U.S. patent application number 16/185397 was filed with the patent office on 2019-05-23 for compositions comprising bacillus strains and methods of use to suppress the activities and growth of fungal plant pathogens.
The applicant listed for this patent is BIO-CAT Microbials LLC. Invention is credited to Sebhat GEBRECHRISTOS, Steve C. Lamb, Christopher SCHULER, Jessica SPEARS.
Application Number | 20190150454 16/185397 |
Document ID | / |
Family ID | 52468624 |
Filed Date | 2019-05-23 |
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United States Patent
Application |
20190150454 |
Kind Code |
A1 |
SPEARS; Jessica ; et
al. |
May 23, 2019 |
Compositions Comprising Bacillus Strains and Methods of Use to
Suppress The Activities and Growth of Fungal Plant Pathogens
Abstract
This invention provides compositions of Bacillus strains and
methods for using such compositions to inhibit the activity and/or
growth of fungal pathogens of plants. In one embodiment, this
invention provides a composition comprising Bacillus bacteria
selected from the group consisting of Brevibacillus laterosporus
strain CM-3, Brevibacillus laterosporus strain CM-33, Bacillus
amyloliquefaciens BCM-CM5, Bacillus licheniformis ATCC-11946,
Bacillus mojavensis BCM-01, Bacillus pumilus NRRL-1875, Bacillus
subtilis 10 DSM-10, Bacillus subtilis NRRL-1650, Bacillus
megaterium BCM-07, Paenibacillus polymyxa DSM-36, Paenibacillus
chitinolyticus DSM-11030, and combinations thereof. In another
embodiment, this invention provides a method for preparing a
bacterial composition comprising one or more Bacillus strains by
growing Bacillus strain bacteria until the bacteria form spores,
collecting said spores, and formulating said composition.
Inventors: |
SPEARS; Jessica;
(Minnetonka, MN) ; Lamb; Steve C.; (Shakopee,
MN) ; GEBRECHRISTOS; Sebhat; (New Hope, MN) ;
SCHULER; Christopher; (Charlottesville, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIO-CAT Microbials LLC |
Shakopee |
MN |
US |
|
|
Family ID: |
52468624 |
Appl. No.: |
16/185397 |
Filed: |
November 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14911971 |
Feb 12, 2016 |
10154670 |
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PCT/US2014/050710 |
Aug 12, 2014 |
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16185397 |
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62016855 |
Jun 25, 2014 |
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61958994 |
Aug 12, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 63/00 20130101;
C12N 1/20 20130101; C12Q 1/18 20130101; A01N 63/00 20130101; A01N
63/00 20130101; A01N 63/00 20130101; A01N 63/00 20130101 |
International
Class: |
A01N 63/00 20060101
A01N063/00; C12N 1/20 20060101 C12N001/20; C12Q 1/18 20060101
C12Q001/18 |
Claims
1-88. (canceled)
89. A liquid composition comprising Bacillus mojavensis BCM-01
(PTA-121389), water, and a microbial stabilizer.
90. The liquid composition of claim 89, further comprising Bacillus
licheniformis (ATCC-11946).
91. The liquid composition of claim 90, further comprising
Brevibacillus laterosporus strain CM-3 (PTA-3593) and Brevibacillus
laterosporus strain CM-33 (PTA-3592).
92. The liquid composition of claim 89, wherein the Bacillus
mojavensis BCM-01 (PTA-121389) is in a concentration of
1.times.10.sup.8 to 1.times.10.sup.12 colony forming units
(CFU)/gram.
93. The liquid composition of claim 90, wherein each of the
Bacillus mojavensis BCM-01 (PTA-121389) and Bacillus licheniformis
(ATCC-11946) are in a concentration of 1.times.10.sup.8 to
1.times.10.sup.12 CFU/gram.
94. The liquid composition of claim 93, wherein each of the
Bacillus mojavensis BCM-01 (PTA-121389), Bacillus licheniformis
(ATCC-11946), Brevibacillus laterosporus strain CM-3 (PTA-3593) and
Brevibacillus laterosporus strain CM-33 (PTA-3592) are in a
concentration of 1.times.10.sup.8 to 1.times.10.sup.12
CFU/gram.
95. The liquid composition of claim 89, wherein the composition is
a concentrate.
96. A method for inoculating a plant, seed, or soil comprising
applying the liquid composition of claim 89 to a plant, seed, or
soil.
97. The method of claim 96, wherein the liquid composition is
applied to soil, plant foliage, plant seeds, during sowing of plant
seeds, or after plants germinate.
98. The method of claim 96, wherein the liquid composition is
applied by spraying plants or mixing into soil.
99. The method of claim 96, wherein the liquid composition is
applied around the seed and/or to the root zone.
100. The method of claim 96, wherein the composition is applied by
seed coating, spraying in planting furrow with seeds, or foliar
spray.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This International Patent Application claims priority to
U.S. Provisional Patent Application No. 61/958,994, filed Aug. 12,
2013, and U.S. Provisional Patent Application No. 62/016,855, filed
Jun. 25, 2014, the disclosures of both of which are herein
incorporated in their entireties.
FIELD OF THE INVENTION
[0002] The present invention is directed to the use of a mixture of
Bacillus strain concentrates to inhibit the activities and growth
of plant pathogens.
BACKGROUND OF THE INVENTION
[0003] The use of viable microorganisms as root-zone inoculants,
particularly beneficial bacteria, has expanded to include many food
crops including fruits, vegetables, root crops and grains. The
emerging science, referred to as probiotics, is based in part on
the observation that certain soils, which contain specific cultures
of microorganisms that aggressively colonize root surfaces,
suppress a variety of plant diseases. It is postulated that
colonization of root surfaces with deleterious microorganisms can
be prevented by pre-colonization with probiotic microorganisms,
which is referred to as competitive exclusion (CE). Schroth, et al.
(1982) entitled "Disease-Suppressive Soil and Root-Colonizing
Bacteria", Science, Vol. 216: 1376-1381 (1982). In this review,
gram-negative Pseudomonas bacterial species were discussed as being
the most effective in CE, and their ability to produce iron-binding
compounds (called "siderophores") was postulated as the potential
mode-of-action.
[0004] U.S. Pat. No. 5,503,651 discusses plant growth promoting
rhizobacteria (referred to therein as "PGPR"), and in a listing of
41 PGPR bacterial species, 37 of them are Pseudomonas species and
strains. Since strains of these same Pseudomonas species are plant
pathogens, and since plasmid transfer within a bacterial species is
commonplace, there is a concern that there could be transfer of
genetic material from a pathogenic strain, to convert a previously
harmless strain into a pathogenic strain. Accordingly, it is
preferred to use gram-positive bacteria, such as Bacillus, and not
gram-negative Pseudomonas, for probiotics.
[0005] U.S. Pat. No. 4,877,738 discusses a seed inoculum for
application to seeds to be protected from "damping off" fungal
plant disease, and this patent also discusses a method of
protecting growing plants from damping off and root rot fungal
plant disease with a similar composition. The composition includes
a carrier and an effective quantity of protective bacteria,
including Bacillus cereus ATCC 53522, a mutant of Bacillus cereus
ATCC 53522 retaining the capability to produce a plant protecting
toxin effective against Phytophthora megasperma, a mixture of such
mutants, and a mixture of Bacillus cereus ATCC 53522 and such
mutants wherein the inoculum is substantially soil-free. There is
no indication that testing of any other Bacillus species for such
purposes had the same effect.
[0006] U.S. Pat. No. 4,952,229 discusses a microbial plant
supplement and method for increasing plant productivity and
quality, which includes a mixture of microbes with various in vivo
properties. This patent also states that the microbes should be
used with certain organic acids, and with trace metals and
minerals.
[0007] U.S. Pat. No. 4,952,229 describes commercialization hurdles
for mixtures of microbial strains, because it would be difficult
and expensive to insure uniform end-products due to the
difficulties associated with consistently combining a plurality of
microorganisms. Without a consistent and uniform end-product, it
would be difficult to obtain the regulatory permits required for
sales and marketing of such products. It is indicated to be
preferable for a single strain of a single species is the only
active ingredient in a commercial product.
[0008] U.S. Pat. No. 5,441,735 discusses the use of the
microorganism Erwina carotovora subsp. carotovora (E234M403 strain)
which has been modified by mutagenesis to eliminate its soft rot
pathology in rice. When applied to rice plants, this modified
strain competitively excludes pathogenic strains of the same
species. The disadvantage with this strain is the same as discussed
above with Pseudomonas, i.e., a reversion to pathology is possible
since this microorganism is pathogenic prior to mutation. Also, it
is clear that this microorganism is of no benefit to rice that is
not experiencing a soft rot infection.
[0009] U.S. Pat. No. 5,157,207 discusses a method of inoculating
bacteria into rice by introducing a bacterial cell into the seed or
plant, such bacteria belonging to the species Calvibacter xyli.
This creates a modified rice plant that demonstrates a slight yield
improvement (4.81 kg/ha treated vs. 4.66 kg/ha control). Microbial
invasion into rice plant tissue is not preferred, however, as it
raises possible health and regulatory concerns.
[0010] There is a need for new enhancing yields in rice farming
beyond those achieved with modern "high yielding" rice varieties.
From 1964 to 1990, irrigated rice field yields in Asia increased
from 3.0 to 5.8 metric tons/ha. This was largely the result of the
introduction of the higher yielding IR varieties of rice developed
by the International Rice Research Institute in the Philippines,
starting with IR-8 in 1966. At the time of introduction, IR-8
yielded 10 metric tons/ha in the Philippines and up to 14 metric
tons/ha in certain temperate regions of China, where fewer overcast
days resulted in enhanced photosynthesis. Yields from variety IR-8,
as well as other IR varieties, have decreased at a rate of 0.2
metric tons/ha/yr. Pingali, et al., C.A.B. International &
International Rice Research Institute (1997), "Asian rice bowls:
The returning crisis?" New York: CAB International. Yields of 6
metric tons/ha are seldom achieved by Asian farmers. New rice
varieties are being selected more for disease resistance, shorter
photoperiod, and grain quality than for yield. It has become
generally accepted within the industry that yield increases from
advances in plant genetics have been effectively maximized, and
further increases can only be achieved by other means. A similar
need exists for other crops due to pressures on the environment and
increased demand for food production.
