U.S. patent application number 16/561694 was filed with the patent office on 2019-12-26 for methods and compositions for controlling root lesion nematodes.
The applicant listed for this patent is NewLeaf Symbiotics, Inc.. Invention is credited to Gregg Bogosian, Natalie Breakfield.
Application Number | 20190387747 16/561694 |
Document ID | / |
Family ID | 59018265 |
Filed Date | 2019-12-26 |
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United States Patent
Application |
20190387747 |
Kind Code |
A1 |
Breakfield; Natalie ; et
al. |
December 26, 2019 |
Methods and Compositions for Controlling Root Lesion Nematodes
Abstract
The present disclosure provides compositions comprising
Root-Lesion Nematode (RLN)-inhibitory Methylobacterium sp., as well
as cell-free culture supernatants, fractions, and concentrates
obtained from the Root-Lesion Nematode (RLN)-inhibitory
Methylobacterium sp. Also provided are related methods for
controlling RLN infections of plants, and methods of making the
compositions.
Inventors: |
Breakfield; Natalie; (St.
Louis, MO) ; Bogosian; Gregg; (St. Louis,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NewLeaf Symbiotics, Inc. |
St. Louis |
MO |
US |
|
|
Family ID: |
59018265 |
Appl. No.: |
16/561694 |
Filed: |
September 5, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15373203 |
Dec 8, 2016 |
10448645 |
|
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16561694 |
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62266115 |
Dec 11, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 25/00 20130101;
A01N 63/00 20130101; A01N 63/00 20130101; A01N 63/00 20130101 |
International
Class: |
A01N 63/00 20060101
A01N063/00 |
Claims
1. A method for reducing Pratylenchus sp. damage to a plant that
comprises (a) applying a composition comprising a Methylobacterium
selected from the group consisting of NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto and an agriculturally acceptable
excipient and/or an agriculturally acceptable adjuvant to a plant
part to obtain a treated plant part; and (b) growing the plant from
said treated plant part in the presence of Pratylenchus sp.,
wherein Pratylenchus sp. damage to the plant is reduced in
comparison to a control plant from a control plant part that is not
treated with the Methylobacterium and that is grown in the presence
of Pratylenchus sp.
2. The method of claim 1, wherein the Methylobacterium is present
on said treated plant part in an amount of at least about
1.times.10.sup.3 colony forming units (CFU) of said
Methylobacterium per treated plant part.
3. The method of claim 1, wherein the NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, or NLS0089 derivative is obtained by
mutagenizing or transforming Methylobacterium NLS0021, NLS0038,
NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089, respectively.
4. The method of claim 1, wherein the NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, or NLS0089-related Methylobacterium is
characterized by having a gene encoding a 16S RNA that has at least
95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence
identity across the entire length of SEQ ID NO: 1, 2, 3, 4, 5, 6,
or 7, respectively.
5. The method of claim 1, wherein the Methylobacterium is
heterologous to the plant part.
6. The method of claim 1, wherein the Methylobacterium is: (i)
NLS0021 and the plant part is not a lettuce plant part; (ii)
NLS0038 and the plant part is not a tomato plant part; (iii)
NLS0042 or NLS0934 and the plant part is not a soybean plant part;
(iv) NLS0062 or NLS0069 and the plant part is not a corn plant
part; or (v) NLS0089 and the plant part is not a broccoli plant
part.
7. The method of claim 1, wherein the Pratylenchus sp. damage is
selected from the group consisting of a reduction in plant growth,
yield, water-deficit tolerance, chlorosis, produce quality, and
combinations thereof.
8. The method of claim 1, wherein the reduction in damage is
evidenced by an increase in shoot weight, root weight, or the
combination thereof in the plant in comparison to the control
plant.
9. The method of claim 1, wherein the plant part is a seed, leaf,
tuber, or root.
10. The method of any one of claims 1 to 9, wherein the
Pratylenchus sp. is selected from the group consisting of
Pratylenchus brachyurus, Pratylenchus coffeae, P. neglectus,
Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus
thornei, Pratylenchus vulnus, and Pratylenchus zeae.
11. The method of any one of claims 1 to 9, wherein the plant part
is selected from the group consisting of a Brassica sp. corn,
wheat, rye, rice, alfalfa, rye, sorghum, millet, soybean, tobacco,
potato, peanut, carrot, cotton, coffee, coconut, pineapple, sugar
beet, strawberry, oat, barley, tomato, lettuce, pepper, pea, onion,
green bean, and cucurbit plant part.
12. A plant part that is at least partially coated with a
composition comprising a Methylobacterium selected from the group
consisting of NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069,
NLS0089, derivatives thereof, and Methylobacterium related thereto
and an agriculturally acceptable excipient and/or an agriculturally
acceptable adjuvant, wherein said composition is provided on said
plant part in an amount that reduces Pratylenchus sp. damage to a
plant grown from the plant part in comparison to a control plant
grown from a control plant part that is not treated with the
Methylobacterium.
13. The plant part of claim 12, wherein the amount of
Methylobacterium present on said plant part is at least about
1.times.10.sup.3 colony forming units (CFU) of said
Methylobacterium per plant part.
14. The plant part of claim 12, wherein the plant part is a seed,
leaf, stem, root, or tuber.
15. The plant part of claim 12, wherein the NLS0021, NLS0038,
NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089 derivative is
obtained by mutagenizing or transforming Methylobacterium NLS0021,
NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089,
respectively.
16. The plant part of claim 12, wherein the NLS0021, NLS0038,
NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089-related
Methylobacterium has a gene encoding a 16S RNA that has at least
95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence
identity across the entire length of SEQ ID NO: 1, 2, 3, 4, 5, 6,
or 7, respectively.
17. The plant part of any one of claims 12 to 16, wherein the
Methylobacterium is heterologous to the seed, tuber, or
seedling.
18. The plant part of claim 17, wherein the Methylobacterium is:
(i) NLS0021 and the plant part is not a lettuce plant part; (ii)
NLS0038 and the plant part is not a tomato plant part; (iii)
NLS0042 or NLS0934 and the plant part is not a soybean plant part;
(iv) NLS0062 or NLS0069 and the plant part is not a corn plant
part; or (v) NLS0089 and the plant part is not a broccoli plant
part.
19. The plant part of any one of claims 12 to 16, wherein the
Pratylenchus sp. damage is selected from the group consisting of a
reduction in plant growth, yield, water-deficit tolerance,
chlorosis, produce quality, and combinations thereof.
20. The plant part of claim 19, wherein the reduction in damage is
evidenced by an increase in shoot weight, root weight, or the
combination thereof in the plant in comparison to the control
plant.
21. The plant part of any one of claims 12 to 16, wherein the
Pratylenchus sp. is selected from the group consisting of
Pratylenchus brachyurus, Pratylenchus coffeae, P. neglectus,
Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus
thornei, Pratylenchus vulnus, and Pratylenchus zeae.
22. The plant part of any one of claims 12 to 16, wherein the plant
part is selected from the group consisting of a Brassica sp. corn,
wheat, rye, rice, alfalfa, rice, rye, sorghum, millet, soybean,
tobacco, potato, peanut, carrot, cotton, coffee, coconut,
pineapple, sugar beet, strawberry, oat, barley, tomato, lettuce,
pepper, pea, onion, green bean, and cucurbit plant part.
23. A method for obtaining a Pratylenchus-inhibitory cell-free
culture supernatant comprising growing a Methylobacterium selected
from the group consisting of NLS0021, NLS0038, NLS0042, NLS0934,
NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto in a culture comprising a liquid
or an emulsion and separating the Methylobacterium from the culture
supernatant, thereby obtaining a Pratylenchus-inhibitory cell-free
culture supernatant.
24. The method of claim 23, further comprising the step of
concentrating the cell-free supernatant.
25. The method of claim 23, further comprising the step of
fractionating to cell-free supernatant.
26. The method of claim 23, further comprising the step of
concentrating the cell-free supernatant.
27. The method of any one of claims 23-26, wherein the culture
comprising a liquid or an emulsion further comprises a solid
substance.
28. A Pratylenchus-inhibitory cell-free culture supernatant,
fraction thereof, or concentrate therefrom obtained by the method
of any one of claims 23-27.
29. A method for controlling Root Lesion Nematode (RLN) damage to a
plant comprising: (i) applying a composition comprising an
RLN-inhibitory Methylobacterium sp. isolate selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0042, NLS0934,
NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto to soil where a plant is growing
or will be grown; and, (ii) growing a plant or a plant from seed in
soil subjected to the application of the composition and in the
presence of RLN, wherein RLN damage sustained by the plant grown in
the presence of the RLN is reduced in comparison to a control plant
grown in the presence of the RLN.
30. The method of claim 29, wherein the composition comprises a
solid substance with adherent RLN-inhibitory Methylobacterium grown
thereon at a titer of about 5.times.10.sup.8, 1.times.10.sup.9, or
1.times.10.sup.10 colony-forming units per gram to about
5.times.10.sup.13 colony-forming units of Methylobacterium per
gram, wherein the adherent RLN-inhibitory Methylobacterium is
selected from the group consisting of Methylobacterium NLS0021,
NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives
thereof, and Methylobacterium related thereto.
31. The method of claim 29, wherein the composition comprises the
RLN-inhibitory Methylobacterium sp. at a titer of about
1.times.10.sup.4 or 1.times.10.sup.5 colony-forming units per ml to
about 1.times.10.sup.9, 1.times.10.sup.10, 1.times.10.sup.11,
5.times.10.sup.11, or 1.times.10.sup.12 colony-forming units of
Methylobacterium per mL of a liquid or emulsion.
32. The method of claim 29, wherein the RLN-inhibitory
Methylobacterium sp. is a Methylobacterium isolate selected from
the group consisting of Methylobacterium NLS0021, NLS0038, NLS0069,
derivatives thereof, and Methylobacterium related thereto.
33. The method of any one of claims 29-32, wherein the composition
is applied to the soil by broadcasting the composition, by
drenching the soil with the composition, or by depositing the
composition in furrow.
34. The method of claim 33, wherein composition is deposited in
furrow and wherein the depositing in furrow is performed prior to
placing seed in the furrow, at the same time as placing seed in the
furrow, or after placing seed in the furrow.
35. The method of any one of claims 29-32, wherein the composition
further comprises a nematicide that provides for inhibition of RLN
growth, motility, and/or reproduction and/or reductions in
RLN-mediated plant damage.
36. The method of claim 35, wherein the nematicide is selected from
the group consisting of an organophosphate, biological, and a
carbamate nematicide.
37. A method for reducing the populations of Root Lesion Nematodes
(RLN) in soil comprising: (a) applying a composition comprising a
Methylobacterium selected from the group consisting of NLS0021,
NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives
thereof, and Methylobacterium related thereto and an agriculturally
acceptable excipient and/or an agriculturally acceptable adjuvant
to a plant part to obtain a treated plant part; and (b) growing the
plant from said treated plant part in the presence of RLN, wherein
RLN populations are reduced in soil where the plant was grown in
comparison to soil where a control plant from a control plant part
that is not treated with the Methylobacterium was grown.
38. The method of claim 37, wherein the NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, or NLS0089 derivative is obtained by
mutagenizing or transforming Methylobacterium NLS0021, NLS0038,
NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089, respectively.
39. The method of claim 37, wherein the NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, or NLS0089-related Methylobacterium is
characterized by having a gene encoding a 16S RNA that has at least
95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence
identity across the entire length of SEQ ID NO: 1, 2, 3, 4, 5, 6,
or 7, respectively.
40. The method of claim 37, wherein the Methylobacterium is
heterologous to the plant part.
41. The method of claim 37, wherein the Methylobacterium is: (i)
NLS0021 and the plant part is not a lettuce plant part; (ii)
NLS0038 and the plant part is not a tomato plant part; (iii)
NLS0042 or NLS0934 and the plant part is not a soybean plant part;
(iv) NLS0062 or NLS0069 and the plant part is not a corn plant
part; or (v) NLS0089 and the plant part is not a broccoli plant
part.
42. The method of claim 37, wherein the plant part is selected from
the group consisting of a Brassica sp. corn, wheat, rye, rice,
alfalfa, sorghum, millet, soybean, tobacco, potato, peanut, carrot,
cotton, coffee, coconut, pineapple, sugar beet, strawberry, oat,
barley, tomato, lettuce, pepper, pea, onion, green bean, and
cucurbit plant part.
43. The method of any one of claims 37-42, wherein RLN populations
are reduced in soil at least about 5%, at least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 75%, at least about 85%, or at least about 95%
in comparison to soil where a control plant from a control plant
part that is not treated with the Methylobacterium was grown.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. non-provisional patent
application Ser. No. 15/373,203, filed Dec. 8, 2016 and
incorporated herein by reference in its entirety, which claims the
benefit of U.S. provisional patent application Ser. No. 62/266,115,
filed Dec. 11, 2015 and incorporated herein by reference in its
entirety.
SEQUENCE LISTING STATEMENT
[0002] A sequence listing containing the file named
53907_160860_SEQ_LST_ST25.txt which is 14,455 bytes (measured in
MS-Windows.RTM.) and created on Dec. 1, 2016, comprises 7
sequences, is provided herewith via the USPTO's EFS system, and is
incorporated herein by reference in its entirety.
BACKGROUND
[0003] One-carbon organic compounds such as methane and methanol
are found extensively in nature, and are utilized as carbon sources
by bacteria classified as methanotrophs and methylotrophs.
Methanotrophic bacteria include species in the genera
Methylobacter, Methylomonas, Methylomicrobium, Methylococcus,
Methylosinus, Methylocystis, Methylosphaera, Methylocaldum, and
Methylocella (Lidstrom, 2006). Methanotrophs possess the enzyme
methane monooxygenase, that incorporates an atom of oxygen from
O.sub.2 into methane, forming methanol. All methanotrophs are
obligate one-carbon utilizers that are unable to use compounds
containing carbon-carbon bonds. Methylotrophs, on the other hand,
can also utilize more complex organic compounds, such as organic
acids, higher alcohols, sugars, and the like. Thus, methylotrophic
bacteria are facultative methylotrophs. Methylotrophic bacteria
include species in the genera Methylobacterium, Hyphomicrobium,
Methylophilus, Methylobacillus, Methylophaga, Aminobacter,
Methylorhabdus, Methylopila, Methylosulfonomonas,
Marinosulfonomonas, Paracoccus, Xanthobacter, Ancylobacter (also
known as Microcyclus), Thiobacillus, Rhodopseudomonas, Rhodobacter,
Acetobacter, Bacillus, Mycobacterium, Arthobacter, and Nocardia
(Lidstrom, 2006).
[0004] Most methylotrophic bacteria of the genus Methylobacterium
are pink-pigmented. They are conventionally referred to as PPFM
bacteria, being pink-pigmented facultative methylotrophs. Green
(2005, 2006) identified twelve validated species in the genus
Methylobacterium, specifically M. aminovorans, M. chloromethanicum,
M. dichloromethanicum, M. extorquens, M. fujisawaense, M
mesophilicum, M. organophilum, M. radiotolerans, M. rhodesianum, M.
rhodinum, M. thiocyanatum, and M. zatmanii. However, M. nidulans is
a nitrogen-fixing Methylobacterium that is not a PPFM (Sy et al.,
2001). Methylobacterium are ubiquitous in nature, being found in
soil, dust, fresh water, sediments, and leaf surfaces, as well as
in industrial and clinical environments (Green, 2006).
SUMMARY
[0005] Provided herein are isolated Root Lesion Nematode
(RLN)-inhibitory Methylobacterium sp., compositions comprising
RLN-inhibitory Methylobacterium sp. and/or RLN-inhibitory cell-free
culture supernatants, fractions, or concentrates obtained
therefrom, methods of using the compositions to control RLN damage
to plants, plant parts, and plants derived therefrom, methods of
using the compositions to reduce populations of RLN in soil, and
methods of making the compositions. Such RLN-inhibitory
Methylobacterium sp. are in certain instances referred to herein as
simply "Methylobacterium" or as "PPFM" (pink-pigmented facultative
methylotrophs). In certain embodiments, the RLN-inhibitory
Methylobacterium sp. is aMethylobacterium isolate selected from the
group consisting ofMethylobacterium NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto.
[0006] Methods for reducing Pratylenchus sp. damage to a plant that
comprise applying a composition comprising at least one of a
Methylobacterium selected from the group consisting of NLS0021,
NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives
thereof, Methylobacterium related thereto, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof obtained therefrom, and any combination thereof and an
agriculturally acceptable excipient and/or an agriculturally
acceptable adjuvant to a plant part to obtain a treated plant part;
and growing the plant from said treated plant part in the presence
of Pratylenchus sp., wherein Pratylenchus sp. damage to the plant
is reduced in comparison to a control plant from a control plant
part that is not treated with the Methylobacterium and that is
grown in the presence of Pratylenchus sp. are provided. Such
methods may also be used for reducing the populations of RLN in
soil. In certain embodiments, the Methylobacterium is present on
said treated plant part in an amount of at least about
1.times.10.sup.2 or 1.times.10.sup.3 colony forming units (CFU) of
said Methylobacterium per treated plant part. In certain
embodiments, about 1.times.10.sup.2, 1.times.10.sup.3, or
1.times.10.sup.4 CFU to about 1.times.10.sup.8 or 1.times.10.sup.9
CFU of the RLN-inhibitory Methylobacterium sp. are provided on a
100 mm.sup.2 surface of a treated plant part. In certain
embodiments of the methods, the composition comprises a solid
substance with adherent RLN-inhibitory Methylobacterium grown
thereon or an emulsion having RLN-inhibitory Methylobacterium grown
therein. In certain embodiments of the methods, the composition
comprises the RLN-inhibitory Methylobacterium sp. at a titer of
about 1.times.10.sup.4 or 1.times.10.sup.5 colony-forming units per
ml to about 1.times.10.sup.9, 1.times.10.sup.10, 6.times.10.sup.10,
1.times.10.sup.11, 5.times.10.sup.11, or 1.times.10.sup.12
colony-forming units of Methylobacterium per mL of a liquid or
emulsion. In certain embodiments of the methods, the composition
comprises the RLN-inhibitory Methylobacterium sp. at a titer of
about 5.times.10.sup.8, 1.times.10.sup.9, or 1.times.10.sup.10
colony-forming units per gram (CFU/gm) to about 1.times.10.sup.12
or 5.times.10.sup.13 colony-forming units of Methylobacterium per
gram of a solid substance to which the Methylobacterium is adhered
or at a titer of about 1.times.10.sup.6 CFU/mL to about
1.times.10.sup.9 CFU/mL of the Methylobacterium in an emulsion. In
certain embodiments, the NLS0021, NLS0038, NLS0042, NLS0934,
NLS0062, NLS0069, or NLS0089 derivative is obtained by mutagenizing
or transforming Methylobacterium NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, or NLS0089, respectively. In certain
embodiments, the NLS0021, NLS0038, NLS0042, NLS0934, NLS0062,
NLS0069, or NLS0089-related Methylobacterium is characterized by
having a gene encoding a 16S RNA that has at least 95%, 96%, 97%,
98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the
entire length of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, respectively.
In certain embodiments of any of the aforementioned methods, the
Methylobacterium is heterologous to the plant part. In certain
embodiments of any of the aforementioned methods, the
Methylobacterium is: (i) NLS0021 and the plant part is not a
lettuce plant part; (ii) NLS0038 and the plant part is not a tomato
plant part; (iii) NLS0042 or NLS0934 and the plant part is not a
soybean plant part; (iv) NLS0062 or NLS0069 and the plant part is
not a corn plant part; or (v) NLS0089 and the plant part is not a
broccoli plant part. In certain embodiments of any of the
aforementioned methods, the Pratylenchus sp. damage is selected
from the group consisting of a reduction in plant growth, yield,
water-deficit tolerance, chlorosis, produce quality, and
combinations thereof. In certain embodiments of the aforementioned
methods, the reduction in damage is evidenced by an increase in
shoot weight, root weight, or the combination thereof in the plant
in comparison to the control plant. In certain embodiments, such
produce is a plant part. In certain embodiments of any of the
aforementioned methods, the plant part is a seed, leaf, tuber, or
root. In certain embodiments of any of the aforementioned methods,
the applied composition coats or partially coats the plant part. In
certain embodiments of the aforementioned methods, the composition
is applied to the seed. In certain embodiments of any of the
aforementioned methods, the Pratylenchus sp. is selected from the
group consisting of Pratylenchus brachyurus, Pratylenchus coffeae,
P. neglectus Pratylenchus penetrans, Pratylenchus scribneri,
Pratylenchus thornei, Pratylenchus vulnus, and Pratylenchus zeae.
