U.S. patent application number 16/560620 was filed with the patent office on 2020-01-09 for use of pectin or pectin-related saccharides to enhance efficacy of plant growth-promoting rhizobacteria (pgpr) strains for promo.
This patent application is currently assigned to Auburn University. The applicant listed for this patent is Auburn University. Invention is credited to Joseph Kloepper, Mark R. Liles.
Application Number | 20200009200 16/560620 |
Document ID | / |
Family ID | 54292953 |
Filed Date | 2020-01-09 |
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United States Patent
Application |
20200009200 |
Kind Code |
A1 |
Liles; Mark R. ; et
al. |
January 9, 2020 |
USE OF PECTIN OR PECTIN-RELATED SACCHARIDES TO ENHANCE EFFICACY OF
PLANT GROWTH-PROMOTING RHIZOBACTERIA (PGPR) STRAINS FOR PROMOTING
GROWTH AND HEALTH IN PLANTS AND ANIMALS
Abstract
Disclosed are compositions and methods that include or utilize
plant growth promoting rhizobacteria (PGPR) for improving growth
and health in plants and animals. The compositions and methods
include or utilize a plant growth promoting rhizobacteria (PGPR)
that expresses a protein associated with pectin metabolism, and a
saccharide comprising pectin or a pectin-related saccharide.
Inventors: |
Liles; Mark R.; (Auburn,
AL) ; Kloepper; Joseph; (Auburn, AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Auburn University |
Auburn |
AL |
US |
|
|
Assignee: |
Auburn University
Auburn
AL
|
Family ID: |
54292953 |
Appl. No.: |
16/560620 |
Filed: |
September 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15473975 |
Mar 30, 2017 |
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16560620 |
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PCT/US2015/053239 |
Sep 30, 2015 |
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15473975 |
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62057667 |
Sep 30, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/00 20180101; A61K
31/732 20130101; A61K 35/742 20130101; A61K 35/74 20130101; A23K
10/18 20160501; A01H 3/00 20130101; A01N 63/00 20130101; A23K 50/80
20160501; A61K 48/00 20130101; A23K 20/163 20160501; A61K 31/7004
20130101; A61K 31/732 20130101; A61K 2300/00 20130101; A61K 31/7004
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/742 20060101
A61K035/742; A01N 63/00 20060101 A01N063/00; A23K 20/163 20060101
A23K020/163; A23K 50/80 20060101 A23K050/80; A23K 10/18 20060101
A23K010/18; A61K 31/732 20060101 A61K031/732 |
Claims
1. An inoculant comprising: (a) Bacillus amyloliquefaciens
subspecies plantarum (BAP), and (b) a saccharide comprising pectin
or a pectin-related saccharide.
2. The inoculant of claim 1, wherein the saccharide is a
heteropolysaccharide comprising D-galacturonate monomers which
represents at least 50% of all monomers of the
heteropolysaccharide.
3. The inoculant of claim 1, wherein the saccharide is a
heterogeneous mixture of polysaccharides or monosaccharides
comprising D-galacturonate monomers or D-glucuronate monomers, and
the sum of D-galacturonate monomers and D-glucuronate monomers in
the mixture represent at least 50% of the total monomers of the
mixture.
4. The inoculant of claim 1, wherein the saccharide is pectin
having an average molecular weight of at least about 30000
g/mol.
5. The inoculant of claim 1, further comprising a nitrogen-fixing
bacteria.
6. Plant seeds coated with the inoculant of claim 1.
7. The plant seeds of claim 6, wherein the seeds comprise at least
two layers, the two layers comprising at least: (i) a first layer
comprising the Bacillus amyloliquefaciens subspecies plantarum
(BAP); and at least (ii) a second layer comprising the pectin or
the pectin-related saccharide, wherein the second layer is interior
relative to the first layer.
8. Animal feed comprising the inoculant of claim 1.
9. The animal feed of claim 8, wherein the feed comprises
10.sup.4-10.sup.7 CFU of the BAP per gram.
10. The animal feed of claim 1, wherein the feed comprises pectin
at a concentration (w/w) of at least about 0.05%.
11. The animal feed of claim 8 formulated for farmed fish.
12. The animal feed of claim 8, wherein the animal feed is coated
with the inoculant.
13. The animal feed of claim 8, wherein the animal feed is prepared
by forming a mixture of the animal feed and the inoculant and
forming a compressed or pelleted animal feed from the mixture.
14. A method for increasing nodulation in a legume, the method
comprising: (a) treating the legume, seeds of the legume, or soil
surrounding the legume with Bacillus amyloliquefaciens subspecies
plantarum (BAP) and (b) treating the legume, the seeds of the
legume, or the soil surrounding the legume with a saccharide
comprising pectin or a pectin-related saccharide.
15. The method of claim 14, wherein the legume, the seeds of the
legume, or the soil surrounding the legume are treated concurrently
with the BAP and the saccharide.
16. The method of claim 14, wherein the legume, the seeds of the
legume, or the soil surrounding the legume are treated first with
the BAP and subsequently the legume, the seeds of the legume, or
the soil surrounding the legume are treated with the
saccharide.
17. The method of claim 14, wherein the legume, the seeds of the
legume, or the soil surrounding the legume are treated first with
the saccharide and subsequently the legume, the seeds of the
legume, or the soil surrounding the legume are treated with the
BAP.
18. The method of claim 14, comprising administering a composition
comprising the BAP to the soil surrounding the legume at a rate
that delivers 10.sup.3-10.sup.5 CFU per gram soil.
19. A method for improving growth or health of a plant, the method
comprising: (a) treating the plant, seeds of the plant, or soil
surrounding the plant with Bacillus amyloliquefaciens subspecies
plantarum (BAP) and (b) treating the plant, the seeds of the plant,
or the soil surrounding the plant with a saccharide comprising
pectin or a pectin-related saccharide.
20. A method for improving growth or health in an animal, the
method comprising: (a) administering to the animal Bacillus
amyloliquefaciens subspecies plantarum and (b) administering to the
animal a saccharide comprising pectin or a pectin-related
saccharide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part (CIP)
under 35 U.S.C. .sctn. 365(c) of International Application No.
PCT/US2015/053239, filed on Sep. 30, 2015, which international
application claims the benefit of priority under 35 U.S.C. .sctn.
119(e) to U.S. provisional application No. 62/057,667, filed on
Sep. 30, 2014, the content of which applications are incorporated
herein by reference in their entireties.
FIELD
[0002] The presently disclosed subject matter relates to the field
of plant growth-promoting rhizobacteria (PGPR). In particular, the
present subject matter relates to the use of pectin or
pectin-related saccharide to enhance the efficacy of PGPR in regard
to promoting growth and health in plants and animals.
BACKGROUND
[0003] Plant-associated microorganisms have been extensively
examined for their roles in natural and induced suppressiveness of
soilborne diseases. Among the many groups of such organisms are
root-associated bacteria, which generally represent a subset of
soil bacteria. Rhizobacteria are a subset of total rhizosphere
bacteria which have the capacity, upon re-introduction to seeds or
vegetative plant parts (such as potato seed pieces), to colonize
the developing root system in the presence of competing soil
microflora. Root colonization is typically examined by quantifying
bacterial populations on root surfaces; however, some rhizobacteria
can also enter roots and establish at least a limited endophytic
phase. Hence, root colonization may be viewed as a continuum from
the rhizosphere to the rhizoplane to internal tissues of roots.
[0004] Rhizobacteria which exert a beneficial effect on the plant
being colonized are termed "plant-growth promoting rhizobacteria"
or "PGPR." PGPR may benefit the host by causing plant growth
promotion or biological disease control. The same strain of PGPR
may cause both growth promotion and biological control. Among the
soilborne pathogens shown to be negatively affected by PGPR are
Aphanomyces spp., Fusarium oxysporum, Gaeumannomyces graminis,
Phytophthora spp., Pythium spp., Rhizoctonia solani, Sclerotium
rolfsii, Thielaviopsis basicola, and Verticillium spp. In most of
these cases, biological control results from bacterial production
of metabolites which directly inhibit the pathogen, such as
antibiotics, hydrogen cyanide, iron-chelating siderophores, and
cell wall-degrading enzymes. Plant growth promotion by PGPR may
also be an indirect mechanism of biological control, leading to a
reduction in the probability of a plant contracting a disease when
the growth promotion results in shortening the time that a plant is
in a susceptible state, e.g. in the case where PGPR cause enhanced
seedling emergence rate, thereby reducing the susceptible time for
pre-emergence damping-off. An alternative mechanism for biological
control by PGPR is induced systemic resistance. PGPR and uses
thereof are disclosed in the prior art. (See, e.g., U.S. Pat. Nos.
8,445,255; 6,524,998; 5,935,839; 5,640,803; 5,503,652; and
5,503,651; the contents of which are incorporated herein by
reference in their entirety).
[0005] In addition to their observed association in nature with
plants, PGPR also may be utilized as probiotics for animals in
order to improve animal growth or animal health. For example,
Bacillus amyloliquefaciens subsp. plantarum AP193 has been
described as a probiotic for fish. (See U.S. Published Application
No. 2012/0328572).
[0006] In swine, probiotics have been used to have a positive
influence on gut microbiota balance, intestinal epithelium
integrity and maturation of gut-associated tissue. (See
Corcionivoshi et al., Animal Science and Biotechnologies, 2010,
43(1)). In poultry, probiotics have been used to maintain digestive
microbial balance and to reduce potential pathogenic bacteria which
results in improving growth, egg production, and feed conversion.
(See id.). In cattle, probiotics have been used to prevent and
combat digestive disorders such as diarrhea during lactation, to
influence ruminal metabolism of nutrients, which helps maintain
health and improve productive performance. (See id.). In sheep,
probiotics have been used to prevent and combat pathological
conditions that arise from digestive balance. (See id.).
[0007] Therefore, new compositions and methods of use for PGPR in
promoting growth and health in plants and animals are
desirable.
SUMMARY
[0008] Disclosed are compositions and methods that include or
utilize plant growth promoting rhizobacteria (PGPR) for improving
growth and health in plants and animals. The compositions and
methods include or utilize a plant growth promoting rhizobacteria
(PGPR) that expresses a protein associated with pectin metabolism,
and a saccharide comprising pectin or a pectin-related
saccharide.
[0009] The disclosed compositions may include inoculants which
comprise: (a) a plant growth promoting rhizobacteria (PGPR) that
expresses a protein associated with pectin metabolism; and (b) a
saccharide comprising pectin or a pectin-related saccharide.
Suitable PGPR may include Bacillus species such as Bacillus
amyloliquefaciens subspecies plantarum. The pectin or
pectin-related saccharides may include pectin-derived saccharides
such as hydrolyzed pectin, D-galacturonate, D-glucuronate, or
mixtures thereof. Optionally, the pectin or pectin-related
saccharide functions as a carrier for the PGPR and/or the inoculant
includes a carrier other than the pectin or pectin-related
saccharide.
[0010] The disclosed compositions may be used to treat plants,
seeds, and soils in order to improve plant growth or plant health.
The disclosed compositions may be formulated as a plant treatment
composition, a coating for seeds, or a soil amendment
composition.
[0011] The disclosed compositions also may be administered to
animals in order to improve animal growth or animal health. The
disclosed compositions may be formulated as an animal feed, such as
a pelleted animal feed.
[0012] Also disclosed are methods of using pectin or pectin-related
saccharides and PGPR in regard to promoting growth or health in
plants and animals. The disclosed methods for improving plant
growth or plant health may include: (a) treating plants, seeds, or
soil with a plant growth promoting rhizobacteria (PGPR) that
expresses a protein associated with pectin metabolism and (b)
treating the plants, seeds, or soil with a saccharide comprising
pectin or a pectin-related saccharide, where the plants, seeds, or
soil may be treated with the PGPR and the saccharide concurrently
or are treated with the PGPR and saccharide non-currently in either
order.
[0013] The disclosed methods for improving animal growth or animal
health may include (a) administering to an animal a plant growth
promoting rhizobacteria (PGPR) that expresses a protein associated
with pectin metabolism and (b) administering to the animal a
saccharide comprising pectin or a pectin-related saccharide, where
the animals may be administered the PGPR and the saccharide
concurrently or are treated with the PGPR and saccharide
non-currently in either order.
[0014] Also disclosed are methods of using pectin or pectin-related
saccharides and PGPR in regard to promoting and/or increasing
nodulation in nitrogen-fixing plants such as nitrogen-fixing
legumes. The disclosed methods for promoting and/or increasing
nodulation in a legume may include: (a) treating the legume, seeds
of the legume, or soil surrounding the legume with a plant growth
promoting rhizobacteria (PGPR) that expresses a protein associated
with pectin metabolism and (b) treating the legume, seeds of the
legume, or soil surrounding the legume with a saccharide comprising
pectin or a pectin-related saccharide, where the legume, seeds of
the legume, or soil surrounding the legume may be treated with the
PGPR and the saccharide concurrently or the legume, seeds of the
legume, or soil surrounding the legume are treated with the PGPR
and saccharide non-currently in either order.
[0015] Also disclosed are methods of using pectin or pectin-related
saccharides to prepare compositions and inoculants as disclosed
herein. The methods may include combining PGPR and pectin, which
has been extracted from pectin-containing plant material, or
pectin-related saccharides to prepare the disclosed compositions
and inoculants. Optionally, a carrier may be combined with the PGPR
and pectin or pectin-related saccharides to prepare the disclosed
compositions and inoculants.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1. Expression of a pectin lyase activity by PGPR Bap
strain AP193. Note the cleared halo around the growth of the Bap
strain due to pectin degradation.
[0017] FIG. 2. Use of 1% pectin as a sole C source by PGPR strains
AP143 and AP193 in TSS medium. The small increase in OD.sub.600 by
the non-PGPR strain HD73 was due to residual nutrients present from
the previous TSB culture.
[0018] FIG. 3. Effect of PGPR and/or 0.1% (w/w) pectin soil
amendment on soybean root and growth after 4 weeks. By strains
AP143 and AP193 were applied as seed inoculants with 10.sup.6
CFU/seed and plants (n=11/treatment) were grown in a greenhouse.
Treatment groups assigned different letters are significantly
different (P<0.5).
[0019] FIG. 4. A. Representative soybean roots grown in the
presence of absence of 0.1% pectin amended to soil and seed
inoculated with 10.sup.6 CFU By strain AP142. B. Effect of
treatment groups that include 10.sup.6 CFU/seed By strains, with
and without 0.1% pectin, on soybean nodulation (n=11). Treatment
groups assigned different letters are significantly different
(P<0.5).
DETAILED DESCRIPTION
[0020] The disclosed subject matter of the invention may be
described using various terms as described below.
[0021] Unless otherwise specified or indicated by context, the
terms "a", "an", and "the" mean "one or more." For example, "a
sugar" should be interpreted to mean "one or more sugars" unless
otherwise specified or indicated by context.
[0022] As used herein, "about", "approximately," "substantially,"
and "significantly" will be understood by persons of ordinary skill
in the art and will vary to some extent on the context in which
they are used. If there are uses of the term which are not clear to
persons of ordinary skill in the art given the context in which it
is used, "about" and "approximately" will mean plus or minus
.ltoreq.10% of the particular term and "substantially" and
"significantly" will mean plus or minus >10% of the particular
term.
[0023] As used herein, "about", "approximately," "substantially,"
and "significantly" will be understood by persons of ordinary skill
in the art and will vary to some extent on the context in which
they are used. If there are uses of the term which are not clear to
persons of ordinary skill in the art given the context in which it
is used, "about" and "approximately" will mean plus or minus
.ltoreq.10% of the particular term and "substantially" and
"significantly" will mean plus or minus >10% of the particular
term.
[0024] As used herein, the terms "include" and "including" have the
same meaning as the terms "comprise" and "comprising." The terms
"comprise" and "comprising" should be interpreted as being "open"
transitional terms that permit the inclusion of additional
components further to those components recited in the claims. The
terms "consist" and "consisting of" should be interpreted as being
"closed" transitional terms that do not permit the inclusion of
additional components other than the components recited in the
claims. The term "consisting essentially of" should be interpreted
to be partially closed and allowing the inclusion only of
additional components that do not fundamentally alter the nature of
the claimed subject matter.
[0025] The term "plant" as utilized herein should be interpreted
broadly and may include angiosperms and gymnosperms, dicots and
monocots, and trees. Examples of angiosperm dicots may include, but
are not limited to tomato, tobacco, cotton, rapeseed, field beans,
soybeans, peppers, lettuce, peas, alfalfa, clover, cabbage,
broccoli, cauliflower, brussel sprouts), radish, carrot, beets,
eggplant, spinach, cucumber, squash, melons, cantaloupe, and
sunflowers. Example of angiosperm monocots may include, but are not
limited to asparagus, field and sweet com, barley, wheat, rice,
sorghum, onion, pearl millet, rye, oats, and sugar cane. Woody
plants may include, but are not limited to fruit trees, acacia,
alder, aspen, beech, birch, sweet gum, sycamore, poplar, willow,
fir, pine, spruce, larch, cedar, and hemlock.
[0026] The term "plant" may include nitrogen-fixing plants such as
nitrogen-fixing legumes. As is understood in the art, a "legume" is
a plant belonging to the family Facaceae or Leguminosae. Most
legumes have root nodules comprising symbiotic nitrogen-fixing
bacteria. The symbiotic nitrogen-fixing bacteria of root nodules
are capable of taking atmospheric nitrogen (N.sub.2) and reducing
the atmospheric N.sub.2 to ammonia via the reaction:
N.sub.2+8H.sup.++8e.sup.-.fwdarw.2NH.sub.3+H.sub.2. The ammonia
thus produced can be further reduced to ammonium by the following
reaction: NH.sub.3+H.sup.+.fwdarw.NH.sub.4.sup.+. The ammonium thus
produced can be used by the legume as a nitrogen source for growth.
As such, root nodules and the symbiotic nitrogen-fixing bacteria
therewithin are important for plant growth and methods and
compositions that promote and/or increase nodulation are
desirable.
[0027] The term "animal" as utilized herein should be interpreted
broadly and may include mammals and non-mammals. Mammals may
include human and non-human mammals, such as cows, pigs, sheep, and
the like. Non-mammals may include birds (e.g., chickens, turkeys,
ducks, and the like) and fish.
[0028] Non-human animals may include aquatic animals. In
particular, aquatic animals may include farmed fish (e.g. catfish
or tilapia) and crustaceans (e.g., shrimp).
[0029] The present inventors have identified a collection of plant
growth-promoting rhizobacteria (PGPR) that are capable of improving
the growth of plants, and also have disease- and pest-controlling
activity. From an analysis of genome sequences from the
best-performing Bacillus amyloliquefaciens subspecies plantarum
PGPR strains, the inventors identified some genetically encoded
functions that are always present within these Bacillus PGPR
strains and are not present in other Bacillus species that are not
plant-related. In particular, these PGPR strains can use sugars
derived from plant pectin as a carbon and/or energy source. By
supplementing pectin on plant seeds that are inoculated with
Bacillus spores, or by supplementing the amount of pectin available
for Bacillus PGPR strain post-seed germination, this will result in
an enhancement of 1) the Bacillus strain colonization of the plant
rhizosphere and/or 2) better persistence of Bacillus within the
plant rhizosphere and/or 3) better plant growth performance in
response to PGPR strain+pectin administration and/or 4) better
biological control of disease (e.g., bacteria, fungi, viruses) or
pests (e.g., nematodes) as a result of PGPR strain+pectin
administration.
[0030] PGPR
[0031] The term "plant growth promoting rhizobacteria" or "PGPR"
refers to a group of bacteria that colonize plant roots, and in
doing so, promote plant growth and/or reduce disease or damage from
predators. Bacteria that are PGPR may belong to genera including,
but not limited to Actinobacter, Alcaligenes, Bacillus,
Burkholderia, Buttiauxella, Enterobacter, Klebsiella, Kluyvera,
Pseudomonas, Rahnella, Ralstonia, Rhizobium, Serratia,
Stenotrophomonas, Paenibacillus, and Lysinibacillus. The PGPR
utilized in the disclosed methods and composition may be a single
strain, species, or genus of bacteria or may comprise a mixture of
bacterial strains, species, or genera. For example, the PGPR may be
selected from genera including, but not limited to, Actinobacter,
Alcaligenes, Bacillus, Burkholderia, Buttiauxella, Enterobacter,
Klebsiella, Kluyvera, Pseudomonas, Rahnella, Ralstonia, Rhizobium,
Serratia, Stenotrophomonas, Paenibacillus, and Lysinibacillus.
[0032] The genus Bacillus as used herein refers to a genus of
Gram-positive, rod-shaped bacteria which are members of the
division Firmicutes. Under stressful environmental conditions, the
Bacillus bacteria produce oval endospores that can stay dormant for
extended periods. Bacillus bacteria may be characterized and
identified based on the nucleotide sequence of their 16S rRNA or a
fragment thereof (e.g., approximately a 1000 nt, 1100 nt, 1200 nt,
1300 nt, 1400 nt, or 1500 nt fragment of 16S rRNA or rDNA
nucleotide sequence). Bacillus bacteria may include, but are not
limited to B. acidiceler, B. acidicola, B. acidiproducens, B.
aeolius, B. aerius, B. aerophilus, B. agaradhaerens, B.
aidingensis, B. akibai, B. alcalophilus, B. algicola, B.
alkalinitrilicus, B. alkalisediminis, B. alkalitelluris, B.
altitudinis, B. alveayuensis, B. amyloliquefaciens, B. anthracis,
B. aquimaris, B. arsenicus, B. aryabhattai, B. asahii, B.
atrophaeus, B. aurantiacus, B. azotoformans, B. badius, B.
barbaricus, B. bataviensis, B. beijingensis, B. benzoevorans, B.
beveridgei, B. bogoriensis, B. boroniphilus, B. butanolivorans, B.
canaveralius, B. carboniphilus, B. cecembensis, B.
cellulosilyticus, B. cereus, B. chagannorensis, B. chungangensis,
B. cibi, B. circulans, B. clarkii, B. clausii, B. coagulans, B.
coahuilensis, B. cohnii, B. decisifrondis, B. decolorationis, B.
drentensis, B. farraginis, B. fastidiosus, B. firmus, B. flexus, B.
foraminis, B. fordii, B. fortis, B. fumarioli, B. funiculus, B.
galactosidilyticus, B. galliciensis, B. gelatini, B. gibsonii, B.
ginsengi, B. ginsengihumi, B. graminis, B. halmapalus, B.
halochares, B. halodurans, B. hemicellulosilyticus, B.
herbertsteinensis, B. horikoshi, B. horneckiae, B. horti, B. humi,
B. hwajinpoensis, B. idriensis, B. indicus, B. infantis, B.
infernus, B. isabeliae, B. isronensis, B. jeotgali, B. koreensis,
B. korlensis, B. kribbensis, B. krulwichiae, B. lehensis, B.
lentus, B. licheniformis, B. litoralis, B. locisalis, B.
luciferensis, B. luteolus, B. macauensis, B. macyae, B.
mannanilyticus, B. marisflavi, B. marmarensis, B. massiliensis, B.
megaterium, B. methanolicus, B. methylotrophicus, B. mojavensis, B.
muralis, B. murimartini, B. mycoides, B. nanhaiensis, B.
nanhaiisediminis, B. nealsonii, B. neizhouensis, B. niabensis, B.
niacini, B. novalis, B. oceanisediminis, B. odysseyi, B. okhensis,
B. okuhidensis, B. oleronius, B. oshimensis, B. panaciterrae, B.
patagoniensis, B. persepolensis, B. plakortidis, B. pocheonensis,
B. polygoni, B. pseudoalcaliphilus, B. pseudofirmus, B.
pseudomycoides, B. psychrosaccharolyticus, B. pumilus, B.
qingdaonensis, B. rigui, B. runs, B. safensis, B. salarius, B.
saliphilus, B. schlegelii, B. selenatarsenatis, B.
selenitireducens, B. seohaeanensis, B. shackletonii, B. siamensis,
B. simplex, B. siralis, B. smithii, B. soli, B. solisalsi, B.
sonorensis, B. sporothermodurans, B. stratosphericus, B.
subterraneus, B. subtilis, B. taeansis, B. tequilensis, B.
thermantarcticus, B. thermoamylovorans, B. thermocloacae, B.
thermolactis, B. thioparans, B. thuringiensis, B. tripoxylicola, B.
tusciae, B. vallismortis, B. vedderi, B. vietnamensis, B. vireti,
B. wakoensis, B. weihenstephanensis, B. xiaoxiensis, and mixtures
or blends thereof.
[0033] The PGPR and inoculants thereof disclosed herein may include
B. amyloliquefaciens or a Bacillus species that is closely related
to B. amyloliquefaciens. A Bacillus species that is closely related
to B. amyloliquefaciens may be defined as a species having a 16S
rDNA sequence comprising SEQ ID NO:26 or comprising a 16S rDNA
sequence having at least about 98% or 99% sequence identity to SEQ
ID NO:26. The PGPR preferably is B. amyloliquefaciens subspecies
plantarum or a Bacillus species that is closely related to B.
amyloliquefaciens subspecies plantarum. B. amyloliquefaciens
subspecies plantarum is a subspecies of B. amyloliquefaciens which
is colonizes plant roots and typically exhibits amylase activity.
Suitable PGPR strains for the disclosed methods and compositions
may include PGPR strains having a gyrB gene that exhibits sequence
identity to the gyrB gene from strains of Bacillus
amyloliquefaciens subspecies plantarum. In some embodiment, the
PGPR strain utilized in the disclosed methods and compositions has
at gyrB gene having at least about 80%, 90%, 95%, 96%, 97%, 98%,
99% or 100% sequence identity to the polynucleotide sequence of SEQ
ID NO:25, which is the polynucleotide sequence of the gyrB gene
from strains of Bacillus amyloliquefaciens subspecies
plantarum.
[0034] Suitable strains of B. amyloliquefaciens subspecies
plantarum for use in the disclosed compositions and methods may
include but are not limited to Bacillus amyloliquefaciens subsp.
plantarum AS43.3, Bacillus amyloliquefaciens subsp. plantarum
TrigoCor1448, Bacillus amyloliquefaciens subsp. plantarum UCMB5033,
Bacillus amyloliquefaciens subsp. plantarum UCMB5113, Bacillus
amyloliquefaciens subsp. plantarum EBL11, Bacillus
amyloliquefaciens subsp. plantarum W2, Bacillus amyloliquefaciens
subsp. plantarum UCMB5036, Bacillus amyloliquefaciens subsp.
plantarum IT-45, Bacillus amyloliquefaciens subsp. plantarum UASWS
BA1, Bacillus amyloliquefaciens subsp. plantarum LFB 112, Bacillus
amyloliquefaciens subsp. plantarum CAUB946, Bacillus
amyloliquefaciens subsp. plantarum M27, Bacillus amyloliquefaciens
subsp. plantarum B1895, Bacillus amyloliquefaciens subsp. plantarum
SQR9, Bacillus amyloliquefaciens subsp. plantarum AH159-1, Bacillus
amyloliquefaciens subsp. plantarum DC-12, Bacillus
amyloliquefaciens subsp. plantarum YAU B9601-Y2, Bacillus
amyloliquefaciens subsp. plantarum Y2, Bacillus amyloliquefaciens
subsp. plantarum EGD_AQ14, Bacillus amyloliquefaciens subsp.
plantarum NAU-B3, Bacillus amyloliquefaciens subsp. plantarum
FZB42, Bacillus amyloliquefaciens subsp. plantarum CC 178, Bacillus
amyloliquefaciens subsp. plantarum AP79, Bacillus amyloliquefaciens
subsp. plantarum AP71, Bacillus amyloliquefaciens subsp. plantarum
AP143, Bacillus amyloliquefaciens subsp. plantarum AP193, Bacillus
amyloliquefaciens subsp. plantarum AB01, and Bacillus
amyloliquefaciens subsp. plantarum GB03.
[0035] Suitable PGPR strains and inoculants thereof for the methods
and compositions disclosed herein may include PGPR strains that
express one or more proteins associated with pectin metabolism. In
some embodiments, the PGPR strain may express one or more proteins
associated with pectin metabolism, which may include but are not
limited to proteins encoded by a gene selected from the group
consisting of uxaA (altronate dehydratase), uxaB (altronate
oxidoreductase), uxaC (uronate isomerase), uxaA (mannonate
dehydratase, uxuB (D-mannonate oxidoreductase), kdgA
(4-hydroxy-2-oxoglutarate aldolase), kdgK
(2-dehydro-3-deoxygluconokinase), exuR (hexuronate utilization
operon transcriptional repressor), exuT (hexuronate transporter),
and combinations thereof. In some embodiments, the PGPR strain may
express one or more pectinase enzymes selected from a group
consisting of pectin lyase, pectate lyase, polygalacturonase, and
pectin esterase.
[0036] The uxaA gene encodes an enzyme which is an altronate
dehydratase (EC:4.2.1.7) which converts D-altronate to
2-dehydro-3-deoxy-D-gluconate and water. Therefore, suitable PGPR
strains and inoculants thereof for the methods and composition
disclosed herein may include a PGPR strain that expresses altronate
dehydratase. SEQ ID NO:1 provides the polynucleotide sequence
encoding for altronate dehydratase. SEQ ID NO:2 provides the amino
acid sequence for altronate dehydratase.
[0037] The uxaB gene encodes an enzyme which is an altronate
oxidoreductase (EC:5.3.1.12) which converts D-altronate and
NAD.sup.+ to D-tagaturonate and NADH. Therefore, suitable PGPR
strains and inoculants thereof for the methods and composition
disclosed herein may include a PGPR strain that expresses altronate
oxidoreductase. SEQ ID NO:3 provides the polynucleotide sequence
encoding for altronate oxidoreductase. SEQ ID NO:4 provides the
amino acid sequence for altronate oxidoreductase.
[0038] The uxaC gene encodes an enzyme which is an uronate
isomerase (EC:1.3.1.12) which converts D-glucuronate to
D-fructuronate and which converts D-galacturonate to
D-tagaturonate. Therefore, suitable PGPR strains and inoculants
thereof for the methods and composition disclosed herein may
include a PGPR strain that expresses uronate isomerase. SEQ ID NO:5
provides the polynucleotide sequence encoding for altronate
oxidoreductase. SEQ ID NO:6 provides the amino acid sequence for
altronate oxidoreductase.
[0039] The uxuA gene encodes an enzyme which is a mannonate
dehydratase (EC:4.2.1.8) which converts D-mannonate to
2-dehydro-3-deoxy-D-gluconate. Therefore, suitable PGPR strains and
inoculants thereof for the methods and composition disclosed herein
may include a PGPR strain that expresses mannonate dehydratase. SEQ
ID NO:7 provides the polynucleotide sequence encoding for mannonate
dehydratase. SEQ ID NO:8 provides the amino acid sequence for
mannonate dehydratase.
[0040] The uxuB gene encodes an enzyme which is a D-mannonate
oxidoreductase (EC:1.1.1.57) which converts D-mannonate and
NAD.sup.+ to D-fructuronate and NADH. Therefore, suitable PGPR
strains and inoculants thereof for the methods and composition
disclosed herein may include a PGPR strain that expresses
D-mannonate oxidoreductase. SEQ ID NO:9 provides the polynucleotide
sequence encoding for altronate oxidoreductase. SEQ ID NO:10
provides the amino acid sequence for altronate oxidoreductase.
[0041] The kdgA gene encodes an enzyme which is a
4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16) which converts
4-hydroxy-2-oxoglutarate to pyruvate and glyoxylate, and which
converts 2-dehydro-3-deoxy-6-phosphate-D-gluconate to pyruvate and
D-glyceraldehyde 3-phosphate. Therefore, suitable PGPR strains and
inoculants thereof for the methods and composition disclosed herein
may include a PGPR strain that expresses 4-hydroxy-2-oxoglutarate
aldolase. SEQ ID NO:11 provides the polynucleotide sequence
encoding for 4-hydroxy-2-oxoglutarate aldolase. SEQ ID NO:12
provides the amino acid sequence for 4-hydroxy-2-oxoglutarate
aldolase.