[0011] Tomato-Tone.RTM. (plant fertilizer) made by Esporma
comprises a fertilizer and Bacillus species bacteria for use as an
organic fertilizer. Serenade.RTM. Garden Disease Control
(anti-fungal spray for plants) contains Bacillus subtilis, a
soil-dwelling bacterium that controls leaf blight, black mold,
powdery mildew and many other diseases. However, both products
contain relatively low amounts of Bacillus and are designed for
small-scale use.
[0012] Serenade.RTM. (microbial control agent) is a microbial
biological control agent comprising Bacillus subtilis strain QST
713 which protects against fungal and bacterial plant pathogens.
Bacillus subtilis strain QST 713 is a naturally occurring
widespread bacterium that can be used to control plant diseases
including blight, scab, gray mold, and several types of mildew.
SERENADE SOIL.RTM. (biofungicide) is a fungicide designed to
protect young plants against the effects of soil diseases like
Pythium, Rhizoctonia, Fusarium and Phytophthora.
[0013] Annual crop losses due to pre- and post-harvest fungal
diseases exceed $260 Billion annually. About 15,000 fungal species
cause disease in plants. The majority of these fungal plant
pathogens belong to the Ascomycetes and Basidiomycetes.
Gonzalez-Fernandez, et al. Journal of Biomedicine and Biotechnology
Vol. 2010, Article ID 932527, 26 pages, 2010. Plant pathogens can
have many devastating effects on a variety of commercial crops.
Thus there exists in the art a need for compositions and methods
for controlling plant fungal pathogens.
SUMMARY OF THE INVENTION
[0014] This invention provides compositions of Bacillus strains and
methods for inhibiting the activity and/or growth of plant fungal
pathogens.
[0015] In one embodiment, this invention provides a composition
comprising Bacillus bacteria selected from the group consisting of
Brevibacillus laterosporus strain CM-3, Brevibacillus laterosporus
strain CM-33, Bacillus amyloliquefaciens BCM-CM5, Bacillus
licheniformis ATCC-11946, Bacillus mojavensis BCM-01, Bacillus
pumilus NRRL-1875, Bacillus subtilis 10 DSM-10, Bacillus subtilis
NRRL-1650, Bacillus megaterium BCM-07, Paenibacillus polymyxa
DSM-36, Paenibacillus chitinolyticus DSM-11030, and combinations
thereof. Preferably, the composition comprises at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, or 11 of said strains, more preferably the
composition comprises at least 2, 3, 4, 5, 6 or 7 of said strains.
In one mode of this embodiment, the composition comprises spores or
live cells of Bacillus strains, preferably the Bacillus strain
bacteria are in spore form, and the spores may be formulated in a
suspension comprising water, which in turn may be substantially
chlorine-free. The composition may also comprise nutrient organic
compounds, trace minerals, vitamins, growth factors, and/or
adjuvants. Typically, the Bacillus strain bacteria are in a
concentration of 1.times.10.sup.3 to 1.times.10.sup.12 colony
forming units (CFU)/mL. In one mode of this embodiment, the
composition is spray-dried; in another mode, the composition is
lyophilized. In another embodiment, the composition is a
liquid.
[0016] In another embodiment, this invention provides a method for
preparing a bacterial composition comprising one or more Bacillus
strains by growing Bacillus strain bacteria until the bacteria form
spores, collecting said spores, and formulating said composition.
Preferably, the spores are obtained by ultra-filtration,
centrifugation, spray-drying, freeze-drying, or combinations
thereof. Preferably, the spores will germinate and colonize soil,
particularly the rhizosphere.
[0017] In yet another embodiment, the invention provides a method
for inhibiting the growth and/or activity of fungal plant pathogens
comprising applying a bacterial composition comprising one or more
Bacillus strains to a plant, seed for plant, or soil adjacent to a
plant, and the fungal plant pathogens may be members of the
Fusarium species, optionally Fusarium graminearum, Fusarium
oxysporum, Fusarium solani, Fusarium verticilliodes, and Fusarium
virguliforme; Phytophthora species, optionally Phytophthora
medicaginis and Phytophthora sojae; Pythium species, optionally
Pythium aphanidermatum and Pythium ultimum, Rhizoctonia species,
optionally Rhizoctonia solani; and Sclerotinia species, optionally
Sclerotinia sclerotiorum. The composition may be applied to the
soil, to the plant foliage, to the plant seeds, during sowing of
the plant seeds, within 10 days of sowing of the plant seeds,
optionally within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of sowing
the seeds. The composition may be applied to the soil and/or to the
plant foliage after the plants germinate, optionally within 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 days after germination. The composition
may be applied to the soil, to the plant foliage, to the plant
seeds, before or after planting or germination. The composition may
be applied by seed coating, spraying in planting furrow with seeds,
or foliar spray. The composition may be admixed with a soil and
then the soil/composition mixture may be applied to the soil, to
the plant foliage, to the plant seeds, before or after germination.
The composition may be applied after a period of rain or watering
of the plants, and preferably, the composition is applied to the
plant or soil when the temperature is over 65.degree. F. In one
mode of this embodiment, the composition is applied around the seed
of the plant. In another mode of this embodiment, the composition
is applied by spraying plants or mixing into soil; preferably, the
composition is applied to the root zone. The composition is
preferably applied within 2 weeks of plant emergence. The
composition may be applied within 10 days of sowing of the plant
seeds, optionally within 3, 5, or 7 days of sowing the seeds.
[0018] Alternatively, the composition is applied after a fungal
pathogen is present. In a preferred mode, the composition comprises
spores, and the spores germinate and colonize the soil. Typically
the composition comprises between at least about 1.times.10.sup.3
to 1.times.10.sup.12 CFU/mL.
[0019] In still another embodiment, this invention provides a
method for increasing the yields of a plant or protecting a plant
from fungal pathogens comprising applying the bacterial composition
of this invention to the plant, to seeds for the plant or to soil
adjacent to the plant. The plant may be a grain crop, optionally
barley, sorghum, millet, rice, corn, oats, wheat, barley, or hops.
The plant may be an ornamental flower, optionally an annual or
perennial; preferably the ornamental flower is a geranium, petunia,
or daffodil. The plant may be a legume, optionally alfalfa, clover,
peas, beans, lentils, lupins, mesquite, carob, soybeans, peanuts,
or tamarind; preferably, the plant is soybean. The plant may be a
fruit tree, optionally apple, peach, pear, or plum, or the plant
may be a fruit bush, optionally grape, raspberry, blueberry,
strawberry, or blackberry. The plant may be a vegetable, optionally
tomatoes, beans, peas, broccoli, or cauliflower, or the plant may
be a root vegetable, optionally potato, carrot, or beet. The plant
may be a decorative tree, optionally poplar, or the plant may be an
evergreen tree, optionally pine. The plant may be a vine vegetable,
optionally cucumber, pumpkins, or zucchini. In a preferred mode,
the composition of this invention comprises Bacillus strains which
inhibit fungal plant pathogens. Typically, the fungal plant
pathogen is a species of the Fusarium, Phytophthora, Pythium,
Rhizoctonia, or Sclerotinia genera; preferably, the fungal plant
pathogen is one or more of Fusarium graminearum, Fusarium
oxysporum, Fusarium solani, Fusarium verticilliodes, Fusarium
virguliforme, Phytophthora medicaginis, Phytophthora sojae, Pythium
aphanidermatum, Pythium ultimum, Rhizoctonia solani, and/or
Sclerotinia sclerotiorum. More preferably, substantially all of the
fungal plant pathogens are inhibited by one or more of the Bacillus
strains in the composition. More preferably, none of the Bacillus
strains of the composition inhibit the growth of Bradyrhizobium,
Rhizobium, or Trichoderma species. Even more preferably, the
Bacillus strains of the composition secrete anti-fungal
metabolites, and the method of this invention does not require cell
to cell contact of the Bacillus with the pathogen for the
suppression of the fungal pathogen activity or growth.
[0020] In yet another embodiment, the invention provides a method
for inhibiting the growth and/or activity of fungal plant pathogens
by applying a composition comprising aerobic or faculatively
aerobic, Gram-positive, spore-forming rods of Class Bacilli, Order
Bacillales, Family Bacillaceae or Paenibacillaceae, preferably at
least three bacterial strains from species of genus Bacillus,
Brevibacillus, and/or Paenibacillus, where each strain produces a
fungal inhibition zone of at least 1 mm for at least two fungal
strains of different genera selected from Fusarium, Phytophthora,
Pythium, Rhizoctonia, and Sclerotinia. In an alternative
embodiment, the invention provides a method for inhibiting the
growth and/or activity of fungal plant pathogens comprising
applying a composition comprising aerobic or faculatively aerobic,
Gram-positive, spore-forming rods of Class Bacilli, Order
Bacillales, Family Bacillaceae or Paenibacillaceae, preferably
three or more bacterial strains from species of genus Bacillus,
Brevibacillus, and/or Paenibacillus, where each strain is selected
on the basis of at least a 2 mm zone of inhibition against at least
two pathogenic fungal genera while maintaining compatibility (<1
mm zone of inhibition) against beneficial soil organisms,
optionally Bradyrhizobium and/or Trichoderma. Typically, the
composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11
Bacillus strains. Preferably, the composition comprises at least 2,
3, 4, or 5 Bacillus strains. More preferably, composition comprises
four Bacillus strains. In a preferred mode, at least three
bacterial strains are selected from the group consisting of
Brevibacillus laterosporus strain CM-3, Brevibacillus laterosporus
strain CM-33, Bacillus amyloliquefaciens BCM-CM5 (PTA-121388),
Bacillus licheniformis ATCC-11946, Bacillus mojavensis BCM-01
(PTA-121389), Bacillus pumilus NRRL-1875, Bacillus subtilis 10
DSM-10, Bacillus subtilis NRRL-1650, Bacillus megaterium BCM-07
(PTA-121390), Paenibacillus polymyxa DSM-36, Paenibacillus
chitinolyticus DSM-11030, and combinations thereof. In another
preferred mode, the at least three bacterial strains are: (a)
Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus
subtilis 10, and Brevibacillus laterosporus (CM3 and/or CM33); (b)
Bacillus licheniformis, Brevibacillus laterosporus (CM3 and/or
CM33), and Bacillus mojavensis; (c) Bacillus amyloliquefaciens,
Brevibacillus laterosporus (CM3 and/or CM33), and Bacillus pumilus
or (d) Bacillus amyloliquefaciens, Brevibacillus laterosporus (CM3
and/or CM33), Bacillus pumilus, and Paenibacillus polymyxa. In
another embodiment, the composition may comprise BCM-CM5
(PTA-121388), Bacillus mojavensis BCM-01 (PTA-121389), Bacillus
megaterium BCM-07 (PTA-121390), or combinations thereof.