In certain embodiments of any of the aforementioned methods, the
plant part is selected from the group consisting of a Brassica sp.
corn, wheat, rye, rice, alfalfa, rice, rye, sorghum, millet,
soybean, tobacco, potato, peanut, carrot, cotton, coffee, coconut,
pineapple, sugar beet, strawberry, oat, barley, tomato, lettuce,
pepper, pea, onion, green bean, and cucurbit plant part. In certain
embodiments of any of the aforementioned methods, the composition
further comprises a nematicide that provides for inhibition of RLN
growth, motility, and/or reproduction and/or reductions in
RLN-mediated plant damage. In certain embodiments where the
composition further comprises a nematicide, the nematicide is
selected from the group consisting of an organophosphate,
biological, and a carbamate nematicide. In certain embodiments of
any of the aforementioned methods, soil in which the plant is to be
grown is surveyed for the presence of Pratylenchus sp. and the
composition is applied to the plant part when Pratylenchus sp. are
present in the soil at a level that can result in reductions in
plant growth, yield, water-deficit tolerance, chlorosis, produce
quality, and combinations thereof to an untreated control plant. In
certain embodiments of the methods, the composition is not applied
to the plant part when the Pratylenchus sp. are present in the soil
below levels that result in reductions in plant growth, yield,
water-deficit tolerance, chlorosis, produce quality, and
combinations thereof to an untreated control plant.
[0007] Plant parts that are at least partially coated with a
composition comprising at least one of a Methylobacterium selected
from the group consisting of Methylobacterium NLS0021, NLS0038,
NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof,
Methylobacterium related thereto, a RLN-inhibitory cell-free
culture supernatant, fraction thereof, concentrate thereof obtained
therefrom, and any combination thereof and an agriculturally
acceptable excipient and/or an agriculturally acceptable adjuvant,
wherein said composition is provided on said plant part in an
amount that reduces Pratylenchus sp. damage to a plant grown from
the plant part in comparison to a control plant grown from a
control plant part that is not treated with the Methylobacterium
are also provided. In certain embodiments, the amount of
Methylobacterium present on said plant part is at least about
1.times.10.sup.3 colony forming units (CFU) of said
Methylobacterium per plant part. In certain embodiments, about
1.times.10.sup.2, 1.times.10.sup.3, or 1.times.10.sup.4 CFU to
about 1.times.10.sup.8 or 1.times.10.sup.9 CFU of the
RLN-inhibitory Methylobacterium sp. are provided on a 100 mm.sup.2
surface of the plant part. In certain embodiments, about
1.times.10.sup.2, 1.times.10.sup.3, or 1.times.10.sup.4 CFU to
about 1.times.10.sup.8 or 1.times.10.sup.9 CFU of the
RLN-inhibitory Methylobacterium sp. are provided on the surface of
a plant part that is a seed. In certain embodiments, the plant part
is a seed, leaf, stem, root, or tuber. In certain embodiments, the
NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089
derivative is obtained by mutagenizing or transforming
Methylobacterium NLS0021, NLS0038, NLS0042, NLS0934, NLS0062,
NLS0069, or NLS0089, respectively. In certain embodiments, the
NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or
NLS0089-related Methylobacterium is characterized by having a gene
encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%,
99.7%, 99.9%, or 100% sequence identity across the entire length of
SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, respectively. In certain
embodiments of any of the aforementioned plant parts, the
Methylobacterium is heterologous to the seed, tuber, or seedling.
In certain embodiments of any of the aforementioned methods, the
Methylobacterium is: (i) NLS0021 and the plant part is not a
lettuce plant part; (ii) NLS0038 and the plant part is not a tomato
plant part; (iii) NLS0042 or NLS0934 and the plant part is not a
soybean plant part; (iv) NLS0062 or NLS0069 and the plant part is
not a corn plant part; or (v) NLS0089 and the plant part is not a
broccoli plant part. In certain embodiments of any of the
aforementioned plant parts, the Pratylenchus sp. damage is selected
from the group consisting of a reduction in plant growth, yield,
water-deficit tolerance, chlorosis, produce quality, and
combinations thereof. In certain embodiments of the aforementioned
plant parts, the reduction in damage is evidenced by an increase in
shoot weight, root weight, or the combination thereof in the plant
in comparison to the control plant. In certain embodiments, such
produce is a plant part. In certain embodiments of any of the
aforementioned plant parts, the Pratylenchus sp. is selected from
the group consisting of Pratylenchus brachyurus, Pratylenchus
coffeae, P. neglectus, Pratylenchus penetrans, Pratylenchus
scribneri, Pratylenchus thornei, Pratylenchus vulnus, and
Pratylenchus zeae. In certain embodiments of any of the
aforementioned plant parts, the plant part is selected from the
group consisting of a Brassica sp. corn, wheat, rye, rice, alfalfa,
rice, rye, sorghum, millet, soybean, tobacco, potato, peanut,
carrot, cotton, coffee, coconut, pineapple, sugar beet, strawberry,
oat, barley, tomato, lettuce, pepper, pea, onion, green bean, and
cucurbit plant part.
[0008] Also provided are methods for controlling Root Lesion
Nematode (RLN) damage to a plant that comprise: (i) applying a
composition comprising at least one of an RLN-inhibitory
Methylobacterium sp., a RLN-inhibitory cell-free culture
supernatant, fraction thereof, concentrate thereof, and any
combination thereof to soil where a plant is growing or will be
grown. In certain embodiments, the composition comprises a solid
substance with adherent RLN-active Methylobacterium grown thereon
or an emulsion having RLN-inhibitory Methylobacterium grown
therein; and, (ii) growing a plant or a plant from seed in soil
subjected to the application of the composition and in the presence
of RLN. Such methods may also be used for reducing the populations
of RLN in soil. In certain embodiments of the methods, RLN damage
sustained by the plant grown in the presence of the RLN is reduced
in comparison to a control plant grown in the presence of the RLN.
In certain embodiments of the methods, the composition comprises
the RLN-inhibitory Methylobacterium sp. at a titer of about
5.times.10.sup.8, 1.times.10.sup.9, or 1.times.10.sup.10
colony-forming units per gram of the solid substance to about
5.times.10.sup.13 colony-forming units of Methylobacterium per gram
of the solid substance or at a titer of about 1.times.10.sup.6
CFU/mL to about 1.times.10.sup.9 CFU/mL for the emulsion. In
certain embodiments of the methods, the composition that is applied
comprises the RLN-inhibitory Methylobacterium sp. at a titer of
about 1.times.10.sup.4 or 1.times.10.sup.5 colony-forming units per
ml to about 1.times.10.sup.9, 1.times.10.sup.10, 1.times.10.sup.11,
5.times.10.sup.11, or 1.times.10.sup.12 colony-forming units of
Methylobacterium per mL of a liquid or emulsion. In certain
embodiments, about 1.times.10.sup.2, 1.times.10.sup.3, or
1.times.10.sup.4 CFU to about 1.times.10.sup.8 or 1.times.10.sup.9
CFU of the RLN-inhibitory Methylobacterium sp. are provided on a
100 mm.sup.2 surface of a plant part. In certain embodiments, about
1.times.10.sup.2, 1.times.10.sup.3, or 1.times.10.sup.4 CFU to
about 1.times.10.sup.8 or 1.times.10.sup.9 CFU of the
RLN-inhibitory Methylobacterium sp. are provided on the surface of
a seed. In certain embodiments of the methods, the RLN-inhibitory
Methylobacterium sp. is a Methylobacterium isolate selected from
the group consisting of Methylobacterium NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto. In certain embodiments of any of
the aforementioned methods, the composition is applied to the soil
by broadcasting the composition, by drenching the soil with the
composition, and/or by depositing the composition in furrow. In
certain embodiments of the methods, the depositing in furrow is
performed prior to placing seed in the furrow, at the same time as
placing seed in the furrow, or after placing seed in the furrow. In
certain embodiments of any of the aforementioned methods, the
composition further comprises a nematicide that provides for
inhibition of RLN growth, motility, and/or reproduction and/or
reductions in RLN-mediated plant damage. In certain embodiments
where the composition further comprises a nematicide, the
nematicide is selected from the group consisting of an
organophosphate, biological, and a carbamate nematicide. In certain
embodiments of any of the aforementioned methods, soil in which the
plant is to be grown is surveyed for the presence of RLN and the
composition is applied to the soil when the RLN are present in the
soil at a level that can result in reductions in plant growth,
yield, water-deficit tolerance, chlorosis, produce quality, and
combinations thereof to an untreated control plant. In certain
embodiments of the methods, the composition is not applied to the
soil when the RLN are present in the soil below levels that result
in reductions in plant growth, yield, water-deficit tolerance,
chlorosis, produce quality, and combinations thereof to an
untreated control plant.
[0009] Methods for treating a plant seed that can provide a Root
Lesion Nematodes (RLN) tolerant plant that comprises applying a
composition comprising at least one of a RLN-inhibitory
Methylobacterium sp., a RLN-inhibitory cell-free culture
supernatant, fraction thereof, concentrates thereof, or any
combination thereof to a seed, thereby obtaining a treated seed
that can provide a RLN tolerant plant are also provided. In certain
embodiments of the methods, RLN damage sustained by the RLN
tolerant plant grown from the treated seed and in the presence of
the RLN is reduced in comparison to RLN damage sustained by a
control plant grown from an untreated seed in the presence of RLN.
In certain embodiments of the methods, the composition comprises a
solid substance with adherent RLN-inhibitory Methylobacterium grown
thereon or an emulsion having RLN-inhibitory Methylobacterium grown
therein. In certain embodiments of the methods, the composition
comprises the RLN-inhibitory Methylobacterium sp. at a titer of
about 5.times.10.sup.8, 1.times.10.sup.9, or 1.times.10.sup.10
colony-forming units per gram of the solid substance to about
5.times.10.sup.13 colony-forming units of Methylobacterium per gram
of the solid substance or at a titer of about 1.times.10.sup.6
CFU/mL to about 1.times.10.sup.9 CFU/mL for the emulsion. In
certain embodiments of the methods, the composition comprises the
RLN-inhibitory Methylobacterium sp. at a titer of about
1.times.10.sup.4 or 1.times.10.sup.5 colony-forming units per ml to
about 1.times.10.sup.9, 1.times.10.sup.10, 6.times.10.sup.10,
1.times.10.sup.11, 5.times.10.sup.11, or 1.times.10.sup.12
colony-forming units of Methylobacterium per mL of a liquid or
emulsion. In certain embodiments, about 1.times.10.sup.2,
1.times.10.sup.3, or 1.times.10.sup.4 CFU to about 1.times.10.sup.8
or 1.times.10.sup.9 CFU of the RLN-inhibitory Methylobacterium sp.
are provided on the surface of the seed. In certain embodiments of
the methods, the RLN-inhibitory Methylobacterium sp. is
aMethylobacterium isolate selected from the group consisting of
Methylobacterium NLS0021, NLS0038, NLS0042, NLS0934, NLS0062,
NLS0069, NLS0089, derivatives thereof, and Methylobacterium related
thereto. In certain embodiments of any of the aforementioned
methods, the applied composition coats or partially coats the seed.
Also provided herein are treated seeds obtained by any of the
aforementioned methods. In certain embodiments of any of the
aforementioned methods, the composition further comprises a
nematicide that provides for inhibition of RLN growth, motility,
and/or reproduction and/or reductions in RLN-mediated plant damage.
In certain embodiments where the composition further comprises a
nematicide, the nematicide is selected from the group consisting of
an organophosphate, biological, and a carbamate nematicide.
[0010] Also provided herein are methods for controlling Root Lesion
Nematodes (RLN) damage to a plant that comprise: (i) planting a
seed that has been treated with a composition comprising at least
one of a RLN-inhibitory Methylobacterium sp., a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof; and, (ii) growing a
RLN-tolerant plant from the treated seed in the presence of RLN. In
certain embodiments of the methods, the RLN damage sustained by the
RLN-tolerant plant grown in the presence of the RLN is reduced in
comparison to RLN damage sustained by a control plant grown from
untreated seed in the presence of RLN. In certain embodiments of
the methods, the seed was treated with a composition that comprises
a solid substance with adherent RLN-inhibitory Methylobacterium
grown thereon or an emulsion having RLN-inhibitory Methylobacterium
grown therein. In certain embodiments of the methods, the
composition comprises the RLN-inhibitory Methylobacterium sp. at a
titer of about 5.times.10.sup.8, 1.times.10.sup.9, or
1.times.10.sup.10 colony-forming units per gram of the solid
substance to about 5.times.10.sup.13 colony-forming units of
Methylobacterium per gram of the solid substance or at a titer of
about 1.times.10.sup.6 CFU/mL to about 1.times.10.sup.9 CFU/mL for
the emulsion. In certain embodiments of the methods, the
composition comprises the RLN-inhibitory Methylobacterium sp. at a
titer of about 1.times.10.sup.4 or 1.times.10.sup.5 colony-forming
units per ml to about 1.times.10.sup.9, 1.times.10.sup.10,
1.times.10.sup.11, 5.times.10.sup.11, or 1.times.10.sup.12
colony-forming units of Methylobacterium per mL of a liquid or
emulsion. In certain embodiments, about 1.times.10.sup.2,
1.times.10.sup.3, or 1.times.10.sup.4 CFU to about 1.times.10.sup.8
or 1.times.10.sup.9 CFU of the RLN-inhibitory Methylobacterium sp.
are provided on the surface of the seed. In certain embodiments of
the methods, the RLN-inhibitory Methylobacterium sp. is
aMethylobacterium isolate selected from the group consisting of
Methylobacterium NLS0021, NLS0038, NLS0042, NLS0934, NLS0062,
NLS0069, NLS0089, derivatives thereof, and Methylobacterium related
thereto. In certain embodiments of any of the aforementioned
methods, the applied composition coats or partially coats the seed.
In certain embodiments of any of the aforementioned methods, the
composition further comprises a nematicide that provides for
inhibition of RLN growth, motility, and/or reproduction and/or
reductions in RLN-mediated plant damage. In certain embodiments
where the composition further comprises a nematicide, the
nematicide is selected from the group consisting of a
organophosphate, biological, and a carbamate nematicide.
[0011] Also provided are compositions comprising at least one of a
RLN-inhibitory Methylobacterium sp., a RLN-inhibitory cell-free
culture supernatant, fraction thereof, concentrate thereof, or any
combination thereof and an agriculturally acceptable adjuvant
and/or and agriculturally acceptable excipient. In certain
embodiments, the composition comprises a solid substance with
adherent RLN-inhibitory Methylobacterium grown thereon or an
emulsion having RLN-inhibitory Methylobacterium grown therein. In
certain embodiments, the composition comprises the RLN-inhibitory
Methylobacterium sp. at a titer of about 5.times.10.sup.8,
1.times.10.sup.9, or 1.times.10.sup.10 colony-forming units per
gram of the solid substance to about 5.times.10.sup.13
colony-forming units of Methylobacterium per gram of the solid
substance or at a titer of about 1.times.10.sup.6 CFU/mL to about
1.times.10.sup.9 CFU/mL for the emulsion. In certain embodiments,
the composition comprises the RLN-inhibitory Methylobacterium sp.
at a titer of about 1.times.10.sup.4 or 1.times.10.sup.5
colony-forming units per ml to about 1.times.10.sup.9,
1.times.10.sup.10, or 6.times.10.sup.10 colony-forming units of
Methylobacterium per mL of a liquid or emulsion. In certain
embodiments, about 1.times.10.sup.2, 1.times.10.sup.3, or
1.times.10.sup.4 CFU to about 1.times.10.sup.8 or 1.times.10.sup.9
CFU of the RLN-inhibitory Methylobacterium sp. are provided on a
100 mm.sup.2 surface of a plant part. In certain embodiments, about
1.times.10.sup.2, 1.times.10.sup.3, or 1.times.10.sup.4 CFU to
about 1.times.10.sup.8 or 1.times.10.sup.9 CFU of the
RLN-inhibitory Methylobacterium sp. are provided on the surface of
a seed. In certain embodiments, the RLN-inhibitory Methylobacterium
sp. is selected from the group consisting of Methylobacterium
NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089,
derivatives thereof, and Methylobacterium related thereto. In
certain embodiments, the composition further comprises a nematicide
that provides for inhibition of RLN growth, motility, and/or
reproduction and/or reductions in RLN-mediated plant damage. In
certain embodiments, the nematicide is selected from the group
consisting of an organophosphate, biological, and a carbamate
nematicide. In any of the aforementioned compositions, the
composition can be in a liquid form or in a dry form. In certain
embodiments, the composition is in a dry, lyophilized form and
further comprises a cryoprotectant. In certain embodiments, the
compositions will be essentially free of contaminating
microorganisms.
[0012] In certain embodiments of any of the aforementioned
compositions comprising at least one of a RLN-inhibitory
Methylobacterium sp., a RLN-inhibitory cell-free culture
supernatant, fraction thereof, concentrate thereof, or any
combination thereof, plants or plant part that is coated or
partially coated with the composition, methods of using the
compositions to control RLN damage to plants, plant parts, and
plants derived therefrom, and methods of making the compositions,
the RLN-inhibitory Methylobacterium sp. is heterologous to the
plant or plant part to which it is applied.
[0013] In certain embodiments of any of the aforementioned
compositions, plants, or plant part that is coated or partially
coated with the compositions, methods of using the compositions to
control RLN damage to plants, plant parts, and plants derived
therefrom, methods of using the compositions to reduce populations
of RLN in soil, and methods of making the compositions, the
RLN-inhibitory Methylobacterium is selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0042, NLS0934,
NLS0062, NLS0069, NLS0089, and a derivative thereof.
[0014] Also provided are methods for obtaining a RLN-inhibitory
cell-free culture supernatant comprising growing a Methylobacterium
selected from the group consisting of NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto in a culture comprising a liquid
or an emulsion and separating the Methylobacterium from the culture
supernatant, thereby obtaining a RLN-inhibitory cell-free culture
supernatant. In certain embodiments, the methods further comprise
the step of concentrating the cell-free culture supernatant. In
certain embodiments, the methods further comprise the step of
fractionating to cell-free supernatant. In certain embodiments, the
methods further comprise the step of concentrating the cell-free
supernatant. In certain embodiments of any of the aforementioned
methods, the culture comprising a liquid or an emulsion further
comprises a solid substance. Also provided are RLN-inhibitory
cell-free culture supernatants, fractions thereof, or concentrates
therefrom obtained by any of the aforementioned methods.
[0015] In certain embodiments of any of the aforementioned
compositions, methods, plant, or plant parts, the RLN-inhibitory
Methylobacterium sp. has a 16S RNA encoding sequence that has
significant sequence identity to the 16S RNA encoding sequence of a
RLN-inhibitory Methylobacterium sp. provided herein. In certain
embodiments, the RLN-inhibitory Methylobacterium sp. has a 16S RNA
encoding sequence that has at least 95%, 96%, 97%, 98%, 99%, or
99.5% sequence identity across the entire length of the 16S RNA
encoding sequence of the RLN-inhibitory Methylobacterium sp.
isolate NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or
NLS0089 provided herein. A RLN active Methylobacterium sp. that can
be used in any of the composition, plants or plant parts that are
coated or partially coated with the compositions, methods of using
the compositions to control RLN damage to plants, plant parts, and
plants derived therefrom, and methods of making the compositions
can be RLN active Methylobacterium sp. can be at least 95%, 96%,
97%, 98%, 99%, or 99.5% sequence identity across the entire length
of the 16S RNA encoding sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6,
or 7.
[0016] In certain embodiments, methods for reducing the populations
of Root Lesion Nematodes (RLN) in soil comprising: (a) applying a
composition comprising a Methylobacterium selected from the group
consisting of NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069,
NLS0089, derivatives thereof, and Methylobacterium related thereto
and an agriculturally acceptable excipient and/or an agriculturally
acceptable adjuvant to a plant part to obtain a treated plant part;
and (b) growing the plant from said treated plant part in the
presence of RLN, wherein RLN populations are reduced in soil where
the plant was grown in comparison to soil where a control plant
from a control plant part that is not treated with the
Methylobacterium was grown are provided. In certain embodiments,
methods for reducing the populations of Root Lesion Nematodes (RLN)
in soil comprising: (i) applying a composition comprising at least
one of an RLN-inhibitory Methylobacterium sp., a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, and any combination thereof to soil where a plant is
growing or will be grown to obtain a treated soil; and (b) growing
a plant in the treated soil and in the presence of RLN, wherein RLN
populations are reduced in treated soil where the plant was grown
in comparison to untreated soil where a control plant was grown are
provided. In certain embodiments of the methods, the NLS0021,
NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089 derivative
is obtained by mutagenizing or transforming Methylobacterium
NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089,
respectively. In certain embodiments of the methods, the NLS0021,
NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089-related
Methylobacterium is characterized by having a gene encoding a 16S
RNA that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%,
or 100% sequence identity across the entire length of SEQ ID NO: 1,
2, 3, 4, 5, 6, or 7, respectively. In certain embodiments of the
methods, the Methylobacterium is heterologous to the plant or the
plant part. In certain embodiments of the methods, the
Methylobacterium is: (i) NLS0021 and the plant or plant part is not
a lettuce plant or plant part; (ii) NLS0038 and the plant or plant
part is not a tomato plant or plant part; (iii) NLS0042 or NLS0934
and the plant or plant part is not a soybean plant or plant part;
(iv) NLS0062 or NLS0069 and the plant or plant part is not a corn
plant or plant part; or (v) NLS0089 and the plant or plant part is
not a broccoli plant or plant part. In certain embodiments of the
methods, the plant or plant part is selected from the group
consisting of a Brassica sp. corn, wheat, rye, rice, alfalfa,
sorghum, millet, soybean, tobacco, potato, peanut, carrot, cotton,
coffee, coconut, pineapple, sugar beet, strawberry, oat, barley,
tomato, lettuce, pepper, pea, onion, green bean, and cucurbit plant
or plant part. In certain embodiments of the aforementioned
methods, the RLN populations are reduced in soil at least about 5%,
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 75%, at least
about 85%, or at least about 95% in comparison to soil where a
control plant from a control plant part that is not treated with
the Methylobacterium was grown. In certain embodiments of the
aforementioned methods, the RLN populations are reduced in the soil
that had been treated by at least about 5%, at least about 10%, at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 75%, at least about 85%, or at least
about 95% in comparison to soil that had not been treated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Not Applicable.