[0042] The kdgK gene encodes an enzyme which is
2-dehydro-3-deoxygluconokinase (EC 2.7.1.45) which phosphorylates
2-keto-3-deoxygluconate (KDG) to produce
2-keto-3-deoxy-6-phosphogluconate (KDPG). Therefore, suitable PGPR
strains and inoculants thereof for the methods and composition
disclosed herein may include a PGPR strain that expresses
2-dehydro-3-deoxygluconokinase. SEQ ID NO:13 provides the
polynucleotide sequence encoding for
2-dehydro-3-deoxygluconokinase. SEQ ID NO:14 provides the amino
acid sequence for 2-dehydro-3-deoxygluconokinase.
[0043] The exuR gene encodes a hexuronate utilization operon
transcriptional repressor. Therefore, suitable PGPR strains and
inoculants thereof for the methods and composition disclosed herein
may include a PGPR strain that expresses a hexuronate utilization
operon transcriptional repressor. SEQ ID NO:15 provides the
polynucleotide sequence encoding for a hexuronate utilization
operon transcriptional repressor. SEQ ID NO:16 provides the amino
acid sequence for a hexuronate utilization operon transcriptional
repressor.
[0044] The exuT gene encodes a hexuronate transporter which
exhibits hexuronate transmembrane transporter activity. Therefore,
suitable PGPR strains and inoculants thereof for the methods and
composition disclosed herein may include a PGPR strain that
expresses a hexuronate transporter. SEQ ID NO:17 provides the
polynucleotide sequence encoding for a hexuronate transporter. SEQ
ID NO:18 provides the amino acid sequence for a hexuronate
transporter.
[0045] In some embodiments, the PGPR strain may express one or more
pectinase enzymes selected from a group consisting of pectin lyase
(EC 4.2.2.10), pectate lyase (EC 4.2.2.2), polygalacturonase (EC
3.2.1.15), and pectin esterase (EC 3.1.1.11). SEQ ID NO:19 provides
the polynucleotide sequence encoding for a pectate lyase precursor.
SEQ ID NO:20 provides the amino acid sequence for a pectate lyase
precursor. SEQ ID NO:21 provides the polynucleotide sequence
encoding for a pectin-lyase like protein. SEQ ID NO:22 provides the
amino acid sequence for a pectin-lyase like protein. SEQ ID NO:23
provides the polynucleotide sequence encoding for a pectin lyase.
SEQ ID NO:24 provides the amino acid sequence for a pectin
lyase.
[0046] "Percentage sequence identity" may be determined by aligning
two sequences of equivalent length using the Basic Local Alignment
Search Tool (BLAST) available at the National Center for
Biotechnology Information (NCBI) website (i.e., "b12seq" as
described in Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2
sequences--a new tool for comparing protein and nucleotide
sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein
by reference in its entirety). For example, percentage sequence
identity between SEQ ID NO:1 and another sequence for comparison
may be determined by aligning these two sequences using the online
BLAST software provided at the NCBI website.
[0047] "Percentage sequence identity" between two
deoxyribonucleotide sequences may also be determined using the
Kimura 2-parameter distance model which corrects for multiple hits,
taking into account transitional and transversional substitution
rates, while assuming that the four nucleotide frequencies are the
same and that rates of substitution do not vary among sites (Nei
and Kumar, 2000) as implemented in the MEGA 4 (Tamura K, Dudley J,
Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics
Analysis (MEGA) software version 4.0. Molecular Biology and
Evolution 24:1596-1599), preferably version 4.0.2 or later. The gap
opening and extension penalties are set to 15 and 6.66
respectively. Terminal gaps are not penalized. The delay divergent
sequences switch is set to 30. The transition weight score is 35
set to 0.5, as a balance between a complete mismatch and a matched
pair score. The DNA weight matrix used is the IUB scoring matrix
where x's and n's are matches to any IUB ambiguity symbol, and all
matches score 1.9, and all mismatched score O.
[0048] Pectin and Pectin-Related Saccharides
[0049] The disclosed compositions and methods include or utilize
pectin or pectin-derived sugars in order to sugars to enhance the
efficacy of PGPR in regard to promoting plant growth and plant
health. "Pectin" is a heteropolysaccharide found natively in the
primary cell walls of terrestrial plants having a typical molecular
weight of 60,000-130,000 g/mol, which varies based on the origin of
the pectin and the extraction conditions. As used herein, "pectin"
is meant to include extracted pectin that has been extracted from
its native condition (e.g., extracted pectin from primary cell
walls of terrestrial plants).
[0050] The compositions and methods disclosed herein may comprise
and/or utilize a relatively high molecular weight polysaccharide
such as a relatively high molecular weight pectin. In some
embodiments, the compositions and methods disclosed herein comprise
and/or utilize pectin having an average molecular weight of at
least about 1000, 2000, 5000, 10000, 15000, 20000, 25000, 30000,
35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000,
80000, 85000, 90000, 95000, 100000, 105000, 110000, 115000, 120000,
125000, 130000 g/mol or higher. In some embodiments, the relatively
high molecular weight polysaccharide represents at least about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher of the total
amount of carbohydrate in a composition as contemplated herein.
[0051] The disclosed composition and methods may include and/or
utilize pecting and/or pectin derived sugars in any form. In some
embodiments, the pectin and/or pectin derivated sugars are in
powder form. The powder form may be utilized to prepare a solution
of the pectin and/or pectin derivated sugars. Solutions of pectin
prepared for use in the presently disclosed methods may have a
concentration (w/w) of about 0.0001%, 0.0005%, 0.001%, 0.005%,
0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%, or solutions of
pectin prepared for use in the presently disclosed methods may have
a concentation of pectin within a range bounded by any of these
percentage values (e.g., within a range of 0.01%-1%). A solution
thus prepared may be utilized in the methods disclosed herein,
including methods for treating plants, increasing nodulation in
legumes, and/or treating animals.
[0052] The pectin or pectin-related saccharides utilized in the
disclosed composition and methods may be isolated or substantially
purified. The terms "isolated" or "substantially purified" refers
to pectin or pectin-related saccharides that have been removed from
a natural environment and have been isolated or separated, and are
at least 60% free, preferably at least 75% free, and more
preferably at least 90% free, even more preferably at least 95%
free, and most preferably at least 100% free from other components
with which they were naturally associated, which other components
may include but are not limited to cellulose or other non-pectin
polysaccharides.
[0053] Although the composition of pectin may vary among plants,
pectin typically has a composition in which D-galacturonic acid is
the main monomeric constituent (i.e., typically D-galacturonic acid
represents >50% of the monomeric constituents of pectin). The
D-galacturonic residues of pectin optionally may be substituted
with D-xylose or D-apiose to form xylogalacturonan and
apiogalacturonan, respectively, branching from a D-galacturonic
acid residue. So-called "rhamnogalcturonan pectins" contain a
backbone of repeating disaccharides of D-galacturonic acid and
L-rhamnose. Pectins and pectin-derived products suitable for use in
the presently disclosed compositions and methods may include pectin
in which D-galacturonic acid represents >50% of the monomeric
constituents of the pectin, optionally where one or more of the
D-galacturonic residues of pectin are substituted with D-xylose or
D-apiose to form xylogalacturonan and apiogalacturonan,
respectively, branching from a D-galacturonic acid residue. Pectins
and pectin-derived products suitable for use in the presently
disclosed compositions and methods may include so-called
"rhamnogalcturonan pectins" that contain a backbone of repeating
disaccharides of D-galacturonic acid and L-rhamnose.
[0054] In nature, the majority of carboxyl groups of galacturonic
acid in pectin are esterified with methanol (i.e., >50% and as
much as 80% of the carboxyl groups of galacturonic acid in pectin
are esterified with methanol). During extraction, this percentage
may decrease where extraction may result in hydrolysis of the ester
bond, and extracted pectins may be categorized as high-ester versus
low-ester pectins having <50% of galacturonic acid residues
being esterified. Non-esterified galacturonic acid units can be
either free acids (i.e., carboxyl groups) or salts with sodium,
potassium, or calcium (i.e., galacturonate salts). Pectins and
pectin-derived products suitable for use in the presently disclosed
compositions and methods may include pectins in which the majority
of carboxyl groups of galacturonic acid in pectin are esterified
with methanol (i.e., >50% and as much as 80% of the carboxyl
groups of galacturonic acid in pectin are esterified with
methanol). After extraction, pectins and pectin-derived products
suitable for use in the presently disclosed compositions and
methods may include extracted pectins (e.g., high-ester pectins or
low-ester pectins having <50% of galacturonic acid residues
being esterified).
[0055] In nature, D-galacturonic acid may be synthesized from
D-gluconoric acid derivatives (e.g., from UDP-D-glucuronate via
4-epimerization) and conversely, D-galacturonic acid in pectin may
be metabolized to form D-gluconoric acid derivatives (e.g.,
5-dehydro-4-deoxy-D-glucuronate via oligogalacturonate lysis). As
used herein, pectin-related saccharides include pectin-derived
saccharides such as hydrolyzed pectin, D-galacturonic acid (or
D-galacturonate salts), and D-gluconoric acid (or D-gluconorate
salts), polymers thereof, or combinations thereof.
[0056] The compositions and methods disclosed herein may include or
utilize a saccharide that is a substrate for an enzyme or
transporter encoded by a gene selected from the group consisting of
uxaA (altronate dehydratase), uxaB (altronate oxidoreductase), uxaC
(uronate isomerase), uxuA (mannonate dehydratase), uxuB
(D-mannonate oxidoreductase), kdgA (4-hydroxy-2-oxoglutarate
aldolase), kdgK (2-dehydro-3-deoxygluconokinase), exuR (hexuronate
utilization operon transcriptional repressor), exuT (hexuronate
transporter), and combinations thereof. The compositions and
methods disclosed herein may include or utilize a saccharide that
is a substrate for a pectinase enzyme (e.g., a pectinase enzyme
selected from a group consisting of pectin lyase, pectate lyase,
polygalacturonase, and pectin esterase).
[0057] Substrates as such may include but are not limited to
saccharides derived from pectin such as D-galacturonate and
D-glucuronate. The saccharide may comprise a mixture of sugars or
the saccharide may comprise a heteropolysaccharide. In embodiments
in which the saccharide is a heterogeneous mixture of sugars or the
saccharide is a heteropolysaccharide, preferably D-galacturonate
monomeric units, D-glucuronate monomeric units, or the sum of
D-galacturonate monomeric units and D-glucuronate monomeric units
represent >50%, >60%, >70%, >80%, >90%, or >95%
of total monomeric units in the heterogeneous mixture of sugars or
the heteropolysaccharide.
[0058] The disclosed pectin and pectin-related substances may
include synthetic pectin. Synthetic pectin may include pectin
synthesized by polymerizing pectin monomers (e.g., uronic acid) in
vitro to form pectin-like substance referred to as synthetic
pectin. (See, e.g., U.S. Pat. No. 2,156,223. Furthermore, the
disclosed pectin and pectin-related substances may include
naturally and non-naturally occurring polyuronic acids.
[0059] In the disclosed methods and compositions, pectin may be
present at a desirable concentration, for example, in soil
surrounding a plant, in a seed coating, or in animal feed. When
pectin is administered to soil, the pectin may be administered, for
example, to achieve a concentration in soil (w/w) of about 0.0001%,
0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%,
or 5%, or to achieve a concentration in soil within a concentration
range bounded by any of these percentage values (e.g., within a
range of 0.001%-0.01%). When pectin is present in a seed coating,
the pectin may be present at a concentration (w/w) in the coating
of about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%,
20%, 30%, 40%, 50%, or higher, or the pectin may be present at
concentration (w/w) in the coating within a concentration range
bounded by any two of these values (e.g., within a range of
0.1%-1%). When pectin is present in animal feed, the pectin may be
present at a concentration (w/w) in the animal feed of about
0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10% or higher
or the pectin may be present in the animal feed at within range
concentration bounded by any two of these values (e.g., within a
range of 0.1%-1%).
[0060] Inoculants
[0061] The presently disclosed PGPR may be formulated as an
inoculant for a plant. The term "inoculant" means a preparation
that includes an isolated culture of a PGPR and optionally a
carrier. Inoculants comprising PGPR and carriers are known in the
art. (See, e.g., Bashan, "Inoculants of Plant Growth-Promoting
Bacteria for use in Agriculture," Biotechnology Advances, Vol. 16,
No. 4, pp. 729-770, 1998). PGPR inoculants may be administered to
plants (e.g. to the roots of plants), to seeds (e.g., as a coating
for the seed or at the time that the seed is planted), or to soil
(e.g., to soil surrounding plants to be treated).
[0062] A PGPR inoculant may be described as a formulation
containing one or more PRPR species in a carrier material, which
may be an organic carrier, an inorganic carrier, or a carrier
synthesized from defined molecules. Optionally, the carrier may be
sterile or sterilized prior to be formulated with the PGPR to form
the PGPR inoculant. Preferably, the carrier is nontoxic,
biodegradable and nonpolluting. In the disclosed inoculants
comprising a pectin saccharide, the pectin saccharide optionally
may function as a carrier or optionally the inoculants may comprise
a carrier other than the pectin saccharide.
[0063] The carrier of the PGPR inoculant is the delivery vehicle
for the live PGPR to the plant, seeds, or soil. The carrier
represent is the major portion by volume or weight of the
inoculant. Suitable carriers may include liquids, powders (e.g.,
having an average effective particle diameter of 0.075 to 0.25 mm),
granulars (e.g., having an average effective particle diameter of
0.35 to 1.18 mm), and slurries which have the capacity to deliver a
sufficient number of viable PGPR cells to the plant, seeds, or
soil. Preferably, the carrier extends the shelf-life of the PGPR
(e.g., such that the PGPR has a shelf-life of at least 1 or 2 years
at room temperature). Examples of carriers include but are not
limited to peat, coal, clays, inorganic soil material, plant waste
materials, composts, farmyard manure, soybean meal, soybean oil,
peanut oil, wheat bran, inert materials such as vermiculite,
perlite, phosphate, polyacrylamide, alginate beads, oil-dried
bacteria. In some embodiments, the PGPR may be encapsulated by a
carrier, for example, where the carrier is a carbohydrate that
forms a matrix around the PGPR.
[0064] The PGPR utilized in the disclosed composition and methods
may be isolated or substantially purified. The terms "isolated" or
"substantially purified" refers to PGPR that have been removed from
a natural environment and have been isolated or separated, and are
at least 60% free, preferably at least 75% free, and more
preferably at least 90% free, even more preferably at least 95%
free, and most preferably at least 100% free from other components
with which they were naturally associated. An "isolated culture"
refers to a culture of the PGPR that does not include significant
amounts of other materials such as other materials which normally
are found in soil in which the PGPR grows and/or from which the
PGPR normally may be obtained. An "isolated culture" may be a
culture that does not include any other biological, microorganism,
and/or bacterial species in quantities sufficient to interfere with
the replication of the "isolated culture." Isolated cultures of
PGPR may be combined to prepare a mixed culture of PGPR.
[0065] The inoculant typically includes a suitable amount of PGPR
relative to carrier. In some embodiments, the inoculant includes
10.sup.2-10.sup.12 cfu PGPR per ml carrier (or per gram carrier),
or 10.sup.4-10.sup.10 cfu PGPR per ml carrier (or per gram
carrier), or 10.sup.6-10.sup.8 cfu PGPR per ml carrier (or per gram
carrier). The composition may include additional additives
including buffering agents, surfactants, adjuvants, or coating
agents. Suitable carriers may include, but are not limited to,
water or other aqueous solutions, slurries, solids (e.g., peat,
wheat, bran, vermiculite, and pasteurized soil) or dry powders.
[0066] In the disclosed methods and compositions, PGPR may be
present at a desirable concentration, for example, in soil
surrounding a plant, in a seed coating, or in animal feed. In some
embodiments where PGPR is applied to soil, PGPR may be applied as a
seedling root-dip or as a soil drench at a concentration of about
10.sup.2-10.sup.12 cfu/ml, 10.sup.4-10.sup.10 cfu/ml, or about
10.sup.6-10.sup.8 cfu/ml. In some embodiments where PGPR is present
as a coating on a seed, suitable application concentrations may be
between 10.sup.2-10.sup.8 cfu per seed, preferably
10.sup.4-10.sup.7 cfu per seed. In some embodiments where PGPR is
present in animal feed, the PGPR may be presented at a
concentration of at least about 10.sup.4 CFU/g of feed. More
preferably, the spore-forming strain of the genus Bacillus is
present in the composition at a concentration of at least about
10.sup.5 CFU/g of feed. Even more preferably, the spore-forming
strain of the genus Bacillus is present in the composition at a
concentration of at least about 10.sup.6 CFU/g of feed or per ml of
water or at least about 10.sup.7 CFU/g of feed or per ml of water.
A suitable concentration range may include 10.sup.4-10.sup.7 CFU/g
of feed or per ml of water or sub-ranges there within.
[0067] The disclosed inoculants and compositions may include
additional agents for promoting plant growth and health, including
additional agents for promoting nodulation. The additional agents
may include additional bacterial inoculants, including, but not
limited to, additional rhizobacteria such as a nitrogen-fixing
bacteria and/or a phosphate-solubilizing bacteria. Additional
agents may include fungal inoculants, for example mycorrhizae.
Additional agents may include plant nutrients such as nitrogen
salts and/or phosphate salts and/or potassium salts.
[0068] Methods of Treating Plants, Seeds, or Soil
[0069] Also disclosed are methods of using pectin or pectin-related
saccharides to improve the efficacy of PGPR in regard to promoting
growth or health in plant. The disclosed methods for promoting
growth or health in plant may include, but are not limited to,
methods of increasing nodulation in legumes. The disclosed methods
may include administering the above-described inoculants comprising
a PGPR and a pectin saccharide to plants, seeds, or soil. In some
embodiments, the disclosed methods for improving plant growth or
plant health may include: (a) treating plants, seeds, or soil with
a plant growth promoting rhizobacteria (PGPR) that expresses a
protein associated with pectin metabolism and (b) treating the
plants, seeds, or soil with a saccharide comprising pectin or a
pectin-related saccharide (e.g., hydrolyzed pectin,
D-galacturonate, D-glucuronate, or mixtures thereof), where the
plants, seeds, or soil may be treated with the PGPR and the
saccharide concurrently or in either order (i.e., the PGPR may be
administered before, concurrently with, or after the saccharide is
administered). The PGPR and pectin saccharide may be formulated as
an inoculant and administered concurrently to treat plants (e.g.,
administered to the roots of plants), to seeds (e.g., as a coating
for seeds), or to soil (e.g., as a soil amendment).
[0070] The disclosed methods may be utilized to improve plant
growth or plant health by controlling soil-borne pests. Soil-borne
pests controlled by the disclosed methods may include but are not
limited to nematodes and herbivorous insects. The disclosed methods
may be utilized to improve plant growth or plant health by
controlling or treating a disease. Disease controlled or treated by
the disclosed methods may include but are not limited to a
bacterial disease, a fungal disease, and a viral disease.
[0071] The presently disclosed PGPR and pectin saccharide may be
administered as an inoculant for treating plants. The methods of
treatment contemplated herein may include treating a plant directly
including treating leaves, stems, or roots of the plant directly.
The methods of treatment contemplated herein may include treating
seeds of the plant, e.g., coating the seeds prior to the seeds
being planted to produce a treated plant. The methods contemplated
herein also may include treating a plant indirectly, for example,
by treating soil or the environment surrounding the plant (e.g.,
in-furrow granular or liquid applications). Suitable methods of
treatment may include applying an inoculant including the PGPR and
the saccharide via high or low pressure spraying, drenching, and/or
injection. Plant seeds may be treated by applying low or high
pressure spraying, coating, immersion, and/or injection. After
plant seeds have been thusly treated, the seeds may be planted and
cultivated to produce plants. Plants propagated from such seeds may
be further treated with one or more applications. Suitable
application concentrations may be determined empirically. In some
embodiments where the PGPR and pectin saccharide are applied as a
spray to plants, suitable application concentrations may include
spraying 10.sup.6-10.sup.18 colony forming units (cfu) per hectare
of plants, more commonly 10.sup.7-10.sup.15 cfu per hectare. For
coated seeds, in some embodiments, suitable application
concentrations may be between 10.sup.2-10.sup.8 cfu per seed,
preferably 10.sup.4-10.sup.7 cfu per seed. In other embodiments,
the PGPR and pectin saccharide may be applied as a seedling
root-dip or as a soil drench at a concentration of about
10.sup.2-10.sup.12 cfu/ml, 10.sup.4-10.sup.10 cfu/ml, or about
10.sup.6-10.sup.8 cfu/ml.
[0072] Methods of Treating Animals
[0073] Bacillus species isolates cultured from plant rhizospheres
have the ability to utilize complex plant polysaccharides as a
carbon and energy source. Increasingly, animal feeds are plant
based and many of the plant-derived polysaccharides and other
compounds (e.g. phytic acid) are not readily degraded or utilized
by fish, poultry or livestock. In fact, in many cases these
plant-derived compounds such as phytic acid serve as an
anti-nutrient that can make animals anemic. Using Bacillus or other
species that can degrade complex plant polysaccharides can promote
feed conversion efficiency and these rhizosphere isolates are
ideally suited to help improve animal feeds and degrade phytic acid
to improve animal nutrition.
[0074] As such, also disclosed are methods of using pectin or
pectin-related saccharides to improve the efficacy of PGPR in
regard to promoting growth or health in animals. The disclosed
methods may include administering the afore-described inoculants
comprising a PGPR and a pectin saccharide to animals (e.g., in the
form of an animal feed composition such as a pelleted feed
composition comprising the afore-described inoculants). In some
embodiments, the disclosed methods for improving animal growth or
animal health may include: (a) administering to an animal a plant
growth promoting rhizobacteria (PGPR) that expresses a protein
associated with pectin metabolism and (b) administering to the
animal a pectin saccharide comprising pectin or a pectin-related
saccharides (e.g., hydrolyzed pectin, D-galacturonate,
D-glucuronate, or mixtures thereof), where the animals may be
administered the PGPR and the pectin saccharide concurrently or in
either order (i.e., the PGPR may be administered before,
concurrently with, or after the saccharide is administered).
[0075] Feed compositions comprising the PGPR and pectin saccharide
may be administered to animals orally. Oral administration
includes, but is not limited to, delivery in feed, water, by oral
gavage or aerosol spray. If supplied in an animal feed, the feed
may comprise between 10.sup.4 and 10.sup.9 cfu PGPR/gm of finished
feed. Suitably the feed comprises between 10.sup.5 and
5.times.10.sup.7 cfu PGPR/gm feed. The PGPR and pectin saccharide
may be added to the feed during production, after production by the
supplier, or by the person feeding the animals, just prior to
providing the food to the animals.
[0076] An animal feed composition may be prepared by forming a
mixture of the animal feed and an inoculant as discussed above, and
then optionally forming a compressed or pelleted animal feed from
the mixture. Animal feed suitable for preparing animal feed
compositions as disclosed herein may include animal feed comprising
plant material (e.g., hay straw, silage, grains (e.g., maize,
soybean, wheat, oats, barley, sorghum, and rice), and legumes).
Animal feed suitable for preraing animal feed compositions as
disclosed herein amy include fish products (e.g., fish oils and
fish proteins).
[0077] The disclosed methods for promoting growth or health in
animals may be practiced in order to increase overall
gastrointestinal health, improve production performance, and reduce
enteric bacterial pathogens of importance to both animal health and
human food safety. These PGPR and pectin saccharide may be added to
animal diets at the rate of about 10.sup.4 to 10.sup.9 PGPR per
gram of finished feed for optimal inclusion rate, if the bacteria
or probiotic compositions being administered continuously, and a
higher inclusion rate may be necessary if the PGPR or the
compositions are provided intermittently. While administration
though the feed is a preferred route of administration, the PGPR
and pectin saccharide may also be administered via drinking water,
through course spray, through aerosol spray, or through any other
means by which the agricultural animals may ingest these PGPR and
pectin saccharide.
[0078] The disclosed methods may include methods of promoting
growth or health in aquatic animals, which may include farmed fish
(e.g. catfish or tilapia) and crustaceans (e.g., shrimp). The term
"catfish" refers to a fish belonging to the genus Ictaluri. Catfish
may include the species Ictaluri punctatus Rafinesque.
[0079] Methods for Preparing the Disclosed Compositions and
Inoculants
[0080] Also disclosed are methods of using pectin or pectin-related
saccharides to prepare compositions and inoculants as disclosed
herein. The methods may include combining PGPR and pectin, which
has been extracted from pectin-containing plant material, or
pectin-related saccharides to prepare the disclosed compositions
and inoculants. Optionally, a carrier may be combined with the PGPR
and pectin or pectin-related saccharides to prepare the disclosed
compositions and inoculants.
[0081] In some embodiments, the methods may include combining
10.sup.2-10.sup.12 cfu PGPR per ml carrier (or per gram carrier),
or 10.sup.4-10.sup.10 cfu PGPR per ml carrier (or per gram
carrier), or 10.sup.6-10.sup.8 cfu PGPR per ml carrier (or per gram
carrier). In some embodiments, the methods may include combining
pectin, which has been extracted from pectin-containing plant
material, or pectin-related saccharides may be combined with PGPR
and optionally a carrier to prepare the disclosed compositions and
inoculants, wherein the pectin or pectin-related saccharides are
present in the prepared compositions and inoculants at a
concentration of at least about 0.1%, 0.5%, 1.0%, 1.5%, or 2.0%
(w/w or w/v) to about 0.5%, 1.0%, 1.5%, 2.0%, or 5.0% (w/w or w/v).
In some embodiments, the methods may include combining PGPR and
pectin at a concentration of about at least about 10.sup.2,
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9, 10.sup.10 , 10.sup.11, 10.sup.12, 10.sup.13, or 10.sup.14
cfu PGPR per gram pectin or pectin-related saccharides, to about
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14,
10.sup.15 cfu PGPR per gram pectin or pectin-related saccharides
(e.g., ranges such as 10.sup.7 to 10.sup.12 cfu PGPR per gram
pectin or pectin-related saccharides are contemplated herein). In
the methods, additional additives including buffering agents,
surfactants, adjuvants, and coating agents may be combined with the
PGPR, pectin or pectin-related saccharides, and optional carrier in
order to prepare the disclosed compositions and inoculants.
Compositions and inoculants prepared by the afore-disclosed methods
also are contemplated herein.
EXAMPLES
[0082] The following Examples are illustrative and are not intended
to limit the scope of the claimed subject matter.
Example 1--Deciphering the Conserved Genetic Loci Implicated in
Plant Disease through Comparative Genomics of Bacillus
amyloliquefaciens subsp. plantarum Strains
[0083] Reference is made to Hossain et al., "Deciphering the
conserved genetic loci implicated in plant disease through
comparative genomics of Bacillus amyloliquefaciens subsp. plantarum
strains," Frontiers in Plant Science, 2015 Aug. 17; 6:631 doi:
10.3389/fpls.2015.00631. eCollection 2015, (hereinafter referred to
as "Hossain et al., Frontiers Plant Science 2015), the content of
which is incorporated herein by reference in its entirety.
[0084] Abstract
[0085] To understand the growth-promoting and disease-inhibiting
activities of plant growth-promoting rhizobacteria (PGPR) strains,
the genomes of 12 Bacillus subtilis group strains with PGPR
activity were sequenced and analyzed. These B. subtilis strains
exhibited high genomic diversity, whereas the genomes of B.
amyloliquefaciens strains (a member of the B. subtilis group) are
highly conserved. A pairwise BLASTp matrix revealed that gene
family similarity among Bacillus genomes ranges from 32-90%, with
2,839 genes within the core genome of B. amyloliquefaciens subsp.
plantarum. Comparative genomic analyses of B. amyloliquefaciens
strains identified genes that are linked with biological control
and colonization of roots and/or leaves, including 73 genes
uniquely associated with subsp. plantarum strains that have
predicted functions related to signaling, transportation, secondary
metabolite production, and carbon source utilization. Although B.
amyloliquefaciens subsp. plantarum strains contain gene clusters
that encode many different secondary metabolites, only polyketide
biosynthetic clusters that encode difficidin and macrolactin are
conserved within this subspecies. To evaluate their role in plant
pathogen biocontrol, genes involved in secondary metabolite
biosynthesis were deleted in B. amyloliquefaciens subsp. plantarum
strain, revealing that difficidin expression is critical in
reducing the severity of disease, caused by Xanthomonas axonopodis
pv. vesicatoria in tomato plants. This Example defines genomic
features of PGPR strains and links them with biocontrol activity
and with host colonization.
[0086] Introduction
[0087] Bacteria associated with plant roots that exert beneficial
effects on plant growth and development are referred to as plant
growth-promoting rhizobacteria (PGPR) (Kloepper and Schroth, 1978;
Kloepper et al., 2004). Bacillus and Pseudomonas spp. are
predominant among the diverse bacterial genera that have been
linked with PGPR activity (Podile and Kishore, 2006). Members of
the B. subtilis group, including B. subtilis, B. licheniformis, B.
pumilus, B. amyloliquefaciens, B. atrophaeus, B. mojavensis, B.
vallismortis, B. sonorensis, and B. tequilensis have been
identified as PGPR strains for their capacity to stimulate plant
growth and suppress pathogens within rhizosphere and phyllosphere
(Kloepper et al., 2004; Hao et al., 2012; Kim et al., 2012).
Strains of B. amyloliquefaciens are widely used for their positive
effects on plant growth (Idriss et al., 2002). Reva et al. (Reva et
al., 2004) reported that seven Bacillus isolates from plants or
soil are closely related yet distinct from B. amyloliquefaciens
type strain DSM7.sup.T. In addition, these strains are more
proficient for rhizosphere colonization than other members of the
B. subtilis group. GB03 (Nakkeeran et al., 2005), INR7
(Kokalis-Burelle et al., 2002) and FZB42 (Chen et al., 2007a) are
PGPR strains within the Bacillus subtilis group that have been
widely used in different commercial formulations to promote plant
growth.
[0088] In addition to promoting plant growth, PGPR strains may
exhibit biological control of plant diseases. Antibiosis, through
the production of inhibitory bioactive compounds, and induced
systemic resistance are widely reported biological control
mechanisms of Bacillus spp. PGPR strains (Ryu et al., 2004). PGPR
Bacillus spp. strains produce diverse antimicrobial compounds
including antibiotics (Emmert et al., 2004), volatile organic
compounds (VOCs) (Yuan et al., 2012), and lipopeptides (Ongena et
al., 2007) that are associated with the observed biocontrol
activity against plant pathogens. For example, B. amyloliquefaciens
NJN-6 produces 11 VOCs that provide antifungal activity against
Fusarium oxysporum f. sp. cubense (Yuan et al., 2012). Similarly,
B. subtils strains produce lipopeptides (e.g. surfactin and
fengycin), that induce systemic resistance in bean plants (Ongena
et al., 2007). PGPR strains usually need to colonize plant roots
extensively to exert plant growth promoting effects using both
direct and indirect mechanisms (Lugtenberg and Kamilova, 2009),
extensive root colonization is not required for induced systemic
resistance (ISR) (Kamilova et al., 2005). In some PGPR strains,
root colonization is a prerequisite for biocontrol activity through
antibiosis (Chin et al., 2000). For example, B. amyloliquefaciens
subsp. plantarum FZB42 exerts growth promoting activities through
efficient colonization of plant roots (Fan et al., 2011).
Previously, it has been demonstrated that over-expression of genes
involved in phosphorylation of DegU, a two-component response
regulator of B. amyloliquefaciens strain SQR9, positively
influences root colonization as well as other growth-promoting
activities by PGPR strains for controlling cucumber wilt disease
(Xu et al., 2014). Moreover, the root colonization capacity of a
poor root colonizer can be improved by cloning genes that are
required for efficient root colonization (Dekkers et al., 2000).
Competitive root colonization by PGPR are controlled by many genes
and/or genetic cluster(s) (Dietel et al., 2013), so identification
of these genetic loci involved in competitive root colonization are
challenging if genome sequences are lacking for those PGPR strains
(Lugtenberg and Kamilova, 2009). Analysis of additional PGPR
strains will help elucidate the mechanisms of competitive root
colonization, antibiosis and ISR of PGPR strains and form a
foundation for genetic engineering and other strategies to increase
the plant-growth promoting capacity of these bacteria.
