Compositions Comprising Bacillus Strains and Methods of Use to Suppress The Activities and Growth of Fungal Plant Pathogens

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.

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Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

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

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

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.

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.

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.

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.

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.

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.

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.

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.

Tomato-Tone® (plant fertilizer) made by Esporma comprises a fertilizer and Bacillus species bacteria for use as an organic fertilizer. Serenade® 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.

Serenade® (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® (biofungicide) is a fungicide designed to protect young plants against the effects of soil diseases like Pythium, Rhizoctonia, Fusarium and Phytophthora.

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. González-Fernández, 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

This invention provides compositions of Bacillus strains and methods for inhibiting the activity and/or growth of plant fungal pathogens.

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 1×103 to 1×1012 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.

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.

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° 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.

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 1×103 to 1×1012 CFU/mL.

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.

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.

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

FIG. 1 depicts the “zone of inhibition” measuring method.

FIG. 2 A-K depicts the mean inhibition zone sizes of plant pathogen species by Bacillus strains.

FIG. 3A-K depicts the percent inhibition of plant pathogen species by Bacillus strains.

FIG. 4 depicts the percent fungal pathogens inhibited by Bacillus strains.

FIG. 5 shows that the Bacillus concentrate does not inhibit the growth of beneficial Bradyrhizobium and depicts the Bacillus and Bradyrhizobium compatibility.

FIG. 6A-B depicts the Bacillus and Trichoderma compatibility.

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.

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.

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.

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.

FIG. 11 is a photograph of poplar trees three weeks after dip treatment with (B) and without Bacillus treatment (A).

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

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

There are five major fungal genera that cause significant losses across the commercial cash crops. These include:

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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

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.

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

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.

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).

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.

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.

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.

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.

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 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).

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.

A composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×1010 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° 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° 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° 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.

The CFU/ml of each Bacillus strain in the formulated Bacillus strain concentrates can vary from 1×103 CFU/ml up to 1×1012 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 1×103 CFU/gram of soil with a range of 1×103 CFU/gram soil up to 1×1011/gram soil. For seed coating applications the minimum dose of each Bacillus strain in the Bacillus strain concentrates should be a minimum of 1×103 CFU/seed with a range of 1×103 CFU/seed up to 1×1010 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.

A liquid composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×10′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 9×1011 colony forming units per mL (CFU/mL). A liquid composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×1010 colony forming units per mL (CFU/mL). A liquid composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×109 colony forming units per mL (CFU/mL). A liquid composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×108 colony forming units per mL (CFU/mL). A liquid composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×107 colony forming units per mL (CFU/mL). A liquid composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×106 colony forming units per mL (CFU/mL). A liquid composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×105 colony forming units per mL (CFU/mL). A liquid composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×104 colony forming units per mL (CFU/mL). A liquid composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×103 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.

Particularly preferred ranges for liquid composition may comprise between at least about 0.1-1×109 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 106-1010 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 1×107-1×109 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 1×108-1×109 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 1×106-1×108 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 1×107-1×108 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 1×108-1×1010 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 1×103-1×106 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 1×104-1×1011 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 1×105-1×1012 colony forming units per mL (CFU/mL). A liquid composition may comprise between at least about 1×103-1×1012 colony forming units per mL (CFU/mL).

A dried powder composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9×1012 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 9×10″ 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 9×1010 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 9×109 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 9×108 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 9×107 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 9×106 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 9×105 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 9×104 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 9×103 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.

Particularly preferred ranges for a dried powder composition may comprise between at least about 0.1-1×109 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×106-1×109 colony forming units per gram (CFU/gram). A lyophilized composition may comprise between at least about 1×106-1×109 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×107-1×109 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×108-1×109 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×106-1×1010 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×106-1×108 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×107-1×108 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×108-1×1010 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×103-1×106 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×104-1×1011 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×105-1×1012 colony forming units per gram (CFU/gram). A dried powder composition may comprise between at least about 1×103-1×1012 colony forming units per gram (CFU/gram).