DESCRIPTION
Definitions
[0018] As used herein, the phrases "adhered thereto" and "adherent"
refer to Methylobacterium that are associated with a solid
substance by growing, or having been grown, on a solid
substance.
[0019] As used herein, the phrase "agriculturally acceptable
adjuvant" refers to a substance that enhances the performance of an
active agent in a composition for treatment of plants and/or plant
parts. In certain compositions, an active agent can comprise a
mono-culture or co-culture of Methylobacterium.
[0020] As used herein, the phrase "agriculturally acceptable
excipient" refers to an essentially inert substance that can be
used as a diluent and/or carrier for an active agent in a
composition for treatment of plants and/or plant parts. In certain
compositions, an active agent can comprise a mono-culture or
co-culture of Methylobacterium.
[0021] As used herein, the term "Methylobacterium" refers to
bacteria that are facultative methylotrophs of the genus
Methylobacterium. The term Methylobacterium, as used herein, thus
does not encompass species in the genera Methylobacter,
Methylomonas, Methylomicrobium, Methylococcus, Methylosinus,
Methylocystis, Methylosphaera, Methylocaldum, and Methylocella,
which are obligate methanotrophs.
[0022] As used herein, the phrase "control plant" refers to a plant
that had not received treatment with a RLN-inhibitory
Methylobacterium, a RLN-inhibitory cell-free culture supernatant,
fraction thereof, concentrate thereof, any combination thereof, or
composition comprising the same at either the seed or any
subsequent stage of the control plant's development. Control plants
include, but are not limited to, un-treated plants, plants treated
with compositions lacking any RLN-inhibitory agents, non-transgenic
plants, transgenic plants having a transgene-conferred RLN
resistance trait, and plants treated with, or grown in soil treated
with, an insecticidal compound or other agent that can protect a
plant from RLN feeding.
[0023] As used herein, the terms "Root Lesion Nematodes" and "RLN"
are used interchangeable to refer to the juvenile or adult forms of
any nematode of the genus Pratylenchus.
[0024] As used herein, the phrase "co-culture of Methylobacterium"
refers to a Methylobacterium culture comprising at least two
strains of Methylobacterium or at least two species of
Methylobacterium.
[0025] As used herein, the phrase "contaminating microorganism"
refers to microorganisms in a culture, fermentation broth,
fermentation broth product, or composition that were not identified
prior to introduction into the culture, fermentation broth,
fermentation broth product, or composition.
[0026] As used herein, the phrase "derivatives thereof", when used
in the context of a Methylobacterium isolate, refers to any strain
that is obtained from the Methylobacterium isolate. Derivatives of
aMethylobacterium isolate include, but are not limited to, variants
of the strain obtained by selection, variants of the strain
selected by mutagenesis and selection, and genetically transformed
strains obtained from the Methylobacterium isolate.
[0027] As used herein, the term "emulsion" refers to a colloidal
mixture of two immiscible liquids wherein one liquid is the
continuous phase and the other liquid is the dispersed phase. In
certain embodiments, the continuous phase is an aqueous liquid and
the dispersed phase is liquid that is not miscible, or partially
miscible, in the aqueous liquid.
[0028] As used herein, the phrase "essentially free of
contaminating microorganisms" refers to a culture, fermentation
broth, fermentation product, or composition where at least about
95% of the microorganisms present by amount or type in the culture,
fermentation broth, fermentation product, or composition are the
desired Methylobacterium or other desired microorganisms of
pre-determined identity.
[0029] As used herein, the term "heterologous", when used in the
context of Methylobacterium, cell-free culture supernatant,
fraction thereof, or concentrate thereof that at least partially
coats a plant or plant part, refers to a Methylobacterium,
cell-free culture supernatant, fraction thereof, or concentrate
thereof that is not naturally associated with a plant or plant part
of the same species as the plant or plant part that is at least
partially coated with the Methylobacterium, cell-free culture
supernatant, fraction thereof, or concentrate thereof. In certain
embodiments, the heterologous Methylobacterium that is used to at
least partially coat a plant or plant part of a first plant species
is a Methylobacterium that was isolated, or can be isolated, from a
second and distinct plant species. In certain embodiments, the
heterologous Methylobacterium cell-free culture supernatant,
fraction thereof, or concentrate thereof that is used to at least
partially coat a plant or plant part of a first plant species is
obtained from aMethylobacterium that was isolated, or can be
isolated, from a second and distinct plant species.
[0030] As used herein, the phrase "inanimate solid substance"
refers to a substance which is insoluble or partially soluble in
water or aqueous solutions and which is either non-living or which
is not a part of a still-living organism from which it was
derived.
[0031] As used herein, the phrase "mono-culture of
Methylobacterium" refers to a Methylobacterium culture consisting
of a single strain of Methylobacterium.
[0032] As used herein, the phrase "partially coated", when used in
the context of a composition comprising a RLN-inhibitory
Methylobacterium sp. and a plant part (e.g., a seed), refers to a
plant part where at least 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%,
or 95% of the surface area of the plant part is coated with the
composition.
[0033] As used herein, the term "peptide" refers to any polypeptide
of 50 amino acid residues or less.
[0034] As used herein, the term "protein" refers to any polypeptide
having 51 or more amino acid residues.
[0035] As used herein, a "pesticide" refers to an agent that is
insecticidal, fungicidal, nematocidal, bacteriocidal, or any
combination thereof.
[0036] As used herein, the phrase "bacteriostatic agent" refers to
agents that inhibit growth of bacteria but do not kill the
bacteria.
[0037] As used herein, the phrase "pesticide does not substantially
inhibit growth of said Methylobacterium" refers to any pesticide
that when provided in a composition comprising a fermentation
product comprising a solid substance wherein a mono-culture or
co-culture of Methylobacterium is adhered thereto, results in no
more than a 50% inhibition of Methylobacterium growth when the
composition is applied to a plant or plant part in comparison to a
composition lacking the pesticide. In certain embodiments, the
pesticide results in no more than a 40%, 20%, 10%, 5%, or 1%
inhibition of Methylobacterium growth when the composition is
applied to a plant or plant part in comparison to a composition
lacking the pesticide.
[0038] As used herein, the term "PPFM bacteria" refers without
limitation to bacterial species in the genus Methylobacterium other
than M. nodulans.
[0039] As used herein, the phrase "solid substance" refers to a
substance which is insoluble or partially soluble in water or
aqueous solutions.
[0040] As used herein, the phrase "solid phase that can be
suspended therein" refers to a solid substance that can be
distributed throughout a liquid by agitation.
[0041] As used herein, the term "non-regenerable" refers to either
a plant part or processed plant product that cannot be regenerated
into a whole plant.
[0042] To the extent to which any of the preceding definitions is
inconsistent with definitions provided in any patent or non-patent
reference incorporated herein by reference, any patent or
non-patent reference cited herein, or in any patent or non-patent
reference found elsewhere, it is understood that the preceding
definition will be used herein.
RLN-Inhibitory Methylobacterium, Compositions Comprising
RLN-Inhibitory Methylobacterium and/or RLN-Inhibitory Cell Free
Culture Supernatants, Fractions, or Concentrates Thereof, Methods
of their Use, and Methods of Making
[0043] Various RLN-inhibitory Methylobacterium isolates,
compositions comprising these Methylobacterium, and/or
RLN-inhibitory cell free culture supernatants, fractions, or
concentrates thereof, methods of using the compositions to inhibit
RLN growth, motility, or reproduction and/or reduce RLN damage to a
plant, and methods of making the compositions are provided herein.
As used herein, inhibition of the growth, motility, or reproduction
of a RLN includes any measurable decrease in RLN growth, motility,
and/or reproduction, where RLN growth, motility, and/or
reproduction includes, but is not limited to, any measurable
increase in the juvenile weight, any measurable movement, and/or
any progression through juvenile development stages or from
juvenile to adult development. As used herein, inhibition of RLN
growth, motility, and/or reproduction and/or reduction of RLN
damage to a plant are also understood to include any measurable
decrease in RLN infection and/or the adverse effects or damage
caused by RLN feeding on a plant. Adverse effects of RLN infection
on a plant include, but are not limited to, deformation and/or
reductions in root systems, reductions in top growth, any type of
tissue damage or necrosis, increased incidence of fungal or
bacterial disease, any type of yield reduction, and/or decreased
water-deficit tolerance.
[0044] Isolated RLN-inhibitory Methylobacterium sp. are provided
herein. In certain embodiments, the RLN-inhibitory Methylobacterium
isolate is selected from the group consisting of Methylobacterium
NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089,
derivatives thereof, and Methylobacterium related thereto. In
certain embodiments, the RLN-inhibitory Methylobacterium isolate is
selected from the group consisting of Methylobacterium NLS0021,
NLS0038, NLS0069, and a derivative thereof. In certain embodiments,
the RLN-inhibitory Methylobacterium provides for at least about
25%, at least about 50%, or at least about 75% reductions in RLN
damage to a treated plant, plant arising from a treated seed, or
plant grown in soil treated with the RLN in comparison to untreated
control plants, plants arising from untreated seeds, or plants
grown in untreated soils upon exposure to a RLN. In certain
embodiments, the RLN-inhibitory Methylobacterium provides for
increased yield, shoot weight, or root weight in the treated plant,
plant part, or a plant derived therefrom relative to an untreated
control plant, plant part, or a plant derived therefrom. In certain
embodiments, a cell-free culture supernatant or active ingredient
contained therein from an RLN-inhibitory Methylobacterium provides
for decreased motility or viability of RLN obtained from the
treated plant, plant part, or a plant derived therefrom relative to
an untreated control plant, plant part, or a plant derived
therefrom. In certain embodiments, the RLN-inhibitory
Methylobacterium, a cell-free culture supernatant obtained
therefrom, or an active ingredient contained in the cell-free
culture supernatant provides for increased yield, shoot weight, or
root weight in the treated plant, plant part, or a plant derived
therefrom relative to an untreated control plant, plant part, or a
plant derived therefrom. In certain embodiments, the RLN that is
inhibited is selected from the group consisting of a Pratylenchus
brachyurus, Pratylenchus coffeae, P. neglectus, Pratylenchus
penetrans, Pratylenchus scribneri, Pratylenchus thornei,
Pratylenchus vulnus, and Pratylenchus zeae.
[0045] In certain embodiments, the RLN-inhibitory Methylobacterium,
a cell-free culture supernatant obtained therefrom, fraction
thereof, concentrate thereof, or an active ingredient contained in
the cell-free culture supernatant provides for at least about 25%,
at least about 50%, or at least about 75% reductions in RLN
motility, viability, growth, and/or reproduction on a treated
plant, plant arising from a treated seed, or plant grown in soil
treated with the RLN-inhibitory Methylobacterium in comparison to a
untreated control plants, plants arising from untreated seeds, or
plants grown in untreated soils upon exposure to a RLN. In certain
embodiments, the RLN-inhibitory Methylobacterium is a
Methylobacterium that inhibits a Pratylenchus sp. is selected from
the group consisting of a Pratylenchus brachyurus, Pratylenchus
coffeae, P. neglectus, Pratylenchus penetrans, Pratylenchus
scribneri, Pratylenchus thornei, Pratylenchus vulnus, and
Pratylenchus zeae species. In certain embodiments of any of the
aforementioned compositions, the composition comprises a solid
substance wherein a mono-culture or co-culture of Methylobacterium
is adhered thereto. In certain embodiments where the
Methylobacterium is adhered to a solid substance, the composition
comprises a colloid formed by the solid substance wherein a
mono-culture or co-culture of Methylobacterium is adhered thereto
and a liquid. In certain embodiments, the colloid is a gel. In
certain embodiments of certain aforementioned compositions,
composition is an emulsion that does not contain a solid substance.
In certain embodiments of any of the aforementioned compositions,
the RLN-inhibitory Methylobacterium is selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0042, NLS0934,
NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto. In certain embodiments of any of
the aforementioned compositions, the RLN-inhibitory
Methylobacterium is selected from the group consisting of
Methylobacterium NLS0021, NLS0038, NLS0069, and a derivative
thereof.
[0046] In certain embodiments, isolated RLN-inhibitory
Methylobacterium sp., a cell-free culture supernatant obtained
therefrom, fraction thereof, or concentrate thereof can be
identified by treating a plant, a seed, soil in which the plant or
a plant arising from the seed are grown, or other plant growth
media in which the plant or a plant arising from the seed are grown
and assaying for either reductions in RLN damage, RLN growth, RLN
reproduction, RLN feeding activity, RLN motility, numbers of
recovered RLN, and combinations thereof. In still other
embodiments, the RLN-inhibitory Methylobacterium sp., compositions
comprising the same, fermentation products comprising the same,
cell free culture supernatants therefrom, fractions therefrom,
concentrates therefrom, or compounds derived therefrom can be
exposed to juvenile RLN and assayed for inhibition of juvenile
growth, development, behavior, motility, or feeding activity.
Various assays for determining quantity and/or activity of RLN that
can be adapted for use in identifying RLN-inhibitory cell-free
culture supernatants, fractions therefrom, or concentrates thereof,
and RLN-inhibitory Methylobacterium sp. have been disclosed
(Hollaway et al. Australasian Plant Pathology, 2003, 32(1):73-79;
Kimura et al. Agric. Biol. Chem., 1981, 45 (1), 249-251).
[0047] In certain embodiments, the RLN-inhibitory Methylobacterium
sp. has a 16S RNA encoding sequence that has significant sequence
identity to the 16S RNA encoding sequence of a RLN-inhibitory
Methylobacterium sp. provided herein. In certain embodiments, the
RLN-inhibitory Methylobacterium sp. has a 16S RNA encoding sequence
that has at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence
identity across the entire length of the 16S RNA encoding sequence
of an RLN-inhibitory Methylobacterium sp. isolate NLS0021, NLS0038,
NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089. A RLN-inhibitory
Methylobacterium sp. that can be used in any of the composition,
plants or plant parts that are coated or partially coated with the
compositions, methods of using the compositions to control RLN
damage to plants, plant parts, and plants derived therefrom, and
methods of making the compositions can be RLN-inhibitory
Methylobacterium sp. can be at least 95%, 96%, 97%, 98%, 99%, or
99.5% sequence identity across the entire length of the 16S RNA
encoding sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7. The 16S
RNA encoding sequence of SEQ ID NO: 1-7 are set forth in Table
1.