[0089] In this Example, we sequenced the genomes of 12 Bacillus
subtilis group isolates from diverse locales. Comparative genomic
analyses of PGPR strains and control strains of the B. subtilis
group without any reported biocontrol activity against plant
pathogens provides insight into genomic features involved in PGPR
activity. PGPR strain AP193, which inhibits growth of plant and
animal bacterial pathogens (Ran et al., 2012), is an ideal
candidate to evaluate the relative contribution of genes that are
predicted to be involved in the biosynthesis of bioactive secondary
metabolites that could contribute to biocontrol activity,
specifically difficidin (dfnD mutant), surfactin (srfAA mutant), as
well as all polyketides and lipopeptides produced by non-ribosomal
peptide synthesis, including difficidin (sfp mutant). Mutants were
then tested for their ability to inhibit plant pathogens in vitro
and control bacterial spot disease in tomato.
[0090] Materials and Methods
[0091] Bacterial strains, plasmids and growth conditions. Bacterial
strains and plasmids used in this Example are listed in Table 1. E.
coli and Bacillus strains were grown in Luria-Bertani (LB) medium;
however, for electrocompetent cell preparation, Bacillus
amyloliquefaciens subsp. plantarum AP193 was grown in NCM medium
(17.4 g K.sub.2HPO.sub.4, 11.6 g NaCl, 5 g glucose, 5 g tryptone, 1
g yeast extract, 0.3 g trisodium citrate, 0.05 g
MgSO.sub.4.7H.sub.2O and 91.1 g sorbitol in 1 L deionized water, pH
7.2). For production of secondary metabolites, Bacillus cultures
were grown for 48 h at 30.degree. C. in Tryptic Soy broth (TSB). In
addition, ampicillin (100 .mu.g/ml), chloramphenicol (12.5
.mu.g/ml) or erythromycin (200 .mu.g/ml for E. coli or 5 .mu.g/ml
for Bacillus) were used as selective agents in growth media as
required.
[0092] Sequencing, assembly and annotation. Next-generation
sequencing of Bacillus spp. genomes was performed using Illumina
and Roche 454 sequencing platforms. Indexed Illumina libraries were
prepared for strains AP71, AP79, and ABO1 using Nextera DNA Sample
Prep Kit (Epicentre, Madison, Wis.) and sequences were generated
using an Illumina MiSeq with a 2.times.250 paired end sequencing
kit. Barcoded Illumina libraries for strains AP143, AP193, and
AP254 were constructed using a NxSeq.RTM. DNA Sample Prep Kit
(Lucigen, Middleton, Wis.) and sequenced at EnGenCore (Univ. of
South Carolina) using the 454-pyrosequencing platform. Genomic DNA
library construction and sequencing for Bacillus subtilis GB03,
Bacillus pumilus INR7, B. mojavensis KCTC 3706T, B. tequilensis
KCTC 13622T, Bacillus siamensis KCTC 13613T, and B. sonorensis KCTC
13918T were conducted at the National Instrument Center for
Environmental Management (Seoul, Republic of Korea), using the
Illumina HiSeq 2000 sequencing platform. Sequence reads were
trimmed for quality then assembled de novo using the CLC Genomics
Workbench (CLCBio, Cambridge, Mass.). Gene prediction and
annotation were performed using GeneMark (Lukashin and Borodovsky,
1998) and the RAST annotation server (Aziz et al., 2008),
respectively. The identity of individual open reading frames (ORFs)
from secondary metabolite biosynthesis gene clusters was confirmed
by BLASTx against the GenBank database. Genome sequence reads for
strains AB01, AP71, AP79, AP143, AP193, AP254, GB03 (Choi et al.,
2014), INR7 (Jeong et al., 2014), KCTC 3706T, KCTC 13613T (Jeong et
al., 2012), KCTC 13918T, and KCTC 13622T were deposited into the
Short Read Archive (SRA) at NCBI under the accession numbers
SRR1176001, SRR1176002, SRR1176003, SRR1176004, SRR1176085, and
SRR1176086, SRR1034787, SRR1141652, SRR1141654, SRR1144835,
SRR1144836, and SRR1144837, respectively.
[0093] Determination of average nucleotide identity. Average
nucleotide identities (ANI) between genomes were calculated using
an ANI calculator that estimates ANI according to the methods
described previously (Goris et al., 2007).
[0094] Phylogenetic analysis of Bacillus species. For phylogenetic
analysis, the gyrB gene sequence for each strain was retrieved from
sequence data. (See Hossain et al., Frontiers Plant Science 2015,
FIG. 1). Strains AS43.3, FZB42, YAU B9601-Y2, CAU B946, and 5B6
were used as representative strains of B. amyloliquefaciens subsp.
plantarum; strains DSM7, LL3 and TA208 were used as representative
strains of B. amyloliquefaciens subsp. amyloliquefaciens. The gyrB
phylogenetic tree was inferred with MEGA5.05 (Tamura et al., 2011)
using Neighbor-Joining (Saitou and Nei, 1987) and Maximum
Likelihood (ML) methods (Felsenstein, 1981). All positions that
contained gaps or missing data were eliminated from the final
dataset, resulting in 1911 bp positions of gyrB sequence. We used
729,383 bp of DNA to represent the conserved core genome found
across 25 strains of the B. subtilis group, to generate a
phylogenomic tree using RAxML (v 7.2.7) (Pfeiffer and Stamatakis,
2010). The phylogenomic tree was then visualized with iTOL
(http://itol.embl.de) (Letunic and Bork, 2011).
[0095] BLAST matrix. The BLAST matrix algorithm was used for
pairwise comparison of Bacillus PGPR strain proteomes, using
methods described previously (Friis et al., 2010). The BLAST matrix
determines the average percent similarity between proteomes by
measuring the ratio of conserved gene families shared between
strains to the total number of gene families within each strain.
The absolute number of shared and combined gene families for each
strain was displayed in matrix output. This matrix shows the number
of proteins shared between each proteome.
[0096] Core-genome analysis. The core-genome of 13 Bacillus spp.
strains was generated using coding and non-coding sequences. Whole
genome sequences from these strains were aligned using progressive
Mauve (Darling et al., 2004), which identifies and aligns locally
collinear blocks (LCBs) in the XMFA format. LCBs from alignments
were collected using stripSubsetLCB s
(http://gel.ahab.wise.edu/mauve/snapshots/), using minimum lengths
of 500bp. All LCBs were concatenated and converted to multifasta
format using a perl script. The same protocol was used to obtain
all core sequences, with the exception that the minimum lengths of
LCBs were 50 bp, instead of 500 bp. The Bacillus spp. core genome
was obtained from the comparative alignment of all complete
Bacillus spp. genomes available in the GenBank as of August 2014
(n=81 genomes). The core genome of the B. subtilis group was
obtained from comparative analysis of 53 whole genomes of B.
subtilis strains that included 41 genomes obtained from GenBank and
12 PGPR genomes sequenced in this Example. B. amyloliquefaciens
species-level and B. amyloliquefaciens subsp. plantarum-level core
genomes were generated from 32 B. amyloliquefaciens and 28 subsp.
plantarum genomes. Core genomes were exported to the CLC Genomics
Workbench (v 4.9) for evaluation of alignments and annotation using
the RAST server (Aziz et al., 2008). The list of Bacillus spp.
strains used for core genome determination is provided in Table ?.
Additionally, to identify GPR-specific core genes, raw sequence
reads of PGPR strains sequenced in this Example were sequentially
reference mapped against the genome sequence of non-PGPR strain B.
subtilis subsp. subtilis str. 168 according to methods described
previously (Hossain et al., 2013).
[0097] Identification of core genes uniquely present in B.
amyloliquefaciens subsp. plantarum strains. The aligned genome
sequences of 32 B. amyloliquefaciens strains and 28 B.
amyloliquefaciens subsp. plantarum strains (which were included
within the B. amyloliquefaciens strains) were analyzed using CLC
Genomics Workbench to obtain the respective species- and
subsp.-level core genomes. Trimmed sequence reads of subsp.
plantarum strain AP193 were reference mapped against the subsp.
plantarum core genome to obtain core genome-specific sequence
reads. The parameters of reference mapping were as follows:
mismatch cost=2, insertion cost=3, deletion cost=3, length
fraction=0.5, and similarity=0.8. Sequence reads mapped to the
subsp. plantarum core genome were then mapped against the species
amyloliquefaciens core genome to obtain unmapped sequence reads.
These unmapped sequence reads, represent the subsp. plantarum core
genome that is absent in the amyloliquefaciens species-level core
genome, were assembled de novo using CLC Genomics Workbench then
the resulting contigs were uploaded to RAST for gene prediction and
annotation. Each ORF, exclusively encoded by the plantarum core
genome, was further confirmed for uniqueness using BLASTn analysis
against the genome sequences of 28 B. amyloliquefaciens subsp.
plantarum and four B. amyloliquefaciens subsp. amyloliquefaciens
strains listed in Supplementary Table 1.
[0098] Prediction of secondary metabolite biosynthesis gene
clusters in PGPR strain AP193. Secondary metabolite biosynthesis
gene clusters for strain AP193 were predicted using the secondary
metabolite identification tool antiSMASH (Blin et al., 2013).
Primer-walking PCR was used to fill gaps between contigs containing
gene clusters encoding secondary metabolite biosynthesis. Gene
prediction and annotation were carried out by GeneMark (Lukashin
and Borodovsky, 1998) and BLASTx (NCBI), respectively.
[0099] DNA manipulation and plasmid construction for PGPR strain
AP193 mutagenesis. Chromosomal DNA was isolated with the E.Z.N.A.
Bacterial DNA Isolation Kit (Omega Biotek, Atlanta, Ga.) and
plasmids were isolated with the E.Z.N.A. Plasmids Mini Kit II
(Omega Biotek). Primers used in this Example are listed in Table 2.
Gene deletion constructs were assembled using splicing through
overlap extension PCR (Horton et al., 1989). The assembled products
were gel purified with Gel/PCR DNA Fragments Extraction Kit (IBI),
digested with appropriate restriction enzymes, and cloned into a
pNZT1 vector to construct the delivery plasmids for gene
replacement.
[0100] In vitro plasmid methylation using cell free extract of
Bacillus amyloliquefaciens subsp. plantarum AP193. To methylate
plasmids prior to transformation into B. amyloliquefaciens subsp.
plantarum AP193, the method developed for Lactobacillus plantarum
was used with minor modifications (Alegre et al., 2004). Cells from
a 100 ml overnight culture of strain AP193 (OD.sub.600=1.3-1.5)
were pelleted by centrifugation (8000 xg), washed with 100 ml of
chilled PENP buffer (10 mM potassium phosphate, 10 mM EDTA, 50 mM
NaCl and 0.2 mM PMSF, pH 7.0), and then re-suspended to a final
volume of 4 ml. Cells were disrupted by performing two bursts
(amplitude 50, pulse 3 and watts 25-30) for 5 min each with a pause
of 2 min, using a Vibra-Cell sonicator, and cooled with ice to
prevent overheating. Cell debris was removed by centrifugation
(8000 x g) at 4.degree. C. and the extract was collected through
decanting. Three ml aliquots of extract were mixed with 3 ml of
glycerol (100% v/v) and 0.6 ml of bovine serum albumin (1 mg/ml),
then stored at -20.degree. C.
[0101] The DNA modification assay was performed in a final volume
of 100 .mu.l of the following: 53 .mu.l TNE buffer [50 mM Tris (pH
7.5), 50 mM NaCl, 10 mM EDTA], 10 .mu.l S-adenosylmethionine (0.8
mM), 2 .mu.l BSA (5 mg/ml), 25 .mu.l cell free extract derived from
strain AP193 and 10 .mu.l plasmid DNA extracted from E. coli K12
ER2925 (0.5-1 .mu.g/.mu.l). The mixture was incubated at 37.degree.
C. for 16 h. Methylated DNA was extracted with a DNA Clean &
Concentrator Kit (Zymo Research, Calif.), then re-suspended in
water and stored at -20.degree. C.
[0102] Electrotransformation of B. amyloliquefaciens subsp.
plantarum AP193. For preparation of electrocompetent cells, strain
AP193 was grown overnight in TSB, then diluted 100-fold in NCM to
inoculate a subculture. The culture was grown at 37.degree. C. on a
rotary shaker until the OD.sub.600 reached 0.7. The cell culture
was cooled on ice for 15 min and subjected to centrifugation at
8000.times.g for 5 min at 4.degree. C. After washing four times
with ice cold ETM buffer (0.5 M sorbitol, 0.5 M mannitol, and 10%
glycerol), electrocompetent cells were re-suspended in 1/100 volume
of the original culture (Zhang et al., 2011). For electroporation,
100 .mu.l of cells were mixed with 100 ng of plasmid DNA in an
ice-cold electroporation cuvette (1 mm electrode gap). Cells were
exposed to a single 21 kV/cm pulse generated by Gene-Pulser
(Bio-Rad Laboratories) with the resistance and capacitance set as
200.OMEGA. and 3 .mu.F, respectively. The cells were immediately
diluted into 1 ml of recovery medium (NCM plus 0.38M mannitol)
(Zhang et al., 2011) and shaken gently at 30.degree. C. or
37.degree. C. for 3 h to allow expression of the antibiotic
resistance genes. Aliquots of the recovery culture were then spread
onto LB agar supplemented with appropriate antibiotics.
[0103] Two-step replacement recombination procedure for the
modification of the strain AP193 genome. A two-step replacement
recombinationwas performed as previously described, with minor
modifications (Zakataeva et al., 2010). To integrate the plasmid
into AP193's chromosome, a single crossover between the target gene
and the homologous sequence on the plasmid must occur. To do this,
AP193 that contained a delivery plasmid with thedeletion construct
was first grown in LB broth for 24 h at 37.degree. C. (a
non-permissive temperature for plasmid replication). Next, the
culture was serially diluted, plated onto LB agar plates with
erythromycin, and incubated at 37.degree. C. Clones were screened
by colony PCR using two sets of primers. Each set of primers
anneals sequences specific to one of the homologous fragments and
to the chromosomal region just outside of the other homologous
fragment. If PCR products had a reduced size, relative to the
wild-type genotype for either primer set, this indicated successful
chromosomal integration of the plasmid. In the second step, clones
of the integrant were cultured with aeration in LB at 30.degree. C.
for 24-48 h to initiate the second single-crossover event,
resulting in excision of the plasmid, yielding erythromycin
sensitive (EmS) clones with either a parental or a mutant allele on
the chromosome. Colony PCR was used to examine the presence of
desired mutations by primer sets that flank the deleted
sequence.
[0104] Construction of strain AP193 mutants defective in secondary
metabolite biosynthesis. All mutant strains generated in this
Example are indicated in Table 1. The disruption of the dfnD gene
was achieved as follows: DNA fragments corresponding to positions
-867 to +247 and +643 to +1570 with respect to the dfnD translation
initiation site were PCR amplified using AP193 genomic DNA as a
template. The two fragments were then assembled by fusion PCR. A
frameshift mutation was introduced during fusion to ensure complete
disruption of the gene. The deletion construct was digested with
Xhol and Spel, then cloned into pNZT1, yielding pNZ-dif. The
plasmid was methylated in vitro as described above and introduced
into strain AP193 by electroporation. Once introduced into strain
AP193, plasmid pNZ-dif generated the isogenic mutant
AP193.DELTA.dfnD by two-step replacement recombination.
[0105] To generate the sfp deletion mutant, DNA fragments
corresponding to positions -781 to +29, with respect to the sfp
translation initiation site, and +95 to +935, with respect to the
sfp translation termination site, were PCR amplified using AP193
genomic DNA as template, assembled by fusion PCR, digested with
HindIII and Pstl, and cloned into pNZT1 to construct pNZ-sfp. The
plasmid pNZ-sfp was used to generate mutant AP193.DELTA.sfp using
procedures described above.
[0106] The .DELTA.srfAA mutant was obtained as follows: DNA
fragments corresponding to positions +5375 to +6091 and +6627 to
+7366, with respect to the srfAA translation initiation site, were
PCR-amplified, fused by fusion PCR, digested with HindIII and Pstl
and cloned into pNZT1 as pNZ-srf. Similarly, a frameshift mutation
was introduced during the fusion of the upstream and downstream
fragments of the target deletion sequence to ensure complete
disruption of the gene. The plasmid pNZ-srf was used to generate
mutant AP193.DELTA.srfAA using procedures described above.
[0107] In vitro antimicrobial activities of PGPR strain AP193 and
its mutants against plant pathogens. Plant pathogens Pseudomonas
syringe pv. tabaci, Rhizobium radiobacter, Xanthomonas axonopodis
pv. vesicatoria, and Xanthomonas axonopodis pv. campestris were
grown in TSB until the OD.sub.600 reached 1.0. The wild type strain
AP193, as well as the three isogenic mutants .DELTA.dfnD,
.DELTA.sfp, and .DELTA.srfAA developed in this Example, were grown
at 30.degree. C. in TSB for 48 h at 220 rpm. Cultures were then
centrifuged at 10,000.times.g for 2 min then supernatant was passed
through a 0.2 .mu.m nylon filter (VWR, PA). For antibiosis assays,
100 .mu.l of an overnight culture for each plant pathogen was
spread onto TSA plates (Thermo Scientific, N.Y.) separately then
sterile cork borers (10 mm diameter) were used to bore wells in
agar plates. Filtered supernatant of AP193 and its three mutants
were separately added to fill wells. Plates were allowed to dry and
then incubated at 30.degree. C. overnight. Zones of inhibition were
measured and compared between mutants and wild-type strain AP193 to
determine their antimicrobial activities against plant
pathogens.
[0108] LC-MS analysis of bacterial supernatants. Bacterial cultures
were grown in 2 ml TSB for 72 hours and then cells were removed by
centrifugation at 10,000 x g for 10 min, followed by 0.2 .mu.m
filtration of the culture supernatant. Samples were analyzed by
direct injection from m/z 50-1200 on a ultra-high pressure liquid
chromatography/QTof-mass spectrometer (Waters Acquity UPLC and
Q-Tof Premier, Milford, MA) operated at a spray voltage of 3.03 kv
and the source temperature of 100.degree. C. The MS analysis was
conducted in negative ion mode with a mobile phase of 95%
acetonitrile, 5% water and 0.1% formic acid.
[0109] In vivo antibiosis of strain AP193 and its mutants against a
plant pathogen. Rutgers tomato seeds (Park Seed, USA) were sown in
Styrofoam trays. Three weeks after planting, seedlings were
transplanted into a 4.5 inch square pot with commercial potting
substrate (Sunshine mix, Sun Gro Horticulture, Agawam, Maine).
Three days after transplanting, plants were sprayed with sterile
water or PGPR cell suspensions (10.sup.6 CFU/ml) that had been
washed three times prior to being resuspended in sterile water and
normalized at an OD.sub.600=1.0 before being serially diluted.
PGPR-inoculated plants were placed into a dew chamber at 100%
humidity in the dark for two days at 24.degree. C. then transferred
to the greenhouse. One day later, plants were challenge-inoculated
with X. axonopodis pv. vesicatoria by spraying approximately 10 ml
of a 10.sup.7 CFU/ml pathogen suspension over each plant.
Pathogen-inoculated plants were placed in the dew chamber for two
days then placed in the greenhouse. Plants were watered once daily.
Disease severity ratings and harvest were conducted after 14 days
of challenge-inoculation. For disease severity rating, four
compound leafs were selected from the bottom of each plant. The
disease severity of each of the compound leaves was determined by
rating the disease severity of each leaflet and calculating the
average rating for the compound leaf. Leaflets were rated using a
0-4 rating scale, where 0=healthy leaflet, 1=<20% necrotic area
of the leaflet, 2=20-50% necrotic area of the leaflet, 3=51-80%
necrotic area of the leaflet, 4=80-100% necrotic area of the
leaflet. In addition, dry shoot and root weights were determined.
The experimental design was a randomized complete block with ten
replications per treatment. The experiment was conducted twice.
[0110] Data analysis. All data were analyzed by an analysis of
variance (ANOVA), and the treatment means were separated by using
Fisher's protected least significant difference (LSD) test at
P=0.05 using SAS 9.3 (SAS Institute, Gary, N.C., USA).
[0111] Results
[0112] Genome Statistics and genetic relatedness of Bacillus
species. Genome sequences of 12 different PGPR Bacillus spp.
strains were determined using next-generation sequencing. The
summary statistics for each Bacillus spp. genome sequences and
their assemblies are presented in Table 2. The approximate sizes of
Bacillus spp. genomes ranged from 2.95-4.43 Mbp with an average
genome size of 3.93 Mbp, which is similar to the 4.09 Mbp average
genome size of complete B. subtilis genomes available in GenBank
(April, 2015). The percent G+C content of the 12 PGPR Bacillus spp.
strains ranged from 41.3-46.6%, averaging 45.15% , which is similar
to the average percent G+C content of the B. subtilis genome
sequences available in GenBank (43.72%) (March, 2015). Pairwise
average nucleotide identities (ANI), a newly proposed standard for
species definition in prokaryotes (Richter and Rossello-Mora,
2009), were calculated for 13 Bacillus PGPR strains to determine
their interspecies relatedness among Bacillus species. The ANI
values for PGPR Bacillus spp. strains AB01, AP71, AP79, AP143,
AP193, and GB03 against B. amyloliquefaciens FZB42 (Chen et al.,
2007a) were greater than 98% (data not shown), indicating that
these PGPR strains are affiliated with the B. amyloliquefaciens
species. The 98.88% ANI of PGPR strain AP254 to B. subtilis subsp.
subtilis strain 168 suggests that AP254 is affiliated with B.
subtilis (data not shown). The pairwise ANI comparison of PGPR
strains INR7, KCTC 3706T, KCTC 13613T, KCTC 13918T, and KCTC 13622T
against each other produce ANI values less than 95% (data not
shown) suggests that they are distantly related to each other and
represent diverse Bacillus species.
[0113] Phylogenetic relationship of Bacillus strains. A
phylogenetic analysis based on gyrB gene sequences showed
sufficient resolution among Bacillus taxa and was consistent with
ANI comparisons. Strains AP71, AP79, AP143, AP193, AB01, and GB03
were grouped together with reference strains of B.
amyloliquefaciens subsp. plantarum with high bootstrap support,
indicating that they are affiliated with subsp. plantarum. The
three strains of B. amyloliquefaciens subsp. amyloliquefaciens
DSM7, TA208, and LL3 clustered as a single clade, separated from
strains of subsp. plantarum, supporting the division of two
subspecies in B. amyloliquefaciens (Borriss et al., 2011). The
placement of strain AP254 with B. subtilis subsp. subtilis strain
168 as a single clade with strong bootstrap support suggests its
affiliation with members of the B. subtilis group. (See Hossain et
al., Frontiers Plant Science 2015, FIG. 1A). A gyrB gene based
phylogenetic tree constructed using Maximum Likelihood (ML) methods
was also concordant with the phylogeny constructed using
Neighbor-Joining methods (data not shown). In addition to the
gyrB-based phylogeny, we constructed a phylogenomic tree using
729,383 bp of core genome sequences present within the genome of 25
B. subtilis group isolates to provide a more refined phylogenetic
placement of PGPR strains. The topology and allocation of strains
to clades in the gyrB phylogeny was similar to the phylogenomic
tree (See Hossain et al., Frontiers Plant Science 2015, FIG. 1B).
One notable difference is that the topology of the tree regarding
the position of strain B. siamensis KCTC13613 differs significantly
between the gyrB-based tree and the phylogenomic tree, with the
gyrB based phylogeny placing KCTC13613 in a separate clade whereas
the phylogenomic tree included it within a monophyletic group that
includes strains of B. amyloliquefaciens subsp. plantarum.
[0114] BLAST matrix. Genome wide proteome comparisons of 13 PGPR
Bacillus strains using an all-against-all BLASTp approach
demonstrated that PGPR Bacillus spp. strains are highly diverse, as
indicated by gene family similarity between PGPR Bacillus spp.
genomes ranging from 32-90% (data not shown). Consistent with the
phylogenetic analysis, high similarity was found among strains
AP71, AP79, AP193, AB01, GB03, and FZB42, with proteomic similarity
ranging from 70-90%.
[0115] Core-genome analysis. Analysis of genome sequence alignment
using progressive Mauve determined that the core genome of 13 PGPR
Bacillus spp. strains contains 1,407,980 bp of genomic DNA which
encode 1,454 ORFs (data not shown). Comparison of core genome
sequences of the genus Bacillus, subgroup B. subtilis, species B.
amyloliquefaciens, and subspecies plantarum demonstrated that as
the number of genomes increases, the number of different subsystems
within each respective core genome decreases. (See Hossain et al.,
Frontiers Plant Science 2015, FIG. 2A-D). The highest numbers of
subsystems in each of the core genome categories, except for the
genus Bacillus core genome, was devoted to carbohydrate metabolism.
These findings suggest that strains from the genus Bacillus use
diverse carbon sources. In addition, the core genome for the genus
Bacillus has more subsystems devoted to RNA, DNA, and protein
metabolism compared to carbohydrate metabolism. (See Hossain et
al., Frontiers Plant Science 2015, FIG. 2A-D).
[0116] The genome alignment from 28 different subsp. plantarum
strains, including six subsp. plantarum strains sequenced in this
Example, identified 2,550,854 bp of core genome sequence that is
predicted to encode 2,839 ORFs. The genome alignment of 32 B.
amyloliquefaciens strains, including 28 subsp. plantarum strains,
identified 2,418,042 bp of core genome sequence predicted to encode
2,773 ORFs.
[0117] The genome alignment of 53 strains of B. subtilis group,
including the 12 strains sequenced in this Example, identified
578,872 bp of core genome sequence predicted to encode 674 ORFs.
The number of protein coding genes present within the genome of
Bacillus spp. (.about.4,000) and the low number of ORFs (674)
encoded by their core genomes suggests a large amount of genomic
plasticity among Bacillus genomes that experience frequent gene
acquisitions and losses. It was observed that the B.
amyloliquefaciens core genome was devoid of mobile genetic
elements, such as prophages, transposable elements, and plasmids
(data not shown). Furthermore, the B. subtilis core genome was also
devoid of genes or genetic clusters linked with iron acquisition
and metabolism, secondary metabolite biosynthesis, signal
transduction and phosphorus metabolism. (See Hossain et al.,
Frontiers Plant Science 2015, FIG. 2A-D).
[0118] In this Example, the genus Bacillus core genome was also
determined by analyzing all complete genome sequences from the
genus Bacillus currently available in GenBank. We determined that
the genus Bacillus contains 194,686 bp of core sequence predicted
to encode 201 different ORFs. The predicted functions present in
all Bacillus strains are limited to the following subsystem
features: cofactor synthesis, vitamin synthesis, prosthetic groups
and pigments biogenesis, cell wall and capsule biogenesis, membrane
transport, RNA metabolism, nucleoside metabolism, protein
metabolism, regulation and cell signaling, DNA metabolism,
respiration, amino acids and derivatives, sulfur metabolism, and
carbohydrate utilization
[0119] Comparative analysis of core genes uniquely present in B.
amyloliquefaciens subsp. plantarum. Comparison of PGPR-specific
genomes with that of non-PGPR B. subtilis subsp. subtilis str. 168
did not identify any genes other than essential housekeeping genes
that were conserved within the genomes of PGPR strains (data not
shown). Comparative analysis of core genomes from 28 B.
amyloliquefaciens subsp. plantarum and 32 B. amyloliquefaciens
species identified 193,952 bp of sequences that are present within
the subsp. plantarum core genome but absent in the B.
amyloliquefaciens core genome. Among these genetic loci there were
73 genes shared by all 28 plantarum strains but were not present in
any strains of subsp. amyloliquefaciens. The putative functions of
these genes includes transportation (7 genes), regulation (7
genes), signaling (1 gene), carbon degradation (10 genes),
synthesis of secondary metabolites (19 genes), and hypothetical
proteins (12 genes). (See Hossain et al., Frontiers Plant Science
2015, FIG. 2D). Some of these gene products may be involved in
interactions with plants and rhizosphere competence of subsp.
plantarum strains (e.g., pectin utilization). For instance, genes
required for uptake and use of D-galacturonate and D-glucuronate
are shared among genomes of B. amyloliquefaciens subsp. plantarum
strains. These include uxuA (mannonate dehydratase (EC 4.2.1.8)),
kdgA (4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16)), kdgK
(2-dehydro-3-deoxygluconate kinase (EC 2.7.1.45)), exuT (hexuronate
transporter), exuR (hexuronate utilization operon transcriptional
repressor), and uxuB (D-mannonate oxidoreductase (EC 1.1.1.57)). In
addition, genes required for biosynthesis of the polyketides
difficidin and macrolactin were consistently found in PGPR subsp.
plantarum strains, suggesting their relevance in the biocontrol
activities of these strains.
[0120] Gene clusters encoding secondary metabolite biosynthesis and
natural competency in strain AP193. Due to our observations of
beneficial interactions between PGPR strain AP193 and both plant
and animal hosts (Ran et al., 2012), we selected this strain for
more intensive genome analysis. Assembly of strain AP193 genome
sequences de novo resulted in 152 contigs larger than 1 kb, with a
combined length of 4,121,826 bp. Analysis of AP193 contig
sequences, using the antiSMASH secondary metabolite prediction
program, suggests that gene clusters were present that are
responsible for synthesis of three different polyketides:
bacillaene, macrolactin and difficidin. In order to provide
complete sequences for these biosynthesis pathways, the gaps
between contigs 5 and 6, contigs 33 and 38, as well as contigs 27
and 28 were filled using PCR, followed by DNA sequencing. Each of
the gene clusters in AP193 are collinear to their counterparts in
B. amyloliquefaciens FZB42; a naturally competent plant
root-colonizing B. amyloliquefaciens isolate with the ability to
promote plant growth and suppress plant pathogens (Chen et al.,
2007a). The percent amino acid identities of the proteins encoded
by those clusters were within the range of 98-100% when compared
with those of FZB42. Secondary metabolite biosynthesis gene
clusters involved in non-ribosomal synthesis of cyclic lipopeptides
surfactins, fengycin and bacillomycin D and of the antimicrobial
dipeptide bacilysin present in FZB42 were also detected in the
AP193 genome. The percent amino acid identities of the AP193
proteins encoded on those clusters to the FZB42 homologs ranged
from 98% to 100%. The lack of natural competency of the PGPR strain
AP193 prompted us to determine the presence of competence-related
genes within this strain. We searched the AP193 genome sequences
for the presence of competence related genes found within the
genome of FZB42, and observed that all of the genes required for
encoding the structural components of the competence system found
in strain FZB42 are present within the genome of AP193 with 98 to
100% identity (data not shown); however, genes comQ, comX, and comP
are involved in regulating quorum-sensing in B. amyloliquefaciens
FZB42 (Chen et al., 2007a) were absent within the genome of strain
AP193 (data not shown). The absence of comQ, comX, and comP may be
responsible for the lack of natural competency for strain
AP193.
[0121] AP193 secondary metabolites inhibit the growth of multiple
bacterial plant pathogens in vitro. Antimicrobial activities of
strain AP193 and its mutants AP193.DELTA.dfnD (deficient in the
production of difficidin), AP193.DELTA.srfAA (deficient in
surfactin production), and AP193.DELTA.sfp (unable to produce
polyketide or lipopepetide due to a deletion of sfp gene encoding
4'-phosphopantetheinyl transferase) were tested against plant
pathogens Pseudomonas syringe pv. tabaci, Rhizobium radiobacter,
Xanthomonas axonopodis pv. vesicatoria, and Xanthomonas axonopodis
pv. campestris. The AP193 wild type strain demonstrated strong
antimicrobial activity, whereas the AP193.DELTA.sfp mutant was
devoid of an inhibitory effect against those plant pathogens. (See
Hossain et al., Frontiers Plant Science 2015, FIG. 3), underlining
the contribution of lipopeptides and/or polyketides in the
bioactivity of AP193. This also indicates that the dipeptide
bacilysin, whose synthesis is independent of Sfp, was not involved
in antagonistic activity expressed in vitro. The AP193.DELTA.srfAA
mutant conferred antimicrobial activity similar to wild-type to P.
syringe pv. tabaci, R. radiobacter, X. axonopodis pv. vesicatoria,
and X. axonopodis pv. Campestris (see
[0122] Hossain et al., Frontiers Plant Science 2015, FIG. 3),
suggesting that surfactin has no putative role in the antibacterial
activity of AP193 against those plant pathogens under the
conditions tested in this Example. These findings also demonstrated
that surfactin neither influences the antimicrobial compound
biosynthesis in AP193 nor does it inhibit antibacterial activities
of the antibacterial compounds produced by AP193. Difficidin acts
as the major antibiotic in antagonism of AP193 against plant
pathogens P. syringe pv. tabaci, R. radiobacter, X. axonopodis pv.
vesicatoria, and X. axonopodis pv. campestris as indicated by the
lack of the inhibitory effect of the AP193.DELTA.dfnD mutant
against those plant pathogens. (See Hossain et al., Frontiers Plant
Science 2015, FIG. 4).