The CFU/ml or gm of the formulated Bacillus strain concentrates can vary from 1×103 CFU/ml or/gm up to 1×1012 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 1×103 CFU/gm of soil with a range of 1×103 CFU/gm soil up to 1×1011/gm soil.

For seed coating applications the minimum dose of the Bacillus strain concentrates should be a minimum of 1×103 CFU/seed with a range of 1×103 CFU/seed up to 1×1010 CFU/seed.

Preparing a Spore Suspension

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° 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° 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 108/mL will be reached within 48 hours.

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° C. to preserve it for later use, such spore suspensions refrigerated at 5° 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° C.).

Protocol for Generation of Spores

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° 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° 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° 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 1×109 to 1×1010 CFU/mL.

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.

For quality assurance, all products, dry and liquid, are assayed for viable total population using CFU/mL.

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

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.

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.

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

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 1×103 CFU/ml or/gm up to 1×1012 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 1×101 CFU/gm of soil with a range of 1×103 CFU/gm soil up to 1×1011/gm soil. For seed coating applications the minimum dose of the Bacillus strain concentrates should be a minimum of 1×103 CFU/seed with a range of 1×103 CFU/seed up to 1×1010 CFU/seed.

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.

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.

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° 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 250×106 to 5×109 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.

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

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 1×103 to 1×1012 cells/mL or 1×103 to 1×1012 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.

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 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

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.

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.

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

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.

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° C. until used. Bacillus strains were subcultured in LB broth overnight at 37° C. with shaking at 200 rpm. The subculture (0.5 ml) was used to inoculate 50 ml LB broth and grown overnight at 37° 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 μl 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° 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.

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.

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.

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:

    • #1: Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis 10, and Brevibacillus laterosporus (CM3 and/or CM 33)
    • #2: Bacillus licheniformis, Brevibacillus laterosporus (CM3 and/or CM 33), and Bacillus mojavensis
    • #3: Bacillus amyloliquefaciens, Brevibacillus laterosporus (CM3 and/or CM 33), and Bacillus pumilus
    • #4: Bacillus amyloliquefaciens, Brevibacillus laterosporus (CM3 and/or CM 33), Bacillus pumilus and Paenibacillus polymxa

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.

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

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.

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.

From FIG. 5 it is clear that there is no inhibition of growth of Bradyrhizobium by the Bacillus cultures.

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° C. for, and monitored every 12-18 hours until fungus covered the entire plate. Additionally, another plate was incubated at 37° 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° C. In neither case was there inhibition of the Trichoderma by Bacillus.

From FIGS. 5 and 6 it is clear that these Bacillus strains do not inhibit either Bradyrhizobium or Trichoderma.

Example 3 Field Study

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

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.

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 ½ 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° C. Germination counts were recorded on day 7 post planting (T=+7 days) and the experiment terminated at T=+14 days.

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.

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

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 1×103 CFU/ml final liquid up to 1×1011 CFU/ml final liquid but is more generally in the range of 1×108 CFU/ml of final liquid up to 1×1010 CFU/ml of final liquid.

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.

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° C. for 5 minutes, quenched in an ice bath, and then plated on standard Tryptic Soy Agar. After incubation at 37° 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

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 1×106 CFU/gm final powder up to 1×1012 CFU/gm final powder but is more generally in the range of 1×109 CFU/gm final powder up to 1×1011 CFU/gm final powder.

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.

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° C. for 5 minutes, quenched in an ice bath, and then plated on standard Tryptic Soy Agar. After incubation at 37° 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

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

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

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

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.

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

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

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.

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.

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 1×108 to 1×1012 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 1×108 to 1×1012 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 1×108 to 1×1012 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.

Patent History
Publication number: 20190150454
Type: Application
Filed: Nov 9, 2018
Publication Date: May 23, 2019
Inventors: Jessica SPEARS (Minnetonka, MN), Steve C. Lamb (Shakopee, MN), Sebhat GEBRECHRISTOS (New Hope, MN), Christopher SCHULER (Charlottesville, VA)
Application Number: 16/185,397
Classifications
International Classification: A01N 63/00 (20060101); C12N 1/20 (20060101); C12Q 1/18 (20060101);