TABLE-US-00001 TABLE 1 16S RNA encoding sequences Isolate SEQ ID
DNA (NLS No.) NO: sequence encoding the 16S RNA NLS0021 SEQ ID
GAGTTTGATCCTGGCTCAGAGCGAACGCT NO: 1 GGCGGCAGGCTTAACACATGCAAGTCGA
ACGGGCTTCTTCGGAAGTCAGTGGCAGAC GGGTGAGTAACACGTGGGAACGTGCCCTT
CGGTTCGGAATAACTCAGGGAAACTTGA GCTAATACCGGATACGCCCTTATGGGGAA
AGGTTTACTGCCGAAGGATCGGCCCGCGT CTGATTAGCTTGTTGGTGGGGTAACGGCC
TACCAAGGCGACGATCAGTAGCTGGTCTG AGAGGATGATCAGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCA GCAGTGGGGAATATTGGACAATGGGCGC
AAGCCTGATCCAGCCATGCCGCGTGAGTG ATGAAGGCCTTAGGGTTGTAAAGCTCTTT
TGTCCGGGACGATAATGACGGTACCGGA AGAATAAGCCCCGGCTAACTTCGTGCCAG
CAGCCGCGGTAATACGAAGGGGGCTAGC GTTGCTCGGAATCACTGGGCGTAAAGGGC
GCGTAGGCGGCCGATTAAGTCGGGGGTG AAAGCCTGTGGCTCAACCACAGAATTGCC
TTCGATACTGGTTGGCTTGAGACCGGAAG AGGACAGCGGAACTGCGAGTGTAGAGGT
GAAATTCGTAGATATTCGCAAGAACACCA GTGGCGAAGGCGGCTGTCTGGTCCGGTTC
TGACGCTGAGGCGCGAAAGCGTGGGGAG CAAACAGGATTAGATACCCTGGTAGTCCA
CGCCGTAAACGATGAATGCCAGCCGTTGG TCTGCTTGCAGGTCAGTGGCGCCGCTAAC
GCATTAAGCATTCCGCCTGGGGAGTACGG TCGCAAGATTAAAACTCAAAGGAATTGA
CGGGGGCCCGCACAAGCGGTGGAGCATG TGGTTTAATTCGAAGCAACGCGCAGAACC
TTACCATCCCTTGACATGGCATGTTACCT CGAGAGATCGGGGATCCTCTTCGGAGGC
GTGCACACAGGTGCTGCATGGCTGTCGTC AGCTCGTGTCGTGAGATGTTGGGTTAAGT
CCCGCAACGAGCGCAACCCACGTCCTTAG TTGCCATCATTCAGTTGGGCACTCTAGGG
AGACTGCCGGTGATAAGCCGCGAGGAAG GTGTGGATGACGTCAAGTCCTCATGGCCC
TTACGGGATGGGCTACACACGTGCTACAA TGGCGGTGACAGTGGGACGCGAAACCGC
GAGGTTGAGCAAATCCCCAAAAGCCGTCT CAGTTCGGATTGCACTCTGCAACTCGGGT
GCATGAAGGCGGAATCGCTAGTAATCGT GGATCAGCACGCCACGGTGAATACGTTCC
CGGGCCTTGTACACACCGCCCGTCACACC ATGGGAGTTGGTCTTACCCGACGGCGCTG
CGCCAACCGCAAGGGGGCAGGCGACCAC GGTAGGGTCAGCGACTGGGGTGAAGTCG
TAACAAGGTAGCCGTAGGGGAACCTGCG GCTGGATCACCT NLS0038 SEQ ID
GGTGATCCAGCCGCAGGTTCCCCTACGGC NO: 2 TACCTTGTTACGACTTCACCCCAGTCGCT
GACCCTACCGTGGTCGCCTGCCTCCTTGC GGTTGGCGCAGCGCCGTCGGGTAAGACC
AACTCCCATGGTGTGACGGGCGGTGTGTA CAAGGCCCGGGAACGTATTCACCGTGGC
GTGCTGATCCACGATTACTAGCGATTCCG CCTTCATGCACCCGAGTTGCAGAGTGCAA
TCCGAACTGAGACGGTTTTTGGGGATTTG CTCCACCTCGCGGCTTCGCGTCCCACTGT
CACCGCCATTGTAGCACGTGTGTAGCCCA TCCCGTAAGGGCCATGAGGACTTGACGTC
ATCCACACCTTCCTCGCGGCTTATCACCG GCAGTCTCCCTAGAGTGCCCAACTGAATG
ATGGCAACTAAGGACGTGGGTTGCGCTCG TTGCGGGACTTAACCCAACATCTCACGAC
ACGAGCTGACGACAGCCATGCAGCACCT GTGTGCACGCCTCCGAAGAGGATCCCCGA
TCTCTCGAGGTAACATGCCATGTCAAGGG ATGGTAAGGTTCTGCGCGTTGCTTCGAAT
TAAACCACATGCTCCACCGCTTGTGCGGG CCCCCGTCAATTCCTTTGAGTTTTAATCTT
GCGACCGTACTCCCCAGGCGGAATGCTTA ATGCGTTAGCGGCGCCACTGACCTGCAAG
CAGGCCAACGGCTGGCATTCATCGTTTAC GGCGTGGACTACCAGGGTATCTAATCCTG
TTTGCTCCCCACGCTTTCGCGCCTCAGCGT CAGAACCGGACCAGACAGCCGCCTTCGC
CACTGGTGTTCTTGCGAATATCTACGAAT TTCACCTCTACACTCGCAGTTCCGCTGTCC
TCTTCCGGTCTCAAGCCAACCAGTATCGA AGGCAATTCTGTGGTTGAGCCACAGGCTT
TCACCCCCGACTTAATCGGCCGCCTACGC GCCCTTTACGCCCAGTGATTCCGAGCAAC
GCTAGCCCCCTTCGTATTACCGCGGCTGC TGGCACGAAGTTAGCCGGGGCTTATTCTT
CCGGTACCGTCATTATCGTCCCGGACAAA AGAGCTTTACAACCCTAAGGCCTTCATCA
CTCACGCGGCATGGCTGGATCAGGCTTGC GCCCATTGTCCAATATTCCCCACTGCTGC
CTCCCGTAGGAGTCTGGGCCGTGTCTCAG TCCCAGTGTGGCTGATCATCCTCTCAGAC
CAGCTACTGATCGTCGCCTTGGTAGGCCG TTACCCCACCAACAAGCTAATCAGACGCG
GGCCGATCCTTCGGCAGTAAACCTTTCCC CAAAAGGGCGTATCCGGTATTAGCTCAAG
TTTCCCTGAGTTATTCCGAACCGAAGGGT ACGTTCCCACGTGTTACTCACCCGTCTGC
CACTGACACCCGAAGGTGCCCGTTCGACT TGCATGTGTTAAGCCTGCCGCCAGCGTTC
GCTCTGAGCCAGGATCAAACTCT NLS0042 SEQ ID
GAGTTTGATCCTGGCTCAGAGCGAACGCT NO: 3 GGCGGCAGGCTTAACACATGCAAGTCGA
ACGGGCACCTTCGGGTGTCAGTGGCAGAC GGGTGAGTAACACGTGGGAACGTACCCTT
CGGTTCGGAATAACTCAGGGAAACTTGA GCTAATACCGGATACGCCCTTTTGGGGAA
AGGTTTACTGCCGAAGGATCGGCCCGCGT CTGATTAGCTTGTTGGTGGGGTAACGGCC
TACCAAGGCGACGATCAGTAGCTGGTCTG AGAGGATGATCAGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCA GCAGTGGGGAATATTGGACAATGGGCGC
AAGCCTGATCCAGCCATGCCGCGTGAGTG ATGAAGGCCTTAGGGTTGTAAAGCTCTTT
TGTCCGGGACGATAATGACGGTACCGGA AGAATAAGCCCCGGCTAACTTCGTGCCAG
CAGCCGCGGTAATACGAAGGGGGCTAGC GTTGCTCGGAATCACTGGGCGTAAAGGGC
GCGTAGGCGGCCGATTAAGTCGGGGGTG AAAGCCTGTGGCTCAACCACAGAATTGCC
TTCGATACTGGTTGGCTTGAGACCGGAAG AGGACAGCGGAACTGCGAGTGTAGAGGT
GAAATTCGTAGATATTCGCAAGAACACCA GTGGCGAAGGCGGCTGTCTGGTCCGGTTC
TGACGCTGAGGCGCGAAAGCGTGGGGAG CAAACAGGATTAGATACCCTGGTAGTCCA
CGCCGTAAACGATGAATGCCAGCCGTTGG CCTGCTTGCAGGTCAGTGGCGCCGCTAAC
GCATTAAGCATTCCGCCTGGGGAGTACGG TCGCAAGATTAAAACTCAAAGGAATTGA
CGGGGGCCCGCACAAGCGGTGGAGCATG TGGTTTAATTCGAAGCAACGCGCAGAACC
TTACCATCCCTTGACATGGCATGTTACCT CGAGAGATCGGGGATCCTCTTCGGAGGC
GTGCACACAGGTGCTGCATGGCTGTCGTC AGCTCGTGTCGTGAGATGTTGGGTTAAGT
CCCGCAACGAGCGCAACCCACGTCCTTAG TTGCCATCATTCAGTTGGGCACTCTAGGG
AGACTGCCGGTGATAAGCCGCGAGGAAG GTGTGGATGACGTCAAGTCCTCATGGCCC
TTACGGGATGGGCTACACACGTGCTACAA TGGCGGTGACAGTGGGACGCGAAGCCGC
GAGGTGGAGCAAATCCCCAAAAACCGTC TCAGTTCGGATTGCACTCTGCAACTCGGG
TGCATGAAGGCGGAATCGCTAGTAATCGT GGATCAGCACGCCACGGTGAATACGTTCC
CGGGCCTTGTACACACCGCCCGTCACACC ATGGGAGTTGGTCTTACCCGACGGCGCTG
CGCCAACCGCAAGGAGGCAGGCGACCAC GGTAGGGTCAGCGACTGGGGTGAAGTCG
TAACAAGGTAGCCGTAGGGGAACCTGCG GCTGGATCACCT NLS0934 SEQ ID
GAGTTTGATCCTGGCTCAGAGCGAACGCT NO: 4 GGCGGCAGGCTTAACACATGCAAGTCGA
ACGCACCGCAAGGTGAGTGGCAGACGGG TGAGTAACACGTGGGAACGTGCCCTCCGG
TCTGGGATAACCCTGGGAAACTAGGGCTA ATACCGGATACGTGCTTTGGCAGAAAGGT
TTACTGCCGGAGGATCGGCCCGCGTCTGA TTAGCTTGTTGGTGGGGTAACGGCCTACC
AAGGCGACGATCAGTAGCTGGTCTGAGA GGATGATCAGCCACACTGGGACTGAGAC
ACGGCCCAGACTCCTACGGGAGGCAGCA GTGGGGAATATTGGACAATGGGCGCAAG
CCTGATCCAGCCATGCCGCGTGAGTGATG ACGGCCTTAGGGTTGTAAAGCTCTTTTCT
CCGGGACGATAATGACGGTACCGGAGGA ATAAGCCCCGGCTAACTTCGTGCCAGCAG
CCGCGGTAATACGAAGGGGGCTAGCGTT GCTCGGAATCACTGGGCGTAAAGGGCGC
GTAGGCGGCGTTTTAAGTCGGGGGTGAA AGCCTGTGGCTCAACCACAGAATGGCCTT
CGATACTGGGACGCTTGAGTATGGTAGAG GTTGGTGGAACTGCGAGTGTAGAGGTGA
AATTCGTAGATATTCGCAAGAACACCGGT GGCGAAGGCGGCCAACTGGACCATTACT
GACGCTGAGGCGCGAAAGCGTGGGGAGC AAACAGGATTAGATACCCTGGTAGTCCAC
GCCGTAAACGATGAATGCTAGCTGTTGGG GTGCATGCACCGCAGTAGCGCAGCTAAC
GCATTAAGCATTCCGCCTGGGGAGTACGG TCGCAAGATTAAAACTCAAAGGAATTGA
CGGGGGCCCGCACAAGCGGTGGAGCATG TGGTTTAATTCGAAGCAACGCGCAGAACC
TTACCATCCTTTGACATGGCGTGTTACTG GGAGAGATTCCAGGTCCCCTTCGGGGGCG
CGCACACAGGTGCTGCATGGCTGTCGTCA GCTCGTGTCGTGAGATGTTGGGTTAAGTC
CCGCAACGAGCGCAACCCACGTCCTTAGT TGCCATCATTTGGTTGGGCACTCTAGGGA
GACTGCCGGTGATAAGCCGCGAGGAAGG TGTGGATGACGTCAAGTCCTCATGGCCCT
TACGGGATGGGCTACACACGTGCTACAAT GGCGGTGACAGTGGGACGCGAAGGGGTG
ACCCGGAGCCAATCTCCAAAAGCCGTCTC AGTTCGGATTGCACGCTGCAACTCGCGTG
CATGAAGGCGGAATCGCTAGTAATCGTG GATCAGCATGCCACGGTGAATACGTTCCC
GGGCCTTGTACACACCGCCCGTCACACCA TGGGAGTTGGTCTTACCCGACGGCGCTGC
GCCAACCAAATCGAGCTTGCTCGACTGGA GGCAGGCGACCACGGTAGGGTCAGCGAC
TGGGGTGAAGTCGTAACAAGGTAGCCGT AGGGGAACCTGCGGCTGGATCACCT NLS0062 SEQ
ID GGTGATCCAGCCGCAGGTTCCCCTACGGC NO: 5
TACCTTGTTACGACTTCACCCCAGTCGCT GACCCTACCGTGGTCGCCTGCCTCCTTGC
GGTTGGCGCAGCGCCGTCGGGTAAGACC AACTCCCATGGTGTGACGGGCGGTGTGTA
CAAGGCCCGGGAACGTATTCACCGTGGC GTGCTGATCCACGATTACTAGCGATTCCG
CCTTCATGCACCCGAGTTGCAGAGTGCAA TCCGAACTGAGACGGTTTTTGGGGATTTG
CTCCACCTCGCGGCTTCGCGTCCCACTGT CACCGCCATTGTAGCACGTGTGTAGCCCA
TCCCGTAAGGGCCATGAGGACTTGACGTC ATCCACACCTTCCTCGCGGCTTATCACCG
GCAGTCTCCCTAGAGTGCCCAACTGAATG ATGGCAACTAAGGACGTGGGTTGCGCTCG
TTGCGGGACTTAACCCAACATCTCACGAC ACGAGCTGACGACAGCCATGCAGCACCT
GTGTGCACGCCTCCGAAGAGGATCCCCGA TCTCTCGAGGTAACATGCCATGTCAAGGG
ATGGTAAGGTTCTGCGCGTTGCTTCGAAT TAAACCACATGCTCCACCGCTTGTGCGGG
CCCCCGTCAATTCCTTTGAGTTTTAATCTT GCGACCGTACTCCCCAGGCGGAATGCTTA
ATGCGTTAGCGGCGCCACTGACCTGCAAG CAGGCCAACGGCTGGCATTCATCGTTTAC
GGCGTGGACTACCAGGGTATCTAATCCTG TTTGCTCCCCACGCTTTCGCGCCTCAGCGT
CAGAACCGGACCAGACAGCCGCCTTCGC CACTGGTGTTCTTGCGAATATCTACGAAT
TTCACCTCTACACTCGCAGTTCCGCTGTCC TCTTCCGGTCTCAAGCCAACCAGTATCGA
AGGCAATTCTGTGGTTGAGCCACAGGCTT TCACCCCCGACTTAATCGGCCGCCTACGC
GCCCTTTACGCCCAGTGATTCCGAGCAAC GCTAGCCCCCTTCGTATTACCGCGGCTGC
TGGCACGAAGTTAGCCGGGGCTTATTCTT CCGGTACCGTCATTATCGTCCCGGACAAA
AGAGCTTTACAACCCTAAGGCCTTCATCA CTCACGCGGCATGGCTGGATCAGGCTTGC
GCCCATTGTCCAATATTCCCCACTGCTGC CTCCCGTAGGAGTCTGGGCCGTGTCTCAG
TCCCAGTGTGGCTGATCATCCTCTCAGAC CAGCTACTGATCGTCGCCTTGGTAGGCCG
TTACCCCACCAACAAGCTAATCAGACGCG GGCCGATCCTTCGGCAGTAAACCTTTCCC
CAAAAGGGCGTATCCGGTATTAGCTCAAG TTTCCCTGAGTTATTCCGAACCGAAGGGT
ACGTTCCCACGTGTTACTCACCCGTCTGC CACTGACACCCGAAGGTGCCCGTTCGACT
TGCATGTGTTAAGCCTGCCGCCAGCGTTC GCTCTGAGCCAGGATCAAACTCT NLS0069 SEQ
ID GGTGATCCAGCCGCAGGTTCCCCTACGGC NO: 6
TACCTTGTTACGACTTCACCCCAGTCGCT GACCCTACCGTGGTCGCCTGCCTCCTTGC
GGTTGGCGCAGCGCCGTCGGGTAAGACC AACTCCCATGGTGTGACGGGCGGTGTGTA
CAAGGCCCGGGAACGTATTCACCGTGGC ATGCTGATCCACGATTACTAGCGATTCCG
CCTTCATGCACCCGAGTTGCAGAGTGCAA TCCGAACTGAGACGGCTTTTGGGGATTTG
CTCAACCTCGCGGTTTCGCGTCCCACTGT CACCGCCATTGTAGCACGTGTGTAGCCCA
TCCCGTAAGGGCCATGAGGACTTGACGTC ATCCACACCTTCCTCGCGGCTTATCACCG
GCAGTCTCCCTAGAGTGCCCAACTGAATG ATGGCAACTAAGGACGTGGGTTGCGCTCG
TTGCGGGACTTAACCCAACATCTCACGAC ACGAGCTGACGACAGCCATGCAGCACCT
GTGTGCAGGTCCCCGAAGGGAACGACCG ATCTCTCGGACAATCCTGCCATGTCAAGG
GATGGTAAGGTTCTGCGCGTTGCTTCGAA TTAAACCACATGCTCCACCGCTTGTGCGG
GCCCCCGTCAATTCCTTTGAGTTTTAATCT TGCGACCGTACTCCCCAGGCGGAATGCTT
AATGCGTTAGCGGCGCCACTGACCTGCAA GCAGACCAACGGCTGGCATTCATCGTTTA
CGGCGTGGACTACCAGGGTATCTAATCCT GTTTGCTCCCCACGCTTTCGCGCCTCAGC
GTCAGAACCGGACCAGACAGCCGCCTTC GCCACTGGTGTTCTTGCGAATATCTACGA
ATTTCACCTCTACACTCGCAGTTCCGCTGT CCTCTTCCGGTCTCAAGCTTTCCAGTATCG
AAGGCAATTCTGTGGTTGAGCCACAGGCT TTCACCCCCGACTTAAAAAGCCGCCTACG
CGCCCTTTACGCCCAGTGATTCCGAGCAA CGCTAGCCCCCTTCGTATTACCGCGGCTG
CTGGCACGAAGTTAGCCGGGGCTTATTCT TCCGGTACCGTCATTATCGTCCCGGACAA
AAGAGCTTTACAACCCTAAGGCCTTCATC ACTCACGCGGCATGGCTGGATCAGGCTTG
CGCCCATTGTCCAATATTCCCCACTGCTG CCTCCCGTAGGAGTCTGGGCCGTGTCTCA
GTCCCAGTGTGGCTGATCATCCTCTCAGA CCAGCTACTGATCGTCGCCTTGGTAGGCC
GTTACCCCACCAACTAGCTAATCAGACGC GGGCCGATCCTTCGGCAGTAAACCTTTCC
CCAAAAGGGCGTATCCGGTATTAGCTCAA GTTTCCCTGAGTTATTCCGAACCGAAGGG
CACGTTCCCACGTGTTACTCACCCGTCTG CCGCTGACCCCGAAGGGCCCGCTCGACTT
GCATGTGTTAAGCCTGCCGCCAGCGTTCG CTCTGAGCCAGGATCAAACTCT NLS0089 SEQ ID
GAGTTTGATCCTGGCTCAGAGCGAACGCT NO: 7 GGCGGCAGGCTTAACACATGCAAGTCGA
ACGGGCTTCTTCGGAAGTCAGTGGCAGAC GGGTGAGTAACACGTGGGAACGTGCCCTT
CGGTTCGGAATAACTCAGGGAAACTTGA GCTAATACCGGATACGCCCTTACGGGGAA
AGGTTTACTGCCGAAGGATCGGCCCGCGT CTGATTAGCTTGTTGGTGGGGTAACGGCC
TACCAAGGCGACGATCAGTAGCTGGTCTG AGAGGATGATCAGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCA GCAGTGGGGAATATTGGACAATGGGCGC
AAGCCTGATCCAGCCATGCCGCGTGAGTG ATGAAGGCCTTAGGGTTGTAAAGCTCTTT
TGTCCGGGACGATAATGACGGTACCGGA AGAATAAGCCCCGGCTAACTTCGTGCCAG
CAGCCGCGGTAATACGAAGGGGGCTAGC GTTGCTCGGAATCACTGGGCGTAAAGGGC
GCGTAGGCGGCCGATTAAGTCGGGGGTG AAAGCCTGTGGCTCAACCACAGAATTGCC
TTCGATACTGGTTGGCTTGAGACCGGAAG AGGACAGCGGAACTGCGAGTGTAGAGGT
GAAATTCGTAGATATTCGCAAGAACACCA GTGGCGAAGGCGGCTGTCTGGTCCGGTTC
TGACGCTGAGGCGCGAAAGCGTGGGGAG CAAACAGGATTAGATACCCTGGTAGTCCA
CGCCGTAAACGATGAATGCCAGCCGTTGG TCTGCTTGCAGGTCAGTGGCGCCGCTAAC
GCATTAAGCATTCCGCCTGGGGAGTACGG TCGCAAGATTAAAACTCAAAGGAATTGA
CGGGGGCCCGCACAAGCGGTGGAGCATG TGGTTTAATTCGAAGCAACGCGCAGAACC
TTACCATCCCTTGACATGGCATGTTACCT CGAGAGATCGGGGATCCTCTTCGGAGGC
GTGCACACAGGTGCTGCATGGCTGTCGTC AGCTCGTGTCGTGAGATGTTGGGTTAAGT
CCCGCAACGAGCGCAACCCACGTCCTTAG TTGCCATCATTCAGTTGGGCACTCTAGGG
AGACTGCCGGTGATAAGCCGCGAGGAAG GTGTGGATGACGTCAAGTCCTCATGGCCC
TTACGGGATGGGCTACACACGTGCTACAA TGGCGGTGACAGTGGGACGCGAAACCGC
GAGGTTGAGCAAATCCCCAAAAGCCGTCT CAGTTCGGATTGCACTCTGCAACTCGGGT
GCATGAAGGCGGAATCGCTAGTAATCGT GGATCAGCACGCCACGGTGAATACGTTCC
CGGGCCTTGTACACACCGCCCGTCACACC ATGGGAGTTGGTCTTACCCGACGGCGCTG
CGCCAACCGCAAGGGGGCAGGCGACCAC GGTAGGGTCAGCGACTGGGGTGAAGTCG
TAACAAGGTAGCCGTAGGGGAACCTGCG GCTGGATCACCT
[0048] Various Methylobacterium sp. isolates provided herein are
disclosed in Table 2.
TABLE-US-00002 TABLE 2 Methylobacterium sp. Isolates NLS Plant host
of original USDA ARS No. isolate NRRL No..sup.1 NLS0021 lettuce
NRRL B-50939 NLS0038 tomato NRRL B-50942 NLS0042 soybean NRRL
B-50932 NLS0934 soybean NRRL B-67341 NLS0062 corn NRRL B-50937
NLS0069 corn NRRL B-50936 NLS0089 broccoli NRRL B-50933 NLS0037
NRRL B-50941 NLS0064 NRRL B-50938 NLS0017 NRRL B-50931 NLS0065 NRRL
B-50935 NLS0068 NRRL B-50934 .sup.1Deposit number for strains
deposited with the AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION
(NRRL) of the National Center for Agricultural Utilization
Research, Agricultural Research Service, U.S. Department of
Agriculture, 1815 North University Street, Peoria, Illinois 61604
U.S.A. under the terms of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure. Subject to 37 CFR .sctn.1.808(b), all
restrictions imposed by the depositor on the availability to the
public of the deposited material will be irrevocably removed upon
the granting of any patent from this patent application.
[0049] Also provided herein are methods for controlling RLN that
comprise applying any of the aforementioned compositions provided
herein to a plant or a plant part in an amount that provides for
inhibition of RLN damage in the plant, plant part, or a plant
obtained therefrom relative to infection of, or damage in, a
control plant, plant part, or plant obtained therefrom that had not
received an application of the composition. In certain embodiments,
application of the composition provides for at least about 5%, at
least about 10%, at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 75%, at least about
85%, or at least about 95% reduction of RLN damage in the plant,
plant part, or a plant derived therefrom relative to RLN damage of
the control plant, plant part, or plant obtained therefrom. In
certain embodiments, application of the composition provides for at
least about 5%, at least about 10%, at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
75%, at least about 85%, or at least about 95% reduction of RLN
reproduction, viability, or motility in the plant, plant part, or a
plant derived therefrom relative to RLN reproduction, viability, or
motility in the control plant, plant part, or plant obtained
therefrom. In certain embodiments, application of the composition
provides for at least about 5%, at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 75%, at least about 85%, or at least about 95%
reduction of RLN recovered from the plant, plant part, or a plant
derived therefrom relative to RLN recovered from the control plant,
plant part, or plant obtained therefrom. In certain embodiments,
the methods provide for a decrease in numbers of RLN obtained from
the treated plant, plant part, or a plant derived therefrom
relative to an untreated control plant, plant part, or a plant
derived therefrom. In certain embodiments, application of the
composition provides for at least about 5%, at least about 10%, at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 75%, at least about 85%, or at least
about 95% reduction of RLN populations in soil. In certain
embodiments, the plant part is selected from the group consisting
of a leaf, a stem, a flower, a root, a tuber, a pollen grain, and a
seed. In certain embodiments, the method further comprises the step
of harvesting at least one plant part selected from the group
consisting of a leaf, a stem, a flower, a root, a tuber, a pollen
grain, or a seed from the plant or plant part. In certain
embodiments of any of the aforementioned methods, the methods
further comprise obtaining a processed food or feed composition
from the plant or plant part. In certain embodiments, the processed
food or feed composition is a meal or a paste. In certain
embodiments of any of the aforementioned methods, the
RLN-inhibitory Methylobacterium is selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0042, NLS0934,
NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto. In certain embodiments, the
RLN-inhibitory Methylobacterium is selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0069, and a
derivative thereof.