[0123] We further confirmed that the AP193.DELTA.dfnD and
.DELTA.sfp mutants lacked synthesis of difficidin by conducting
LC-MS analysis of the cell-free TSB culture supernatants from
wild-type AP193 and each of these mutants. As reported previously,
only the deprotonated form of oxydifficidin was detectable in
bacterial supernatants using MS in the negative mode
([M-H].sup.-=559.3) (Chen et al., 2006), with a molecular mass of
559.3 detected in supernatants of the wild-type AP193 culture but
not observed from the culture of the .DELTA.dfnD mutant. (See
Hossain et al., Frontiers Plant Science 2015, FIG. 4) or from the
.DELTA.sfp mutant (data not shown). The .DELTA.srfAA mutant
exhibited difficidin synthesis as in the wild-type AP193 culture
(data not shown). These findings demonstrate the importance of
difficidin in the biocontrol activity of subsp. plantarum strains
against plant pathogens.
[0124] Strain AP193 secondary metabolites control bacterial spot
caused by X. axonopodis pv. vesicatoria in tomato plants. To
determine the role of bioactive compounds produced by strain AP193
in providing protection against plant diseases, the AP193 wild-type
strain and its AP193.DELTA.dfnD, AP193.DELTA.sfp and
AP193.DELTA.srfAA mutants were applied to tomato plants several
days before those plants were subsequently inoculated with plant
pathogen X. axonopodis pv. vesicatoria. Both AP193 wild-type and
AP193.DELTA.srfAA significantly (P<0.05) reduced disease
severity of bacterial spot on tomato plants compared to the disease
control (Table 3). Additionally, the application of strain AP193
significantly increased the root dry weight of the plants (Table
3). Unlike AP193 wild-type and its AP193.DELTA.srfAA mutant,
strains AP193.DELTA.sfp and AP193.DELTA.dfnD neither protected
tomato plants from severe bacterial spot caused by X. axonopodis
pv. vesicatoria nor improved plant growth (Table 3), further
supporting the importance of difficidin for plant disease
protection. These findings are in agreement with the in vitro
antibiosis pattern of AP193 wild-type strain and its
AP193.DELTA.dfnD, AP193.DELTA.sfp and AP193.DELTA.srfAA mutants
demonstrated against plant pathogen X. axonopodis pv.
vesicatoria.
[0125] Discussion
[0126] PGPR Bacillus spp. strains are used worldwide to improve
crop yields and to protect against plant diseases. In this Example,
12 PGPR genomes were sequenced, including B. subtilis, B. pumilus,
B. amyloliquefaciens, B. mojavensis, B. siamensis, B. sonorensis,
and B. tequilensis. These data were analyzed using ANI, gyrB-based
phylogenies and core genome-based phylogenies to resolve taxonomic
affiliation of Bacillus spp. strains. Our findings demonstrate that
half of the strains sequenced in this Example are affiliated with
B. amyloliquefaciens subsp. plantarum, including strain GB03 that
was formerly designated as B. subtilis. Previously, B. siamensis
type strain KCTC 13613T was proposed as a novel species (Sumpavapol
et al., 2010), but a Bacillus core genome-based phylogenomic
analysis. (See Hossain et al., Frontiers Plant Science 2015, FIG.
1) revealed that B. siamensis KCTC 13613T is instead affiliated
with B. amyloliquefaciens subsp. plantarum. This finding supports
the results of Jeong et al (Jeong et al., 2012) that determined the
close affiliation of B. siamensis type strain KCTC 13613T to B.
amyloliquefaciens subsp. plantarum based on ANI. These findings
also support the continued use of core genome-based phylogenomic
approaches to provide better phylogenetic resolution than analyses
that use a single housekeeping gene (e.g., gyrB). Phylogenies based
on gyrB and core genome sequences demonstrate that B.
amyloliquefaciens subsp. plantarum are highly similar, but
comparison of their proteomes demonstrates that they are closely
related, yet distinct, and may exert plant growth-promoting
activities through different mechanisms.
[0127] B. amyloliquefaciens subsp. plantarum strain ABO1 was
isolated from the intestine of channel catfish (Ran et al., 2012),
but its affiliation with plant-associated strains may suggest
transient presence within a fish gastrointestinal tract; however,
given that the fish feed is soy-based it is likely that the
plant-based diet was also a factor in the growth of this strain
within a fish intestine. Similarly, B. siamensis type strain KCTC
13613T was found to be closely affiliated with B. amyloliquefaciens
subsp. plantarum and was isolated from salted crab, rather than a
plant-associated source. The efficacy of strains AB01, AP193, and
other plant-associated strains as probiotics in fish shows the
capacity for biocontrol of animal and plant pathogens as well as an
overlap in host colonization (Ran et al., 2012).
[0128] With rapid advances in sequencing technologies it is now
possible to extend genomic analysis beyond individual genomes to
analyze core genomes (Medini et al., 2008). In this Example, core
genomic analyses were conducted on PGPR strains from species
affiliated with the B. subtilis group. This analysis identified 73
genes exclusively present among all subsp. plantarum that are
absent in subsp. amyloliquefaciens strains. This small number of
subsp. plantarum-specific genes agrees with a previous report that
identified 130 subsp. plantarum-specific genes using a limited
number of genome sequences from subsp. plantarum strains (He et
al., 2012). Of these 73 plantarum-specific genes identified in this
Example, many are predicted to be important for plant-associated
and soil-associated functions. For example, genes that are required
for the use of D-galacturonate and D-glucuronate were found in the
pool of B. amyloliquefaciens subsp. plantarum-specific core genes.
This observation is consistent with the absence of these genes in
the genome of B. amyloliquefaciens subsp. amyloliquefaciens DSM7
(Ruckert et al., 2011), a strain without any reported PGPR
activity.Pectin, a complex polymer found in plant tissues, is
broken down to D-glucuronate and D-galacturonate which then serves
as a carbon source for bacterial growth (Nemoz et al., 1976). This
pectin could potentially serve as a nutrient source for efficient
root colonization of PGPR through competitive nutrient uptake.
Therefore, the presence of genes that enable D-galacturonate and
D-glucuronate utilization could be advantageous for B.
amyloliquefaciens subsp. plantarum for plant growth-promoting
activity through efficient root colonization.
[0129] Since many of the PGPR strains are from the B. subtilis
group, the core genome estimation was expanded to include a larger
number of B. subtilis strains. Increasing the number of Bacillus
subtilis genomes analyzed to 53 resulted in a 579,166 bp core
genome that is predicted to encode 674 ORFs. This smaller number of
predicted genes reflects genomic diversity among the B. subtilis
group. This finding demonstrates that the number of ORFs found in
the B. subtilis group core genome is close to the number of B.
subtilis ORFs that are considered as indispensable for growth in
complex media (610 ORFs)
(http://www.minibacilius.org/project#genes).
[0130] To validate a gene's involvement in plant-related processes,
it is essential to construct isogenic mutants that are devoid of
those genes. Therefore, we deleted genes from PGPR strain AP193 to
evaluate the role of secondary metabolite biosynthesis gene
clusters in the biological control of plant pathogens. To do this,
a methylated shuttle vector pNZT1 (Zakataeva et al., 2010) with
gene deletion constructs delivered targeted genetic modifications
to AP193, demonstrating the efficacy of in vitro methylation of
plasmids by cell-free extract in circumventing a restriction system
that was presumed to have prevented transformation through
electroporation.
[0131] Difficidin is a highly unsaturated 22-membered macrocylic
polyene lactone phosphate ester with broad-spectrum antibacterial
activity (Zimmerman et A, 1987). Difficidin expressed by strain
FZB42, together with the dipeptide bacilysin, are antagonistic
against Erwinia amylovora--the causative agent of fire blight
disease in orchard trees (Chen et al.. 2009). This Example using an
isogenic mutant AP193.DELTA.dfnD demonstrated for the first time
that difficidin solely, not in conjunction with any other
polyketides or dipeptides, exerts in vitro antibacterial activity
against plant pathogens, such as Pseudomonas syringe pv. tabaci,
Rhizobium radiobacter, Xanthomonas axonopodis pv. vesicatoria and
Xanthomonas axonopodis pv. campestris. We also demonstrated, by
using isogenic mutant AP193.DELTA.dfnD, that difficidin expression
is responsible for control of bacterial spot disease in tomato
plants caused by X. axonopodis pv. vesicatoria. Taken together,
these findings demonstrate that difficidin is the most important
strain AP193 secondary metabolite for biological control of plant
diseases due to bacterial pathogens. In addition, the construction
of the sfp gene deletion allowed investigation of multiple
secondary metabolites produced by AP193 and their individual
contributions to biocontrol activity. The sfp deletion mutant lost
antagonistic activity against each pathogen that was susceptible to
the AP193 wild-type strain. Mutants with the sfp deletion are
expected to lose the ability to synthesize difficidin in addition
to other metabolites. Because the lack of antimicrobial activity of
AP193.DELTA.sfp is consistent with that of the AP193.DELTA.dfnD
mutant, this therefore suggests that difficidin is the primary
metabolite responsible for in vitro inhibition of bacterial
pathogens. In contrast, the surfactin mutant retained antimicrobial
activity against all plant pathogens tested, demonstrating that
surfactin is neither critical for in vitro antibiotic activity nor
influences the synthesis or secretion of other secondary metabolite
biosynthesis in this Bacillus spp. strain; however, surfactin may
influence plant growth promoting activity since it has been
observed that surfactin of B. subtilis elicits ISR in plants
(Ongena et al., 2007) and is expressed in the plant cells colonized
by FZB42 (Fan et al., 2011).
[0132] By studying the contributions of genetic loci that are
conserved among top-performing PGPR strains we continue to uncover
the relative contributions of genes in plant colonization, growth
promotion, and/or pathogen biocontrol. In particular, future
investigation of genes related to the uptake and use of
pectin-derived sugars will help determine the relative importance
of these genes for colonization of plants and persistence within
this microbiome. Comparative genomic analysis of Bacillus spp. PGPR
strains has led to a better understanding of gene products and
provides a foundation to develop application strategies that result
in greater plant growth promotion and biocontrol activity.
[0133] Tables
TABLE-US-00001 TABLE 1 Bacterial strains and plasmids used in this
Example. Strains or plasmids Relevant characteristics Source or
reference E. coli K12 ER2925 dcm-6 dam13::Tn9 New England Biolabs
B. amyloliquefaciens subsp. Wild type Dr. Joseph Kloepper plantarum
strain AP193 (Department of Entomology and Plant Pathology, Auburn
University) AP193.DELTA.sfp deficient in lipopeptides This study
and polyketides AP193.DELTA.srfAA deficient in surfactin This study
production AP193.DELTA.dfnD deficient in difficidin This study
production Bacillus amyloliquefaciens Wild type (Chen et al.,
2007b) FZB42 pMK4 E. coli-Bacillus shuttle BGSC plasmid, rolling
circle replicative, Cm.sup.R pNZT1 Replication Xiaozhou Zhang,
Virginia thermosensitive derivative Tech of the rolling-circle
plasmid pWV01 (pG.sup.+ replicon, Em.sup.R) pNZ-sfp pNZT1 with
upstream and This study downstream sequences of gene sfp pNZ-srf
pNZT1 with knock-out This study construct of srfAA pNZ-dif pNZT1
with knock-out This study construct of dfnD
TABLE-US-00002 TABLE 2 Summary of draft genomes of Bacillus species
sequenced used in this Example Number of Size (total NCBI NCBI
Short Approx. Number of Contigs bp in BioProject Read Archive
sequence predicted Isolates (>1 kb) assembly) % G + C Number
Accession No. coverage (x) ORFs AB01 20 3,903,296 46.4 PRJNA239317
SRX475739 44 3944 AP71 198 4,278,192 45.7 PRJNA239317 SRX475740 15
4531 AP79 47 4,236,770 45.8 PRJNA239317 SRX475741 31 4368 AP143 146
2,956,670 46.6 PRJNA239317 SRX475742 24 3324 AP193 152 4,121,826
46.3 PRJNA239317 SRX475807 37 4159 AP254 59 4,048,419 43.8
PRJNA239317 SRX475808 29 4717 GB03 26 3,849,547 46.5 PRJNA227787
SRX380920 560 3928 INR7 44 3,681,709 41.3 PRJNA227786 SRX447924 750
3857 KCTC 3706T 17 3,935,582 43.7 PRJNA227789 SRX447926 895 4140
KCTC 13613T 23 3,779,696 46.3 PRJNA161489 SRX450083 500 3915 KCTC
13918T 32 4,428,962 45.5 PRJNA227788 SRX450084 1000 4704 KCTC
13622T 33 3,981,302 43.9 PRJNA227791 SRX450086 1000 4299
TABLE-US-00003 TABLE 3 Effects of plant growth-promoting
rhizobacteria (PGPR) strains on severity of bacterial spot disease
and plant growth Root Dry Strain.sup.ab Disease severity.sup.c
Shoot Dry Weight (g) Weight (g) Disease Control 2.11a 2.07bc 0.378c
AP193 1.30b 2.18b 0.453a AP193.DELTA.srfAA 1.48b 2.16b 0.423abc
AP193.DELTA.sfp 2.31a 2.18b 0.405abc AP193.DELTA.dif 2.06a 2.00c
0.389bc Healthy Control 0.00c 2.38a 0.435ab LSD 0.35 0.15 0.050
Note: .sup.aThe experimental design was a randomized complete block
with ten replications per treatment. The experiment was conducted
twice. Values followed by the same letter were not significantly
different (P = 0.05) according to Fischer's protected LSD.
.sup.bOne plant was in each replication. Plants were sprayed with
PGPR suspension (10.sup.6 CFU/ml) one week after transplanting, and
were challenge-inoculated with pathogen solutions (10.sup.7 CFU/ml)
three days after inoculating PGPR. .sup.cDisease severity ratings
and harvest were done 14 days later. For disease severity rating,
four compound leafs were selected from the bottom of each plant.
The disease severity of each of the compound leaves was determined
by rating the disease severity of each leaflet and calculating the
average rating for the compound leaf. The leaflet was rated using a
0-4 rating scale, where 0 = healthy leaflet, 1 = <20% necrotic
area of the leaflet, 2 = 20-50% necrotic area of the leaflet, 3 =
51-80% necrotic area of the leaflet, 4 = 80-100% necrotic area of
the leaflet, or fully dead leaflet.
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Example 2--Efficiency of Pectin Supplement in Root Colonization and
Plant Growth-Promotion of Soybean Plants by Bacillus
amyloliquefaciens subsp. plantarum (Bap)
[0186] Abstract
[0187] A second greenhouse experiment was conducted to determine
the effects of pectin on root colonization, nodulation by
indigenous soil rhizobia, and plant growth-promotion activity of
Bacillus amyloliquefaciens subsp. plantarum (Bap) rifR strains
AP193 and AP143 on soybean. The overall hypothesis tested is that
the complex carbohydrate pectin will enhance Bap-mediated plant
growth promotion. The experimental design included soybean seeds
planted in soil that contained Bap strains with or without 0.1%
pectin supplement, along with a water control, and after 28 days
the plants were harvested and analyzed for statistical
significance. The data indicate that Bap rifR strains with pectin
supplement enhanced soybean shoot and root length in greenhouse
conditions. Dry shoot and root weights of Bap rifR strains with
pectin supplement also increased, but were not significantly
different compared to Bap rifR strains without pectin supplement.
Bap rifR strains with pectin supplement increased significantly
root nodulation of soybean. However, rhizobacterial populations
recovered from the rhizo sphere of soybean plant roots were not
significantly different.
[0188] Materials and Methods
[0189] Preparation of rhizobacterial cells. Plant growth-promoting
rhizobacteria (PGPR) Bacillus amyloliquefaciens subsp. plantarum
(Bap) strains AP193 and AP143 were selected based on their capacity
for utilization of pectin as a C and energy source. Bacillus
thuringiensis subsp. kurstaki (Btk) strain HD73 was collected from
the USDA-ARS culture collection (Ames, Iowa) that was selected for
inability to degrade and use pectin.
[0190] Three bacterial strains were cultured on TSA (BD Difco.TM.
Agar, USA) with 50 .mu.g/m1 Rifampicin (Sigma, USA) from the cryo
stocks (-80 .degree. C.) at 28.degree. C. for 24 hours. A single
colony of each strain was inoculated in 50 m1 tubes containing 35
ml TSB with rifampicin (50 .mu.g/ml) in tubes wrapped with aluminum
foil to avoid light. Bacterial cultures were incubated for 48 hours
with shaking at 220 rpm at 30.degree. C. The bacterial cells were
then spun down using Sorvall Legend RT centrifuge (The Thermo
Scientific, USA) at 10,000.times.g for 10 minutes and the
supernatant was discarded, and the pellet was washed with sterile
water to remove the media. After the final wash, the supernatant
was discarded and re-suspended in sterile Milli-Q water. The mid
log phase bacterial suspension optical density was calculated at on
optical density of 600 nm (OD600) using a GENESYS.TM. 10S UV-Vis
Spectrophotometer (Thermo Scientific, USA). The OD600 of each
rhizobacterial strain was serially diluted for calculated of
colony-forming units (CFU) per milliliter. Suspensions were diluted
to obtain 1.times.106 CFU/ml for inoculation of soybean seeds.
[0191] Soil preparation and pectin mixture. Field soil sieved to
remove root debris was used for the greenhouse experiment. Soil
(450 g) was placed in each cone-tainer tube (lightweight large
Deepots D40L, Stuewe & Sons, Danville, Ill., USA) that
contained three cotton balls in the bottom to retain soil. Pectin
power (EC No. 232-553-0, Tokyo chemical industry co., Toshima,
Kita-Ku, Tokyo, Japan) from citrus source was mixed thoroughly with
field soil using soil mixing machine at a rate of 1 g per 1,000 g
field soil.
[0192] Soybean seed inoculation and Plant growth measurement.
Soybean seed (`Asgrow 6702 RR`) not treated with chemicals was used
as in the previous experiment. One seed was placed in each
cone-tainer, and 1 ml of 106 CFU was pipetted over each seed. Then,
5 g of soil was placed over each seed. Each cone-tainer rack was
covered by plastic sheet to prevent soil desiccation for 48 hours.
Afterwards, cone-tainer racks were transferred to the greenhouse
chamber and tubes were watered twice daily.
[0193] After 28 days, all the plants were harvested for plant
morphometric measurement. Shoot length was measured from the
growing apical region to the basal region connected to the root.
Root nodules were visually counted from all plants. The soil
(approximately 448 g) was removed gently from the roots of each
plant and placed in a 15 ml falcon tubes. Nine ml sterile water
were added to each conical tube, and tubes were vortexed
thoroughly. Then serial dilutions were made from 1:10 to 1:1000 in
sterile Milli-Q water in microcentrifuge tubes and plated on TSA
plates with rifampicin (50 g/ml) for each dilution and incubated at
28.degree. C. for 24 to 48 hours. Colonies that grew on the rif TSA
plates were counted and expressed in log10 CFU/ml. For root dry
mass measurement, roots were washed to remove soil and dried in
oven 45.degree. C. After washing roots to remove all soil, roots
and shoots were dried in an oven at 45.degree. C. for dry weights.
Root length was measured from the root apex to root-stem
junction.
[0194] Experimental design and statistical data analysis.
Cone-tainers were arranged in a completely randomized design (RCD)
with 8 treatments and with 12 replications, with each replication
being a single plant in a single cone-tainer. The data of shoot
height, root height, dry shoot weight, dry root weight, root
nodules, and rhizobacterial growth were analyzed with SAS 9.4
software (SAS Institute, Cary, N.C.) using the proc GLIMMIX. Each
treatment means was compared using LSMEANS at P=0.05 level of
significance.
[0195] Results
[0196] The effects of Bap strains AP193 and AP143 on shoot length,
root length, dry shoot weight, dry root weight, and root nodulation
of soybean varied with pectin amendment are shown in Table 4. Each
experimental control and treatment had 12 replicates, except for
AP143 treatment which had 11 replicates due to lack of
germination.
TABLE-US-00004 TABLE 4 Effect of pectin amendment on soybean
growth, nodulation by indigenous rhizobia, and root colonization by
Bap strains and Btk. Plant growth metrics Mean no. of Shoot Root
Dry Shoot Dry Root nodulation by Root length length weight weight
indigenous soil colonization Treatment (cm) (cm) (g) (g) rhizobia
(Log CFU/g) Water control 28.71c 18.58b 0.33d 0.07cd 0 0 Pectin
control 38.04b 24.25a 0.39bc 0.07cd 5.41ab 2.4c AP193W 33.91b
25.29a 0.42bc 0.10bc 1.50bcd 4.48a AP193P 46.37a 26.41a 0.64a
0.15ab 7.33a 4.94a AP143W 36.80b 22.89a 0.45b 0.16a 2.16bc 3.76b
AP143P 43.12ab 22.79ab 0.59ab 0.15ab 9.33a 4.10ab HD73W 30.37bc
18.08b 0.35cd 0.06cd 0.66cd 2.03c HD73P 36.70b 23.95a 0.35cd 0.06d
0.91cd 1.43c * Bap.sup.x stain = AP193 & AP143; Btk.sup.y
strain = HD73 * Mean values from the greenhouse experiment with
eight treatments and 12 replications. Mean value in the column
followed by the same letter are not significantly different at P
.ltoreq. 0.05 using Tukey's multiple comparison tests.
[0197] The shoot length was significantly enhanced by pectin
supplement and inoculation with Bap strain AP193 but not by Bap
strain AP143 with pectin supplement. The mean shoot length of Bap
strains AP193 and AP143 with pectin supplement were 46.40 cm and
43.10 cm. Shoot of Bap strain AP193 with pectin supplement was
higher than Bap strain AP193 without pectin supplement. However,
with Bap strain AP143 and pectin supplement, mean shoot length was
slightly higher than treatment without pectin supplement. Mean root
lengths of Bap strains AP193 and AP143 with pectin supplement were
slightly higher but not significantly different compared to Bap
strains AP193 and AP143 without pectin supplement.
[0198] Pectin amendment did not result in significantly higher
shoot weights for treatment with either of the Bap strains. Dry
shoot weight of Bap strain AP143 with pectin supplement were 0.59 g
and not significantly different than Bap strain AP143 without
pectin supplement. Soybean dry root weight of Bap stain AP193 with
pectin supplement were 0.15 g and it was slightly higher than Bap
strain AP193 without pectin supplement. There was no significant
difference found between Bap strain AP193 with pectin supplement
and Bap strain AP193 without pectin supplement.
[0199] Nodulation of soybean roots by native soil rhizobia (Table
4) were present with application of pectin put not in the control
with no pectin and no PGPR strains. Root nodulation by indigenous
rhizobia of Bap strains AP193 and AP143 with pectin supplement were
significantly different than Bap strains AP193 and AP143 without
pectin supplement. Mean root nodulation by native rhizobia of Bap
strains AP193 and AP143 with pectin supplement were 7.33 and 9.33.
Root nodulation were not observed in the water control
treatment.
[0200] The effects of pectin supplement on root colonization by the
inoculated Bap strains are shown in Table 4. The rhizosphere
bacterial populations of Bap strain AP143 with pectin supplement
were recovered within 24 hours after incubation at 28 .degree. C.
Bap strain AP193 with pectin supplement was recovered within 36
hours after incubation at 28 .degree. C. The colony_morphology of
recovered Bap and Btk strains were alike to the applied Bap and Btk
strains. Bap and Btk strains were observed in the pectin control
but not the water control. With all three bacteria, supplementation
with pectin did not significantly increased root colonization.
[0201] Discussion
[0202] The overall results of the greenhouse experiment support the
hypothesis that pectin supplement enhances plant growth-promotion
caused by Bap PGPR strains. In this study, greenhouse results
showed that Bap strains with pectin supplement have the ability to
enhance soybean plant growth by root colonization without causing
damage on the roots, leaves, and shoots. The results (Table 4)
showed that plant responses to pectin amendment with and without
two Bap strains depended on the strain. For example, comparing
effect on plants of Bap strain AP143 with and without pectin, there
was no significant effect on shoot or root length and weights. Bap
strain AP193, shoot length and root weight were significantly
enhanced with pectin amendment, Comparing the effects of pectin
amendments with and without Bap strains on nodulation by native
soil rhizobia, Bap strain AP143 and pectin had enhanced nodulation
compared to pectin alone, but with Bap strain AP193, there was no
significant difference in nodulation with and without pectin.
[0203] Bap strains AP193 and AP143 soil amended with pectin
enhanced soybean shoot and root growth in greenhouse conditions
will be important to understanding how the concentration, source,
and structure of pectin impacts the degradation and utilization by
rhizobacteria to promote plant growth. Pectin composition in
monocots (2-10%) and dicots (35%) vary widely due to plant primary
cell wall structure (Ridley et al., 2001). Recent studies have
found that 0.5% pectin increased biofilm formation on Arabidopsis
thaliana by Bacillus subtilis, but little is known about its effect
on plant growth (Beauregard et al., 2013).
[0204] There have been consistent observations that Bap strains
with combined with pectin amendment increased the frequency of
soybean root nodulation. Soybean root nodulation in Bap strains
AP193 and AP143 with pectin supplement were fivefold and fourfold
greater compared to Bap strains AP193 and AP143 without pectin
supplement. This results indicate that pectin mixed field soil with
Bap strain might induce soybean nodulation substantially than Bap
strain without pectin supplement. There is a report that mixed
inoculation of Bradyrhizobium japonicum with Bacillus
amyloliquefaciens strain LL2012 enhanced soybean nodulation
(Masciarelli et al., 2014). Another study also indicated that
Bacillus cereus UW85 increased soybean nodulation in a growth
chamber and the field conditions without inoculation of
Bradyrhizobia spp. (Halverson and Handelsman, 1991). Bradyrhizobia
spp. in the field soils may have enhanced root nodulation in the
presence of Bap strains with pectin supplement. Co-inoculation of
Bacillus polymyxa or B. subtilis with Azospirillum sp. was shown to
allowed pectin degradation and nitrogen fixation by using pectin as
a sole carbon and energy sources (Khammas, 1992).
[0205] Although, Bap strains with pectin supplement populations
were not significantly different that Bap strains without pectin
supplement populations, these re-isolations of Bap strains were
only conducted using rhizosphere bacterial population counts. The
two BAP strains, that colonization, while not significantly
different, was about 0.5 log greater with pectin supplement, so a
separate study on the effect of colonization at several time point,
will be conducted. Therefore, future studies need to address
whether PGPR bacteria are colonizing on the root rhizosphere,
rhizoplane, or as an endophytic population. It may be possible that
the Bap strains are not increasing in the CFU counts within the
rhizosphere, rhizoplane or endophytically, but are utilizing the
pectin and increasing the production of bioactive metabolites that
are stimulating plant growth. By determining the production of
Bap-derived metabolites in these different treatment groups we will
be able to test the hypothesis.
REFERENCES
[0206] Beauregard, P. B., Chai, Y., Vlamakis, H., Losick, R.,
Kolter, R., 2013. Bacillus subtilis biofilm induction by plant
polysaccharides. Proceedings of the National Academy of Sciences
110, E1621-E1630.
[0207] Halverson, L., Handelsman, J., 1991. Enhancement of soybean
nodulation by Bacillus cereus UW85 in the field and in a growth
chamber. Appl Environ Microbiol 57, 2767-2770.
[0208] Khammas, K. K., P, 1992. Pectin decomposition and associated
nitrogen fixation by mixed cultures of Azospirillum and Bacillus
species. Canadian journal of microbiology 38, 794-797.
[0209] Masciarelli, O., Llanes, A., Luna, V., 2014. A new PGPR
co-inoculated with Bradyrhizobium japonicum enhances soybean
nodulation. Microbiological research 169, 609-615.
[0210] Ridley, B. L., O'Neill, M. A., Mohnen, D., 2001. Pectins:
structure, biosynthesis, and oligogalacturonide-related signaling.
Phytochemistry 57, 929-967.
Example 3--Enhanced Soybean Growth Root Nodulation from PGPR and
Pectin Application
[0211] In recent years, PGPR have been developed as biofertilizers
to promote plant growth (Calvo, 2014; Yan, 2002; Kloepper, 1994)
and provide biological control of plant diseases (Liu et al., 2016a
& b). co-PI Prof. Kloepper has a collection of PGPR strains
that have shown plant growth-promoting activity in field soils on
corn, soybean, wheat, canola, and several vegetable crops including
tomato, pepper, and cabbage. Many of these PGPR strains have been
identified as Bacillus velezensis (Note: their phylogenetic
affiliation was previously described as B. amyloliquefaciens subsp.
plantarum; Hossain et al., 2015; Bashan et al., 2014). Bacillus
spp. strains can induce plant growth by diverse mechanisms
including solubilizing phosphate and production of plant hormones
such as indole acetic acid (Niazi et al., 2014). Many PGPR strains
also produce many metabolites that protect plants against bacterial
and fungal pathogens, or can induce systemic resistance (Adesemoye
et al., 2009; Avdeeva et al., 2014, Liu et al., 2016a & b).
[0212] Our labs conducted a comparative genomic study on our 28
best-performing B. velezensis (Bv) strains, and found 73 genes
consistently present in these genomes, but not present in other
Bacillus species that lacked PGPR activity. These PGPR-specific
gene products include the uptake and utilization of pectin-derived
sugars (Hossain et al., 2015). We subsequently found that all 59 of
the B. velezensis strains in our PGPR collection could use pectin
as a carbon and energy source (data not shown). This led to our
central hypothesis that pectin amendment to soil will increase root
colonization by By PGPR strains leading to improved efficacy of
plant growth promotion. There are no previous publications on the
use of pectin to enhance rhizobacteria-mediated plant growth
promotion or to induce legume nodulation. Our greenhouse trials to
test our central hypothesis showed a significant 2-fold increase in
soybean root weight when both By and pectin were amended to an
agricultural soil compared to the water control (FIG. 3).
Interestingly, we also observed an over 10-fold increase in soybean
nodulation (FIG. 4). In addition, the size of the nodules produced
in plants that had a PGPR inoculum and pectin supplementation
greatly increased compared to a By strain alone (FIG. 4A). We
hypothesize that increased pectinolytic activity or other By
metabolites stimulated by pectin amendment enhances nodulation of
soybean roots by native Bradyrhizobium japonicum present in soils
previously cropped with soybean. We predict that the larger and
more numerous nodules present on the more extensive soybean root
systems will result in significant soybean yield increases. The
greenhouse experiments represented in the above figures have been
replicated and showed the same significant increases in root mass
and nodulation size and frequency when pectin and PGPR were amended
to soil (data not shown). These studies provide strong support for
extending this research into field studies to evaluate soybean
yield. We further anticipate that the use of pectin and PGPR
inoculants will be a cost-effective and sustainable method to
promote plant growth and reduce the variability inherent in the use
of beneficial PGPR strains in field soils.
REFERENCES
[0213] Adesemoye A O, Torbert H A, & Kloepper J W. 2009. Plant
Growth-Promoting Rhizobacteria Allow Reduced Application Rates of
Chemical Fertilizers. Microbial Ecology 58:921-929.
[0214] Avdeeva L V, Dragovoz I V, Korzh lu V, Leonova N O,
Iutinskaia G A, Berezhnaia A V, Kuptsov V N, Mandrik M N, &
Kolomiets E I. 2014. Antagonistic activity of Bacillus
amyloliquefaciens subsp. plantarum IMV B-7404 and BIM B-439D
strains towards pathogenic bacteria and micromycetes]. Mikrobiol Z.
76:27-33.
[0215] Bashan Y, de-Bashan L E, Prabhu S R, & Hernandez J P.
2014. Advances in plant growth-promoting bacterial inoculant
technology: formulations and practical perspectives (1998-2013).
Plant Soil. 378 :1-33.
[0216] Calvo P, Nelson L, & Kloepper J W.2014. Agricultural
uses of plant biostimulants. Plant Soil. 383:3-41.