Preferably, the composition comprises spores or live cells of
bacterial strains. More preferably, the bacteria strains are in
spore form. The spores may be formulated in a suspension comprising
water, which preferably is substantially chlorine-free. The
composition may further comprise nutrient organic compounds, trace
minerals, vitamins, growth factors, or adjuvants. Preferably the
composition inhibits fungal plant pathogens which are members of
the Fusarium species, optionally Fusarium graminearum, Fusarium
oxysporum, Fusarium solani, Fusarium verticilliodes, and/or
Fusarium virguliforme; Phytophthora species, optionally
Phytophthora medicaginis and/or Phytophthora sojae; Pythium
species, optionally Pythium aphanidermatum and/or Pythium ultimum,
Rhizoctonia species, optionally Rhizoctonia solani; and/or
Sclerotinia species, optionally Sclerotinia sclerotiorum. More
preferably, substantially all of these species of fungal plant
pathogens are inhibited. Preferably, the Bacillus strains do not
inhibit the growth of beneficial rhizosphere microbes. More
preferably, the Bacillus strains do not inhibit the growth of a
Bradyrhizobium or Trichoderma species. In a preferred mode, the
Bacillus strains of the composition secrete anti-fungal
metabolites. The composition may be applied within 10 days of
sowing of the plant seeds, optionally within 3, 5, or 7 days of
sowing the seeds. The composition may be applied within 10 days of
sowing of the plant seeds, optionally within 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 days of sowing the seeds. The composition may be
applied before the seeds germinate. The composition may be applied
to the soil or to the plant foliage after germination, optionally
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after germination.
[0021] In still another embodiment, this invention provides a
method for selecting a bacterial strain comprising selecting at
least three strains from genera of aerobic, spore-formers selected
from the group consisting of Bacillus, Brevibacillus, and
Paenibacillus and testing whether each of the selected Bacillus
strains produces a fungal inhibition zone on an agar plate of at
least one mm for at least two fungal plant pathogen species
selected from the Fusarium genus, Phytophthora genus, Pythium
genus, Rhizoctonia genus, and Sclerotinia genus. Preferably, each
plant pathogen fungal species is represented by multiple variant
isolates from different geographically located infected field sites
and each bacterial strain will exhibit inhibition of multiple
variant isolates of the minimum two fungal pathogen species. More
preferably, the method further comprises selecting bacteria that
have complementary inhibition patterns where the selected bacteria,
when combined, collectively inhibit multiple strain variants of all
the species of all the plant pathogen fungal genera. Even more
preferably, the method further comprises selecting bacteria which
do not inhibit the growth of at least one beneficial soil microbe,
optionally Bradyrhizobium or Trichoderma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 depicts the "zone of inhibition" measuring
method.
[0023] FIG. 2 A-K depicts the mean inhibition zone sizes of plant
pathogen species by Bacillus strains.
[0024] FIG. 3A-K depicts the percent inhibition of plant pathogen
species by Bacillus strains.
[0025] FIG. 4 depicts the percent fungal pathogens inhibited by
Bacillus strains.
[0026] FIG. 5 shows that the Bacillus concentrate does not inhibit
the growth of beneficial Bradyrhizobium and depicts the Bacillus
and Bradyrhizobium compatibility.
[0027] FIG. 6A-B depicts the Bacillus and Trichoderma
compatibility.
[0028] FIG. 7 depicts the roots of soybeans showing plants treated
with Bacillus and plants not treated with Bacillus. The treated
soybean plants show naturally occurring Rhizobium developing
nitrogen nodules of greater number and larger size on the
roots.
[0029] FIG. 8 is a photograph of rows of untreated (no Bacillus)
soybeans compared to rows of treated soybeans (treated with
Bacillus). The treated soybeans are larger plants with darker
foliage.
[0030] FIG. 9 is a photograph of corn stalks (4 weeks after
germination) showing Bacillus treated on the right (B); untreated
on left (A). The treated plant (B) shows a healthier root system
than the untreated plant.
[0031] FIG. 10 is a photograph of ornamental flowers showing
Bacillus treated on the right (B); untreated on left (A). The
treated plants in (B) show a larger plants with greater foliage
than the untreated plant.
[0032] FIG. 11 is a photograph of poplar trees three weeks after
dip treatment with (B) and without Bacillus treatment (A).
[0033] FIG. 12 (A) depicts the percent of pea seeds germinated on
Day T=7 (384 seeds per treatment) and (B) depicts the percent of
pea seeds germinated on Day T=7 (288 seeds per treatment).
DETAILED DESCRIPTION OF THE INVENTION
[0034] The current invention develops and applies a new model for
biocontrol in which novel concentrates comprised of specific
combinations of Bacillus strains are used to suppress the
activities and growth of an extremely wide spectrum of fungal plant
pathogens while maintaining compatibility with beneficial plant
microbes such as Bradyrhizobium and Trichoderma.
Plant Fungal Pathogens
[0035] There are five major fungal genera that cause significant
losses across the commercial cash crops. These include:
[0036] Fusarium: Fusarium is extremely ubiquitous and can survive
for long periods in the soil increasing its ability to cause
significant crop loss in corn, soy, wheat, and barley. Fusarium
infects roots and seeds as well as seedlings and can act as a
pathogen complex. The species Fusarium oxysporum affects a wide
variety of hosts of any age. Tomato, tobacco, legumes, cucurbits,
sweet potatoes and banana are a few of the most susceptible plants,
but it will also infect other herbaceous plants. Fusarium oxysporum
generally produces symptoms such as wilting, chlorosis, necrosis,
premature leaf drop, browning of the vascular system, stunting, and
damping-off--the killing of newly emerged or emerging
seedlings.
[0037] Pythium: Pythium also has a large host range including soy
and corn. Pythium infects and rots seeds and seedlings and can
cause the common crop disease root rot. This pathogen can cause
both prior and post emergent damage making it a common problem for
fields as well as greenhouses.
[0038] Phytophthora: Phytophthora much like Pythium can damage and
kill plants throughout the growing season. Phytophthora is capable
of causing enormous economic losses on crops worldwide. Members of
the Phytophthora genus are mostly pathogens of dicotyledons, and
are relatively host-specific parasites of considerable economic
importance. Among the plants that are commonly infected by
Phytophthora are soybeans, potatoes, strawberries, cucumbers,
squash and oak and alder trees.
[0039] Rhizoctonia: Rhizoctonia is common in many crops and does
the most damage to plant seedlings, stunting plant growth leading
to significant yield loss. Rhizoctonia solani causes a wide range
of commercially significant plant diseases. It is one of the fungi
responsible for Brown patch (a turf grass disease), damping off in
seedlings, as well as black scurf of potatoes, bare patch of
cereals, root rot of sugar beet, belly rot of cucumber, sheath
blight of rice, and many other pathogenic conditions.
[0040] Sclerotinia: Sclerotinia--"white mold"--is commonly
destructive in the upper Midwest. Lesions develop at stem nodes
during or after flowering. Sclerotinia sclerotiorum can also be
known as cottony rot, watery soft rot, stem rot, drop, crown rot
and blossom blight. The host range is over 400 species including
major agricultural and horticultural plants; among the most
susceptible hosts are soy, snap beans, and sunflowers.
[0041] Plant fungal pathogens are typically controlled by the
application of chemical fungicides either on the seed, into the
soil or by foliar spray. A limited number of commercial biocontrol
agents are in use in the current market, but these are single
strains of bacteria primarily targeted toward a specific fungal
pathogen. The approach thus far for developing biological products
to control plant fungal pathogens has been built on and is based on
the chemical fungicide model. The standard approach has been and
continues to be to isolate and apply a single strain of microbe
which exhibits specific activity against a narrow list of target
plant specific pathogens.
[0042] The present invention applies a new model for biocontrol in
which novel concentrates comprising specific combinations of
bacterial strains are used to suppress the activities and growth of
an extremely wide spectrum of fungal plant pathogens, while
maintaining compatibility with beneficial plant microbes such as
Bradyrhizobium and Trichoderma.
[0043] The present invention provides compositions comprising
mixtures of bacterial strain concentrates selected to inhibit the
activities and growth of a broad spectrum of plant pathogens
including but not limited to five fungal genera. The compositions
of this invention have been successfully tested against thirteen
fungal species and fifty-nine distinct fungal pathogen isolates,
all of which have been isolated from infected field sites.
[0044] For the methods described herein, fungal genera and species
representative of both the Ascomycetes and Basidiomycetes were
used. See Table 1. From the Ascomycetes the Fusarium genus (5
species, 23 variant isolates) and the Sclerotinia genus (1 species,
4 variant isolates) were used. From the Basidiomycetes the
Rhizoctonia genus (1 species, 15 variant isolates), the
Phytophthora genus (2 species, 10 variant isolates) and the Pythium
genus (4 species, 7 variant isolates) were used. These 5 genera
comprised of 13 species encompassing 59 different variant isolates
is representative of fungal pathogens that infect essentially all
plants of commercial importance.