[0050] Also provided are methods of making the compositions useful
for controlling RLN that comprise combining at least one of a
RLN-inhibitory Methylobacterium, a RLN-inhibitory cell-free culture
supernatant, fraction thereof, concentrate thereof, or any
combination thereof with an agriculturally acceptable excipient
and/or with an agriculturally acceptable adjuvant. In certain
embodiments of the methods, the Methylobacterium is adhered to a
solid substance. In certain embodiments of the methods, the
Methylobacterium is adhered to the solid substance is combined with
a liquid to form a composition that is a colloid. In certain
embodiments of the methods, the colloid is a gel. In certain
embodiments of the methods, the Methylobacterium adhered to the
solid substance is provided by culturing the Methylobacterium in
the presence of the solid substance. In certain embodiments of the
methods, the composition comprises an emulsion. In certain
embodiments of the methods, the Methylobacterium is provided by
culturing the Methylobacterium in an emulsion. In certain
embodiments of any of the aforementioned methods, the
RLN-inhibitory Methylobacterium is selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0042, NLS0934,
NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto. In certain embodiments, the
RLN-inhibitory Methylobacterium is selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0069, and a
derivative thereof.
[0051] Methods where Methylobacterium are cultured in biphasic
media comprising a liquid phase and a solid substance have been
found to significantly increase the resultant yield of
Methylobacterium relative to methods where the Methylobacterium are
cultured in liquid media alone. In certain embodiments, the methods
can comprise growing the Methylobacterium in liquid media with a
particulate solid substance that can be suspended in the liquid by
agitation under conditions that provide for Methylobacterium
growth. In certain embodiments where particulate solid substances
are used, at least substantially all of the solid phase can thus be
suspended in the liquid phase upon agitation. Such particulate
solid substances can comprise materials that are about 1 millimeter
or less in length or diameter. In certain embodiments, the degree
of agitation is sufficient to provide for uniform distribution of
the particulate solid substance in the liquid phase and/or optimal
levels of culture aeration. However, in other embodiments provided
herein, at least substantially all of the solid phase is not
suspended in the liquid phase, or portions of the solid phase are
suspended in the liquid phase and portions of the solid phase are
not suspended in the liquid phase. Non-particulate solid substances
can be used in certain biphasic media where the solid phase is not
suspended in the liquid phase. Such non-particulate solid
substances include, but are not limited to, materials that are
greater than about 1 millimeter in length or diameter. Such
particulate and non-particulate solid substances also include, but
are not limited to, materials that are porous, fibrous, or
otherwise configured to provide for increased surface areas for
adherent growth of the Methylobacterium. Biphasic media where
portions of the solid phase are suspended in the liquid phase and
portions of the solid phase are not suspended in the liquid phase
can comprise a mixture of particulate and non-particulate solid
substances. Such particulate and non-particulate solid substances
used in any of the aforementioned biphasic media also include, but
are not limited to, materials that are porous, fibrous, or
otherwise configured to provide for increased surface areas for
adherent growth of the Methylobacterium. In certain embodiments,
the media comprises a colloid formed by a solid and a liquid phase.
A colloid comprising a solid and a liquid can be pre-formed and
added to liquid media or can be formed in media containing a solid
and a liquid. Colloids comprising a solid and a liquid can be
formed by subjecting certain solid substances to a chemical and/or
thermal change. In certain embodiments, the colloid is a gel. In
certain embodiments, the liquid phase of the media is an emulsion.
In certain embodiments, the emulsion comprises an aqueous liquid
and a liquid that is not miscible, or only partially miscible, in
the aqueous liquid. Liquids that are not miscible, or only
partially miscible, in water include, but are not limited to, any
of the following: (1) liquids having a miscibility in water that is
equal to or less than that of pentanol, hexanol, or heptanol at 25
degrees C.; (2) liquids comprising an alcohol, an aldehyde, a
ketone, a fatty acid, a phospholipid, or any combination thereof;
(3) alcohols selected from the group consisting of aliphatic
alcohols containing at least 5 carbons and sterols; (4) an animal
oil, microbial oil, synthetic oil, plant oil, or combination
thereof; and/or, (5) a plant oil is selected from the group
consisting of, soybean, cotton, peanut, sunflower, olive, flax,
coconut, palm, rapeseed, sesame seed, safflower, and combinations
thereof. In certain embodiments, the immiscible or partially
immiscible liquid can comprises at least about 0.02% to about 20%
of the liquid phase by mass. In certain embodiments, the methods
can comprise obtaining a biphasic culture media comprising the
liquid, the solid, and Methylobacterium and incubating the culture
under conditions that provide for growth of the Methylobacterium.
Biphasic culture medias comprising the liquid, the solid, and
Methylobacterium can be obtained by a variety of methods that
include, but are not limited to, any of: (a) inoculating a biphasic
media comprising the liquid and the solid substance with
Methylobacterium; (b) inoculating the solid substance with
Methylobacterium and then introducing the solid substance
comprising the Methylobacterium into the liquid media; (c)
inoculating the solid substance with Methylobacterium, incubating
the Methylobacterium on the solid substance, and then introducing
the solid substance comprising the Methylobacterium into the liquid
media; or (d) any combination of (a), (b), or (c). Methods and
compositions for growing Methylobacterium in biphasic media
comprising a liquid and a solid are disclosed in co-assigned U.S.
Pat. No. 9,181,541, issued Nov. 10, 2015, which is incorporated
herein by reference in its entirety. Compositions comprising dried
formulations of Methylobacterium that are adhered to solid
substances, methods for making such compositions, and methods of
applying those compositions to plants and plant parts including
seeds are disclosed in co-assigned U.S. patent application Ser. No.
14/856,020, filed Sep. 16, 2015, and which is incorporated herein
by reference in its entirety.
[0052] Methods where Methylobacterium are cultured in media
comprising an emulsion have also been found to significantly
increase the resultant yield of Methylobacterium relative to
methods where the Methylobacterium are cultured in liquid media
alone. In certain embodiments, the methods for making the
compositions provided herein can comprise growing the
RLN-inhibitory Methylobacterium agent in an emulsion under
conditions that provide for Methylobacterium growth. Media
comprising the emulsion and RLN-inhibitory Methylobacterium can be
obtained by a variety of methods that include, but are not limited
to, any of: (a) inoculating a media comprising the emulsion with
Methylobacterium; (b) inoculating the aqueous liquid with the
Methylobacterium, introducing the non-aqueous liquid, and mixing to
form an emulsion; (c) inoculating the aqueous liquid with the
Methylobacterium, introducing the non-aqueous liquid, and mixing to
form an emulsion; or (d) any combination of (a), (b), or (c). In
certain embodiments, the emulsion comprises an aqueous liquid and a
liquid that is not miscible, or only partially miscible, in the
aqueous liquid. Non-aqueous liquids that are not miscible, or only
partially miscible, in water include, but are not limited to, any
of the following: (1) liquids having a miscibility in water that is
equal to or less than that of n-pentanol, n-hexanol, or n-heptanol
at 25 degrees C.; (2) liquids comprising an alcohol, an aldehyde, a
ketone, a fatty acid, a phospholipid, or any combination thereof;
(3) alcohols is selected from the group consisting of aliphatic
alcohols containing at least 5, 6, or 7 carbons and sterols; (4) an
animal oil, microbial oil, synthetic oil, plant oil, or combination
thereof; and/or, (5) a plant oil is selected from the group
consisting of, soybean, cotton, peanut, sunflower, olive, flax,
coconut, palm, rapeseed, sesame seed, safflower, and combinations
thereof. In certain embodiments, the immiscible or partially
immiscible non-aqueous liquid can comprise at least about 0.02% to
about 20% of the emulsion by mass. In certain embodiments, the
immiscible or partially immiscible non-aqueous liquid can comprise
at least about any of about 0.05%, 0.1%, 0.5%, or 1% to about 3%,
5%, 10%, or 20% of the emulsion by mass. Methods and compositions
for growing Methylobacterium in media comprising an emulsion are
disclosed in co-assigned International Patent Application
PCT/US14/40218, filed May 30, 2014, and US Patent Application
Publication No. 20160120188, which is incorporated herein by
reference in its entirety.
[0053] In certain embodiments, compositions that comprise at least
one of a RLN-inhibitory Methylobacterium, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof can further comprise one or
more introduced microorganisms of pre-determined identity other
than Methylobacterium. Other microorganisms that can be added
include, but are not limited to, microorganisms that are
biopesticidal or provide some other benefit when applied to a plant
or plant part. Biopesticidal or otherwise beneficial microorganisms
thus include, but are not limited to, various Bacillus sp.,
Pseudomonas sp., Coniothyrium sp., Pantoea sp., Streptomyces sp.,
and Trichoderma sp. Microbial biopesticides can be a bacterium,
fungus, virus, or protozoan. Particularly useful biopesticidal
microorganisms include various Bacillus subtilis, Bacillus
thuringiensis, Bacillus pumilis (e.g., Bacillus pumilis strain
QST2808), Pseudomonas syringae, Trichoderma harzianum, Trichoderma
virens, and Streptomyces lydicus strains. Biopesticidal
microorganisms that can be used include, but are not limited to,
the Bacillus pumilis strains described in U.S. Pat. No. 9,023,339,
and the Bacillus cereus and Bacillus firmus strains disclosed in
U.S. Pat. No. 6,406,690, each of which are incorporated herein by
reference in their entireties. Other microorganisms that are added
can be genetically engineered or other isolates that are available
as pure cultures. In certain embodiments, it is anticipated that
the bacterial or fungal microorganism can be provided in the
fermentation broth, fermentation broth product, or composition in
the form of a spore.
[0054] In certain embodiments, a RLN-inhibitory Methylobacterium, a
RLN-inhibitory cell-free culture supernatant, fraction thereof,
concentrate thereof, or any combination thereof that are provided
herein can be used in conjunction with transgenic plants that
express gene products that are inhibitory to growth of certain RLN.
Such transgenic plants include, but are not limited to, those
expressing interfering RNA molecules that suppress endogenous RLN
genes such as those disclosed in U.S. Patent Appl. Publication No.
US 20150135363, which is incorporated herein by reference in its
entirety.
[0055] In certain embodiments, the compositions comprising at least
one of a RLN-inhibitory Methylobacterium, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof that are provided herein can be
used in conjunction with plants that comprise one or more genetic
loci that can confer resistance to RLN. Such RLN resistant plants
include, but are not limited to, wheat plants comprising the Rlnnl
gene (Theoretical and Applied Genetics, 2002, 104 (5): 874-879) or
alfalfa plants (Baldridge et al. Plant Molecular Biology, 1998,
38(6): 999-1010).
[0056] In certain embodiments, the compositions comprising at least
one of a RLN-inhibitory Methylobacterium, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof that are provided herein can be
used in conjunction with, or comprise, nematicides that also
provide for inhibition of RLN growth and/or reproduction and/or
reductions in RLN-mediated plant damage. Such nematicides can be
used in soil treatments (drenches, in furrow deposits, and the
like) and/or in seed treatments. In certain embodiments, the
nematicide is selected from the group consisting of
organophosphate, biological, and carbamate nematicides. In certain
embodiments, the seed is treated with one or more of the
aforementioned nematicides (U.S. Pat. Nos. 6,660,690 and 8,080,496,
each incorporated herein by reference in their entireties).
Commercial soil applied nematicide formulations that can be used in
conjunction with the RLN-inhibitory Methylobacterium sp. provided
herein include, but are not limited to, formulations containing the
carbamates aldicarb, aldoxycarb, oxamyl, carbofuran, and
cleothocarb, and/or the organophosphates thionazin, ethoprophos,
fenamiphos, fensulfothion, and/or terbufos formulations.
Combinations of the aforementioned nematicides and the
aforementioned transgenic plants that provide for inhibition of RLN
growth and/or reproduction and/or reductions in RLN-mediated plant
damage can also be used in conjunction with the RLN-inhibitory
Methylobacterium sp. provided herein.
[0057] In certain embodiments, any of the aforementioned
compositions comprising RLN-inhibitory Methylobacterium sp., a
RLN-inhibitory cell-free culture supernatant, fraction thereof,
concentrate thereof, or any combination thereof that are provided
herein are selectively applied to plant parts or to soil or other
media in which a plant is or is to be grown, or plant parts
subjected to such applications are used in soil or media, following
a determination that RLN levels in the soil or other media in which
the plants are grown are above levels that would result in RLN
damage in the absence of such applications or use. Soil or media in
which the plant is to be grown can be surveyed for the presence of
RLN and the composition is applied to the plant part, soil, or
media when the RLN are present in the soil or media at a level that
can result in reductions in plant growth, yield, water-deficit
tolerance, chlorosis, produce quality, and combinations thereof to
an untreated control plant. In certain embodiments, the composition
is not applied to the plant part, soil, or media when the RLN are
present in the soil or media below levels that can result in
reductions in plant growth, yield, water-deficit tolerance,
chlorosis, produce quality, and combinations thereof to an
untreated control plant. Any survey method that provides for a
determination of RLN levels that result in plant damage can be
used. In certain embodiments, plants having yellow lower leaves and
loss of secondary branching in the root system that are found in
patches in a field can indicate that RLN are present (Johnson et
al. Root Lesion Nematodes in Wheat, Montana State University
Extension Publication MT200801AG, February 2008) and prompt use of
the compositions provided herein. In other embodiments, samples are
taken from the soil or media in which the plants are to be grown
and levels of RLN are determined (Todd et al. Plant Health Progress
15(3):112, 2014). In certain embodiments where the RLN are present
in soil above threshold levels that result in yield loss, the
compositions provided herein are used.
[0058] In certain embodiments, the liquid medium that can be used
in the methods and compositions that provide for the efficient
growth of Methylobacterium is prepared from inexpensive and readily
available components, including, but not limited to, inorganic
salts such as potassium phosphate, magnesium sulfate and the like,
carbon sources such as glycerol, methanol, glutamic acid, aspartic
acid, succinic acid and the like, and amino acid blends such as
peptone, tryptone, and the like. Non-limiting examples of liquid
media that can be used include, but are not limited to, ammonium
mineral salts (AMS) medium (Whittenbury et al., 1970), Vogel-Bonner
(VB) minimal culture medium (Vogel and Bonner, 1956), and LB broth
("Luria-Bertani Broth").
[0059] In general, the solid substance that can in certain
embodiments be used in the methods and compositions that provide
for the efficient growth of Methylobacterium can be any suitable
solid substance which is insoluble or only partially soluble in
water or aqueous solutions. Such suitable solid substances are also
non-bacteriocidal or non-bacteriostatic with respect to
RLN-inhibitory Methylobacterium sp. when the solid substances are
provided in the liquid culture media. In certain embodiments, such
suitable solid substances are also solid substances that are
readily obtained in sterile form or rendered sterile. Solid
substances used herein can be sterilized by any method that
provides for removal of contaminating microorganisms and thus
include, but are not limited to, methods such as autoclaving,
irradiation, chemical treatment, and any combination thereof. These
solid substances include substances of animal, plant, microbial,
fungal, or mineral origin, manmade substances, or combinations of
substances of animal, plant, microbial, fungal, or mineral origin
and manmade substances. In certain embodiments, the solid
substances are inanimate solid substances. Inanimate solid
substances of animal, plant, microbial, or fungal origin can be
obtained from animals, plants, microbes, or fungi that are inviable
(i.e. no longer living) or that have been rendered inviable. Diatom
shells are thus inanimate solid substances when previously
associated diatom algae have been removed or otherwise rendered
inviable. Since diatom shells are inanimate solid substances, they
are not considered to be photosynthetic organisms or photosynthetic
microorganisms. In certain embodiments, solid substances include,
but are not limited to, sand, silt, soil, clay, ash, charcoal,
diatomaceous earth and other similar minerals, ground glass or
glass beads, ground ceramic materials, ceramic beads, bentonite,
kaolin, talc, perlite, mica, vermiculite, silicas, quartz powder,
montmorillonite, and combinations thereof. In certain embodiments,
the solid substance can be a polymer or polymeric beads. Polymers
that can be used as a solid substance include, but are not limited
to, various polysaccharides such as cellulosic polymers and
chitinous polymers which are insoluble or only partially soluble in
water or aqueous solutions, agar (i.e. galactans), and combinations
thereof. In certain embodiments, the solid substance can be an
insoluble or only partially soluble salt crystal. Salt crystals
that can be used include, but are not limited to, insoluble or only
partially soluble carbonates, chromates, sulfites, phosphates,
hydroxides, oxides, and sulfides. In certain embodiments, the solid
substance can be a microbial cell, fungal cell, microbial spore, or
fungal spore. In certain embodiments, the solid substance can be a
microbial cell or microbial spore wherein the microbial cell or
microbial spore is not a photosynthetic microorganism. In still
other embodiments, the solid substance can be an inactivated (i.e.
inviable) microbial cell, fungal cell, microbial spore, or fungal
spore. In still other embodiments, the solid substance can be a
quiescent (i.e. viable but not actively dividing) microbial cell,
fungal cell, microbial spore, or fungal spore. In still other
embodiments, the solid substance can be cellular debris of
microbial origin. In still other embodiments, the solid substance
can be particulate matter from any part of a plant. Plant parts
that can be used to obtain the solid substance include, but are not
limited to, cobs, husks, hulls, leaves, roots, flowers, stems,
barks, seeds, and combinations thereof. Products obtained from
processed plant parts including, but not limited to, bagasse, wheat
bran, soy grits, crushed seed cake, stover, and the like can also
be used. Such plant parts, processed plants, and/or processed plant
parts can be milled to obtain the solid material in a particulate
form that can be used. In certain embodiments, wood or a wood
product including, but not limited to, wood pulp, sawdust,
shavings, and the like can be used. In certain embodiments, the
solid substance can be a particulate matter from an animal(s),
including, but not limited to, bone meal, gelatin, ground or
powdered shells, hair, macerated hide, and the like.
[0060] In certain embodiments, the solid substance is provided in a
particulate form that provides for distribution of the solid
substance in the culture media. In certain embodiments, the solid
substance is comprised of particle of about 2 microns to about 1000
microns in average length or average diameter. In certain
embodiments, the solid substance is comprised of particle of about
1 microns to about 1000 microns in average length or average
diameter. In certain embodiments, the solid substance is a particle
of about 1, 2, 4, 10, 20, or 40 microns to any of about 100, 200,
500, 750, or 1000 microns in average length or average diameter.
Desirable characteristics of particles used in the methods and
compositions provided herein include suitable wettability such that
the particles can be suspended throughout the media upon
agitation.
[0061] In certain embodiments, the solid substance is provided in
the media as a colloid wherein the continuous phase is a liquid and
the dispersed phase is the solid. Suitable solids that can be used
to form colloids in liquid media used to grow RLN-inhibitory
Methylobacterium sp. include, but are not limited to, various
solids that are referred to as hydrocolloids. Such hydrocolloids
used in the media, methods and compositions provided herein can be
hydrophilic polymers, of plant, animal, microbial, or synthetic
origin. Hydrocolloid polymers used in the methods can contain many
hydroxyl groups and/or can be polyelectrolytes. Hydrocolloid
polymers used in the compositions and methods provided herein
include, but are not limited to, agar, alginate, arabinoxylan,
carrageenan, carboxymethylcellulose, cellulose, curdlan, gelatin,
gellan, (3-glucan, guar gum, gum arabic, locust bean gum, pectin,
starch, xanthan gum, and mixtures thereof. In certain embodiments,
the colloid used in the media, methods, and compositions provided
herein can comprise a hydrocolloid polymer and one or more
proteins.