[0217] Corbin, E J., Brockwell, j., and Gault, R. R. 1977.
Nodulation studies on chickpea (Cicer arietinum). Australian
Journal of Experimental Agriculture and Animal Husbandry, 17:
126-134.
[0218] Hassan, M K. 2016. The Role of Pectin Utilization in Root
Colonization and Plant Growth-Promotion by Bacillus
amyloliquefaciens subsp. plantarum. Thesis submitted to University
of Auburn.
[0219] Hossain M J, Ran C, Liu K, Ryu C M, Rasmussen-Ivey C R,
Williams M A, Hassan M K, Choi S K, Jeong H, Newman M, Kloepper J
W, & Liles M R. 2015. Deciphering the conserved genetic loci
implicated in plant disease control through comparative genomics of
B. amyloliquefaciens subsp plantarum. Frontiers in Plant Science
6.
[0220] ISTA. 1993. Proceedings of the International Seed Testing
Association, International Rules for Seed Testing. Seed Sci.
Technol. 21:25-30.
[0221] Liu, K., Garrett, C., Fadamiro, H., Kloepper, J W. 2016a.
Antagonism of black rot in cabbage by mixtures of plant
growth-promoting rhizobacteria (PGPR) BioControl. 61: 605-613.
[0222] Liu, K., Garrett, C., Fadamiro, H., Kloepper, J W. 2016b.
Induction of systemic resistance in Chinese cabbage against black
rot by plant growth-promoting rhizobacteria. Biological Control.
99: 8-13.
[0223] Kjeldahl, J. 1883. Neue Methode zur Bestimmung des
Stickstoffs in organischen Korpern. Zeitschrift fur analytische
Chemie, 22: 366-383.
[0224] Kloepper J W. 1994. Plant-growth-promoting rhizobacteria
(other systems). Azospirillum/plant associations, ed Okon Y (CRC
Press, Boca Raton), pp 139-154.
[0225] Kloepper J W, Ryu C M, & Zhang S A. 2004. Induced
systemic resistance and promotion of plant growth by Bacillus spp.
Phytopathology 94:1259-1266.
[0226] Kloepper, J W and Schroth, M N. 1981. Development of a
powder formulation of rhizobacteria for inoculation of potato seed
pieces. Phytopathology 71:590-592.
[0227] Niazi, A., Manzoor, S., Asari, S., Bejai, S., Meijer, J.,
Bongcam-Rudloff, E. 2014. Genome Analysis of Bacillus
amyloliquefaciens Subsp. plantarum UCMB5113: A Rhizobacterium that
improves plant growth and stress management. PLOS ONE. 9:
e104651.
[0228] Yan Z N, Reddy M S, Ryu C M, Mclnroy J A, Wilson M, &
Kloepper J W. 2002. Induced systemic protection against tomato late
blight elicited by plant growth-promoting rhizobacteria.
Phytopathology. 92:1329-1333.
Example 4--Grant Proposal for "Use of Pectin to Enhance Efficacy of
Plant Growth-Promoting Rhizobacteria (PGPR) Strains to Improve
Agricultural Productivity"
[0229] Introduction
[0230] Plant growth-promoting rhizobacteria (PGPR) have been
identified that control plant diseases and promote overall plant
growth. "Rhizobacteria" means root-colonizing bacteria, and hence,
root colonization is essential for plant growth promotion by PGPR
strains. Plant roots exude various organic compounds, including
sugars, and successful bacterial colonization hinges on nutrient
uptake from the host plants through extracellular enzymatic
activity. Strains of Bacillus amyloliquefaciens subsp. plantarum
(Bap) colonize plant roots, and have been used as biofertilizers or
biocontrol agents during the past decades. Some of the
best-performing PGPR Bap strains at Auburn have been subjected to a
comparative genomic analysis, which indicates that the use of
pectin is a conserved trait among these sequenced strains. As a
structural component of the plant cell wall, much is known
regarding pectin biochemistry and plant synthesis; however, little
is known about the possible role of pectin in root colonization. In
fact, the current scientific paradigm regards pectin utilization as
a function expressed by plant pathogens, and not as a potentially
useful characteristic expressed by plant-associated PGPR strains.
We now have experimental evidence that our best-performing PGPR Bap
strains can obtain carbon and energy via 1) production of an
extracellular pectinolytic enzyme(s) that degrades plant pectin
into hexuronate sugars, 2) transport of pectin-derived sugars, and
3) utilization of these pectin-derived sugars for bacterial
respiration. While these PGPR Bap strains consistently perform well
under lab or greenhouse conditions, field trials are more variable
in PGPR efficacy. We hypothesize that supplementing pectin levels
on seeds or in the plant rhizosphere will improve the efficacy of
PGPR strains in stimulating plant growth and disease control.
[0231] Introduction
[0232] There is a growing need for environmentally sustainable
methods to promote plant growth in agriculture that has prompted
the search for methods, like the probiotic strategies described in
this proposal, to cost-effectively enhance plant growth. Plant
growth-promoting rhizobacteria (PGPR) have been developed as
biofertilizers to promote plant growth (8-11). While many species
of bacteria are classified as PGPR strains, Bacillus species have
been closely studied due to their spore-forming activity that
confers a longer shelf life and greater viability in commercial
formulations. Within this genus, strains of Bacillus
amyloliquefaciens subsp. plantarum (Bap) have emerged as especially
effective PGPR strains that lack any potential for pathogenesis
(12, 13). At Auburn University, co-PI Kloepper has collected over
1,000 rhizobacteria isolates, of which over 300 have shown plant
growth promoting activity in field soils on corn, soybean and wheat
plants and 59 of these PGPR strains have been identified as Bap
strains.
[0233] Some PGPR strains, including many Bap strains, can be used
as biostimulants to promote plant growth (14). For example,
microbial inoculants can solubilize phosphorus and/or fix nitrogen
that can then be absorbed by plant roots, directly stimulating
plant growth. There is a large body of literature on the use of
bacterial inoculants for nitrogen fixation (15), and many Bacillus
spp. strains have been identified as phosphate-solubilizing
bacteria with commercial potential as biofertilizers (16). In
addition, PGPR strains have been found to produce many secondary
metabolites that have antibiotic activity against bacterial and/or
fungal pathogens (17-19), including our discovery of the novel and
potent antibiotic Bacillusin A produced by a Bap strain (20). PGPR
strains can also induce the control of plant disease through
production of compounds that induce plant systemic acquired
resistance (SAR; mediated by salicylic acid) and induced systemic
resistance (ISR; jasmonic acid-dependent) (8, 21, 22).
[0234] The earliest reported studies of seed bacterization for
agricultural purposes dates to the use of Rhizobium inoculants on
legumes in 1895 (23). Yield increases for cereal crops after
bacterial inoculants were applied were observed in a variety of
Soviet and Indian studies throughout the 1960s and early 1970s
(23). However, field studies have consistently produced lower
yields than greenhouse studies, suggesting that the introduced
microbial population declines rapidly after soil inoculation (23,
24). This decline was likely due to an inability of the PGPR
strains to compete and thrive within the rhizosphere. As described
by Hawes et al. (26), "Efforts to improve plant health by adding
exogenous populations of beneficial microorganisms (biological
control) are notoriously unreliable" (25). Despite the inherent
difficulties of using bacterial inoculants, the North American
market for biostimulants, which includes PGPR, is estimated to grow
to $490 million by 2018 (26). There is therefore strong interest in
strategies that can enhance the efficacy of PGPR strains to improve
agricultural productivity.
[0235] Our labs have conducted a comparative genomic study on our
best-performing PGPR Bap strains, and we were able to identify 73
genes that were consistently present within all 28 genomes
surveyed, but not present in other strains of that B.
amyloliquefaciens were known not to have PGPR activity (7).
Importantly, we found that genes related to the uptake and
utilization of pectin-derived sugars were always observed within
these 28 PGPR Bap strains (7). This led to the hypothesis that
root-derived pectin is important for beneficial rhizobacteria to
colonize roots, produce bioactive metabolites and provide nutrients
to plants, resulting in improved efficacy of plant growth
promotion.
[0236] There is a significant knowledge base for pectin
biochemistry that can benefit this project. Henri Braconnot
discovered pectin in 1825 (27), and pectic substances are now known
to be highly complex heteropolysaccharides that comprise a major
component of plant primary cell walls in addition to cellulose and
hemicellulose (28). For example, the primary cell wall of Sycamore
is composed of 34% pectin, 24% hemicellulose, 23% cellulose, and
19% hydroxyproline-rich glycoprotein (29). The highest levels of
pectin occur in the fruits, leaves, and roots of plants, so this is
consistent with the potential for pectin to provide a needed
root-derived nutrient source for beneficial microorganisms. Pectin
is found in the middle lamella between cells, where it helps to
bind cells together, and the availability and structure of pectins
(polygalacturonans) varies among plant species (30). The
demethylesterification of pectins, which is mediated through the
action of pectin methylesterases, is an important process in seed
germination (31) and in release of root border cells (32) which are
living cells programmed to separate from roots into the external
soil environment (26). Importantly, the mucilage produced by root
border cells is rich in pectin and there is a "dramatic increase in
the levels of soluble, de-esterified pectin in the root tip during
border cell development" (32). The pectin-rich mucilage associated
with root border cell release has been shown to be important for
root penetration into soil, as well as binding metal cations such
as aluminum to prevent toxicity (33, 34). However, to our knowledge
no studies have investigated the contribution of root-derived
pectin for beneficial plant-microbial interactions.
[0237] Pectin degradation occurs through the action of many
different pectinolytic enzymes that are found in bacteria, fungi
and higher plants. Protopectinases hydrolyze protopectin that
exists in an insoluble form within plant tissues, resulting in
soluble pectin, and have been observed in a wide range of Bacillus
sp. (35). Many bacteria are known to secrete pectin lyases to
degrade plant pectin. This pathway was first reported in
Escherichia coli (36) and pectinolytic activity has been shown in
the following bacterial genera: Achromobacter, Arthrobacter,
Agrobacterium, Bacillus, Clostridium, Erwinia, Pseudomonas, and
Xanthomonas (35). Many of these bacteria are recognized as plant
pathogens, and the degradation of pectin is a characteristic of
soft rot disease as caused by Erwinia species. Among soft rot
pathogens there is evidence that pectinolytic activity is inducible
and highly expressed compared to low-level constitutive expression
of pectinolytic activities by non-soft rot bacteria (37);
therefore, the competition for pectin as a nutrient source within
rhizospheres could be a mechanism by which beneficial PGPR strains
(e.g., Bap) antagonize plant pathogens without themselves causing
plant damage. We have experimental evidence that our sequenced Bap
strains encode and express a pectin lyase activity (FIG. 1).
[0238] By producing and secreting pectinolytic enzymes, bacteria
can degrade pectin and uptake the pectin-derived sugars
D-glucuronate, D-galacturonate and D-mannose (38), which can be
taken up by bacteria via a hexuronate transporter (exuT). We found
that the exuT gene is conserved among all sequenced PGPR Bap
strains (7), and using an exuT-specific primer set we also found
that this hexuronate transporter gene was present in all of the Bap
strains in the Auburn collection (data not shown). A similar
approach confirmed the universal presence within these Bap strains
of the uxuB gene, which encodes D-fructuronate oxidoreductase that
is one of the enzymes responsible for utilizing pectin-derived
sugars (39). Furthermore, we have experimental evidence that our
sequenced PGPR strains encode and express pectinolytic activity,
and can use the resulting monosaccharides as a sole carbon source
(data not shown).
[0239] Since the acquisition of carbon is essential for bacterial
physiology we hypothesized that increased availability of pectin
could promote the survival, persistence and metabolic activity of
PGPR strains within plant rhizospheres, leading to improved plant
growth and disease control. There have been two previous reports on
the use of exogenously supplied pectin to stimulate
plant-associated bacteria. In the case of nitrogen-fixing
Azospirillum isolates, soils supplemented with pectin showed an
increase in Azospirillum growth (40). There is also evidence that
plant polysaccharides can induce biofilm formation in B. subtilis
strains (5). However, in previous studies there was no attempt to
evaluate any benefit for plant growth. In our preliminary studies,
we have observed that there is a synergy between PGPR strains and
soil amendment with pectin, resulting in statistically significant
increases in root and shoot weight (FIG. 3). The soybean root and
shoot growth enhancement with both Bap and pectin amendment was
observed with both PGPR strains, with greater than a 2-fold
increase in root weight (dry or wet) compared to the water
control.
[0240] Interestingly, we also observed an over 10-fold increase in
nodulation (FIG. 4B), with the size of the nodules produced in
plants that had a PGPR inoculum and pectin supplementation greatly
increased compared to a Bap strain alone (FIG. 4A). Whereas we had
predicted the increased rate of root and shoot growth, the finding
that nodulation was enhanced was a serendipitous discovery. We
hypothesize that increased pectinolytic activity from the Bap plus
pectin combination may result in enhanced Bradyrhizobium infection
of soybean roots, since it has been shown before in the model
legume Lotus japonicus that a mutant lacking pectate lyase activity
was also deficient in nodulation (41). Based on the increased
nodule frequency and size (FIG. 4A), we predict that the rhizobia
metabolic activity within the nodules is increased relative to the
no pectin controls, and that this results in greater nitrogen
fixation rates. We further anticipate that the use of a pectin-rich
soil amendment together with PGPR inoculants will be a
cost-effective and sustainable method to promote plant growth and
reduce the variability inherent in the use of beneficial PGPR
strains in field soils.
[0241] Research Plan
[0242] (Objective 1) Identify & characterize novel plant root
carbohvdrate-utilizing and pectin-utilizing microorganism.
Surprisingly, a search of the literature for a microbial growth
medium containing a "root extract" only yields a single publication
in which soluble root exudates were used to enrich for
root-associated Archaea (42). While citrus pectin-degrading
microbes have been previously identified from soils (43, 44), to
our knowledge no previous research effort has used plant
root-derived complex carbohydrates incorporated into a growth
medium to culture plant root-associated microorganisms. We will use
a minimal medium containing either root extract or purified root
pectin as a sole carbon and energy source, and will use long-term
incubation conditions as have been done previously by our research
group and others to culture soil bacteria that have not been
previously cultivated under laboratory conditions (45, 46). In a
preliminary study using soybean root incorporated into a minimal
medium and inoculating agar plates with serially diluted soybean
rhizosphere samples, we have isolated 184 bacterial isolates that
formed colonies after 3 months of incubation. Among these, we have
completed sequencing the 16S rRNA gene from 144 isolates, of which
39% have a 16S rRNA gene with a % identity <97% to their top hit
in the GenBank database! These bacterial isolates include
representatives from multiple bacterial phyla (i.e.,
alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria,
Bacteroidetes, and other phyla) and genera (e.g., Inquilinus,
Chitinophaga, Herbaspirillum, Flavobacterium, Delftia, Ralstonia,
Burkholderia, and Dyella). Interestingly, of the 22 unique isolates
that have <97% identity to the top BLAST hit, 10 (45%) had top
BLAST hits to previously uncultured soil bacteria. In this proposed
study we will culture from multiple soil types and use root
homogenates and root pectin to greatly expand our culture
collection to identify microbial isolates with potential as PGPR
strains. In a preliminary experiment, we have observed that two of
our previously sequenced PGPR Bap strains, AP143 and AP193, could
utilize pectin as a C source for growth (FIG. 2). As a negative
control, a non-PGPR Bacillus thuringiensis subsp. kurstaki strain
HD73 was obtained from the USDA ARS culture collection (Ames, Iowa)
that was identified as a non-pectin utilizing strain based on its
genome sequence. This strain was not observed to grow using pectin
as a sole C source (FIG. 2).
[0243] (1A) Culture rhizosphere microorganisms: using a root
extract medium.
[0244] Hypothesis: The use of plant root-derived complex
carbohydrates as a C source will enable cultivation of diverse
endophytic and rhizosphere microorganisms.
[0245] Experimental Methods: The bacterial and fungal assemblages
associated with plant roots will be cultured using long-term
incubation on a minimal medium containing either a plant root
extract or purified pectin.
[0246] Root extract preparation: Root extract will be prepared
using 10 corn (Zea mays) or soybean (Glycine max) roots grown in
sand in pots in a greenhouse for 21 days. The washed roots will be
homogenized with a mortar and pestle after freezing under liquid
nitrogen, and then the homogenized roots will be suspended in
sterile water in a 50 ml centrifuge tube. After centrifugation, the
supernatant containing soluble root exudates (simple sugars and
amino acids) will be removed. This washing will be repeated twice,
and the root homogenate will be dried in a clean hood. The dry
weight of the root homogenate will be used to prepare a suspension
of 1 g root tissue per 10 ml sterile water. The root suspension
will then be thoroughly homogenized using a T10 Basic Ultra-Turrax
dispersion unit (IKA Works, Inc., Wilmington, Del.) which is
capable of generating sub-micron sized particulates suitable as a
microbial growth substrate.
[0247] Pectin purification: Commercially available pectin is
derived from citrus or apple sources, and is chemically different
from root pectin (47). Therefore, we will extract pectin from corn
or soybean roots, using approximately 50 corn or soybean plants
grown at the same time as the plants above. The washed roots will
be shipped to the Complex Carbohydrate Research Center (CCRC) where
the root pectin will be extracted by using a 0.5% ammonium oxalate
buffer containing 0.1% NaBH.sub.4 (pH 4) in a boiling water bath
for 1 h each and pooling the supernatants as described previously
(48).
[0248] Root extract medium (REM) preparation: The root or pectin
extract will be added to a M9 salts minimal defined medium at a
final concentration of 4 mg per ml based on preliminary experiments
with a range of root extract concentrations (data not shown). After
the addition of 1.5% agar and autoclaving the suspension, trace
elements (FeCl.sub.3, ZnCl.sub.2, CuCl.sub.2, CoCl.sub.2,
H.sub.3BO.sub.3, and MnCl.sub.2) will be added to the REM according
to the recommended concentrations (49) and plates will be thickly
poured (30 ml per plate) after magnetic stirring. There will be a
total of 4 different media prepared (corn root, corn pectin,
soybean root, soybean pectin) for cultivation efforts.
[0249] Cultivation of microorganisms: Soils will be sampled from
actively growing corn or soybean plants in distinct soil types
(clay, sandy and loam soils) within proximity to Auburn University.
The sandy soil will be collected from the Cullars Rotation in plots
that have not received K or P inputs for over 100 years, but yet
have diverse microbial communities (50). Intact roots with
associated rhizosphere will be sampled from five plants per site
and then pooled together. In order to access both endophytic and
rhizosphere microorganisms, sub-samples will be suspended in
sterile water (10% w/v) and homogenized to produce a fine
suspension of microbial cells. This microbial suspension will be
serially diluted, and the 10.sup.-4, 10.sup.-5 and 10.sup.-6
dilutions will be plated onto the four REM medium types with
10.times. replication (a total of 30 plates per media type per soil
site; 120 total plates per soil site). From previous experience,
the 10.sup.-5 and 10.sup.-6 dilutions provide isolated colonies on
a soybean REM whereas the 10.sup.-4 dilution tends to be overgrown
with microbial colonies. The inoculated plates will be wrapped in
parafilm and maintained in containers in which humidity is kept
elevated to avoid plate desiccation. The plates will be incubated
for 2-3 months with regular monitoring for the appearance of
microbial colonies. After incubation, colonies will be passaged
onto the same REM type as their original isolation to obtain
isolated colonies from pure cultures. Pure cultures will be
documented for their colony type by taking digital photos using a
digital microscope and will then be cryopreserved in REM broth with
20% glycerol, prior to storing replicate tubes at -80.degree. C.
The primary isolation of plant root carbohydrate-utilizing
microorganisms will be repeated as necessary to achieve several
thousand pure microbial cultures in our collection.
[0250] Expected results: A large culture collection of
plant-associated microorganisms that can utilize plant root-derived
carbohydrates will be obtained that include many novel microbial
taxa.
[0251] Anticipated problems and their solutions: Based on our
previous efforts to cultivate previously uncultured bacterial taxa
from soils (46), a principle challenge is maintaining the viability
of laboratory cultures. Sequential passaging may lead to loss of
some cultures due to insufficient nutrient availability or other
unknown factors. Therefore, each culture will be cryopreserved at
passage one when a pure culture has been obtained so that if a
culture losses viability that it may be resuscitated on a different
growth medium, especially if there is promising data generated
regarding phylogenetic affiliation or beneficial plant
interactions. It is also anticipated to be difficult to continually
prepare root extract medium agar plates because this requires
growing corn and soybean plants for medium preparation. We will
therefore explore the use of commercially available root extracts
that are known to be pectin-rich (e.g., beet pulp) to prepare REM
agar and broth in order to maintain laboratory cultures for
subsequent experiments.
[0252] (IB) Conduct a phylogenetic analysis of cultured microbial
isolates. Hypothesis: The bacterial and fungal isolates obtained
from Obj. 1A will include recognized plant pathogens, beneficial
microbes as well as novel cultured isolates that have not been
previously been characterized for their plant interactions.
[0253] Experimental Methods: Each of the pure microbial cultures
obtained in Obj. lA will be subjected to a molecular phylogenetic
analysis based on a comparison with 16S rRNA sequences for Bacteria
(or Archaea, should these be isolated) and internal transcribed
spacer (ITS) analysis for fungal isolates. A portion of the pure
culture from passage one will be swabbed from the REM agar plate
into 1 ml of sterile water within a microcentrifuge tube, and after
centrifugation at 10,000.times.g the microbial pellet will be used
to isolate genomic DNA using an E.Z.N.A. genomic DNA isolation kit
(Omega Biotek, Atlanta, Ga.). The genomic DNA will be used as a
template for a PCR in which `universal` bacterial 16S-specific (27F
and 1492R (51)) or fungal ITS-specific (ITS86F and ITS4 (52, 53))
primer sets are used in order to generate an amplicon from each
isolate. The PCR product will be purified over a column and used
for Sanger sequencing with a forward or reverse primer (e.g., 27F
and 907R) sufficient to provide a consensus sequence over a large
portion of the amplicon. The consensus 16S rRNA gene or ITS
sequence will be generated using the CLC Genomics Workbench
(Qiagen, Cambridge, Mass.) and these will be subjected to a
multiple sequence alignment and maximum likelihood analysis (1000
iterations using RAxML (54)). Each of the unique isolate sequences
will be compared against the databases at GenBank (nr/nt) and
Ribosomal Database Project (RDP) in order to identify the nearest
neighbor for each of the microbial taxa. A record of the %
identity, nearest neighbor, and its phylogenetic affiliation will
be recorded for each isolate within a lab PlantMicrobiomeNet
database (see Data Management Plan), and if the isolate corresponds
to a known pathogen (e.g., Erwinia sp.) or beneficial microbe (B.
subtilis) this will also be indicated within the database. Of
particular interest will be isolates that do not fall into either
of the latter categories and have a relatively low % identity
(<97%) to known cultured microorganisms within GenBank or RDP.
Based on these results we will assemble 96-well plates containing a
glycerol stock of each of the unique isolates, in triplicate, that
is not affiliated with a known pathogen. We will group the isolates
based on their relative rate of growth so that each 96-well plate
contains isolates that will take approximately the same amount of
time to reach the stationary phase of growth.
[0254] Expected results: The phylogenetic affiliation for each of
the pure cultures obtained in Obj. 1A. Other outputs from this
objective will be a collection of genomic DNAs from each isolate,
an indication of whether each culture represents a taxon that has
not been previously cultured in vitro or been associated with
plants, and a 96-well plate formatted culture collection.
[0255] Anticipated problems and their solutions: This objective has
a high probability of success. If an isolate does not provide a PCR
amplicon, which could be due to the primer set not amplifying from
unique microbial taxa, alternative primer sets will be used; if
necessary, genomic DNA will be further purified using a method
developed by the PI (55).
[0256] (1C) Screen cultures far plant hormone synthesis: and the
ability to promote plant growth. Hypothesis: Many of the microbial
isolates will have the ability to induce plant hormone expression
and will be able to stimulate plant growth.
[0257] Experimental Methods: Each of the pure cultures that have
been identified as non-pathogenic in Obj. 1B will be used to
inoculate A. thaliana that express green fluorescent protein (GFP)
in response to plant hormones, and to test for soybean growth
promotion.
[0258] Broth culture of microorganisms: Each 96-well cryopreserved
plate containing the unique, non-pathogenic microbial cultures
(bacterial and fungal) in triplicate will be used to inoculate a
96-well, deep-well plate with each well containing 1 ml of REM
broth. Each plate will also contain controls inoculated in
triplicate wells, the positive control will be B. amyloliquefaciens
FZB42 which has been previously shown to produce auxin (56), and
the negative control will be Bacillus thuringiensis HD73 that does
not produce any plant hormone. A breathable, sterile film will be
placed over each 96-well plate which will be shaken at 200 rpm for
approximately 1 week. The exact length of time will depend on
preliminary experiments to establish the rate of growth for
representative isolates in this REM broth medium. Each plate will
be monitored for the growth of the cultures and used when the
cultures have attained sufficient turbidity (OD.sub.600>0.5) to
inoculate wells containing the A. thaliana bioassay.
[0259] Bioassay for plant auxin or cytokinin response: Auxin and
cytokinin are essential plant hormones involved in root growth and
are likely to be involved in the promotion of plant growth by PGPR
strains. Since direct measurements of these plant hormones is both
difficult and time consuming, indirect fluorescent reporter lines
have been generated and are standardly used to analyze auxin (DR5)
and cytokinin (pTCSn) (57, 58). We have bred each reporter line
linked to a different fluorescence protein together to generate a
single plant wherein both auxin and cytokinin response can be
examined simultaneously. This hormone responsive line contains both
DR5-YFP and pTCSn-GFP reporters. Seeds of this hormone-responsive
line will be added to 96-well plates in which 0.5 ml of molten
(42.degree. C.) 1.4% agar in water has been added to each well, and
then a multichannel pipettor will be used to transfer 0.5 ml of the
respective microbial culture to each well and will be mixed by
pipetting. The seedlings will be grown for five days under standard
growth conditions and then roots will be analyzed for changes in
response to auxin and cytokinin levels based on altered
fluorescence using an epifluorescence microscope. Both positive
(hormone treated) and negative (blank) controls will be examined in
parallel and replicated to determine the results for this
objective. Positive results of hormone response will be confirmed
using qPCR expression analysis of routinely examine auxin and
cytokinin responsive genes (e.g. IAAs and type-ARRs) as described
previously (59).
[0260] Greenhouse bioassay for plant growth promotion: The
microbial isolates will also be tested for their respective ability
to promote soybean plant growth in an Alabama field soil in a
greenhouse. The experiments will be designed in a randomized
complete block design in triplicate. Conetainers that are 2.5
inches in diameter and 8.25 inches deep will be filled with the
same weight of thoroughly mixed field soil. Prior to planting, the
soil water content in conetainers will be adjusted to 60% field
capacity. At planting, one soybean seed will be placed into the
center of each conetainer at a depth of 1 inch, followed by
pipetting 0.5 ml of the respective microbial suspension over the
top of each seed, then covering the soybean seed with soil.
Watering conetainers will begin 24 hr after planting. PGPR Bap
strain AP193 used as a positive control; B. thuringiensis strain
HD73 and water only will be used as negative controls. Plants will
be transferred to the greenhouse and watered daily for 21 days,
after which the dry shoot and root weights will be determined. For
each isolate's impact on plant root and shoot growth the variation
will be compared to controls and analyzed using ANOVA at 5% level
of significance.
[0261] Expected results: Identification of specific microbial
cultures that produce plant hormones and/or induce plant growth
that could be used as PGPR strains.
[0262] Anticipated problems and their solutions: There is inherent
variability in bioassays that may result in false negative or
positive results. By conducting both laboratory (A. thaliana) and
greenhouse assays in this objective we will increase the likelihood
of observing biologically meaningful plant growth stimulation. The
isolates that promote plant growth and/or produce plant hormones
will be further tested in a potato slice assay (60) to eliminate
any isolates with the potential for inducing soft rot. We will
select specific strains based on their positive effects on plant
growth to advance to field studies to assess their potential for
plant growth promotion and/or disease biocontrol, and these studies
will be conducted using other funding sources.
[0263] (Objective 2) Evaluate the ability of exogenous pectin
supplemention to enhance PGPR-mediated plant growth.
[0264] In preliminary experiments we have observed that amending
soil with citrus-derived pectin can significantly enhance
PGPR-mediated plant growth (FIG. 3) and nodulation frequency and
size (FIG. 4). In this objective we will investigate the effect of
pectin concentration on PGPR strain growth in soils; furthermore,
we will assess the effects of soil type and pectin source on
PGPR-mediated growth enhancement of corn and soybean plants.
Lastly, we will determine the impact of A. thaliana mutants that
modulate root pectin levels on PGPR-mediated growth promotion.
These studies will advance the agricultural applications of this
science and foster our understanding of the role of pectin in
mediating plant-microbial interactions.
[0265] (2A) Determine pectin dose-dependent enhancement of PGPR
strain growth.
[0266] Hypothesis: There will be a pectin dose-dependent increase
in Bap growth within the rhizosphere.
[0267] It is important to first determine the concentration of
pectin to use in subsequent plant growth experiments. Our initial
experiments evaluated different pectin (citrus source)
concentrations from 0.001% to 5% on soybean seed germination, and
observed slightly inhibited germination rates at concentrations
>1%, and our initial greenhouse trials have therefore been
conducted using 0.1% pectin amended as a dry ingredient to
soils.
[0268] Experimental Methods: In order to track strain colonization
in soils we have identified rifampicin (Rif)-resistant mutants
using 50.quadrature. g/ml Rif for Bap strains AP143 and AP193, and
these Rif.sup.R mutants have similar growth and PGPR activity
compared to the wild-type strains (data not shown). A negative
control strain, B. thuringiensis strain HD73, which does not use
pectin as a carbon source (61) and does not have PGPR activity
(data not shown), was also used to select a Rif.sup.R mutant. The
experimental system for assessing root colonization by the
Rif.sup.R mutants will be the same greenhouse tests as described
above in Obj. 1C using soybean seeds grown in field soil. Seeds
will be inoculated at the time of planting with 1.0 ml of AP143,
AP193 or HD73 Rif.sup.R spores (10.sup.6 colony forming units
(CFU)/seedling) applied in soils that receive 1) no amendment, 2)
0.01% (w/w) pectin 3) 0.025% pectin, 4) 0.05% pectin, or 5) 0.1%
pectin, with 8 replications for each treatment group. At 21 days
post-inoculation, 10 g of rhizosphere soil will be sampled from
each of the pots, and the dry shoot and root weights will be
determined. The numbers of nodules formed per soybean plant will
also be determined along with the average nodule mass. The soil
will be homogenized in 90 ml of sterile water (10.sup.-1 dilution)
and then serially diluted to 10.sup.-6 dilution and each of the
dilutions from 10.sup.-1 to 10.sup.-6 will be plated onto TSA
plates containing 50 .quadrature. g/ml Rif in order to determine
the number of Rif.sup.R CFU/g of soil for each of the PGPR strains
and the negative control strain.
[0269] Expected results: Determination of the pectin dose-dependent
increase in growth of the PGPR Bap strains when amended into soil.
The results of this experiment are expected to show a significant
pectin dose-dependent increase in the CFU/g of Bap strains AP143
and AP193, with no significant increase observed for
pectin-incompetent strain HD73. These results will enable the
selection of a pectin concentration to use in Obj. 2B. We will also
evaluate the degree of nodulation enhancement and future studies
will investigate the molecular basis for the increase in nodulation
frequency and metabolic activity associated with pectin and PGPR
inoculation.
[0270] Anticipated problems and their solutions: To confirm that
the CFU counts of the Rif.sup.R are due to the inoculated PGPR Bap
strains, representative colonies will be selected to conduct PCR
using an AP143 or AP193 strain-specific PCR primer set that has
been developed in the Liles lab (data not shown). Our preliminary
experiments have not shown any Rif.sup.R colonies identified from
plants that have not been inoculated with a Bacillus Rif.sup.R
strain.
[0271] (2) Evaluate pectin enhancement of PGPR-mediated plant
growth promotion using multiple pectin sources, plants and soils.
Hypotheses: There will be a significant increase in plant growth in
response to PGPR and pectin amendment compared to PGPR inoculation
alone. Soil amendment with beet pulp will provide similar results
in enhancing PGPR-mediated plant growth as with citrus-derived
pectin.