[0045] Another characteristic of this invention is that the
selected antifungal strains all secrete agar-diffusible anti-fungal
metabolites, as demonstrated by a distinct no-growth zone
surrounding the Bacillus growth colony. No cell to cell contact of
the Bacillus cells with the pathogen is necessary for the
suppression of the fungal pathogen activity or growth.
[0046] The bacterial strains described herein may be used to
inhibit the growth and/or activity of fungal plant pathogens. For
example, the Bacillus strain compositions may be used in methods of
inhibiting the growth and/or activity of Fusarium species,
including but not limited to Fusarium graminearum, Fusarium
oxysporum, Fusarium solani, Fusarium verticilliodes, and Fusarium
virguliforme; Phytophthora species, including but not limited to
Phytophthora medicaginis and Phytophthora sojae; Pythium species,
including but not limited to Pythium aphanidermatum and Pythium
ultimum, Rhizoctonia species, including but not limited to
Rhizoctonia solani; and Sclerotinia species, including but not
limited to Sclerotinia sclerotiorum.
TABLE-US-00001 TABLE 1 Fungal plant pathogen species and number of
isolates of each tested. No. of Isolates Fungal Pathogen species
tested Fusarium graminearum 6 Fusarium oxysporum 5 Fusarium solani
6 Fusarium verticilliodes 1 Fusarium virguliforme 5 Phytophthora
medicaginis 9 Phytophthora sojae 1 Pythium aphanidermatum 2 Pythium
ultimum 3 Pythium undefined species 1 1 Pythium undefined species 2
1 Rhizoctonia solani 15 Sclerotinia sclerotiorum 4 Total pathogen
isolates 59 *All fungal pathogen are naturally occurring and were
isolated from infected plants/fields.
[0047] The bacterial strains of this invention show a high level of
inhibition of a broad spectrum of plant pathogens comprising five
different fungal genera, thirteen different species and isolates of
these fifteen species of pathogenic fungi from fifty-nine different
infected field sites. These bacterial strains are combined in
complementary ways such that substantially all of the selected
virulent plant pathogens are inhibited by the bacterial
concentrate. The bacterial strains described herein do not inhibit
the growth of beneficial rhizosphere microbes such as
Bradyrhizobium which is key to symbiotic nitrogen fixation in
legumes and Trichoderma which is a known endophytic beneficial soil
fungus.
Benefits of Bradyrhizobium and Trichoderma to Soil/Plant Health
[0048] Bradyrhizobium is a soil bacterium belonging to the larger
bacterial group Rhizobia which fixes nitrogen inside the root
nodules of legumes such soy, peas, and beans. This symbiotic
relationship between bacteria and plant is critical because plants
cannot readily utilize atmospheric nitrogen and Rhizobia cannot fix
nitrogen independently of a plant host. Adding further importance
to this relationship, Rhizobia are the only known nitrogen-fixing
bacteria able to establish a symbiotic relationship with legume
nodules. Overall, the increased root nodules and useable nitrogen
source increase the total plant yield. Because the overuse of
nitrogen-containing fertilizers poses a significant environmental
threat, the need for nitrogen-fixing Rhizobium has become
increasingly more important.
[0049] Like Rhizobia, Trichoderma species are plant symbionts whose
presence also increase total plant productivity. This increase in
plant productivity is due, at least in part, to increased root
growth and induced systemic resistance in the presence of
Trichoderma. Harman, et al. Nature Reviews Microbiology 2, 43-56
(January 2004).
Bacillus Microbes
[0050] Rich, fertile, biologically active soil contains many
diverse species of microorganisms which are essential to plant
growth and vigor. Among the most common naturally occurring soil
microbes are members of the Bacillus genus. Bacillus are a diverse
group of bacteria which can grow aerobically (need air) or
facultatively (can grow in presence or absence of air). Bacillus
are all capable of entering a dormant state by sporulation (forming
spores). Dormant spores can be thought of as "bacterial seeds"
except that unlike plants, the Bacillus becomes the spore not as
part of the regular succession of stages in their life cycle but
rather in response to stress, which in the soil is most commonly
due to nutrient limitation, drought, or temperature extremes.
[0051] Compositions according to the present invention contain
bacteria which are Gram-positive, aerobic or facultatively aerobic,
spore-forming rods. These bacteria will be referred to as,
"Bacillus," although recent taxonomy has expanded the
classification to identify some of the species as belonging to the
genera Brevibacillus or Paenibacillus (See Table 2); however the
term "Bacillus" as used in this application should be understood to
include all three genera. Bacillus, which are the subject of the
present invention, are added to the soil as soil or seed
inoculants. Bacillus spores are in essence encapsulated naturally.
In addition to having a stable shelf life in product form, the
Bacillus spores will lie dormant in the soil or on the seed until
physical conditions (temperature, moisture, nutrient levels) become
favorable to seed germination, at which time the spores will also
germinate and grow in the rhizosphere (the soil surrounding the
emerging plant roots).
[0052] As the plant grows, Bacillus vegetative cells, which are
progeny of the germinated spores, will grow and propagate in the
root zone, exerting their many unique properties in the soil and in
interaction with the plant roots. If adverse conditions arise in
the soil, such as drought, the Bacillus are capable of
re-sporulation, followed by re-germination when conditions return
to favorable. This ensures that the spore-forming Bacillus will
have an extended presence in the root zone through the growing
season. Non-spore forming soil microbes such as Actinomyces and
Pseudomonas cannot form spores and thus may not survive transient
adverse soil conditions. See also U.S. Patent Application
Publication No. 2003/004528.
[0053] The Bacillus of the current invention can be used in
combination with other beneficial soil microorganisms, including
but not limited to symbiotic nitrogen fixing bacteria of the
Rhizobium and Bradyrhizobium genera, free living beneficial soil
bacteria of the Actinomyces and Streptomyces genera, beneficial
filamentous fungi of the Trichoderma genus, and Micorrhizal fungi
of the Glomus genus.
[0054] The inventors surprisingly found that the use of a suitable
mixture of Bacillus strains alone could produce anti-fungal
activities and increased growth of plants in the absence of any
chemical fertilizers.
[0055] The Bacillus strains that may be used in the compositions
and methods described herein include but are not limited to
Brevibacillus laterosporus strain CM-3 [ATCC Accession No.
PTA-3593], Brevibacillus laterosporus strain CM-33 [ATCC Accession
No. PTA-3592], Bacillus amyloliquefaciens BCM-Cm5, Bacillus
licheniformis ATCC-11946, Bacillus mojavensis BCM-01, Bacillus
pumilus NRRL-1875, Bacillus subtilis 10 DSM-10, Bacillus subtilis
NRRL-1650, Bacillus megaterium BCM-07, Paenibacillus polymyxa
DSM-36, Paenibacillus chitinolyticus DSM-11030, and combinations
thereof. Alternative designations for these strains are shown in
Table 2 herein.
[0056] A composition may comprise at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, or 11 bacterial strains. A composition may consist of at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 strains. A composition
may comprise 2, 3, 4, or 5 strains. A composition may comprise 3
strains. A composition may comprise 4 strains. A composition may
comprise strains of at least three different Bacillus spp. A
composition may comprise strains of two different genera, three
different genera, or at least four different Bacillus spp.
TABLE-US-00002 TABLE 2 Bacillus strains screened during fungal
plant pathogen study. Bacillus Strain* Other designations Bacillus
amyloliquefaciens BCM-CM5 BCM strain deposited as PTA-121388
Bacillus licheniformis ATCC-11946 (Weigmann) Chester, 1333[B-1001]
Bacillus mojavensis BCM-01 BCM strain deposited as PTA-121389
Bacillus pumilus NRRL-1875 B-1875, C-1479, NRS-2003, 2003 is Smith
number; 1479 is NCA number ATCC 6051, CCM 2216, IAM 12118, IFO
13719, Bacillus subtills 10 DSM-10 JCM 1465, LMG 7135, NBRC 13719,
NCIB 3610, NCTC 3610, NRS 744 Bacillus subtilis 1650 NRRL-1650
B-1650 Bacillus megaterium BCM-07 BCM strain deposited as
PTA-121390 Brevibucillus laterosporus BCM-CM3 BCM strain deposited
as ATCC PTA-3593 Brevibacitlus laterosporus BCM-CM33 BCM strain
deposited as ATCC PTA-3592 Paenibacillus polymyxa DSM-36 ATCC 842,
BUCSAV 162, CCM 1459, JCM 2507, LMG 13294, NCIB 8158, NCTC 10343
Paenibacillus chitinalyticus DSM-11030 IFO 15660, NBRC 15660
*Strains designated DSM, ATCC, and NRRL are strains obtained from
culture collections. Strains designated BCM are naturally occurring
bacillus strains obtained by BCM (not from culture
collections).
[0057] Bacillus strains designated BCM-CM5, BCM-01, and BCM-07 were
deposited on Jul. 15, 2014 with the American Type Culture
Collection (ATCC), located at 10801 University Boulevard, Manassas,
Va., 20110-2209, USA under terms of the Budapest Treaty, and
assigned Accession Numbers PTA-121388, PTA-121389, and PTA-121390,
accordingly.
[0058] A composition may comprise at least about 1, 2, 3, 4, 5, 6,
7, 8, or 9.times.10.sup.10 colony forming units per mL (CFU/mL). A
colony forming unit (CFU) is an estimation of the total population
of viable cells (bacterial or fungal) capable of growing and
replicating giving rise to a single colony. This estimation is
based upon the assumption a single cell (or spore) gives rise to a
single colony--thus a colony forming unit. Because spores will
germinate, grow, and replicate on solid media, CFUs can be an
estimate of viable cell or spores. CFUs in the total population may
be estimated by serially diluting the given culture or solution and
evenly spreading a single dilution on a solid complex medium such
as Tryptic Soy Agar (TSA) and incubating at 37.degree. C.
overnight. The number of colonies which grow overnight multiplied
by the total dilution factor will give the number of CFUs/mL, an
estimate of the number of viable spores and/or cells/mL. Spore
estimates may be done in the same protocol with the added step of
holding the dilution at 80.degree. C. for 5 minutes before
spreading on solid media; this step ensures all vegetative cells
are killed. Spores, which are able to withstand high heat, remain
unharmed during this 80.degree. C. incubation. Where a composition
comprises more than one Bacillus strain, the same protocol may be
used, and the concentration of individual strains may be determined
from their distinct and differentiable colony morphology.