[0062] In certain embodiments, the solid substance can be a solid
substance that provides for adherent growth of the RLN-inhibitory
Methylobacterium sp. on the solid substance. RLN-inhibitory
Methylobacterium sp. that are adhered to a solid substance are
Methylobacterium that cannot be substantially removed by simply
washing the solid substance with the adherent RLN-inhibitory
Methylobacterium sp. with growth media whereas non-adherent
Methylobacterium can be substantially removed by washing the solid
substance with liquid growth media. In this context, "substantially
removed" means that at least about 30%, 40%, 50%, 60%, 70%, or 80%
the Methylobacterium present are removed when the solid substance
is washed with three volumes of liquid growth media. Such washing
can be effected by a variety of methods including, but not limited
to, decanting liquid from a washed solid phase or passing liquid
through a solid phase on a filter that permits flow through of
bacteria in the liquid. In certain embodiments, the adherent
RLN-inhibitory Methylobacterium sp. that are associated with the
solid can include both Methylobacterium that are directly attached
to the solid and/or Methylobacterium that are indirectly attached
to the solid substance. Methylobacterium that are indirectly
attached to the solid substance include, but are not limited to,
Methylobacterium that are attached to another Methylobacterium or
to another microorganism that is attached to the solid substance,
Methylobacterium that are attached to the solid substance by being
attached to another substance that is attached to the solid
substance, and the like. In certain embodiments, at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of
the Methylobacterium in the fermentation broth, fermentation broth
product, or compositions are Methylobacterium that are adhered to
the solid substance. In certain embodiments, adherent
RLN-inhibitory Methylobacterium sp. can be present on the surface
of the solid substance in the fermentation broth, fermentation
broth product, or composition at a density of at least about 1
Methylobacterium/20 square micrometers, of at least about 1
Methylobacterium/10 square micrometers, of at least about 1
Methylobacterium/10 square micrometers, of at least about 1
Methylobacterium/5 square micrometers, of at least about 1
Methylobacterium/2 square micrometers, or of at least about 1
Methylobacterium/square micrometer. In certain embodiments,
adherent RLN-inhibitory Methylobacterium sp. can be present on the
surface of the solid substance in the fermentation broth,
fermentation broth product, or composition at a density of at least
about 1 Methylobacterium/20 square micrometers to about 1
Methylobacterium/square micrometer, of at least about 1
Methylobacterium/10 square micrometers to about 1
Methylobacterium/square micrometer, of at least about 1
Methylobacterium/10 square micrometers to about 1
Methylobacterium/square micrometer, of at least about 1
Methylobacterium/5 square micrometers to about 1
Methylobacterium/square micrometer, or of at least about 1
Methylobacterium/2 square micrometers to about 1
Methylobacterium/square micrometer. In certain embodiments,
adherent RLN-inhibitory Methylobacterium sp. can be present on the
surface of the solid substance in the fermentation broth,
fermentation broth product, or composition at a density of at least
about 1 Methylobacterium/20 square micrometers to about 1
Methylobacterium/2 square micrometers, of at least about 1
Methylobacterium/10 square micrometers to about 1
Methylobacterium/2 square micrometers, of at least about 1
Methylobacterium/10 square micrometers to about 1
Methylobacterium/2 square micrometers, or of at least about 1
Methylobacterium/5 square micrometers to about 1 Methylobacterium/2
square micrometers. Biphasic fermentation broths provided herein
can comprise a liquid phase that contains non-adherent
Methylobacterium. In certain embodiments, titers of non-adherent
Methylobacterium in the liquid phase can be less than about
100,000, 10,000, or 1,000 CFU/ml. In certain embodiments, the
RLN-inhibitory Methylobacterium is selected from the group
consisting ofMethylobacterium NLS0021, NLS0038, NLS0042, NLS0934,
NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto. In certain embodiments, the
RLN-inhibitory Methylobacterium is selected from the group
consisting ofMethylobacterium NLS0037 and a derivative thereof.
[0063] Biphasic culture methods provided can yield fermentation
broths with RLN-inhibitory Methylobacterium sp. at a titer of
greater than about 5.times.10.sup.8 colony-forming units per
milliliter, at a titer of greater than about 1.times.10.sup.9
colony-forming units per milliliter, at a titer of greater than
about 1.times.10.sup.10 colony-forming units per milliliter, at a
titer of at least about 3.times.10.sup.10 colony-forming units per
milliliter. In certain embodiments, fermentation broths provided
herein can comprise RLN-inhibitory Methylobacterium sp. at a titer
of at least about 5.times.10.sup.8 colony-forming units per
milliliter to at least about 3.times.10.sup.10 colony-forming units
per milliliter, at least about 5.times.10.sup.8 colony-forming
units per milliliter to at least about 4.times.10.sup.10
colony-forming units per milliliter, or at least about
5.times.10.sup.8 colony-forming units per milliliter to at least
about 6.times.10.sup.10 colony-forming units per milliliter. In
certain embodiments, fermentation broths provided herein can
comprise RLN-inhibitory Methylobacterium sp. at a titer of at least
about 1.times.10.sup.9 colony-forming units per milliliter to at
least about 3.times.10.sup.10 colony-forming units per milliliter,
at least about 1.times.10.sup.9 colony-forming units per milliliter
to at least about 4.times.10.sup.10 colony-forming units per
milliliter, or at least about 1.times.10.sup.9 colony-forming units
per milliliter to at least about 6.times.10.sup.10 colony-forming
units per milliliter. In certain embodiments, fermentation broths
provided herein will comprise RLN-inhibitory Methylobacterium sp.
at a titer of at least about 1.times.10.sup.10 colony-forming units
per milliliter to at least about 3.times.10.sup.10 colony-forming
units per milliliter, at least about 1.times.10.sup.10
colony-forming units per milliliter to at least about
4.times.10.sup.10 colony-forming units per milliliter, or at least
about 1.times.10.sup.10 colony-forming units per milliliter to at
least about 6.times.10.sup.10 colony-forming units per milliliter.
In certain embodiments, fermentation broths provided herein will
comprise RLN-inhibitory Methylobacterium sp. at a titer of, at
least about 3.times.10.sup.10 colony-forming units per milliliter
to at least about 4.times.10.sup.10 colony-forming units per
milliliter, or at least about 3.times.10.sup.10 colony-forming
units per milliliter to at least about 6.times.10.sup.10
colony-forming units per milliliter. In certain embodiments, the
RLN-inhibitory Methylobacterium is selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0042, NLS0934,
NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto. In certain embodiments, the
RLN-inhibitory Methylobacterium is selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0069, and a
derivative thereof.
[0064] Solid substances with adherent RLN-inhibitory
Methylobacterium sp. obtained as fermentation products can be used
to make various compositions useful for treating plants or plant
parts to inhibit RLN growth and/or reproduction or reduce RLN
damage to a plant. Such compositions can comprise at least one of a
RLN-inhibitory Methylobacterium sp., a solid substance with
adherent RLN-inhibitory Methylobacterium sp., a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof. Compositions provided herein
comprising RLN-inhibitory Methylobacterium sp., solid substances
with RLN-inhibitory Methylobacterium sp. grown thereon, or
comprising emulsions with RLN-inhibitory Methylobacterium sp. grown
therein can be used to treat plants or plant parts. Plants, plant
parts, and, in particular, plant seeds that have been at least
partially coated or coated with the aforementioned compositions are
thus provided. Also provided are processed plant products that
contain any of the aforementioned compositions. Solid substances
with adherent RLN-inhibitory Methylobacterium sp. can be used to
make various compositions that are particularly useful for treating
plant seeds. Seeds that have been at least partially coated with
any of the aforementioned compositions are thus provided. Also
provided are processed seed products, including, but not limited
to, meal, flour, feed, and flakes that contain the fermentation
broth products or compositions provided herein. In certain
embodiments, the processed plant product will be non-regenerable
(i.e. will be incapable of developing into a plant). In certain
embodiments, the solid substance used in the fermentation product
or composition that at least partially coats the plant, plant part,
or plant seed or that is contained in the processed plant, plant
part, or seed product comprises a solid substance and associated or
adherent RLN-inhibitory Methylobacterium sp. that can be readily
identified by comparing a treated and an untreated plant, plant
part, plant seed, or processed product thereof. In certain
embodiments, the RLN-inhibitory Methylobacterium is selected from
the group consisting of Methylobacterium NLS0021, NLS0038, NLS0042,
NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and
Methylobacterium related thereto. In certain embodiments, the
RLN-inhibitory Methylobacterium is selected from the group
consisting of Methylobacterium NLS0021, NLS0038, NLS0069, and a
derivative thereof.
[0065] Compositions useful for treating plants or plant parts that
comprise at least one of a RLN-inhibitory Methylobacterium sp., a
solid substance with adherent RLN-inhibitory Methylobacterium sp.,
emulsions with RLN-inhibitory Methylobacterium sp. grown therein, a
RLN-inhibitory cell-free culture supernatant, fraction thereof,
concentrate thereof, or any combination thereof, can also further
comprise an agriculturally acceptable adjuvant or an agriculturally
acceptable excipient. Such compositions can be in a liquid or dry
form. An agriculturally acceptable adjuvant or an agriculturally
acceptable excipient is typically an ingredient that does not cause
undue phytotoxicity or other adverse effects when exposed to a
plant or plant part. In certain embodiments, the solid substance
can itself be an agriculturally acceptable adjuvant or an
agriculturally acceptable excipient so long as it is not
bacteriocidal or bacteriostatic to the Methylobacterium. In other
embodiments, the composition further comprises at least one of an
agriculturally acceptable adjuvant or an agriculturally acceptable
excipient. Any of the aforementioned compositions can also further
comprise a pesticide. Pesticides used in the composition include,
but are not limited to, a nematicide, a fungicide, and a
bacteriocide. In certain embodiments, the pesticide used in the
composition is a pesticide that does not substantially inhibit
growth of the Methylobacterium. As Methylobacterium are gram
negative bacteria, suitable bacteriocides used in the compositions
can include, but are not limited to, bacteriocides that exhibit
activity against gram positive bacteria but not gram negative
bacteria. Compositions provided herein can also comprise a
bacteriostatic agent that does not substantially inhibit growth of
the Methylobacterium. Bacteriostatic agents suitable for use in
compositions provided herein include, but are not limited to, those
that exhibit activity against gram positive bacteria but not gram
negative bacteria. Any of the aforementioned compositions can also
be an essentially dry product (i.e. having about 5% or less water
content), a mixture of the composition with an emulsion, or a
suspension.
[0066] Agriculturally acceptable adjuvants used in the compositions
that comprise at least one of a RLN-inhibitory Methylobacterium
sp., a solid substance with adherent RLN-inhibitory
Methylobacterium sp., emulsions with RLN-inhibitory
Methylobacterium sp. grown therein, a RLN-inhibitory cell-free
culture supernatant, fraction thereof, concentrate thereof, or any
combination thereof include, but are not limited to, components
that enhance product efficacy and/or products that enhance ease of
product application. Adjuvants that enhance product efficacy can
include various wetters/spreaders that promote adhesion to and
spreading of the composition on plant parts, stickers that promote
adhesion to the plant part, penetrants that can promote contact of
the active agent with interior tissues, extenders that increase the
half-life of the active agent by inhibiting environmental
degradation, and humectants that increase the density or drying
time of sprayed compositions. Wetters/spreaders used in the
compositions can include, but are not limited to, non-ionic
surfactants, anionic surfactants, cationic surfactants, amphoteric
surfactants, organo-silicate surfactants, and/or acidified
surfactants. Stickers used in the compositions can include, but are
not limited to, latex-based substances, terpene/pinolene, and
pyrrolidone-based substances. Penetrants can include mineral oil,
vegetable oil, esterified vegetable oil, organo-silicate
surfactants, and acidified surfactants. Extenders used in the
compositions can include, but are not limited to, ammonium
sulphate, or menthene-based substances. Humectants used in the
compositions can include, but are not limited to, glycerol,
propylene glycol, and diethyl glycol. Adjuvants that improve ease
of product application include, but are not limited to,
acidifying/buffering agents, anti-foaming/de-foaming agents,
compatibility agents, drift-reducing agents, dyes, and water
conditioners. Anti-foaming/de-foaming agents used in the
compositions can include, but are not limited to,
dimethopolysiloxane. Compatibility agents used in the compositions
can include, but are not limited to, ammonium sulphate.
Drift-reducing agents used in the compositions can include, but are
not limited to, polyacrylamides, and polysaccharides. Water
conditioners used in the compositions can include, but are not
limited to, ammonium sulphate.
[0067] Methods of treating plants and/or plant parts with the
compositions comprising at least one of a RLN-inhibitory
Methylobacterium sp., a solid substance with adherent
RLN-inhibitory Methylobacterium sp., emulsions with RLN-inhibitory
Methylobacterium sp. grown therein, a RLN-inhibitory cell-free
culture supernatant, fraction thereof, concentrate thereof, or any
combination thereof are also provided herein. Treated plants, and
treated plant parts obtained therefrom, include, but are not
limited to, Brassica sp. (e.g., B. napus, B. rapa, B. juncea),
corn, wheat, rye, rice, alfalfa, rice, rye, sorghum, millet (e.g.,
pearl millet (Pennisetum glaucum), proso millet (Panicum
miliaceum), foxtail millet (Setaria italica), finger millet
(Eleusine coracana), sunflower, safflower, soybean, tobacco,
potato, peanuts, carrot, cotton, sweet potato (Ipomoea batatus),
cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea,
banana, avocado, fig, guava, mango, olive, papaya, cashew,
macadamia, almond, sugar beets, sugarcane, strawberry, oats,
barley, tomato, lettuce, pepper, green beans, lima beans, peas,
cucurbits such as cucumber, cantaloupe, and musk melon, turf,
ornamentals, and conifers. Plant parts that are treated include,
but are not limited to, leaves, stems, flowers, roots, seeds,
fruit, tubers, coleoptiles, and the like. Ornamental plants and
plant parts that can be treated include, but are not limited to
azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias,
carnation, poinsettia, and chrysanthemum. Seeds or other propagules
of any of the aforementioned plants can be treated with the
fermentation broths, fermentation broth products, fermentation
products, and/or compositions provided herein.
[0068] In certain embodiments, plants and/or plant parts are
treated by applying compositions that comprise at least one of a
RLN-inhibitory Methylobacterium sp., a solid substance with
adherent RLN-inhibitory Methylobacterium sp., emulsions with
RLN-inhibitory Methylobacterium sp. grown therein, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof as a spray. Such spray
applications include, but are not limited to, treatments of a
single plant part or any combination of plant parts. Spraying can
be achieved with any device that will distribute the fermentation
broths, fermentation broth products, fermentation products, and
compositions to the plant and/or plant part(s). Useful spray
devices include a boom sprayer, a hand or backpack sprayer, crop
dusters (i.e. aerial spraying), and the like. Spraying devices and
or methods providing for application of the fermentation broths,
fermentation broth products, fermentation products, and
compositions to either one or both of the adaxial surface and/or
abaxial surface can also be used. Plants and/or plant parts that
are at least partially coated with any of the aforementioned
compositions are also provided herein. Also provided herein are
processed plant products that comprise any of the aforementioned
compositions.
[0069] In certain embodiments, plants and/or plant parts are
treated by applying compositions that comprise at least one of a
RLN-inhibitory Methylobacterium sp., a solid substance with
adherent RLN-inhibitory Methylobacterium sp., emulsions with
RLN-inhibitory Methylobacterium sp. grown therein, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof as a soil drench, soil
injection, and/or in-furrow deposit. Such soil drench, soil
injections, and/or in-furrow deposit applications include, but are
not limited to, treatments of a single plant part or any
combination of plant parts. Soil drench, soil injections and/or
in-furrow deposits can be achieved with any device that will
distribute the compositions to the plant and/or plant part(s)
and/or to the soil that the plant or plant parts have or will
contact. Useful devices include, but are not limited to, soil
injectors, in-furrow applicators, and the like.
[0070] In certain embodiments, seeds are treated by exposing the
seeds to compositions that comprise at least one of a
RLN-inhibitory Methylobacterium sp., a solid substance with
adherent RLN-inhibitory Methylobacterium sp., emulsions with
RLN-inhibitory Methylobacterium sp. grown therein, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof. Seeds can be treated with the
compositions provided herein by methods including, but not limited
to, imbibition, coating, spraying, and the like. Seed treatments
can be effected with both continuous and/or a batch seed treaters.
In certain embodiments, the coated seeds can be prepared by
slurrying seeds with a coating composition containing at least one
of a RLN-inhibitory Methylobacterium sp., a solid substance with
adherent RLN-inhibitory Methylobacterium sp., emulsions with
RLN-inhibitory Methylobacterium sp. grown therein, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof and air drying the resulting
product. Air drying can be accomplished at any temperature that is
not deleterious to the seed or the Methylobacterium, but will
typically not be greater than 30 degrees Centigrade. The proportion
of coating that comprises a solid substance and RLN-inhibitory
Methylobacterium sp. includes, but is not limited to, a range of
0.1 to 25% by weight of the seed, 0.5 to 5% by weight of the seed,
and 0.5 to 2.5% by weight of seed. In certain embodiments, a solid
substance used in the seed coating or treatment will have
RLN-inhibitory Methylobacterium sp. adhered thereon. In certain
embodiments, a solid substance used in the seed coating or
treatment will be associated with RLN-inhibitory Methylobacterium
sp. and will be a fermentation broth, fermentation broth product,
or composition obtained by the methods provided herein. Various
seed treatment compositions and methods for seed treatment
disclosed in U.S. Pat. Nos. 5,106,648, 5,512,069, and 8,181,388 are
incorporated herein by reference in their entireties and can be
adapted for use with an active agent comprising the compositions
provided herein. In certain embodiments, the composition used to
treat the seed can contain agriculturally acceptable excipients
that include, but are not limited to, woodflours, clays, activated
carbon, diatomaceous earth, fine-grain inorganic solids, calcium
carbonate and the like. Clays and inorganic solids that can be used
with the fermentation broths, fermentation broth products, or
compositions provided herein include, but are not limited to,
calcium bentonite, kaolin, china clay, talc, perlite, mica,
vermiculite, silicas, quartz powder, montmorillonite and mixtures
thereof. Agriculturally acceptable adjuvants that promote sticking
to the seed that can be used include, but are not limited to,
polyvinyl acetates, polyvinyl acetate copolymers, hydrolyzed
polyvinyl acetates, polyvinylpyrrolidone-vinyl acetate copolymer,
polyvinyl alcohols, polyvinyl alcohol copolymers, polyvinyl methyl
ether, polyvinyl methyl ether-maleic anhydride copolymer, waxes,
latex polymers, celluloses including ethylcelluloses and
methylcelluloses, hydroxy methylcelluloses, hydroxypropylcellulose,
hydroxymethylpropylcelluloses, polyvinyl pyrrolidones, alginates,
dextrins, malto-dextrins, polysaccharides, fats, oils, proteins,
karaya gum, jaguar gum, tragacanth gum, polysaccharide gums,
mucilage, gum arabics, shellacs, vinylidene chloride polymers and
copolymers, soybean-based protein polymers and copolymers,
lignosulfonates, acrylic copolymers, starches, polyvinylacrylates,
zeins, gelatin, carboxymethylcellulose, chitosan, polyethylene
oxide, acrylamide polymers and copolymers, polyhydroxyethyl
acrylate, methylacrylamide monomers, alginate, ethylcellulose,
polychloroprene and syrups or mixtures thereof. Other useful
agriculturally acceptable adjuvants that can promote coating
include, but are not limited to, polymers and copolymers of vinyl
acetate, polyvinylpyrrolidone-vinyl acetate copolymer and
water-soluble waxes. Various surfactants, dispersants,
anticaking-agents, foam-control agents, and dyes disclosed herein
and in U.S. Pat. No. 8,181,388 can be adapted for use with an
active agent comprising the fermentation broths, fermentation broth
products, or compositions provided herein.
[0071] Provided herein are compositions that comprise at least one
of a RLN-inhibitory Methylobacterium sp., a solid substance with
adherent RLN-inhibitory Methylobacterium sp., emulsions with
RLN-inhibitory Methylobacterium sp. grown therein, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof that provide control of RLN
damage to plants, plant parts, and plants obtained therefrom
relative to untreated plants, plant parts, and plants obtained
therefrom that have not been exposed to the compositions. In
certain embodiments, plant parts, including, but not limited to, a
seed, a leaf, a fruit, a stem, a root, a tuber, a pollen grain, or
a coleoptile can be treated with the compositions provided herein
to inhibit of RLN growth and/or reproduction and/or reduce of RLN
damage to a plant. Treatments or applications can include, but are
not limited to, spraying, coating, partially coating, immersing,
and/or imbibing the plant or plant parts with the compositions
provided herein. In certain embodiments, a seed, a leaf, a fruit, a
stem, a root, a tuber, or a coleoptile can be immersed and/or
imbibed with a liquid, semi-liquid, emulsion, or slurry of a
composition provided herein. Such seed immersion or imbibition can
be sufficient to provide for inhibition of RLN growth and/or
reductions in RLN damage in a treated plant or plant part in
comparison to an untreated plant or plant part. Such inhibition of
RLN growth and/or reductions in RLN damage includes, but is not
limited to inhibition of RLN development and/or reproduction,
disruption of RLN feeding behaviors, inhibition of motility, and/or
reductions in damage to roots, tubers, or other plant parts
relative to untreated plants. In certain embodiments, plant seeds
can be immersed and/or imbibed for at least 1, 2, 3, 4, 5, or 6
hours. Such immersion and/or imbibition can, in certain
embodiments, be conducted at temperatures that are not deleterious
to the plant seed or the Methylobacterium. In certain embodiments,
the seeds can be treated at about 15 to about 30 degrees Centigrade
or at about 20 to about 25 degrees Centigrade. In certain
embodiments, seed imbibition and/or immersion can be performed with
gentle agitation.
[0072] Amounts of the compositions that comprise at least one of a
RLN-inhibitory Methylobacterium sp., a solid substance with
adherent RLN-inhibitory Methylobacterium sp., emulsions with
RLN-inhibitory Methylobacterium sp. grown therein, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof sufficient to provide for a
reduction in RLN damage of a plant or plant part can thus be
determined by measuring any or all of changes in RLN feeding
behavior, RLN growth, RLN reproduction, RLN motility, numbers of
recovered RLN, numbers of viable RLN, and/or the adverse effects of
RLN feeding in treated plants or plant parts relative to untreated
plants or plant parts. Adverse effects of RLN growth and/or
reproduction in a plant that can be measured include any type of
plant tissue damage or necrosis (e.g., damage of plant parts
including but not limited to roots and tubers), any type of plant
yield reduction, any reduction in the value of the crop plant
product, and/or increases in fungal disease incidence. In certain
embodiments, an RLN damage rating scale can be used to assess
inhibition of RLN growth and/or reproduction and/or reductions in
damage to a plant or plant part.
