments of
six strains plus the control--four main plots comprised 0, 15, 85,
and 100% of maximum return to nitrogen (MRTN) with local
verification. The control (UTC only) was conducted with 10 100%
MRTN plus, 5, 10, or 15 pounds. Treatments had four
replications.
[0613] Plots of corn (minimum) were 4 rows of 30 feet in length,
with 124 plots per location. All observations were taken from the
center two rows of the plots, and all destructive sampling was
taken from the outside rows. Seed samples were refrigerated until
1.5 to 2 hours prior to use.
[0614] Local Agricultural Practice: The seed was a commercial corn
without conventional fungicide and insecticide treatment. All seed
treatments were applied by a single seed treatment specialist to
assure uniformity. Planting date, seeding rate, weed/insect
management, etc. were left to local agricultural practices. With
the exception of fungicide applications, standard management
practices were followed.
[0615] Soil Characterization: Soil texture and soil fertility were
evaluated. Soil samples were pre-planted for each replicate to
insure residual nitrate levels lower than 50 lbs/Ac. Soil cores
were taken from 0 cm to 30 cm. The soil was further characterized
for pH, CEC, total K and P.
[0616] Assessments: The initial plant population was assessed 14
days after planting (DAP)/acre, and were further assessed for: (1)
vigor (1 to 10 scale, w/10=excellent) 14 DAP & V10; (2)
recordation of disease ratings any time symptoms are evident in the
plots; (3) record any differences in lodging if lodging occurs in
the plots; (4) yield (Bu/acre), adjusted to standard moisture pct;
(5) test weight; and (6) grain moisture percentage.
[0617] Sampling Requirements: The soil was sampled at three
timepoints (prior to trial initiation, V10-VT, 1 week
post-harvest). All six locations and all plots were sampled at 10
grams per sample (124 plots.times.3 timepoints.times.6
locations).
[0618] Colonization Sampling: Colonization samples were collected
at two timepoints (V10 and VT) for five locations and six
timepoints (V4, V8, V10, VT, R5, and Post-Harvest). Samples were
collected as follows: (1) from 0% and 100% MRTN, 60 plots per
location; (2) 4 plants per plot randomly selected from the outside
rows; (3) 5 grams of root, 8 inches of stalk, and top three
leaves--bagged and IDed each separately--12/bags per plot; (4) five
locations (60 plots.times.2 timepoints.times.12 bags/plot); and one
location (60 plots.times.6 timepoints.times.12 bags/plot.
[0619] Normalized difference vegetation index (NDVI) determination
was made using a Greenseeker instrument at two timepoints (V4-V6
and VT). Assessed each plot at all six locations (124 plots.times.2
timepoints.times.6 locations).
[0620] Root analysis was performed with Win Rhizo from one location
that best illustrated treatment differentiation. Ten plants per
plot were randomly sampled (5 adjacent from each outside row; V3-V4
stage plants were preferred) and gently washed to remove as much
dirt as reasonable. Ten roots were placed in a plastic bag and
labelled. Analyzed with WinRhizo Root Analysis.
[0621] Stalk Characteristics were measured at all six locations
between R2 and R5. The stalk diameter of ten plants per plot at the
6'' height were recorded, as was the length of the first internode
above the 6'' mark. Ten plants were monitored; five consecutive
plants from the center of the two inside rows. Six locations were
evaluated (124 plots.times.2 measures.times.6 locations).
[0622] The tissue nitrates were analyzed from all plots and all
locations. An 8'' segment of stalk beginning 6'' above the soil
when the corn is between one and three weeks after black layer
formation; leaf sheaths were removed. All locations and plots were
evaluated (6 locations.times.124 plots).
[0623] The following weather data was recorded for all locations
from planting to harvest: daily maximum and minimum temperatures,
soil temperature at seeding, daily rainfall plus irrigation (if
applied), and any unusual weather events such as excessive rain,
wind, cold, or heat.
[0624] Yield data across all six locations is presented in Table
26. Nitrogen rate had a significant impact on yield, but strains
across nitrogen rates did not. However, at the lowest nitrogen
rate, strains C1006, CM029, and C1019 numerically out-yielded the
UTC by 4 to 6 bu/acre. Yield was also numerically increased 2 to 4
bu/acre by strains CM029, C1019, and CM081 at 15% MRTN.
TABLE-US-00027 TABLE 26 Yield data across all six locations Stalk
Internode YLD (bu) Vigor_E Vigor_L Diameter (mm) Length (in)
NDVI_Veg NDVI_Rep MRTN % 0 143.9 7.0 5.7 18.87 7.18 64.0 70.6 15
165.9 7.2 6.3 19.27 7.28 65.8 72.5 85 196.6 7.1 7.1 20.00 7.31 67.1
74.3 100 197.3 7.2 7.2 20.23 7.37 66.3 72.4 Strain CI006 (1) 176.6
7.2 6.6 19.56 18.78 66.1 72.3 CM029 (2) 176.5 7.1 6.5 19.54 18.61
65.4 71.9 CM038 (3) 175.5 7.2 6.5 19.58 18.69 65.7 72.8 CI019 (4)
176.0 7.1 6.6 19.51 18.69 65.5 72.9 CM081 (5) 176.2 7.1 6.6 19.57
18.69 65.8 73.1 CM029/CM081 (6) 174.3 7.1 6.6 19.83 18.79 66.2 72.5
UTC (7) 176.4 7.1 6.6 19.54 18.71 65.9 71.7 MRTN/Strain 0 1 145.6
7.0 5.6 19.07 7.12 63.5 70.3 0 2 147.0 7.0 5.5 18.74 7.16 64.4 70.4
0 3 143.9 7.0 5.5 18.83 7.37 64.6 70.5 0 4 146.0 6.9 5.7 18.86 7.15
63.4 70.7 0 5 141.7 7.0 5.8 18.82 7.05 63.6 70.9 0 6 142.2 7.2 5.8
19.12 7.09 64.7 69.9 0 7 141.2 7.0 5.8 18.64 7.32 64.0 71.4 15 1
164.2 7.3 6.1 19.09 7.21 66.1 71.5 15 2 167.3 7.2 6.3 19.32 7.29
65.5 72.7 15 3 165.6 7.3 6.3 19.36 7.23 64.8 72.5 15 4 167.9 7.3
6.4 19.31 7.51 66.1 72.3 15 5 169.3 7.2 6.2 19.05 7.32 66.0 72.8 15
6 161.9 7.1 6.3 19.45 7.20 66.2 72.2 15 7 165.1 7.3 6.4 19.30 7.18
66.0 73.3 85 1 199.4 7.3 7.2 19.70 7.32 67.2 74.0 85 2 195.1 7.1
7.2 19.99 7.09 66.5 74.4 85 3 195.0 7.0 7.0 20.05 7.26 67.3 74.6 85
4 195.6 7.2 7.1 20.04 7.29 66.4 74.4 85 5 196.4 7.2 7.0 19.87 7.39
67.3 74.5 85 6 195.1 7.0 6.9 20.35 7.34 67.4 74.4 85 7 199.5 6.9
7.2 19.97 7.48 67.4 74.1 100 1 197.1 7.2 7.3 20.38 7.68 67.5 73.4
100 2 196.5 7.0 7.1 20.11 7.21 65.3 70.2 100 3 197.6 7.5 7.3 20.08
7.42 66.3 73.4 100 4 194.6 7.1 7.1 19.83 7.40 66.1 74.1 100 5 197.4
7.2 7.3 20.53 7.36 66.2 74.3 100 6 198.1 7.2 7.4 20.40 7.16 66.6
73.6 100 7 199.9 7.2 7.2 20.26 7.32 66.2 68.1
[0625] Another analysis approach is presented in Table 27. The
table comprises the four locations where the response to nitrogen
was the greatest which suggests that available residual nitrogen
was lowest. This approach does not alter the assessment that the
nitrogen rate significantly impacted yield, which strains did not
when averaged across all nitrogen rates. However, the numerical
yield advantage at the lowest N rate is more pronounced for all
strains, particularly C1006, CM029, and CM029/CM081 where yields
were increased from 8 to 10 bu/acre. At 15% MRTN, strain CM081
outyielded UTC by 5 bu.
TABLE-US-00028 TABLE 27 Yield data across four locations 4 Location
Average - SGS, Agldea, Bennett, RFR Stalk Diameter Internode YLD
(bu) Vigor_E Vigor_L (mm) Length (in) MRTN % 0 137.8 7.3 5.84 18.10
5.36 15 162.1 7.5 6.63 18.75 5.40 85 199.2 7.4 7.93 19.58 5.62 100
203.5 7.5 8.14 19.83 5.65 Strain CI006 (1) 175.4 7.5 7.08 19.03
5.59 CM029 (2) 176.1 7.4 7.08 19.09 5.39 CM038 (3) 175.3 7.5 7.05
19.01 5.59 CI019 (4) 174.8 7.5 7.16 19.02 5.45 CM081 (5) 176.7 7.4
7.16 19.00 5.53 CM029/CM081 (6) 175.1 7.4 7.17 19.33 5.46 UTC (7)
176.0 7.3 7.27 18.98 5.55 MRTN/Strain 0 1 140.0 7.3 5.69 18.32 5.28
0 2 140.7 7.4 5.69 18.19 5.23 0 3 135.5 7.3 5.63 17.95 5.50 0 4
138.8 7.3 5.81 17.99 5.36 0 5 136.3 7.3 6.06 18.05 5.34 0 6 141.4
7.5 6.00 18.43 5.30 0 7 131.9 7.3 6.00 17.75 5.48 15 1 158.0 7.6
6.44 18.53 5.34 15 2 164.1 7.5 6.56 19.13 5.42 15 3 164.3 7.6 6.63
18.68 5.51 15 4 163.5 7.6 6.81 18.84 5.34 15 5 166.8 7.5 6.63 18.60
5.39 15 6 156.6 7.4 6.56 18.86 5.41 15 7 161.3 7.5 6.81 18.62 5.42
85 1 199.4 7.6 8.00 19.15 5.63 85 2 199.0 7.4 8.09 19.49 5.46 85 3
198.2 7.4 7.75 19.88 5.69 85 4 196.8 7.4 8.00 19.65 5.60 85 5 199.5
7.4 7.75 19.26 5.70 85 6 198.7 7.3 7.81 19.99 5.61 85 7 202.8 7.2
8.13 19.66 5.65 100 1 204.3 7.4 8.19 20.11 6.10 100 2 200.6 7.3
8.00 19.53 5.46 100 3 203.3 7.7 8.19 19.55 5.67 100 4 200.2 7.6
8.00 19.59 5.49 100 5 203.9 7.4 8.19 20.08 5.68 100 6 203.8 7.5
8.31 20.05 5.52 100 7 208.1 7.4 8.13 19.90 5.63
[0626] The results from the field trial are also illustrated in
FIGS. 9-15. The results indicate that the microbes of the
disclosure are able to increase plant yield, which points to the
ability of the taught microbes to increase nitrogen fixation in an
important agricultural crop, i.e. corn.
[0627] The field based results further validate the disclosed
methods of non-intergenerically modifying the genome of selected
microbial strains, in order to bring about agriculturally relevant
results in a field setting when applying said engineered strains to
a crop.
[0628] FIG. 6 depicts the lineage of modified remodeled strains
that were derived from strain C1006 (WT Kosakonia sacchari). The
field data demonstrates that an engineered derivative of the C1006
WT strain, i.e. CM029, is able to bring about numerically relevant
results in a field setting. For example, Table 26 illustrates that
at 0% MRTN CM029 yielded 147.0 bu/acre compared to untreated
control at 141.2 bu/acre (an increase of 5.8 bu/acre). Table 26
also illustrates that at 15% MRTN CM029 yielded 167.3 bu/acre
compared to untreated control at 165.1 bu/acre (an increase of 2.2
bu/acre). Table 27 is supportive of these conclusions and
illustrates that at 0% MRTN CM029 yielded 140.7 bu/acre compared to
untreated control at 131.9 bu/acre (an increase of 8.8 bu/acre).
Table 27 also illustrates that at 15% MRTN CM029 yielded 164.1
bu/acre compared to untreated control at 161.3 bu/acre (an increase
of 2.8 bu/acre).
[0629] FIG. 7 depicts the lineage of modified remodeled strains
that were derived from strain C1019 (WT Rahnella aquatilis). The
field data demonstrates that an engineered derivative of the C1019
WT strain, i.e. CM081, is able to bring about numerically relevant
results in a field setting. For example, Table 26 illustrates that
at 15% MRTN CM081 yielded 169.3 bu/acre compared to untreated
control at 165.1 bu/acre (an increase of 4.2 bu/acre). Table 27 is
supportive of these conclusions and illustrates that at 0% MRTN
CM081 yielded 136.3 bu/acre compared to untreated control at 131.9
bu/acre (an increase of 4.4 bu/acre). Table 27 also illustrates
that at 15% MRTN CM081 yielded 166.8 bu/acre compared to untreated
control at 161.3 bu/acre (an increase of 5.5 bu/acre).
[0630] Further, one can see in Table 27 that the combination of
CM029/CM081 at 0% MRTN yielded 141.4 bu/acre compared to untreated
control at 131.9 bu/acre (an increase of 9.5 bu/acre).
Example 4: Field Trials with Remodeled Microbes of the
Disclosure--Summer 2017
[0631] In order to evaluate the efficacy of remodeled strains of
the present disclosure on corn growth and productivity under
varying nitrogen regimes, field trials were conducted. The below
field data demonstrates that the non-intergeneric microbes of the
disclosure are able to fix atmospheric nitrogen and deliver said
nitrogen to a plant--resulting in increased yields--in both a
nitrogen limiting environment, as well as a non-nitrogen limiting
environment.
[0632] Trials were conducted at seven locations across the United
States with six geographically diverse Midwestern locations. Five
nitrogen regimes were used for fertilizer treatments: 100% of
standard agricultural practice of the site/region, 100% minus 25
pounds, 100% minus 50 pounds, 100% minus 75 pounds, and 0%; all per
acre. The pounds of nitrogen per acre for the 100% regime depended
upon the standard agricultural practices of the site/region. The
aforementioned nitrogen regimes ranged from about 153 pounds per
acre to about 180 pounds per acre, with an average of about 164
pounds of nitrogen per acre.
[0633] Within each fertilizer regime, there were 14 treatments.
Each regime had six replications, and a split plot design was
utilized. The 14 treatments included: 12 different microbes, 1 UTC
with the same fertilizer rate as the main plot, and 1 UTC with 100%
nitrogen. In the 100% nitrogen regime the 2nd UTC is 100 plus 25
pounds.
[0634] Plots of corn, at a minimum, were 4 rows of 30 feet in
length (30 inches between rows) with 420 plots per location. All
observations, unless otherwise noted, were taken from the center
two rows of the plants, and all destructive sampling was taken from
the outside rows. Seed samples were refrigerated until 1.5 to 2
hours prior to use.
[0635] Local Agricultural Practice: The seed was a commercial corn
applied with a commercial seed treatment with no biological
co-application. The seeding rate, planting date, weed/insect
management, harvest times, and other standard management practices
were left to the norms of local agricultural practices for the
regions, with the exception of fungicide application (if
required).
[0636] Microbe Application: The microbes were applied to the seed
in a seed treatment over seeds that had already received a normal
chemical treatment. The seed were coated with fermentation broth
comprising the microbes.
[0637] Soil Characterization: Soil texture and soil fertility were
evaluated. Standard soil sampling procedures were utilized, which
included soil cores of depths from 0-30 cm and 30-60 cm. The
standard soil sampling included a determination of nitrate
nitrogen, ammonium nitrogen, total nitrogen, organic matter, and
CEC. Standard soil sampling further included a determination of pH,
total potassium, and total phosphorous. To determine the nitrogen
fertilizer levels, preplant soil samples from each location were
taken to ensure that the 0-12'' and potentially the 12'' to 24''
soil regions for nitrate nitrogen.
[0638] Prior to planting and fertilization, 2 ml soil samples were
collected from 0 to 6-12'' from the UTC. One sample per replicate
per nitrogen region was collected using the middle of the row. (5
fertilizer regimes.times.6 replicates=thirty soil samples).
[0639] Post-planting (V4-V6), 2 ml soil samples were collected from
0 to 6-12'' from the UTC. One sample per replicate per nitrogen
region was collected using the middle of the row. (5 fertilizer
regimes.times.6 replicates=thirty soil samples).
[0640] Post-harvest (V4-V6), 2 ml soil samples were collected from
0 to 6-12'' from the UTC. One sample per replicate per nitrogen
region was collected using the middle of the row. Additional
post-harvest soil sample collected at 0-12'' from the UTC and
potentially 12-24'' from the UTC (5 fertilizer regimes.times.6
replicates=thirty soil samples).
[0641] A V6-V10 soil sample from each fertilizer regime (excluding
the treatment of 100% and 100%+25 lbs [in the 100% block] for all
fertilizer regimes at 0-12'' and 12-24''. (5 fertilizer
regimes.times.2 depths=10 samples per location).
[0642] Post-harvest soil sample from each fertilizer regime
(excluding the treatment of 100% and 100%+25 lbs [in the 100%
block] for all fertilizer regimes at 0-12'' and 12-24''. (5
fertilizer regimes.times.2 depths=10 samples per location).
[0643] Assessments: The initial plant population was assessed at
.about.50% UTC and the final plant population was assessed prior to
harvest. Assessment included (1) potentially temperature
(temperature probe); (2) vigor (1-10 scale with 10=excellent) at V4
and V8-V10; (3) plant height at V8-V10 and V14; (4) yield
(bushels/acre) adjusted to standard moisture percentage; (5) test
weight; (6) grain moisture percentage; (7) stalk nitrate tests at
black layer (420 plots.times.7 locations); (8) colonization with 1
plant per plot in zip lock bag at 0% and 100% fertilizer at V4-V6
(1 plant.times.14 treatments.times.6 replicates.times.2 fertilizer
regimes=168 plants); (9) transcriptomics with 1 plant per plot in
zip lock bag at 0% and 100% fertilizer at V4-V6 (1 plant.times.14
treatments.times.6 replicates.times.2 fertilizer regimes=168
plants); (10) Normalized difference vegetative index (NDVI) or
normalized difference red edge (NDRE) determination using a
Greenseeker instrument at two time points (V4-V6 and VT) to assess
each plot at all 7 locations (420 plots.times.2 time points.times.7
locations=5,880 data points); (11) stalk characteristics measured
at all 7 locations between R2 and R5 by recording the stalk
diameter of 10 plants/plot at 6'' height, record length of first
internode above the 6'' mark, 10 plants monitored (5 consecutive
plants from center of two inside rows) (420 plots.times.10
plants.times.7 locations=29,400 data points).