[0059] The CFU/ml of each Bacillus strain in the formulated
Bacillus strain concentrates can vary from 1.times.10.sup.3 CFU/ml
up to 1.times.10.sup.12 CFU/ml. The dose of each Bacillus strain in
the Bacillus strain concentrates, when applied to soil or seed,
should be such that the concentration in the Rhizosphere (root
zone) near the seed is a minimum per Bacillus strain of
1.times.10.sup.3 CFU/gram of soil with a range of 1.times.10.sup.3
CFU/gram soil up to 1.times.10.sup.11/gram soil. For seed coating
applications the minimum dose of each Bacillus strain in the
Bacillus strain concentrates should be a minimum of
1.times.10.sup.3 CFU/seed with a range of 1.times.10.sup.3 CFU/seed
up to 1.times.10.sup.10 CFU/seed. The total number of CFU in the
product, in the Rhizosphere, and/or on the seed will be the sum of
the CFU for each strain present.
[0060] A liquid composition may comprise at least about 1, 2, 3, 4,
5, 6, 7, 8, or 9.times.10'.sup.2 colony forming units per mL
(CFU/mL). A liquid composition may comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, or 9.times.10.sup.11 colony forming units per mL
(CFU/mL). A liquid composition may comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, or 9.times.10.sup.10 colony forming units per mL
(CFU/mL). A liquid composition may comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, or 9.times.10.sup.9 colony forming units per mL
(CFU/mL). A liquid composition may comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, or 9.times.10.sup.8 colony forming units per mL
(CFU/mL). A liquid composition may comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, or 9.times.10.sup.7 colony forming units per mL
(CFU/mL). A liquid composition may comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, or 9.times.10.sup.6 colony forming units per mL
(CFU/mL). A liquid composition may comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, or 9.times.10.sup.5 colony forming units per mL
(CFU/mL). A liquid composition may comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, or 9.times.10.sup.4 colony forming units per mL
(CFU/mL). A liquid composition may comprise at least about 1, 2, 3,
4, 5, 6, 7, 8, or 9.times.10.sup.3 colony forming units per mL
(CFU/mL). Preferred ranges of CFU concentration according to this
invention may be the range between any two concentration levels
identified in this paragraph.
[0061] Particularly preferred ranges for liquid composition may
comprise between at least about 0.1-1.times.10.sup.9 colony forming
units per mL (CFU/mL). A liquid composition may comprise between at
least about 10.sup.6-10.sup.10 colony forming units per mL
(CFU/mL). A liquid composition may comprise between at least about
1.times.10.sup.7-1.times.10.sup.9 colony forming units per mL
(CFU/mL). A liquid composition may comprise between at least about
1.times.10.sup.8-1.times.10.sup.9 colony forming units per mL
(CFU/mL). A liquid composition may comprise between at least about
1.times.10.sup.6-1.times.10.sup.8 colony forming units per mL
(CFU/mL). A liquid composition may comprise between at least about
1.times.10.sup.7-1.times.10.sup.8 colony forming units per mL
(CFU/mL). A liquid composition may comprise between at least about
1.times.10.sup.8-1.times.10.sup.10 colony forming units per mL
(CFU/mL). A liquid composition may comprise between at least about
1.times.10.sup.3-1.times.10.sup.6 colony forming units per mL
(CFU/mL). A liquid composition may comprise between at least about
1.times.10.sup.4-1.times.10.sup.11 colony forming units per mL
(CFU/mL). A liquid composition may comprise between at least about
1.times.10.sup.5-1.times.10.sup.12 colony forming units per mL
(CFU/mL). A liquid composition may comprise between at least about
1.times.10.sup.3-1.times.10.sup.12 colony forming units per mL
(CFU/mL).
[0062] A dried powder composition may comprise at least about 1, 2,
3, 4, 5, 6, 7, 8, or 9.times.10.sup.12 colony forming units per
gram (CFU/gram). A dried powder composition may comprise at least
about 1, 2, 3, 4, 5, 6, 7, 8, or 9.times.10'' colony forming units
per gram (CFU/gram). A dried powder composition may comprise at
least about 1, 2, 3, 4, 5, 6, 7, 8, or 9.times.10.sup.10 colony
forming units per gram (CFU/gram). A dried powder composition may
comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9.times.10.sup.9
colony forming units per gram (CFU/gram). A dried powder
composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or
9.times.10.sup.8 colony forming units per gram (CFU/gram). A dried
powder composition may comprise at least about 1, 2, 3, 4, 5, 6, 7,
8, or 9.times.10.sup.7 colony forming units per gram (CFU/gram). A
dried powder composition may comprise at least about 1, 2, 3, 4, 5,
6, 7, 8, or 9.times.10.sup.6 colony forming units per gram
(CFU/gram). A dried powder composition may comprise at least about
1, 2, 3, 4, 5, 6, 7, 8, or 9.times.10.sup.5 colony forming units
per gram (CFU/gram). A dried powder composition may comprise at
least about 1, 2, 3, 4, 5, 6, 7, 8, or 9.times.10.sup.4 colony
forming units per gram (CFU/gram). A dried powder composition may
comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9.times.10.sup.3
colony forming units per gram (CFU/gram). Preferred ranges of CFU
concentration according to this invention may be formed between any
two concentration levels identified in this paragraph.
[0063] Particularly preferred ranges for a dried powder composition
may comprise between at least about 0.1-1.times.10.sup.9 colony
forming units per gram (CFU/gram). A dried powder composition may
comprise between at least about 1.times.10.sup.6-1.times.10.sup.9
colony forming units per gram (CFU/gram). A lyophilized composition
may comprise between at least about
1.times.10.sup.6-1.times.10.sup.9 colony forming units per gram
(CFU/gram). A dried powder composition may comprise between at
least about 1.times.10.sup.7-1.times.10.sup.9 colony forming units
per gram (CFU/gram). A dried powder composition may comprise
between at least about 1.times.10.sup.8-1.times.10.sup.9 colony
forming units per gram (CFU/gram). A dried powder composition may
comprise between at least about 1.times.10.sup.6-1.times.10.sup.10
colony forming units per gram (CFU/gram). A dried powder
composition may comprise between at least about
1.times.10.sup.6-1.times.10.sup.8 colony forming units per gram
(CFU/gram). A dried powder composition may comprise between at
least about 1.times.10.sup.7-1.times.10.sup.8 colony forming units
per gram (CFU/gram). A dried powder composition may comprise
between at least about 1.times.10.sup.8-1.times.10.sup.10 colony
forming units per gram (CFU/gram). A dried powder composition may
comprise between at least about 1.times.10.sup.3-1.times.10.sup.6
colony forming units per gram (CFU/gram). A dried powder
composition may comprise between at least about
1.times.10.sup.4-1.times.10.sup.11 colony forming units per gram
(CFU/gram). A dried powder composition may comprise between at
least about 1.times.10.sup.5-1.times.10.sup.12 colony forming units
per gram (CFU/gram). A dried powder composition may comprise
between at least about 1.times.10.sup.3-1.times.10.sup.12 colony
forming units per gram (CFU/gram).
[0064] The CFU/ml or gm of the formulated Bacillus strain
concentrates can vary from 1.times.10.sup.3 CFU/ml or/gm up to
1.times.10.sup.12 CFU/ml or/gm. The dose of the Bacillus strain
concentrates when applied to soil or seed should be such that the
concentration in the Rhizosphere (root zone) near the seed is a
minimum per Bacillus strain of 1.times.10.sup.3 CFU/gm of soil with
a range of 1.times.10.sup.3 CFU/gm soil up to 1.times.10.sup.11/gm
soil.
[0065] For seed coating applications the minimum dose of the
Bacillus strain concentrates should be a minimum of
1.times.10.sup.3 CFU/seed with a range of 1.times.10.sup.3 CFU/seed
up to 1.times.10.sup.10 CFU/seed.
Preparing a Spore Suspension
[0066] The CFUs in the compositions of this invention are
obtainable by growing cells of the respective Bacillus strains in
liquid monoculture using well-known techniques for bacterial
culture. The cells are grown to high density to induce sporulation.
Suitable microbiological media for the cultivation of Bacillus
strain spores include Tryptic Soy Broth (TSB) and Schaeffer's
Sporulation Medium, as discussed in Biology of Bacilli (Doi, et al.
Butterworth-Heinemann, 1992). In one embodiment, the medium of
choice is prepared in baffled Erlenmeyer flasks and sterilized at
121.degree. C. under 15 psig for 30 minutes, or until rendered
sterile. One may under fill the Erlenmeyer flasks to optimize
aeration during shaking; 200 ml of medium works well in a 4 liter
Erlenmeyer flask. The flask may be fitted with a sterile filter cap
that allows the contents to breadth without becoming contaminated.
The sterile medium is inoculated from a slant culture on tryptic
soy agar, preferably by having a slant medium with good colony
growth melted and poured into the Erlenmeyer flask. The inoculated
medium is then shaken on a rotary orbital shaker at 100-200 rpm and
incubated at 32.degree. C. for 48 hours. Thus prepared, the
Bacillus strains may be 90% sporulated by 48 hours. If vegetative
cells are required, a sample thereof can be taken from the
suspension at 18-24 hours after inoculation. Typically, when using
TSB as the medium, a viable spore count of about 10.sup.8/mL will
be reached within 48 hours.