[0073] Compositions provided herein comprising at least one of a
RLN-inhibitory Methylobacterium sp., a solid substance with
adherent RLN-inhibitory Methylobacterium sp., emulsions with
RLN-inhibitory Methylobacterium sp. grown therein, a RLN-inhibitory
cell-free culture supernatant, fraction thereof, concentrate
thereof, or any combination thereof are therefore expected to be
useful in inhibiting RLN growth, motility, and/or reproduction
and/or reducing RLN damage in a wide variety of plants, including,
but not limited to: Brassica sp. (e.g., B. napus, B. rapa, B.
juncea), corn, wheat, rye, rice, alfalfa, rice, rye, sorghum,
millet (e.g., pearl millet (Pennisetum glaucum), proso millet
(Panicum miliaceum), foxtail millet (Setaria italica), finger
millet (Eleusine coracana), sunflower, safflower, soybean, tobacco,
potato, peanuts, carrot, cotton, sweet potato (Ipomoea batatus),
cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea,
banana, avocado, fig, guava, mango, olive, papaya, cashew,
macadamia, almond, sugar beets, sugarcane, strawberry, oats, okra,
onion, barley, tomato, lettuce, pepper, green beans, lima beans,
peas, cucurbits such as cucumber, cantaloupe, and musk melon, turf,
ornamentals, and conifers. Compositions provided herein are also
expected to be useful in inhibiting growth and/or reducing damage
caused by Pratylenchus brachyurus, Pratylenchus coffeae, P.
neglectus, Pratylenchus penetrans, Pratylenchus scribneri,
Pratylenchus thornei, Pratylenchus vulnus, and Pratylenchus
zeae.
[0074] In certain embodiments, an amount of a composition provided
herein that is sufficient to provide for inhibition of RLN damage
in a plant or plant part can be a composition with RLN-inhibitory
Methylobacterium sp. at a titer of at least about 1.times.10.sup.4
colony-forming units per milliliter, at least about
1.times.10.sup.5 colony-forming units per milliliter, at least
about 1.times.10.sup.6 colony-forming units per milliliter, at
least about 5.times.10.sup.6 colony-forming units per milliliter,
at least about 1.times.10.sup.7 colony-forming units per
milliliter, at least about 5.times.10.sup.8 colony-forming units
per milliliter, at least about 1.times.10.sup.9 colony-forming
units per milliliter, at least about 1.times.10.sup.10
colony-forming units per milliliter, or at least about
3.times.10.sup.10 colony-forming units per milliliter. In certain
embodiments, an amount of a composition provided herein that is
sufficient to provide for inhibition of RLN growth and/or
reproduction and/or reduction of RLN damage to a plant or plant
part can be a composition with RLN-inhibitory Methylobacterium sp.
at a titer of at least about 1.times.10.sup.4 colony-forming units
per milliliter, at least about 1.times.10.sup.5 colony-forming
units per milliliter, about least about 1.times.10.sup.6
colony-forming units per milliliter, at least about
5.times.10.sup.6 colony-forming units per milliliter, at least
about 1.times.10.sup.7 colony-forming units per milliliter, or at
least about 5.times.10.sup.8 colony-forming units per milliliter to
at least about 6.times.10.sup.10, 1.times.10.sup.11,
5.times.10.sup.11, or 1.times.10.sup.12 colony-forming units per
milliliter of a liquid or an emulsion. In certain embodiments, an
amount of a composition provided herein that is sufficient to
provide for inhibition of RLN growth and/or reproduction and/or
reduction of RLN damage to a plant or plant part can be a
fermentation broth product with a RLN-inhibitory Methylobacterium
sp. titer of a solid phase of that product is at least about
1.times.10.sup.4 colony-forming units per gram, at least about
1.times.10.sup.5 colony-forming units per gram, at least about
1.times.10.sup.6 colony-forming units per gram, at least about
5.times.10.sup.6 colony-forming units per gram, at least about
1.times.10.sup.7 colony-forming units per gram, or at least about
5.times.10.sup.8 colony-forming units per gram to at least about
6.times.10.sup.10 colony-forming units of Methylobacterium per
gram, at least about 1.times.10.sup.11 colony-forming units of
Methylobacterium per gram, at least about 1.times.10.sup.12
colony-forming units of Methylobacterium per gram, at least about
1.times.10.sup.13 colony-forming units of Methylobacterium per
gram, or at least about 5.times.10.sup.13 colony-forming units of
Methylobacterium per gram of the solid phase. In certain
embodiments, an amount of a composition provided herein that is
sufficient to provide for inhibition of RLN growth, motility,
and/or reproduction and/or reduction of RLN damage to a plant or
plant part can be a composition with a Methylobacterium titer of at
least about 1.times.10.sup.6 colony-forming units per gram, at
least about 5.times.10.sup.6 colony-forming units per gram, at
least about 1.times.10.sup.7 colony-forming units per gram, or at
least about 5.times.10.sup.8 colony-forming units per gram to at
least about 6.times.10.sup.10 colony-forming units of
Methylobacterium per gram, at least about 1.times.10.sup.11
colony-forming units of Methylobacterium per gram, at least about
1.times.10.sup.12 colony-forming units of Methylobacterium per
gram, at least about 1.times.10.sup.13 colony-forming units of
Methylobacterium per gram, or at least about 5.times.10.sup.13
colony-forming units of Methylobacterium per gram of particles in
the composition containing the particles that comprise a solid
substance wherein a mono-culture or co-culture of RLN-inhibitory
Methylobacterium sp. is adhered thereto. In certain embodiments, an
amount of a composition provided herein that is sufficient to
provide for inhibition of RLN growth, motility, and/or reproduction
and/or reduction of RLN damage to a plant or plant part can be a
composition with aMethylobacterium titer of at least about
1.times.10.sup.6 colony-forming units per mL, at least about
5.times.10.sup.6 colony-forming units per mL, at least about
1.times.10.sup.7 colony-forming units per mL, or at least about
5.times.10.sup.8 colony-forming units per mL to at least about
6.times.10.sup.10 colony-forming units of Methylobacterium per mL
in a composition comprising an emulsion wherein a mono-culture or
co-culture of a RLN-inhibitory Methylobacterium sp. adhered to a
solid substance is provided therein or grown therein. In certain
embodiments, an amount of a composition provided herein that is
sufficient to provide for inhibition of RLN growth motility, and/or
reproduction and/or reduction of RLN damage to a plant or plant
part can be a composition with a Methylobacterium titer of at least
about 1.times.10.sup.6 colony-forming units per mL, at least about
5.times.10.sup.6 colony-forming units per mL, at least about
1.times.10.sup.7 colony-forming units per mL, or at least about
5.times.10.sup.8 colony-forming units per mL to at least about
6.times.10.sup.10 colony-forming units of Methylobacterium per mL
of in a composition comprising an emulsion wherein a mono-culture
or co-culture of a RLN-inhibitory Methylobacterium sp. is provided
therein or grown therein.
EXAMPLES
[0075] The following examples are included to demonstrate certain
embodiments. It will be appreciated by those of skill in the art
that the techniques disclosed in the following examples represent
techniques determined by the Applicants to function well in the
practice of the disclosure. However, those of skill in the art
should, in light of the instant disclosure, appreciate that many
changes can be made in the specific embodiments that are disclosed,
while still obtaining like or similar results, without departing
from the scope of the invention.
Example 1. Root Lesion Nematodes (RLN) In Vitro Assay
[0076] Pratylenchus penetrans (WI isolate) in all vermiform stages
were purchased from the MacGuidwin Nematology Lab, University of
Wisconsin-Madison. (available on the http internet site
"labs.russell.wisc. edu/macguidwinlab/category/uncategorized").
[0077] The growth medium was a liquid base medium amended with a
solid substance. Thus, the medium was comprised of both a liquid
phase and a solid phase.
[0078] The liquid base medium was Ammonium Mineral Salts (AMS)
containing peptone, glutamate as the carbon source, and
diatomaceous earth as the solid substance.
[0079] To prepare this medium, three stock solutions were made as
follows.
[0080] Stock solution I was made by dissolving 35 grams of
anhydrous dibasic potassium phosphate and 27 grams of anhydrous
monobasic potassium phosphate in a final volume of 1 liter of
distilled water. Stock solution I was at a 50.times.
concentration.
[0081] Stock solution II was made by dissolving 50 grams of
magnesium sulfate heptahydrate and 25 grams of anhydrous ammonium
chloride in a final volume of 1 liter of distilled water. Stock
Solution II was at a 50.times. concentration.
[0082] Stock solution III was made by dissolving 10 grams calcium
chloride dihydrate in one liter of distilled water. Stock Solution
III was at 50.times. concentration.
[0083] Iron (II) sulfate stock solution was made by dissolving 5 g
iron (II) sulfate heptahydrate in one liter of distilled water.
Iron (II) sulfate stock solution was at 1000.times.
concentration.
[0084] To prepare one liter of growth medium, 20 ml of stock
solution I, 20 ml of stock solution II, 20 ml of stock solution
III, 1 ml iron (II) stock solution, 10 grams of peptone, 15 grams
of sodium glutamate, and 2 grams of diatomaceous earth were added
to 940 ml of distilled water. The growth medium was autoclaved
before use.
[0085] Single PPFM colonies were used to inoculate cultures. The
cultures were grown for 4 days at 30 degrees centigrade with
shaking at 200 rpm to titer of 1.times.10.sup.7 to 1.times.10.sup.8
CFU/ml.
[0086] Two ml of each culture was spun down at 10,000.times.g for 5
min, and the supernatant was passed through a 0.2 micron acrodisc
syringe filter (Pall Corporation, Port Washington, N.Y.) to obtain
a cell-free culture supernatant. An antibiotic solution with final
concentrations of 0.025 mg/ml chlorotetracycline (Sigma-Aldrich,
St. Louis, Mo.), 0.025 mg/ml chloramphenicol (Sigma-Aldrich, St.
Louis, Mo.), 0.05 mg/ml Nystatin (Sigma-Aldrich, St. Louis, Mo.),
and 2.5% ethanol was used to control growth of microorganisms over
the course of the experiment. 2:1:1 supernatant: antibiotic
solution: 200 P. penetrans nematodes in water were added to each
well of a 96 well plate. Each plate was divided into 5 complete
randomized blocks, with each isolate and control repeated once per
block. The activity of the P. penetrans nematodes was rated after 3
days under a dissecting microscope. The rating system was 1=no
movement, nematodes mostly straight; 2=some moving, curly;
3=most/all moving, curly.
[0087] The experiment was repeated twice. A linear mixed model was
fitted using the "lmer" function in the lme4 package (Bates, D.,
Maechler, M., Bolker, B., & Walker, S. (2014). lme4: Linear
mixed-effects models using Eigen and S4. R package version 1.1-7,
Available on the http internet site
"CRAN.R-project.org/package=lme4")) in R (R Core Team (2013). R: A
language and environment for statistical computing. R Foundation
for Statistical Computing, Vienna, Austria, available on the world
wide web internet site "R-project.com") with random effect of
experiment. P values were estimated using a normal distribution.
Results are shown in Table 3.
TABLE-US-00003 TABLE 3 Effect of PPFM cell-free culture
supernatants on RLN activity. Average Difference from Strain
Activity Score Control (%) p value Control 1.550 NLS0017 1.500 -3%
0.778 NLS0020 1.833 18% 0.120 NLS0021 1.000 -35% 0.002 NLS0037
1.611 4% 0.738 NLS0038 1.100 -29% 0.011 NLS0042 1.000 -35% 0.002
NLS0934 1.150 -26% 0.024 NLS0046 1.889 22% 0.063 NLS0062 1.200 -23%
0.049 NLS0064 1.450 -6% 0.573 NLS0065 1.250 -19% 0.091 NLS0066
1.650 6% 0.573 NLS0068 1.300 -16% 0.159 NLS0069 1.100 -29% 0.011
NLS0089 1.150 -26% 0.024
[0088] Each line m the table is the results of replicates in each
of two separate experiments. A linear mixed model was fitted with
the random effect of experiment.
[0089] Each line in the table is the results of inverse variance
meta-analysis combining the results of six independent experiments.
Each strain was tested in at least three separate experiments.
[0090] Cell-free culture supernatants from strains NLS0021,
NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, and NLS0089 show
significant decreases in activity scores for P. penetrans.
Cell-free culture supernatants from strains NLS0017, NLS0037,
NLS0064, and NLS0066 showed negligible effects on activity scores
for P. penetrans.
Example 2. Root Lesion Nematodes (RLN) in Planta Assay
[0091] Pratylenchus penetrans (WI isolate) in all vermiform stages
were purchased from the MacGuidwin Nematology Lab, University of
Wisconsin-Madison. (available on the http internet site
"labs.russell.wisc. edu/macguidwinlab/category/uncategorized").
[0092] Tomato seeds (Solanum lycopersicum variety "Charger" or
"Sweet Olive", Johnny's Selected Seeds, Winslow, Me.) in horticubes
were treated with 0.25 ml PPFM solution in water at concentration
of 1.times.10.sup.7 to 1.times.108 CFU/ml. This treatment simulates
an in-furrow treatment applied at planting. Control seeds were
treated with 0.25 ml water. Plants were grown for approximately two
weeks in the greenhouse before transplanting to autoclaved 9:1
sand:soil mixture in pots. Plants were watered as needed with 2.5
g/L Jack's Professional 15-16-17 Peat-Lite Fertilizer. Plants were
grown for approximately one additional week before inoculation with
P. penetrans nematodes. Inoculum was prepared by diluting the
purchased nematode solution to concentration of 100 nematodes/ml
and adding 5 ml to holes around the roots in each pot (500
nematodes/pot total). Plants were grown approximately 6 additional
weeks before harvest.
[0093] At harvest, the plant height was measured. Shoots were dried
2-3 days and dry weights were measured. Roots were extracted from
the sandy soil, rinsed, and blotted dry. Root fresh and dry weights
were measured. 250 ml of sandy soil was soaked in 800 ml water for
approximately 1 hour with occasional stirring. The mixture was
stirred and allowed to settle 1 min before decanting the water
through #40 sieve. The flowthrough was stirred and allowed to
settle before decanting through #170 sieve. The material caught on
the #170 screen was backwashed into a clean beaker. The flowthrough
was decanted through #325 sieve, and the material caught on the
#325 screen was backwashed into the same clean beaker. The volume
of this solution was measured and the number of nematodes in 10
microliters was counted three times under a dissecting microscope
to calculate the total number of nematodes.
[0094] Each experiment contained 6 replicates per isolate arranged
in 3 randomized complete blocks. The values obtained from each
experiment were normalized to the average values obtained from the
control plants. A linear mixed model was fitted using the "lmer"
function in the lme4 package (Bates, D., Maechler, M., Bolker, B.,
& Walker, S. (2014). lme4: Linear mixed-effects models using
Eigen and S4. R package version 1.1-7, available on the http
internet site "CRAN.R-project.org/package=lme4") in R (R Core Team
(2013). R: A language and environment for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria, available on
the world wide web internet site "R-project.com") with random
effect of block. An effect size and p value for each strain was
calculated from results of at least three separate experiments in a
total of five separate experiments using the inverse variance
meta-analysis method (Lipsey, M. W., & Wilson, D. B. (2001).
Practical meta-analysis. Thousand Oaks, Calif.: Sage Publications,
available on the http internet site
mason.gmu.edu/-dwilsonb/ma.html."). Results are shown in Table
4.
TABLE-US-00004 TABLE 4 Effects of seed treatments (simulated
in-furrow treatment) on tomato growth parameters and RLN counts.
Fresh Root RLN/mg Fresh Shoot Weight Weight Total RLN Root Effect p
Effect p Effect p p Strain Size value Size value Size value Effect
Size value NLS0021 4% 0.326 14% 0.056 9% 0.218 -13% 0.088 NLS0037
-1% 0.469 -3% 0.358 4% 0.384 15% 0.101 NLS0038 11% 0.227 24% 0.064
-10% 0.224 -39% 0.001 NLS0069 24% 0.054 28% 0.038 11% 0.211 -21%
0.048
[0095] Each line in the table is the results of inverse variance
meta-analysis combining the results of five independent
experiments. Each strain was tested in at least three separate
experiments.
[0096] Treatment with NLS0069 produces a statistically significant
increase in both shoot weight and fresh root weight, while
treatment with NLS0021 and NLS0038 have statistically significant
increases on fresh root weight. NLS0038 and NLS0069 also
significantly reduce the amount of RLN per mg root. NLS0037 does
not have an inhibitory effect on the amount of RLN. These results
are in agreement with the results of the RLN in vitro assay
presented in Table 1.
Example 3. Field Tests
[0097] A field trial was conducted during the summer of 2016 to
determine the ability of PPFMs to suppress root lesion nematode
(RLN; Pratylenchus penetrans) on spring wheat (Triticum aestivum
L.; var. RB07, supplied by Johnson Grain Inc.). The trial was
planted on May 30, 2016 and harvested on Oct. 11, 2016. Metrics
collected include: Stand, vigor at 7-days post planting, vigor at
21-days post planting, plot height at Feekes 10.5 growth stage,
flowering at Feekes 10.5, lodging, yield, pre-plant nematode counts
and in-season nematode counts for RLN, stunt nematode
(Tylenchorhynchus spp.), and total plant parasitic nematodes. The
experiment was designed as a randomized complete block with five
treatments (Table 5) and four replications. Each experimental plot
consisted of four 10-foot rows, with 50 grams of seed planted per
plot. The entire experiment was surrounded by two border rows to
mitigate edge effects.
[0098] Wheat seed for the trial was treated at NewLeaf Symbiotics'
laboratory facilities in St. Louis, Mo. and shipped to Moccasin,
Mont., where the field trial was conducted. All PPFM treatments
were applied at a rate of 14 mL PPFM culture concentrate per pound
of seed. PPFM culture concentrates had a concentration in the range
of 1.times.10.sup.9 to 1.times.10.sup.10 colony forming units
(CFUs) per mL of concentrate. A portion of treated seed was
retained at the NewLeaf Symbiotic's facilities for PPFM
enumeration. Enumeration confirmed that treatment was successful,
with on-seed concentrations remaining at or above 1.times.10.sup.6
colony forming units per seed until weeks after the treatment
date.
TABLE-US-00005 TABLE 5 2016 Spring Wheat Lesion Nematode Trial
Treatments Treatment Number Treatment Application Timing Rate 1
Untreated control N/A N/A 2 NLS0021 Seed treatment 14 mL/lb 3
NLS0038 Seed treatment 14 mL/lb 4 NLS0066 Seed treatment 14 mL/lb 5
NLS0069 Seed treatment 14 mL/lb
[0099] Prior to planting, root lesion and stunt nematode
populations were enumerated for each repetition by collecting 4
soil cores of .about.25 g wet weight and counting the number of
nematodes within each core. The average count from the four cores
collected per block was calculated to provide baseline estimates
for root lesion, stunt, and total plant parasitic nematode
populations (Table 6). Root and stunt nematode population were
again enumerated after planting, at the Feekes 3 and Feekes 7
growth stages. Multiple 25 g subsamples were collected per plot and
the counts in these subsamples were averaged and used to calculate
number of nematodes per kg of soil within the plot (Table 7).
Throughout the growing season, various data were collected,
including stand, vigor, plant height, flowering, time, and yield
(Table 8).
TABLE-US-00006 TABLE 6 2016 Spring Wheat Plant Parasitic Nematode
Pre-Plant Counts Repetition Root lesion/kg soil Stunt/kg soil Total
PP.sup.1 Nem/kg soil Rep 1 274 704 978 Rep 2 588 1412 2001 Rep 3
2359 580 2939 Rep 4 1056 313 1369 Average 1069 752 1822 .sup.1PP
Nem = Plant Parasitic Nematode (Root lesion + stunt)
TABLE-US-00007 TABLE 7 2016 Spring Wheat Root Lesion Nematode Trial
In-season Counts Treatment Pre-Plant Feekes 3 Feekes 7 UTC 1069.00
2930.00 4300.00 A.sup.2 NLS0021 1069.00 500.00 2330.00 B NLS0038
1069.00 2890.00 1980.00 B NLS0066 1069.00 3110.00 2840.00 AB
NLS0069 1069.00 2070.00 1430.00 B .sup.2Counts followed by the same
letter do not differ significantly from one-another by Fisher's
LSD; No significant differences were found between counts taken at
the Feekes 3 growth stage.
TABLE-US-00008 TABLE 8 2016 Spring Wheat Root Lesion Nematode Trial
Data Summary Height 7DAE 21DAE 7DAE 21DAE Feekes Flowering Yield
Stand Stand Vigor Vigor 10.5 Feekes 10.5 (kg/A) Trt (SEM) (SEM)
(SEM) (SEM) (SEM) (SEM (SEM) NLS0021 21.38 46.25 8.25 8.5 45.6
45.60 205.50 (1.09) (5.13) (0.75) (0.29) (0.29) (0.29) (29.58)
NLS0038 24.63 46.50 8.25 8.50 44.25 44.25 203.85 (0.80) (2.89)
(0.25) (0.50) (0.99) (0.99) (25.86) NLS0066 18.50 34.13 7.75 8.75
47.25 47.25 230.35 (4.46) (1.84) (0.25) (0.25) (2.08) (2.08)
(30.23) NLS0069 20.88 38.88 8.00 8.50 44.60 44.60 209.45 (2.60)
(1.91) (0.82) (0.29) (0.37) (0.37) (13.64) UTC 20.63 42.13 7.50 9.0
45.85 45.85 213.42 (3.79) (3.07) (0.29) (0.00) (1.37) (1.37)
(15.57)
[0100] Three of four PPFM treatments applied to plants in this
trial, NLS0021, NLS0038, and NLS0069, significantly suppressed root
lesion nematode populations in spring wheat at growth stage Feekes
7. Data were analyzed using the JMP statistical analysis software
package (version 12.0; SAS Institute, Cary, N.C., 1989-2016). A
mixed model was fit in the `Fit Model` functionality with
`Repetition` as a random factor and treatment as a fixed factor.