[0644] Monitoring Schedule: Practitioners visited all trials at
V3-V4 stage to assess early-season response to treatments and
during reproductive growth stage to monitor maturity. Local
cooperator visited research trial on an on-going basis.
[0645] Weather Information: Weather data spanning from planting to
harvest was collected and consisted of daily minimum and maximum
temperatures, soil temperature at seeding, daily rainfall plus
irrigation (if applied), and unusual weather events such as
excessive wind, rain, cold, heat.
[0646] Data Reporting: Including the data indicated above, the
field trials generated data points including soil textures; row
spacing; plot sizes; irrigation; tillage; previous crop; seeding
rate; plant population; seasonal fertilizer inputs including
source, rate, timing, and placement; harvest area dimensions,
method of harvest, such as by hand or machine and measurement tools
used (scales, yield monitor, etc.)
[0647] Results: Select results from the aforementioned field trial
are reported in FIG. 16 and FIG. 17.
[0648] In FIG. 16, it can be seen that a remodeled microbe of the
disclosure (i.e. 6-403) resulted in a higher yield than the wild
type strain (WT) and a higher yield than the untreated control
(UTC). The "-25 lbs N" treatment utilizes 25 lbs less N per acre
than standard agricultural practices of the region. The "100% N"
UTC treatment is meant to depict standard agricultural practices of
the region, in which 100% of the standard utilization of N is
deployed by the farmer. The microbe "6-403" was deposited as NCMA
201708004 and can be found in Table 1. This is a mutant Kosakonia
sacchari (also called CM037) and is a progeny mutant strain from
CI006 WT.
[0649] In FIG. 17, the yield results obtained demonstrate that the
remodeled microbes of the disclosure perform consistently across
locations. Furthermore, the yield results demonstrate that the
microbes of the disclosure perform well in both a nitrogen stressed
environment (i.e. a nitrogen limiting environment), as well as an
environment that has sufficient supplies of nitrogen (i.e. a
non-nitrogen-limiting condition). The microbe "6-881" (also known
as CM094, PBC6.94), and which is a progeny mutant Kosakonia
sacchari strain from CI006 WT, was deposited as NCMA 201708002 and
can be found in Table 1. The microbe "137-1034," which is a progeny
mutant Klebsiella variicola strain from CI137 WT, was deposited as
NCMA 201712001 and can be found in Table 1. The microbe "137-1036,"
which is a progeny mutant Klebsiella variicola strain from CI137
WT, was deposited as NCMA 201712002 and can be found in Table 1.
The microbe "6-404" (also known as CM38, PBC6.38), and which is a
progeny mutant Kosakonia sacchari strain from CI006 WT, was
deposited as NCMA 201708003 and can be found in Table 1.
Example 5: Genus of Non-Intergeneric Remodeled Microbes Beneficial
for Agricultural Systems
[0650] The remodeled microbes of the present disclosure were
evaluated and compared against one another for the production of
nitrogen produced in an acre across a season. See FIG. 8, FIG. 24,
and FIG. 25.
[0651] It is hypothesized by the inventors that in order for a
population of engineered non-intergeneric microbes to be beneficial
in a modern row crop agricultural system, then the population of
microbes needs to produce at least one pound or more of nitrogen
per acre per season.
[0652] To that end, the inventors have surprisingly discovered a
functional genus of microbes that are able to contribute, inter
alia, to: increasing yields in non-leguminous crops; and/or
lessening a farmer's dependence upon exogenous nitrogen
application; and/or the ability to produce at least one pound of
nitrogen per acre per season, even in non-nitrogen-limiting
environments, said genus being defined by the product of
colonization ability.times.mmol of N produced per microbe per hour
(i.e. the line partitioning FIGS. 8, 24, and 25).
[0653] With respect to FIGS. 8, 24, and 25, certain data utilizing
microbes of the disclosure was aggregated, in order to depict a
heatmap of the pounds of nitrogen delivered per acre-season by
microbes of the disclosure, which are recorded as a function of
microbes per g-fresh weight by mmol of nitrogen/microbe-hr. Below
the thin line that transects the larger images are the microbes
that deliver less than one pound of nitrogen per acre-season, and
above the line are the microbes that deliver greater than one pound
of nitrogen per acre-season.
[0654] Field Data & Wild Type Colonization Heatmap: The
microbes utilized in the FIG. 8 heatmap were assayed for N
production in corn. For the WT strains CI006 and CI019, corn root
colonization data was taken from a single field site. For the
remaining strains, colonization was assumed to be the same as the
WT field level. N-fixation activity was determined using an in
vitro ARA assay at 5 mM glutamine. The table below the heatmap in
FIG. 8 gives the precise value of mmol N produced per microbe per
hour (mmol N/Microbe hr) along with the precise CFU per gram of
fresh weight (CFU/g fw) for each microbe shown in the heatmap.
[0655] Field Data Heatmap: The data utilized in the FIG. 24 heatmap
is derived from microbial strains assayed for N production in corn
in field conditions. Each point represents lb N/acre produced by a
microbe using corn root colonization data from a single field site.
N-fixation activity was determined using in vitro ARA assay at 5 mM
N in the form of glutamine or ammonium phosphate. The below Table
28 gives the precise value of mmol N produced per microbe per hour
(mmol N/Microbe hr) along with the precise CFU per gram of fresh
weight (CFU/g fw) for each microbe shown in the heatmap of FIG.
24.
[0656] Greenhouse & Laboratory Data Heatmap: The data utilized
in the FIG. 25 heatmap is derived from microbial strains assayed
for N production in corn in laboratory and greenhouse conditions.
Each point represents lb N/acre produced by a single strain. White
points represent strains in which corn root colonization data was
gathered in greenhouse conditions. Black points represent mutant
strains for which corn root colonization levels are derived from
average field corn root colonization levels of the wild-type parent
strain. Hatched points represent the wild type parent strains at
their average field corn root colonization levels. In all cases,
N-fixation activity was determined by in vitro ARA assay at 5 mM N
in the form of glutamine or ammonium phosphate. The below Table 29
gives the precise value of mmol N produced per microbe per hour
(mmol N/Microbe hr) along with the precise CFU per gram of fresh
weight (CFU/g fw) for each microbe shown in the heatmap of FIG.
25.
TABLE-US-00029 TABLE 28 FIG. 24 - Field Data Heatmap Activity Peak
Strain (mmol N/ Colonization N Produced/ Name Microbe hr) (CFU/g
fw) acre season Taxonomic Designation CI006 3.88E-16 1.50E+07 0.24
Kosakonia sacchari 6-404 1.61E-13 3.50E+05 2.28 Kosakonia sacchari
6-848 1.80E-13 2.70E+05 1.97 Kosakonia sacchari 6-881 1.58E-13
5.00E+05 3.20 Kosakonia sacchari 6-412 4.80E-14 1.30E+06 2.53
Kosakonia sacchari 6-403 1.90E-13 1.30E+06 10.00 Kosakonia sacchari
CI019 5.33E-17 2.40E+06 0.01 Rahnella aquatilis 19-806 6.65E-14
2.90E+06 7.80 Rahnella aquatilis 19-750 8.90E-14 2.60E+05 0.94
Rahnella aquatilis 19-804 1.72E-14 4.10E+05 0.29 Rahnella aquatilis
CI137 3.24E-15 6.50E+06 0.85 Klebsiella variicola 137-1034 1.16E-14
6.30E+06 2.96 Klebsiella variicola 137-1036 3.47E-13 1.30E+07
182.56 Klebsiella variicola 137-1314 1.70E-13 1.99E+04 0.14
Klebsiella variicola 137-1329 1.65E-13 7.25E+04 0.48 Klebsiella
variicola 63 3.60E-17 3.11E+05 0.00 Rahnella aquatilis 63-1146
1.90E-14 5.10E+05 0.39 Rahnella aquatilis 1021 1.77E-14 2.69E+07
19.25 Kosakonia pseudosacchari 728 5.56E-14 1445240.09 3.25
Klebsiella variicola
TABLE-US-00030 TABLE 29 FIG. 25 - Greenhouse & Laboratory Data
Heatmap Activity Peak Strain (mmol N/ Colonization N Produced/ Name
Microbe hr) (CFU/g fw) acre season Taxonomic Designation CI006
3.88E-16 1.50E+07 0.24 Kosakonia sacchari 6-400 2.72E-13 1.79E+05
1.97 Kosakonia sacchari 6-397 1.14E-14 1.79E+05 0.08 Kosakonia
sacchari CI137 3.24E-15 6.50E+06 0.85 Klebsiella variicola 137-1586
1.10E-13 1.82E+06 8.10 Klebsiella variicola 137-1382 4.81E-12
1.82E+06 354.60 Klebsiella variicola 1021 1.77E-14 2.69E+07 19.25
Kosakonia pseudosacchari 1021-1615 1.20E-13 2.69E+07 130.75
Kosakonia pseudosacchari 1021-1619 3.93E-14 2.69E+07 42.86
Kosakonia pseudosacchari 1021-1612 1.20E-13 2.69E+07 130.75
Kosakonia pseudosacchari 1021-1623 4.73E-17 2.69E+07 0.05 Kosakonia
pseudosacchari 1293 5.44E-17 8.70E+08 1.92 Azospirillum lipoferum
1116 1.05E-14 1.37E+07 5.79 Enterobacter sp. 1113 8.05E-15 4.13E+07
13.45 Enterobacter sp. 910 1.19E-13 1.34E+06 6.46 Kluyvera
intermedia 910-1246 2.16E-13 1.34E+06 11.69 Kluyvera intermedia 850
7.2301E-16 1.17E+06 0.03 Achromobacter spiritinus 852 5.96E-16
1.07E+06 0.03 Achromobacter marplatensis 853 6.42E-16 2.55E+06 0.07
Microbacterium murale
[0657] Conclusions: The data in FIGS. 8, 24, 25, and Tables 28 and
29, illustrates more than a dozen representative members of the
described genus (i.e. microbes to the right of the line in the
figures). Further, these numerous representative members come from
a diverse array of taxonomic genera, which can be found in the
above Tables 28 and 29. Further still, the inventors have
discovered numerous genetic attributes that depict a
structure/function relationship that is found in many of the
microbes. These genetic relationships can be found in the numerous
tables of the disclosure setting forth the genetic modifications
introduced by the inventors, which include introducing at least one
genetic variation into at least one gene, or non-coding
polynucleotide, of the nitrogen fixation or assimilation genetic
regulatory network.
[0658] Consequently, the newly discovered genus is supported by:
(1) a robust dataset, (2) over a dozen representative members, (3)
members from diverse taxonomic genera, and (4) classes of genetic
modifications that define a structure/function relationship, in the
underlying genetic architecture of the genus members.
Example 6: Methods and Assays for Detection of Non-Intergeneric
Remodeled Microbes
[0659] The present disclosure teaches primers, probes, and assays
that are useful for detecting the microbes utilized in the various
aforementioned Examples. The assays are able to detect the
non-natural nucleotide "junction" sequences in the derived/mutant
non-intergeneric remodeled microbes. These non-naturally occurring
nucleotide junctions can be used as a type of diagnostic that is
indicative of the presence of a particular genetic alteration in a
microbe.
[0660] The present techniques are able to detect these
non-naturally occurring nucleotide junctions via the utilization of
specialized quantitative PCR methods, including uniquely designed
primers and probes. The probes can bind to the non-naturally
occurring nucleotide junction sequences. That is, sequence-specific
DNA probes consisting of oligonucleotides that are labelled with a
fluorescent reporter, which permits detection only after
hybridization of the probe with its complementary sequence can be
used. The quantitative methods can ensure that only the
non-naturally occurring nucleotide junction will be amplified via
the taught primers, and consequently can be detected via either a
non-specific dye, or via the utilization of a specific
hybridization probe. Another aspect of the method is to choose
primers such that the primers flank either side of a junction
sequence, such that if an amplification reaction occurs, then said
junction sequence is present.
[0661] Consequently, genomic DNA can be extracted from samples and
used to quantify the presence of microbes of the disclosure by
using qPCR. The primers utilized in the qPCR reaction can be
primers designed by Primer Blast
(www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify unique
regions of the wild-type genome or unique regions of the engineered
non-intergeneric mutant strains. The qPCR reaction can be carried
out using the SYBR GreenER qPCR SuperMix Universal (Thermo Fisher
P/N 11762100) kit, using only forward and reverse amplification
primers; alternatively, the Kapa Probe Force kit (Kapa Biosystems
P/N KK4301) can be used with amplification primers and a TaqMan
probe containing a FAM dye label at the 5' end, an internal ZEN
quencher, and a minor groove binder and fluorescent quencher at the
3' end (Integrated DNA Technologies).
[0662] Certain primer, probe, and non-native junction
sequences--which can be used in the qPCR methods--are listed in the
below Table 30. Specifically, the non-native junction sequences can
be found in SEQ ID NOs: 372-405 and 425-430.
TABLE-US-00031 TABLE 30 Microbial Detection up/ down 100 bp
Junction Junc- stream SEQ 100 bp SEQ downstream SEQ SEQ "/" F R
base tion junc- ID upstream of ID of ID indicating Junction primer
primer Probe CI Name tion NO junction NO junction NO junction des.
SEQ SEQ SEQ 1021 ds1131 up 304 TGGTGTCCGGGC 338 TTCTTGGTTCTCT 372
5'- disrupted N/A N/A N/A GAACGTCGCCAG GGAGCGCTTTAT TGGTGTCCGGGC
nifL gene/ GTGGCACAAATT CGGCATCCTGAC GAACGTCGCCAG PinfC
GTCAGAACTACG TGAAGAATTTGC GTGGCACAAATT ACACGACTAACC AGGCTTCTTCCCA
GTCAGAACTACG GACCGCAGGAGT ACCTGGCTTGCA ACACGACTAACC GTGCGATGACCC
CCCGTGCAGGTA GACCGCAGGAGT TGAATATGATGA GTTGTGATGAAC GTGCGATGACCC
TGGA AT TGAATATGATGA TGGA/ TTCTTGGTTCTC TGGAGCGCTTTA TCGGCATCCTGA
CTGAAGAATTTG CAGGCTTCTTCC CAACCTGGCTTG CACCCGTGCAGG TAGTTGTGATGA
ACAT-3' 1021 ds1131 down 305 CGGAAAACGAGT 339 GCGATAGAACTC 373 5'-
PinfC/ N/A N/A N/A TCAAACGGCGCG ACTTCACGCCCC CGGAAAACGAGT disrupted
TCCCAATCGTATT GAAGGGGGAAGC TCAAACGGCGCG nifL gene AATGGCGAGATT
TGCCTGACCCTAC TCCCAATCGTAT CGCGCCACGGAA GATTCCCGCTATT TAATGGCGAGAT
GTTCGCTTAACAG TCATTCACTGACC TCGCGCCACGGA GTCTGGAAGGCG GGAGGTTCAAAA
AGTTCGCTTAAC AGCAGCTTGGTA TGACCCAGCGAA AGGTCTGGAAGG TT C
CGAGCAGCTTGG TATT/ GCGATAGAACTC ACTTCACGCCCC GAAGGGGGAAGC
TGCCTGACCCTA CGATTCCCGCTA TTTCATTCACTG ACCGGAGGTTCA AAATGACCCAGC
GAAC-3' 1021 ds1133 N/A 306 CGCCAGAGAGTT 340 TCCCTGTGCGCCG 374 5'-
5' UTR N/A N/A N/A GAAATCGAACAT CGTCGCCGATGG CGCCAGAGAGTT and ATG/
TTCCGTAATACCG TGGCCAGCCAAC GAAATCGAACAT truncated CCATTACCCAGG
TGGCGCGCTACC TTCCGTAATACC glnE gene AGCCGTTCTGGTT CGATCCTGCTCG
GCCATTACCCAG GCACAGCGGAAA ATGAACTGCTCG GAGCCGTTCTGG ACGTTAACGAAA
ACCCGAACACGC TTGCACAGCGGA GGATATTTCGCAT TCTATCAACCGA AAACGTTAACGA G
CGG AAGGATATTTCG CATG/ TCCCTGTGCGCC GCGTCGCCGATG GTGGCCAGCCAA
CTGGCGCGCTAC CCGATCCTGCTC GATGAACTGCTC GACCCGAACACG CTCTATCAACCG
ACGG-3' 1021 ds1145 up 307 CGGGCGAACGTC 341 CGTTCTGTAATAA 375 5'-
disrupted N/A N/A N/A GCCAGGTGGCAC TAACCGGACAAT CGGGCGAACGTC nifL
gene/ AAATTGTCAGAA TCGGACTGATTA GCCAGGTGGCAC Prm1 CTACGACACGAC
AAAAAGCGCCCT AAATTGTCAGAA TAACCGACCGCA CGCGGCGCTTTTT CTACGACACGAC
GGAGTGTGCGAT TTATATTCTCGAC TAACCGACCGCA GACCCTGAATAT TCCATTTAAAATA
GGAGTGTGCGAT GATGATGGATGC AAAAATCCAATC GACCCTGAATAT CAGC
GATGATGGATGC CAGC/ CGTTCTGTAATA ATAACCGGACAA TTCGGACTGATT
AAAAAAGCGCCC TCGCGGCGCTTT TTTTATATTCTC GACTCCATTTAA AATAAAAAATCC
AATC-3' 1021 ds1145 down 308 TCAACCTAAAAA 342 AACTCACTTCAC 376 5'-
Prm1/ N/A N/A N/A AGTTTGTGTAATA GCCCCGAAGGGG TCAACCTAAAAA disrupted
CTTGTAACGCTAC GAAGCTGCCTGA AGTTTGTGTAAT nifL gene ATGGAGATTAAC
CCCTACGATTCCC ACTTGTAACGCT TCAATCTAGAGG GCTATTTCATTCA ACATGGAGATTA
GTATTAATAATG CTGACCGGAGGT ACTCAATCTAGA AATCGTACTAAA TCAAAATGACCC
GGGTATTAATAA CTGGTACTGGGC AGCGAACCGAGT TGAATCGTACTA GC CG
AACTGGTACTGG GCGC/ AACTCACTTCAC GCCCCGAAGGGG GAAGCTGCCTGA
CCCTACGATTCC CGCTATTTCATT CACTGACCGGAG GTTCAAAATGAC CCAGCGAACCGA
GTCG-3' 1021 ds1148 up 309 CGGGCGAACGTC 343 CGCGTCAGGTTG 377 5'-
disrupted N/A N/A N/A GCCAGGTGGCAC AACGTAAAAAAG CGGGCGAACGTC nifL
gene/ AAATTGTCAGAA TCGGTCTGCGCA GCCAGGTGGCAC Prm7 CTACGACACGAC
AAGCACGTCGTC AAATTGTCAGAA TAACCGACCGCA GTCCGCAGTTCTC CTACGACACGAC
GGAGTGTGCGAT CAAACGTTAATT TAACCGACCGCA GACCCTGAATAT GGTTTCTGCTTCG
GGAGTGTGCGAT GATGATGGATGC GCAGAACGATTG GACCCTGAATAT CAGC GC
GATGATGGATGC CAGC/ CGCGTCAGGTTG AACGTAAAAAAG TCGGTCTGCGCA
AAGCACGTCGTC GTCCGCAGTTCT CCAAACGTTAAT TGGTTTCTGCTT CGGCAGAACGAT
TGGC-3' 1021 ds1148 down 310 AATTTTCTGCCCA 344 AACTCACTTCAC 378 5'-
Prm4/ N/A N/A N/A AATGGCTGGGAT GCCCCGAAGGGG AATTTTCTGCCC disrupted
TGTTCATTTTTTG GAAGCTGCCTGA AAATGGCTGGGA nifL gene TTTGCCTTACAAC
CCCTACGATTCCC TTGTTCATTTTT GAGAGTGACAGT GCTATTTCATTCA TGTTTGCCTTAC
ACGCGCGGGTAG CTGACCGGAGGT AACGAGAGTGAC TTAACTCAACATC TCAAAATGACCC
AGTACGCGCGGG TGACCGGTCGAT AGCGAACCGAGT TAGTTAACTCAA CG CATCTGACCGGT
CGAT/ AACTCACTTCAC GCCCCGAAGGGG GAAGCTGCCTGA CCCTACGATTCC
CGCTATTTCATT CACTGACCGGAG GTTCAAAATGAC CCAGCGAACCGA GTCG-3' CI006
ds126 N/A 311 GTAACCAATAAA 345 CCGATCCCCATC 379 5'- 5' UTR up N/A
N/A N/A GGCCACCACGCC ACTGTGTGTCTTG GTAACCAATAAA to ATG-
AGACCACACGAT TATTACAGTGCC GGCCACCACGCC 4 bp of AGTGATGGCAAC
GCTTCGTCGGCTT AGACCACACGAT amtB gene/ ACTTTCCAGCTGC CGCCGGTACGAA
AGTGATGGCAAC disrupted ACCAGCACCTGA TACGAATGACGC ACTTTCCAGCTG amtB
gene TGGCCCATGGTC GTTGCAGCTCAG CACCAGCACCTG ACACCTTCAGCG
CAACGAAAATTT ATGGCCCATGGT AAA TG CACACCTTCAGC GAAA/ CCGATCCCCATC
ACTGTGTGTCTT GTATTACAGTGC CGCTTCGTCGGC TTCGCCGGTACG AATACGAATGAC
GCGTTGCAGCTC AGCAACGAAAAT TTTG-3' CI019 ds172 down 312
TGGTATTGTCAGT 346 CCGTCTCTGAAG 380 5'- Prm1.2/ SEQ SEQ N/A
CTGAATGAAGCT CTCTCGGTGAAC TGGTATTGTCAG disrupted ID ID CTTGAAAAAGCT
ATTGTTGCGAGG TCTGAATGAAGC nifL gene NO: NO: GAGGAAGCGGGC
CAGGATGCGAGC TCTTGAAAAAGC 406 407 GTCGATTTAGTAG TGGTTGTGTTTTG
TGAGGAAGCGGG CAAG TGCC AAATCAGTCCGA ACATTACCGATA CGTCGATTTAGT AAGT
TCGC ATGCCGAGCCGC ATGTGCCGCGTG AGAAATCAGTCC TCGC AACA CAGTTTGTCGAAT
AACGGGTGCGTT GAATGCCGAGCC CTCA ATGT C ATG GCCAGTTTGTCG CAGG TCAC
AATC/ CCGTCTCTGAAG CTCTCGGTGAAC ATTGTTGCGAGG CAGGATGCGAGC
TGGTTGTGTTTT GACATTACCGAT AATGTGCCGCGT GAACGGGTGCGT TATG-3' CI019
ds172 up 313 ACCGATCCGCAG 347 TGAACATCACTG 381 5'- disrupted N/A
N/A N/A GCGCGCATTTGTT ATGCACAAGCTA ACCGATCCGCAG nifL gene/
ATGCCAATCCGG CCTATGTCGAAG GCGCGCATTTGT Prm1.2 CATTCTGCCGCCA
AATTAACTAAAA TATGCCAATCCG GACGGGTTTTGC AACTGCAAGATG GCATTCTGCCGC
ACTTGAGACACTT CAGGCATTCGCG CAGACGGGTTTT TTGGGCGAGAAC TTAAAGCCGACT
GCACTTGAGACA CACCGTCTGCTGG TGAGAAATGAGA CTTTTGGGCGAG AGAT
AACCACCGTCTG CTGG/ TGAACATCACTG ATGCACAAGCTA CCTATGTCGAAG
AATTAACTAAAA AACTGCAAGATG CAGGCATTCGCG TTAAAGCCGACT TGAGAAATGAGA
AGAT-3' CI019 ds175 down 314 CGGGAACCGGTG 348 CCGTCTCTGAAG 382 5'-
Prm3.1/ SEQ SEQ SEQ TTATAATGCCGCG CTCTCGGTGAAC CGGGAACCGGTG
disrupted ID ID ID CCCTCATATTGTG ATTGTTGCGAGG TTATAATGCCGC nifL
gene NO: NO: NO: GGGATTTCTTAAT CAGGATGCGAGC GCCCTCATATTG 408 409
410/ GACCTATCCTGG TGGTTGTGTTTTG TGGGGATTTCTT CGCC GGCA 56-
GTCCTAAAGTTGT ACATTACCGATA AATGACCTATCC CTCA TAAC FAM/ AGTTGACATTAG
ATGTGCCGCGTG TGGGTCCTAAAG TATT GCAC TA CGGAGCACTAAC AACGGGTGCGTT
TTGTAGTTGACA GTGG CCGT ACC ATG TTAGCGGAGCAC GGAT TCA CGT TAAC/ C/
CCGTCTCTGAAG ZEN/ CTCTCGGTGAAC TCTG ATTGTTGCGAGG AAG CAGGATGCGAGC
CTC TGGTTGTGTTTT TCG GACATTACCGAT GT/ AATGTGCCGCGT 3IAB
GAACGGGTGCGT kFQ/ TATG-3' CI019 ds175 up 315 ACCGATCCGCAG 349
TACAGTAGCGCC 383 5'- disrupted N/A N/A N/A GCGCGCATTTGTT
TCTCAAAAATAG ACCGATCCGCAG nifL gene/ ATGCCAATCCGG ATAAACGGCTCA
GCGCGCATTTGT Prm3.1 CATTCTGCCGCCA TGTACGTGGGCC TATGCCAATCCG
GACGGGTTTTGC GTTTATTTTTTCT GCATTCTGCCGC ACTTGAGACACTT ACCCATAATCGG
CAGACGGGTTTT TTGGGCGAGAAC GAACCGGTGTTA GCACTTGAGACA CACCGTCTGCTGG
TAATGCCGCGCC CTTTTGGGCGAG
CTC AACCACCGTCTG CTGG/ TACAGTAGCGCC TCTCAAAAATAG ATAAACGGCTCA
TGTACGTGGGCC GTTTATTTTTTC TACCCATAATCG GGAACCGGTGTT ATAATGCCGCGC
CCTC-3' CI006 ds20 down 316 TCAACCTAAAAA 350 AACTCACTTCAC 384 5'-
Prm1/ SEQ SEQ SEQ AGTTTGTGTAATA ACCCCGAAGGGG TCAACCTAAAAA disrupted
ID ID ID CTTGTAACGCTAC GAAGTTGCCTGA AGTTTGTGTAAT nifL gene NO: NO:
NO: ATGGAGATTAAC CCCTACGATTCCC ACTTGTAACGCT 411 412 413
TCAATCTAGAGG GCTATTTCATTCA ACATGGAGATTA TAAA CAAA /56- GTATTAATAATG
CTGACCGGAGGT ACTCAATCTAGA CTGG TCGA FAM/ AATCGTACTAAA TCAAAATGACCC
GGGTATTAATAA TACT AGCG AAG CTGGTACTGGGC AGCGAACCGAGT TGAATCGTACTA
GGGC CCAG TTGC GC CG AACTGGTACTGG GCAA ACGG CT/ GCGC/ CT TAT ZEN/
AACTCACTTCAC GACC ACCCCGAAGGGG CTAC GAAGTTGCCTGA GATT CCCTACGATTCC
CCC/ CGCTATTTCATT 3IAB CACTGACCGGAG kFQ/ GTTCAAAATGAC CCAGCGAACCGA
GTCG-3' CI006 ds20 up 317 GGGCGACAAACG 351 CGTCCTGTAATA 385 5'-
disrupted N/A N/A N/A GCCTGGTGGCAC ATAACCGGACAA GGGCGACAAACG nifL
gene/ AAATTGTCAGAA TTCGGACTGATTA GCCTGGTGGCAC Prm1 CTACGACACGAC
AAAAAGCGCCCT AAATTGTCAGAA TAACTGACCGCA TGTGGCGCTTTTT CTACGACACGAC
GGAGTGTGCGAT TTATATTCCCGCC TAACTGACCGCA GACCCTGAATAT TCCATTTAAAATA
GGAGTGTGCGAT GATGATGGATGC AAAAATCCAATC GACCCTGAATAT CGGC
GATGATGGATGC CGGC/ CGTCCTGTAATA ATAACCGGACAA TTCGGACTGATT
AAAAAAGCGCCC TTGTGGCGCTTT TTTTATATTCCC GCCTCCATTTAA AATAAAAAATCC
AATC-3' CI006 ds24 up 318 GGGCGACAAACG 352 GGACATCATCGC 386 5'-
disrupted SEQ SEQ SEQ GCCTGGTGGCAC GACAAACAATAT GGGCGACAAACG nifL
gene/ ID ID ID AAATTGTCAGAA TAATACCGGCAA GCCTGGTGGCAC Prm5 NO: NO:
NO: CTACGACACGAC CCACACCGGCAA AAATTGTCAGAA 414 415 416 TAACTGACCGCA
TTTACGAGACTG CTACGACACGAC GGTG GCGC /56- GGAGTGTGCGAT CGCAGGCATCCT
TAACTGACCGCA CACT AGTC FAM/ GACCCTGAATAT TTCTCCCGTCAAT GGAGTGTGCGAT
CTTT TCGT CA GATGATGGATGC TTCTGTCAAATAA GACCCTGAATAT GCAT AAAT GGA
CGGC AG GATGATGGATGC GGTT TGCC GTG CGGC/ T/ GGACATCATCGC ZEN/
GACAAACAATAT GCGA TAATACCGGCAA TGA CCACACCGGCAA CCC TTTACGAGACTG
TGA CGCAGGCATCCT AT/ TTCTCCCGTCAA 3IAB TTTCTGTCAAAT kFQ AAAG-3'
CI006 ds24 down 319 TAAGAATTATCTG 353 AACTCACTTCAC 387 5'- Prm5/
N/A N/A N/A GATGAATGTGCC ACCCCGAAGGGG TAAGAATTATCT disrupted
ATTAAATGCGCA GAAGTTGCCTGA GGATGAATGTGC nifL gene GCATAATGGTGC
CCCTACGATTCCC CATTAAATGCGC GTTGTGCGGGAA GCTATTTCATTCA AGCATAATGGTG
AACTGCTTTTTTT CTGACCGGAGGT CGTTGTGCGGGA TGAAAGGGTTGG TCAAAATGACCC
AAACTGCTTTTT TCAGTAGCGGAA AGCGAACCGAGT TTTGAAAGGGTT AC CG
GGTCAGTAGCGG AAAC/ AACTCACTTCAC ACCCCGAAGGGG GAAGTTGCCTGA
CCCTACGATTCC CGCTATTTCATT CACTGACCGGAG GTTCAAAATGAC CCAGCGAACCGA
GTCG-3' CI006 ds30 N/A 320 CGCCAGAGAGTC 354 TTTAACGATCTGA 388 5'-
5' UTR N/A N/A N/A GAAATCGAACAT TTGGCGATGATG CGCCAGAGAGTC and ATG/
TTCCGTAATACCG AAACGGATTCGC GAAATCGAACAT truncated CGATTACCCAGG
CGGAAGATGCGC TTCCGTAATACC glnE gene AGCCGTTCTGGTT TTTCTGAGAGCTG
GCGATTACCCAG GCACAGCGGAAA GCGCGAATTGTG GAGCCGTTCTGG ACGTTAACGAAA
GCAGGATGCGTT TTGCACAGCGGA GGATATTTCGCAT GCAGGAGGAGGA AAACGTTAACGA G
TT AAGGATATTTCG CATG/ TTTAACGATCTG ATTGGCGATGAT GAAACGGATTCG
CCGGAAGATGCG CTTTCTGAGAGC TGGCGCGAATTG TGGCAGGATGCG TTGCAGGAGGAG
GATT-3' CI006 ds31 N/A 321 CGCCAGAGAGTC 355 GCACTGAAACAC 389 5'- 5'
UTR N/A N/A N/A GAAATCGAACAT CTCATTTCCCTGT CGCCAGAGAGTC and ATG/
TTCCGTAATACCG GTGCCGCGTCGC GAAATCGAACAT truncated CGATTACCCAGG
CGATGGTTGCCA TTCCGTAATACC glnE gene AGCCGTTCTGGTT GTCAGCTGGCGC
GCGATTACCCAG GCACAGCGGAAA GCTACCCGATCCT GAGCCGTTCTGG ACGTTAACGAAA
GCTTGATGAATT TTGCACAGCGGA GGATATTTCGCAT GCTCGACCCGAA AAACGTTAACGA G
TA AAGGATATTTCG CATG/ GCACTGAAACAC CTCATTTCCCTG TGTGCCGCGTCG
CCGATGGTTGCC AGTCAGCTGGCG CGCTACCCGATC CTGCTTGATGAA TTGCTCGACCCG
AATA-3' CI019 ds34 N/A 322 GATGATGGATGC 356 GCGCTCAAACAG 390 5'- 5'
UTR N/A N/A N/A TTTCTGGTTAAAC TTAATCCGTCTGT GATGATGGATGC and ATG/
GGGCAACCTCGT GTGCCGCCTCGC TTTCTGGTTAAA truncated TAACTGACTGACT
CGATGGTCGCGA CGGGCAACCTCG glnE gene AGCCTGGGCAAA CACAACTTGCAC
TTAACTGACTGA CTGCCCGGGCTTT GTCATCCTTTATT CTAGCCTGGGCA TTTTTGCAAGGAA
GCTCGATGAACT AACTGCCCGGGC TCTGATTTCATG GCTCGACCCGCG TTTTTTTTGCAA CA
GGAATCTGATTT CATG/ GCGCTCAAACAG TTAATCCGTCTG TGTGCCGCCTCG
CCGATGGTCGCG ACACAACTTGCA CGTCATCCTTTA TTGCTCGATGAA CTGCTCGACCCG
CGCA-3' CI019 ds70 up 323 ACCGATCCGCAG 357 AGTCTGAACTCA 391 5'-
disrupted N/A N/A N/A GCGCGCATTTGTT TCCTGCGGCAGT ACCGATCCGCAG nifL
gene/ ATGCCAATCCGG CGGTGAGACGTA GCGCGCATTTGT Prm4 CATTCTGCCGCCA
TTTTTGACCAAAG TATGCCAATCCG GACGGGTTTTGC AGTGATCTACAT GCATTCTGCCGC
ACTTGAGACACTT CACGGAATTTTGT CAGACGGGTTTT TTGGGCGAGAAC GGTTGTTGCTGCT
GCACTTGAGACA CACCGTCTGCTGG TAAAAGGGCAAA CTTTTGGGCGAG T AACCACCGTCTG
CTGG/ AGTCTGAACTCA TCCTGCGGCAGT CGGTGAGACGTA TTTTTGACCAAA
GAGTGATCTACA TCACGGAATTTT GTGGTTGTTGCT GCTTAAAAGGGC AAAT-3' CI019
ds70 down 324 CATCGGACACCA 358 CCGTCTCTGAAG 392 5'- Prm4/ N/A N/A
N/A CCAGCTTACAAA CTCTCGGTGAAC CATCGGACACCA disrupted TTGCCTGATTGCG
ATTGTTGCGAGG CCAGCTTACAAA nifL gene GCCCCGATGGCC CAGGATGCGAGC
TTGCCTGATTGC GGTATCACTGAC TGGTTGTGTTTTG GGCCCCGATGGC CGACCATTTCGTG
ACATTACCGATA CGGTATCACTGA CCTTATGTCATGC ATGTGCCGCGTG CCGACCATTTCG
GATGGGGGCTGG AACGGGTGCGTT TGCCTTATGTCA G ATG TGCGATGGGGGC TGGG/
CCGTCTCTGAAG CTCTCGGTGAAC ATTGTTGCGAGG CAGGATGCGAGC TGGTTGTGTTTT
GACATTACCGAT AATGTGCCGCGT GAACGGGTGCGT TATG-3' 137 ds799 down 325
TCTTCAACAACTG 359 GCCATTGAGCTG 393 5'- PinfC/ SEQ SEQ SEQ
GAGGAATAAGGT GCTTCCCGACCG TCTTCAACAACT disrupted ID ID ID
ATTAAAGGCGGA CAGGGCGGCACC GGAGGAATAAGG nifL gene NO: NO: NO:
AAACGAGTTCAA TGCCTGACCCTGC TATTAAAGGCGG 417 418 419/ ACGGCACGTCCG
GTTTCCCGCTGTT AAAACGAGTTCA CTCG AGGG 56- AATCGTATCAAT TAACACCCTGAC
AACGGCACGTCC GCAG TGTT FAM/ GGCGAGATTCGC CGGAGGTGAAGC GAATCGTATCAA
CATG AAAC AA GCCCTGGAAGTT ATGATCCCTGAA TGGCGAGATTCG GACG AGCG CGG
CGC TC CGCCCTGGAAGT TAA GGAA CAC TCGC/ A G/ GCCATTGAGCTG ZEN/
GCTTCCCGACCG TCCG CAGGGCGGCACC AAT TGCCTGACCCTG CGT CGTTTCCCGCTG
ATC TTTAACACCCTG AA/ ACCGGAGGTGAA 3IAB GCATGATCCCTG kFQ/ AATC-3'
137 ds799 up 326 TCCGGGTTCGGCT 360 AGCGTCAGGTAC 394 5'- disrupted
N/A N/A N/A TACCCCGCCGCGT CGGTCATGATTC TCCGGGTTCGGC nifL gene/
TTTGCGCACGGTG ACCGTGCGATTCT TTACCCCGCCGC PinfC TCGGACAATTTGT
CGGTTCCCTGGA GTTTTGCGCACG CATAACTGCGAC GCGCTTCATTGGC GTGTCGGACAAT
ACAGGAGTTTGC ATCCTGACCGAA TTGTCATAACTG GATGACCCTGAA GAGTTCGCTGGC
CGACACAGGAGT TATGATGCTCGA TTCTTCCCAACCT TTGCGATGACCC G TGAATATGATGC
TCGA/ AGCGTCAGGTAC CGGTCATGATTC ACCGTGCGATTC TCGGTTCCCTGG
AGCGCTTCATTG GCATCCTGACCG AAGAGTTCGCTG GCTTCTTCCCAA CCTG-3' 137
ds809 N/A 327 ATCGCAGCGTCTT 361 GCGCTGAAGCAC 395 5'- 5' UTR SEQ SEQ
SEQ TGAATATTTCCGT CTGATCACGCTCT ATCGCAGCGTCT and ATG/ ID ID ID
CGCCAGGCGCTG GCGCGGCGTCGC TTGAATATTTCC truncated NO: NO: NO:
GCTGCCGAGCCG CGATGGTCGCCA GTCGCCAGGCGC glnE gene 420 421 422/
TTCTGGCTGCATA GCCAGCTGGCGC TGGCTGCCGAGC GAGC GCCG 56- GTGGAAAACGAT
GCCACCCGCTGC CGTTCTGGCTGC CGTT TCGG FAM/ AATTTCAGGCCA TGCTGGATGAGC
ATAGTGGAAAAC CTGG CTGA TTAT
GGGAGCCCTTAT TGCTGGATCCCA GATAATTTCAGG CTGC TAGA GGC G ACA
CCAGGGAGCCCT ATAG GG GC/ TATG/ ZEN/ GCGCTGAAGCAC TGAA CTGATCACGCTC
GCA TGCGCGGCGTCG CCTG CCGATGGTCGCC ATC AGCCAGCTGGCG A/ CGCCACCCGCTG
3IAB CTGCTGGATGAG kFQ/ CTGCTGGATCCC AACA-3' 137 ds843 up 328
TCCGGGTTCGGCT 362 GCCCGCTGACCG 396 5'- disrupted N/A N/A N/A
TACCCCGCCGCGT ACCAGAACTTCC TCCGGGTTCGGC nifL gene/ TTTGCGCACGGTG
ACCTTGGACTCG TTACCCCGCCGC Prm1.2 TCGGACAATTTGT GCTATACCCTTGG
GTTTTGCGCACG CATAACTGCGAC CGTGACGGCGCG GTGTCGGACAAT ACAGGAGTTTGC
CGATAACTGGGA TTGTCATAACTG GATGACCCTGAA CTACATCCCCATT CGACACAGGAGT
TATGATGCTCGA CCGGTGATCTTAC TTGCGATGACCC C TGAATATGATGC TCGA/
GCCCGCTGACCG ACCAGAACTTCC ACCTTGGACTCG GCTATACCCTTG GCGTGACGGCGC
GCGATAACTGGG ACTACATCCCCA TTCCGGTGATCT TACC-3' 137 ds843 down 329
TCACTTTTTAGCA 363 GCCATTGAGCTG 397 5'- Prm1.2/ N/A N/A N/A
AAGTTGCACTGG GCTTCCCGACCG TCACTTTTTAGC disrupted ACAAAAGGTACC
CAGGGCGGCACC AAAGTTGCACTG nifL gene ACAATTGGTGTA TGCCTGACCCTGC
GACAAAAGGTAC CTGATACTCGAC GTTTCCCGCTGTT CACAATTGGTGT ACAGCATTAGTG
TAACACCCTGAC ACTGATACTCGA TCGATTTTTCATA CGGAGGTGAAGC CACAGCATTAGT
TAAAGGTAATTTT ATGATCCCTGAA GTCGATTTTTCA G TC TATAAAGGTAAT TTTG/
GCCATTGAGCTG GCTTCCCGACCG CAGGGCGGCACC TGCCTGACCCTG CGTTTCCCGCTG
TTTAACACCCTG ACCGGAGGTGAA GCATGATCCCTG AATC-3' 137 ds853 up 330
TCCGGGTTCGGCT 364 GCTAAAGTTCTC 398 5'- disrupted N/A N/A N/A
TACCCCGCCGCGT GGCTAATCGCTG TCCGGGTTCGGC nifL gene/ TTTGCGCACGGTG
ATAACATTTGAC TTACCCCGCCGC Prm6.2 TCGGACAATTTGT GCAATGCGCAAT
GTTTTGCGCACG CATAACTGCGAC AAAAGGGCATCA GTGTCGGACAAT ACAGGAGTTTGC
TTTGATGCCCTTT TTGTCATAACTG GATGACCCTGAA TTGCACGCTTTCA CGACACAGGAGT
TATGATGCTCGA TACCAGAACCTG TTGCGATGACCC GC TGAATATGATGC TCGA/
GCTAAAGTTCTC GGCTAATCGCTG ATAACATTTGAC GCAATGCGCAAT AAAAGGGCATCA
TTTGATGCCCTT TTTGCACGCTTT CATACCAGAACC TGGC-3' 137 ds853 down 331
GTTCTCCTTTGCA 365 GCCATTGAGCTG 399 5'- Prm6.2/ N/A N/A N/A
ATAGCAGGGAAG GCTTCCCGACCG GTTCTCCTTTGC disrupted AGGCGCCAGAAC
CAGGGCGGCACC AATAGCAGGGAA nifL gene CGCCAGCGTTGA TGCCTGACCCTGC
GAGGCGCCAGAA AGCAGTTTGAAC GTTTCCCGCTGTT CCGCCAGCGTTG GCGTTCAGTGTAT
TAACACCCTGAC AAGCAGTTTGAA AATCCGAAACTT CGGAGGTGAAGC CGCGTTCAGTGT
AATTTCGGTTTGG ATGATCCCTGAA ATAATCCGAAAC A TC TTAATTTCGGTT TGGA/
GCCATTGAGCTG GCTTCCCGACCG CAGGGCGGCACC TGCCTGACCCTG CGTTTCCCGCTG
TTTAACACCCTG ACCGGAGGTGAA GCATGATCCCTG AATC-3' 137 ds857 up 332
TCCGGGTTCGGCT 366 CGCCGTCCTCGC 400 5'- disrupted N/A N/A N/A
TACCCCGCCGCGT AGTACCATTGCA TCCGGGTTCGGC nifL gene/ TTTGCGCACGGTG
ACCGACTTTACA TTACCCCGCCGC Prm8.2 TCGGACAATTTGT GCAAGAAGTGAT
GTTTTGCGCACG CATAACTGCGAC TCTGGCACGCAT GTGTCGGACAAT ACAGGAGTTTGC
GGAACAAATTCT TTGTCATAACTG GATGACCCTGAA TGCCAGTCGGGC CGACACAGGAGT
TATGATGCTCGA TTTATCCGATGAC TTGCGATGACCC GAA TGAATATGATGC TCGA/
CGCCGTCCTCGC AGTACCATTGCA ACCGACTTTACA GCAAGAAGTGAT TCTGGCACGCAT
GGAACAAATTCT TGCCAGTCGGGC TTTATCCGATGA CGAA-3' 137 ds857 down 333
GATATGCCTGAA 367 GCCATTGAGCTG 401 5'- Prm8.2/ N/A N/A N/A
GTATTCAATTACT GCTTCCCGACCG GATATGCCTGAA disrupted TAGGCATTTACTT
CAGGGCGGCACC GTATTCAATTAC nifL gene AACGCAGGCAGG TGCCTGACCCTGC
TTAGGCATTTAC CAATTTTGATGCT GTTTCCCGCTGTT TTAACGCAGGCA GCCTATGAAGCG
TAACACCCTGAC GGCAATTTTGAT TTTGATTCTGTAC CGGAGGTGAAGC GCTGCCTATGAA
TTGAGCTTGATC ATGATCCCTGAA GCGTTTGATTCT TC GTACTTGAGCTT GATC/
GCCATTGAGCTG GCTTCCCGACCG CAGGGCGGCACC TGCCTGACCCTG CGTTTCCCGCTG
TTTAACACCCTG ACCGGAGGTGAA GCATGATCCCTG AATC-3' 63 ds908 down 334
TGGTATTGTCAGT 368 TCTTTAGATCTCT 402 5'- PinfC/ SEQ SEQ N/A
CTGAATGAAGCT CGGTCCGCCCTG TGGTATTGTCAG disrupted ID ID CTTGAAAAAGCT
ATGGCGGCACCT TCTGAATGAAGC nifL gene NO: NO: GAGGAAGCGGGC
TGCTGACGTTAC TCTTGAAAAAGC 423 424 GTCGATTTAGTAG GCCTGCCGGTAC
TGAGGAAGCGGG GGAA GGGC AAATCAGTCCGA AGCAGGTTATCA CGTCGATTTAGT AACG
GGAC ATGCCGAGCCGC CCGGAGGCTTAA AGAAATCAGTCC AGTT CGAG CAGTTTGTCGAAT
AATGACCCAGTT GAATGCCGAGCC CAAC AGAT C ACC GCCAGTTTGTCG CGGC CTAA
AATC/ TCTTTAGATCTC TCGGTCCGCCCT GATGGCGGCACC TTGCTGACGTTA
CGCCTGCCGGTA CAGCAGGTTATC ACCGGAGGCTTA AAATGACCCAGT TACC-3' 63
ds908 up 335 TGCAAATTGCAC 369 TGAATATCACTG 403 5'- disrupted N/A
N/A N/A GGTTATTCCGGGT ACTCACAAGCTA TGCAAATTGCAC nifL gene/
GAGTATATGTGT CCTATGTCGAAG GGTTATTCCGGG PinfC GATTTGGGTTCCG
AATTAACTAAAA TGAGTATATGTG GCATTGCGCAAT AACTGCAAGATG TGATTTGGGTTC
AAAGGGGAGAAA CAGGCATTCGCG CGGCATTGCGCA GACATGAGCATC TTAAAGCCGACT
ATAAAGGGGAGA ACGGCGTTATCA TGAGAAATGAGA AAGACATGAGCA GC AGAT
TCACGGCGTTAT CAGC/ TGAATATCACTG ACTCACAAGCTA CCTATGTCGAAG
AATTAACTAAAA AACTGCAAGATG CAGGCATTCGCG TTAAAGCCGACT TGAGAAATGAGA
AGAT-3' 910 ds960 up 336 TCAGGGCTGCGG 370 CTGGGGTCACTG 404 5'-
disrupted N/A N/A N/A ATGTCGGGCGTTT GAGCGCTTTATC TCAGGGCTGCGG nifL
gene/ CACAACACAAAA GGCATCCTGACC ATGTCGGGCGTT PinfC TGTTGTAAATGCG
GAAGAATTTGCC TCACAACACAAA ACACAGCCGGGC GGTTTCTTCCCGA ATGTTGTAAATG
CTGAAACCAGGA CCTGGCTGGCCC CGACACAGCCGG GCGTGTGATGAC CTGTTCAGGTTGT
GCCTGAAACCAG CTTTAATATGATG GGTGATGAATAT GAGCGTGTGATG C CA
ACCTTTAATATG ATGC/ CTGGGGTCACTG GAGCGCTTTATC GGCATCCTGACC
GAAGAATTTGCC GGTTTCTTCCCG ACCTGGCTGGCC CCTGTTCAGGTT GTGGTGATGAAT
ATCA-3' 910 ds960 down 337 CGGAAAACGAGT 371 GCAATAGAACTA 405 5'-
PinfC/ N/A N/A N/A TCAAACGGCACG ACTACCCGCCCT CGGAAAACGAGT disrupted
TCCGAATCGTATC GAAGGCGGTACC TCAAACGGCACG nifL gene AATGGCGAGATT
TGCCTGACCCTGC TCCGAATCGTAT CGCGCCCAGGAA GATTCCCGTTATT CAATGGCGAGAT
GTTCGCTTAACTG TCATTCACTGACC TCGCGCCCAGGA GTCTGGAAGGTG GGAGGCCCACGA
AGTTCGCTTAAC AGCAGCTGGGTA TGACCCAGCGAC TGGTCTGGAAGG TT C
TGAGCAGCTGGG TATT/ GCAATAGAACTA ACTACCCGCCCT GAAGGCGGTACC
TGCCTGACCCTG CGATTCCCGTTA TTTCATTCACTG ACCGGAGGCCCA CGATGACCCAGC
GACC-3' 137 ds2551 up 425 CTTCGGCGGCGT 427 GCCCGCTGACCG 429 5'- 3'
end of N/A N/A N/A GAAGAAGAGTGG ACCAGAACTTCC CTTCGGCGGCGT sad
TTTTGGTCGCGAG ACCTTGGACTCG GAAGAAGAGTGG (Succinate CTGTCACATTTTG
GCTATACCCTTGG TTTTGGTCGCGA semialdehyde GTCTGCACGAGTT CGTGACGGCGCG
GCTGTCACATTT dehydro- CTGTAATGCGCA CGATAACTGGGA TGGTCTGCACGA
genase)/ GACCGTCTGGAA CTACATCCCCATT GTTCTGTAATGC Prm1.2
AGACCGTCGCTA CCGGTGATCTTAC GCAGACCGTCTG A C GAAAGACCGTCG CTAA/
GCCCGCTGACCG ACCAGAACTTCC ACCTTGGACTCG GCTATACCCTTG GCGTGACGGCGC
GCGATAACTGGG ACTACATCCCCA TTCCGGTGATCT TACC-3' 137 ds2551 down 426
TCACTTTTTAGCA 428 ATGGCGGCGGTG 430 5'- Prm1.2/ N/A N/A N/A
AAGTTGCACTGG ATCAATAACGCA TCACTTTTTAGC glsA2 ACAAAAGGTACC
ATGCTGGAAGCC AAAGTTGCACTG ACAATTGGTGTA ATTCTGGCAGAA GACAAAAGGTAC
CTGATACTCGAC ATCAGGCCGCTG CACAATTGGTGT ACAGCATTAGTG ATTGGCCGCGGT
ACTGATACTCGA
TCGATTTTTCATA AAAGTGGCGGAT CACAGCATTAGT TAAAGGTAATTTT TACATTCCGGCG
GTCGATTTTTCA G CTGG TATAAAGGTAAT TTTG/ ATGGCGGCGGTG ATCAATAACGCA
ATGCTGGAAGCC ATTCTGGCAGAA ATCAGGCCGCTG ATTGGCCGCGGT AAAGTGGCGGAT
TACATTCCGGCG CTGG-3'
TABLE-US-00032 TABLE 31 Remodeled Non-intergeneric Microbes Strain
Name Genotype SEQ ID NO CI006 16S rDNA - contig 5 62 CI006 16S rDNA
- contig 8 63 CI019 16S rDNA 64 CI006 nifH 65 CI006 nifD 66 CI006
nifK 67 CI006 nifL 68 CI006 nifA 69 CI019 nifH 70 CI019 nifD 71
CI019 nifK 72 CI019 nifL 73 CI019 nifA 74 CI006 Prm5 with 500bp 75
flanking regions CI006 nifLA operon - upstream 76 intergenic region
plus nifL and nifA CDSs CI006 nifL (Amino Acid) 77 CI006 nifA
(Amino Acid) 78 CI006 glnE 79 CI006 glnE_KO1 80 CI006 glnE (Amino
Acid) 81 CI006 glnE_KO1 (Amino Acid) 82 CI006 GlnE ATase domain 83
(Amino Acid) CM029 Prm5 inserted into nifL 84 region
TABLE-US-00033 TABLE 32 Remodeled Non-intergeneric Microbes
Associated Novel Strain SEQ Junction If Strain ID ID NO Genotype
Description Applicable CI63; 63 SEQ ID 16S N/A N/A CI063 NO 85
CI63; 63 SEQ ID nifH N/A N/A CI063 NO 86 CI63; 63 SEQ ID nifD1 1 of
2 unique genes annotated as nifD N/A CI063 NO 87 in 63 genome CI63;
63 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A CI063 NO
88 in 63 genome CI63; 63 SEQ ID nifK1 1 of 2 unique genes annotated
as nifK N/A CI063 NO 89 in 63 genome CI63; 63 SEQ ID nifK2 2 of 2
unique genes annotated as nifK N/A CI063 NO 90 in 63 genome CI63;
63 SEQ ID nifL N/A N/A CI063 NO 91 CI63; 63 SEQ ID nifA N/A N/A
CI063 NO 92 CI63; 63 SEQ ID glnE N/A N/A CI063 NO 93 CI63; 63 SEQ
ID amtB N/A N/A CI063 NO 94 CI63; 63 SEQ ID PinfC 500bp immediately
upstream of the N/A CI063 NO 95 ATG start codon of the infC gene
CI137 137 SEQ ID 16S N/A N/A NO 96 CI137 137 SEQ ID nifH1 1 of 2
unique genes annotated as nifH N/A NO 97 in 137 genome CI137 137
SEQ ID nifH2 2 of 2 unique genes annotated as nifH N/A NO 98 in 137
genome CI137 137 SEQ ID nifD1 1 of 2 unique genes annotated as nifD
N/A NO 99 in 137 genome CI137 137 SEQ ID nifD2 2 of 2 unique genes
annotated as nifD N/A NO 100 in 137 genome CI137 137 SEQ ID nifK1 1
of 2 unique genes annotated as nifK N/A NO 101 in 137 genome CI137
137 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A NO 102
in 137 genome CI137 137 SEQ ID nifL N/A N/A NO 103 CI137 137 SEQ ID
nifA N/A N/A NO 104 CI137 137 SEQ ID glnE N/A N/A NO 105 CI137 137
SEQ ID PinfC 500bp immediately upstream of the N/A NO 106 TTG start
codon of infC CI137 137 SEQ ID amtB N/A N/A NO 107 CI137 137 SEQ ID
Prm8.2 internal promoter located in nlpI gene; N/A NO 108 299bp
starting at 81bp after the A of the ATG of the nlpI gene CI137 137
SEQ ID Prm6.2 300bp upstream of the secE gene N/A NO 109 starting
at 57bp upstream of the A of the ATG of secE CI137 137 SEQ ID
Prm1.2 400bp immediately upstream of the N/A NO 110 ATG of cspE
gene none 728 SEQ ID 16S N/A N/A NO 111 none 728 SEQ ID nifH N/A
N/A NO 112 none 728 SEQ ID nifD1 1 of 2 unique genes annotated as
nifD N/A NO 113 in 728 genome none 728 SEQ ID nifD2 2 of 2 unique
genes annotated as nifD N/A NO 114 in 728 genome none 728 SEQ ID
nifK1 1 of 2 unique genes annotated as nifK N/A NO 115 in 728
genome none 728 SEQ ID nifK2 2 of 2 unique genes annotated as nifK
N/A NO 116 in 728 genome none 728 SEQ ID nifL N/A N/A NO 117 none
728 SEQ ID nifA N/A N/A NO 118 none 728 SEQ ID glnE N/A N/A NO 119
none 728 SEQ ID amtB N/A N/A NO 120 none 850 SEQ ID 16S N/A N/A NO
121 none 852 SEQ ID 16S N/A N/A NO 122 none 853 SEQ ID 16S N/A N/A
NO 123 none 910 SEQ ID 16S N/A N/A NO 124 none 910 SEQ ID nifH N/A
N/A NO 125 none 910 SEQ ID Dinitrogenase iron- N/A N/A NO 126
molybdenum cofactor CDS none 910 SEQ ID nifD1 N/A N/A NO 127 none
910 SEQ ID nifD2 N/A N/A NO 128 none 910 SEQ ID nifK1 N/A N/A NO
129 none 910 SEQ ID nifK2 N/A N/A NO 130 none 910 SEQ ID nifL N/A
N/A NO 131 none 910 SEQ ID nifA N/A N/A NO 132 none 910 SEQ ID glnE
N/A N/A NO 133 none 910 SEQ ID amtB N/A N/A NO 134 none 910 SEQ ID
PinfC 498bp immediately upstream of the N/A NO 135 ATG of the infC
gene none 1021 SEQ ID 16S N/A N/A NO 136 none 1021 SEQ ID nifH N/A
N/A NO 137 none 1021 SEQ ID nifD1 1 of 2 unique genes annotated as
nifD N/A NO 138 in 910 genome none 1021 SEQ ID nifD2 2 of 2 unique
genes annotated as nifD N/A NO 139 in 910 genome none 1021 SEQ ID
nifK1 1 of 2 unique genes annotated as nifK N/A NO 140 in 910
genome none 1021 SEQ ID nifK2 2 of 2 unique genes annotated as nifK
N/A NO 141 in 910 genome none 1021 SEQ ID nifL N/A N/A NO 142 none
1021 SEQ ID nifA N/A N/A NO 143 none 1021 SEQ ID glnE N/A N/A NO
144 none 1021 SEQ ID amtB N/A N/A NO 145 none 1021 SEQ ID PinfC
500bp immediately upstream of the N/A NO 146 ATG start codon of the
infC gene none 1021 SEQ ID Prm1 348bp includes the 319bp
immediately N/A NO 147 upstream of the ATG start codon of the lpp
gene and the first 29bp of the lpp gene none 1021 SEQ ID Prm7 339bp
upstream of the sspA gene, N/A NO 148 ending at 46bp upstream of
the ATG of the sspA gene none 1113 SEQ ID 16S N/A N/A NO 149 none
1113 SEQ ID nifH N/A N/A NO 150 none 1113 SEQ ID nifD1 1 of 2
unique genes annotated as nifD N/A NO 151 in 1113 genome none 1113
SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 152 in
1113 genome none 1113 SEQ ID nifK N/A N/A NO 153 none 1113 SEQ ID
nifL N/A N/A NO 154 none 1113 SEQ ID nifA partial gene due to a gap
in the sequence assembly, N/A NO 155 we can only identify a partial
gene from the 1113 genome none 1113 SEQ ID glnE N/A N/A NO 156 none
1116 SEQ ID 16S N/A NO 157 none 1116 SEQ ID nifH N/A NO 158 none
1116 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A NO 159
in 1116 genome none 1116 SEQ ID nifD2 2 of 2 unique genes annotated
as nifD N/A NO 160 in 1116 genome none 1116 SEQ ID nifK1 1 of 2
unique genes annotated as nifK N/A NO 161 in 1116 genome none 1116
SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A NO 162 in
1116 genome none 1116 SEQ ID nifL N/A N/A NO 163 none 1116 SEQ ID
nifA N/A N/A NO 164 none 1116 SEQ ID glnE N/A N/A NO 165 none 1116
SEQ ID amtB N/A N/A NO 166 none 1293 SEQ ID 16S N/A N/A NO 167 none
1293 SEQ ID nifH N/A N/A NO 168 none 1293 SEQ ID nifD1 1 of 2
unique genes annotated as nifD N/A NO 169 in 1293 genome none 1293
SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 170 in
1293 genome none 1293 SEQ ID nifK 1 of 2 unique genes annotated as
nifK N/A NO 171 in 1293 genome none 1293 SEQ ID nifK1 2 of 2 unique
genes annotated as nifK N/A NO 172 in 1293 genome none 1293 SEQ ID
nifA N/A N/A NO 173 none 1293 SEQ ID glnE N/A N/A NO 174 none 1293
SEQ ID amtB1 1 of 2 unique genes annotated as amtB N/A NO 175 in
1293 genome none 1293 SEQ ID amtB2 2 of 2 unique genes annotated as
amtB N/A NO 176 in 1293 genome none 1021-1612 SEQ ID
.DELTA.nifL::PinfC starting at 24bp after the A of the ATG ds1131
NO 177 start codon, 1375bp of nifL have been deleted and replaced
with the 1021 PinfC promoter sequence none 1021-1612 SEQ ID
.DELTA.nifL::PinfC with starting at 24bp after the A of the ATG
ds1131 NO 178 500bp flank start codon, 1375bp of nifL have been
deleted and replaced with the 1021 PinfC promoter sequence; 500bp
flanking the nifL gene upstream and downstream are included none
1021-1612 SEQ ID glnE.DELTA.AR-2 glnE gene with 1673bp immediately
ds1133 NO 179 downstream of the ATG start codon deleted, resulting
in a truncated glnE protein lacking the adenylyl-removing (AR)
domain none 1021-1612 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1673bp immediately ds1133 NO 180 500bp flank downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain; 500bp flanking the glnE gene
upstream and downstream are included none 1021-1615 SEQ ID
.DELTA.nifL::Prm1 starting at 24bp after the A of the ATG ds1145 NO
181 start codon, 1375bp of nifL have been deleted and replaced with
the 1021 Prm1 promoter sequence none 1021-1615 SEQ ID
.DELTA.nifL::Prm1 with starting at 24bp after the A of the ATG
ds1145 NO 182 500bp flank start codon, 1375bp of nifL have been
deleted and replaced with the 1021 rm1 promoter sequence; 500bp
flanking the nifL gene upstream and downstream are included none
1021-1615 SEQ ID glnE.DELTA.AR-2 glnE gene with 1673bp immediately
ds1133 NO 183 downstream of the ATG start codon deleted, resulting
in a truncated glnE protein lacking the adenylyl-removing (AR)
domain none 1021-1615 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1673bp immediately ds1133 NO 184 500bp flank downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain; 500bp flanking the glnE
gene upstream and downstream are included none 1021-1619 SEQ ID
.DELTA.nifL::Prm1 starting at 24bp after the A of the ATG ds1145 NO
185 start codon, 1375bp of nifL have been deleted and replaced with
the 1021 Prm1 promoter sequence none 1021-1619 SEQ ID
.DELTA.nifL::Prm1 with starting at 24bp after the A of the ATG
ds1145 NO 186 500bp flank start codon, 1375bp of nifL have been
deleted and replaced with the 1021 rm1 promoter sequence; 500bp
flanking the nifL gene upstream and downstream are included none
1021-1623 SEQ ID glnE.DELTA.AR-2 glnE gene with 1673bp immediately
ds1133 NO 187 downstream of the ATG start codon deleted, resulting
in a truncated glnE protein lacking the adenylyl-removing (AR)
domain none 1021-1623 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1673bp immediately ds1133 NO 188 500bp flank downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain; 500bp flanking the glnE gene
upstream and downstream are included none 1021-1623 SEQ ID
.DELTA.nifL::Prm7 starting at 24bp after the A of the ATG ds1148 NO
189 start codon, 1375bp of nifL have been deleted and replaced with
the 1021 Prm7 promoter sequence none 1021-1623 SEQ ID
.DELTA.nifL::Prm7 with starting at 24bp after the A of the ATG
ds1148 NO 190 500bp flank start codon, 1375bp of nifL have been
deleted and replaced with the 1021 rm7 promoter sequence; 500bp
flanking the nifL gene upstream and downstream are included none
137-1034 SEQ ID glnE.DELTA.AR-2 glnE gene with 1290bp immediately
ds809 NO 191 downstream of the ATG start codon deleted, resulting
in a truncated glnE protein lacking the adenylyl-removing (AR)
domain none 137-1034 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1290bp immediately ds809 NO 192 500bp flank downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain; 500bp flanking the glnE gene
upstream and downstream are included none 137-1036 SEQ ID
.DELTA.nifL::PinfC starting at 24bp after the A of the ATG ds799 NO
193 start codon, 1372bp of nifL have been deleted and replaced with
the 137 PinfC promoter sequence none 137-1036 SEQ ID
.DELTA.nifL::PinfC with starting at 24bp after the A of the ATG
ds799 NO 194 500bp flank start codon, 1372bp of nifL have been
deleted and replaced with the 137 PinfC promoter sequence; 500bp
flanking the nifL gene upstream and downstream are included none
137-1314 SEQ ID glnE.DELTA.AR-2 36bp glnE gene with 1290bp
immediately none NO 195 deletion downstream of the ATG start codon
deleted AND 36bp deleted beginning at 1472bp downstream of the
start codon, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain none 137-1314 SEQ ID glnE.DELTA.AR-2
36bp glnE gene with 1290bp immediately none NO 196 deletion
downstream of the ATG start codon deleted AND 36bp deleted
beginning at 1472bp downstream of the start codon, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain;
500bp flanking the nifL gene upstream and downstream are included
none 137-1314 SEQ ID .DELTA.nifL::Prm8.2 starting at 24bp after the
A of the ATG ds857 NO 197 start codon, 1372bp of nifL have been
deleted and replaced with the 137 Prm8.2 promoter sequence none
137-1314 SEQ ID .DELTA.nifL::Prm8.2 with starting at 24bp after the
A of the ATG ds857 NO 198 500bp flank start codon, 1372bp of nifL
have been deleted and replaced with the 137 Prm8.2 promoter
sequence; 500bp flanking the nifL gene upstream and downstream are
included none 137-1329 SEQ ID glnE.DELTA.AR-2 36bp glnE gene with
1290bp immediately none NO 199 deletion downstream of the ATG start
codon deleted AND 36bp deleted beginning at 1472bp downstream of
the start codon, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain none 137-1329 SEQ ID glnE.DELTA.AR-2
36bp glnE gene with 1290bp immediately none NO 200 deletion
downstream of the ATG start codon deleted AND 36bp deleted
beginning at 1472bp downstream of the start codon, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain;
500bp flanking the nifL gene upstream and downstream are included
none 137-1329 SEQ ID .DELTA.nifL::Prm6.2 starting at 24bp after the
A of the ATG ds853 NO 201 start codon, 1372bp of nifL have been
deleted and replaced with the 137 Prm6.2 promoter sequence none
137-1329 SEQ ID .DELTA.nifL::Prm6.2 with starting at 24bp after the
A of the ATG ds853 NO 202 500bp flank start codon, 1372bp of nifL
have been deleted and replaced with the 137 Prm6.2 promoter
sequence; 500bp flanking the nifL gene upstream and downstream are
included none 137-1382 SEQ ID .DELTA.nifL::Prm1.2 starting at 24bp
after the A of the ATG ds843 NO 203 start codon, 1372bp of nifL
have been deleted and replaced with the 137 Prm1.2 promoter
sequence none 137-1382 SEQ ID .DELTA.nifL::Prm1.2 with starting at
24bp after the A of the ATG ds843 NO 204 500bp flank start codon,
1372bp of nifL have been deleted and replaced with the 137 Prm1.2
promoter sequence; 500bp flanking the nifL gene upstream and
downstream are included none 137-1382 SEQ ID glnE.DELTA.AR-2 36bp
glnE gene with 1290bp immediately none NO 205 deletion downstream
of the ATG start codon deleted AND 36bp deleted beginning at 1472bp
downstream of the start codon, resulting in a truncated glnE
protein lacking the adenylyl-removing (AR) domain none 137-1382 SEQ
ID glnE.DELTA.AR-2 36bp glnE gene with 1290bp immediately none NO
206 deletion downstream of the ATG start codon deleted AND 36bp
deleted beginning at 1472bp downstream of the start codon,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain; 500bp flanking the nifL gene upstream and downstream
are included none 137-1586 SEQ ID .DELTA.nifL::PinfC starting at
24bp after the A of the ATG ds799 NO 207 start codon, 1372bp of
nifL have been deleted and replaced with the 137 PinfC promoter
sequence none 137-1586 SEQ ID .DELTA.nifL::PinfC with starting at
24bp after the A of the ATG ds799 NO 208 500bp flank start codon,
1372bp of nifL have been deleted and replaced with the 137 PinfC
promoter sequence; 500bp flanking the nifL gene upstream and
downstream are included none 137-1586 SEQ ID glnE.DELTA.AR-2 glnE
gene with 1290bp immediately ds809 NO 209 downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain none 137-1586 SEQ ID
glnE.DELTA.AR-2 with glnE gene with 1290bp immediately ds809 NO 210
500bp flank downstream of the ATG start codon deleted, resulting in
a truncated glnE protein lacking the adenylyl-removing (AR) domain;
500bp flanking the glnE gene upstream and downstream are included
none 19-594 SEQ ID glnE.DELTA.AR-2 glnE gene with 1650bp
immediately ds34 NO 211 downstream of the ATG start codon deleted,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain none 19-594 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1650bp immediately ds34 NO 212 500bp flank downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain; 500bp flanking the glnE gene
upstream and downstream are included none 19-594 SEQ ID
.DELTA.nifL::Prm6.1 starting at 221bp after the A of the ds180 NO
213 ATG start codon, 845bp of nifL have been deleted and replaced
with the CI019 Prm6.1 promoter sequence none 19-594 SEQ ID
.DELTA.nifL::Prm6.1 with starting at 221bp after the A of the ds180
NO 214 500bp flank ATG start codon, 845bp of nifL have been deleted
and replaced with the CI019 Prm6.1promoter sequence; 500bp flanking
the nifL gene upstream and downstream are included none 19-714 SEQ
ID .DELTA.nifL::Prm6.1 starting at 221bp after the A of the ds180
NO 215 ATG start codon, 845bp of nifL have been deleted and
replaced with the CI019 Prm6.1 promoter sequence none 19-714 SEQ ID
.DELTA.nifL::Prm6.1 with starting at 221bp after the A of the ds180
NO 216 500bp flank ATG start codon, 845bp of nifL have been deleted
and replaced with the CI019 Prm6.1promoter sequence; 500bp flanking
the nifL gene upstream and downstream are included none 19-715 SEQ
ID .DELTA.nifL::Prm7.1 starting at 221bp after the A of the ds181
NO 217 ATG start codon, 845bp of nifL have been deleted and
replaced with the CI019 Prm7.1 promoter sequence none 19-715 SEQ ID
.DELTA.nifL::Prm7.1 with starting at 221bp after the A of the ds181
NO 218 500bp flank ATG start codon, 845bp of nifL have been deleted
and replaced with the CI019 Prm76.1promoter sequence; 500bp
flanking the nifL gene upstream and downstream are included 19-713
19-750 SEQ ID .DELTA.nifL::Prm1.2 starting at 221bp after the A of
the ds172 NO 219 ATG start codon, 845bp of nifL have been deleted
and replaced with the CI019 Prm1.2 promoter sequence 19-713 19-750
SEQ ID .DELTA.nifL::Prm1.2 with starting at 221bp after the A of
the ds172 NO 220 500bp flank ATG start codon, 845bp of nifL have
been deleted and replaced with the CI019 Prm1.2 promoter sequence;
500bp flanking the nifL gene upstream and downstream are included
17-724 19-804 SEQ ID .DELTA.nifL::Prm1.2 starting at 221bp after
the A of the ds172 NO 221 ATG start codon, 845bp of nifL have been
deleted and replaced with the CI019 Prm1.2 promoter sequence 17-724
19-804 SEQ ID .DELTA.nifL::Prm1.2 with starting at 221bp after the
A of the ds172 NO 222 500bp flank ATG start codon, 845bp of nifL
have been deleted and replaced with the CI019 Prm1.2 promoter
sequence; 500bp flanking the nifL gene upstream
and downstream are included 17-724 19-804 SEQ ID glnE.DELTA.AR-2
glnE gene with 1650bp immediately ds34 NO 223 downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain 17-724 19-804 SEQ ID
glnE.DELTA.AR-2 with glnE gene with 1650bp immediately ds34 NO 224
500bp flank downstream of the ATG start codon deleted, resulting in
a truncated glnE protein lacking the adenylyl-removing (AR) domain;
500bp flanking the glnE gene upstream and downstream are included
19-590 19-806 SEQ ID .DELTA.nifL::Prm3.1 starting at 221bp after
the A of the ds175 NO 225 ATG start codon, 845bp of nifL have been
deleted and replaced with the CI019 Prm3.1 promoter sequence 19-590
19-806 SEQ ID .DELTA.nifL::Prm3.1 with starting at 221bp after the
A of the ds175 NO 226 500bp flank ATG start codon, 845bp of nifL
have been deleted and replaced with the CI019 Prm3.1 promoter
sequence; 500bp flanking the nifL gene upstream and downstream are
included 19-590 19-806 SEQ ID glnE.DELTA.AR-2 glnE gene with 1650bp
immediately ds34 NO 227 downstream of the ATG start codon deleted,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain 19-590 19-806 SEQ ID glnE.DELTA.AR-2 with glnE gene
with 1650bp immediately ds34 NO 228 500bp flank downstream of the
ATG start codon deleted, resulting in a truncated glnE protein
lacking the adenylyl-removing (AR) domain; 500bp flanking the glnE
gene upstream and downstream are included none 63-1146 SEQ ID
.DELTA.nifL::PinfC starting at 24bp after the A of the ATG ds908 NO
229 start codon, 1375bp of nifL have been deleted and replaced with
the 63 PinfC promoter sequence none 63-1146 SEQ ID
.DELTA.nifL::PinfC with starting at 24bp after the A of the ATG
ds908 NO 230 500bp flank start codon, 1375bp of nifL have been
deleted and replaced with the 63 PinfC promoter sequence; 500bp
flanking the nifL gene upstream and downstream are included CM015;
6-397 SEQ ID .DELTA.nifL::Prm5 starting at 31bp after the A of the
ATG ds24 PBC6.15 NO 231 start codon, 1375bp of nifL have been
deleted and replaced with the CI006 Prm5 promoter sequence CM015;
6-397 SEQ ID .DELTA.nifL::Prm5 with starting at 31bp after the A of
the ATG ds24 PBC6.15 NO 232 500bp flank start codon, 1375bp of nifL
have been deleted and replaced with the CI006 Prm5 promoter
sequence; 500bp flanking the nifL gene upstream and downstream are
included CM014 6-400 SEQ ID .DELTA.nifL::Prm1 starting at 31bp
after the A of the ATG ds20 NO 233 start codon, 1375bp of nifL have
been deleted and replaced with the CI006 Prm1 promoter sequence
CM014 6-400 SEQ ID .DELTA.nifL::Prm1 with starting at 31bp after
the A of the ATG ds20 NO 234 500bp flank start codon, 1375bp of
nifL have been deleted and replaced with the CI006 Prm1 promoter
sequence; 500bp flanking the nifL gene upstream and downstream are
included CM037; 6-403 SEQ ID .DELTA.nifL::Prm1 starting at 31bp
after the A of the ATG ds20 PBC6.37 NO 235 start codon, 1375bp of
nifL have been deleted and replaced with the CI006 Prm1 promoter
sequence CM037; 6-403 SEQ ID .DELTA.nifL::Prm1 with starting at
31bp after the A of the ATG ds20 PBC6.38 NO 236 500bp flank start
codon, 1375bp of nifL have been deleted and replaced with the CI006
Prm1 promoter sequence; 500bp flanking the nifL gene upstream and
downstream are included CM037; 6-403 SEQ ID glnE.DELTA.AR-2 glnE
gene with 1644bp immediately ds31 PBC6.39 NO 237 downstream of the
ATG start codon deleted, resulting in a truncated glnE protein
lacking the adenylyl-removing (AR) domain CM037; 6-403 SEQ ID
glnE.DELTA.AR-2 with glnE gene with 1644bp immediately ds31 PBC6.40
NO 238 500bp flank downstream of the ATG start codon deleted,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain; 500bp flanking the glnE gene upstream and downstream
are included CM038; 6-404 SEQ ID glnE.DELTA.AR-1 glnE gene with
1287bp immediately ds30 PBC6.38 NO 239 downstream of the ATG start
codon deleted, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain CM038; 6-404 SEQ ID .DELTA.nifL::Prm1
starting at 31bp after the A of the ATG ds20 PBC6.38 NO 240 start
codon, 1375bp of nifL have been deleted and replaced with the CI006
Prm1 promoter sequence CM038; 6-404 SEQ ID .DELTA.nifL::Prm1 with
starting at 31bp after the A of the ATG ds20 PBC6.38 NO 241 500bp
flank start codon, 1375bp of nifL have been deleted and replaced
with the CI006 Prm1 promoter sequence; 500bp flanking the nifL gene
upstream and downstream are included CM038; 6-404 SEQ ID
glnE.DELTA.AR-1 with glnE gene with 1287bp immediately ds30 PBC6.38
NO 242 500bp flank downstream of the ATG start codon deleted,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain; 500bp flanking the glnE gene upstream and downstream
are included CM029; 6-412 SEQ ID glnE.DELTA.AR-1 glnE gene with
1287bp immediately ds30 PBC6.29 NO 243 downstream of the ATG start
codon deleted, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain CM029; 6-412 SEQ ID glnE.DELTA.AR-1
with glnE gene with 1287bp immediately ds30 PBC6.29 NO 244 500bp
flank downstream of the ATG start codon deleted, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain;
500bp flanking the glnE gene upstream and downstream are included
CM029; 6-412 SEQ ID .DELTA.nifL::Prm5 starting at 31bp after the A
of the ATG ds24 PBC6.29 NO 245 start codon, 1375bp of nifL have
been deleted and replaced with the CI006 Prm5 promoter sequence
CM029; 6-412 SEQ ID .DELTA.nifL::Prm5 with starting at 31bp after
the A of the ATG ds24 PBC6.29 NO 246 500bp flank start codon,
1375bp of nifL have been deleted and replaced with the CI006 Prm5
promoter sequence; 500bp flanking the nifL gene upstream and
downstream are included CM093; 6-848 SEQ ID .DELTA.nifL::Prm1
starting at 31bp after the A of the ATG ds20 PBC6.93 NO 247 start
codon, 1375bp of nifL have been deleted and replaced with the CI006
Prm1 promoter sequence CM093; 6-848 SEQ ID .DELTA.nifL::Prm1 with
starting at 31bp after the A of the ATG ds20 PBC6.93 NO 248 500bp
flank start codon, 1375bp of nifL have been deleted and replaced
with the CI006 Prm1 promoter sequence; 500bp flanking the nifL gene
upstream and downstream are included CM093; 6-848 SEQ ID
glnE.DELTA.AR-2 glnE gene with 1644bp immediately ds31 PBC6.93 NO
249 downstream of the ATG start codon deleted, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain
CM093; 6-848 SEQ ID glnE.DELTA.AR-2 with glnE gene with 1644bp
immediately ds31 PBC6.93 NO 250 500bp flank downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain; 500bp flanking the glnE gene
upstream and downstream are included CM093; 6-848 SEQ ID
.DELTA.amtB First 1088bp of amtB gene and 4bp ds126 PBC6.93 NO 251
upstream of start codon deleted; 199bp of gene remaining lacks a
start codon; no amtB protein is translated CM093; 6-848 SEQ ID
.DELTA.amtB with 500bp First 1088bp of amtB gene and 4bp ds126
PBC6.93 NO 252 flank upstream of start codon deleted; 199bp of gene
remaining lacks a start codon; no amtB protein is translated CM094:
6-881 SEQ ID glnE.DELTA.AR-1 glnE gene with 1287bp immediately ds30
PBC6.94 NO 253 downstream of the ATG start codon deleted, resulting
in a truncated glnE protein lacking the adenylyl-removing (AR)
domain CM094; 6-881 SEQ ID glnE.DELTA.AR-1 with glnE gene with
1287bp immediately ds30 PBC6.94 NO 254 500bp flank downstream of
the ATG start codon deleted, resulting in a truncated glnE protein
lacking the adenylyl-removing (AR) domain; 500bp flanking the glnE
gene upstream and downstream are included CM094; 6-881 SEQ ID
.DELTA.nifL::Prm1 starting at 31bp after the A of the ATG ds20
PBC6.94 NO 255 start codon, 1375bp of nifL have been deleted and
replaced with the CI006 Prm1 promoter sequence CM094; 6-881 SEQ ID
.DELTA.nifL::Prm1 with starting at 31bp after the A of the ATG ds20
PBC6.94 NO 256 500bp flank start codon, 1375bp of nifL have been
deleted and replaced with the CI006 Prm1 promoter sequence; 500bp
flanking the nifL gene upstream and downstream are included CM094;
6-881 SEQ ID .DELTA.amtB First 1088bp of amtB gene and 4bp ds126
PBC6.94 NO 257 upstream of start codon deleted; 199bp of gene
remaining lacks a start codon; no amtB protein is translated CM094;
6-881 SEQ ID .DELTA.amtB with 500bp First 1088bp of amtB gene and
4bp ds126 PBC6.94 NO 258 flank upstream of start codon deleted;
199bp of gene remaining lacks a start codon; no amtB protein is
translated none 910-1246 SEQ ID .DELTA.nifL::PinfC starting at 20bp
after the A of the ATG ds960 NO 259 start codon, 1379bp of nifL
have been deleted and replaced with the 910 PinfC promoter sequence
none 910-1246 SEQ ID .DELTA.nifL::PinfC with starting at 20bp after
the A of the ATG ds960 NO 260 500bp flank start codon, 1379bp of
nifL have been deleted and replaced with the 910 PinfC promoter
sequence; 500bp flanking the nifL gene upstream and downstream are
included PBC6.1, CI006 SEQ ID 16S-1 1 of 3 unique 16S rDNA genes in
the N/A 6, CI6 NO 261 CI006 genome PBC6.1, CI006 SEQ ID 16S-2 2 of
3 unique 16S rDNA genes in the N/A 6. CI6 NO 262 CI006 genome
PBC6.1. CI006 SEQ ID nifH N/A N/A 6, CI6 NO 263 PBC6.1, CI006 SEQ
ID nifD2 2 of 2 unique genes annotated as nifD N/A 6, CI6 NO 264 in
CI006 genome PBC6.1, CI006 SEQ ID nifK2 2 of 2 unique genes
annotated as nifK N/A 6. CI6 NO 265 in CI006 genome PBC6.1, CI006
SEQ ID nifL N/A N/A 6,06 NO 266 PBC6.1. CI006 SEQ ID nifA N/A N/A
6, CI6 NO 267
PBC6.1, CI006 SEQ ID glnE N/A N/A 6, CI6 NO 268 PBC6.1, CI006 SEQ
ID 16S-3 3 of 3 unique 16S rDNA genes in the N/A 6,06 NO 269 CI006
genome PBC6.1, CI006 SEQ ID nifD1 1 of 2 unique genes annotated as
nifD N/A 6, CI6 NO 270 in CI006 genome PBC6.1. CI006 SEQ ID nifK1 1
of 2 unique genes annotated as nifK N/A 6, CI6 NO 271 in CI006
genome PBC6.1, CI006 SEQ ID amtB N/A N/A 6, CI6 NO 272 PBC6.1,
CI006 SEQ ID Prm1 348bp includes the 319bp immediately N/A 6, CI6
NO 273 upstream of the ATG start codon of the lpp gene and the
first 29bp of the lpp gene PBC6.1, CI006 SEQ ID Prm5 313bp starting
at 432bp upstream of the N/A 6, CI6 NO 274 ATG start codon of the
ompX gene and ending 119bp upstream of the ATG start codon of the
ompX gene 19, CI19 CI019 SEQ ID nifL N/A N/A NO 275 19, CI19 CI019
SEQ ID nifA N/A N/A NO 276 19, CI19 CI019 SEQ ID 16S-1 1 of 7
unique 16S rDNA genes in the N/A NO 277 CI019 genome 19, CI19 CI019
SEQ ID 16S-2 2 of 7 unique 16S rDNA genes in the N/A NO 278 CI019
genome 19, CI19 CI019 SEQ ID 16S-3 3 of 7 unique 16S rDNA genes in
the N/A NO 279 CI019 genome 19, CI19 CI019 SEQ ID 16S-4 4 of 7
unique 16S rDNA genes in the N/A NO 280 CI019 genome 19, CI19 CI019
SEQ ID 16S-5 5 of 7 unique 16S rDNA genes in the N/A NO 281 CI019
genome 19, CI19 CI019 SEQ ID 16S-6 6 of 7 unique 16S rDNA genes in
the N/A NO 282 CI019 genome 19, CI19 CI019 SEQ ID 16S-7 7 of 7
unique 16S rDNA genes in the N/A NO 283 CI019 genome 19, CI19 CI019
SEQ ID nifH1 1 of 2 unique genes annotated as nifH N/A NO 284 in
CI019 genome 19, CI19 CI019 SEQ ID nifH2 2 of 2 unique genes
annotated as nifH N/A NO 285 in CI019 genome 19, CI19 CI019 SEQ ID
nifD1 1 of 2 unique genes annotated as nifD N/A NO 286 in CI019
genome 19, CI19 CI019 SEQ ID nifD2 2 of 2 unique genes annotated as
nifD N/A NO 287 in CI019 genome 19, CI19 CI019 SEQ ID nifK1 1 of 2
unique genes annotated as nifK N/A NO 288 in CI019 genome 19, CI19
CI019 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A NO 289
in CI019 genome 19, CI19 CI019 SEQ ID glnE N/A N/A NO 290 19, CI19
CI019 SEQ ID Prm4 449bp immediately upstream of the N/A NO 291 ATG
of the dscC 2 gene 19, CI19 CI019 SEQ ID Prm1.2 500bp immediately
upstream of the N/A NO 292 TTG start codon of the infC gene 19,
CI19 CI019 SEQ ID Prm3.1 170 bp immediately upstream of the N/A NO
293 ATG start codon of the rplN gene 19, CI20 CI020 SEQ ID Prm6.1
142bp immediately upstream of the N/A NO 294 ATG of a
highly-expressed hypothetical protein (annotated as PROKKA_00662 in
CI019 assembly 82) 19, CI21 CI021 SEQ ID Prm7.1 293bp immediately
upstream of the N/A NO 295 ATG of the lpp gene 19-375, CM67 SEQ ID
glnE.DELTA.AR-2 glnE gene with 1650bp immediately ds34 19-417, NO
296 downstream of the ATG start codon CM067 deleted, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain
19-375, CM67 SEQ ID glnE.DELTA.AR-2 with glnE gene with 1650bp
immediately ds34 19-417, NO 297 500bp flank downstream of the ATG
start codon CM067 deleted, resulting in a truncated glnE protein
lacking the adenylyl-removing (AR) domain; 500bp flanking the glnE
gene upstream and downstream are included 19-375, CM67 SEQ ID
.DELTA.nifL::null-v1 starting at 221bp after the A of the none
19-417, NO 298 ATG start codon, 845bp of nifL have CM067 been
deleted and replaced with the 31bp sequence
"GGAGTCTGAACTCATCCTGCGATGGGGGCTG" 19-375, CM67 SEQ ID
.DELTA.nifL::null-v1 with starting at 221bp after the A of the none
19-417, NO 299 500bp flank ATG start codon, 845bp of nifL have
CM067 been deleted and replaced with the 31bp sequence
"GGAGTCTGAACTCATCCTGCGATGGGGGCTG"; 500bp flanking the nifL gene
upstream and downstream are included 19-377, CM69 SEQ ID
.DELTA.nifL::null-v2 starting at 221bp after the A of the none
CM069 NO 300 ATG start codon, 845bp of nifL have been deleted and
replaced with the 5bp sequence "TTAAA" 19-377, CM69 SEQ ID
.DELTA.nifL::null-v2 with starting at 221bp after the A of the none
CM069 NO 301 500bp flank ATG start codon, 845bp of nifL have been
deleted and replaced with the 5bp sequence "TTAAA"; 500bp flanking
the nifL gene upstream and downstream are included 19-389, CM81 SEQ
ID .DELTA.nifL::Prm4 starting at 221bp after the A of the ds70
19-418, NO 302 ATG start codon, 845bp of nifL have CM081 been
deleted and replaced with the CI19 Prm4 sequence 19-389, CM81 SEQ
ID .DELTA.nifL::Prm4 with starting at 221bp after the A of the ds70
19-418, NO 303 500bp flank ATG start codon, 845bp of nifL have
CM081 been deleted and replaced with the CI19 Prm4 sequence; 500bp
flanking the nifL gene upstream and downstream are included
137-2084 SEQ ID glnE_KO2 glnE gene with 1290bp immediately ds809 NO
191 downstream of the ATG start codon deleted, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain
137-2084 SEQ ID glnE_KO2 with glnE gene with 1290bp immediately
ds809 NO 192 500bp flank downstream of the ATG start codon deleted,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain; 500bp flanking the glnE gene upstream and downstream
are included 137-2084 SEQ ID .DELTA.nifL-Prm1.2 starting at 24bp
after the A of the ATG ds843 NO 203 start codon, 1372bp of nifL
have been deleted and replaced with the 137 Prm1.2 promoter
sequence 137-2084 SEQ ID .DELTA.nifL-Prm1.2 with starting at 24bp
after the A of the ATG ds843 NO 204 500bp flank start codon, 1372bp
of nifL have been deleted and replaced with the 137 Prm1.2 promoter
sequence; 500bp flanking the nifL gene upstream and downstream are
included 137-2237 SEQ ID glnE_KO2 glnE gene with 1290bp immediately
ds809 NO 191 downstream of the ATG start codon deleted, resulting
in a truncated glnE protein lacking the adenylyl-removing (AR)
domain 137-2237 SEQ ID glnE_KO2 with glnE gene with 1290bp
immediately ds809 NO 192 500bp flank downstream of the ATG start
codon deleted, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain; 500bp flanking the glnE gene
upstream and downstream are included 137-2237 SEQ ID
.DELTA.nifL-Prm1.2 starting at 24bp after the A of the ATG ds843 NO
203 start codon, 1372bp of nifL have been deleted and replaced with
the 137 Prm1.2 promoter sequence 137-2237 SEQ ID .DELTA.nifL-Prm1.2
with starting at 24bp after the A of the ATG ds843 NO 204 500bp
flank start codon, 1372bp of nifL have been deleted and replaced
with the 137 Prm1.2 promoter sequence; 500bp flanking the nifL gene
upstream and downstream are included 137-2237 SEQ ID
glsA2::Prm1.2_AT 81bp immediately upstream of the ds2551 NO 431
G-start ATG start codon were deleted and replaced with the 137
Prm1.2 promoter sequence 137-2237 SEQ ID glsA2::Prm1.2_AT 81bp
immediately upstream of the ds2551 NO 432 G-start with 500bp ATG
start codon were deleted and flank replaced with the 137 Prm1.2
promoter sequence; 500bp flanking the Prm1.2 promoter and glsA2
gene upstream and downstream are included
Example 7: Guided Microbial Remodeling Campaign for the Rational
Improvement of Multiple Phenotypic Traits
[0663] In this example, Steps A-F described in Example 1 were used
to generate several non-transgenic derivative strains of Klebsiella
variicola Wild type (WT) strain, CI137. First, the WT strain,
CI137, was isolated from a rhizosphere, characterized, and
domesticated using the approaches described in steps A-C of Example
1.
[0664] Then using the approaches described in steps D-F of Example
1, multiple phenotypic traits of CI137 were rationally improved
without the use of transgenes. For example, to test whether the
nitrogen fixation trait of the WT strain can be improved, various
genes involved in nitrogen fixation as described throughout this
application were targeted to engineer non-intergeneric mutations,
the engineered/remodeled microbes were analyzed for nitrogen
fixation, and the engineering and the analytics steps were iterated
to test whether further improvements can be made in the nitrogen
fixation ability. Once substantial improvement in the nitrogen
fixation ability was achieved for a remodeled strain of CI137, the
microbes were assessed for the colonization trait. As the remodeled
strains with improved nitrogen fixation showed lower colonization
than the WT strain, genes involved in colonization as described in
this application were targeted to engineer non-intergeneric
mutations to improve colonization, the engineered/remodeled
microbes were analyzed for colonization, and the engineering and
the analytics steps were iterated.
[0665] To generate non-intergeneric mutations through the iterative
remodeling process, two approaches were employed to remodel the
underlying genetic architecture of the microbe: (1) creating
markerless deletions of genomic sequences encoding protein domains
or whole genes, and (2) rewiring regulatory networks by
intragenomic promoter rearrangement.
[0666] The remodeled strains of CI137 obtained through this
iterative remodeling process that showed improvement in nitrogen
fixation and/or colonization are described below.
[0667] To test whether the nitrogen fixation trait of the WT
strain, CI137, can be improved, a 1647 basepair region after the
start codon of the glnE gene was deleted to produce a truncated
GlnE protein that does not comprise the adenylyl-removing (AR)
domain resulting in a constitutively adenylylated glutamine
synthetase. This remodeled CI137 is referred to herein as 137-1034.
137-1034 could excrete ammonia whereas the WT strain did not
excrete ammonia (FIG. 26). However, 137-1034 showed reduced
colonization compared to the WT strain (FIGS. 27-28). The step of
generating non-intergeneric mutations was iterated to further
remodel 137-1034 and to test if any improvement in ammonia
excretion can be obtained. For this, the nifL gene was deleted from
20 bp after the ATG start codon to 87 bp before the TGA stop codon
and a 400 bp fragment from the region upstream of the cspE gene
containing a promoter of the cspE gene was inserted (Prm1.2)
upstream of nifA replacing the deleted portion. This remodeled
microbe is referred to herein as 137-2084. This strain showed
improved ammonia excretion over 137-1034 (FIG. 26). When tested for
colonization potential, 137-2084 showed slightly improved
colonization compared to 137-1034; however, its colonization
potential was still lower by .about.10-fold compared to the WT
strain (FIGS. 27-28). The step of generating non-intergeneric
mutations was iterated/repeated to test whether both the
colonization potential and ammonia excretion could be improved.
Specifically, the nifL gene was deleted from 20 bp after the ATG
start codon to 87 bp before the TGA stop codon; a 400 bp fragment
from the region upstream of the cspE gene containing a promoter of
the cspE gene was inserted (Prm1.2) upstream of nifA replacing the
deleted portion; and a 400 bp fragment from the region upstream of
the cspE gene containing a promoter of the cspE gene was inserted
(Prm1.2) upstream of the glsA2 gene replacing the native promoter
of the gene. This strain is referred to herein as 137-2237.
137-2237 showed similar levels of improved ammonia excretion as
137-2084 (FIG. 26) and at the same time, the colonization potential
of 137-2237 was similar or slightly improved compared to the WT
strain (FIGS. 27-28).
[0668] The above description illustrates how steps D-F described in
Example 1 can be used to iterate non-intergeneric mutations to
stack improvements in multiple traits in remodeled microbes.
[0669] The ammonia excretion assay used here is as described in
Example 2.
Colonization Assay
[0670] Briefly, strains were grown overnight in rich media (SOB).
Optical densities of culture media containing respective strains
were measured, normalized to 1 and and 1 mL of the culture medium
was used to inoculate a corn seed during planting in a grow-pot.
Plants were grown for 3 weeks, then de-potted, and roots were
washed to remove loose sand and debris. Samples of root tissue were
taken from the seminal root and first node (found 1-2 inches below
the crown), washed in PBS, and a gDNA prep was done on the
solution.
[0671] Cells per gram/fresh weight was determined by running the
gDNA preps through qPCR using strain specific primers. A more
detailed description of the assay can be found in a PCT
publication, WO/2019/032926, incorporated by reference herein in
its entirety.
[0672] The above-described mutations made to the C1137 WT strain
are summarized in Table 33 below.
TABLE-US-00034 TABLE 33 List of isolated and derivative K.
variicola strains Strain ID Genotype Mutation Mutation Description
137 WT WT Wild type Klebsiella variicola strain. 137-1034
.DELTA.glnE.sub.AR-KO2 .DELTA.glnE.sub.AR-KO2 Deletion of 1647bp
after the start codon of the glnE gene. 137-2084
.DELTA.nifL::Prm1.2, .DELTA.nifL::Prm1.2 Deletion of the nifL gene
from 20bp after the ATG .DELTA.glnE.sub.AR-KO2, (start) to 87bp
before the TGA (stop) of the gene. A 400bp fragment from the region
upstream of the cspE gene containing a promoter of the cspE gene
was inserted (Prm1.2) upstream of nifA replacing the deleted
portion. .DELTA.glnE.sub.AR-KO2 Deletion of 1647bp after the start
codon of the glnE gene. 137-2337 .DELTA.nifL::Prm1.2,
.DELTA.nifL::Prm1.2 Deletion of the nifL gene from 20bp after the
ATG .DELTA.glnE.sub.AR-KO2, (start) to 87bp before the TGA (stop)
of the gene. glsA2::Prm1.2 A 400bp fragment from the region
upstream of the cspE gene containing a promoter of the cspE gene
was inserted (Prm1.2) upstream of nifA replacing the deleted
portion. .DELTA.glnE.sub.AR-KO2 Deletion of 1647bp after the start
codon of the glnE gene. glsA2::Prm1.2 A 400bp fragment from the
region upstream of the cspE gene containing a promoter of the cspE
gene was inserted (Prm1.2) upstream of the glsA2 gene replacing the
native promoter of the gene.
Numbered Embodiments of the Disclosure
[0673] Notwithstanding the appended claims, the disclosure sets
forth the following numbered embodiments: [0674] 1. A guided
microbial remodeling method for the rational improvement of
plant-associated microbes to perform plant-beneficial functions,
comprising: [0675] a. providing a plurality of microbial species
that are associated with a target plant of interest; [0676] b.
sequencing the genomes of the plurality of microbial species and
characterizing one or more genomic pathways, or sets of genes, that
are associated with a plant-beneficial function of interest; [0677]
c. assaying the plurality of microbial species for: colonization
metrics, transcriptionally active genes under metabolically
relevant environmental conditions, and ability to perform a
plant-beneficial function of interest; [0678] d. selecting a
candidate microbial species from the plurality of assayed microbial
species; [0679] e. transforming the candidate microbial species
with a transformation plasmid comprising: [0680] i. a selection
marker, [0681] ii. a counterselection marker, [0682] iii. a DNA
fragment comprising: a non-intergeneric genetic variation to be
introduced into the candidate microbial species at a target genomic
locus in one or more genomic pathways, or sets of genes, that are
associated with a plant-beneficial function of interest, and
homology arms to the target genomic locus flanking the
non-intergeneric genetic variation; and [0683] iv. plasmid
backbone, [0684] f. selecting for a candidate microbial species
that has undergone an initial homologous recombination and has the
non-intergeneric genetic variation integrated into the target
genomic locus based on the presence of the selection marker in the
genome; [0685] g. selecting for a candidate microbial species that
has the non-intergeneric genetic variation integrated into the
target genomic locus, but has undergone an additional homologous
recombination that loops-out the plasmid backbone, based on the
absence of the counterselection marker; [0686] h. sequencing the
genome of the transformed candidate microbial species and
confirming integration of the non-intergeneric genetic variation at
the target genomic locus and absence of any genetic sequence from
the transformation plasmid; and [0687] i. repeating steps e)-h) one
or more times, until a candidate microbial species has acquired an
improved ability to perform the plant-beneficial function of
interest. [0688] 2. The guided microbial remodeling method
according to embodiment 1, wherein the plant-beneficial function of
interest is nitrogen fixation. [0689] 3. The guided microbial
remodeling method according to embodiment 1, wherein the
plant-beneficial function of interest is phosphate solubilization.
[0690] 4. The guided microbial remodeling method according to
embodiment 1, wherein the plant-beneficial function of interest is
microbial colonization. [0691] 5. The guided microbial remodeling
method according to any one of the embodiments 1-4, wherein step b)
comprises whole genome sequencing. [0692] 6. The guided microbial
remodeling method according to any one of the embodiments 1-5,
wherein step b) comprises characterizing the nitrogen fixation
pathway. [0693] 7. The guided microbial remodeling method according
to any one of the embodiments 1-6, wherein step b) comprises
characterizing a set of genes selected from the group consisting
of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine
synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding
glutaminase, glnD, glnE, nifJ nifH, nifD, nifK, nifY, nifE, nifN,
nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene
associated with biosynthesis of a nitrogenase enzyme, and
combinations thereof. [0694] 8. The guided microbial remodeling
method according to any one of the embodiments 1-7, wherein step c)
comprises assaying the plurality of microbial species for
colonization metrics under greenhouse or lab based conditions.
[0695] 9. The guided microbial remodeling method according to any
one of the embodiments 1-7, wherein step c) comprises assaying the
plurality of microbial species for colonization metrics under field
conditions. [0696] 10. The guided microbial remodeling method
according to any one of the embodiments 1-7, wherein step c)
comprises assaying the plurality of microbial species for
colonization metrics under greenhouse or lab based conditions, and
also under field conditions. [0697] 11. The guided microbial
remodeling method according to any one of the embodiments 1-10,
wherein a colonization metric assayed in step c) comprises at least
one of the following: spatial colonization patterns, temporal
colonization dynamics, density of colonization, or combinations
thereof. [0698] 12. The guided microbial remodeling method
according to any one of the embodiments 1-11, wherein assaying the
plurality of microbial species for transcriptionally active genes
under metabolically relevant environmental conditions in step c)
occurs under greenhouse or lab based conditions. [0699] 13. The
guided microbial remodeling method according to any one of the
embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs under
greenhouse or lab based conditions and comprises measuring the
transcriptomic profile of the microbial species. [0700] 14. The
guided microbial remodeling method according to any one of the
embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs under
greenhouse or lab based conditions and comprises measuring the
transcriptomic activity of genes associated with the microbial
species ability to perform a plant-beneficial function of interest.
[0701] 15. The guided microbial remodeling method according to any
one of the embodiments 1-11, wherein assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions in step c) occurs
under greenhouse or lab based conditions and comprises measuring
the transcriptomic activity of regulatory gene sequences. [0702]
16. The guided microbial remodeling method according to any one of
the embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs under
greenhouse or lab based conditions and comprises measuring the
transcriptomic activity of promoter sequences. [0703] 17. The
guided microbial remodeling method according to any one of the
embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs under
greenhouse or lab based conditions and comprises measuring the
transcriptomic activity of promoter sequences in the presence of
exogenous nitrogen. [0704] 18. The guided microbial remodeling
method according to any one of the embodiments 1-11, wherein
assaying the plurality of microbial species for transcriptionally
active genes under metabolically relevant environmental conditions
in step c) occurs under greenhouse or lab based conditions and
comprises measuring the transcriptomic activity of promoter
sequences in the presence of exogenous nitrogen, wherein said
transcriptomic activity of the promoter sequences is measured by
quantifying the expression of a regulated gene. [0705] 19. The
guided microbial remodeling method according to any one of the
embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs under field
conditions. [0706] 20. The guided microbial remodeling method
according to any one of the embodiments 1-11, wherein assaying the
plurality of microbial species for transcriptionally active genes
under metabolically relevant environmental conditions in step c)
occurs under field conditions and comprises measuring the
transcriptomic profile of the microbial species. [0707] 21. The
guided microbial remodeling method according to any one of the
embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs under field
conditions and comprises measuring the transcriptomic activity of
genes associated with the microbial species ability to perform a
plant-beneficial function of interest. [0708] 22. The guided
microbial remodeling method according to any one of the embodiments
1-11, wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions in step c) occurs under field conditions
and comprises measuring the transcriptomic activity of regulatory
gene sequences. [0709] 23. The guided microbial remodeling method
according to any one of the embodiments 1-11, wherein assaying the
plurality of microbial species for transcriptionally active genes
under metabolically relevant environmental conditions in step c)
occurs under field conditions and comprises measuring the
transcriptomic activity of promoter sequences. [0710] 24. The
guided microbial remodeling method according to any one of the
embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs under field
conditions and comprises measuring the transcriptomic activity of
promoter sequences in the presence of exogenous nitrogen. [0711]
25. The guided microbial remodeling method according to any one of
the embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs under field
conditions and comprises measuring the transcriptomic activity of
promoter sequences in the presence of exogenous nitrogen, wherein
said transcriptomic activity of the promoter sequences is measured
by quantifying the expression of a regulated gene. [0712] 26. The
guided microbial remodeling method according to any one of the
embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs in vitro.
[0713] 27. The guided microbial remodeling method according to any
one of the embodiments 1-11, wherein assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions in step c) occurs
in vitro and comprises measuring the transcriptomic profile of the
microbial species. [0714] 28. The guided microbial remodeling
method according to any one of the embodiments 1-11, wherein
assaying the plurality of microbial species for transcriptionally
active genes under metabolically relevant environmental conditions
in step c) occurs in vitro and comprises measuring the
transcriptomic activity of genes associated with the microbial
species ability to perform a plant-beneficial function of interest.
[0715] 29. The guided microbial remodeling method according to any
one of the embodiments 1-11, wherein assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions in step c) occurs
in vitro and comprises measuring the transcriptomic activity of
regulatory gene sequences. [0716] 30. The guided microbial
remodeling method according to any one of the embodiments 1-11,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions in step c) occurs in vitro and comprises
measuring the transcriptomic activity of promoter sequences. [0717]
31. The guided microbial remodeling method according to any one of
the embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs in vitro and
comprises measuring the transcriptomic activity of promoter
sequences in N-depleted and N-replete conditions. [0718] 32. The
guided microbial remodeling method according to any one of the
embodiments 1-11, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions in step c) occurs in vitro and
comprises measuring the transcriptomic activity of promoter
sequences in N-depleted and N-replete conditions, wherein said
transcriptomic activity of the promoter sequences is measured by
quantifying the expression of a regulated gene. [0719] 33. The
guided microbial remodeling method according to any one of the
embodiments 1-7, wherein assaying the plurality of microbial
species for colonization metrics, comprises: growing said plurality
of microbial species in intimate association with a target plant.
[0720] 34. The guided microbial remodeling method according to any
one of the embodiments 1-7, wherein assaying the plurality of
microbial species for colonization metrics, comprises: growing said
plurality of microbial species in intimate association with a
target plant under greenhouse or lab based conditions. [0721] 35.
The guided microbial remodeling method according to any one of the
embodiments 1-7, wherein assaying the plurality of microbial
species for colonization metrics, comprises: growing said plurality
of microbial species in intimate association with a target plant
under field conditions. [0722] 36. The guided microbial remodeling
method according to any one of the embodiments 1-7, wherein
assaying the plurality of microbial species for transcriptionally
active genes under metabolically relevant environmental conditions,
comprises: growing said plurality of microbial species in intimate
association with a target plant. [0723] 37. The guided microbial
remodeling method according to any one of the embodiments 1-7,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions, comprises: growing said plurality of
microbial species in intimate association with a target plant under
greenhouse or lab based conditions. [0724] 38. The guided microbial
remodeling method according to any one of the embodiments 1-7,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions, comprises: growing said plurality of
microbial species in intimate association with a target plant under
field conditions. [0725] 39. The guided microbial remodeling method
according to any one of the embodiments 1-7, wherein assaying the
plurality of microbial species for colonization metrics and
transcriptionally active genes under metabolically relevant
environmental conditions, comprises: growing said plurality of
microbial species in intimate association with a target plant under
field conditions.
[0726] 40. The guided microbial remodeling method according to any
one of the embodiments 1-7, wherein step c) comprises assaying the
plurality of microbial species for ability to perform a
plant-beneficial function of interest under greenhouse or lab based
conditions. [0727] 41. The guided microbial remodeling method
according to any one of the embodiments 1-7, wherein step c)
comprises assaying the plurality of microbial species for nitrogen
fixation activity. [0728] 42. The guided microbial remodeling
method according to any one of the embodiments 1-7, wherein step c)
comprises assaying the plurality of microbial species for nitrogen
fixation activity in an acetylene reduction assay or ammonium
excretion assay. [0729] 43. The guided microbial remodeling method
according to any one of the embodiments 1-42, wherein the
transformation plasmid of step e) is a suicide plasmid. [0730] 44.
The guided microbial remodeling method according to any one of the
embodiments 1-43, wherein step h) comprises whole genome
sequencing. [0731] 45. A guided microbial remodeling method for the
rational improvement of plant-associated microbes to perform
plant-beneficial functions, comprising: [0732] a. providing a
plurality of microbial species; [0733] b. assaying the plurality of
microbial species for: colonization metrics, transcriptionally
active genes under metabolically relevant environmental conditions,
and ability to perform a plant-beneficial function of interest;
[0734] c. selecting a candidate microbial species from the
plurality of assayed microbial species; [0735] d. introducing one
or more targeted non-intergeneric genetic variations into the
candidate microbial species at a target genomic locus in one or
more genomic pathways, or sets of genes, that are associated with a
plant-beneficial function of interest, said non-intergeneric
genetic variations selected from the group consisting of: full gene
deletions, partial gene deletions, promoter insertions, single base
pair changes, and combinations thereof; [0736] e. sequencing the
genome of the candidate microbial species and confirming
integration of the non-intergeneric genetic variation at the target
genomic locus and absence of any transgenic genetic sequence; and
[0737] f. repeating steps d)-e) one or more times, until a
candidate microbial species has acquired an improved ability to
perform the plant-beneficial function of interest. [0738] 46. A
guided microbial remodeling method for the rational improvement of
plant-associated microbes to perform plant-beneficial functions,
comprising: [0739] a. providing a plurality of microbial species;
[0740] b. assaying the plurality of microbial species for:
colonization metrics, transcriptionally active genes under
metabolically relevant environmental conditions, and ability to
perform a plant-beneficial function of interest; [0741] c.
selecting a candidate microbial species from the plurality of
assayed microbial species; [0742] d. introducing two or more
targeted non-intergeneric genetic variations into the candidate
microbial species at two or more target genomic loci, in one or
more genomic pathways, or sets of genes, that are associated with a
plant-beneficial function of interest, said non-intergeneric
genetic variations selected from the group consisting of: full gene
deletions, partial gene deletions, promoter insertions, single base
pair changes, and combinations thereof; and [0743] e. sequencing
the genome of the candidate microbial species and confirming
introduction of the non-intergeneric genetic variations at the
target genomic loci and absence of any transgenic genetic sequence.
[0744] 47. A computationally guided microbial remodeling method for
the rational improvement of plant-associated microbes to perform
plant-beneficial functions, comprising: [0745] a. accessing a
plurality of microbial whole genome sequences; [0746] b.
identifying a plurality of regulatory gene sequences that actively
regulate the transcription of a gene under a metabolically relevant
environmental condition; [0747] c. identifying a plurality of genes
associated with a plant-beneficial function; [0748] d. selecting a
regulatory gene sequence and a gene associated with a
plant-beneficial function from said pluralities; wherein steps
a)-d) occur in silico; and [0749] e. manufacturing, in vivo, a
remodeled microbial cell that has the selected regulatory gene
sequence operably linked to the selected gene associated with a
plant-beneficial function, thereby improving the expression of the
gene associated with a plant-beneficial function. [0750] 48. A
computationally guided microbial remodeling system for the rational
improvement of plant-associated microbes to perform
plant-beneficial functions, comprising: [0751] a. one or more
processors; and [0752] b. one or more memories operatively coupled
to the one or more processors and having instructions stored
thereon, that when executed by the one or more processors, cause
the system to: [0753] i. access a plurality of microbial whole
genome sequences; [0754] ii. identify a plurality of regulatory
gene sequences that actively regulate the transcription of a gene
under a metabolically relevant environmental condition; [0755] iii.
identify a plurality of genes associated with a plant-beneficial
function; and [0756] iv. select a regulatory gene sequence and a
gene associated with a plant-beneficial function from said
pluralities. [0757] 49. A computationally guided microbial
remodeling method for the rational improvement of plant-associated
microbes to perform plant-beneficial functions, comprising: [0758]
a. activating a computer system having: one or more processors and
one or more memories operatively coupled to the one or more
processors and having instructions stored thereon, thereby causing
the one or more processors to execute the instructions, and cause
the system to: [0759] i. access a plurality of microbial whole
genome sequences; [0760] ii. identify a plurality of regulatory
gene sequences that actively regulate the transcription of a gene
under a metabolically relevant environmental condition; [0761] iii.
identify a plurality of genes associated with a plant-beneficial
function; [0762] iv. select a regulatory gene sequence and a gene
associated with a plant-beneficial function from said pluralities;
and [0763] b. manufacturing, in vivo, a remodeled microbial cell
that has the selected regulatory gene sequence operably linked to
the selected gene associated with a plant-beneficial function,
thereby improving the expression of the gene associated with a
plant-beneficial function. [0764] 50. A guided microbial remodeling
method for the rational improvement of plant-associated microbes to
perform plant-beneficial functions, comprising: [0765] a. providing
a plurality of microbial species that are associated with a target
plant of interest; [0766] b. assaying the plurality of microbial
species for: colonization metrics, and ability to perform a
plant-beneficial function of interest; [0767] c. selecting a
candidate microbial species from the plurality of assayed microbial
species; [0768] d. introducing one or more targeted
non-intergeneric genetic variations into the candidate microbial
species; [0769] e. confirming integration of the non-intergeneric
genetic variation at the target genomic locus and absence of any
transgenetic sequence; and [0770] f. repeating steps d) and e) one
or more times, until the candidate microbial species has acquired
an improved ability to perform the plant-beneficial function of
interest. [0771] 51. The guided microbial remodeling method
according to embodiment 50, wherein the step b) comprises assaying
the plurality of microbial species for transcriptionally active
genes under metabolically relevant environmental conditions. [0772]
52. The guided microbial remodeling method according to embodiment
50, comprising a step of sequencing the genomes of the plurality of
microbial species obtained in step a) and characterizing one or
more genomic pathways, or sets of genes, that are associated with a
plant-beneficial function of interest. [0773] 53. The guided
microbial remodeling method according to embodiment 50, wherein the
step d) of introducing one or more targeted non-intergeneric
genetic variations into the candidate microbial species comprises:
[0774] a. transforming the candidate microbial species with a
transformation plasmid comprising: [0775] i. a selection marker,
[0776] ii. a counterselection marker, [0777] iii. a DNA fragment
comprising: a non-intergeneric genetic variation to be introduced
into the candidate microbial species at a target genomic locus in
one or more genomic pathways, or sets of genes, that are associated
with a plant-beneficial function of interest, and homology arms to
the target genomic locus flanking the non-intergeneric genetic
variation, and [0778] iv. plasmid backbone; [0779] b. selecting for
a candidate microbial species that has undergone an initial
homologous recombination and has the non-intergeneric genetic
variation integrated into the target genomic locus based on the
presence of the selection marker in the genome; and [0780] c.
selecting for a candidate microbial species that has the
non-intergeneric genetic variation integrated into the target
genomic locus, but has undergone an additional homologous
recombination that loops-out the plasmid backbone, based on the
absence of the counterselection marker. [0781] 54. The guided
microbial remodeling method according to embodiment 50, wherein the
step e) of confirming integration of the non-intergeneric genetic
variation at the target genomic locus and absence of any
transgenetic sequence comprises sequencing the genome of the
transformed candidate microbial species. [0782] 55. The guided
microbial remodeling method according to embodiment 50, wherein the
step e) comprises confirming absence of any transgenetic sequence
from the transformation plasmid. [0783] 56. The guided microbial
remodeling method according to embodiment 50, wherein the
plant-beneficial function of interest is nitrogen fixation. [0784]
57. The guided microbial remodeling method according to embodiment
50, wherein the plant-beneficial function of interest is phosphate
solubilization. [0785] 58. The guided microbial remodeling method
according to embodiment 50, wherein the plant-beneficial function
of interest is microbial colonization. [0786] 59. The guided
microbial remodeling method according to embodiment 52, wherein the
step of sequencing comprises whole genome sequencing. [0787] 60.
The guided microbial remodeling method according to embodiment 52,
comprising characterizing the nitrogen fixation pathway. [0788] 61.
The guided microbial remodeling method according to embodiment 52,
comprising characterizing a set of genes selected from the group
consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding
glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide
encoding glutaminase, glnD, glnE, nifJ nifH, nifD, nifK, nifY,
nifE, nifty, nifU, nifS, nif, nifW, nifZ, nifM, nifF, nifB, nifA, a
gene associated with biosynthesis of a nitrogenase enzyme, and
combinations thereof. [0789] 62. The guided microbial remodeling
method according to embodiment 52, comprising characterizing one or
more genes involved in a pathway selected from the group consisting
of: exopolysaccharide production, endo-polygalaturonase production,
trehalose production, and glutamine conversion. [0790] 63. The
guided microbial remodeling method according to embodiment 52,
comprising characterizing one or more genes selected from the group
consisting of: bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2,
and combinations thereof [0791] 64. The guided microbial remodeling
method according to embodiment 52, comprising characterizing one or
more genes selected from the group consisting of: nifA, nifL, ntrB,
ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB,
glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE,
nifJ, nifH, nifD, nifK, nifY, nifE, nifty, nifU, nifS, nifV, nifW,
nifZ, nifM, nifF, nifB, nifA, a gene associated with biosynthesis
of a nitrogenase enzyme, bcsii, bcsiii, yjbE, fhaB, pehA, otsB,
treZ, glsA2, and combinations thereof [0792] 65. The guided
microbial remodeling method according to embodiment 50, wherein
step b) comprises assaying the plurality of microbial species for
colonization metrics under greenhouse or lab based conditions.
[0793] 66. The guided microbial remodeling method according to
embodiment 50, wherein step b) comprises assaying the plurality of
microbial species for colonization metrics under field conditions.
[0794] 67. The guided microbial remodeling method according to
embodiment 50, wherein step b) comprises assaying the plurality of
microbial species for colonization metrics under greenhouse or lab
based conditions, and also under field conditions. [0795] 68. The
guided microbial remodeling method according to embodiment 50,
wherein a colonization metric assayed in step b) comprises at least
one of the following: spatial colonization patterns, temporal
colonization dynamics, density of colonization, or combinations
thereof [0796] 69. The guided microbial remodeling method according
to embodiment 51, wherein assaying the plurality of microbial
species for transcriptionally active genes under metabolically
relevant environmental conditions occurs under greenhouse or lab
based conditions. [0797] 70. The guided microbial remodeling method
according to embodiment 51, wherein assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions occurs under
greenhouse or lab based conditions and comprises measuring the
transcriptomic profile of the microbial species. [0798] 71. The
guided microbial remodeling method according to embodiment 51,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs under greenhouse or lab based
conditions and comprises measuring the transcriptomic activity of
genes associated with the microbial species ability to perform a
plant-beneficial function of interest. [0799] 72. The guided
microbial remodeling method according to embodiment 51, wherein
assaying the plurality of microbial species for transcriptionally
active genes under metabolically relevant environmental conditions
occurs under greenhouse or lab based conditions and comprises
measuring the transcriptomic activity of regulatory gene sequences.
[0800] 73. The guided microbial remodeling method according to
embodiment 51, wherein assaying the plurality of microbial species
for transcriptionally active genes under metabolically relevant
environmental conditions occurs under greenhouse or lab based
conditions and comprises measuring the transcriptomic activity of
promoter sequences. [0801] 74. The guided microbial remodeling
method according to embodiment 51, wherein assaying the plurality
of microbial species for transcriptionally active genes under
metabolically relevant environmental conditions occurs under
greenhouse or lab based conditions and comprises measuring the
transcriptomic activity of promoter sequences in the presence of
exogenous nitrogen.
[0802] 75. The guided microbial remodeling method according to
embodiment 51, wherein assaying the plurality of microbial species
for transcriptionally active genes under metabolically relevant
environmental conditions occurs under greenhouse or lab based
conditions and comprises measuring the transcriptomic activity of
promoter sequences in the presence of exogenous nitrogen, wherein
said transcriptomic activity of the promoter sequences is measured
by quantifying the expression of a regulated gene. [0803] 76. The
guided microbial remodeling method according to embodiment 51,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs under field conditions. [0804] 77.
The guided microbial remodeling method according to embodiment 51,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs under field conditions and
comprises measuring the transcriptomic profile of the microbial
species. [0805] 78. The guided microbial remodeling method
according to embodiment 51, wherein assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions occurs under field
conditions and comprises measuring the transcriptomic activity of
genes associated with the microbial species ability to perform a
plant-beneficial function of interest. [0806] 79. The guided
microbial remodeling method according to embodiment 51, wherein
assaying the plurality of microbial species for transcriptionally
active genes under metabolically relevant environmental conditions
occurs under field conditions and comprises measuring the
transcriptomic activity of regulatory gene sequences. [0807] 80.
The guided microbial remodeling method according to embodiment 51,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs under field conditions and
comprises measuring the transcriptomic activity of promoter
sequences. [0808] 81. The guided microbial remodeling method
according to embodiment 51, wherein assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions occurs under field
conditions and comprises measuring the transcriptomic activity of
promoter sequences in the presence of exogenous nitrogen. [0809]
82. The guided microbial remodeling method according to embodiment
51, wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs under field conditions and
comprises measuring the transcriptomic activity of promoter
sequences in the presence of exogenous nitrogen, wherein said
transcriptomic activity of the promoter sequences is measured by
quantifying the expression of a regulated gene. [0810] 83. The
guided microbial remodeling method according to embodiment 51,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs in vitro. [0811] 84. The guided
microbial remodeling method according to embodiment 51, wherein
assaying the plurality of microbial species for transcriptionally
active genes under metabolically relevant environmental conditions
occurs in vitro and comprises measuring the transcriptomic profile
of the microbial species. [0812] 85. The guided microbial
remodeling method according to embodiment 51, wherein assaying the
plurality of microbial species for transcriptionally active genes
under metabolically relevant environmental conditions occurs in
vitro and comprises measuring the transcriptomic activity of genes
associated with the microbial species ability to perform a
plant-beneficial function of interest. [0813] 86. The guided
microbial remodeling method according to embodiment 51, wherein
assaying the plurality of microbial species for transcriptionally
active genes under metabolically relevant environmental conditions
occurs in vitro and comprises measuring the transcriptomic activity
of regulatory gene sequences. [0814] 87. The guided microbial
remodeling method according to embodiment 51, wherein assaying the
plurality of microbial species for transcriptionally active genes
under metabolically relevant environmental conditions occurs in
vitro and comprises measuring the transcriptomic activity of
promoter sequences. [0815] 88. The guided microbial remodeling
method according to embodiment 51, wherein assaying the plurality
of microbial species for transcriptionally active genes under
metabolically relevant environmental conditions occurs in vitro and
comprises measuring the transcriptomic activity of promoter
sequences in N-depleted and N-replete conditions. [0816] 89. The
guided microbial remodeling method according to embodiment 51,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions occurs in vitro and comprises measuring
the transcriptomic activity of promoter sequences in N-depleted and
N-replete conditions, wherein said transcriptomic activity of the
promoter sequences is measured by quantifying the expression of a
regulated gene. [0817] 90. The guided microbial remodeling method
according to embodiment 50, wherein assaying the plurality of
microbial species for colonization metrics, comprises: growing said
plurality of microbial species in intimate association with a
target plant. [0818] 91. The guided microbial remodeling method
according to embodiment 50, wherein assaying the plurality of
microbial species for colonization metrics, comprises: growing said
plurality of microbial species in intimate association with a
target plant under greenhouse or lab based conditions. [0819] 92.
The guided microbial remodeling method according to embodiment 50,
wherein assaying the plurality of microbial species for
colonization metrics, comprises: growing said plurality of
microbial species in intimate association with a target plant under
field conditions. [0820] 93. The guided microbial remodeling method
according to embodiment 51, wherein assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions, comprises: growing
said plurality of microbial species in intimate association with a
target plant. [0821] 94. The guided microbial remodeling method
according to embodiment 51, wherein assaying the plurality of
microbial species for transcriptionally active genes under
metabolically relevant environmental conditions, comprises: growing
said plurality of microbial species in intimate association with a
target plant under greenhouse or lab based conditions. [0822] 95.
The guided microbial remodeling method according to embodiment 51,
wherein assaying the plurality of microbial species for
transcriptionally active genes under metabolically relevant
environmental conditions, comprises: growing said plurality of
microbial species in intimate association with a target plant under
field conditions. [0823] 96. The guided microbial remodeling method
according to embodiment 51, wherein assaying the plurality of
microbial species for colonization metrics and transcriptionally
active genes under metabolically relevant environmental conditions,
comprises: growing said plurality of microbial species in intimate
association with a target plant under field conditions. [0824] 97.
The guided microbial remodeling method according to embodiment 50,
wherein step b) comprises assaying the plurality of microbial
species for ability to perform a plant-beneficial function of
interest under greenhouse or lab based conditions. [0825] 98. The
guided microbial remodeling method according to embodiment 50,
wherein step b) comprises assaying the plurality of microbial
species for nitrogen fixation activity. [0826] 99. The guided
microbial remodeling method according to embodiment 50, wherein
step b) comprises assaying the plurality of microbial species for
nitrogen fixation activity in an acetylene reduction assay or
ammonium excretion assay. [0827] 100. The guided microbial
remodeling method according to embodiment 50, wherein the
transformation plasmid is a suicide plasmid. [0828] 101. The guided
microbial remodeling method according to embodiment 54, wherein the
sequencing comprises whole genome sequencing. [0829] 102. A guided
microbial remodeling method for the rational improvement of
plant-associated microbes to perform plant-beneficial functions,
comprising: [0830] a. providing a plurality of microbial species;
[0831] b. assaying the plurality of microbial species for:
colonization metrics, and ability to perform a plant-beneficial
function of interest; [0832] c. selecting a candidate microbial
species from the plurality of assayed microbial species; [0833] d.
introducing one or more targeted non-intergeneric genetic
variations into the candidate microbial species at a target genomic
locus in one or more genomic pathways, or sets of genes, that are
associated with a plant-beneficial function of interest; [0834] e.
confirming integration of the non-intergeneric genetic variation at
the target genomic locus and absence of any transgenic genetic
sequence; and [0835] f. repeating steps d)-e) one or more times,
until a candidate microbial species has acquired an improved
ability to perform the plant-beneficial function of interest.
[0836] 103. The guided microbial remodeling method according to
embodiment 102, wherein the step b) comprises assaying
transcriptionally active genes under metabolically relevant
environmental conditions. [0837] 104. The guided microbial
remodeling method according to embodiment 102, wherein in step d),
said non-intergeneric genetic variations are selected from the
group consisting of: full gene deletions, partial gene deletions,
promoter insertions, single base pair changes, and combinations
thereof. [0838] 105. The guided microbial remodeling method
according to embodiment 102, wherein step e) comprises sequencing
the genome of the candidate microbial species. [0839] 106. A guided
microbial remodeling method for the rational improvement of
plant-associated microbes to perform plant-beneficial functions,
comprising: [0840] a. providing a plurality of microbial species;
[0841] b. assaying the plurality of microbial species for:
colonization metrics, and ability to perform a plant-beneficial
function of interest; [0842] c. selecting a candidate microbial
species from the plurality of assayed microbial species; [0843] d.
introducing two or more targeted non-intergeneric genetic
variations into the candidate microbial species at two or more
target genomic loci, in one or more genomic pathways, or sets of
genes, that are associated with a plant-beneficial function of
interest; and [0844] e. confirming introduction of the
non-intergeneric genetic variations at the target genomic loci and
absence of any transgenic genetic sequence. [0845] 107. The guided
microbial remodeling method according to embodiment 106, wherein
step b) comprises assaying transcriptionally active genes under
metabolically relevant environmental conditions. [0846] 108. The
guided microbial remodeling method according to embodiment 106,
wherein in step d), said non-intergeneric genetic variations are
selected from the group consisting of: full gene deletions, partial
gene deletions, promoter insertions, single base pair changes, and
combinations thereof. [0847] 109. The guided microbial remodeling
method according to embodiment 106, wherein step e) comprises
sequencing the genome of the candidate microbial species.
[0848] While preferred embodiments of the present disclosure have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
disclosure. It should be understood that various alternatives to
the embodiments of the disclosure described herein may be employed
in practicing the disclosure. It is intended that the following
Claims define the scope of the disclosure and that methods and
structures within the scope of these claims and their equivalents
be covered thereby.
INCORPORATION BY REFERENCE
[0849] All references, articles, publications, patents, patent
publications, and patent applications cited herein are incorporated
by reference in their entireties for all purposes. However, mention
of any reference, article, publication, patent, patent publication,
and patent application cited herein is not, and should not be taken
as, an acknowledgment or any form of suggestion that they
constitute valid prior art or form part of the common general
knowledge in any country in the world. Further, U.S. Pat. No.
9,975,817, issued on May 22, 2018, and entitled: Methods and
Compositions for Improving Plant Traits, is hereby incorporated by
reference. Further, PCT/US2018/013671, filed Jan. 12, 2018, and
entitled: Methods and Compositions for Improving Plant Traits, is
hereby incorporated by reference.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220127627A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220127627A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References