[0067] The resulting spore suspension, without further preparation,
can be applied to rice or other grain plants. If the spore
suspension is not used within one week of preparation, it may be
refrigerated at 5.degree. C. to preserve it for later use, such
spore suspensions refrigerated at 5.degree. C. have a half-life of
about two months when prepared according the above procedure. The
spores may be isolated by spray drying. The dried spores may be
stored at room temperature (e.g., about 25.degree. C.).
Protocol for Generation of Spores
[0068] In an alternative embodiment, suitable microbiological media
for the cultivation of Bacillus spores include complex media
supplemented with glucose (carbon source) and glutamate (nitrogen
source). In one embodiment, the medium of choice is prepared in
baffled Erlenmeyer flasks and sterilized at about 121.degree. C.
under 15 psig for 30 minutes, or until rendered sterile. One may
under fill the Erlenmeyer flasks to optimize aeration during
shaking; 1 liter of medium in a 3 liter baffled Erlenmeyer flask
works well. The flask may be fitted with a sterile sponge cap that
allows the contents to breathe without becoming contaminated. The
sterile medium is inoculated with a single, well isolated, typical
colony from Tryptic Soy Agar (TSA), a complex solid medium well
suited for the propagation of a wide variety of bacteria and fungi.
The inoculated medium is then shaken on a rotary orbital shaker at
about 190-250 rpm and incubated at 37.degree. C. for about 24 to 48
hours. Thus prepared, the Bacillus strains may be about 85-95%
sporulated by 24 to 48 hours. These spores may be recovered by
centrifugation or more commonly used as a "seed." This "seed" may
be used to inoculate large scale production fermentation vessels
filled with similar media. The culture may be fermented under
typical conditions used for growing aerobic bacteria: incubate at
35-38.degree. C. with an air sparge rate of 0.75-1.50 VVM (volume
of air/volume of liquid/minute), and constant agitation via an
impeller. The culture is fermented until the desired spore
population is reached. The spore population will increase as the
cells are starved for a carbon source such as glucose but the final
population of spores attained is somewhat strain specific. For the
present invention the culture is grown to a final spore
concentration 1.times.10.sup.9 to 1.times.10.sup.10 CFU/mL.
[0069] The above spore suspension may be stabilized by dropping the
pH to 4.2-4.5 by adding acid and then concentrated by
centrifugation. The concentrated slurry may be spray-dried at which
point the spores are stable for at least 12 months at room
temperature. Because spores will germinate when proper conditions
(temperature, nutrients) exist--in the soil, for example--they can
be applied to crops directly or mixed with a nutrient solution to
facilitate germination and then applied. The freshly mixed spore
suspension should be mixed thoroughly before application and should
ideally be used within 48 hours.
[0070] For quality assurance, all products, dry and liquid, are
assayed for viable total population using CFU/mL.
[0071] The Bacillus strain spores may also be purified or
concentrated using methods such as ultra-filtration,
centrifugation, spray-drying or freeze-drying to generate a
packaged product.
Formulations
[0072] The composition may be formulated to allow for storage,
transport, and/or application to soil and/or crops. The formulation
of mixtures of the strains may be adjusted to optimize stability
and sporulation. CFUs as spores and/or viable cells should be
presented at the concentrations described herein.
[0073] The spores may be present in a composition that includes
water, or water and additives and excipients that do not have a
deleterious effect on the action of the spores, or water, additives
and excipients and other ingredients conventionally used in spore
preparations, e.g., binders, dry feeds, and the like. The
composition may also include certain nutrient organic compounds and
trace minerals or vitamins, or growth factors and adjuvants,
although it is unknown if all of these additives act to increase
crop yield. Vitamin additives may be selected, for example, from
pantothenic acid, pyridoxine, riboflavin, thiamin, 25-hydroxy
vitamin A, and vitamins B12, C, D, E, K, biotin, choline, folacin
and niacin. Mineral additives may be selected, for example,
magnesium, potassium, sodium, copper, iodine, iron, manganese
calcium, phosphorous, selenium, chlorine and chromium pincolinate.
The concentration of the vitamins and minerals will depend upon the
plant being treated but, in general, will be between about 0.01%
and about 5% by weight of the dry matter.
[0074] The Bacillus strains may also be combined with other
bacterial species, including but not limited to Shroth's
gram-negative Pseudomonas species. This Pseudomonas species has
been described as being effective in producing siderophores, which
compounds are believed to be the mode-of-action for a demonstrated
increase in crop production by application of this Pseudomonas
species. However, since there are strains of Pseudomonas species
that are plant pathogens, and since plasmid transfer within a
bacterial species can be commonplace, there is a concern such
transfer could convert a previously harmless strain into a
pathogenic strain.
Applying Bacillus Strains to Crops
[0075] The Bacillus strain concentrates of this invention can be
applied to the soil, to the seed or as a foliar application in a
variety of forms including liquids and solids of various
formulations, such as those described herein. The CFU/ml or gm of
the formulated Bacillus strain concentrates can vary from
1.times.10.sup.3 CFU/ml or/gm up to 1.times.10.sup.12 CFU/ml or/gm.
The dose of the Bacillus strain concentrates when applied to soil
or seed should be such that the concentration in the Rhizosphere
(root zone) near the seed is a minimum per Bacillus strain of
1.times.10.sup.1 CFU/gm of soil with a range of 1.times.10.sup.3
CFU/gm soil up to 1.times.10.sup.11/gm soil. For seed coating
applications the minimum dose of the Bacillus strain concentrates
should be a minimum of 1.times.10.sup.3 CFU/seed with a range of
1.times.10.sup.3 CFU/seed up to 1.times.10.sup.10 CFU/seed.
[0076] The spores can be applied as an aqueous suspension obtained
directly from the fermentation process described above, or, if the
spores are purified or concentrated using methods such as
ultra-filtration, centrifugation, spray-drying or freeze-drying,
they should be re-suspended in water before application to crops.
When the spores are applied as an aqueous suspension taken directly
from the fermentation broth, other substances present in the broth
will also be applied to the crops. These non-viable substances,
such as bacterial metabolites or un-utilized microbial nutrients,
will be applied to the plants in very small concentrations, such as
100 grams/ha or less. This level of non-viable substance will not
deleteriously affect the crop.
[0077] Bacillus strains as described herein may be applied to any
type of grain, and to both conventional and hybrid varieties.
During grow-out, applications of the spore suspension can be made
manually, by backpack sprayer or by a more sophisticated mode such
as by helicopter spraying or by any mechanical spraying device
known for use in farming practice.
[0078] The Bacillus strains spores can be applied to crops by
direct application to the soil, coating of the seeds prior to
planting, spraying on the soil, spraying on crops after the seeds
germinate, or within 2 weeks of the seedlings emerging. The
composition may be applied to the soil, to the plant foliage, to
the plant seeds, during sowing of said plant seeds, or after said
plants germinate. The composition may be applied after a period of
rain or watering of said plants. The composition may be applied
within 10 days of sowing of the plant seeds, optionally within 3,
5, or 7 days of sowing the seeds. The composition may be applied
before germination, optionally within 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 days of sowing the seeds. The composition may be applied after
germination, optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after
germination. The composition may be applied by spraying plants or
mixing into soil. The composition may be applied to the root zone.
The composition may be around the seed of the plant. The
composition may preferably be applied to a plant or soil when the
air temperature is over 65.degree. F. The composition may be
admixed with a soil. The composition/soil mixture may be applied to
the plants, seeds, or seedlings. The composition may be applied at
any temperature appropriate for field work, because if the
temperature is not suitable for germination, then the spores will
lie dormant until an adequate temperature occurs. The composition
may be applied within 2 weeks of plant emergence. The plants may be
dipped into a liquid spore composition, optionally comprising about
250.times.10.sup.6 to 5.times.10.sup.9 CFU/mL of the Bacillus
strains described herein. The plants may be dipped for about 1-30
seconds or 30 seconds and then planted. The plants may be treated a
second time, by spraying the plants about 14 days after treatment
by dipping.
[0079] The composition may be sprayed directly onto row crops as a
foliar spray. The crops may be a grain crop, optionally rice, corn,
alfalfa, oats, wheat, barley, or hops. The crop may be wheat,
soybeans, cabbage, ornamental flowers, optionally geraniums,
petunias, daffodils, or trees, optionally poplar trees. New
seedling fruit trees or bushes may be dipped into containers
comprising a liquid spore concentrate. Crops whose treatment is
contemplated and suitable application routes, are shown in Table
3
[0080] The crops may be treated with a composition comprising a
Bacillus strain bacteria selected from the group consisting of
Brevibacillus laterosporus strain CM-3, Brevibacillus laterosporus
strain CM-33, Bacillus amyloliquefaciens BCM-CM5, Bacillus
licheniformis ATCC-11946, Bacillus mojavensis BCM-01, Bacillus
pumilus NRRL-1875, Bacillus subtilis 10 DSM-10, Bacillus subtilis
NRRL-1650, Bacillus megaterium BCM-07, Paenibacillus polymyxa
DSM-36, Paenibacillus chitinolyticus DSM-11030, and combinations
thereof. The composition may comprise at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or 11 of said strains. The composition may comprise at
least 2, 3, 4, or 5 of said strains. The composition may comprise
spores or live cells of Bacillus strains. The Bacillus strain
bacteria may be in spore form. The spores may be formulated in a
suspension comprising water including but not limited to
substantially chlorine-free. The composition may further comprise
nutrient organic compounds, trace minerals, vitamins, growth
factors, or adjuvants. The Bacillus strain bacteria may be applied
to the crops in a concentration of 1.times.10.sup.3 to
1.times.10.sup.12 cells/mL or 1.times.10.sup.3 to 1.times.10.sup.12
cells/gram of soil. The composition may be spray-dried or
lyophilized. The spores may be obtained by ultra-filtration,
centrifugation, spray-drying, freeze-drying, or combinations
thereof. The spores will preferably germinate and colonize the
soil.