Treatment means and post-hoc means comparisons were extracted from
this analysis. The finding that NLS0021, NLS0038, and NLS0069
significantly reduced root lesion nematode populations while
NLS0066 did not is consistent with findings from in vitro assays
reported in Example 1 and supports the positive data for isolates
NLS0021, NLS038, and NLS0069 reported in Example 2. Stunt nematodes
were present in the soil but their populations were not
significantly affected by the PPFM treatments.
[0101] Of the four strains tested, none significantly impacted
yield or other growth parameters measured. This indicates that seed
treatment with PPFMs presents no deleterious effects on
germination, vigor, or yield of spring wheat. The trial was planted
later than is ideal for spring wheat in this region of Montana. As
a result, possible benefits from PPFM application to seeds may have
been masked by the late planting date.
[0102] Having illustrated and described the principles of the
present disclosure, it should be apparent to persons skilled in the
art that the disclosure can be modified in arrangement and detail
without departing from such principles.
[0103] Although the materials and methods of this disclosure have
been described in terms of various embodiments and illustrative
examples, it will be apparent to those of skill in the art that
variations can be applied to the materials and methods described
herein without departing from the concept, spirit and scope of the
disclosure. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the disclosure as defined by the appended
claims.
Sequence CWU 1
1
711472DNAMethylobacterium sp. 1gagtttgatc ctggctcaga gcgaacgctg
gcggcaggct taacacatgc aagtcgaacg 60ggcttcttcg gaagtcagtg gcagacgggt
gagtaacacg tgggaacgtg cccttcggtt 120cggaataact cagggaaact
tgagctaata ccggatacgc ccttatgggg aaaggtttac 180tgccgaagga
tcggcccgcg tctgattagc ttgttggtgg ggtaacggcc taccaaggcg
240acgatcagta gctggtctga gaggatgatc agccacactg ggactgagac
acggcccaga 300ctcctacggg aggcagcagt ggggaatatt ggacaatggg
cgcaagcctg atccagccat 360gccgcgtgag tgatgaaggc cttagggttg
taaagctctt ttgtccggga cgataatgac 420ggtaccggaa gaataagccc
cggctaactt cgtgccagca gccgcggtaa tacgaagggg 480gctagcgttg
ctcggaatca ctgggcgtaa agggcgcgta ggcggccgat taagtcgggg
540gtgaaagcct gtggctcaac cacagaattg ccttcgatac tggttggctt
gagaccggaa 600gaggacagcg gaactgcgag tgtagaggtg aaattcgtag
atattcgcaa gaacaccagt 660ggcgaaggcg gctgtctggt ccggttctga
cgctgaggcg cgaaagcgtg gggagcaaac 720aggattagat accctggtag
tccacgccgt aaacgatgaa tgccagccgt tggtctgctt 780gcaggtcagt
ggcgccgcta acgcattaag cattccgcct ggggagtacg gtcgcaagat
840taaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg
tttaattcga 900agcaacgcgc agaaccttac catcccttga catggcatgt
tacctcgaga gatcggggat 960cctcttcgga ggcgtgcaca caggtgctgc
atggctgtcg tcagctcgtg tcgtgagatg 1020ttgggttaag tcccgcaacg
agcgcaaccc acgtccttag ttgccatcat tcagttgggc 1080actctaggga
gactgccggt gataagccgc gaggaaggtg tggatgacgt caagtcctca
1140tggcccttac gggatgggct acacacgtgc tacaatggcg gtgacagtgg
gacgcgaaac 1200cgcgaggttg agcaaatccc caaaagccgt ctcagttcgg
attgcactct gcaactcggg 1260tgcatgaagg cggaatcgct agtaatcgtg
gatcagcacg ccacggtgaa tacgttcccg 1320ggccttgtac acaccgcccg
tcacaccatg ggagttggtc ttacccgacg gcgctgcgcc 1380aaccgcaagg
gggcaggcga ccacggtagg gtcagcgact ggggtgaagt cgtaacaagg
1440tagccgtagg ggaacctgcg gctggatcac ct
147221472DNAMethylobacterium sp. 2ggtgatccag ccgcaggttc ccctacggct
accttgttac gacttcaccc cagtcgctga 60ccctaccgtg gtcgcctgcc tccttgcggt
tggcgcagcg ccgtcgggta agaccaactc 120ccatggtgtg acgggcggtg
tgtacaaggc ccgggaacgt attcaccgtg gcgtgctgat 180ccacgattac
tagcgattcc gccttcatgc acccgagttg cagagtgcaa tccgaactga
240gacggttttt ggggatttgc tccacctcgc ggcttcgcgt cccactgtca
ccgccattgt 300agcacgtgtg tagcccatcc cgtaagggcc atgaggactt
gacgtcatcc acaccttcct 360cgcggcttat caccggcagt ctccctagag
tgcccaactg aatgatggca actaaggacg 420tgggttgcgc tcgttgcggg
acttaaccca acatctcacg acacgagctg acgacagcca 480tgcagcacct
gtgtgcacgc ctccgaagag gatccccgat ctctcgaggt aacatgccat
540gtcaagggat ggtaaggttc tgcgcgttgc ttcgaattaa accacatgct
ccaccgcttg 600tgcgggcccc cgtcaattcc tttgagtttt aatcttgcga
ccgtactccc caggcggaat 660gcttaatgcg ttagcggcgc cactgacctg
caagcaggcc aacggctggc attcatcgtt 720tacggcgtgg actaccaggg
tatctaatcc tgtttgctcc ccacgctttc gcgcctcagc 780gtcagaaccg
gaccagacag ccgccttcgc cactggtgtt cttgcgaata tctacgaatt
840tcacctctac actcgcagtt ccgctgtcct cttccggtct caagccaacc
agtatcgaag 900gcaattctgt ggttgagcca caggctttca cccccgactt
aatcggccgc ctacgcgccc 960tttacgccca gtgattccga gcaacgctag
cccccttcgt attaccgcgg ctgctggcac 1020gaagttagcc ggggcttatt
cttccggtac cgtcattatc gtcccggaca aaagagcttt 1080acaaccctaa
ggccttcatc actcacgcgg catggctgga tcaggcttgc gcccattgtc
1140caatattccc cactgctgcc tcccgtagga gtctgggccg tgtctcagtc
ccagtgtggc 1200tgatcatcct ctcagaccag ctactgatcg tcgccttggt
aggccgttac cccaccaaca 1260agctaatcag acgcgggccg atccttcggc
agtaaacctt tccccaaaag ggcgtatccg 1320gtattagctc aagtttccct
gagttattcc gaaccgaagg gtacgttccc acgtgttact 1380cacccgtctg
ccactgacac ccgaaggtgc ccgttcgact tgcatgtgtt aagcctgccg
1440ccagcgttcg ctctgagcca ggatcaaact ct
147231472DNAMethylobacterium sp. 3gagtttgatc ctggctcaga gcgaacgctg
gcggcaggct taacacatgc aagtcgaacg 60ggcaccttcg ggtgtcagtg gcagacgggt
gagtaacacg tgggaacgta cccttcggtt 120cggaataact cagggaaact
tgagctaata ccggatacgc ccttttgggg aaaggtttac 180tgccgaagga
tcggcccgcg tctgattagc ttgttggtgg ggtaacggcc taccaaggcg
240acgatcagta gctggtctga gaggatgatc agccacactg ggactgagac
acggcccaga 300ctcctacggg aggcagcagt ggggaatatt ggacaatggg
cgcaagcctg atccagccat 360gccgcgtgag tgatgaaggc cttagggttg
taaagctctt ttgtccggga cgataatgac 420ggtaccggaa gaataagccc
cggctaactt cgtgccagca gccgcggtaa tacgaagggg 480gctagcgttg
ctcggaatca ctgggcgtaa agggcgcgta ggcggccgat taagtcgggg
540gtgaaagcct gtggctcaac cacagaattg ccttcgatac tggttggctt
gagaccggaa 600gaggacagcg gaactgcgag tgtagaggtg aaattcgtag
atattcgcaa gaacaccagt 660ggcgaaggcg gctgtctggt ccggttctga
cgctgaggcg cgaaagcgtg gggagcaaac 720aggattagat accctggtag
tccacgccgt aaacgatgaa tgccagccgt tggcctgctt 780gcaggtcagt
ggcgccgcta acgcattaag cattccgcct ggggagtacg gtcgcaagat
840taaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg
tttaattcga 900agcaacgcgc agaaccttac catcccttga catggcatgt
tacctcgaga gatcggggat 960cctcttcgga ggcgtgcaca caggtgctgc
atggctgtcg tcagctcgtg tcgtgagatg 1020ttgggttaag tcccgcaacg
agcgcaaccc acgtccttag ttgccatcat tcagttgggc 1080actctaggga
gactgccggt gataagccgc gaggaaggtg tggatgacgt caagtcctca
1140tggcccttac gggatgggct acacacgtgc tacaatggcg gtgacagtgg
gacgcgaagc 1200cgcgaggtgg agcaaatccc caaaaaccgt ctcagttcgg
attgcactct gcaactcggg 1260tgcatgaagg cggaatcgct agtaatcgtg
gatcagcacg ccacggtgaa tacgttcccg 1320ggccttgtac acaccgcccg
tcacaccatg ggagttggtc ttacccgacg gcgctgcgcc 1380aaccgcaagg
aggcaggcga ccacggtagg gtcagcgact ggggtgaagt cgtaacaagg
1440tagccgtagg ggaacctgcg gctggatcac ct
147241483DNAMethylobacterium sp. 4gagtttgatc ctggctcaga gcgaacgctg
gcggcaggct taacacatgc aagtcgaacg 60caccgcaagg tgagtggcag acgggtgagt
aacacgtggg aacgtgccct ccggtctggg 120ataaccctgg gaaactaggg
ctaataccgg atacgtgctt tggcagaaag gtttactgcc 180ggaggatcgg
cccgcgtctg attagcttgt tggtggggta acggcctacc aaggcgacga
240tcagtagctg gtctgagagg atgatcagcc acactgggac tgagacacgg
cccagactcc 300tacgggaggc agcagtgggg aatattggac aatgggcgca
agcctgatcc agccatgccg 360cgtgagtgat gacggcctta gggttgtaaa
gctcttttct ccgggacgat aatgacggta 420ccggaggaat aagccccggc
taacttcgtg ccagcagccg cggtaatacg aagggggcta 480gcgttgctcg
gaatcactgg gcgtaaaggg cgcgtaggcg gcgttttaag tcgggggtga
540aagcctgtgg ctcaaccaca gaatggcctt cgatactggg acgcttgagt
atggtagagg 600ttggtggaac tgcgagtgta gaggtgaaat tcgtagatat
tcgcaagaac accggtggcg 660aaggcggcca actggaccat tactgacgct
gaggcgcgaa agcgtgggga gcaaacagga 720ttagataccc tggtagtcca
cgccgtaaac gatgaatgct agctgttggg gtgcatgcac 780cgcagtagcg
cagctaacgc attaagcatt ccgcctgggg agtacggtcg caagattaaa
840actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta
attcgaagca 900acgcgcagaa ccttaccatc ctttgacatg gcgtgttact
gggagagatt ccaggtcccc 960ttcgggggcg cgcacacagg tgctgcatgg
ctgtcgtcag ctcgtgtcgt gagatgttgg 1020gttaagtccc gcaacgagcg
caacccacgt ccttagttgc catcatttgg ttgggcactc 1080tagggagact
gccggtgata agccgcgagg aaggtgtgga tgacgtcaag tcctcatggc
1140ccttacggga tgggctacac acgtgctaca atggcggtga cagtgggacg
cgaaggggtg 1200acccggagcc aatctccaaa agccgtctca gttcggattg
cacgctgcaa ctcgcgtgca 1260tgaaggcgga atcgctagta atcgtggatc
agcatgccac ggtgaatacg ttcccgggcc 1320ttgtacacac cgcccgtcac
accatgggag ttggtcttac ccgacggcgc tgcgccaacc 1380aaatcgagct
tgctcgactg gaggcaggcg accacggtag ggtcagcgac tggggtgaag
1440tcgtaacaag gtagccgtag gggaacctgc ggctggatca cct
148351472DNAMethylobacterium sp. 5ggtgatccag ccgcaggttc ccctacggct
accttgttac gacttcaccc cagtcgctga 60ccctaccgtg gtcgcctgcc tccttgcggt
tggcgcagcg ccgtcgggta agaccaactc 120ccatggtgtg acgggcggtg
tgtacaaggc ccgggaacgt attcaccgtg gcgtgctgat 180ccacgattac
tagcgattcc gccttcatgc acccgagttg cagagtgcaa tccgaactga
240gacggttttt ggggatttgc tccacctcgc ggcttcgcgt cccactgtca
ccgccattgt 300agcacgtgtg tagcccatcc cgtaagggcc atgaggactt
gacgtcatcc acaccttcct 360cgcggcttat caccggcagt ctccctagag
tgcccaactg aatgatggca actaaggacg 420tgggttgcgc tcgttgcggg
acttaaccca acatctcacg acacgagctg acgacagcca 480tgcagcacct
gtgtgcacgc ctccgaagag gatccccgat ctctcgaggt aacatgccat
540gtcaagggat ggtaaggttc tgcgcgttgc ttcgaattaa accacatgct
ccaccgcttg 600tgcgggcccc cgtcaattcc tttgagtttt aatcttgcga
ccgtactccc caggcggaat 660gcttaatgcg ttagcggcgc cactgacctg
caagcaggcc aacggctggc attcatcgtt 720tacggcgtgg actaccaggg
tatctaatcc tgtttgctcc ccacgctttc gcgcctcagc 780gtcagaaccg
gaccagacag ccgccttcgc cactggtgtt cttgcgaata tctacgaatt
840tcacctctac actcgcagtt ccgctgtcct cttccggtct caagccaacc
agtatcgaag 900gcaattctgt ggttgagcca caggctttca cccccgactt
aatcggccgc ctacgcgccc 960tttacgccca gtgattccga gcaacgctag
cccccttcgt attaccgcgg ctgctggcac 1020gaagttagcc ggggcttatt
cttccggtac cgtcattatc gtcccggaca aaagagcttt 1080acaaccctaa
ggccttcatc actcacgcgg catggctgga tcaggcttgc gcccattgtc
1140caatattccc cactgctgcc tcccgtagga gtctgggccg tgtctcagtc
ccagtgtggc 1200tgatcatcct ctcagaccag ctactgatcg tcgccttggt
aggccgttac cccaccaaca 1260agctaatcag acgcgggccg atccttcggc
agtaaacctt tccccaaaag ggcgtatccg 1320gtattagctc aagtttccct
gagttattcc gaaccgaagg gtacgttccc acgtgttact 1380cacccgtctg
ccactgacac ccgaaggtgc ccgttcgact tgcatgtgtt aagcctgccg
1440ccagcgttcg ctctgagcca ggatcaaact ct
147261470DNAMethylobacterium sp. 6ggtgatccag ccgcaggttc ccctacggct
accttgttac gacttcaccc cagtcgctga 60ccctaccgtg gtcgcctgcc tccttgcggt
tggcgcagcg ccgtcgggta agaccaactc 120ccatggtgtg acgggcggtg
tgtacaaggc ccgggaacgt attcaccgtg gcatgctgat 180ccacgattac
tagcgattcc gccttcatgc acccgagttg cagagtgcaa tccgaactga
240gacggctttt ggggatttgc tcaacctcgc ggtttcgcgt cccactgtca
ccgccattgt 300agcacgtgtg tagcccatcc cgtaagggcc atgaggactt
gacgtcatcc acaccttcct 360cgcggcttat caccggcagt ctccctagag
tgcccaactg aatgatggca actaaggacg 420tgggttgcgc tcgttgcggg
acttaaccca acatctcacg acacgagctg acgacagcca 480tgcagcacct
gtgtgcaggt ccccgaaggg aacgaccgat ctctcggaca atcctgccat
540gtcaagggat ggtaaggttc tgcgcgttgc ttcgaattaa accacatgct
ccaccgcttg 600tgcgggcccc cgtcaattcc tttgagtttt aatcttgcga
ccgtactccc caggcggaat 660gcttaatgcg ttagcggcgc cactgacctg
caagcagacc aacggctggc attcatcgtt 720tacggcgtgg actaccaggg
tatctaatcc tgtttgctcc ccacgctttc gcgcctcagc 780gtcagaaccg
gaccagacag ccgccttcgc cactggtgtt cttgcgaata tctacgaatt
840tcacctctac actcgcagtt ccgctgtcct cttccggtct caagctttcc
agtatcgaag 900gcaattctgt ggttgagcca caggctttca cccccgactt
aaaaagccgc ctacgcgccc 960tttacgccca gtgattccga gcaacgctag
cccccttcgt attaccgcgg ctgctggcac 1020gaagttagcc ggggcttatt
cttccggtac cgtcattatc gtcccggaca aaagagcttt 1080acaaccctaa
ggccttcatc actcacgcgg catggctgga tcaggcttgc gcccattgtc
1140caatattccc cactgctgcc tcccgtagga gtctgggccg tgtctcagtc
ccagtgtggc 1200tgatcatcct ctcagaccag ctactgatcg tcgccttggt
aggccgttac cccaccaact 1260agctaatcag acgcgggccg atccttcggc
agtaaacctt tccccaaaag ggcgtatccg 1320gtattagctc aagtttccct
gagttattcc gaaccgaagg gcacgttccc acgtgttact 1380cacccgtctg
ccgctgaccc cgaagggccc gctcgacttg catgtgttaa gcctgccgcc
1440agcgttcgct ctgagccagg atcaaactct 147071472DNAMethylobacterium
sp. 7gagtttgatc ctggctcaga gcgaacgctg gcggcaggct taacacatgc
aagtcgaacg 60ggcttcttcg gaagtcagtg gcagacgggt gagtaacacg tgggaacgtg
cccttcggtt 120cggaataact cagggaaact tgagctaata ccggatacgc
ccttacgggg aaaggtttac 180tgccgaagga tcggcccgcg tctgattagc
ttgttggtgg ggtaacggcc taccaaggcg 240acgatcagta gctggtctga
gaggatgatc agccacactg ggactgagac acggcccaga 300ctcctacggg
aggcagcagt ggggaatatt ggacaatggg cgcaagcctg atccagccat
360gccgcgtgag tgatgaaggc cttagggttg taaagctctt ttgtccggga
cgataatgac 420ggtaccggaa gaataagccc cggctaactt cgtgccagca
gccgcggtaa tacgaagggg 480gctagcgttg ctcggaatca ctgggcgtaa
agggcgcgta ggcggccgat taagtcgggg 540gtgaaagcct gtggctcaac
cacagaattg ccttcgatac tggttggctt gagaccggaa 600gaggacagcg
gaactgcgag tgtagaggtg aaattcgtag atattcgcaa gaacaccagt
660ggcgaaggcg gctgtctggt ccggttctga cgctgaggcg cgaaagcgtg
gggagcaaac 720aggattagat accctggtag tccacgccgt aaacgatgaa
tgccagccgt tggtctgctt 780gcaggtcagt ggcgccgcta acgcattaag
cattccgcct ggggagtacg gtcgcaagat 840taaaactcaa aggaattgac
gggggcccgc acaagcggtg gagcatgtgg tttaattcga 900agcaacgcgc
agaaccttac catcccttga catggcatgt tacctcgaga gatcggggat
960cctcttcgga ggcgtgcaca caggtgctgc atggctgtcg tcagctcgtg
tcgtgagatg 1020ttgggttaag tcccgcaacg agcgcaaccc acgtccttag
ttgccatcat tcagttgggc 1080actctaggga gactgccggt gataagccgc
gaggaaggtg tggatgacgt caagtcctca 1140tggcccttac gggatgggct
acacacgtgc tacaatggcg gtgacagtgg gacgcgaaac 1200cgcgaggttg
agcaaatccc caaaagccgt ctcagttcgg attgcactct gcaactcggg
1260tgcatgaagg cggaatcgct agtaatcgtg gatcagcacg ccacggtgaa
tacgttcccg 1320ggccttgtac acaccgcccg tcacaccatg ggagttggtc
ttacccgacg gcgctgcgcc 1380aaccgcaagg gggcaggcga ccacggtagg
gtcagcgact ggggtgaagt cgtaacaagg 1440tagccgtagg ggaacctgcg
gctggatcac ct 1472
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