[0272] Experimental Methods: We will conduct this greenhouse
experiment in separate trials using soybean and corn plants. In
each separate trial, plants will be will be sown in trays that
contain a sandy loam soil (Cullars Rotation soil) or a field soil
used routinely in greenhouse studies, and 3 weeks later
transplanted into 8 inch pots that contain the respective soil. As
with previous experiments we will incorporate pectin into soil as a
dry ingredient, and we will use a concentration depending upon the
results of Obj. 2A. The PGPR strain will be applied as a 1 ml
drench to each seedling three days after transplantation. In
addition to the commercial source of citrus pectin, we will also
evaluate the use of powdered beet pulp as an alternative and
cost-effective, pectin-rich amendment (62). The treatment groups
will include 1) no PGPR in loamy soil, 2) no PGPR in clay soil, 3)
10.sup.6 CFU/seedling PGPR Bap strain AP193 in loamy soil and 4)
10.sup.6 CFU/seedling PGPR Bap strain AP193 in clay soil. These
four treatment groups will each be incubated with soil without
pectin, with citrus pectin or beet pulp (n=12 treatment groups with
8 replicates=96 pots per each experiment). A completely randomized
blocked design will be used. Fresh and dry weight of root and shoot
will be measured at the completion of the greenhouse experiment
after 4 weeks. Root morphology will be analyzed by a WinRHIZO root
scanner (Regent Instruments, Quebec City, Canada). The numbers of
nodules formed per soybean plant will also be determined along with
the average nodule mass. For each parameter the variation of each
treatment will be compared and analyzed using ANOVA at 5% level of
significance (SAS 9.1 software). These experiments will be repeated
with soybean and corn, and if additional low-cost sources of pectin
are identified we will evaluate the use of these alternative soil
amendments to enhance PGPR-mediated plant growth promotion.
[0273] Expected results: The addition of citrus pectin or beet pulp
to Bap-treated soybean seeds will result in an increase in soybean
root and shoot growth relative to non-pectin-treated plants. No
pectin-mediated increase will be observed in the plants without a
PGPR strain inoculum.
[0274] Anticipated problems and their solutions: There can be
unforeseen issues, such as a disease outbreak, in experimental
plants. We will take ample precautions to limit disease incidence,
and plan to repeat this experiment at least once and more if
necessary. The timing of the experiment may need to be extended to
achieve significant differences among treatment groups.
[0275] (2C) Evaluate PGPR colonization and persistence in wild-type
Arabidopsis vs. pectin biosynthesis mutants. Hypothesis: A reduced
PGPR-mediated increase in plant growth performance will be
identified in Arabidopsis that have a defect in pectin biosynthesis
relative to wild-type.
[0276] Because A. thaliana does not exhibit a distinct root border
cell separation as with other plant species (63), it will be of
interest to evaluate PGPR-dependent growth responses for wild-type
and mutant A. thaliana.
[0277] Experimental Methods: As discussed above pectin is a complex
polysaccharide for which there are many biosynthesis and catabolism
pathway steps. Because of this there are numerous genes involved in
these processes, and subsequently in Arabidopsis there are numerous
potential mutants to be examined that could have altered pectin
levels. However, even in the well-studied Arabidopsis system only a
handful of gene mutants have been shown to have altered pectin
levels in root tissues. We will examine the two best characterized
mutants with defects in pectin biosynthesis that are root
expressed, quasimodo1 and 2 (qua1, qua2). The QUA1 gene encodes a
glycosyltransferase (also known as GUAT8) and its mutant qua1 has
been shown to have 25% less homogalacturonan (HG) and have a
reduction in pectin esterification (64). QUA2 is a pectin
methyltransferase and its mutant qua2 has 50% less HG with normal
esterification levels (65). Importantly, both mutants in these
genes show alterations in root border cell separation (reduced cell
adhesion) resulting in distinct border cell separation typically
observed in the other crop species (soybean and corn) examined in
this proposal (63). To our knowledge these are the only cell
adhesion mutants that have pectin defects in roots. The selected
qua1 and qua2 mutants have a dwarfed stature resulting from their
reduction in pectin biosynthesis, although these mutants are still
viable (8, 65, 66). While there are a few other Arabidopsis mutants
in pectin-related biosynthesis genes that are expressed in root
tissues, these either show no change in growth phenotype possibly
due to the large degree of redundancy in this pathway or have
little information regarding changes in pectin levels (67). The Bap
strain AP193 will be inoculated onto wild-type, QUA1, or QUA2
mutant A. thaliana seeds at an inoculum of 10.sup.6 CFU per seed
such that the impact of mutant phenotype on PGPR-mediated growth
response can be assessed and compared to negative controls (water
or B. thuringiensis strain HD73). Inoculated seeds will be
germinated and grown under several standard conditions (96-well
plate as noted above, standard agar plates and soil growth chamber
conditions). Plants will have basic growth (leaf size and number,
silique and seed production) and health (Fv/Fm chlorophyll
fluorescence) measured at distinct stages across development to
determine PGPR-mediated effects. Measurements at all stages will be
performed with a minimum of 10 plants per treatment group and
measurements will be analyzed as noted above.
[0278] Expected results: Reduced PGPR-mediated growth will be
observed in the A. thaliana pectin synthesis mutants compared to
the wild-type control.
[0279] Anticipated problems and their solutions: Some alleles of
the qua1 and qua2 selected mutants have extreme dwarf growth
phenotypes that would make the proposed examinations difficult to
conduct. As such we will select more robust alleles that still have
pectin defects, readily available from the Arabidopsis stock center
to conduct these assessments.
[0280] (Objective 3) Determine the identity and functions
associated with root pectin-metabolizing soil microorganisms using
culture independent approaches.
[0281] The experiments described above rely upon culture-dependent
studies of novel rhizosphere microorganisms and known PGPR strains.
While the use of cultured PGPR strains has great utility for
application in agriculture, we also know that the vast majority of
soil microorganisms are not readily cultured under laboratory
conditions (68). In this objective we will therefore use stable
isotope probing (SIP) with .sup.13C-root pectin in order to study
the phylogenetic diversity and functional contributions of soil
microorganisms that utilize root pectin as a C source. We will
complement this culture-independent approach by studying microbial
genomes recovered from a soil metagenomic library that are
predicted to encode pectin-degrading or -utilizing functions.
[0282] (3A) Determine the phvlogenv and functions of soil microbial
taxa that utilize .sup.13C-pectin. Hypothesis.sub.1: The
pectin-utilizing microorganisms identified will include those
identified in Objective 1 and will also include other microbes that
have not been previously associated within pectin utilization or
characterized as plant-associated. Hypothesis.sub.2: The
pectin-utilizing microbes will express gene products that are
predicted to be important for plant growth.
[0283] Experimental Methods: .sup.13C-Stable Isotope Probing. We
will evaluate the soil microbial use of .sup.13C-root pectin with
and without the presence of the PGPR Bap strain AP193. The Cullars
Rotation soil (described above) will be added to trays in which
corn seeds will be sown. After three weeks the seedlings will be
transplanted into 4.5 inch pots containing soil with and without
0.1% root pectin (w/w) extracted from corn roots by the CCRC (see
above). Additional pots will be planted to guard against loss of
plants from disease and to serve as controls and for method
refinement. For the appropriate treatment groups, three days after
transplantation a 10 ml PGPR inoculum will be applied as a 10 ml
root drench (10.sup.6 CFU/seedling) and then all plants will be
grown for 3 weeks in a greenhouse prior to addition of the
.sup.13C-pectin inoculation. This is comparable to a study in which
.sup.12C-carbohydrate amendments (but not pectin) were added to
soils for 3 weeks prior to addition of the .sup.13C label (69). As
in this study, we will assess CO.sub.2 production from replicate
pots receiving unlabeled root pectin at different concentrations
using headspace-collected CO.sub.2 and will measure CO.sub.2 at the
Auburn University Mass Spec facility using a Shimadzu GC-2014 gas
chromatograph, using multiple time points post-pectin addition in
order to establish the appropriate concentration of .sup.13C-pectin
to add and the time frame for sampling. Our ability to identify the
soil microorganisms that are responsible for actively degrading and
incorporating .sup.13C-pectin will be enabled by isolating RNA from
soils which is more labile and representative of metabolically
active microorganisms. Unlike readily metabolized substrates such
as .sup.13C-glucose in which RNA-SIP is conducted within 12 hours
(reference), a more complex polysaccharide such as pectin should
require a multiple day time frame for this study. There will be two
treatment groups (control, PGPR).times.4 replicates.times.4 time
points for a total of 32 pots. The .sup.13C-labeled root pectin
will be prepared by purchase of 20 g uniformly .sup.13C-labeled [97
atom % .sup.13C] corn root (IsoLife, Wageningen, The Netherlands),
and pectin will be extracted from root tissue by the CCRC as
described above at the scale needed to have sufficient root-derived
pectin for soil amendment (estimated yield .about.4 g .sup.13C
pectin). Each pot will contain .about.100g of soil, therefore our
total needed yield of .sup.13C-pectin will be 0.1 g per pot
.times.32 pots=3.2 g. Four time points will be sampled, at time
zero just prior to a .sup.13C-root pectin inoculum equivalent to
the concentration used in previous experiments (0.1% (w/w)) in a 10
ml volume and then at three post-inoculation time points
(.about.24, 48 and 72 hours, depending on the results of CO.sub.2
production). At each time point we will sample the rhizosphere soil
from each plant, adding approximately 2 g of rhizosphere soil to 5
ml of LifeGuard.TM. Soil Preservation Solution (MoBio, Carlsbad,
Calif.) in a 15 ml collection tube, and a replicate tube will be
frozen at -20.degree. C. which will remain viable for RNA/DNA
isolation for>30 days. Total DNA and RNA will be isolated from 2
g of soil sample using the PowerSoil Total RNA Isolation kit
(MoBio) in a dedicated RNA processing area of the PI' s lab. DNA
will be separately eluted using a DNA Elution Accessory kit (MoBio)
and RNA and DNA samples will be quantified using a Qubit
fluorometer. The RNA samples will be separated on a cesium
trifluoroacetate isopycnic gradient subjected to
ultracentrifugation at 128,000.times.g for 50 hr using a Beckman
ultracentrifuge. Control gradients using a pure bacterial (Bap
AP193) culture grown on .sup.12C-pectin or .sup.13C-pectin will be
conducted in advance to establish conditions that result in good
separation of light from heavy RNA (70), and a syringe pump will be
used in order to provide a low flow rate (.about.200
.quadrature.1/min) for fraction collection in sterile RNAse-free
microcentrifuge tubes. We will quantify the amount of RNA in each
100 .quadrature.1 fraction using a rapid qPCR method targeting
bacterial rRNA (71) using SYBR Green fluorescence in a Bio-Rad CFX
real-time PCR system (Hercules, Calif.) in order to establish
migration of light and heavy RNA. Fractions with abundant extracted
RNA will be pooled for the fractions exhibiting clear separation of
heavy from light RNA, treated with DNase I, and then reverse
transcribed using M-MLV Reverse Transcriptase (Invitrogen,
Carlsbad, Calif.). The resulting cDNA will be used for shotgun
metagenomic sequencing.
[0284] Microbiome analysis: The .sup.13C-labeled cDNA and total DNA
recovered from each sample (32 samples, with cDNA and DNA, for a
total of 64 samples) will be used as a template for 16S rRNA and
gyrB gene PCR amplification. While the 16S rRNA gene is the most
common molecular target in microbiome studies, it produces a
distorted representation of microbial relative abundance due to
differing copy numbers of the rRNA operon within microbial genomes.
We will use a two stage polymerase-exonuclease (PEX) PCR method in
development with our collaborator Dr. Stefan Green (Univ of
Illinois--Chicago) that results in improved evenness of
amplification across mixed templates and allows for greater primer
degeneracy (72). Using the cDNA or DNA templates, an analysis based
on the standard Illumina protocol for 16S rRNA gene sequence
amplicons with the 2-stage PEX-PCR protocol will be conducted for
both 16S rRNA and gyrB sequences. Since gyrB is a housekeeping gene
that is single-copy, we expect that this will result in a less
biased representation of the microbiome, and we have already
generated a novel set of `universal bacteria` gyrB primers that
will be used in this study (data not shown). Using the QIIME
pipeline and custom scripts, the 16S and gyrB sequences will be
trimmed, binned into operational taxonomic units (OTUs) based on
>97% identity, and compared to existing phylogenetic databases
to determine the relative abundance as compared to the known colony
forming units. By conducting principal coordinate analysis from the
16S and gyrB sequence data we will reveal trends in the comparison
of treatment groups at the different phylogenetic resolutions
afforded through 16S and gyrB, respectively. The known 16S rRNA and
gyrB sequences for Bap strain AP193 will be used for
comparison.
[0285] Next-generation sequencing (NGS) and bioinformatics
analyses: In order to reduce the number of samples used for NGS
analysis, we will evaluate the concentration of heavy cDNA to
identify samples in which there was less recovered .sup.13C labeled
cDNA. Of the 32 total samples processed, we expect to be able to
eliminate one of the time points resulting in .about.24
.sup.13C-cDNA samples. For comparison purposes we will also include
the total DNA sample recovered from each sample and each of the
samples will be bar-coded for Illumina sequencing using the Nextera
kit (Illumina, San Diego, Calif.) using a unique index. We will
include 8 samples per Illumina HiSeq lane in order to provide
sufficient coverage per sample in order to better access lower
abundant transcripts. The raw sequence reads will be trimmed for
sequence quality equivalent to a Q score>30 and the trimmed
reads will then be used for de novo assembly using the SPAdes
assembler running on the Auburn University supercomputer. We will
compare the transcriptome from each sample and treatment group
using the CLC Genomics Workbench in order to identify
differentially expressed transcripts in each treatment group and to
determine the highly expressed transcripts present within the
.sup.13C-enriched sample. We will submit the transcripts to the
MG-RAST automated pipeline for microbial metagenomic analysis and
will in particular be seeking information on plant-related
functions. Our specific molecular targets will include transcripts
related to nutrient (NPK) acquisition, plant hormone production,
and secondary metabolite synthesis, among other potential
functions. The contigs from each transcriptome will also be
submitted to the antiSMASH pipeline for biosynthetic cluster
identification to provide enhanced detection of biosynthetic
clusters, with comparisons of the % identity and synteny of each
discovered pathway with known pathways from the GenBank database.
These analyses will lead to testable hypotheses regarding
pectin-utilizing microorganisms and their contributions to plant
growth.
[0286] Expected results: A culture-independent analysis of the
pectin-utilizing microorganisms within the corn rhizosphere. This
will indicate the phylogenetic affiliation and the expressed
functions of the pectin-utilizing microorganisms, with a particular
emphasis on plant-related phenotypes.
[0287] Anticipated problems and their solutions: One potential
problem will be that using a living corn plant will result in
dilution of the .sup.13C enrichment for pectin-utilizing
microorganisms. It will therefore be necessary to carefully monitor
the degree to which providing exogenous pectin stimulates microbial
respiration by the series of control pots using non-labeled pectin
prior to the .sup.13C-pectin experiments. In this way we can
maximize the degree to which the RNA of the pectin-utilizing
microbes has incorporate the .sup.13C label and can be separated
from non-labeled RNA.
[0288] (3B) Mine a soil metagenomie library for pectinolytic
enzymes and associated functions.
[0289] We have previously constructed a large-insert soil
metagenomic library from the same plot of the Cullars Rotation soil
that will be used for the SIP study. High molecular weight DNA
isolated from the soil microbiota (73) was randomly sheared and
ligated into a bacterial artificial chromosome (BAC) vector,
resulting in a metagenomic clone library containing 110 kb average
insert sizes with 19,200 clones. The library consists of 50
384-well plates, and based on the average insert size the total
amount of cloned metagenomic DNA is estimated to exceed 2 Gbp, or
over 500 Escherichia coli genome equivalents (data not shown). A
next generation sequencing strategy was used in which clones were
pooled from each of the 50 plates, 24 columns and 16 rows
separately and each pool was uniquely bar-coded and sequenced using
multiple Illumina HiSeq runs to achieve greater than 100.times. x
average coverage per each of the 19,200 clones. In other words,
each of these metagenomic clones is theoretically represented
within 3 different pools (column, plate and row) and by
bioinformatically comparing these different sequenced library pools
we can identify the gene content and exact library well location
for each clone (manuscript in preparation). The pooled sequences
were processed to achieve high quality sequence reads and generate
assembled, contiguous genomic fragments (contigs). In a preliminary
search for microbial genomic regions involved in pectin-related
functions, we searched for gene sequences related to the
degradation, uptake or utilization of pectin and pectin (or
pectate)-derived sugars. Interestingly, we discovered 75 unique
metagenomic contigs that had significant (E<10.sup.-5) homology
with a Bacteroides pectate lyase gene, and when each of these gene
homologs were queried against the GenBank database these gave a
mean % amino acid identity of 62%, and only one metagenome-derived
gene had a % identity greater than 90%. Several hundred unique
contigs were also identified that have homology with pectin-derived
sugar uptake or utilization genes (data not shown). Collectively,
this indicates that there is a wealth of previously undiscovered
diversity of pectin-associated functions among soil
microorganisms.
[0290] Sequencing of the BAC clones containing pectin-related
functions: Each of the BAC clones identified by querying using
known pectin-related gene sequences (including each gene known to
be required for pectin degradation, uptake and utilization) will be
identified for its exact location within the soil metagenomic
library from a local BLAST search against each of the respective
pool of clones (plate, column and row). To validate that the
correct clone has been identified, the respective E. coli BAC clone
will be grown for isolated colonies onto LB medium containing 12.5
.quadrature.g per ml chloramphenicol and then a clone-specific
primer set will be designed and used to PCR amplify a small genetic
region unique to each clone (<500 bp). Each validated BAC clone
will then be bar-coded using a Nextera kit and included within a
HiSeq run in order to generate a complete insert sequence for each
clone. The clone insert will be generated by trimming the sequence
reads and de novo assembled using the CLC Genomics Workbench, and
then the clone contigs will be submitted to the RAST automated
pipeline for annotation. We will also manually annotate clones to
examine the predicted functions and related genes present on each
genomic region. We will conduct comparative genomic analyses to
examine pathways related to pectin degradation, uptake and
utilization from diverse soil microorganisms, deposit these
sequences in GenBank for public access, and compare these sequences
to those obtained from the SIP results (from the same soil) in
order to link transcriptome data within a larger genomic context.
Because the sequences obtained from the transcriptome analysis are
expected to be short contigs all less than .about.5 kb, the
availability of genomic contigs >100 kb from the same soil will
enable linking encoded functions from abundant and metabolically
active soil microorganisms that metabolize pectin.
[0291] Expected results: A culture-independent metagenomic analysis
of the pectin-utilizing genes recovered from soil microorganisms.
This will result in a unique dataset of large genomic regions
recovered from microorganisms from the same soil used for
transcriptome analysis.
[0292] Anticipated problems and their solutions: Much of the
research has already been completed in order to construct the soil
metagenomic library and to generate an exhaustive sequence database
from column, plate and row pools. This objective is therefore very
low risk because we already know that there are diverse
pectinolytic functions encoded within the library that can be
accessed, sequenced, and compared to the transcriptome data
generated in Objective 3A.
[0293] Broader Impacts
[0294] We expect that this research will provide: 1) New beneficial
microbial cultures that can be used to promote plant growth in many
agricultural crops. These cultures may have novel plant
interactions and produce metabolites that promote plant growth
and/or control plant pathogens. 2) The cost-effective application
of pectin as a soil amendment in order to enhance microbial plant
growth promotion. Field application of PGPR strains is often
ineffective, and we expect that the use of pectin will promote
better efficacy and result in improvement in PGPR-mediated
biological effects. Our study will evaluate cost-effective
pectin-rich soil amendments that could be used for agricultural
application of this technology. 3) New insights into root
pectin-utilizing microbes and their plant-related functions.
Through the use of culture-independent approaches we will provide
information on the diversity of pectin-utilizing microbes, the
plant-related functions they express and provide a database of
their genomic regions associated with pectin use. 4) We will
provide educational opportunities as a component of this
research.
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Example 5 --Proposal: Enhancing Fish Nutrition, Growth Performance
and Disease Resistance in Fish Using a Probiotic Bacillus and
Prebiotic Pectin
[0369] Project Summary
[0370] Aquaculture sustainability is threatened by disease pressure
and the eutrophic conditions resulted from intensive fish farming.
Previous research conducted separately at Auburn University and at
Ocean University-Qingdao has resulted in the identification of
Bacillus amyloliquefaciens strains that have the ability to reduce
mortality due to multiple aquaculture pathogens. Studies in aquaria
and ponds have determined that feeding fish with B.
amyloliquefaciens probiotic-amended feed can enhance catfish growth
performance resulting in a 8-14% increase in final weight as
compared to control fish. Interestingly, in the pond study there
was also an observed improvement in water quality in ponds in which
fish were fed with the probiotic-amended feed, as significant
reductions were found in pond water total phosphorus (19%), total
nitrogen (43%), and nitrate (75%), along with reduced levels of
chlorophyll and 2-methylisoborneol. Recent insights into the
genetics and physiology of the B. amyloliquefaciens probiotic
strains indicates that these bacteria can use plant-derived pectin
as a carbon and energy source. Because aquaculture feeds are
plant-based, and these probiotic bacteria were derived from plant
rhizospheres, the use of plant carbohydrates like pectin by the
probiotic bacteria is expected to enhance probiotic-mediated
effects such as feed conversion efficiency and the production of
secondary metabolites that antagonize aquaculture pathogens. This
proposal will evaluate the hypothesis that this synbiotic approach,
the addition of a probiotic Bacillus together with the prebiotic
pectin to fish feed, will result in improved fish growth
performance and reduced mortality due to infection with an
aquaculture pathogen compared to fish fed with a control diet or
with the probiotic or prebiotic alone.
[0371] Project Narrative and Justification
[0372] Due to their rapid growth rate, low cost, and proficient
reproduction capabilities, catfish species including the channel
catfish (Ictalurus punctatus) in the United States and the Southern
catfish (Silurus meridionalis Chen) in the People's Republic of
China has become one of the more popular and economically important
aquaculture species, particularly in the southeastern United States
(USDA, 2003a; USDA 2003b). For maximized productivity of the
aquaculture system, fish feeding efficiency is extremely important.
Traditionally, forage fisheries have been exploited for generation
of fish meal, but the rapid depletion of wild fisheries (Naylor et
al, 2009) has led to the use of soy meal as an alternative (Tacon,
1987; NRC, 2011). However, feed conversion ratios (FCR) are much
lower in fish with the use of plant protein resulting in up to a
15% deterioration of fish growth performance (Sales, 2008). Due to
the presence of 1-2% phytate content in soy-based feed, over
two-thirds of the phosphorous found in plant protein sources are
unusable by fish, and phytate can serve as an anti-nutrient in
binding iron resulting in anemia (Zhu, 2014). Unused phytate will
ultimately be released as fish waste, contributing to the
eutrophication of the aquaculture pond ecosystem (Lazzari,
2008).
[0373] Eutrophication due to indigestible components of the feed
and excreted nutrients can result in blooms of algae and
cyanobacteria (Shaw et al, 2003). Due to the ability of
Cyanobacteria taxa to synthesize and release toxins into the water
column, they can be devastating to fish production (Rodgers, 2008).
In addition to hepatotoxins and neurotoxins, some Cyanobacteria and
other bacterial taxa produce the metabolites 2-methylisoborneol
(MIB) and geosmin that result in off-flavors in catfish (Tucker and
Ploeg, 1999).
[0374] Phytase is a phosphohydrolase that catalyses the hydrolysis
of phytate, allowing for phosphorous availability for absorption
(Kumar et al., 2012). This enzyme is found in many microorganisms,
which are being exploited for supplementation in feed. To
supplement high feed demands, production facilities have been
created to exponentially ferment phytase from microorganisms, many
of which are already regarded as probiotics (Askelson et al, 2014).
For this reason, providing the fish probiotics in their diet can
potentially reduce eutrophication, induce weight gain, and be a
viable option for preventing economic loss. Probiotic bacteria may
promote the growth of fish by improving feed nutrient quality and
by removing anti-nutrients such as phytate. Some microorganisms
express phytase activity, catalyzing phytate hydrolysis and
allowing for phosphorous absorption (Kumar et al., 2011). Purified
microbial phytases have been used as a feed additive in fish feeds
to promote growth and reduce eutrophication (Kumar et al 2012)).
For this reason, feeding fish with a phytase-expressing probiotic
could be a sustainable management practice to reduce
eutrophication, induce weight gain, and result in an altered
aquaculture pond ecosystem with reduced incidence of disease and
off-flavor.
[0375] Another factor responsible for significant economic losses
in aquaculture is due to loss from disease (FDA, 2012). One
traditional treatment for disease is the use of antibiotics, and
there are currently three approved by the FDA for use in
aquaculture production facilities (FDA, 2011). However, with
growing concern over the use of antibiotics due to the development
of resistance in pathogens, it is important to seek alternative
means of treatment. Probiotics can reduce mortality due to
pathogens by direct antagonism via synthesis of secondary
metabolites, by competitive exclusion, and by activation of the
innate immune system (Balcazar, 2006; Macfarlane, 1999; Wang,
2008). Bacillus spp. have good potential for aquaculture
application due to their ability to form endospores, allowing for a
long shelf life and survival of gastric acid (Casula, 2002; Hong,
2005; Hyronimus, 2000). Furthermore, strains within the B. subtilis
group, which includes B. amyloliquefaciens, have not been
associated with disease.
[0376] Previous research at Auburn University by the PIs evaluated
a collection of 160 Bacillus spp. strains for their antimicrobial
activity against bacterial and fungal fish pathogens (Ran et al.,
2012). The 21 Bacillus spp. strains that showed production of
secondary metabolites that inhibited the growth of Edwardsiella
ictaluri, Aeromonas hydrophila and other pathogens were then tested
for their survival and persistence in the catfish intestine and
protection against E. ictaluri infection (Ran et al., 2012). When
Platydoras armatulus (striped catfish) were fed with spore-amended
feed significant reductions in mortality relative to the control
group was observed after challenge with E. ictaluri (Ran et al.,
2012).
[0377] The four best-performing Bacillus spp. strains (AB01, AP79,
AP143 and AP193) were selected for further study for their
potential for disease control and fish growth promotion. All four
strains were found to be affiliated with B. amyloliquefaciens based
on phylogenetic analyses (Hossain et al., 2015), without any
virulence-related genetic determinants (data not shown). Each of
these B. amyloliquefaciens strains was evaluated separately for
their relative degree of channel catfish growth promotion and
biocontrol activity. Feed amended with B. amyloliquefaciens AP193
provided the greatest degree of fish growth promotion in both
replicated aquaria and pond studies, with a 8.5% (P<0.05) or
21.8% (P <0.1) increase in the average weight gain per fish
compared to fish fed with control feed in aquaria and pond studies,
respectively. Furthermore, the fish fed with strain AP193 had a
mortality due to E. ictaluri infection of 47.8% compared to the
62.1% mortality rate for fish fed with control feed ((P<0.05).
It was previously observed that strain AP193 expresses the
antibiotic difficidin and that the production of this polyketide is
critical for AP193 biocontrol activity in plants (Hossain et al.,
2015). We have observed that strain AP193 mutants deficient in
difficidin synthesis (.quadrature.sfp or .quadrature.dfnD) were
also completely lacking in the ability to inhibit the in vitro
growth of bacterial fish pathogens such as E. ictaluri and A.
hydrophila (data now shown), further supporting the hypothesis that
difficidin production is important for fish disease control while
leaving open the possibility that other mechanisms (e.g.
competitive exclusion, stimulation of immune competence) are also
involved.
[0378] Together with the increase in catfish growth performance and
reduction in fish mortality, significant reductions in total
phosphorus, total nitrogen, and nitrate nitrogen levels were
observed in ponds containing channel catfish fed with
AP193--indicating beneficial, pond-wide effects on water quality
(Table 5).
TABLE-US-00005 TABLE 5 Mean concentrations mg/L of water quality
parameters in control ponds and ponds fed with AP193 amended feed.
Control AP 193 Treatment Water Quality Parameter P-value (Mean .+-.
SD) (Mean .+-. SD) Total Phosphorus 0.014 0.136 .+-. 0.049a 0.110
.+-. 0.066b Total Nitrogen 0.025 0.344 .+-. 0.248a 0.195 .+-.
0.120b Total Ammonia Nitrogen 0.829 0.142 .+-. 0.065a 0.137 .+-.
0.059a Nitrite Nitrogen 0.945 0.004 .+-. 0.004a 0.004 .+-. 0.004a
Nitrate Nitrogen 0.044 0.051 .+-. 0.095a 0.013 .+-. 0.026b
[0379] Excessive concentrations of N and P in ponds can contribute
to dense cyanobacterial or algal blooms that induce toxic
eutrophication and fish "off-flavor" (Boyd, 2015). Soy-based fish
feed contains high levels of phytate, which is
inositol-hexaphosphate (Cao, 2007; Storebakken, 1998). AP193 is
known to encode a phytase (Hossain et al., 2015) and has been
observed to express phytase activity (data not shown). Thus, AP193
has the capacity to degrade the phytate present within feed,
resulting in more iron availability to support fish growth as well
as decreasing phosphate excreted from fish, thereby preventing the
release of phosphorus into the water that can result in
eutrophication. Furthermore, previous research has determined that
A. hydrophila has the ability to use myo-inositol as a sole C
source and suggests that the presence of high levels of inositol in
the diet could contribute to A. hydrophila pathogenesis (Hossain et
al., 2013). By expressing a phytase activity, strain AP193 may be
improving not only fish growth and water quality, but also removing
a key nutrient (inositol) that may contribute to A. hydrophila
pathogenesis. This study will therefore investigate the benefit of
feeding fish with feed amended with AP193 in reducing mortality
associated with virulent A. hydrophila that is known to cause
severe losses to the aquaculture industries in China and the United
States (Rasmussen-Ivey et al., 2016).
[0380] The probiotic effects observed to date support the potential
benefit of this strategy for improving the sustainability of fish
farming in China and the United States. While promising, there is
inherent variability in the water quality and microbiology
associated with aquaculture practices in both countries, and there
is a need to increase the efficacy of probiotics so that they can
perform under various conditions. Our previous genomic study that
investigated the predicted functions associated with these
probiotic strains found that one trait that the B.
amyloliquefaciens strains have in common is the ability to use the
plant carbohydrate pectin as a carbon and energy source (Hossain et
al., 2015). We subsequently found that the ability to use
pectin-derived sugars was a universal characteristic of the 59 B.
amyloliquefaciens strains that had been used to promote the growth
of plants (Adesemoye et al., 2009) and/or fish (Ran et al., 2012)
in the Auburn collection. This led to the hypothesis that adding
pectin to the fish diet could enhance the growth of the probiotic
bacteria and thereby improve fish growth performance, water
quality, and disease resistance. We have now tested this hypothesis
in soybean model together with Prof. Joseph Kloepper (Auburn
University), and have observed a strong synergy between strains and
B. amyloliquefaciens pectin, resulting in statistically significant
increases in soybean root and shoot weight (data not shown). The
soybean root enhancement using bacterial and pectin amendment was
observed with multiple B. amyloliquefaciens strains, including
strain AP193, with significant root growth and nodulation
enhancement observed when both bacterial spores and pectin were
added together compared to the bacterial treatment alone (FIGS. 3
and 4), and no effect on root growth was observed when pectin was
applied alone (data not shown).
[0381] Objective
[0382] This proposal will test the ability of pectin as a prebiotic
to enhance the probiotic effects we have previously established in
catfish, which is referred to as a "synbiotic" approach.
Specifically, different dosage rates of pectin incorporated into
the diet will be evaluated as to how probiotics respond to this
addition in the diet and by promoting fish survival after bacterial
challenge with virulent Aeromonas hydrophila. This could directly
benefit the sustainability and productivity of aquaculture as
practiced in both China and the United States.
[0383] Experimental design: Parallel studies will be carried out in
the US and China using Channel Catfish and the Southern catfish in
conjunction with specific strains of B. amyloliquefaciens. All
trials will be designed to determine the efficacy of select
prebiotic and probiotic combinations on growth, nutrient retention
and disease resistance under controlled conditions.
[0384] Aquaria Trials: At both institutes a commercial type basal
plant-based diet will be formulated and produced in house using
typical feed manufacturing methods. The basal diet will then be
modified by adding graded level of pectin and suitable levels of
the probiotic. The test diets will then be offered to five
replicate groups (15-20 fish/tank) of juvenile fish over a 10-week
culture period. The fish will be offered feed twice daily at a
level approximating satiation. The fish will be maintained in a
re-circulating culture system designed to maintain suitable water
quality parameters. Each system will consist of culture tank, UV
sterilization of water, solids filtration, biological filtration,
circulation pump and supplemental aeration. At the conclusion of
the growth trial survival, final weight, thermal growth
coefficient, percent weight gain and feed conversion efficiency
will be determined. Additionally, a sub-sample of fish will be
analyzed for proximate composition using standard AOAC procedures
and the date used to determine apparent net protein, phosphorus and
iron retention. Meanwhile, the intestinal morphology will be
analyzed, including the dimensions of both jejunum wall thickness
and villus height in different groups.
TABLE-US-00006 TABLE 6 Aquaria Trials Channel Catfish (US) B.
amyloliquefaciens Southern Catfish (China) Treatment Diet type
AP193 Diet type B. amyloliquefaciens 1 Basal diet (BD) Basal diet
(BD) 2 BD + 0.05% BD + 0.05% pectin pectin 3 BD + 0.10% BD + 0.10%
pectin pectin 4 BD + 0.50% BD + 0.50% pectin pectin 5 Basal diet
(BD) 10.sup.6 CFU/g Basal diet (BD) 10.sup.6 CFU/g 6 BD + 0.05%
10.sup.6 CFU/g BD + 0.05% pectin 10.sup.6 CFU/g pectin 7 BD + 0.10%
10.sup.6 CFU/g BD + 0.10% pectin 10.sup.6 CFU/g pectin 8 BD + 0.50%
10.sup.6 CFU/g BD + 0.50% pectin 10.sup.6 CFU/g pectin
[0385] Disease challenge with virulent A. hydrophila ML09-119: All
fish will be transferred to the disease challenge laboratory
maintaining fish within each replicate aquaria. Fish will be
allowed to acclimate for one week while being offered the same
treatment diet as above. The fish will then be challenged by
immersion with vAh using an immersion protocol developed by the
USDA-ARS in which fish have their adipose fin clipped and are then
immersed in 10.sup.7 CFU/ml vAh for 1 hr, typically resulting in
.about.50% mortality within 24 hrs. The percent mortality in each
treatment group will be determined and confirmed with
representative vAh cases using a vAh-specific diagnostic tests.
Statistical comparisons among treatment groups will be conducted
with one-way ANOVAs with significance assessed at P <0.05.
[0386] Timeline of Events: At about 0-5 months, the growth and
disease experimental trial will be performed. At about 4-9 months,
tissue and histological preparation and evaluation will be
performed. At about 10-12 months, data analysis and report
preparation will be performed
[0387] Expected Results and Outcomes
[0388] As pectin levels are increased within diets containing the
probiotic, we expect higher growth rates and feed conversion
efficiency, along with better survival after pathogen challenge as
a result of increased proliferation of the probiotic and increased
production of probiotic-derived secondary metabolites (e.g.
difficidin) within the intestines. We do not anticipate any affect
on growth performance or survival upon bacterial challenge due to
pectin being in diet alone. Additionally, we do not anticipate any
adverse affects in the intestinal lining due to the increased
levels of beneficial bacteria, being consistent with previous
studies conducted in our laboratories.
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[0437] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, limitation or limitations which is not specifically
disclosed herein. The terms and expressions which have been
employed are used as terms of description and not of limitation,
and there is no intention in the use of such terms and expressions
of excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention. Thus, it should be
understood that although the present invention has been illustrated
by specific embodiments and optional features, modification and/or
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this
invention.
[0438] Citations to a number of patent and non-patent references
are made herein. The cited references are incorporated by reference
herein in their entireties. In the event that there is an
inconsistency between a definition of a term in the specification
as compared to a definition of the term in a cited reference, the
term should be interpreted based on the definition in the
specification.
Sequence CWU 1
1
261759DNABacillus Amyloliquefaciens 1atgaaagagg tcataaaaat
ccataaaaac gataacgtgc ttctcgccat gcgccatttt 60aacaaagggg agcggctgca
tacagacgga ctgacgattg aagtgaagga ccctgtaaag 120cgggggcata
aaatcgcgct gcaaaccatt gaagaaaacg gcagcatcat taaatacggt
180ttttcaatcg ggcgtgcgac aaggcggatt tcggccggcg agcatattca
tacccataat 240ctgcaaacga atttatccga tgtgcaagaa tacacgtatt
caccgcgctt cgaggacaat 300ccttttacaa atgagaaccg gacatttaaa
ggctacagaa gagaaaacgg tacatccggc 360gttcggaatg aactgtggat
cgtgcctaca gtcggctgcg taaacggcgt cgctgagaaa 420atcctgcagc
gtttcgtaaa agaggcgaaa gacattgcgc cctttgacaa tgtgctggtt
480ttaaagcacc aatacggctg ctcccagctt ggcgatgatc atgaaaatac
gaaacaaatg 540ctgatgaacg ccatccgtca tccaaatgcg ggcggtgtgc
tggttttagg gctcggctgt 600gaaaataatg agctggcggc gattagggaa
acgctttctg aagtgaacgg agatcgggtg 660aaatttctcg aatcacaagc
tgtcacagct gagactgaca gcagatcaac tcgaaatgcg 720tctaagactt
cttggcattt caaatataaa cacaattaa 7592252PRTBacillus
Amyloliquefaciens 2Met Lys Glu Val Ile Lys Ile His Lys Asn Asp Asn
Val Leu Leu Ala1 5 10 15Met Arg His Phe Asn Lys Gly Glu Arg Leu His
Thr Asp Gly Leu Thr 20 25 30Ile Glu Val Lys Asp Pro Val Lys Arg Gly
His Lys Ile Ala Leu Gln 35 40 45Thr Ile Glu Glu Asn Gly Ser Ile Ile
Lys Tyr Gly Phe Ser Ile Gly 50 55 60Arg Ala Thr Arg Arg Ile Ser Ala
Gly Glu His Ile His Thr His Asn65 70 75 80Leu Gln Thr Asn Leu Ser
Asp Val Gln Glu Tyr Thr Tyr Ser Pro Arg 85 90 95Phe Glu Asp Asn Pro
Phe Thr Asn Glu Asn Arg Thr Phe Lys Gly Tyr 100 105 110Arg Arg Glu
Asn Gly Thr Ser Gly Val Arg Asn Glu Leu Trp Ile Val 115 120 125Pro
Thr Val Gly Cys Val Asn Gly Val Ala Glu Lys Ile Leu Gln Arg 130 135
140Phe Val Lys Glu Ala Lys Asp Ile Ala Pro Phe Asp Asn Val Leu
Val145 150 155 160Leu Lys His Gln Tyr Gly Cys Ser Gln Leu Gly Asp
Asp His Glu Asn 165 170 175Thr Lys Gln Met Leu Met Asn Ala Ile Arg
His Pro Asn Ala Gly Gly 180 185 190Val Leu Val Leu Gly Leu Gly Cys
Glu Asn Asn Glu Leu Ala Ala Ile 195 200 205Arg Glu Thr Leu Ser Glu
Val Asn Gly Asp Arg Val Lys Phe Leu Glu 210 215 220Ser Gln Ala Val
Thr Ala Glu Thr Asp Ser Arg Ser Thr Arg Asn Ala225 230 235 240Ser
Lys Thr Ser Trp His Phe Lys Tyr Lys His Asn 245 25031446DNABacillus
Amyloliquefaciens 3ttgcggaagc tgaataaaaa gatgtgcggt gaatacaatc
aatatcctga aaaagtcctt 60cagttcggcg aaggcaactt tttgcgggcc ttcgcagact
ggcagatcga tcaattgaat 120caactgacgg attttaacgg aagcgttgtc
gccgtacagc cgagaggctc tgagaaaatc 180aaacgtttaa acgagcagga
cggtttattt acgctttttt tgcaggggat aaaggacgga 240aaaccggctg
aagaacacat gatcgtcaaa tccatcagcc gcggcattga tcttttttct
300gactatgatt cctttaaacg gctggcagca caagaggagc ttcgttttat
catttccaat 360acgacagaag cgggaatcac gtttgagaaa ggcgaccggc
ttgaggatag acctcaaaaa 420acattccccg ggaaattagc ggcgttcctt
tatttccgct ataatgcatt tgcgggtgac 480gccgagaaag ggtgcattgt
gcttccatgt gaactggtcg aagaaaacgg acggaaactg 540aaggcggccg
tgcttcaata tgccgggctg tgggagctgg aacctggttt tactcaatgg
600attcatgacg cgaacatctt ctgcaatacc ctcgtggatc ggattgttcc
cggatttcct 660gttgataccg cggaagaact taccgatttt ctcggatacg
aagaccgtct gctggtggtt 720ggtgagcatt attatttgtg ggtgatagaa
gggccggaac agcttcagaa tgagcttcct 780tttgctgagg ccgggctgaa
cgcactgatc acgtctgacc ttacgtctta caggacaaaa 840aaggtgagaa
tcttaaacgg cgctcacacg gcattggcgc cagtggcttt actatacgga
900ctgcaaaccg tccgggaagc ggctgagcac gaggtgacgg gcagattcat
tgaagagctg 960atcaatgaag aaatcctccc tgtgctgcaa atggagggtg
tcacccaata cgcggcagat 1020gtgttacagc ggtttaaaaa cccttatatc
caccattatc tgcaaagcat tacccttaac 1080gccgccgcga aattcaaaac
aagaaacctg ccgacgctga aggcttatat cgaacaggag 1140ggccggctac
ctgagaagct ggtattttcg ttcagcgcca tgatctattc atactggaac
1200ggaccgaaca aaccggctga cggcgagggg gttcttaacc gttttcaaat
ggcgcggtcc 1260cgttgcaaag atgatatgtt tcaagcggca tctgccatat
taggagaaga ccggttgtgg 1320ggaaagaatc tgaaccatct tccggggctg
actgaacgaa cggctttttt tctgtctgtc 1380attcataagc ggggtatgaa
gcatgcgtta catgaatgct gcggaaaaaa gggggaagtg 1440aaatga
14464481PRTBacillus Amyloliquefaciens 4Met Arg Lys Leu Asn Lys Lys
Met Cys Gly Glu Tyr Asn Gln Tyr Pro1 5 10 15Glu Lys Val Leu Gln Phe
Gly Glu Gly Asn Phe Leu Arg Ala Phe Ala 20 25 30Asp Trp Gln Ile Asp
Gln Leu Asn Gln Leu Thr Asp Phe Asn Gly Ser 35 40 45Val Val Ala Val
Gln Pro Arg Gly Ser Glu Lys Ile Lys Arg Leu Asn 50 55 60Glu Gln Asp
Gly Leu Phe Thr Leu Phe Leu Gln Gly Ile Lys Asp Gly65 70 75 80Lys
Pro Ala Glu Glu His Met Ile Val Lys Ser Ile Ser Arg Gly Ile 85 90
95Asp Leu Phe Ser Asp Tyr Asp Ser Phe Lys Arg Leu Ala Ala Gln Glu
100 105 110Glu Leu Arg Phe Ile Ile Ser Asn Thr Thr Glu Ala Gly Ile
Thr Phe 115 120 125Glu Lys Gly Asp Arg Leu Glu Asp Arg Pro Gln Lys
Thr Phe Pro Gly 130 135 140Lys Leu Ala Ala Phe Leu Tyr Phe Arg Tyr
Asn Ala Phe Ala Gly Asp145 150 155 160Ala Glu Lys Gly Cys Ile Val
Leu Pro Cys Glu Leu Val Glu Glu Asn 165 170 175Gly Arg Lys Leu Lys
Ala Ala Val Leu Gln Tyr Ala Gly Leu Trp Glu 180 185 190Leu Glu Pro
Gly Phe Thr Gln Trp Ile His Asp Ala Asn Ile Phe Cys 195 200 205Asn
Thr Leu Val Asp Arg Ile Val Pro Gly Phe Pro Val Asp Thr Ala 210 215
220Glu Glu Leu Thr Asp Phe Leu Gly Tyr Glu Asp Arg Leu Leu Val
Val225 230 235 240Gly Glu His Tyr Tyr Leu Trp Val Ile Glu Gly Pro
Glu Gln Leu Gln 245 250 255Asn Glu Leu Pro Phe Ala Glu Ala Gly Leu
Asn Ala Leu Ile Thr Ser 260 265 270Asp Leu Thr Ser Tyr Arg Thr Lys
Lys Val Arg Ile Leu Asn Gly Ala 275 280 285His Thr Ala Leu Ala Pro
Val Ala Leu Leu Tyr Gly Leu Gln Thr Val 290 295 300Arg Glu Ala Ala
Glu His Glu Val Thr Gly Arg Phe Ile Glu Glu Leu305 310 315 320Ile
Asn Glu Glu Ile Leu Pro Val Leu Gln Met Glu Gly Val Thr Gln 325 330
335Tyr Ala Ala Asp Val Leu Gln Arg Phe Lys Asn Pro Tyr Ile His His
340 345 350Tyr Leu Gln Ser Ile Thr Leu Asn Ala Ala Ala Lys Phe Lys
Thr Arg 355 360 365Asn Leu Pro Thr Leu Lys Ala Tyr Ile Glu Gln Glu
Gly Arg Leu Pro 370 375 380Glu Lys Leu Val Phe Ser Phe Ser Ala Met
Ile Tyr Ser Tyr Trp Asn385 390 395 400Gly Pro Asn Lys Pro Ala Asp
Gly Glu Gly Val Leu Asn Arg Phe Gln 405 410 415Met Ala Arg Ser Arg
Cys Lys Asp Asp Met Phe Gln Ala Ala Ser Ala 420 425 430Ile Leu Gly
Glu Asp Arg Leu Trp Gly Lys Asn Leu Asn His Leu Pro 435 440 445Gly
Leu Thr Glu Arg Thr Ala Phe Phe Leu Ser Val Ile His Lys Arg 450 455
460Gly Met Lys His Ala Leu His Glu Cys Cys Gly Lys Lys Gly Glu
Val465 470 475 480Lys51206DNABacillus Amyloliquefaciens 5atgagggcca
acggcattga agagcgctgc attaccggaa acgcttcaga tgaagagaag 60tttttcgcct
gggcaaaaac ggtgccgatg gcgataggca atccgctgta cagctggacg
120catttagagc tgcagcgctg gttcggcatt tatgacgtat tgaatgaaaa
aacggccgcg 180gcaatttgga aaaaaacgaa tgagctcctg cagggagacg
ggtttggcgc aagggatctc 240attctgaaat caaaagtcaa agtcatttgc
acaaccgacg atcctgccga tgcgctgaca 300taccacgaac tgttgaaaga
aagtgatttt ccggttcaag tgctgcccgg atttcggcct 360gacaaaggac
tggacatcag cagcccgggt tttgccgatt gggtccgttc gctggagagc
420gcttccggaa tagctgtcac cacctaccaa tcgtatttag atgcgttaga
atcccgcgtc 480cgcttttttc acaatgccgg gggaagagtg tcggatcatg
cgttagatca aatggtttat 540gccgagacga ccgaagagga agcggcccgg
atttttgccg ccggattaag cggagagcat 600gtgtcttttg aagatgaaaa
gaaattcaaa acacggacgc tgcaatatct ttgcggtctt 660tacgccgaac
tggactgggc gatgcagttc catattaacg cgttaagaaa taccaataca
720aataagttca gcagcctcgg tcctgatacg ggatatgact caattaatga
tgaacgtatc 780gcaaagccgc ttgcccggct gttagattcc gccgaaaaaa
agcggcaatt gcctaaaacg 840attttgtatt ctttgaatcc gaatgacaac
tatattatcg ccagcatgat caacagcttt 900caggatggga aaacaccggg
gaaaattcaa ttcggcacag cctggtggtt caatgacacg 960aaagatggaa
tgcttcagca aatgaaagcg ttatcaaaca tgggcctgtt cagccgtttt
1020atcgggatgc tgacggattc aagaagtttc ctgtcctacc cccgccatga
atacttcagg 1080cggcttgtct gcacgctgat cggcggctgg gcggaacaag
gcgaagcgcc ttacgatatg 1140gagcttttag gaaggattgt cgagggaatt
tgttaccgga atgctgagga atatttccgc 1200ttttaa 12066401PRTBacillus
Amyloliquefaciens 6Met Arg Ala Asn Gly Ile Glu Glu Arg Cys Ile Thr
Gly Asn Ala Ser1 5 10 15Asp Glu Glu Lys Phe Phe Ala Trp Ala Lys Thr
Val Pro Met Ala Ile 20 25 30Gly Asn Pro Leu Tyr Ser Trp Thr His Leu
Glu Leu Gln Arg Trp Phe 35 40 45Gly Ile Tyr Asp Val Leu Asn Glu Lys
Thr Ala Ala Ala Ile Trp Lys 50 55 60Lys Thr Asn Glu Leu Leu Gln Gly
Asp Gly Phe Gly Ala Arg Asp Leu65 70 75 80Ile Leu Lys Ser Lys Val
Lys Val Ile Cys Thr Thr Asp Asp Pro Ala 85 90 95Asp Ala Leu Thr Tyr
His Glu Leu Leu Lys Glu Ser Asp Phe Pro Val 100 105 110Gln Val Leu
Pro Gly Phe Arg Pro Asp Lys Gly Leu Asp Ile Ser Ser 115 120 125Pro
Gly Phe Ala Asp Trp Val Arg Ser Leu Glu Ser Ala Ser Gly Ile 130 135
140Ala Val Thr Thr Tyr Gln Ser Tyr Leu Asp Ala Leu Glu Ser Arg
Val145 150 155 160Arg Phe Phe His Asn Ala Gly Gly Arg Val Ser Asp
His Ala Leu Asp 165 170 175Gln Met Val Tyr Ala Glu Thr Thr Glu Glu
Glu Ala Ala Arg Ile Phe 180 185 190Ala Ala Gly Leu Ser Gly Glu His
Val Ser Phe Glu Asp Glu Lys Lys 195 200 205Phe Lys Thr Arg Thr Leu
Gln Tyr Leu Cys Gly Leu Tyr Ala Glu Leu 210 215 220Asp Trp Ala Met
Gln Phe His Ile Asn Ala Leu Arg Asn Thr Asn Thr225 230 235 240Asn
Lys Phe Ser Ser Leu Gly Pro Asp Thr Gly Tyr Asp Ser Ile Asn 245 250
255Asp Glu Arg Ile Ala Lys Pro Leu Ala Arg Leu Leu Asp Ser Ala Glu
260 265 270Lys Lys Arg Gln Leu Pro Lys Thr Ile Leu Tyr Ser Leu Asn
Pro Asn 275 280 285Asp Asn Tyr Ile Ile Ala Ser Met Ile Asn Ser Phe
Gln Asp Gly Lys 290 295 300Thr Pro Gly Lys Ile Gln Phe Gly Thr Ala
Trp Trp Phe Asn Asp Thr305 310 315 320Lys Asp Gly Met Leu Gln Gln
Met Lys Ala Leu Ser Asn Met Gly Leu 325 330 335Phe Ser Arg Phe Ile
Gly Met Leu Thr Asp Ser Arg Ser Phe Leu Ser 340 345 350Tyr Pro Arg
His Glu Tyr Phe Arg Arg Leu Val Cys Thr Leu Ile Gly 355 360 365Gly
Trp Ala Glu Gln Gly Glu Ala Pro Tyr Asp Met Glu Leu Leu Gly 370 375
380Arg Ile Val Glu Gly Ile Cys Tyr Arg Asn Ala Glu Glu Tyr Phe
Arg385 390 395 400Phe71077DNABacillus Amyloliquefaciens 7atgaatatca
cgtttcgctg gtacggccaa ggtaatgata cggtgactct tgaacatgtc 60agacaaattc
ccggggtcaa aggcatcgtc tgggctcttc accagaagca cgcgggcgaa
120gtatgggaaa aggaggacat tgaagcggaa gtcagataca ttcaatccta
tgggtttcat 180gcagaggtcg tggaaagcgt gaatgttcat gaagccatta
aactcggtca tgaagaacgc 240ggacaatata ttgagcacta taaacagacc
attcgcaatc tgtccgaatt cggcgtgaaa 300gtcatctgct acaatttcat
gccgatattt gactggacgc gtaccgatat gttccgcgcg 360ctggaggatg
ggtcaaccgc actgtttttt gaaaaagcga aagtggaaag tctccctccg
420gaggaactga tccgcacggt tgaagaatct tctgatttga cgcttcccgg
gtgggagccg 480gagaaaatgg ccaggatcaa ggaactgttt gaggcctatc
aaacggttga tgaaaaacag 540ctttgggcca atcttgagtt ctttttacat
gaaatccttc ctgtagcgga ggaacacggc 600attcaaatgg ccattcatcc
ggatgatccg ccctggtcaa tttttggcct gccccgtatt 660atcaccggtg
agaaaagtta tgaaaagctg caagagattt cagattcacc cgccaactgt
720attacgttgt gcacgggttc gatgggagcg gatcccgcca atgatatggt
gaagatcacc 780aaacactatg cgggagctgc gccgtttgcc catatacgaa
atgtgaaaat ctgcgataac 840ggtgatttta cggaaacttc gcatcttacc
agtgatgggt ccattgatat taccggtgtt 900gtaaaagaat tgcacggcca
agactataaa ggatatgtac ggcctgatca cggccgtcat 960atctggggcg
aacaatgccg gccgggttat ggtttatatg atcgtgcatt aggaattatg
1020tatttgaacg gactgtggga tgcgttttcg tctcatgaga aaggggtgaa gagatga
10778358PRTBacillus Amyloliquefaciens 8Met Asn Ile Thr Phe Arg Trp
Tyr Gly Gln Gly Asn Asp Thr Val Thr1 5 10 15Leu Glu His Val Arg Gln
Ile Pro Gly Val Lys Gly Ile Val Trp Ala 20 25 30Leu His Gln Lys His
Ala Gly Glu Val Trp Glu Lys Glu Asp Ile Glu 35 40 45Ala Glu Val Arg
Tyr Ile Gln Ser Tyr Gly Phe His Ala Glu Val Val 50 55 60Glu Ser Val
Asn Val His Glu Ala Ile Lys Leu Gly His Glu Glu Arg65 70 75 80Gly
Gln Tyr Ile Glu His Tyr Lys Gln Thr Ile Arg Asn Leu Ser Glu 85 90
95Phe Gly Val Lys Val Ile Cys Tyr Asn Phe Met Pro Ile Phe Asp Trp
100 105 110Thr Arg Thr Asp Met Phe Arg Ala Leu Glu Asp Gly Ser Thr
Ala Leu 115 120 125Phe Phe Glu Lys Ala Lys Val Glu Ser Leu Pro Pro
Glu Glu Leu Ile 130 135 140Arg Thr Val Glu Glu Ser Ser Asp Leu Thr
Leu Pro Gly Trp Glu Pro145 150 155 160Glu Lys Met Ala Arg Ile Lys
Glu Leu Phe Glu Ala Tyr Gln Thr Val 165 170 175Asp Glu Lys Gln Leu
Trp Ala Asn Leu Glu Phe Phe Leu His Glu Ile 180 185 190Leu Pro Val
Ala Glu Glu His Gly Ile Gln Met Ala Ile His Pro Asp 195 200 205Asp
Pro Pro Trp Ser Ile Phe Gly Leu Pro Arg Ile Ile Thr Gly Glu 210 215
220Lys Ser Tyr Glu Lys Leu Gln Glu Ile Ser Asp Ser Pro Ala Asn
Cys225 230 235 240Ile Thr Leu Cys Thr Gly Ser Met Gly Ala Asp Pro
Ala Asn Asp Met 245 250 255Val Lys Ile Thr Lys His Tyr Ala Gly Ala
Ala Pro Phe Ala His Ile 260 265 270Arg Asn Val Lys Ile Cys Asp Asn
Gly Asp Phe Thr Glu Thr Ser His 275 280 285Leu Thr Ser Asp Gly Ser
Ile Asp Ile Thr Gly Val Val Lys Glu Leu 290 295 300His Gly Gln Asp
Tyr Lys Gly Tyr Val Arg Pro Asp His Gly Arg His305 310 315 320Ile
Trp Gly Glu Gln Cys Arg Pro Gly Tyr Gly Leu Tyr Asp Arg Ala 325 330
335Leu Gly Ile Met Tyr Leu Asn Gly Leu Trp Asp Ala Phe Ser Ser His
340 345 350Glu Lys Gly Val Lys Arg 3559477DNABacillus
Amyloliquefaciens 9atggacgaaa cggggttttc cagcgtattt tcaaccaact
ttaccggtgc atttctggca 60tcgcaagtgt ttggaaaaga ccttttgaag gcggaatgtc
ccgcgattat taatatttct 120tctatgagtg cgtatacacc gatgacaaag
gtgcccgcct acagcgcggc caaggcggcg 180gttcagaatt ttacgatgtg
gatggctgtt catttcgcaa aaaaagggct gcgggtgaat 240gccatcgctc
cgggtttctt tttgacagcg caaaaccgtg agttgttatt acaggaagat
300ggaagtctga cagaccgatc caataagatt atggcacata caccgatgaa
acgtttcggg 360aaaccggaag accttctggg aacgctcctg tggctttcgg
atgattcatg ctcaggcttc 420gtcaccgggg tcaccgttcc cgtcgacgga
ggttttatgg cttattccgg agtgtga 47710158PRTBacillus Amyloliquefaciens
10Met Asp Glu Thr Gly Phe Ser Ser Val Phe Ser Thr Asn Phe Thr Gly1
5 10 15Ala Phe Leu Ala Ser Gln Val Phe Gly Lys Asp Leu Leu Lys Ala
Glu 20 25 30Cys Pro Ala Ile Ile Asn Ile Ser Ser Met Ser Ala Tyr Thr
Pro Met 35 40 45Thr Lys Val Pro Ala Tyr Ser Ala Ala Lys Ala Ala Val
Gln Asn Phe 50 55 60Thr Met Trp Met Ala Val His Phe Ala Lys Lys Gly
Leu Arg Val Asn65 70 75 80Ala Ile Ala Pro Gly Phe Phe Leu Thr Ala
Gln Asn Arg Glu Leu Leu 85 90
95Leu Gln Glu Asp Gly Ser Leu Thr Asp Arg Ser Asn Lys Ile Met Ala
100 105 110His Thr Pro Met Lys Arg Phe Gly Lys Pro Glu Asp Leu Leu
Gly Thr 115 120 125Leu Leu Trp Leu Ser Asp Asp Ser Cys Ser Gly Phe
Val Thr Gly Val 130 135 140Thr Val Pro Val Asp Gly Gly Phe Met Ala
Tyr Ser Gly Val145 150 15511636DNABacillus Amyloliquefaciens
11atgctgccta agtacgccat ttacgaaaaa cttctgcatg cacaagtggt ggctgtcata
60aggggacaaa acagccaaga ggcgcttgaa gtttcaaaag cggccatttc aggaggtatc
120agcgccattg agcttacata cacaactcct gaagtggaag atgtctttaa
ggaactccgg 180catcaagata ttcttcttgg agccggctct gtaatggata
cagaaacggc gagacacgcc 240attttatcag gcgcctcatt tatcgtcagc
tctcattttg taaaagagat agcatctcta 300tgcaaccagt acagcgtacc
gtatcttccg ggatgcatga gtgtgtctga tatggctttg 360gcgctcgaag
ccggatgtga tgtggtaaag ctgttccccg ctaattcgtt tgacccttca
420tttatcaagt cggtcaacgg ccctctgccg aatgtccgca ttatgccgac
cgggggtatc 480tcattaaaca gcatgaatga ctggctctct gccggagcgg
tcgcggtcgg tgtgggaagc 540gacttgacaa aagcatatca aaagggcggc
tatgaggctg ccgtatccct cagcaaagaa 600tacgtctgcc gaaaaaacga
atatacgggg gtgtaa 63612211PRTBacillus Amyloliquefaciens 12Met Leu
Pro Lys Tyr Ala Ile Tyr Glu Lys Leu Leu His Ala Gln Val1 5 10 15Val
Ala Val Ile Arg Gly Gln Asn Ser Gln Glu Ala Leu Glu Val Ser 20 25
30Lys Ala Ala Ile Ser Gly Gly Ile Ser Ala Ile Glu Leu Thr Tyr Thr
35 40 45Thr Pro Glu Val Glu Asp Val Phe Lys Glu Leu Arg His Gln Asp
Ile 50 55 60Leu Leu Gly Ala Gly Ser Val Met Asp Thr Glu Thr Ala Arg
His Ala65 70 75 80Ile Leu Ser Gly Ala Ser Phe Ile Val Ser Ser His
Phe Val Lys Glu 85 90 95Ile Ala Ser Leu Cys Asn Gln Tyr Ser Val Pro
Tyr Leu Pro Gly Cys 100 105 110Met Ser Val Ser Asp Met Ala Leu Ala
Leu Glu Ala Gly Cys Asp Val 115 120 125Val Lys Leu Phe Pro Ala Asn
Ser Phe Asp Pro Ser Phe Ile Lys Ser 130 135 140Val Asn Gly Pro Leu
Pro Asn Val Arg Ile Met Pro Thr Gly Gly Ile145 150 155 160Ser Leu
Asn Ser Met Asn Asp Trp Leu Ser Ala Gly Ala Val Ala Val 165 170
175Gly Val Gly Ser Asp Leu Thr Lys Ala Tyr Gln Lys Gly Gly Tyr Glu
180 185 190Ala Ala Val Ser Leu Ser Lys Glu Tyr Val Cys Arg Lys Asn
Glu Tyr 195 200 205Thr Gly Val 21013957DNABacillus
Amyloliquefaciens 13gtggcttctg attatggcat ttttacgatc ggtgatgcga
tgatcacctt aaatcccgca 60agcacaggac ctttgcgttt tgttacccat tttgaacgaa
aagcaggagg agctgaactg 120aattttgcga ttgcatgcgc cagattgaat
ctgcactcaa agtgggtcag cagactgggg 180aaggatgagt tcggcagagt
gatttatcag tttgccagag gagaaggaat tgatgtgact 240gacgtctcct
atgttgaagg ctttcctaca tctttaaatg tcaaagagat cagaaatgac
300ggttcaggca agacgtttta ttatcgtttc aattcaccaa ttctgactct
tgcaccggaa 360gatataacag agaatatgtt tgacggcatt cacatcgttc
atcttaccgg tgtgttcctc 420gcgcttgata agaaaaatct tgaaattgct
gaaagggtgc tttccatcgc caaagcaagg 480gggattcccg tatcttttga
tccgaatatc aggctgaagc tgtggacgat tgaggaggcg 540cgtgcggcat
ttcatcaaat ctttccgcat actgatattc ttctggcggg ccgggacgaa
600atgaaacagc tgacaggtat aggggaaaat gaagcattag cccggtttgc
agagacctat 660tcaatcagtc agcttgtcat aaaagacggt gaagcaggtt
ctgcgctgta tcatgatcat 720aagtggattc acaaagaggc tttcccggtt
gcgcctgttg acacggtagg agcgggtgac 780ggctttaatg ccggctacct
gtacagttat ctgcatggtt ttgaaccgga aaaacgtctg 840gagtttgcca
atgcggtcgg tgcgctcgtc accacagttt ccggtgacaa tgaagggctt
900ccctatctag aagaggttct gtcatttgta aacaaagaga ctgtgattga acggtag
95714318PRTBacillus Amyloliquefaciens 14Met Ala Ser Asp Tyr Gly Ile
Phe Thr Ile Gly Asp Ala Met Ile Thr1 5 10 15Leu Asn Pro Ala Ser Thr
Gly Pro Leu Arg Phe Val Thr His Phe Glu 20 25 30Arg Lys Ala Gly Gly
Ala Glu Leu Asn Phe Ala Ile Ala Cys Ala Arg 35 40 45Leu Asn Leu His
Ser Lys Trp Val Ser Arg Leu Gly Lys Asp Glu Phe 50 55 60Gly Arg Val
Ile Tyr Gln Phe Ala Arg Gly Glu Gly Ile Asp Val Thr65 70 75 80Asp
Val Ser Tyr Val Glu Gly Phe Pro Thr Ser Leu Asn Val Lys Glu 85 90
95Ile Arg Asn Asp Gly Ser Gly Lys Thr Phe Tyr Tyr Arg Phe Asn Ser
100 105 110Pro Ile Leu Thr Leu Ala Pro Glu Asp Ile Thr Glu Asn Met
Phe Asp 115 120 125Gly Ile His Ile Val His Leu Thr Gly Val Phe Leu
Ala Leu Asp Lys 130 135 140Lys Asn Leu Glu Ile Ala Glu Arg Val Leu
Ser Ile Ala Lys Ala Arg145 150 155 160Gly Ile Pro Val Ser Phe Asp
Pro Asn Ile Arg Leu Lys Leu Trp Thr 165 170 175Ile Glu Glu Ala Arg
Ala Ala Phe His Gln Ile Phe Pro His Thr Asp 180 185 190Ile Leu Leu
Ala Gly Arg Asp Glu Met Lys Gln Leu Thr Gly Ile Gly 195 200 205Glu
Asn Glu Ala Leu Ala Arg Phe Ala Glu Thr Tyr Ser Ile Ser Gln 210 215
220Leu Val Ile Lys Asp Gly Glu Ala Gly Ser Ala Leu Tyr His Asp
His225 230 235 240Lys Trp Ile His Lys Glu Ala Phe Pro Val Ala Pro
Val Asp Thr Val 245 250 255Gly Ala Gly Asp Gly Phe Asn Ala Gly Tyr
Leu Tyr Ser Tyr Leu His 260 265 270Gly Phe Glu Pro Glu Lys Arg Leu
Glu Phe Ala Asn Ala Val Gly Ala 275 280 285Leu Val Thr Thr Val Ser
Gly Asp Asn Glu Gly Leu Pro Tyr Leu Glu 290 295 300Glu Val Leu Ser
Phe Val Asn Lys Glu Thr Val Ile Glu Arg305 310 315151002DNABacillus
Amyloliquefaciens 15atgaaaacca ttacaatttc tgatgtggcg aaatatgcaa
acgtatctaa aagcaccgta 60tctcaatttt taaatcagcg ctatgactat atgagcgaga
aaacgaaaca aaaaatagaa 120gcggccattc aagagctgaa ctatcagccg
aatttcgtcg cccgcagctt aaaacagaaa 180tcaacattta cggtaggtgt
ggtggtggct aatattcttc acactttttc cactcaggtc 240atcagggcgg
tggaagacta ttttcatgaa caaggatttc atattattgt ctgcaatgcg
300gatgatgaaa cggagaaaga aaagaaatat attgaaatgc tgcgggctaa
gcaggtggac 360ggaattatta tttttccgac gggcgaaaat ctctctttat
atgaaaagat gaaacgcgat 420acgtttccgg ttgtgtttat ggacagaacc
attgaagaac tcggaattcc gacagtgatg 480cttgataatc atcatgcagc
cggtcttgcc gttgaccggt ttatcgaaag cggcattaag 540agaatcgcaa
ttattacgac gtctattatt cgggaaatca gtccgagggt agaaaggatt
600gaagggtata aaaaggcgct tgaccgtcac ggaatgatgg tgcgggacga
atatatcaaa 660acagccgatg cggctgatat ttccaatgtt ttgtcagagc
tgttctcact tgaggagcct 720ccgaaagcga tattggcggc aaacgatatc
gttcttgttg aggtgctgaa gtatatgaag 780gagcatgata tgacaattcc
ggataaggca gccgtcatcg gaattgatga agttcctttt 840gcgggttttt
ttactcctcc gattacgacg atcgtccagc ctgcggcaga aatggcgcga
900aaagcgggaa gccttcttct tcgccagata caagaaaaag atggcggtga
aaaacggatt 960caccgctata aaccggcgct gctcgcaagg caatccggat aa
100216333PRTBacillus Amyloliquefaciens 16Met Lys Thr Ile Thr Ile
Ser Asp Val Ala Lys Tyr Ala Asn Val Ser1 5 10 15Lys Ser Thr Val Ser
Gln Phe Leu Asn Gln Arg Tyr Asp Tyr Met Ser 20 25 30Glu Lys Thr Lys
Gln Lys Ile Glu Ala Ala Ile Gln Glu Leu Asn Tyr 35 40 45Gln Pro Asn
Phe Val Ala Arg Ser Leu Lys Gln Lys Ser Thr Phe Thr 50 55 60Val Gly
Val Val Val Ala Asn Ile Leu His Thr Phe Ser Thr Gln Val65 70 75
80Ile Arg Ala Val Glu Asp Tyr Phe His Glu Gln Gly Phe His Ile Ile
85 90 95Val Cys Asn Ala Asp Asp Glu Thr Glu Lys Glu Lys Lys Tyr Ile
Glu 100 105 110Met Leu Arg Ala Lys Gln Val Asp Gly Ile Ile Ile Phe
Pro Thr Gly 115 120 125Glu Asn Leu Ser Leu Tyr Glu Lys Met Lys Arg
Asp Thr Phe Pro Val 130 135 140Val Phe Met Asp Arg Thr Ile Glu Glu
Leu Gly Ile Pro Thr Val Met145 150 155 160Leu Asp Asn His His Ala
Ala Gly Leu Ala Val Asp Arg Phe Ile Glu 165 170 175Ser Gly Ile Lys
Arg Ile Ala Ile Ile Thr Thr Ser Ile Ile Arg Glu 180 185 190Ile Ser
Pro Arg Val Glu Arg Ile Glu Gly Tyr Lys Lys Ala Leu Asp 195 200
205Arg His Gly Met Met Val Arg Asp Glu Tyr Ile Lys Thr Ala Asp Ala
210 215 220Ala Asp Ile Ser Asn Val Leu Ser Glu Leu Phe Ser Leu Glu
Glu Pro225 230 235 240Pro Lys Ala Ile Leu Ala Ala Asn Asp Ile Val
Leu Val Glu Val Leu 245 250 255Lys Tyr Met Lys Glu His Asp Met Thr
Ile Pro Asp Lys Ala Ala Val 260 265 270Ile Gly Ile Asp Glu Val Pro
Phe Ala Gly Phe Phe Thr Pro Pro Ile 275 280 285Thr Thr Ile Val Gln
Pro Ala Ala Glu Met Ala Arg Lys Ala Gly Ser 290 295 300Leu Leu Leu
Arg Gln Ile Gln Glu Lys Asp Gly Gly Glu Lys Arg Ile305 310 315
320His Arg Tyr Lys Pro Ala Leu Leu Ala Arg Gln Ser Gly 325
33017489DNABacillus Amyloliquefaciens 17atgttctcaa aagatagatt
ccctattatt cttcttttat tttcagccgg ggtgattaac 60tatttagacc gctcagcgct
gtctattgca gcgccgttca tacagcagga tctcacattg 120tctgcaaccc
aaatggggct gattttcagc agtttttctg tcggatacgc tgtgtttaac
180tttctcggcg gtgttgcatc tgaccggtat ggggctaaat tgacattgtg
tacggcgatg 240atcgtttggt ctcttttcag cggagcggtt gcgcttgctt
tcggatttgt aagtcttttg 300attatccgtg ttttattcgg tatgggagag
gggccgctgt cggctgccat aagcaaaatg 360gtcaacaatt ggtttcctcc
gtctcagcgc gccacggtca tcggcctgac aaacagcggc 420acgccgctcg
gccggttttg gattgatgct tggttaattg gatggcggag cttggcagtg 480ccgcgatga
48918162PRTBacillus Amyloliquefaciens 18Met Phe Ser Lys Asp Arg Phe
Pro Ile Ile Leu Leu Leu Phe Ser Ala1 5 10 15Gly Val Ile Asn Tyr Leu
Asp Arg Ser Ala Leu Ser Ile Ala Ala Pro 20 25 30Phe Ile Gln Gln Asp
Leu Thr Leu Ser Ala Thr Gln Met Gly Leu Ile 35 40 45Phe Ser Ser Phe
Ser Val Gly Tyr Ala Val Phe Asn Phe Leu Gly Gly 50 55 60Val Ala Ser
Asp Arg Tyr Gly Ala Lys Leu Thr Leu Cys Thr Ala Met65 70 75 80Ile
Val Trp Ser Leu Phe Ser Gly Ala Val Ala Leu Ala Phe Gly Phe 85 90
95Val Ser Leu Leu Ile Ile Arg Val Leu Phe Gly Met Gly Glu Gly Pro
100 105 110Leu Ser Ala Ala Ile Ser Lys Met Val Asn Asn Trp Phe Pro
Pro Ser 115 120 125Gln Arg Ala Thr Val Ile Gly Leu Thr Asn Ser Gly
Thr Pro Leu Gly 130 135 140Arg Phe Trp Ile Asp Ala Trp Leu Ile Gly
Trp Arg Ser Leu Ala Val145 150 155 160Pro Arg191266DNABacillus
Amyloliquefaciens 19atgaaaaaaa tgttatttat gctggcggtt tgtttatgta
tgatccctgc ggacgtctat 60gccgctgatc tgggacggca gacgttagga acaaatgacg
gctggggagc cgcttccggc 120gggacaaccg gcggcgcgaa agcctcttca
tcgaatgtat acaccgtatc aaacagacag 180cagctcgttt ctgcattagg
gggaagcgcc aacagcacac cgaagattat ttacatacaa 240ggcacaatca
atatgaatac ggacgaccgc aataaaacgc tcggctttga tgattataaa
300gatccggcat atgatccgaa cgcctatttg aaggcgtacg acccggacaa
atggggtaaa 360aaagcgccgt caggcaccca ggaagacgcc agacagcgat
cgcaaaaaaa ccagaaggca 420cgggtcgtgg ttgacattcc ttcaaatacg
acgatcatcg gctcgggttc aaatgccaaa 480gtgacaggcg ggaattttaa
tattaagaac ggcgtcgaca atgtcatcat ccgcaacatt 540gaatttcaag
acgcttacga ctattttccg caatgggacc cgacggacgg cagcagcggc
600aactggaact cggaatatga caatatcacc attaacggcg cgacgcacat
ctggatcgat 660cattgcacct ttaatgacgg atcgaatccc gacagcagtt
ttccctacta ttacgggaga 720aaatatcagc accatgacgg ccagacggat
atagccaacg gggcgaatta tattacattg 780tcctataata aatatcatga
ccatgacaaa ggctccgtca tcggaaacag cgacagcaag 840acgtcggatg
aaggaaaact gaaggtgacc attcaccata actattacca gaacatcgtc
900cagcgcgcac cgcgggtccg gtacgggcag gttcatattt ataataattt
ttacgcaggc 960tctaaaagcg ccgcataccc gttcagctat gcgtggggcg
cgggacacgc gtcaaaaata 1020tatgctcaga ataatgtctt tgaagtgccg
ggtctggctg ctgataaggt catcagcgtc 1080ttcagcggcg gaaaagcgct
tcatgaagac ggcactcttt taaacggcgc cgccattaac 1140gcgtcagccg
ccaacggatt aagtcagtcc gtcggctgga caccgtcatt gcacggttct
1200atcggctcat catcaaatgt aaaatcagat gttatatcta aagccggagc
gggcatatta 1260aaataa 126620421PRTBacillus Amyloliquefaciens 20Met
Lys Lys Met Leu Phe Met Leu Ala Val Cys Leu Cys Met Ile Pro1 5 10
15Ala Asp Val Tyr Ala Ala Asp Leu Gly Arg Gln Thr Leu Gly Thr Asn
20 25 30Asp Gly Trp Gly Ala Ala Ser Gly Gly Thr Thr Gly Gly Ala Lys
Ala 35 40 45Ser Ser Ser Asn Val Tyr Thr Val Ser Asn Arg Gln Gln Leu
Val Ser 50 55 60Ala Leu Gly Gly Ser Ala Asn Ser Thr Pro Lys Ile Ile
Tyr Ile Gln65 70 75 80Gly Thr Ile Asn Met Asn Thr Asp Asp Arg Asn
Lys Thr Leu Gly Phe 85 90 95Asp Asp Tyr Lys Asp Pro Ala Tyr Asp Pro
Asn Ala Tyr Leu Lys Ala 100 105 110Tyr Asp Pro Asp Lys Trp Gly Lys
Lys Ala Pro Ser Gly Thr Gln Glu 115 120 125Asp Ala Arg Gln Arg Ser
Gln Lys Asn Gln Lys Ala Arg Val Val Val 130 135 140Asp Ile Pro Ser
Asn Thr Thr Ile Ile Gly Ser Gly Ser Asn Ala Lys145 150 155 160Val
Thr Gly Gly Asn Phe Asn Ile Lys Asn Gly Val Asp Asn Val Ile 165 170
175Ile Arg Asn Ile Glu Phe Gln Asp Ala Tyr Asp Tyr Phe Pro Gln Trp
180 185 190Asp Pro Thr Asp Gly Ser Ser Gly Asn Trp Asn Ser Glu Tyr
Asp Asn 195 200 205Ile Thr Ile Asn Gly Ala Thr His Ile Trp Ile Asp
His Cys Thr Phe 210 215 220Asn Asp Gly Ser Asn Pro Asp Ser Ser Phe
Pro Tyr Tyr Tyr Gly Arg225 230 235 240Lys Tyr Gln His His Asp Gly
Gln Thr Asp Ile Ala Asn Gly Ala Asn 245 250 255Tyr Ile Thr Leu Ser
Tyr Asn Lys Tyr His Asp His Asp Lys Gly Ser 260 265 270Val Ile Gly
Asn Ser Asp Ser Lys Thr Ser Asp Glu Gly Lys Leu Lys 275 280 285Val
Thr Ile His His Asn Tyr Tyr Gln Asn Ile Val Gln Arg Ala Pro 290 295
300Arg Val Arg Tyr Gly Gln Val His Ile Tyr Asn Asn Phe Tyr Ala
Gly305 310 315 320Ser Lys Ser Ala Ala Tyr Pro Phe Ser Tyr Ala Trp
Gly Ala Gly His 325 330 335Ala Ser Lys Ile Tyr Ala Gln Asn Asn Val
Phe Glu Val Pro Gly Leu 340 345 350Ala Ala Asp Lys Val Ile Ser Val
Phe Ser Gly Gly Lys Ala Leu His 355 360 365Glu Asp Gly Thr Leu Leu
Asn Gly Ala Ala Ile Asn Ala Ser Ala Ala 370 375 380Asn Gly Leu Ser
Gln Ser Val Gly Trp Thr Pro Ser Leu His Gly Ser385 390 395 400Ile
Gly Ser Ser Ser Asn Val Lys Ser Asp Val Ile Ser Lys Ala Gly 405 410
415Ala Gly Ile Leu Lys 420211407DNABacillus Amyloliquefaciens
21atgaaaataa aattatcgat tttatccgct gcggttctgg ccgcaggcat taccgccttt
60gtctggccaa aaacagaggc caatgaaaaa acgcagacgg acgccgtgta tgtttctccc
120gcaggcagcg accaaaatga aggaacgtta gaaaagccgt tccgcacatt
aaaacatgcg 180gctgaaaagg cagaggccgg aacaaccgta ttgattcggg
aagggacgta cgatgaaacg 240tttgaagtaa agcacagcgg cacggccgaa
aagccgatta catttcggaa ctatcagaat 300gagcatgtct caatcagcgg
caaatctgcg ccaaaaagcg attctgaaac tccgttaatt 360caaatccgca
ataaacagta cattaccatt cacggcgtta cgcttgagaa cctctccgta
420tcatcagaag atgcgactgc catggggatc tgcgtcaccg gatcaagcag
tcatatcaat 480attgagggca accatatccg aaacattaaa accacggctg
atgagggaaa tgcacacggc 540atcgcgtttt acggcacagg cgccatgaag
gatgtcagca tcacaaataa tacggttgaa 600aaactgacgc tcggcgcaag
cgaagcggtc gtgctgaacg ggaatgttga cggatttaag 660attgccggca
ataccattcg ggacaataac aatatcggga ttgacgtgat cggatacgaa
720ggaacgtcca agcaaaacga ctatgcgcga aacggcgtca ttgaaaacaa
tacggtgagc 780cataactctt cttacggaaa tcccgcttac ggagatgaat
attcagcggg cggtatttac 840gttgacggcg cagaacatgt ggacattaag
aagaacaccg tttacaacaa tgacctcggc 900attgaagcca cttccgaaca
tagagggaaa tacgcacgag atatccggat tacggataac 960aaggtgtacg
gcaatgctta caccggtatt tcaatcggag gctatgacac gaaacgcggc
1020ggcaccatca attcggtgat cgctcataac
attatgtatc gaaatgacac gaaggacctt 1080gacggcggcc agctgctgct
gcagtacggc acaaaaggga acacgatcga aaaaaacatc 1140atgacggcaa
gcggttcacg gatttttatc gcaaatgatt atacgaaaaa cgaaggcaat
1200actgttaatc ataacgtcta tcacaaagaa gccggaaaag acggcatttg
gaattggaag 1260aacagagaat acgactcatt ctctgcttac cgaaaaggaa
cggcaaacga ttcggattcc 1320atttatgcgg acccgatgta ccgtgacgaa
tcgtcttatg actttacatt aaagcccgga 1380tcaccggccc tgcccgtcat tcagtaa
140722468PRTBacillus Amyloliquefaciens 22Met Lys Ile Lys Leu Ser
Ile Leu Ser Ala Ala Val Leu Ala Ala Gly1 5 10 15Ile Thr Ala Phe Val
Trp Pro Lys Thr Glu Ala Asn Glu Lys Thr Gln 20 25 30Thr Asp Ala Val
Tyr Val Ser Pro Ala Gly Ser Asp Gln Asn Glu Gly 35 40 45Thr Leu Glu
Lys Pro Phe Arg Thr Leu Lys His Ala Ala Glu Lys Ala 50 55 60Glu Ala
Gly Thr Thr Val Leu Ile Arg Glu Gly Thr Tyr Asp Glu Thr65 70 75
80Phe Glu Val Lys His Ser Gly Thr Ala Glu Lys Pro Ile Thr Phe Arg
85 90 95Asn Tyr Gln Asn Glu His Val Ser Ile Ser Gly Lys Ser Ala Pro
Lys 100 105 110Ser Asp Ser Glu Thr Pro Leu Ile Gln Ile Arg Asn Lys
Gln Tyr Ile 115 120 125Thr Ile His Gly Val Thr Leu Glu Asn Leu Ser
Val Ser Ser Glu Asp 130 135 140Ala Thr Ala Met Gly Ile Cys Val Thr
Gly Ser Ser Ser His Ile Asn145 150 155 160Ile Glu Gly Asn His Ile
Arg Asn Ile Lys Thr Thr Ala Asp Glu Gly 165 170 175Asn Ala His Gly
Ile Ala Phe Tyr Gly Thr Gly Ala Met Lys Asp Val 180 185 190Ser Ile
Thr Asn Asn Thr Val Glu Lys Leu Thr Leu Gly Ala Ser Glu 195 200
205Ala Val Val Leu Asn Gly Asn Val Asp Gly Phe Lys Ile Ala Gly Asn
210 215 220Thr Ile Arg Asp Asn Asn Asn Ile Gly Ile Asp Val Ile Gly
Tyr Glu225 230 235 240Gly Thr Ser Lys Gln Asn Asp Tyr Ala Arg Asn
Gly Val Ile Glu Asn 245 250 255Asn Thr Val Ser His Asn Ser Ser Tyr
Gly Asn Pro Ala Tyr Gly Asp 260 265 270Glu Tyr Ser Ala Gly Gly Ile
Tyr Val Asp Gly Ala Glu His Val Asp 275 280 285Ile Lys Lys Asn Thr
Val Tyr Asn Asn Asp Leu Gly Ile Glu Ala Thr 290 295 300Ser Glu His
Arg Gly Lys Tyr Ala Arg Asp Ile Arg Ile Thr Asp Asn305 310 315
320Lys Val Tyr Gly Asn Ala Tyr Thr Gly Ile Ser Ile Gly Gly Tyr Asp
325 330 335Thr Lys Arg Gly Gly Thr Ile Asn Ser Val Ile Ala His Asn
Ile Met 340 345 350Tyr Arg Asn Asp Thr Lys Asp Leu Asp Gly Gly Gln
Leu Leu Leu Gln 355 360 365Tyr Gly Thr Lys Gly Asn Thr Ile Glu Lys
Asn Ile Met Thr Ala Ser 370 375 380Gly Ser Arg Ile Phe Ile Ala Asn
Asp Tyr Thr Lys Asn Glu Gly Asn385 390 395 400Thr Val Asn His Asn
Val Tyr His Lys Glu Ala Gly Lys Asp Gly Ile 405 410 415Trp Asn Trp
Lys Asn Arg Glu Tyr Asp Ser Phe Ser Ala Tyr Arg Lys 420 425 430Gly
Thr Ala Asn Asp Ser Asp Ser Ile Tyr Ala Asp Pro Met Tyr Arg 435 440
445Asp Glu Ser Ser Tyr Asp Phe Thr Leu Lys Pro Gly Ser Pro Ala Leu
450 455 460Pro Val Ile Gln465231065DNABacillus Amyloliquefaciens
23atgataaaga agacgcgtca tctcgctttc ttggctgcac tcggctttgc gctttgtctg
60gccatcgtat gttccgcatc aaagcaggca gaagcggcag cggcttatcc ggatgtgatg
120cagggactta ccggattcgc gggaaatgcg aaggatcata acggaaaagc
aaaatccgcc 180gtaacgggag gccagggcgg ccccgtcgtc tacgtcagca
atctcaatga tttgaaaaac 240aatgcgggcg gaacggaccg caaaaccatc
gtcatcacaa gtgatatttc ttccccgggt 300aaagccgtcg tgacagtcgg
cgccaataaa accatcgtcg gctcttatac cagccacagg 360ctgaccaaca
tctatctgac caccggctcc ggctcaaaca atgtgatttt caaaaatctc
420attatcagcc atagtgccgc cattacgggc aacaacgaca ttccgatgta
catagcgaac 480ggacagaatt actggattga ccacgtgtgg tttgaaggac
acagctacaa tccgaacagc 540cacagtgatt tgggaaagct tctgtatgtc
ggcgccaaag cggattttgt gacgctgtct 600aactctaaat tcacagacca
tctgtacggg ctgattctcg gttatccgaa tgatgataac 660gaaggacgca
attatatcgg ctatccgcat atgacgatca ccaacaacta ttttaataac
720gtctatgtgc gttcaccggg cctgatgaga tacggctatt ttcacgcgaa
aaacaactat 780gtgaccaatt tcaatttagg attcaccatt catacaaacg
cgactgtctt ctcagaagcc 840aactatttcg gtaacggaaa tgaaaaaggc
ggaatgatag acgactacgg gacggcccaa 900ttcaccgaca ccggttcatt
cccgtcgctc aaggccccga aatcaccgcg caccggctgg 960aatccgagat
caaattacag ctacggcacc ctttcagccc aggacgccaa aaacttcgcc
1020caatcttacg cgggagcgca aaatacaaat ctccgctatc cataa
106524354PRTBacillus Amyloliquefaciens 24Met Ile Lys Lys Thr Arg
His Leu Ala Phe Leu Ala Ala Leu Gly Phe1 5 10 15Ala Leu Cys Leu Ala
Ile Val Cys Ser Ala Ser Lys Gln Ala Glu Ala 20 25 30Ala Ala Ala Tyr
Pro Asp Val Met Gln Gly Leu Thr Gly Phe Ala Gly 35 40 45Asn Ala Lys
Asp His Asn Gly Lys Ala Lys Ser Ala Val Thr Gly Gly 50 55 60Gln Gly
Gly Pro Val Val Tyr Val Ser Asn Leu Asn Asp Leu Lys Asn65 70 75
80Asn Ala Gly Gly Thr Asp Arg Lys Thr Ile Val Ile Thr Ser Asp Ile
85 90 95Ser Ser Pro Gly Lys Ala Val Val Thr Val Gly Ala Asn Lys Thr
Ile 100 105 110Val Gly Ser Tyr Thr Ser His Arg Leu Thr Asn Ile Tyr
Leu Thr Thr 115 120 125Gly Ser Gly Ser Asn Asn Val Ile Phe Lys Asn
Leu Ile Ile Ser His 130 135 140Ser Ala Ala Ile Thr Gly Asn Asn Asp
Ile Pro Met Tyr Ile Ala Asn145 150 155 160Gly Gln Asn Tyr Trp Ile
Asp His Val Trp Phe Glu Gly His Ser Tyr 165 170 175Asn Pro Asn Ser
His Ser Asp Leu Gly Lys Leu Leu Tyr Val Gly Ala 180 185 190Lys Ala
Asp Phe Val Thr Leu Ser Asn Ser Lys Phe Thr Asp His Leu 195 200
205Tyr Gly Leu Ile Leu Gly Tyr Pro Asn Asp Asp Asn Glu Gly Arg Asn
210 215 220Tyr Ile Gly Tyr Pro His Met Thr Ile Thr Asn Asn Tyr Phe
Asn Asn225 230 235 240Val Tyr Val Arg Ser Pro Gly Leu Met Arg Tyr
Gly Tyr Phe His Ala 245 250 255Lys Asn Asn Tyr Val Thr Asn Phe Asn
Leu Gly Phe Thr Ile His Thr 260 265 270Asn Ala Thr Val Phe Ser Glu
Ala Asn Tyr Phe Gly Asn Gly Asn Glu 275 280 285Lys Gly Gly Met Ile
Asp Asp Tyr Gly Thr Ala Gln Phe Thr Asp Thr 290 295 300Gly Ser Phe
Pro Ser Leu Lys Ala Pro Lys Ser Pro Arg Thr Gly Trp305 310 315
320Asn Pro Arg Ser Asn Tyr Ser Tyr Gly Thr Leu Ser Ala Gln Asp Ala
325 330 335Lys Asn Phe Ala Gln Ser Tyr Ala Gly Ala Gln Asn Thr Asn
Leu Arg 340 345 350Tyr Pro251577DNABacillus Amyloliquefaciens
25gatcctggct caggacgaac gctggcggcg tgcctaatac atgcaagtcg agcggacaga
60tgggagcttg ctccctgatg ttagcggcgg acgggtgagt aacacgtggg taacctgcct
120gtaagactgg gataactccg ggaaaccggg gctaataccg gatggttgtc
tgaaccgcat 180ggttcagaca taaaaggtgg cttcggctac cacttacaga
tggacccgcg gcgcattagc 240tagttggtga ggtaacggct caccaaggcg
acgatgcgta gccgacctga gagggtgatc 300ggccacactg ggactgagac
acggcccaga ctcctacggg aggcagcagt agggaatctt 360ccgcaatgga
cgaaagtctg acggagcaac gccgcgtgag tgatgaaggt tttcggatcg
420taaagctctg ttgttaggga agaacaagtg ccgttcaaat agggcggcac
cttgacggta 480cctaaccaga aagccacggc taactacgtg ccagagccgc
gggtaatacg taggtggcaa 540gcgttgtccg gaattattgg gcgtaaaggg
ctcgcaagcg ttttcttaag tctgatgtga 600aacccccggg ctcaaccggg
gagggtcatt ggaaaccgag gaacttgagt gcagaagagg 660agagtggaat
tccacgtgta gcggtgaaat gcgtagagat gtggaggaac accagtggcg
720aaggcgactc tctgttctgt aactgacgct gagagagcga agcgtgggga
gcgaacagaa 780ttagataccc tggtagtcca cgccgtaaac gatgagtgct
aagtgttagg gggtttccgc 840cccttagtgc tgcagctaac gcattaagca
ctccgcctgg ggagtacggt cgcaagactg 900aaactcaaag gaattgacgg
gggcccgcac aagcggtgga gcatgtggtt taattcgaag 960caacgcgaag
aaccttacca ggtcttgaca tcctctgaca atcctagaga taggacgtcc
1020ccttcggggg cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc
gtgagatgtt 1080gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt
gccagcattc agttgggcac 1140tctaaggtga ctgccggtga caaaccggag
gaaggtgggg atgacgtcaa atcatcatgc 1200cccttatgac ctgggctaca
cacgtgctac aatggacaga acaaagggca gcgaaaccgc 1260gaggttaagc
caatcccaca aatctgttct cagttcggat cgcagtctgc aactcgactg
1320cgtgaagctg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata
cgttcccggg 1380ccttgtacac accgcccgtc acaccacgag agtttgtaac
acccgaagtc ggtgaggtaa 1440cctttatgga gccagccgcc gaaggtggga
cagatgattg gggtgaagtc gtaacaaggt 1500agccgtatcg gaaggtgcgg
ctggatcacc tcctttctaa ggattttaac ggaatataag 1560accttgggtc ttataac
1577261836DNABacillus Amyloliquefaciens 26atgtacatcg gatcaactaa
cagcaaaggc cttcaccact tggtgtggga aatcgtcgac 60aacagtattg acgaagccct
ggccggttat tgtacagata ttaacatcga gattgaaaaa 120gataacagca
ttaccgttaa ggacaacggg cgcggaattc cggtcggtat ccaggagaag
180atgggccgcc ctgcggttga agtcatcatg accgttctcc acgccggcgg
taaatttgac 240ggaagcggat ataaagtatc cggcggtctt cacggtgtag
gggcatctgt cgtaaacgcc 300ttgtcgacca ctcttgacgt tacggttcat
cgtgacggaa aaatccacta tcaggcgtac 360gagcgcggtg tacctgtggc
cgatcttgaa gtgatcggtg atactgataa gaccggaacg 420attacgcact
tcgttccgga tccggaaatc ttcaaagaaa caatcgtata cgactatgat
480ctgctttcaa accgtgtccg ggaattggcc ttcctgacaa aaggcgtaaa
catcacgatt 540gaagacaaac gtgatggaca agaacggaaa aacgagtacc
actacgaagg cggaatcaaa 600agctatgttg agtacttaaa ccgttccaaa
gaagtcgttc atgaagagcc gatttatatc 660gaaggcgaga aagacggcat
aacggttgaa gttgcattgc aatacaacga cagctataca 720agcaacattt
attctttcac aaataatatc aacacttacg aaggcgggac gcacgaagcc
780ggatttaaaa ccggtctgac ccgtgtcata aacgactatg caagaagaaa
agggattttc 840aaagaaaatg atccgaactt aagcggggat gatgtgagag
aagggctgac tgccattatt 900tcaattaagc accctgatcc gcaattcgaa
gggcagacga aaaccaagct cggcaactcc 960gaagcgagaa cgatcactga
tacgctgttt tcttctgcgc tggaaacatt ccttcttgaa 1020aatccggact
cagcccgcaa aattgttgaa aaaggtttaa tggccgcaag agcgcggatg
1080gcagcgaaaa aagcacggga attgacccgg cgcaaaagtg cgcttgagat
ttccaatctg 1140ccgggcaaac tggcggactg ttcttctaaa gatccgagca
tttccgagct gtatatcgta 1200gagggtgact ctgcgggcgg atcagcgaaa
cagggacggg accgtcattt ccaagctatt 1260ctgccgctgc gcggtaagat
tctgaacgtt gagaaagcca gacttgataa gattctctca 1320aacaatgagg
tcagatcaat gatcacggcc ctcggaacag gaatcggaga agattttaat
1380cttgaaaaag cccgttatca taaagtggtc atcatgacgg atgccgatgt
tgacggcgcc 1440cacatcagaa cgcttttatt aacgttcttc tacagataca
tgcgggaaat catcgaaaac 1500ggctatgtct acattgccca gccgccgctt
tataaagtgc agcagggaaa acgggtggaa 1560tacgcttata acgataagca
gcttgatgag ctgttaaaag aacttccgca atcacctaag 1620cccggcctcc
agcgttataa aggtcttgga gaaatgaacg cgactcagct ttgggaaacg
1680acaatggacc ctgcgaccag aacgcttctg caagtcaatc ttgaagatgc
aatggacgct 1740gacgagactt ttgaaatgct gatgggtgac aaagtagaac
cgcggagaaa cttcatagaa 1800gcaaacgcca gatacgtgaa aaaccttgat atttaa
1836
* * * * *
References