[0081] The application of the Bacillus composition may inhibit the
growth and/or activity of fungal plant pathogens, optionally a
member of the Fusarium species, optionally Fusarium graminearum,
Fusarium oxysporum, Fusarium solani, Fusarium verticilliodes, and
Fusarium virguliforme; Phytophthora species, optionally
Phytophthora medicaginis and Phytophthora sojae; Pythium species,
optionally Pythium aphanidermatum and Pythium ultimum, Rhizoctonia
species, optionally Rhizoctonia solani; and Sclerotinia species,
optionally Sclerotinia sclerotiorum. The composition may be applied
after a fungal pathogen is present.
TABLE-US-00003 TABLE 3 Crops and Exemplary Applications Routes
FOLIAR- IN FURROW SEED SEEDLING YOUNG FOLIAR CROP APPLICATION
TREATMENT ROOT DIP PLANT MATURE Soybeans and other legumes X X X X
including peanuts Corn, maize X X X Wheat, rye, barley and other X
X X grasses Ornamental flowers X X X Fruit trees (apple, peaches, X
X X X pears, plums etc) Fruit bushes (grapes, X X X raspberries,
blueberries, strawberries, blackberries etc) Vegetables (tomatoes,
all X X X beans, peas, broccoli, cauloflower) Root vegetables
(potatoes, X X X carrots, beets) Decorative trees such as poplar X
X Vine vegetables such as X X X cucumbers, pumpkins, zucchini
[0082] All publications (e.g., Non-Patent Literature), patents,
patent application publications, and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains. All such
publications (e.g., Non-Patent Literature), patents, patent
application publications, and patent applications are herein
incorporated by reference to the same extent as if each individual
publication, patent, patent application publication, or patent
application was specifically and individually indicated to be
incorporated by reference.
[0083] Although methods and materials similar or equivalent to
those described herein may be used in the invention or testing of
the present invention, suitable methods and materials are described
herein. The materials, methods and examples are illustrative only,
and are not intended to be limiting.
[0084] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
EXAMPLES
Example 1
Screening for Fungal Inhibition
[0085] A standard agar-plate-based zone-of-inhibition method was
used to screen select members of the Bacillus, Brevibacillus, or
Paenibacillus genera comprising 9 different species and a total of
11 different strains. The 11 strains of Bacillus, Brevibacillus, or
Paenibacillus and their identity are given in Table 2. The 13
species of fungal plant pathogens tested are listed in Table 1.
[0086] The zone-of-inhibition screening methodology is given in
FIG. 1. Fungal pathogen species were grown on Potato Dextrose Agar
(PDA) except for Phytophthora which was grown on V8 Agar. All fungi
were stored at 4.degree. C. until used. Bacillus strains were
subcultured in LB broth overnight at 37.degree. C. with shaking at
200 rpm. The subculture (0.5 ml) was used to inoculate 50 ml LB
broth and grown overnight at 37.degree. C. with shaking at 200 rpm.
An 8 mm plug from the center of an agar plate (TSA, 6.5 pH) was
removed. An 8 mm plug of pathogen was placed in the empty hole. 10
.mu.l of the Bacillus strain was added at appropriate time to the
perimeter of the plate (up to 3 Bacillus strains per plate). Plates
were incubated at room temperature until the pathogen covered the
plate. Pathogen inhibition zones were measured with calipers at
90.degree. angle as shown in FIG. 1. The mean of two zone
measurements were reported and scored only measured if they were 1
mm or greater. Experiments were performed in duplicate.
[0087] The mean inhibition zone sizes (in mm) of plant pathogens
when grown in the presence of Bacillus strains are shown
graphically in FIG. 2. Each Bacillus strain shows a distinct
profile in ability to inhibit fungal plant pathogens. While species
such as Bacillus subtilis inhibits at least one member of every
fungal genera, others such as Bacillus megaterium and Paenibacillus
chitinolyticus only inhibit one or two fungal genera.
[0088] The percent of plant pathogen species inhibited when grown
in the presence of Bacillus strains are shown graphically in FIG.
3. Each Bacillus strain shows a distinct profile in ability to
inhibit fungal plant pathogens. While species such as Bacillus
subtilis inhibit nearly all 59 pathogen isolates, others such as
Bacillus megaterium and Paenibacillus chitinolyticus only inhibit a
couple of isolates.
[0089] FIG. 4 highlights the percent of fungal plant pathogen
isolates inhibited by each Bacillus strain. The combination of all
data figures leads to the following novel combination of Bacillus
strain concentrates to control fungal plant pathogen activity and
growth: [0090] #1: Bacillus amyloliquefaciens, Bacillus
licheniformis, Bacillus subtilis 10, and Brevibacillus laterosporus
(CM3 and/or CM 33) [0091] #2: Bacillus licheniformis, Brevibacillus
laterosporus (CM3 and/or CM 33), and Bacillus mojavensis [0092] #3:
Bacillus amyloliquefaciens, Brevibacillus laterosporus (CM3 and/or
CM 33), and Bacillus pumilus [0093] #4: Bacillus amyloliquefaciens,
Brevibacillus laterosporus (CM3 and/or CM 33), Bacillus pumilus and
Paenibacillus polymxa
[0094] The initial screen for bacillus antifungal properties were
completed on solid agar media (TSA). Because there was no actual
contact between the Bacillus and the fungus, the zone of inhibition
of fungal growth observed in the presence of Bacillus had to be due
to an agar diffusible compound produced and excreted by the
Bacillus.
[0095] This test may be used to evaluate the antifungal activity of
the compositions and methods described herein. Combinations of
strains/species may be selected based on their efficacy against
pathogens in vitro as shown in this test. This test allows for the
selection of combinations of Bacillus to target multiple fungal
pathogens. For example, a combination may be selected to combine
different Bacillus species/strains that have antifungal activity to
target a larger group of pathogens together than the Bacillus
species/strains would individually. The inventors surprisingly
discovered that combinations of Bacillus strains/species described
herein shown unexpected improved antifungal properties as compared
to single strains (or species).
Example 2
Compatibility of Bacillus Concentrates with Bradyrhizobium and
Trichoderma
[0096] Selected Bacillus concentrates and individual Bacillus
cultures were tested for inhibition of two beneficial soil microbes
Bradyrhizobium, a naturally occurring bacteria of critical
importance in legume symbiotic nitrogen fixation, and Trichoderma,
a naturally occurring beneficial soil fungus. Commercially
available soil inoculant products were used as sources for the two
microbes.
[0097] For Bradyrhizobium a cross streak assay was used in which a
center streak of Bradyrhizobium was made and then cross streaks of
the Bacillus cultures to be tested were streaked perpendicular to
and just touching the center Bradyrhizobium streak. Bacillus cross
streaks were done at 0, 2, 4 and 6 days after the initial
Bradyrhizobium streak and the plates were incubated until good
growth was obtained for both Bradyrhizobium and Bacillus.
[0098] From FIG. 5 it is clear that there is no inhibition of
growth of Bradyrhizobium by the Bacillus cultures.
[0099] Experiments were conducted by placing discs containing equal
amount of each cultures (0.05 ml) on agar plates as shown in FIG.
6. The Trichoderma was added to the middle of each Tryptic Soy Agar
(TSA) plate. The plate was the incubated at 37.degree. C. for, and
monitored every 12-18 hours until fungus covered the entire plate.
Additionally, another plate was incubated at 37.degree. C. for 3
days with the Bacillus strains alone. The Trichoderma was then
added to the middle of those plates and incubated again at
37.degree. C. In neither case was there inhibition of the
Trichoderma by Bacillus.
[0100] From FIGS. 5 and 6 it is clear that these Bacillus strains
do not inhibit either Bradyrhizobium or Trichoderma.
Example 3
Field Study
[0101] In vitro results may be further confirmed by in vivo
greenhouse and/or field trials. During the greenhouse trials each
set of 6 plants will be infected with a representative Fusarium,
Phytophthora, Pythium, Rhizoctonia, or Sclerotinia in the presence
and absence Bacillus strain concentrate. Total plant growth, root
mass, and fungal population will be assessed for all sets of
plants.
Example 4
Greenhouse Study
[0102] In vitro results of Examples 1 and 2 were confirmed by in
vivo greenhouse trials. Planting medium starter bricks were
rehydrated with water at T=-5 days. On T=-3 days the hydrated
planting medium was inoculated by direct mixing into the hydrated
planting medium starter bricks with freshly prepared fungal
inoculum (either a mix of 3 Pythium ultimum isolates or a mix of 3
Rhizoctonia solani isolates), fungal pathogen and Bacillus Blend 1
(Brevibacillus laterosporus 3 and 33, Bacillus licheniformis, and
Bacillus mojavensis) or Bacillus Blend 2 (Brevibacillus
laterosporus 3 and 33, Bacillus licheniformis, Bacillus subtilis
10, and Bacillus amyloliquefaciens), or equivalent volume of water
as the control.
[0103] On planting day (T=0) inoculated soil medium was separately
distributed into several 24 well starter flats beginning with the
control. Four pea seeds were planted into each well at the depth of
1/2 inch and starter trays were placed onto an indoor growth light
table with enclosing cover and checked daily for germination.
Temperature was maintained at a constant 31.degree. C. Germination
counts were recorded on day 7 post planting (T=+7 days) and the
experiment terminated at T=+14 days.
[0104] Observations for growth were recorded at T=7 days. In both
test cases, Pythium and Rhizoctonia caused damping off, observed as
low germination and stunted growth. Both Bacillus Blend 1 and
Bacillus Blend 2 were able to suppress the effects of the fungal
pathogen observed as higher germination numbers (FIGS. 12A and 12B)
and larger, healthier plants (FIG. 1). Observations at T=14 days
showed no increase of disease or seedling die-off other than that
observed at T=7 days.
[0105] From this data it is clear that the mixtures of Bacillus
inhibit the growth and activity of Pythium and Rhizoctonia plant
pathogens, leading to higher germination and larger, healthier
plants. Accordingly, mixtures of Bacillus cells as described
herein, including but not limited to the mixtures described in this
Example, as well as Examples 1 and 2, may be expected to lead to
higher germination and larger, healthier plants as described
herein.
Example 5
Formulation of a Liquid Concentrate of Six Bacillus Strains
[0106] Spores from six (6) strains of Bacillus are grown in
monoculture as described herein, and the liquid concentrates from
the respective centrifugation steps are stabilized as described
herein. An amount from each of the six Bacillus strain liquid
concentrates is mixed into a diluting liquid with a standard
multiple blade, flat blade impeller at a sufficient RPM such that
the desired final concentration of each Bacillus strain was
attained. The equipment for liquid mixing and blending can be any
liquid mixing equipment standard and known to one skilled in the
art of liquid formulation. Sufficient power per volume must be used
to ensure good hydration of all solid components and good mixing to
attain a homogeneous blend. The final concentration of each of the
Bacillus strains can range from 1.times.10.sup.3 CFU/ml final
liquid up to 1.times.10.sup.11 CFU/ml final liquid but is more
generally in the range of 1.times.10.sup.8 CFU/ml of final liquid
up to 1.times.10.sup.10 CFU/ml of final liquid.
[0107] The composition of the diluting liquid can be water, or
water, additives and excipients that do not have a deleterious
effect on the action of the spores or water, additives and
excipients and other ingredients conventionally used in spore
preparations, e.g., microbial stabilizers, thickeners,
hydrocolloids, pH buffers and the like. The composition may also
include certain nutrient organic compounds, trace minerals,
vitamins and growth factors. The concentration of these nutrient
additives will depend on the type of additive and the plant and
soil being treated but, in general, will be between about 0.01% and
about 5% by weight of final liquid formulation.
[0108] The CFU/ml of spores in the formulated liquid concentrate is
determined by doing a total spore count where the appropriate
serial dilution is prepared by methods well known to those skilled
in the art, and the final dilution is then subjected to 80.degree.
C. for 5 minutes, quenched in an ice bath, and then plated on
standard Tryptic Soy Agar. After incubation at 37.degree. C. for 18
to 24 hours, the colonies per plate are counted, and the spore
count is calculated by multiplying the colonies per plate by the
total dilution factor to obtain the CFU/ml in the formulated
Bacillus concentrate liquid. Since each Bacillus strain has
distinct and differentiable colony morphology, the individual
Bacillus strains can be quantified in each Bacillus concentrate by
counting the respective numbers of colony types on each plate.
Example 6
Formulation of a Dry Concentrate of Six Bacillus Strains
[0109] Spores from six (6) strains of Bacillus are grown in
monoculture as described herein and the respective liquid
concentrates are spray dried as described herein. An amount from
each of the six Bacillus strain dry powder concentrates is weighed
into powder blending equipment along with an inert powder
carrier/diluent, and blended using a V-blender such that the
desired final concentration of each Bacillus strain is attained.
The equipment for powder mixing and blending can be any powder
mixing equipment standard and known to one skilled in the art of
powder blending and formulation, including but not limited to
rotating blenders such as a V-Blender or a ribbon blender.
Sufficient component inter-mixing must be attained to ensure a
homogeneous blend. The final concentration for each of the Bacillus
strain can range from 1.times.10.sup.6 CFU/gm final powder up to
1.times.10.sup.12 CFU/gm final powder but is more generally in the
range of 1.times.10.sup.9 CFU/gm final powder up to
1.times.10.sup.11 CFU/gm final powder.
[0110] The composition of the powder/diluents can be any inert
powdered diluent standard to one skilled in the art of powder
formulations, any inert powdered diluent, dry additives and dry
excipients that do not have a deleterious effect on the action of
the spores, or any inert powdered diluent, additives and excipients
and other ingredients conventionally used in powered spore
preparations, e.g., anti-caking agents, flow agents, desiccants and
the like. The composition may also include certain powdered
nutrient organic compounds, trace minerals, vitamins and growth
factors. The concentration of these nutrient additives will depend
on the type of additive and the plant and soil being treated but,
in general, will be between about 0.01% and about 5% by dry weight
of final powder formulation.
[0111] The CFU/gm of spores in the formulated powder concentrate is
determined by doing a total spore count where the appropriate
serial dilution is prepared by methods well known to those skilled
in the art, and the final dilution is then subjected to 80.degree.
C. for 5 minutes, quenched in an ice bath, and then plated on
standard Tryptic Soy Agar. After incubation at 37.degree. C. for 18
to 24 hours, the colonies per plate are counted, and the spore
count is calculated by multiplying the colonies per plate by the
total dilution factor to obtain the CFU/gram in the formulated
Bacillus concentrate powder. Since each Bacillus strain has
distinct and differentiable colony morphology, the individual
Bacillus strains can be quantified in each Bacillus concentrate by
counting the respective numbers of colony types on each plate.
Example 7
Application of Bacillus to Soybeans
[0112] Solution of Bacillus spores in liquid form and containing
four strains of Bacillus containing a minimum of 250 million CFU/ml
is applied at the rate of 1 gallon per acre through mechanical
spraying apparatus commonly found on U.S. farms. The liquid
suspension may be sprayed onto any typical row crop seeds such as
soybeans, corn, wheat, maize directly into the furrow of soil onto
the seed as it is planted. Spore concentrate may be applied
simultaneously as the seed is deposited into the furrow.
Concentration of spores can be adjusted as high as 5 billion
CFU/mL, and dose applied to seeds at planting can be adjusted to as
low as 32 fluid ounces per acre. Seeds treated in furrow should be
conventional seeds with no additional materials added, such as
pesticides, fertilizers and the like. Plants can be examined for
evidence of fungal pathogens from time of germination through
harvest. The pictures in FIGS. 7 and 8 demonstrate the
effectiveness of the Bacillus blend acting in synergy with
naturally occurring Rhizobium at developing nitrogen nodules on
soybeans. No additional Rhizobium was added to the field, and the
only treatment was addition of the Bacillus at time of planting the
seeds.
Example 8
Application of Bacillus to Row Crops
[0113] Solution of Bacillus spores (containing four strains) in dry
form (spray dried) containing concentration of up to 500 billion
per gram CFU's is dissolved in 50 to 250 gallons of water in
typical liquid holding/spray vessels used on farms; material is
slightly mixed to develop a liquid suspension. The liquid
suspension may be sprayed onto any typical row crop seeds such as
soybeans, corn, wheat, maize etc. directly into the furrow of soil
onto the seed as it is planted. Spore concentrate may be applied
simultaneously as the seed is deposited into the furrow. The rate
of application can range from 1 gallon per acre to 100 milliliters
per acre depending on level of pathogen control required. FIGS. 7
and 9 show the beneficial effects of Bacillus applied to soybeans
and corn, respectively.
Example 9
Application of Bacillus by Seed Coating for Row Crops
[0114] A liquid suspension comprising four Bacillus strains and
ranging in total CFU/ml of 50-100 billion CFU/ml may be applied
directly to row crop seeds through any number of application
methods as a coating and then dried to form a micro layer of dried
Bacillus spores on the seed. Seeds are then planted per usual
farming practices. Seed coating can be applied in a variety of ways
including adding liquid spores to seed in a rotary drum type drying
mechanism and rotated for 2-15 minutes to ensure adequate
distribution; spores can be applied in a thin mist spray across a
conveyor full of seeds and air dried to achieve coating effect or
sprayed onto surface of seeds as one of multiple spray ports as
seeds are passed through a rotating screw type conveyor. Final
spore concentrate on seeds may range from 0.1 ounce per 50 pounds
up to 2 fluid ounce per 50 pounds.
Example 10
Application of Bacillus to Row Crops by Foliar Spray
[0115] At first onset of visible pathogen infestation, liquid
Bacillus spore concentrate containing 5-11 strains (250 million
CFU/ml up to 5 billion CFU/ml) can be sprayed directly onto row
crops as a foliar spray. Application rate can be varied to achieve
a dose rate of anywhere from 1 gallon to 1 quart per acre. Higher
concentrations of spores can be diluted in water to achieve a more
uniform distribution. Concentrations of 50-100 billion CFU/ml can
diluted in 50-250 gallons of water prior to spraying. Spraying as
foliar application can be applied to many row crops such as
soybeans, corn and wheat along with a wide variety of vegetable
products such as tomatoes, peppers, beans, broccoli, cauliflower,
cucumbers, zucchini, and eggplant. Foliar spray can also be applied
to fruit shrubs and plants such as grapes, raspberries,
strawberries, and blueberries.
[0116] Additionally, in cases of fruit trees such as apple, pear,
peaches and the like, this same liquid concentrate or diluted with
water can be applied to young and mature trees to prevent pathogen
damage or to aid in the trees recovery from a pathogen
infection
Example 11
Application of Bacillus to Fruit Trees or Bushes by Dipping
[0117] New seedling fruit trees or bushes may be dipped into
containers in which liquid spore concentrate has been added, either
as a low active concentrate (250 million CFU/ml up to 5 billion
CFU/ml) or high active concentrate diluted in water (50 to 100
billion CFU/ml; 8-32 fl oz into 3-5 gallons of water). Seedlings
may be held for no more than 30 seconds in the solution then
immediately planted. Within 7 days of planting, trees are treated a
second time similar to what was outlined in Example 9. FIG. 11
shows the beneficial effect of Bacillus applied to poplar tree
seedlings.
Example 12
Application of Bacillus to Ornamental Flowers
[0118] Liquid Bacillus spore concentrate at concentrations
identical to those described in Example 4, 5 and 9 above is sprayed
onto ornamental flowers (both annuals and perennials) such as
geraniums, petunias, daffodils, either at germination of seeds or
as foliar spray within 3-5 days of germination as preventive
measure for pathogen occurrence. If pathogen infestation is
detected prior to spraying, treatments should be repeated every day
by suspending the spore concentrate into the nursery irrigation
water so a low dose (100-200,000 CFU/ml is delivered each day
through normal watering procedures. This treatment should continue
for 7 days. FIG. 10 shows the effect on treated and untreated
ornamental flowers.
[0119] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *