Genetically modified bacteria for improved pest control in plants

Abstract

The disclosure relates to edited (genetically-modified) microorganisms including microbial consortia, and agricultural compositions comprising the same, for the improvement of plant tolerance to nematode pests and / or by directly reducing the nematode population in the soil or roots. Furthermore, the disclosure teaches methods of utilizing the described microorganisms, microbial consortia, and agricultural compositions comprising the same, in methods for imparting beneficial properties to target plant species. In particular aspects, the disclosure provides methods of increasing desirable plant traits in agronomically important crop species, and reducing undesirable pests that impact crop performance.

Description

GENETICALLY MODIFIED BACTERIA FOR IMPROVED PEST CONTROL IN PLANTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 581,192 filed 07 September 2023, herein incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The official copy of the sequence listing is submitted electronically as a WIPO ST26 compliant XML sequence listing with a file named 23072_SeqListing.xml created on 29 July 2024 and having a size of 134,714 bytes and is filed concurrently with the specification. The sequence listing comprised in this document is part of the specification and is herein incorporated by reference in its entirety.FIELD

[0003] The instant disclosure relates generally to the field of biology, specifically genetically engineered microbes and microbial compositions for the improvement of plant health.BACKGROUND

[0004] According to the United Nations World Food Program, there are close to 900 million malnourished people in the world. The malnourishment epidemic is particularly striking in the developing nations of the world, where one in six children is underweight. The paucity of available food can be attributed to many socioeconomic factors; however, regardless of ultimate cause, the fact remains that there is a shortage of food available to feed a growing world population, which is expected to reach 9 billion people by 2050. The United Nations estimates that agricultural yields must increase by 70-100% to feed the projected global population in 2050.

[0005] These startling world population and malnutrition figures highlight the importance of agricultural efficiency and productivity in sustaining the world’s growing population. The technological advancements achieved by modem row crop agriculture, which has led to never before seen crop yields, are impressive. However, despite the advancements made by technological innovations such as genetically engineered crops and new novel pesticidal andherbicidal compounds, there is a need for improved crop performance, in order to meet the demands of an exponentially increasing global population.

[0006] Scientists have estimated that if the global agricultural “yield gap” (which is the difference between the best observed yield and results elsewhere) could be closed, then worldwide crop production would rise by 45-70%. That is, if all farmers, regardless of worldwide location, could achieve the highest attainable yield expected for their respective regions, then a great majority of the deficiencies in worldwide food production could be addressed. However, solving the problem of how to achieve higher yields across a heterogenous worldwide landscape are difficult.

[0007] Often, yield gaps can be explained by inadequate water, substandard farming practices, inadequate fertilizers, and the non-availability of herbicides and pesticides. However, to vastly increase the worldwide use of water, fertilizers, herbicides, and pesticides, would not only be economically infeasible for most of the world, but would have negative environmental consequences.

[0008] Nematodes are one of the most damaging pests to commercial crops, causing an estimated annual global economic loss of over 173 billion US dollars.

[0009] There is therefore an urgent need in the art for improved methods of increasing crop performance and imparting beneficial traits to both plants and microbes to improve crop health.SUMMARY

[0010] Included are genetically-engineered microorganisms that have application, inter alia, in agriculture. The disclosed microorganisms can be utilized in their isolated and biologically pure states, as well as being formulated into agriculturally acceptable compositions. Further provided are agriculturally beneficial microbial consortia, comprising at least two members of the disclosed microorganisms, as well as methods of utilizing said consortia in agricultural applications. In some aspects, genomic modification of the microbes (individual, consortia, and / or communities) are contemplated, for the improvement of microbial traits and the improvement of microbe-associated plants.

[0011] The solution to increasing crop performance and increasing yield proffered by the present disclosure is not detrimental to the earth’s resources, as it does not rely upon increased water consumption or increased input of synthetic chemicals into a system. Rather, the presentdisclosure utilizes microbes to impart beneficial properties, including increased yields, to desirable plants.

[0012] The disclosure therefore offers an environmentally sustainable solution that allows farmers to increase yields of important crops, which is not reliant upon increased utilization of synthetic and pesticides.

[0013] In embodiments, the disclosure provides for an efficient and broadly applicable agricultural platform utilizing microbes and microbial consortia (a plurality of microbes, in some aspects a plurality that improves the health or desired phenotype of the plant, such as an agronomic trait, with which it is associated) that promote one or more desirable plant properties.

[0014] The microbes disclosed herein improve the performance of plants, such as crop plants, by both direct and indirect mechanisms. In some aspects, the microbe becomes symbiotic with the plant. In some aspects, the microbe produces a compound (e.g., a metabolite) that confers a benefit to the plant or that the plant can use for improved characteristics. In some aspects, the microbe improves the solubility of one or more compositions, such as a nutrient, thereby benefitting the plant. In some aspects, the microbe imparts a tolerance to the plant to an exogenous substance such as an herbicide or a pesticide. In some aspects, the microbe produces a composition that is detrimental to a plant pest, such as a nematode.

[0015] In some aspects, the invention comprises an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl', and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA) wherein the engineered bacterial cell has improved toxicity activity against a nematode as compared to a control bacterial cell of the same genetic background that has not been engineered.

[0016] In some aspects, the invention comprises an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least oneheterologous polynucleotide selected from the group consisting of: appbBal , cry2Ab35, cry5B, cry!2A, cry21Aa, cry5Aa, cry55al, crylAa, cry 1 Ba, cry Ila, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and crySBbl', and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA); wherein the engineered bacterial cell has improved toxicity activity against a nematode as compared to a control bacterial cell of the same genetic background that has not been engineered; wherein the at least one gene in a toxin biosynthetic pathway comprises a sequence sharing at least 95%, between 95% and 96%, at least 96%, between 96% and 97%, at least 97%, between 97% and 98%, at least 98%, between 98% and 99%, at least 99%, or even greater than 99% identity with at least 100, between 100 and 125, at least 125, between 125 and 150, at least 150, between 150 and 175, at least 175, between 175 and 200, at least 200, or even greater than 200 nucleotides with at least one sequence selected from the group consisting of: SEQID NOs: 20, 51, and 52.

[0017] In some aspects, the invention comprises an engineered bacterial strain, wherein a heterologous polynucleotide has been inserted into the genome of the cell in a coding sequence located in a gene cluster. In some embodiments, the gene cluster includes one more genes that encode for a toxin, such as thuringiensin, for example but not limited to thuE. In some embodiments, the gene cluster includes one more genes that encode for a protease, such as but not limited to a serine alkaline protease (e.g., Subtilisin E, aprE) or neutral protease A (nprA). In some embodiments, more than one gene is altered such that expression of more than one toxin and / or protease is reduced.

[0018] In some aspects, the invention comprises an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, cry Ila, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl', and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA) wherein the engineered bacterial cell has improved toxicity activity against a nematode as compared to a control bacterial cell of the same genetic background that has not beenengineered; wherein the at least one heterologous polynucleotide of (a) or (b) comprises a sequence at least 95%, between 95% and 96%, at least 96%, between 96% and 97%, at least 97%, between 97% and 98%, at least 98%, between 98% and 99%, at least 99%, or even greater than 99% identity with at least 100, between 100 and 125, at least 125, between 125 and 150, at least 150, between 150 and 175, at least 175, between 175 and 200, at least 200, or even greater than 200 nucleotides with at least one sequence selected from the group consisting of: SEQID NOs: 1-16.

[0019] In some aspects, the invention comprises an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl', and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA); wherein the engineered bacterial cell has improved toxicity activity against a nematode as compared to a control bacterial cell of the same genetic background that has not been engineered; wherein the at least one heterologous polynucleotide of (a) or (b) encodes a polypeptide sharing at least 95%, between 95% and 96%, at least 96%, between 96% and 97%, at least 97%, between 97% and 98%, at least 98%, between 98% and 99%, at least 99%, or even greater than 99% identity with at least 100, between 100 and 125, at least 125, between 125 and 150, at least 150, between 150 and 175, at least 175, between 175 and 200, at least 200, or even greater than 200 amino acids with at least one sequence selected from the group consisting of: SEQID NOs: 21-36.

[0020] In some aspects, the invention comprises an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl', and / or (c) reduced or abolished expression of at least one gene in abiosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA); wherein the engineered bacterial cell has improved toxicity activity against a nematode as compared to a control bacterial cell of the same genetic background that has not been engineered; wherein the heterologous polynucleotide of (a) or (b) is operably linked to a heterologous promoter.

[0021] A synthetic composition comprising an engineered bacterial, or an exudate therefrom, or culture broth therefrom, or spore therefrom, or any plurality or combination of the preceding; further comprising at least one composition selected from the group consisting of: a plant element, a formulation component, an agricultural composition, and any plurality and / or combination of the preceding; and wherein the plant element is selected from the group consisting of: a seed, a leaf, a root, a whole plant, and / or any plurality and / or combination of the preceding; wherein the formulation component is selected from the group consisting of: a compound that improves the stability of the microbe, a preservative, a carrier, a surfactant, an anticomplex agent, and any combination and / or plurality of the preceding; and wherein the agricultural composition is selected from the group consisting of: comprises a fungicide, a nematicide, a bactericide, an insecticide, an herbicide, a growth medium, and any combination and / or plurality of the preceding; and wherein the engineered bacterial cell is present at a concentration of at least about 10A2 CFU / mL in a liquid formulation, or at least about 10A2 Colony Forming Units per gram in a non-liquid formulation; and wherein the engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cry!2A, cry21Aa, cry5Aa, cry55al, crylAa, cry 1 Ba, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl; and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA).

[0022] A synthetic composition comprising an engineered bacterial, or an exudate therefrom, or culture broth therefrom, or spore therefrom, or any plurality or combination of the preceding; further comprising at least one composition selected from the group consisting of: a plant element, a formulation component, an agricultural composition, and any plurality and / or combination of the preceding; and wherein the engineered bacterial cell of the genus Bacillus, wherein the engineeredbacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, cry! Ba, cry Ila, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry Bb and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA).

[0023] A synthetic composition comprising an engineered bacterial, or an exudate therefrom, or culture broth therefrom, or spore therefrom, or any plurality or combination of the preceding; further comprising at least one composition selected from the group consisting of: a plant element, a formulation component, an agricultural composition, and any plurality and / or combination of the preceding; and wherein the plant element is selected from the group consisting of: a seed, a leaf, a root, a whole plant, and / or any plurality and / or combination of the preceding; and wherein the engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, cry Ila, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3BbI and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA).

[0024] A synthetic composition comprising an engineered bacterial, or an exudate therefrom, or culture broth therefrom, or spore therefrom, or any plurality or combination of the preceding; further comprising at least one composition selected from the group consisting of: a plant element, a formulation component, an agricultural composition, and any plurality and / or combination of the preceding; wherein the formulation component is selected from the group consisting of: a compound that improves the stability of the microbe, a preservative, a carrier, a surfactant, an anticomplex agent, and any combination and / or plurality of the preceding; and wherein the engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin;(b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cry!2A, cry21Aa, cry5Aa, cry55al, crylAa, cry] Ba, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl'. and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE. nprA).

[0025] A synthetic composition comprising an engineered bacterial, or an exudate therefrom, or culture broth therefrom, or spore therefrom, or any plurality or combination of the preceding; further comprising at least one composition selected from the group consisting of: a plant element, a formulation component, an agricultural composition, and any plurality and / or combination of the preceding; and wherein the agricultural composition is selected from the group consisting of: comprises a fungicide, a nematicide, a bactericide, an insecticide, an herbicide, a growth medium, and any combination and / or plurality of the preceding; and wherein the engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl', and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA).

[0026] A synthetic composition comprising an engineered bacterial, or an exudate therefrom, or culture broth therefrom, or spore therefrom, or any plurality or combination of the preceding; further comprising at least one composition selected from the group consisting of: a plant element, a formulation component, an agricultural composition, and any plurality and / or combination of the preceding; and wherein the plant element is selected from the group consisting of: a seed, a leaf, a root, a whole plant, and / or any plurality and / or combination of the preceding; and wherein the engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa,cry2Ab, cry6Aa, app4al , cry5Ba, and cry3Bbl‘, and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprAy, wherein the plant element comprises a transgene.

[0027] A plurality of synthetic compositions, wherein each synthetic composition comprises a genetically-modified bacterium of the genus Bacillus that is heterologously-disposed to a plant element, wherein the genetically-modified bacterium comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cry!2A, cry21Aa, cry5Aa, cry55al , crylAa, cry 1 Ba, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and crySBbl'. and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprAy

[0028] A plurality of synthetic compositions, wherein each synthetic composition comprises a genetically-modified bacterium of the genus Bacillus that is heterologously-disposed to a plant element, wherein the genetically-modified bacterium comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl ; and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA wherein said synthetic compositions are substantially confined within an object selected from the group consisting of: a tube, a bottle, ajar, an ampule, a package, a vessel, a bag, a box, a bin, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and a case.

[0029] A plurality of synthetic compositions, wherein each synthetic composition comprises a genetically-modified bacterium of the genus Bacillus that is heterologously-disposed to a plant element, wherein the genetically-modified bacterium comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of atleast one heterologous polynucleotide selected from the group consisting of: app6Bal , cry2Ab35, cry5B, cry!2A, cry21Aa, cry5Aa, cry55al, crylAa, cry 1 Ba, cry Ila, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl'. and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprA) wherein the plant element is obtained from a plant selected from the group consisting of: alfalfa, apple, avocado, banana, barley, beans, broccoli, canola, carrot, carrots, cassava, cauliflower, celery, chickpea, citrus, coconut, coffee, corn, cotton, crucifers, cucumber, cucurbit, favabean, forest trees, garlic, ginger, grapes, grasses, lemon, lettuce, lime, maize, oat, oats, okra, onion, orange, orchards, ornamental plants, palm, pea, peanuts, pepper, potato, rapeseed, snapbeans, sorghum, soybean, soybeans, strawberries, strawberry, sugarbeets, sugarcane, tea, tomato, trees, vegetables, wheat, and zucchini.

[0030] A plurality of synthetic compositions, wherein each synthetic composition comprises a genetically-modified bacterium of the genus Bacillus that is heterologously-disposed to a plant element, wherein the genetically-modified bacterium comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, cry Ila, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bb and / or (c) reduced or abolished expression of at least one gene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE, nprAy. wherein the plant element is selected from the group consisting of: seed, leaf, stem, root, bud, conn, keikis, shoot, runner, whole plant, and / or any plurality and / or combination of the preceding.

[0031] A plurality of synthetic compositions, wherein each synthetic composition comprises a genetically-modified bacterium of the genus Bacillus that is heterologously-disposed to a plant element, wherein the genetically-modified bacterium comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cry!2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, cry 1 la, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl‘, and / or (c) reduced or abolished expression of at least onegene in a biosynthetic pathway (for example but not limited to one or more of the following: thuE, aprE. nprA) further comprising a growth medium, wherein the growth medium comprises soil, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern with substantially equal spacing between each of the synthetic compositions.

[0032] A method of conferring nematode pest tolerance to a plant, the method comprising: applying to a plant element of said plant a formulation comprising an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, cry 1 Ba, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl‘, and (c) reduced or abolished expression of at least one gene in a biosynthetic pathway.

[0033] A method of conferring nematode pest tolerance to a plant, the method comprising: applying to a plant element of said plant a formulation comprising an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl', and (c) reduced or abolished expression of at least one gene in a biosynthetic pathway; wherein said applying to the plant element is accomplished by an indirect method selected from the group consisting of: in-furrow application, soil drench application, and side-dress application.

[0034] A method of conferring nematode pest tolerance to a plant, the method comprising: applying to a plant element of said plant a formulation comprising an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab,cry6Aa, app4al , cry5Ba, and cry3Bbl‘, and (c) reduced or abolished expression of at least one gene in a biosynthetic pathway; wherein said applying to the plant clement is accomplished by coating said plant element with a liquid formulation of the microbe or exudate therefrom.

[0035] A method of conferring nematode pest tolerance to a plant, the method comprising: applying to a plant element of said plant a formulation comprising an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl'. and (c) reduced or abolished expression of at least one gene in a biosynthetic pathway; wherein said applying to the plant element is accomplished by coating said plant element with a substantially non-liquid formulation of the microbe or exudate therefrom.

[0036] A method of reducing the number of pests in or on a composition, wherein the pests comprise nematodes, nematode eggs, nematode juveniles, or any plurality and / or combination of the preceding, the method comprising: introducing to the composition an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin, and (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl.

[0037] A method of reducing the number of pests in or on a composition, wherein the pests comprise nematodes, nematode eggs, nematode juveniles, or any plurality and / or combination of the preceding, the method comprising: introducing to the composition at an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin, and (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa,cry2Ab, cry6Aa, app4al , cry5Ba, and cry3Bbl', wherein the composition is a growth medium for a plant.

[0038] A method of reducing the number of pests in or on a composition, wherein the pests comprise nematodes, nematode eggs, nematode juveniles, or any plurality and / or combination of the preceding, the method comprising: introducing to the composition at an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin, and (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cry!2A, cry 1Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3BbI'. wherein the composition is a harvested product.

[0039] A method of reducing the number of pests in or on a composition, wherein the pests comprise nematodes, nematode eggs, nematode juveniles, or any plurality and / or combination of the preceding, the method comprising: introducing to the composition at an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding a Cry toxin, and (b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, crylla, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl', wherein the composition is a harvested product; wherein the harvested product is a fruit, a vegetable, a seed, and / or a fiber.

[0040] In any aspect of the compositions and methods described herein, the composition with which the engineered bacterial cell is associated is a plant or plant element.

[0001] In any aspect of the compositions and methods described herein, the engineered bacterial cell is of the genus Bacillus.

[0042] In any aspect of the compositions and methods described herein, the engineered bacterial cell is of the species Bacillus thuringiensis .

[0043] In any aspect of the compositions and methods described herein, the engineered bacterial cell comprises one or more sequence selected from SEQID NO: 37-50.

[0044] In any aspect of the compositions and methods described herein, the engineered bacterial cell is obtained or derived from Bacillus thuringiensis Strain PM39400, deposited as NRRL B- 68090.

[0045] In any aspect of the compositions and methods described herein, at least one additional bacterium may be included with the engineered bacterial cell; wherein the at least one additional bacterium may be wild-type or engineered.

[0046] In any aspect of the compositions and methods described herein, the nematode is of a taxonomy selected from the group consisting of: Meloidogyne. Heterodera. Pralylenchus. Radopholus, Xiphenema. Ditylenchus, and any plurality and / or combination of the preceding.BRIEF DESCRIPTION OF THE DRAWINGS AND THE SEQUENCE LISTING

[0047] The disclosure can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.

[0048] FIG. 1 shows C. elegans mortality under different treatments of the gcnc-cditcd Bacillus strains at days 1, 2, and 3.

[0049] FIG. 2 shows C. elegans mortality under different treatments of the edited Bacillus strains at days 1, 3, and 4.

[0050] FIG. 3 shows reduction in RKN numbers egg masses per gram dry root under different treatments of the edited Bacillus strains.

[0051] FIG. 4 shows reduction in Lesion numbers under different treatments of the edited Bacillus strains.

[0052] FIG. 5 shows the relative fluorescence of different promoters in vectors with GFP reporter fusions (bars), and the observed sporulation of each transformed strain at the indicated timepoint (plus and minus signs for sporulation and not sporulation, respectively).

[0053] The sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§ 1.821 and 1.825. The sequence descriptions comprise the three letter codes for amino acids as defined in 37 C.F.R. §§ 1.821 and 1.825, which are incorporated herein by reference.

[0054] SEQID NO:1 is the app6Bal DNA sequence from Bacillus subtilis.

[0055] SEQID NO:2 is the cry2Ab35 DNA sequence from Bacillus bombysepticus .

[0056] SEQID NO:3 is the cry5B DNA sequence from Bacillus thuringiensis.

[0057] SEQID NO:4 is the cryl2A DNA sequence from Bacillus thuringiensis.

[0058] SEQID NO:5 is the cry21Aa DNA sequence from Bacillus thuringiensis.

[0059] SEQID NO:6 is the cry5Aa DNA sequence from Bacillus thuringiensis.

[0060] SEQID NO:7 is the cry 55 al DNA sequence from Bacillus thuringiensis.

[0061] SEQID NO:8 is the crylAa DNA sequence from Bacillus thuringiensis Strain PM39400.

[0062] SEQID NO:9 is the crylBa DNA sequence from Bacillus thuringiensis Strain PM39400.

[0063] SEQID NO:10 is the crylla DNA sequence from Bacillus thuringiensis Strain PM39400.

[0064] SEQID NO:11 is the cry2Aa DNA sequence from Bacillus thuringiensis Strain PM39400.

[0065] SEQID NO:12 is the cry2Ab DNA sequence from Bacillus thuringiensis Strain PM39400.

[0066] SEQID NO:13 is the cry6Aa DNA sequence from Bacillus thuringiensis.

[0067] SEQID NO:14 is the app4al DNA sequence from Bacillus toyonens A .

[0068] SEQID NO:15 is the cry5Ba DNA sequence from Bacillus thuringiensis.

[0069] SEQID NO:16 is the crySBbl DNA sequence from Bacillus thuringiensis.

[0070] SEQID NO:17 is the dltA DNA sequence from Bacillus thuringiensis Strain PM39400.

[0071] SEQID NO:18 is the ugd DNA sequence from Bacillus thuringiensis Strain PM39400.

[0072] SEQID NO:19 is the xerC DNA sequence from Bacillus thuringiensis Strain PM39400.

[0073] SEQID NO:20 is the thuE DNA sequence from Bacillus thuringiensis Strain PM39400.

[0074] SEQID NO:21 is the App6Bal PRT sequence from Bacillus subtilis.

[0075] SEQID NO:22 is the Cry2Ab35 PRT sequence from Bacillus bombysepticus.

[0076] SEQID NO:23 is the Cry5B PRT sequence from Bacillus thuringiensis.

[0077] SEQID NO:24 is the Cryl2A PRT sequence from Bacillus thuringiensis.

[0078] SEQID NO:25 is the Cry21Aa PRT sequence from Bacillus thuringiensis.

[0079] SEQID NO:26 is the Cry5Aa PRT sequence from Bacillus thuringiensis.

[0080] SEQID NO:27 is the Cry55al PRT sequence from Bacillus thuringiensis.

[0081] SEQID NO:28 is the CrylAa PRT sequence from Bacillus thuringiensis Strain PM39400.

[0082] SEQID NO:29 is the Cry 1 Ba PRT sequence from Bacillus thuringiensis Strain PM39400.

[0083] SEQID NO:30 is the Cry Ila PRT sequence from Bacillus thuringiensis Strain PM39400.

[0084] SEQID NO:31 is the Cry2Aa PRT sequence from Bacillus thuringiensis Strain PM39400.

[0085] SEQID NO:32 is the Cry2Ab PRT sequence from Bacillus thuringiensis Strain PM39400.

[0086] SEQID NO:33 is the Cry6Aa PRT sequence from Bacillus thuringiensis.

[0087] SEQID NO:34 is the App4al PRT sequence from Bacillus tayanensis.

[0088] SEQID NO:35 is the Cry5Ba PRT sequence from Bacillus thuringiensis.

[0089] SEQID NO:36 is the Cry3Bbl PRT sequence from Bacillus thuringiensis.

[0090] SEQID NO:37 is the 16S RNA 1 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0091] SEQID NO:38 is the 16S RNA 2 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0092] SEQID NO:39 is the 16S RNA 3 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0093] SEQID NO:40 is the 16S RNA 4 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0094] SEQID NO:41 is the 16S RNA 5 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0095] SEQID NO:42 is the 16S RNA 6 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0096] SEQID NO:43 is the 16S RNA 7 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0097] SEQID NO:44 is the 16S RNA 8 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0098] SEQID NO:45 is the 16S RNA 9 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0099] SEQID NO:46 is the 16S RNA 10 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0100] SEQID NO:47 is the 16S RNA 11 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0101] SEQID NO:48 is the 16S RNA 12 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0102] SEQID NO:49 is the 16S RNA 13 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0103] SEQID NO:50 is the Strain PM39400 16S RNA 14 DNA sequence from Bacillus thuringiensis Strain PM39400.

[0104] SEQID NO:51 is the aprE DNA sequence from Bacillus thuringiensis.

[0105] SEQID NO:52 is the nprA DNA sequence from Bacillus thuringiensis.

[0106] SEQID NO:53 is the Pcryl DNA sequence from Bacillus thuringiensis.

[0107] SEQID NO:54 is the PaprE DNA sequence from Bacillus thuringiensis.

[0108] The microorganisms described in this Application may be deposited with the Agricultural Research Service Culture Collection (NRRL), which is an International Depositary Authority, located at 1815 North University Street, Peoria, IL 61604, USA.

[0109] The deposits are made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0110] The deposits are made in accordance with, and to satisfy, the criteria set forth in 37 C.F.R. §§ 1.801-1.809 and the Manual of Patent Examining Procedure §§ 2402-2411.05.

[0111] Strain PM39400 was deposited with the NRRL on 26 January 2022 as NRRL B-68090.DETAILED DESCRIPTION

[0112] The term “a” or “an” refers to one or more of that entity, i.e., can refer to a plural referent. As such, the terms “a” or “an”, “one or more” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0113] As used herein, the term "about" refers to up to± 10% of the recited value. For example, the term "about" can refer to ± 1%, ± 2%, ± 3%, ± 4%, ± 5%, ± 6%, ± 7%, ± 8%, ± 9%, ± 10%, of the recited value, or non-integer percentages thereof. By way of additional example, the term "about" may refer to ± 0.2 minutes with respect to the retention times recited herein.

[0114] As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides of any length, cither ribonucleotides or dcoxyribonuclcotidcs, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified nucleic acids such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like. The terms "nucleic acid" and "nucleotide sequence" are used interchangeably.

[0115] As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and / or the regulatory sequences required for their expression. Genes can also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0116] A gene and / or other polynucleotides (coding sequences, non-coding sequences, regulatory elements, and the like) are included in the “genome” of an organism. The term “genome” encompasses all polynucleotides that are naturally-occurring within the cell of a particular organism, irrespective of subcellular location (e.g.. nucleoid, cytoplasm) or compositional superstructure (e.g., chromosome, plasmid). Polynucleotides that are introduced into a cell may be termed “heterologous”, as further described herein.

[0117] As used herein, a “synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence. The term “artificial” may be considered synonymous to “synthetic” in this context.

[0118] As used herein, the term “homologous” or “homologue”, “homolog”, or “ortholog” is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms “homology,” “homologous,” “substantially similar” and “corresponding substantially” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acidfragments of the instant disclosure such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the disclosure encompasses more than the specific exemplary sequences. These terms describe the relationship between a gene found in one species, subspecies, variety, cultivar or strain and the corresponding or equivalent gene in another species, subspecies, variety, cultivar or strain. For purposes of this disclosure homologous sequences are compared.“Homologous sequences” or “homologues” or “orthologs” are thought, believed, or known to be functionally related. A functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and / or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus (Scientific and Educational Software, Pennsylvania) and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), using default parameters.

[0119] As used herein, the term “nucleotide change” refers to, e.g., nucleotide substitution, deletion, insertion, chemical alteration, or any of the preceding, as is well understood in the ail.

[0120] As used herein, the term “protein modification” refers to, e.g., amino acid substitution, amino acid modification, deletion, and / or insertion, as is well understood in the art.

[0121] As used herein, the term “at least a portion” or “fragment” of a nucleic acid or polypeptide means a portion having the minimal size characteristics of such sequences, or any larger fragment of the full length molecule, up to and including the full length molecule. A fragment of a polynucleotide of the disclosure may encode a biologically active portion of a genetic regulatory element. A biologically active portion of a genetic regulatory element can be prepared by isolating a portion of one of the polynucleotides of the disclosure that comprises the genetic regulatory element and assessing activity as described herein. Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on, going up to the full length polypeptide. The length of the portion to be used will depend on the particular application. A portion of a nucleic acid useful as a hybridization probe may be as shortas 12 nucleotides; in some embodiments, it is 20 nucleotides. A portion of a polypeptide useful as an epitope may be as short as 4 amino acids. A portion of a polypeptide that performs the function of the full-length polypeptide would generally be longer than 4 amino acids.

[0122] The term “primer” as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxy ribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and composition (A / T vs. G / C content) of primer. A pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

[0123] The terms “stringency” or “stringent hybridization conditions” refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimized to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na-i- ion, typically about 0.01 to 1.0 M Na + ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes or primers (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or“conditions of reduced stringency” include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C and a wash in 2xSSC at 40° C. Exemplary high stringency conditions include hybridization in 50% formamide, IM NaCl, 1% SDS at 37° C, and a wash in O.lxSSC at 60° C. Hybridization procedures are well known in the art and are described by e.g., Ausubel et al., 1998 and Sambrook et al., 2001. In some embodiments, stringent conditions are hybridization in 0.25 M Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5-20% sodium dodecyl sulfate at 45°C, such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by a wash in 5xSSC, containing 0.1% (w / v) sodium dodecyl sulfate, at 55°C to 65°C.

[0124] As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains Bacteria and Archaea, as well as eukaryotic Fungi and Protist.

[0125] In general, “heterologous” refers to a state conferred by the non-naturally-occurring association of one composition (e.g,, chemical, molecule, seed, plant, gene) with another of the same or different type. Such a non-naturally-occurring combination may further be referred to as a “synthetic combination”. For example, a gene that does not naturally occur in a particular location in a genome of a bacterium may be “heterologous” when placed into the genome of a different bacterium, or when placed into a different location of the genome of the same bacterium. In another example, a microbe may be “heterologous” to a particular plant, if it is associated in a manner not known to be naturally occurring, e.g., a different spatial, temporal, and / or concentration manner. In another example, “heterologous” can refer to a polynucleotide that is not naturally present in a particular strain of microbe but may be present in other strains of the same genus and species. In general, “heterologous” refers to a state conferred by not originating from the same entity or locus.

[0126] As used herein, the term "bacterium" or "bacteria" refers in general to any prokaryotic organism, and may reference an organism from either Kingdom Eubacteria (Bacteria), Kingdom Archaebacteria (Archae), or both. In some cases, bacterial genera or other taxonomic classifications have been reassigned due to various reasons (such as but not limited to the evolving field of whole genome sequencing), and it is understood that such nomenclature reassignments are within the scope of any claimed taxonomy. For example, certain species of the genus Erwinia have been described in the literature as belonging to genus Pantoea (Zhang, Y.,Qiu, S. Examining phylogenetic relationships of Erwinia and Pantoea species using whole genome sequence data. Antonie van Leeuwenhoek 108, 1037-1046 (2015).).

[0127] The term “microbial community” means a group of microbes comprising two or more genera, and / or species, and / or or strains. Unlike microbial consortia, a microbial community does not have to be carrying out a common function, or does not have to be participating in, or leading to, or correlating with, a recognizable parameter or plant phenotypic trait. The community may comprise one or more species, or strains of a species, of microbes. In some instances, the microbes coexist within the community symbiotically.

[0128] The term “microbial consortia” or “microbial consortium” refers to a subset of a microbial community of individual microbes, which can be described as carrying out a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter or plant phenotypic trait. Consortia of microbes identified herein can each provide different aspects of a desired outcome (e.g., plant biotic stress control), and / or can work with one another in an additive fashion (e.g., one microbe providing control of one biotic stressor and another microbe providing control of a different biotic stressor), and / or can work with each other in a synergistic fashion (e.g., two or more microbes providing a level of biotic stress control to a plant greater than the sum of any individual microbe’s effect).

[0129] The term “accelerated microbial selection” or “AMS” is used interchangeably with the term “directed microbial selection” or “DMS” and refers to the iterative selection methodology that was utilized, in some embodiments of the disclosure, to derive the claimed microbial species or consortia of said species.

[0130] As used herein, “isolate,” “isolated,” “isolated microbe,” and like terms, are intended to mean that the one or more microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (for example soil, water, plant tissue).

[0131] Thus, an “isolated microbe” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain) in association with an agricultural carrier.

[0132] In certain aspects of the disclosure, the isolated microbes exist as isolated and biologically pure cultures. It will be appreciated by one of skill in the art, that an isolated andbiologically pure culture of a particular microbe, denotes that said culture is substantially free (within scientific reason) of other living organisms and contains only the individual microbe in question. The culture can contain varying concentrations of said microbe. The present disclosure notes that isolated and biologically pure microbes often “necessarily differ from less pure or impure materials.” See, e.g., In re Bergstrom, 427 F.2d 1394, (CCPA 1970)(discussing purified prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA 1979)(discussing purified microbes), see also, Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned Hand discussing purified adrenaline), aff’d in part, rev’d in part, 196 F. 496 (2d Cir. 1912), each of which are incorporated herein by reference. Furthermore, in some aspects, the disclosure provides for certain quantitative measures of the concentration, or purity limitations, that must be found within an isolated and biologically pure microbial culture. The presence of these purity values, in certain embodiments, is a further attribute that distinguishes the presently disclosed microbes from those microbes existing in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limitations for vitamin B12 produced by microbes), incorporated herein by reference.

[0133] As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms. The phrase should not be taken to indicate the extent to which the microorganism has been isolated or purified. However, “individual isolates” can comprise substantially only one genus, species, or strain, of microorganism.

[0134] The term “growth medium” as used herein, is any medium which is suitable to support growth of a plant. By way of example, the media may be natural or artificial including, but not limited to: soil, potting mixes, bark, vermiculite, hydroponic solutions alone and applied to solid plant support systems, and tissue culture gels. It should be appreciated that the media may be used alone or in combination with one or more other media. It may also be used with or without the addition of exogenous nutrients and physical support systems for roots and foliage.

[0135] In one embodiment, the growth medium is a naturally occurring medium such as soil, sand, mud, clay, humus, regolith, rock, or water. In another embodiment, the growth medium is artificial. Such an artificial growth medium may be constructed to mimic the conditions of a naturally occurring medium; however, this is not necessary. Artificial growth media can be made from one or more of any number and combination of materials including sand, minerals, glass,rock, water, metals, salts, nutrients, water. In one embodiment, the growth medium is sterile. In another embodiment, the growth medium is not sterile.

[0136] The medium may be amended or enriched with additional compounds or components, for example, a component which may assist in the interaction and / or selection of specific groups of microorganisms with the plant and each other. For example, antibiotics (such as penicillin) or sterilants (for example, quaternary ammonium salts and oxidizing agents) could be present and / or the physical conditions (such as salinity, plant nutrients (for example organic and inorganic minerals (such as phosphorus, nitrogenous salts, ammonia, potassium and micronutrients such as cobalt and magnesium), pH, and / or temperature) could be amended.

[0137] The term “plant” generically includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. A “plant element” is intended to reference either a whole plant or a plant component, which may comprise differentiated and / or undifferentiated tissues, for example but not limited to plant tissues, parts, and cell types. In one embodiment, a plant element is one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keiki, shoot, bud, tumor tissue, and various forms of cells and culture (e.g., single cells, protoplasts, embryos, callus tissue). The term “plant organ” refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. As used herein, a “plant part” is synonymous to a “portion” of a plant, and refers to any part of the plant, and can include distinct tissues and / or organs, and may be used interchangeably with the term “tissue” throughout.

[0138] “Progeny” comprises any subsequent generation of an organism, produced via sexual or asexual reproduction.

[0139] As used herein, the term “plant element” refers to plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like, as well as the parts themselves. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, andmutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.

[0140] Similarly, a “plant reproductive element” is intended to generically reference any part of a plant that is able to initiate other plants via either sexual or asexual reproduction of that plant, for example but not limited to: seed, seedling, root, shoot, cutting, scion, graft, stolon, bulb, tuber, corm, keiki, or bud. The plant element may be in plant or in a plant organ, tissue culture, or cell culture.

[0141] The term “monocotyledonous” or “monocot” refers to the subclass of angiosperm plants also known as “monocotyledoneae”, whose seeds typically comprise only one embryonic leaf, or cotyledon. The term includes references to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of the same.

[0142] The term “dicotyledonous” or “dicot” refers to the subclass of angiosperm plants also knows as “dicotyledoneae”, whose seeds typically comprise two embryonic leaves, or cotyledons. The term includes references to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of the same.

[0143] As used herein, the term “cultivar” refers to a variety, strain, or race, of plant that has been produced by horticultural or agronomic techniques and is not normally found in wild populations.

[0144] As used herein, the term “molecular marker”, “marker”, or “genetic marker” refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, micro satellite markers (SSRs), sequence- characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location. Mapping of molecular markers in the vicinity of an allele is a procedure which can be performed by the average person skilled in molecular- biological techniques.

[0145] As used herein, the term “trait” refers to a characteristic or phenotype. For example, in the context of some embodiments of the present disclosure, yield of a crop relates to the amount of marketable biomass produced by a plant (e.g., fruit, fiber, grain). Desirable traits may alsoinclude other plant characteristics, including but not limited to: water use efficiency, nutrient use efficiency, production, mechanical harvestability, fruit maturity, shelf life, pcst / discasc resistance, early plant maturity, tolerance to stresses, etc. A trait may be inherited in a dominant or recessive manner, or in a partial or incomplete- dominant manner. A trait may be monogenic (z.e., determined by a single locus) or polygenic (z.e., determined by more than one locus) or may also result from the interaction of one or more genes with the environment. A trait of a plant may be altered due to the presence or absence of another organism. In one example, the health of a plant may be decreased due to the presence of a pathogen, for example a nematode, fungus, bacterium, or insect. In another example, the health of a plant may be improved due to the presence of a microbe, for example a microbe that is toxic to nematodes may be present on or near a plant, providing the plant a mechanism of tolerance to said nematodes.

[0146] As used herein, the term “phenotype” refers to the observable characteristics of an individual cell, cell culture, organism (e.g., a plant), or group of organisms which results from the interaction between that individual’s genetic makeup (i.e., genotype) and the environment.

[0147] As used herein, “improved” should be taken broadly to encompass improvement of a characteristic of a plant, as compared to a control plant, or as compared to a known average quantity associated with the characteristic in question. For example, “improved” plant biomass associated with application of a beneficial microbe, or consortia, of the disclosure can be demonstrated by comparing the biomass of a plant treated by the microbes taught herein to the biomass of a control plant not treated. Alternatively, one could compare the biomass of a plant treated by the microbes taught herein to the average biomass normally attained by the given plant, as represented in scientific or agricultural publications known to those of skill in the art. In the present disclosure, “improved” does not necessarily demand that the data be statistically significant (e.g., p < 0.05); rather, any quantifiable difference demonstrating that one value (e.g., the average treatment value) is different from another (e.g., the average control value) can rise to the level of “improved.”

[0148] As used herein, “inhibiting and suppressing” and like terms should not be construed to require complete inhibition or suppression, although this may be desired in some embodiments.

[0149] As used herein, the term “genotype” refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.

[0150] The compositions and methods herein may provide for an improved “agronomic trait” or “trait of agronomic importance” or “trait of agronomic interest” to a plant, which may include, but not be limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, insect resistance, nematode resistance, fungal resistance, bacterial resistance, herbivore resistance, pathogen resistance, yield improvement, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of a metabolite, modulation of the proteome, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition, as compared to an isoline plant not comprising a modification derived from the methods or compositions herein. In some aspects, the trait of agronomic importance is achieved by association (physical proximity) of the plant to a microbe that confers a benefit to the plant.

[0151] “Agronomic trait potential” is intended to mean a capability of a plant element for exhibiting a phenotype, preferably an improved agronomic trait, at some point during its life cycle, or conveying said phenotype to another plant element with which it is associated in the same plant.

[0152] In some embodiments, the cell or organism has at least one heterologous trait. As used herein, the term “heterologous trait” refers to a phenotype imparted to a cell or organism by an exogenous molecule or other organism (e.g., a microbe), DNA segment, heterologous polynucleotide or heterologous nucleic acid.

[0153] Various changes in phenotype are of interest to the present disclosure, including but not limited to modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, increasing a plant’s yield of an economically important trait (e.g., grain yield, forage yield, etc.) and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants using the methods and compositions of the present disclosure

[0154] A “synthetic combination” can include a combination of a plant and a microbe, or a plant and a composition, of the disclosure. The combination may be achieved, for example, by coatingthe surface of a seed of a plant, such as an agricultural plant, or host plant tissue (root, stem, leaf, etc.), with a microbe of the disclosure. Further, a “synthetic combination” can include a combination of microbes of various strains or species. Synthetic combinations have at least one variable that distinguishes the combination from any combination that occurs in nature. That variable may be, inter alia, a concentration of microbe on a seed or plant tissue that does not occur naturally, or a combination of microbe and plant that does not naturally occur, or a combination of microbes or strains that do not occur naturally together. In each of these instances, the synthetic combination demonstrates the hand of man and possesses structural and / or functional attributes that are not present when the individual elements of the combination are considered in isolation.

[0155] In some embodiments, a microbe can be “endogenous” to a seed or plant. As used herein, a microbe is considered “endogenous” to a plant or seed, if the microbe is derived from the plant specimen from which it is sourced. That is, if the microbe is naturally found associated with said plant. In embodiments in which an endogenous microbe is applied to a plant, then the endogenous microbe is applied in an amount that differs from the levels found on the plant in nature. Thus, a microbe that is endogenous to a given plant can still form a synthetic combination with the plant, if the microbe is present on said plant at a level that does not occur naturally.

[0156] In some embodiments, a composition (such as a microbe) can be “heterologous” (also termed “exogenous”) to another composition (such as a seed or plant), and in some aspects is referred to herein as a “heterologous composition”. As used herein, a microbe is considered “heterologous” to a plant or seed, if the microbe is not derived from the plant specimen from which it is sourced. That is, if the microbe is not naturally found associated with said plant. For example, a microbe that is normally associated with leaf tissue of a maize plant is considered exogenous to a leaf tissue of another maize plant that naturally lacks said microbe. In another example, a microbe that is normally associated with a maize plant is considered exogenous to a wheat plant that naturally lacks said microbe.

[0157] A composition is “heterologously disposed” when mechanically or manually applied, artificially inoculated, associated with, or disposed onto or into a plant element, seedling, plant or onto or into a plant growth medium or onto or into a treatment formulation so that the treatment exists on or in the plant element, seedling, plant, plant growth medium, or formulation in a manner not found in nature prior to the application of the treatment, e.g., said combination whichis not found in nature in that plant variety, at that stage in plant development, in that plant tissue, in that abundance, or in that growth environment (for example, drought). In some embodiments, such a manner is contemplated to be selected from the group consisting of: the presence of the microbe; presence of the microbe in a different number of cells, concentration, or amount; the presence of the microbe in a different plant element, tissue, cell type, or other physical location in or on the plant; the presence of the microbe at different time period, e.g., developmental phase of the plant or plant element, time of day, time of season, and combinations thereof. In some embodiments, “heterologously disposed” means that the microbe being applied to a different tissue or cell type of the plant element than that in which the microbe is naturally found. In some embodiments, “heterologously disposed” means that the microbe is applied to a developmental stage of the plant element, seedling, or plant in which said microbe is not naturally associated, but may be associated at other stages. For example, if a microbe is normally found at the flowering stage of a plant and no other stage, a microbe applied at the seedling stage may be considered to be heterologously disposed. In some embodiments, a microbe is heterologously disposed the microbe is normally found in the root tissue of a plant element but not in the leaf tissue, and the microbe is applied to the leaf. In another non-limiting example, if a microbe is naturally found in the mesophyll layer of leaf tissue but is being applied to the epithelial layer, the microbe would be considered to be heterologously disposed. In some embodiments, “heterologously disposed” means that the native plant element, seedling, or plant does not contain detectable levels of the microbe in that same plant element, seedling, or plant. In some embodiments, “heterologously disposed” means that the microbe being applied is at a greater concentration, number, or amount of the plant element, seedling, or plant, than that which is naturally found in said plant element, seedling, or plant. For example, a microbe is heterologously disposed when present at a concentration that is at least 1.5 times greater, between 1.5 and 2 times greater, 2 times greater, between 2 and 3 times greater, 3 times greater, between 3 and 5 times greater, 5 times greater, between 5 and 7 times greater, 7 times greater, between 7 and 10 times greater, 10 times greater, or even greater than 10 times higher number, amount, or concentration than the concentration that was present prior to the disposition of said microbe. In another non-limiting example, a microbe that is naturally found in a tissue of a cupressaceous tree would be considered heterologous to tissue of a maize, wheat, cotton, soybean plant. In another example, a microbe that is naturally found in leaf tissue of a maize,spring wheat, cotton, soybean plant is considered heterologous to a leaf tissue of another maize, spring wheat, cotton, soybean plant that naturally lacks said microbe, or comprises the microbe in a different quantity.

[0158] Microbes can also be “heterologously disposed” on a given plant tissue. This means that the microbe is placed upon a plant tissue that it is not naturally found upon. For instance, if a given microbe only naturally occurs on the roots of a given plant, then that microbe could be exogenously applied to the above-ground tissue of a plant and would thereby be “heterologously disposed” upon said plant tissue. As such, a microbe is deemed heterologously disposed, when applied on a plant that does not naturally have the microbe present or does not naturally have the microbe present in the number that is being applied.

[0159] The compositions and methods herein may provide for a “modulated” “agronomic trait” or “trait of agronomic importance” to a host plant, which may include, but not be limited to, the following: altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, and altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, growth improvement, health enhancement, heat tolerance, herbicide tolerance, herbivore resistance, improved nitrogen fixation, improved nitrogen utilization, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, increased yield under water-limited conditions, kernel mass, kernel moisture content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutrition enhancement, pathogen resistance, pest resistance, photosynthetic capability improvement, salinity tolerance, stay-green, vigor improvement, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased length of pods per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, and increased number of non- wilted leaves per plant, a detectable modulation in the level of a metabolite, a detectable modulation in the level of a transcript, and a detectable modulation in the proteome, compared to an isoline plant grown from a seed without said seed treatment formulation. By the term “modulated”, it is intended to refer to a change in a characteristic, such as an agronomic trait, that is changed by virtue of the presence of the microbe(s), exudate, broth,metabolite, etc. In aspects, the modulation provides for the imparting of a trait, such as a trait of agronomic importance.Bacillus thuringiensis

[0160] Aspects of the disclosure include the insertion and modulation of certain polynucleotides, that result in a change of phenotype of the host cell and / or organisms with which the cell are or may become associated.

[0161] Cells of the bacterium Bacillus thuringiensis typically comprise genomic DNA in the form of a primary chromosome as well as several plasmids, each of which comprise various genes that contribute to the function and use of the bacterium.

[0162] Cry toxins, encoded by cry genes, are a family of crystal-forming proteins of the deltaendotoxin class produced by strains of the bacterium Bacillus thuringiensis. Their mode of action is thought to be to create pores that disrupt the gut epithelial membranes of pests, such as insects and nematodes. Genes encoding a spectrum of Cry toxins, including Cry mutants, Cry chimaeras and other Cry derivatives, are used commercially to enhance pest resistance in, for example, genetically modified (GM) crops. Cry toxins tend to be specific for particular insects, for example a particular Cry toxin will target one species of insect but not another.

[0163] Another class of toxins found in strains of the bacterium Bacillus thuringiensis are the beta-endotoxins, also called thuringiensin. Thuringiensin is a small oligosaccharide composed of adenosine, glucose, phosphoric acid, and gluconic diacid at a molecular ratio of 1:1: 1:1. Also called beta-endotoxin, thuringiensin is a non-specific toxin has the potential to affect different organisms and is banned from public use based on World Health Organization advice (World Health Organization. Guidelines Specification for Bacterial Larvicides for Public Health Use. World Health Organization; Geneva, Switzerland: 1999. Publication W. H.O. / CDS / CPC / WHOPES / 99.2. Report of the W.H.O. Informal Consultation.).Microbial-Imparted Traits in Plants

[0164] The present disclosure utilizes genetically-engineered microbes to impart beneficial properties (or beneficial traits) to desirable plant species, such as agronomic species of interest. In the current disclosure, the terminology “beneficial property” or “beneficial trait” is used interchangeably and denotes that a desirable plant phenotypic or genetic property of interest is modulated, by the application of a microbe or microbial consortia as described herein. Asaforementioned, in some aspects, it may very well be that a metabolite produced by a given microbe is ultimately responsible for modulating or imparting a beneficial trait to a given plant.

[0165] There are a vast number of beneficial traits that can be modulated by the application of microbes of the disclosure. For instance, the microbes may have the ability to impart one or more beneficial properties to a plant species, for example: increased growth, increased yield, increased nitrogen utilization efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, enhanced water use efficiency, increased pathogen resistance, modifications to plant architecture that don’t necessarily impact plant yield, but rather address plant functionality, causing the plant to increase production of a metabolite of interest, etc.

[0166] In aspects, the microbes taught herein provide a wide range of agricultural applications, including: improvements in yield of grain, fruit, and flowers, improvements in growth of plant parts, improved ability to utilize nutrients (e.g., nitrogen, phosphate, and the like), improved resistance to disease, biopesticidal effects including improved resistance to fungi, insects, and / or nematodes; improved surviv ability in extreme climate, and improvements in other desired plant phenotypic characteristics.

[0167] In some aspects, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to modulate or alter a plant characteristic such as altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, growth improvement, health enhancement, heat tolerance, herbicide tolerance, herbivore resistance, improved nitrogen fixation, improved nitrogen utilization, improved nutrient utilization (e.g., phosphate, potassium, and the like), improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, increased yield under water-limited conditions, kernel mass, kernel moisture content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutrition enhancement, pathogen resistance, reduced pathogen levels (e.g., via the excretion of metabolites that impair pathogen survival), pest resistance, photosynthetic capability improvement, salinity tolerance, stay-green, vigor improvement, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased length of pods per plant, reduced number of wilted leavesper plant, reduced number of severely wilted leaves per plant, and increased number of nonwilted leaves per plant, a detectable modulation in the level of a metabolite, a detectable modulation in the level of a transcript, and a detectable modulation in the proteome relative to a reference plant.

[0168] In some aspects, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to modulate in a negative way, a particular plant characteristic. For example, in some aspects, the microbes of the disclosure are able to decrease a phenotypic trait of interest, as this functionality can be desirable in some applications. For instance, the microbes of the disclosure may possess the ability to decrease root growth or decrease root length. Or the microbes may possess the ability to decrease shoot growth or decrease the speed at which a plant grows, as these modulations of a plant trait could be desirable in certain applications.

[0169] In some embodiments, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to impart nematode stress tolerance to plants.

[0170] In some embodiments, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to provide bio stimulation (biostimulant effects) to plants. In some embodiments, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to provide disease tolerance to plants.

[0171] In some embodiments, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to directly affect the pest life cycle, pest numbers, or pest population in or around the plants.Nematode Control

[0172] Plant-parasitic nematodes are recognized as one of the greatest threats to crops throughout the world. Plant-parasitic nematodes are microscopic roundworms that live in soil and plant tissues, and feed in or on roots. Several species may occur in the same field. Certain nematode species infest a wide variety of crops and other plants, while other nematodes are only able to feed on a few closely related plants, impairing the plant's ability to take up water and nutrients. Globally, nematodes are responsible for billions of dollars of agricultural yield loss.

[0173] The engineered (genetically-modified) bacteria described herein may find use in controlling nematode pests, by conferring resistance or tolerance to another organism, forexample a plant, to the pest, and / or by directly inhibiting or delaying nematode development, or by controlling nematode population directly, or both.

[0174] Examples of nematodes that may find use with the compositions and methods disclosed herein include major species of the following: Heterodera (Cyst nematodes), such as H. glycines, H. avenae, H. schachtii, H. trifolii, H. gottingiana, H. cajani, H. zeae; Globodera (Cyst nematodes), such as G. rostochiensis, G. pallida, G. tabacum; Meloidogyne (Root-knot nematodes), such as M. arenaria, M. incognita, M. javanica, M. hapla, M. chitwoodi', Ditylenchus (Stem and bulb nematode), such as D. destructor, D. dipsaci, D. angustus;Anguina (Seed gall nematodes), such as A. tritici, A. agrostis, Afrina / Anguina wevelli; Pratylenchus (Lesion nematodes), such as P. penetrans, P. brachyurus, P. coffeae, P. zeae, P. goodeyi, P. thornei, P. vulnus; Radopholus (Burrowing nematodes), such as R. similis with two host races ; Hirschmanniella (Root nematodes), such as H. oryzae, H. mucronata, H. spinicauda; Hoplolaimus (Lance nematodes), such as H. columbus, H. seinhorsti, H. indicus', Rotylenchulus (Reniform nematodes), such as R. reniformis; Tylenchulus (Citrus nematode), such as T. semipenetrans; Helicotylenchus (Spiral nematodes), such as H. multicinctus, H. mucronatus, H. dihystera, H. pseudorobustus'. Criconemella (Ring nematodes), such as C. xenoplax, C. axestis, C. spharocephalum; Xiphinema, Longidorus,Trichodorus & Paratrichodorus (Dagger, needle and stubby root nematodes), such as X. americanum, X. elongatum, L. africanus, P. minor, Aphelenchs (Bud and Leaf & Pine wood nematodes), such as A. fragariae, A. besseyi f, A. ritzemabosi ,Bursaphelenchus xylophilus.

[0175] Particularly crop-specific nematode pests include, without limitation, the following: citrus nematode (Tylenchulus semipenetrans)’, burrowing nematode (Radopholus similis)’ red ring nematode (Rhadinaphelenchus cocophilus) palm weevil (Rhynchophorus palmarum) lesion nematodes; corn cyst nematode (Heterodera zeae) root-knot nematode; reniform nematode (Rotylenchulus reniformis) lesion nematode (Pratylenchus brachyurus) sting nematode (Belonolaimus longicaudatus); Root-knot, cyst and reniform nematodes; pea cyst nematode (Heterodera goellingiana) ; pigeonpea cyst nematode on pigeonpea (Cajanus cajan); peanut root-knot nematode (M. arenaria) Javanese root-knot nematode (M. javanica); northern root-knot nematode (M. hapla) lesion nematode (P. brachyurus); potato cyst nematode Globodera rostochiensis (the "golden" nematode); potato cyst nematode Globodera pallida; root-knot (Meloidogyne), false root-knot (Nacobbus), bulb & stem (Ditylenchus dipsaci), potato-rot (Ditylenchus destructor) and lesion nematodes (Praty enchus)', Columbia root-knot nematode (M. chitwoodi)', white tip nematode (Aphelenchoides besseyi); the rice stem nematode (Dilylenchus angustus) root nematode, Hirschmanniella spp. and the rice cyst nematode (Heterodera oryzae) root-knot nematode (M. graminicola) ; cyst-nematode species infect rice roots (H. oryzae, H. oryzicola, H. elachista and H. saccharic soybean cyst nematode ; sugar-beet cyst nematode ; tobacco cyst nematode; tobacco root knot nematode; tobacco root lesion nematode; tobacco stem nematode; tobacco bulb nematode; tobacco stunt nematode; tobacco spiral nematode; tobacco reniform nematode; vegetable root-knot nematodes; vegetable reniform, stubby root, sugar-beet cyst, false root-knot, sting and stunt nematodes; vegetable stem; vegetable reniform; Potato cyst nematode; cereal cyst nematode, Heterodera avenae; the seed gall or ear-cockle nematode, Anguina tritici, root-knot, stunt, lesion nematodes; wheat gall nematode; Potato-cyst nematode (Globodera pallida), chiefly tuberous Solanum species; G. zelandica, Fuchsiaexcorticata and other native legume plant; rice cyst nematode (Heterodera oryzae)', pigeon-pea cyst nematode (H. cajani), on pigeon-pea (Cajanus cajani); H. mothi, on roots of Cyprus rotundus ; lucerne cyst nematode (H. medicaginis), on lucerne (Medicago sativa) in the Soviet Union; H. mediterranea, on roots of woody plant, lentisc (Pistacia lentiscus); H. pakistanensis, on roots of wheat (Triticum aestivum); H. graminis, from grass (Cynodon dactylori); H. sorghi, on roots of sorghum (Sorghum vulgare); H. raskii, from roots of bulb grass (Cyperus bulbosus); H. spinicauda, from soil around roots of the reed (Phragnutis australis)', Meloidogyne coffeicola, on roots of coffee (Coffea arabica)', M. salasi, on rice (Oryz.a sativa)', M. suginamiensis , from roots of mulberry (Morus alba)', M. sewelli, roots of spike-rush (Eleocharis acicularis); M. microcephala, from roots of tobacco (Nicotiana tabacum); M. arabicida, from coffee (Coffeae arabica)', M. Uni, from roots of rice; M. kongi, from roots of citrus; M. jianyangensis, from roots of mandarin orange; M. vandervegtei, from woody plant; M. hispanica, from roots of peach rootstock, Prunus persica Silvestris', M. brevicauda, from roots of tea (Camellia sinensis)', rice stem nematode (Ditylenchus angustus), in rice-growing areas; seed gall nematode (Afrina wevelli), from galls from amongst seed of Eragrostis curvulva; Afenestrata africana, parasites of grasses (Panicum maximum)', cystoid nematode (Thecavermiculatus andinus), from roots of Oxalis tuberosa .Agricultural Compositions

[0004] In some embodiments, the microbes of the disclosure arc combined with agricultural compositions. Agricultural compositions generally refer to organic and inorganic compounds that can include compositions that promote the cultivation of the microbe and / or the plant element; compositions involved in formulation of microbes for application to plant elements (for example, but not limited to: wetters, compatibilizing agents (also referred to as “compatibility agents”), antifoam agents, cleaning agents, sequestering agents, drift reduction agents, neutralizing agents and buffers, corrosion inhibitors, dyes, odorants, spreading agents (also referred to as “spreaders”), penetration aids (also referred to as “penetrants”), sticking agents (also referred to as “stickers” or “binders”), dispersing agents, thickening agents (also referred to as “thickeners”), stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, and the like); compositions involved in conferring protection to the plant element or plant (for example, but not limited to: pesticides, nematicides, fungicides, bactericides, herbicides, and the like); as well as other compositions that may be of interest for the particular application.

[0005] In some embodiments, the agricultural compositions of the present disclosure are solid. Where solid compositions are used, it may be desired to include one or more carrier materials with the active isolated microbe or consortia. In some embodiments, the present disclosure teaches the use of carriers including, but not limited to: mineral earths such as silicas, silica gels, silicates, talc, kaolin, attaclay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal meals, tree bark meal, wood meal and nutshell meal, cellulose powders, attapulgites, montmorillonites, mica, vermiculites, synthetic silicas and synthetic calcium silicates, or compositions of these.Growth Compositions

[0006] In some embodiments, a composition is provided to the microbe and / or the plant element that promotes the growth and development. Exemplary compositions include liquid (such as broth, media) and / or solid (such as soil, nutrients). Various organic or inorganic compounds may be added to the growth composition to facilitate the health of the microbe, alone or in combination with the plant element, for example but not limited to: amino acids, vitamins, minerals, carbohydrates, simple sugars, lipids.Formulation Compositions

[0007] One or more compositions, in addition to the microbc(s) or microbial-produccd composition, may be combined for various application, stability, activity, and / or storage reasons. The additional compositions may be referred to as “formulation components”.

[0008] In some embodiments, the agricultural compositions of the present disclosure are liquid. Thus in some embodiments, the present disclosure teaches that the agricultural compositions disclosed herein can include compounds or salts such as monoethanolamine salt, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammonium carbamate.

[0009] In some embodiments, the present disclosure teaches that agricultural compositions can include binders such as: polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose, starch, vinylpyrrolidone / vinyl acetate copolymers and polyvinyl acetate, or compositions of these; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or compositions of these; antifoams such as silicone emulsions, long-chain alcohols, phosphoric esters, acetylene diols, fatty acids or organofluorine compounds, and complexing agents such as: salts of ethylenediaminetetraacetic acid (EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoric acids, or compositions of these.

[0010] In some embodiments, the agricultural compositions comprise surface- active agents. In some embodiments, the surface-active agents are added to liquid agricultural compositions. In other embodiments, the surface-active agents are added to solid formulations, especially those designed to be diluted with a carrier before application. Thus, in some embodiments, the agricultural compositions comprise surfactants. Surfactants are sometimes used, either alone or with other additives, such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the microbes on the target. The types of surfactants used for bioenhancement depend generally on the nature and mode of action of the microbes. The surface-active agents can be anionic, cationic, or nonionic in character, and can be employed as emulsifying agents, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactants are non-ionics such as: alky ethoxylates, linear aliphatic alcoholethoxylates, and aliphatic amine ethoxylates. Surfactants conventionally used in the art of formulation and which may also be used in the present formulations arc described, in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, N.J., 1998, and in Encyclopedia of Surfactants, Vol. I-III, Chemical Publishing Co., New York, 1980- 81. In some embodiments, the present disclosure teaches the use of surfactants including alkali metal, alkaline earth metal or ammonium salts of aromatic sulfonic acids, for example, ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and of fatty acids of arylsulfonates, of alkyl ethers, of lauryl ethers, of fatty alcohol sulfates and of fatty alcohol glycol ether sulfates, condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or of the naphthalenesulfonic acids with phenol and formaldehyde, condensates of phenol or phenolsulfonic acid with formaldehyde, condensates of phenol with formaldehyde and sodium sulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-, octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylaryl poly ether alcohols, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenols, salts of phosphated triarylphenolethoxylates, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignin- sulfite waste liquors or methylcellulose, or compositions of these.

[0011] In some embodiments, the present disclosure teaches other suitable surface-active agents, including salts of alkyl sulfates, such as diethanolammonium lauryl sulfate; alkylarylsulfonate salts, such as calcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-C18 ethoxylate; alcohol-alkylene oxide addition products, such as tridecyl alcohol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene- sulfonate salts, such as sodium dibutyl-naphthalenesulfonate; dialkyl esters of sulfosuccinate salts, such as sodium di(2- ethylhexyl)sulfosuccinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as lauryl trimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of mono and dialkyl phosphate esters; vegetable oils such as soybean oil, rapeseed / canola oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil and the like; and esters of the above vegetable oils, particularly methyl esters.

[0012] In some embodiments, the agricultural compositions comprise wetting agents. A wetting agent is a substance that when added to a liquid increases the spreading or penetration power ofthe liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents arc used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank or other vessel to reduce the wetting time of wettable powders and to improve the penetration of water into water-dispersible granules. In some embodiments, examples of wetting agents used in the agricultural compositions of the present disclosure, including wettable powders, suspension concentrates, and water-dispersible granule formulations are: sodium lauryl sulphate; sodium dioctyl sulphosuccinate; alkyl phenol ethoxylates; and aliphatic alcohol ethoxylates.

[0013] In some embodiments, the agricultural compositions of the present disclosure comprise dispersing agents. A dispersing agent is a substance which adsorbs onto the surface of particles and helps to preserve the state of dispersion of the particles and prevents them from reaggregating. In some embodiments, dispersing agents are added to agricultural compositions of the present disclosure to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. In some embodiments, dispersing agents are used in wettable powders, suspension concentrates, and water-dispersible granules.Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to re-aggregation of particles. In some embodiments, the most commonly used surfactants are anionic, non-ionic, or mixtures of the two types.

[0014] In some embodiments, for wettable powder formulations, the most common dispersing agents are sodium lignosulphonates. In some embodiments, suspension concentrates provide very good adsorption and stabilization using poly electrolytes, such as sodium naphthalene sulphonate formaldehyde condensates. In some embodiments, tristyrylphenol ethoxylate phosphate esters are also used. In some embodiments, such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates.

[0015] In some embodiments, the agricultural compositions of the present disclosure comprise polymeric surfactants. In some embodiments, the polymeric surfactants have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a‘comb’ surfactant. In some embodiments, these high molecular weight polymers can give very good long-term stability to suspension concentrates, because the hydrophobic backbones have many anchoring points onto the particle surfaces. In some embodiments, examples of dispersing agents used in agricultural compositions of the present disclosure are: sodium lignosulphonates; sodium naphthalene sulphonate formaldehyde condensates; tristyrylphenol ethoxylate phosphate esters; aliphatic alcohol ethoxylates; alky ethoxylates; EO-PO block copolymers; and graft copolymers.

[0016] In some embodiments, the agricultural compositions of the present disclosure comprise emulsifying agents. An emulsifying agent is a substance, which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent the two liquids would separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkylphenol or aliphatic alcohol with 12 or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzene sulphonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 will normally provide good stable emulsions. In some embodiments, emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant.

[0017] In some embodiments, the agricultural compositions of the present disclosure comprise solubilizing agents. A solubilizing agent is a surfactant, which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials inside the hydrophobic part of the micelle. The types of surfactants usually used for solubilization are non-ionics: sorbitan monooleates; sorbitan monooleate ethoxylates; and methyl oleate esters.

[0018] In some embodiments, the agricultural compositions of the present disclosure comprise organic solvents. Organic solvents are used mainly in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent granular formulations. Sometimes mixtures of solvents are used. In some embodiments, the present disclosure teaches the use of solvents including aliphatic paraffinic oils such as kerosene or refined paraffins. In other embodiments, the present disclosure teaches the use of aromatic solvents such as xylene and higher molecular weight fractions of C9 and CIO aromatic solvents. In some embodiments, chlorinated hydrocarbons are useful as co-solvents to prevent crystallization of pesticides whenthe formulation is emulsified into water. Alcohols are sometimes used as co-solvents to increase solvent power.

[0019] In some embodiments, the agricultural compositions comprise gelling agents. Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. In some embodiments, the agricultural compositions comprise one or more thickeners including, but not limited to: montmorillonite, e.g., bentonite; magnesium aluminum silicate; and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickening agents. The types of polysaccharides most commonly used are natural extracts of seeds and seaweeds or synthetic derivatives of cellulose. Some embodiments utilize xanthan and some embodiments utilize cellulose. In some embodiments, the present disclosure teaches the use of thickening agents including, but are not limited to: guar gum; locust bean gum; carrageenam; alginates; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). In some embodiments, the present disclosure teaches the use of other types of anti-settling agents such as modified starches, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

[0020] In some embodiments, the presence of surfactants, which lower interfacial tension, can cause water-based formulations to foam during mixing operations in production and in application through a spray tank. Thus, in some embodiments, in order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before filling into bottles / spray tanks. Generally, there are two types of anti-foam agents, namely silicones and nonsilicones. Silicones are usually aqueous emulsions of dimethyl poly siloxane, while the nonsilicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the anti-foam agent is to displace the surfactant from the air-water interface.

[0021] In some embodiments, the agricultural compositions comprise a preservative.

[0022] In some embodiments, the agricultural compositions may be formulated as: a soil drench, a foliar spray, a dip treatment, an in-furrow treatment, a soil amendment, granules, a broadcasttreatment, a post-harvest disease control treatment, or a seed treatment. In some embodiments, the agricultural compositions may be applied alone in or in rotation spray programs with other agricultural products.

[0023] In some embodiments, the agricultural compositions may be compatible with tank mixing. In some embodiments, the agricultural compositions may be compatible with tank mixing with other agricultural products. In some embodiments, the agricultural compositions may be compatible with equipment used for ground, aerial, and irrigation applications.

[0024] In some embodiments, the agricultural compositions may be applied to genetically modified seeds or plants.Protective Compositions

[0025] Further, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with known actives available in the agricultural space, such as: pesticide, herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, plant growth regulator, rodenticide, anti-algae agent, biocontrol or beneficial agent. Further, the microbes, microbial consortia, or microbial communities developed according to the disclosed methods can be combined with known fertilizers. Such combinations may exhibit synergistic properties. Further still, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with inert ingredients. Also, in some aspects, the disclosed microbes are combined with biological active agents.

[0026] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with biopesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent. Such biopesticides may be, but are not limited to, macrobial organisms (e.g., beneficial nematodes and the like), microbial organisms (e.g., Serenade, QST713, Bt, and the like), plant extracts (e.g., Timorex Gold and the like), biochemical (e.g., insect pheromones and the like), and / or minerals and oils (e.g., canola oil). Pesticides and Biopesticides

[0027] In some embodiments, the agricultural compositions of the present disclosure comprise pesticides, used in combination with the taught microbes. In some embodiments, the agriculturalcompositions of the present disclosure comprise biopesticides, used in combination with the taught microbes.

[0028] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with known pesticides in the agricultural space, such as; pesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent.

[0029] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with known biopesticides in the agricultural space, such as: biopesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent.

[0030] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients including: macrobial organisms (e.g., beneficial nematodes and the like), microbial organisms (e.g., Serenade, Bt, and the like), plant extracts (e.g., Timorex Gold and the like), biochemical (e.g., insect pheromones and the like), and / or minerals and oils (e.g., canola oil).

[0031] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with an herbicide selected from the group consisting of: an acetamide selected from the group consisting of acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, flufenacet, mefenacet, metolachlor, metazachlor, napropamide, naproanilide, pethoxamid, pretilachlor, propachlor, and thenylchlor; an amino acid derivative selected from the group consisting of bilanafos, glufosinate, and sulfosate; an aryloxyphenoxypropionate selected from the group consisting of clodinafop, cyhalofop-butyl, fenoxaprop, fluazifop, haloxyfop, metamifop, propaquizafop, quizalofop, and quizalo-fop-P-tefuryl; diquat and paraquat; a (thio)carbamate selected from the group consisting of asulam, butylate, carbetamide, desmedipham, dimepiperate, eptam (EPTC), esprocarb, molinate, orbencarb, phenmedipham, prosulfocarb, pyributicarb, thiobencarb, and triallate; a cyclohexanedione selected from the group consisting of butroxydim, clethodim, cycloxydim, profoxydim, sethoxydim, tepraloxydim, and tralkoxydim; a dinitroaniline selected from the group consisting of benfluralin, ethalfluralin, oryzalin, pendimethalin, prodiamine, andtrifluralin; a diphenyl ether selected from the group consisting of acifluorfen, aclonifen, bifenox, diclofop, cthoxyfcn, fomcsafcn, lactofcn, and oxyfluorfcn; a hydroxybenzonitrile selected from the group consisting of bomoxynil, dichlobenil, and ioxynil; an imidazolinone selected from the group consisting of imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, and imazethapyr; a phenoxy acetic acid selected from the group consisting of clomeprop, 2,4- dichlorophenoxyacetic acid (2,4-D), 2,4-DB, dichlorprop, MCPA, MCPA-thioethyl, MCPB, and Mecoprop; a pyrazine selected from the group consisting of chloridazon, flufenpyr-ethyl, fluthiacet, norflurazon, and pyridate; a pyridine selected from the group consisting of aminopyralid, clopyralid, diflufenican, dithiopyr, fluridone, fluroxypyr, picloram, picolinafen, and thiazopyr; a sulfonyl urea selected from the group consisting of amidosulfuron, azimsulfuron, bensulfuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron-methyl, nicosulfuron, oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron, trifloxysulfuron, triflusulfuron, tritosulfuron, and 14(2- chloro-6-propyl-imidazol[l,2]-blpyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxy-pyrimidin-2-yl)urea; a triazine selected from the group consisting of ametryn, atrazine, cyanazine, a dimethametryn, ethiozin, hexazinone, metamitron, metribuzin, prometryn, simazine, terbuthylazine, terbutryn, and triaziflam; a urea compound selected from the group consisting of chlorotoluron, daimuron, diuron, fluometuron, isoproturon, linuron, methabenzthiazuron, and tebuthiuron; an acetolactate synthase inhibitor selected from the group consisting of bispyribac- sodium, cloransulam-methyl, diclosulam, florasulam, flucarbazone, flumetsulam, metosulam, ortho- sulfamuron, penoxsulam, propoxycarbazone, pyribambenz-propyl, pyribenzoxim, pyriftalid, pyriminobac-methyl, pyrimisulfan, pyrithiobac, pyroxasulfone, and pyroxsulam; and a compound selected from the group consisting of amicarbazone, aminotriazole, anilofos, beflubutamid, benazolin, bencarbazone, benfluresate, benzofenap, bentazone, benzobicyclon, bromacil, bromobutide, butafenacil, butamifos, cafenstrole, carfentrazone, cinidon-ethlyl, chlorthal, cinmethylin, clomazone, cumyluron, cyprosulfamide, dicamba, difenzoquat, dillufenzopyr, Drechslera monoceras, endothal, ethofumesate, etobenzanid, fentrazamide, flumiclorac -pentyl, flumioxazin, fhipoxam, flurochloridone, flurtamone, indanofan, isoxaben, isoxaflutole, lenacil, propanil, propyzamide, quinclorac, quinmerac, mesotrione, methyl arsonic acid, naptalam, oxadiargyl,oxadiazon, oxaziclomefone, pentoxazone, pinoxaden, pyraclonil, pyraflufen-ethyl, pyrasulfotole, pyrazoxyfcn, pyrazolynatc, quinoclaminc, saflufcnacil, sulcotrionc, sulfcntrazonc, terbacil, tefuryltrione, tembotrione, thiencarbazone, topramezone, 4-hydroxy-3-[2-(2-methoxy- ethoxymethyl)-6-trifluoromethyl-pyridine-3-carbonyl]-bicyclol[3.2.1]oct-3-en-2-one, (3- [2- chloro-4-fluoro-5-(3-methy 1-2, 6-dioxo-4-trifluoromethyl-3,6-dihydro-2H-pyrimidin- 1-yl)- phenoxyl]-pyridin-2-yloxy)-acetic acid ethyl ester, 6-amino-5-chloro-2-cyclopropyl-pyrimidine- 4-carboxylic acid methyl ester, 6-chloro-3-(2-cyclopropyl-6-methyl-phenoxy)-pyridazin-4-ol, 4- amino-3-chloro-6-(4-chloro-phenyl)-5-lluoro-pyridine-2-carboxylic acid, 4-amino-3-chloro-6-(4- chloro-2-fluoro-3-methoxy-phenyl)-pyridine-2-carboxylic acid methyl ester, and 4-amino-3- chloro-6-(4-chloro-3-dimethylamino-2-fluoro-phenyl)-pyridine-2-carboxylic acid methyl ester.

[0032] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with an insecticide selected from the group consisting of: an organo(thio)phosphate selected from the group consisting of acephate, azamethiphos, azinphos-methyl, chlorpyrifos, chlorpyrifos-methyl, chlorfenvinphos, diazinon, dichlorvos, dicrotophos, dimethoate, disulfoton, ethion, fenitrothion, fenthion, isoxathion, malathion, methamidophos, methidathion, methyl-parathion, mevinphos, monocrotophos, oxydemeton-methyl, paraoxon, parathion, phenthoate, phosalone, phosmet, phosphamidon, phorate, phoxim, pirimiphos-methyl, profenofos, prothiofos, sulprophos, tetrachlorvinphos, terbufos, triazophos, and trichlorfon; a carbamate selected from the group consisting of alanycarb, aldicarb, bendiocarb, benfuracarb, carbaryl, carbofuran, carbosulfan, fenoxycarb, furathiocarb, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, thiodicarb, and triazamate; a pyrethroid selected from the group consisting of allethrin, bifenthrin, cyfluthrin, cyhalothrin, cyphenothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, zeta- cypermethrin, deltamethrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, imiprothrin, lambda-cyhalothrin, permethrin, prallethrin, pyrethrin I and II, resmethrin, silafluofen, taufluvalinate, tefluthrin, tetramethrin, tralomethrin, transfluthrin, profluthrin, and dimefluthrin; an insect growth regulator selected from the group consisting of a) a chitin synthesis inhibitor wherein said chitin synthesis inhibitor is a benzoylurea selected from the group consisting of chlorfluazuron, cyramazin, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, teflubenzuron, triflumuron; buprofezin, diofenolan, hexythiazox, etoxazole, and clofentazine; b) an ecdysone antagonist selected from the group consisting ofhalofenozide, methoxyfenozide, tebufenozide, and azadirachtin; c) a juvenoid selected from the group consisting of pyriproxyfcn, mcthoprcnc, and fcnoxycarb; or d) a lipid biosynthesis inhibitor selected from the group consisting of spirodiclofen, spiromesifen, and spirotetramat; a nicotinic receptor agonist / antagonist compound selected from the group consisting of clothianidin, dinotefuran, imidacloprid, thiamethoxam, nitenpyram, acetamiprid, thiacloprid, and l-(2-chloro-thiazol-5-ylmethyl)-2-nitrimino-3,5-dimethyl-[l,3,5]triazinane; a GABA antagonist compound selected from the group consisting of endosulfan, ethiprole, fipronil, vaniliprole, pyrafluprole, pyriprole, and 5-amino-l-(2,6-dichloro-4-methyl-phenyl)-4-sulfinamoyl-lH- pyrazole-3-c arbothioic acid amide; a macrocyclic lactone insecticide selected from the group consisting of abamectin, emamectin, milbemectin, lepimectin, spinosad, and spinetoram; a mitochondrial electron transport inhibitor (METI) I acaricide selected from the group consisting of fenazaquin, pyridaben, tebufenpyrad, tolfenpyrad, and flufenerim; a METI II and III compound selected from the group consisting of acequinocyl, fluacyprim, and hydramethylnon; chlorfenapyr; an oxidative phosphorylation inhibitor selected from the group consisting of cyhexatin, diafenthiuron, fenbutatin oxide, and propargite; cryomazine; piperonyl butoxide; a sodium channel blocker selected from the group consisting of indoxacarb and metaflumizone; and a compound selected from the group consisting of benclothiaz, bifenazate, cartap, flonicamid, pyridalyl, pymetrozine, sulfur, thiocyclam, flubendiamide, chlorantraniliprole, cyazypyr (HGW86), cyenopyrafen, flupyrazofos, cyflumetofen, amidoflumet, imicyafos, bistrifluron, and pyrifluquinazon.

[0033] In some embodiments, the present invention teaches a synergistic use of the presently disclosed microbes or microbial consortia with known pesticides in the agricultural space, such as: pesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent.

[0034] In some embodiments, the present invention teaches a synergistic use of the presently disclosed microbes or microbial consortia with known biopesticides in the agricultural space, such as: biopesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent.

[0035] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a pesticide one witnesses an additive effect on a plant phenotypic trait of interest. In other embodiments, when the microbe or microbial consortiaidentified according to the taught methods is combined with a pesticide one witness a synergistic effect on a plant phenotypic trait of interest.

[0036] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a biopesticide one witnesses an additive effect on a plant phenotypic trait of interest. In other embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a biopesticide one witness a synergistic effect on a plant phenotypic trait of interest.

[0037] The synergistic effect obtained by the taught methods can be quantified according to Colby’s formula (i.e., (E) =X+Y-(X*Y / 100). See Colby, R. S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967 Weeds, vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, by “synergistic” is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount.

[0038] The isolated microbes and consortia of the present disclosure can synergistically increase the effectiveness of agriculturally active pesticide compounds and also agricultural auxiliary pesticide compounds.

[0039] The isolated microbes and consortia of the present disclosure can synergistically increase the effectiveness of agriculturally active biopesticide compounds and also agricultural auxiliary biopesticide compounds.Plant Growth Regulators and Biostimulants

[0040] In some embodiments, the agricultural compositions of the present disclosure comprise plant growth regulators and / or biostimulants, used in combination with the taught microbes.

[0041] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with known plant growth regulators in the agricultural space, such as: auxins, gibberellins, cytokinins, ethylene generators, growth inhibitors, and growth retardants.

[0042] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients including: ancymidol, butralin, alcohols, chloromequat chloride, cytokinin, daminozide, ethepohon, flurprimidol, giberrelic acid, gibberellin mixtures, indole-3-butryic acid (IB A), maleic hydrazide, mefludide, mepiquat chloride, mepiquat pentaborate, naphthalene-acetic acid (NAA), 1- napthaleneacetemide, (NAD), n-decanol, placlobutrazol, prohexadione calcium, trinexapac-ethyl,uniconazole, salicylic acid, abscisic acid, ethylene, brassinosteroids, jasmonates, polyamines, nitric oxide, strigolactoncs, or karrikins among others.

[0043] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with seed inoculants known in the agricultural space, such as: QUICKROOTS®, VAULT®, RHIZO- STICK®, NODULATOR®, DORMAL®, SABREX®, among others. In some embodiments, a Bradyrhizobium inoculant is utilized in combination with any single microbe or microbial consortia disclosed here. In particular aspects, a synergistic effect is observed when one combines one of the aforementioned inoculants, e.g., QUICKROOTS® or Bradyrhizobium, with a microbe or microbial consortia as taught herein.

[0044] In some embodiments, the agricultural compositions of the present disclosure comprise a plant growth regulator, which contains: kinetin, gibberellic acid, and indole butyric acid, along with copper, manganese, and zinc.

[0045] In some embodiments, the present disclosure teaches agricultural compositions comprising one or more commercially available plant growth regulators, including but not limited to: Abide®, A-Rest®, Butralin®, Fair®, Royaltac M®, Sucker-Plucker®, Off-Shoot®, Contact-85®, Citadel®, Cycocel®, E-Pro®, Conklin®, Culbac®, Cytoplex®, Early Harvest®, Foli-Zyme®, Goldengro®, Happygro®, Incite®, Megagro®, Ascend®, Radiate®, Stimulate®, Suppress®, Validate®, X-Cyte®, B-Nine®, Compress®, Dazide®, Boll Buster®, BollD®, Cerone®, Cotton Quik®, Ethrel®, Finish®, Flash®, Florel®, Mature®, MFX®, Prep®, Proxy®, Quali-Pro®, SA-50®, Setup®, Super Boll®, Whiteout®, Cutless®, Legacy®, Mastiff®, Topflor®, Ascend®, Cytoplex®, Ascend®, Early Harvest®, Falgro®, Florgib®, Foli-Zyme®, GA3®, GibGro®, Green Sol®, Incite®, N-Large®, PGR IV®, Pro-Gibb®, Release®, Rouse®, Ryzup®, Stimulate®, BVB®, Chrysal®, Fascination®, Procone®, Fair®, Rite-Hite®, Royal®, Sucker Stuff®, Embark®, Sta-Lo®, Pix®, Pentia®, DipN Grow®, Goldengro®, Hi-Yield®, Rootone®, Antae®, FST-7®, Royaltac®, Bonzi®, Cambistat®, Cutdown®, Downsize®, Florazol®, Paclo®, Paczol®, Piccolo®, Profile®, Shortstop®, Trimmit®, Turf Enhancer®, Apogee®, Armor Tech®, Goldwing®, Governor®, Groom®, Legacy®, Primeraone®, Primo®, Provair®, Solace®, T-Nex®, T-Pac®, Concise®, and Sumagic®.

[0046] In some embodiments, the present invention teaches a synergistic use of the presently disclosed microbes or microbial consortia with plant growth regulators and / or stimulants such asphytohormones or chemicals that influence the production or disruption of plant growth regulators.

[0047] In some embodiments, the present invention teaches that phytohormones can include: Auxins (e.g., Indole acetic acid IAA), Gibberellins, Cytokinins (e.g., Kinetin), Abscisic acid, Ethylene (and its production as regulated by ACC synthase and disrupted by ACC deaminase).

[0048] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with biostimulants. Such biostimulants may be, but are not limited to, microbial organisms, plant extracts, seaweeds, acids, biochar, and the like.

[0049] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with fertilizers, which may be organic (e.g., manure, blood, fish, urine, seaweed, kelp, and the like), nitrogenbased (e.g., nitrate, ammonium, urea, and the like), phosphate, and potassium. Such fertilizers may also contain micronutrients including, but not limited to, sulfur, iron, zinc, and the like.

[0050] In some embodiments, the present invention teaches additional plant-growth promoting chemicals that may act in synergy with the microbes and microbial consortia disclosed herein, such as: humic acids, fulvic acids, amino acids, polyphenols and protein hydrolysates.

[0051] Thus, in some embodiments, the disclosure provides for the application of the taught microbes in combination with Ascend® upon any crop. Further, the disclosure provides for the application of the taught microbes in combination with Ascend® upon any crop and utilizing any method or application rate.

[0052] In some embodiments, the present disclosure teaches agricultural compositions with bio stimulants.

[0053] As used herein, the term “biostimulant” refers to any substance that acts to stimulate the growth of microorganisms that may be present in soil or other plant growing medium.

[0054] The level of microorganisms in the soil or growing medium is directly correlated to plant health. Microorganisms feed on biodegradable carbon sources, and therefore plant health is also correlated with the quantity of organic matter in the soil. While fertilizers provide nutrients to feed and grow plants, in some embodiments, biostimulants provide biodegradable carbon, e.g., molasses, carbohydrates, e.g., sugars, to feed and grow microorganisms. Unless clearly statedotherwise, a biostimulant may comprise a single ingredient, or a combination of several different ingredients, capable of enhancing microbial activity or plant growth and development, due to the effect of one or more of the ingredients, either acting independently or in combination.

[0055] In some embodiments, biostimulants are compounds that produce non-nutritional plant growth responses. In some embodiments, many important benefits of biostimulants are based on their ability to influence hormonal activity. Hormones in plants (phytohormones) are chemical messengers regulating normal plant development as well as responses to the environment. Root and shoot growth, as well as other growth responses are regulated by phytohormones. In some embodiments, compounds in biostimulants can alter the hormonal status of a plant and exert large influences over its growth and health. Thus, in some embodiments, the present disclosure teaches sea kelp, humic acids, fulvic acids, and B Vitamins as common components of biostimulants. In some embodiments, the biostimulants of the present disclosure enhance antioxidant activity, which increases the plant's defensive system. In some embodiments, vitamin C, vitamin E, and amino acids such as glycine are antioxidants contained in biostimulants.

[0056] In other embodiments, biostimulants may act to stimulate the growth of microorganisms that are present in soil or other plant growing medium. Prior studies have shown that when certain biostimulants comprising specific organic seed extracts (e.g., soybean) were used in combination with a microbial inoculant, the biostimulants were capable of stimulating growth of microbes included in the microbial inoculant. Thus, in some embodiments, the present disclosure teaches one or more biostimulants that, when used with a microbial inoculant, is capable of enhancing the population of both native microbes and inoculant microbes. For a review of some popular uses of biostimulants, please see Calvo et al., 2014, Plant Soil 383:3-41.Combinations of Plant Elements, Microbes, and Agricultural Compositions

[0057] In some embodiments, the present disclosure teaches that the individual microbes, or microbial consortia, or microbial communities, or any combination of the preceding, for example comprising any one or a plurality of microorganisms that include a genetically-modified Bacillus ihuringiensis or any strain comprising a polynucleotide sharing at least 95% identity with any one of SEQ ID NOs. 1-50, may be applied to a plant element, optionally in combination with any agricultural composition, for the improvement of a plant phenotype.

[0058] Isolated microbes or communities or consortia (generally “microbes” or “microbe”, interchangeably) may be applied to a heterologous plant element, creating a syntheticcombination. Microbes are considered heterologous to a plant element if they are not normally associated with the plant element in nature, or if found, arc applied in amounts different than that found in nature. In some embodiments, the microbes may be found naturally in one part of a plant but not another, and introduction of the microbes to another part of the plant is considered a heterologous association.

[0059] It is further contemplated that the microbe, either isolated or in combination with a plant or plant element, may be further associated with one or more agricultural compositions, such as those described above.

[0060] Synthetic combinations of microbes and plant elements, microbes and agricultural compositions, and microbes and plant elements and agricultural compositions are contemplated (generally “synthetic compositions”, compositions that comprise components not typically found associated in nature).Plant Element Treatments

[0061] In some embodiments, the present disclosure also concerns the discovery that treating plant elements before they are sown or planted with a combination of one or more of the microbes or agricultural compositions of the present disclosure can enhance a desired plant trait, e.g., plant growth, plant health, plant yield, plant tolerance to pests. In some aspects, the plant benefits from the microbe imparting a deleterious effect to another organism that affects the plant.

[0062] Thus, in some embodiments, the present disclosure teaches the use of one or more of the microbes or microbial consortia as plant element treatments. The plant element treatment can be a plant element coating applied directly to an untreated and “naked” plant element. However, the plant element treatment can be a plant element overcoat that is applied to a plant element that has already been coated with one or more previous plant element coatings or plant element treatments. The previous plant element treatments may include one or more active compounds, either chemical or biological, and one or more inert ingredients.

[0063] The term “plant element treatment” generally refers to application of a material to a plant element prior to or during the time it is planted in soil. Plant element treatment with microbes, and other agricultural compositions of the present disclosure, has the advantages of delivering the treatments to the locus at which the plant elements are planted shortly before germination of the plant element and emergence of a plant element.

[0064] In other embodiments, the present disclosure also teaches that the use of plant element treatments minimizes the amount of microbe or agricultural composition that is required to successfully treat the plants, and further limits the amount of contact of workers with the microbes and compositions compared to application techniques such as spraying over soil or over emerging plant element.

[0065] Moreover, in some embodiments, the present disclosure teaches that the microbes disclosed herein are important for enhancing the early stages of plant life (e.g., within the first thirty days following emergence of the plant element). Thus, in some embodiments, delivery of the microbes and / or compositions of the present disclosure as a plant element treatment places the microbe at the locus of action at a critical time for its activity.

[0066] In some embodiments, the microbial compositions of the present disclosure are formulated as a plant element treatment. In some embodiments, it is contemplated that the plant elements can be substantially uniformly coated with one or more layers of the microbes and / or agricultural compositions disclosed herein, using conventional methods of mixing, spraying, or a combination thereof through the use of treatment application equipment that is specifically designed and manufactured to accurately, safely, and efficiently apply plant element treatment products to plant elements. Such equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mists, or a combination thereof. Liquid plant element treatments such as those of the present disclosure can be applied via either a spinning “atomizer” disk or a spray nozzle, which evenly distributes the plant element treatment onto the plant element as it moves though the spray pattern. In aspects, the plant element is then mixed or tumbled for an additional period of time to achieve additional treatment distribution and drying.

[0067] The plant elements can be primed or unprimed before coating with the microbial compositions to increase the uniformity of germination and emergence. In an alternative embodiment, a dry powder formulation can be metered onto the moving plant element.

[0068] In some embodiments, the plant elements have at least part of the surface area coated with a microbiological composition, according to the present disclosure. In some embodiments, a plant element coat comprising the microbial composition is applied directly to a naked plant element. In some embodiments, a plant element overcoat comprising the microbial composition is applied to a plant element that already has a plant element coat applied thereon. In someaspects, the plant element may have a plant element coat comprising, e.g., clothianidin and / or Bacillus firmus-I-1582, upon which the present composition will be applied on top of, as a plant element overcoat. In some aspects, the taught microbial compositions are applied as a plant element overcoat to plant elements that have already been treated with PONCHO™ VOTiVO™. In some aspects, the plant element may have a plant element coat comprising, e.g., Metalaxyl, and / or clothianidin, and / or Bacillus firmus-I-1582, upon which the present composition will be applied on top of, as a plant element overcoat. In some aspects, the taught microbial compositions are applied as a plant element overcoat to plant elements that have already been treated with ACCELERON™.

[0069] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A2 to 10A12, 10A2 to 10Al 1, 10A2 to 10A10, 10A2 to 10A9, lA02 to 10A8, 10A2 to 10A7, 10A2 to 10A6, 10A2 to 10A5, 10A2 to 10A4, or 10A2 to 10A3 per plant element.

[0070] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A3 to 10A12, 10A3 to 10Al 1, 10A3 to 10A10, 10A3 to 10A9, 10A3 to 10A8, 10A3 to 10A7, 10A3 to 10A6, 10A3 to 10A5, or 10A3 to 10A4 per plant element.

[0071] In some embodiments, the microorganism- treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A4 to 10A12, 10A4 to 10 11, 10A4 to 10A10, 10A4 to 10A9, 10A4 to 10A8, 10A4 to 10A7, 10A4 to 10A6, or 10A4 to 10A5 per plant element.

[0072] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A5 to 10A12, 10A5 to 10A11, 10A5 to 10A10, 10A5 to 10A9, 10A5 to 10A8, 10A5 to 10A7, or 10A5 to 10A6 per plant element.

[0073] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A5 to 10A9 per plant element.

[0074] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, of at least about: 1 x 10A3, or 1 x 10A4, or 1 x 10A5, or 1 x 10A6, or 1 x 10A7, or 1 x 10A8, or 1 x 10A9 per plant element.

[0075] In some embodiments, the amount of one or more of the microbes and / or agricultural compositions applied to the plant element depend on the final formulation, as well as size or typeof the plant or plant element utilized. Tn some embodiments, one or more of the microbes are present in about 2% w / w / to about 80% w / w of the entire formulation. In some embodiments, the one or more of the microbes employed in the compositions is about 5% w / w to about 65% w / w, or 10% w / w to about 60% w / w by weight of the entire formulation.

[0076] In some embodiments, the plant elements may also have more spores or microbial cells per plant element, such as, for example about 10A2, 10A3, 10A4, 10A5, 10A6, 10A7, 10A8, 10A9, 10A10, 10Al l, 10A12, 10A13, 10A14, 10A15, 10A16, or 10A17 spores or cells per plant element.

[0077] In some embodiments, the plant element coats of the present disclosure can be up to 10pm, 20pm, 30pm, 40pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 110pm, 120pm, 130pm, 140pm, 150pm, 160pm, 170pm, 180pm, 190pm, 200pm, 210pm, 220pm, 230pm, 240pm, 250pm, 260pm, 270pm, 280pm, 290pm, 300pm, 310pm, 320pm, 330pm, 340pm, 350pm, 360pm, 370pm, 380pm, 390pm, 400pm, 410pm, 420pm, 430pm, 440pm, 450pm, 460pm, 470pm, 480pm, 490pm, 500pm, 510pm, 520pm, 530pm, 540pm, 550pm, 560pm, 570pm, 580pm, 590pm, 600pm, 610pm, 620pm, 630pm, 640pm, 650pm, 660pm, 670pm, 680pm, 690pm, 700pm, 710pm, 720pm, 730pm, 740pm, 750pm, 760pm, 770pm, 780pm, 790pm, 800pm, 810pm, 820pm, 830pm, 840pm, 850pm, 860pm, 870pm, 880pm, 890pm, 900pm, 910pm, 920pm, 930pm, 940pm, 950pm, 960pm, 970pm, 980pm, 990pm, 1000pm, 1010pm, 1020pm, 1030pm, 1040pm, 1050pm, 1060pm, 1070pm, 1080pm, 1090pm, 1100pm, 1110pm, 1120pm, 1130pm, 1140pm, 1150pm, 1160pm, 1170pm, 1180pm, 1190pm, 1200pm, 1210pm, 1220pm, 1230pm, 1240pm, 1250pm, 1260pm, 1270pm, 1280pm, 1290pm, 1300pm, 1310pm, 1320pm, 1330pm, 1340pm, 1350pm, 1360pm, 1370pm, 1380pm, 1390pm, 1400pm, 1410pm, 1420pm, 1430pm, 1440pm, 1450pm, 1460pm, 1470pm, 1480pm, 1490pm, 1500pm, 1510pm, 1520pm, 1530pm, 1540pm, 1550pm, 1560pm, 1570pm, 1580pm, 1590pm, 1600pm, 1610pm, 1620pm, 1630pm, 1640pm, 1650pm, 1660pm, 1670pm, 1680pm, 1690pm, 1700pm, 1710pm, 1720pm, 1730pm, 1740pm, 1750pm, 1760pm, 1770pm, 1780pm, 1790pm, 1800pm, 1810pm, 1820pm, 1830pm, 1840pm, 1850pm, 1860pm, 1870pm, 1880pm, 1890pm, 1900pm, 1910pm, 1920pm, 1930pm, 1940pm, 1950pm, 1960pm, 1970pm, 1980pm, 1990pm, 2000pm, 2010pm, 2020pm, 2030pm, 2040pm, 2050pm, 2060pm, 2070pm, 2080pm, 2090pm, 2100pm, 2110pm, 2120pm, 2130pm, 2140pm, 2150pm, 2160pm, 2170pm, 2180pm, 2190pm, 2200pm, 2210pm, 2220pm, 2230pm, 2240pm, 2250pm, 2260pm, 2270pm, 2280pm, 2290pm, 2300pm, 2310pm, 2320pm, 2330pm, 2340pm, 2350pm, 2360pm, 2370pm, 2380pm, 2390pm, 2400pm, 2410pm,2420pm, 2430pm, 2440pm, 2450pm, 2460pm, 2470pm, 2480pm, 2490pm, 2500pm, 2510pm, 2520pm, 2530pm, 2540pm, 2550pm, 2560pm, 2570pm, 2580pm, 2590pm, 2600pm, 2610pm, 2620pm, 2630pm, 2640pm, 2650pm, 2660pm, 2670pm, 2680pm, 2690pm, 2700pm, 2710pm, 2720pm, 2730pm, 2740pm, 2750pm, 2760pm, 2770pm, 2780pm, 2790pm, 2800pm, 2810pm, 2820pm, 2830pm, 2840pm, 2850pm, 2860pm, 2870pm, 2880pm, 2890pm, 2900pm, 2910pm, 2920pm, 2930pm, 2940pm, 2950pm, 2960pm, 2970pm, 2980pm, 2990pm, or 3000pm thick.

[0078] In some embodiments, the plant element coats of the present disclosure can be 0.5mm, 1mm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm, or 5mm thick.

[0079] In some embodiments, the plant element coats of the present disclosure can be at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, or 50% of the uncoated plant element weight.

[0080] In some embodiments, the microbial spores and / or cells can be coated freely onto the plant elements or they can be formulated in a liquid or solid composition before being coated onto the plant elements. For example, a solid composition comprising the microorganisms can be prepared by mixing a solid carrier with a suspension of the spores until the solid carriers are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.

[0081] In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present disclosure further contain functional agents e.g., activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the products or a combination thereof.

[0082] Plant element coating methods and compositions that are known in the art can be particularly useful when they are modified by the addition of one of the embodiments of the present disclosure. Such coating methods and apparatus for their application are disclosed in, forexample: U.S. Pat. Nos. 5,916,029; 5,918,413; 5,554,445; 5,389,399; 4,759,945; 4,465,017, and U.S. Pat. App. NO 13 / 260,310, each of which is incorporated by reference herein.

[0083] Plant element coating compositions are disclosed in, for example: U.S. Pat. Nos. 5,939,356; 5,876,739, 5,849,320; 5,791,084, 5,661,103; 5,580,544, 5,328,942; 4,735,015; 4,634,587; 4,372,080, 4,339,456; and 4,245,432, each of which is incorporated by reference herein.

[0084] In some embodiments, a variety of additives can be added to the plant element treatment formulations comprising the inventive compositions. Binders can be added and include those composed of an adhesive polymer that can be natural or synthetic without phytotoxic effect on the plant element to be coated. The binder may be selected from polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate and chitosans; fats; oils; proteins, including gelatin and zeins; gum arabics; shellacs; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and poly chloroprene .

[0085] Any of a variety of colorants may be employed, including organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that can be added include trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.

[0086] A polymer or other dust control agent can be applied to retain the treatment on the plant element surface.

[0087] In some specific embodiments, in addition to the microbial cells or spores, the coating can further comprise a layer of adherent. The adherent should be non-toxic, biodegradable, and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetates; polyvinyl acetate copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, such as methyl celluloses, hydroxymethyl celluloses, and hydroxymethyl propyl celluloses; dextrins;alginates; sugars; molasses; polyvinyl pyrrolidones; polysaccharides; proteins; fats; oils; gum arabics; gelatins; syrups; and starches. More examples can be found in, for example, U.S. Pat. NO 7,213,367, incorporated herein by reference.

[0088] Various additives, such as adherents, dispersants, surfactants, and nutrient and buffer ingredients, can also be included in the plant element treatment formulation. Other conventional plant element treatment additives include, but are not limited to: coating agents, wetting agents, buffering agents, and polysaccharides. At least one agriculturally acceptable carrier can be added to the plant element treatment formulation such as water, solids, or dry powders. The dry powders can be derived from a variety of materials such as calcium carbonate, gypsum, vermiculite, talc, humus, activated charcoal, and various phosphorous compounds.

[0089] In some embodiments, the plant element coating composition can comprise at least one filler, which is an organic or inorganic, natural or synthetic component with which the active components are combined to facilitate its application onto the plant element. In aspects, the filler is an inert solid such as clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (for example ammonium salts), natural soil minerals, such as kaolins, clays, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earths, or synthetic minerals, such as silica, alumina or silicates, in particular aluminium or magnesium silicates.

[0090] In some embodiments, the plant element treatment formulation may further include one or more of the following ingredients: other pesticides, including compounds that act only below the ground; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of those materials, and the like; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamalcs, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxys, ureas, and benzoic acids; herbicidal safeners such as benzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives; chemical fertilizers; biological fertilizers; and biocontrol agents such as other naturally-occurring or recombinant bacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. These ingredients may be added as a separate layer on the plant element, or alternatively may be added as part of the plant element coating composition of the disclosure.

[0091] In some embodiments, the formulation that is used to treat the plant element in the present disclosure can be in the form of a suspension; emulsion; slurry of particles in an aqueous medium (e.g., water); wettable powder; wettable granules (dry flowable); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation can be about 0.5% to about 99% by weight (w / w), or 5-40%, or as otherwise formulated by those skilled in the art.

[0092] As mentioned above, other conventional inactive or inert ingredients can be incorporated into the formulation. Such inert ingredients include, but are not limited to: conventional sticking agents; dispersing agents such as methylcellulose, for example, serve as combined dispersant / sticking agents for use in plant element treatments; polyvinyl alcohol; lecithin, polymeric dispersants (e.g., polyvinylpyrrolidone / vinyl acetate); thickeners (e.g., clay thickeners to improve viscosity and reduce settling of particle suspensions); emulsion stabilizers; surfactants; antifreeze compounds (e.g., urea), dyes, colorants, and the like. Further inert ingredients useful in the present disclosure can be found in McCutcheon’s, vol. 1, “Emulsifiers and Detergents,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996, incorporated by reference herein.

[0093] The plant element coating formulations of the present disclosure can be applied to plant elements by a variety of methods, including, but not limited to: mixing in a container (e.g., a bottle or bag), mechanical application, tumbling, spraying, and immersion. A variety of active or inert material can be used for contacting plant elements with microbial compositions according to the present disclosure.

[0094] In some embodiments, the amount of the microbes or agricultural composition that is used for the treatment of the plant element will vary depending upon the type of plant element and the type of active ingredients, but the treatment will comprise contacting the plant elements with an agriculturally effective amount of the inventive composition.

[0095] As discussed above, an effective amount means that amount of the inventive composition that is sufficient to affect beneficial or desired results. An effective amount can be administered in one or more administrations.

[0096] In some embodiments, in addition to the coating layer, the plant element may be treated with one or more of the following ingredients: other pesticides including fungicides andherbicides; herbicidal safeners; fertilizers and / or biocontrol agents. These ingredients may be added as a separate layer or alternatively may be added in the coating layer.

[0097] In some embodiments, the plant element coating formulations of the present disclosure may be applied to the plant elements using a variety of techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic plant element treaters, and drum coaters. Other methods, such as spouted beds may also be useful. The plant elements may be presized before coating. After coating, the plant elements are typically dried and then transferred to a sizing machine for sizing. Such procedures are known in the ail.

[0098] In some embodiments, the microorganism-treated plant elements may also be enveloped with a film overcoating to protect the coating. Such overcoatings are known in the art and may be applied using fluidized bed and drum film coating techniques.

[0099] In other embodiments of the present disclosure, compositions according to the present disclosure can be introduced onto a plant element by use of solid matrix priming. For example, a quantity of an inventive composition can be mixed with a solid matrix material and then the plant element can be placed into contact with the solid matrix material for a period to allow the composition to be introduced to the plant element. The plant element can then optionally be separated from the solid matrix material and stored or used, or the mixture of solid matrix material plus plant element can be stored or planted directly. Solid matrix materials which are useful in the present disclosure include polyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, or any other material capable of absorbing or adsorbing the inventive composition for a time and releasing that composition into or onto the plant element. It is useful to make sure that the inventive composition and the solid matrix material are compatible with each other. For example, the solid matrix material should be chosen so that it can release the composition at a reasonable rate, for example over a period of minutes, hours, or days.

[0100] In some embodiments, the present disclosure teaches that the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with any plant biostimulant.

[0101] In some embodiments, the present disclosure teaches agricultural compositions comprising one or more commercially available biostimulants, including but not limited to: Vitazyme®, Diehard™ Biorush®, Diehard™ Biorush® Fe, Diehard™ Soluble Kelp, Diehard™ Humate SP, Phocon®, Foliar Plus™, Plant Plus™, Accomplish LM®, Titan®, Soil Builder™,Nutri Life, Soil Solution™, Seed Coat™, PercPlus™, Plant Power®, CropKarb®, Thrust™, Fast2Grow®, Baccarat®, and Potcntc® among others.

[0102] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with an active chemical agent one witnesses an additive effect on a plant phenotypic trait of interest. In other embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with an active chemical agent one witness a synergistic effect on a plant phenotypic trait of interest.

[0103] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a fertilizer one witnesses an additive effect on a plant phenotypic trait of interest. In other embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a fertilizer one witness a synergistic effect on a plant phenotypic trait of interest.

[0104] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a plant growth regulator, one witnesses an additive effect on a plant phenotypic trait of interest. In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a plant growth regulator, one witnesses a synergistic effect. In some aspects, the microbes of the present disclosure are combined with Ascend® and a synergistic effect is observed for one or more phenotypic traits of interest.

[0105] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a biostimulant, one witnesses an additive effect on a plant phenotypic trait of interest. In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a biostimulant, one witnesses a synergistic effect.

[0106] The synergistic effect obtained by the taught methods can be quantified according to Colby’s formula (i.e., (E) =X+Y-(X*Y / 100). See Colby, R. S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967 Weeds, vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, by “synergistic” is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount.

[0107] The isolated microbes and consortia of the present disclosure can synergistically increase the effectiveness of agricultural active compounds and also agricultural auxiliary compounds.

[0108] In other embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a fertilizer one witnesses a synergistic effect.

[0109] Furthermore, in certain embodiments, the disclosure utilizes synergistic interactions to define microbial consortia. That is, in certain aspects, the disclosure combines together certain isolated microbial species, which act synergistically, into consortia that impart a beneficial trait upon a plant, or which are correlated with increasing a beneficial plant trait.

[0110] The agricultural compositions developed according to the disclosure can be formulated with certain auxiliaries, in order to improve the activity of a known active agricultural compound. This has the advantage that the amounts of active ingredient in the formulation may be reduced while maintaining the efficacy of the active compound, thus allowing costs to be kept as low as possible and any official regulations to be followed. In individual cases, it may also possible to widen the spectrum of action of the active compound since plants, where the treatment with a particular active ingredient without addition was insufficiently successful, can indeed be treated successfully by the addition of certain auxiliaries along with the disclosed microbial isolates and consortia. Moreover, the performance of the active may be increased in individual cases by a suitable formulation when the environmental conditions are not favorable.

[0111] Such auxiliaries that can be used in an agricultural composition can be an adjuvant.Frequently, adjuvants take the form of surface-active or salt-like compounds. Depending on their mode of action, they can roughly be classified as modifiers, activators, fertilizers, pH buffers, and the like. Modifiers affect the wetting, sticking, and spreading properties of a formulation. Activators break up the waxy cuticle of the plant and improve the penetration of the active ingredient into the cuticle, both short-term (over minutes) and long-term (over hours). Fertilizers such as ammonium sulfate, ammonium nitrate or urea improve the absorption and solubility of the active ingredient and may reduce the antagonistic behavior of active ingredients. pH buffers are conventionally used for bringing the formulation to an optimal pH.

[0112] For further embodiments of agricultural compositions of the present disclosure, See “Chemistry and Technology of Agrochemical Formulations,” edited by D. A. Knowles, copyright 1998 by Kluwer Academic Publishers, hereby incorporated by reference.Plants and Agronomic Benefits

[0113] A wide variety of plants, including those cultivated in agriculture, are capable of receiving benefit from the application of microbes, such as those described herein, includingsingle microbes, consortia, and / or compositions produced therefrom, or comprising any of the preceding. Any number of a variety of different plants, including mosses and lichens and algae, may be used in the methods of the disclosure. In embodiments, the plants have economic, social, or environmental value. For example, the plants may include those used as: food crops, fiber crops, oil crops, in the forestry industry, in the pulp and paper industry, as a feedstock for biofuel production, and as ornamental plants.

[0114] In other embodiments, the plants may be economically, socially, or environmentally undesirable, such as weeds. The following is a list of non-limiting examples of the types of plants the methods of the disclosure may be applied to:Food Crops

[0115] Cereals e.g maize, rice, wheat, barley, sorghum, millet, oats, rye, triticale, and buckwheat;

[0116] Leafy vegetables e.g., brassicaceous plants such as cabbages, broccoli, bok choy, rocket; salad greens such as spinach, cress, and lettuce;

[0117] Fruiting and flowering vegetables e.g., avocado, sweet com, feed corn, artichokes; curcubits e.g., squash, cucumbers, melons, courgettes, pumpkins; solanaceous vegetables / fruits e.g., tomatoes, eggplant, and capsicums;

[0118] Podded vegetables e.g., groundnuts, peanuts, peas, soybeans, beans, lentils, chickpea, okra;

[0119] Bulbed and stem vegetables e.g., asparagus, celery, Allium crops e.g garlic, onions, and leeks;

[0120] Roots and tuberous vegetables e.g., carrots, beet, bamboo shoots, cassava, yams, ginger, Jerusalem artichoke, parsnips, radishes, potatoes, sweet potatoes, taro, turnip, and wasabi;

[0121] Sugar crops including sugar beet (Beta vulgaris), sugar cane (Saccharum officinarumf,

[0122] Crops grown for the production of non-alcoholic beverages and stimulants e.g., coffee, black, herbal, and green teas, cocoa, marijuana, and tobacco;

[0123] Fruit crops such as true berry fruits (e.g., kiwifruit, grape, currants, gooseberry, guava, feijoa, pomegranate), citrus fmits (e.g., oranges, lemons, limes, grapefruit), epigynous fruits (e.g., bananas, cranberries, blueberries), aggregate fruit (blackberry, raspberry, boysenberry), multiple fmits (e.g., pineapple, fig), stone fruit crops (e.g., apricot, peach, cherry, plum), pip-fruit (e.g., apples, pears) and others such as strawberries, sunflower seeds;

[0124] Culinary and medicinal herbs e.g., rosemary, basil, bay laurel, coriander, mint, dill, Hypericum, foxglove, aloe vera, rosehips, and cannabis;

[0125] Crop plants producing spices e.g., black pepper, cumin cinnamon, nutmeg, ginger, cloves, saffron, cardamom, mace, paprika, masalas, star anise;

[0126] Crops grown for the production of nuts e.g., almonds and walnuts, Brazil nut, cashew nuts, coconuts, chestnut, macadamia nut, pistachio nuts; peanuts, pecan nuts;

[0127] Crops grown for production of beers, wines and other alcoholic beverages e.g grapes, and hops;

[0128] Oilseed crops e.g., soybean, peanuts, cotton, olives, sunflower, sesame, lupin species and brassicaeous crops (e.g., canola / oilseed rape); and, edible fungi e.g., white mushrooms, Shiitake and oyster mushrooms;Plants used in Pastoral Agriculture

[0129] Legumes: Trifolium species, Medicago species, and Lotus species; White clover (T.repens); Red clover (T. pratense); Caucasian clover (T. ambigum); subterranean clover (T.subterraneum); Alfalfa / Lucerne (Medicago sativum); annual medics; barrel medic; black medic; Sainfoin (Onobrychis viciifolia); Birdsfoot trefoil (Lotus comiculatus); Greater Birdsfoot trefoil (Lotus pedunculatus);

[0130] Seed legumes / pulses including Peas (Pisum sativum), Common bean (Phaseolus vulgaris), Broad beans (Viciafaba), Mung bean (Vigna radiata), Cowpea (Vigna unguiculata), Chick pea (Cicer arietum), Lupins (Lupinus species); Cereals including Maize / com (Zea mays), Sorghum (Sorghum spp. , Millet (Panicum miliaceum, P. sumatrense), Rice (Oryza sativa indica, Oryza sativa japonica), Wheat (Triticum aestivum), Barley (Hordeum vulgare), Rye (Secale cereale), Triticale (Triticum X Secale), Oats (Avena sativa);

[0131] Forage and Amenity grasses: Temperate grasses such as Lolium species; Festuca species; Agrostis spp., Perennial ryegrass (Lolium perenne); hybrid ryegrass (Lolium hybridum); annual ryegrass Lolium multiflorum), tall fescue (Festuca arundinacea); meadow fescue (Festuca pralensis); red fescue (Festuca rubra); Festuca ovina; Fesluloliums (Lolium X Festuca crosses); Cocksfoot (Dactylis glomerata); Kentucky bluegrass Poa pratensis; Poa palustris; Poa nemoralis; Poa trivialis; Poa compresa; Bromus species; Phalaris (Phleum species);Arrhenatherum elatius; Agropyron species; Avena strigosa; Setaria italic;

[0132] Tropical grasses such as: Phalaris species; Brachiaria species; Eragrostis species; Panicum species; Bahai grass (Paspalum notatum); Brachypodium species; and, grasses used for biofuel production such as Switchgrass (Panicum virgatum) and Miscanthus species;Fiber Crops

[0133] Cotton, hemp, jute, coconut, sisal, flax (Linum spp.), New Zealand flax (Phormium spp.); plantation and natural forest species harvested for paper and engineered wood fiber products such as coniferous and broadleaf ed forest species;Tree and shrub species used in plantation forestry and bio-fuel crops

[0134] Pine (Pinus species); Fir (Pseudotsuga species); Spruce (Picea species); Cypress (Cupressus species); Wattle (Acacia species); Alder (Alnus species); Oak species (Quercus species); Redwood (Sequoiadendron species); willow (Salix species); birch (Betula species); Cedar (Cedurus species); Ash (Fraxinus species); Larch (Larix species); Eucalyptus species; Bamboo (Bambuseae species) and Poplars (Populus species).Plants grown for conversion to energy, biofuels or industrial products by extractive, biological, physical or biochemical treatment

[0135] Oil-producing plants such as oil palm, jatropha, com, soybean, cotton, linseed; Latexproducing plants such as the Para Rubber tree, Hevea brasiliensis and the Panama Rubber Tree Castilla elastica; plants used as direct or indirect feedstocks for the production of biofuels i.e., after chemical, physical (e.g., thermal or catalytic) or biochemical (e.g., enzymatic pre-treatment) or biological (e.g., microbial fermentation) transformation during the production of biofuels, industrial solvents or chemical products e.g., ethanol or butanol, propane dials, or other fuel or industrial material including sugar crops (e.g., beet, sugar cane), starch producing crops (e.g., C3 and C4 cereal crops and tuberous crops), cellulosic crops such as forest trees (e.g., Pines, Eucalypts) and Graminaceous and Poaceous plants such as bamboo, switch grass, miscanthus; crops used in energy, biofuel or industrial chemical production via gasification and / or microbial or catalytic conversion of the gas to biofuels or other industrial raw materials such as solvents or plastics, with or without the production of biochai' (e.g., biomass crops such as coniferous, eucalypt, tropical or broadleaf forest trees, graminaceous and poaceous crops such as bamboo, switch grass, miscanthus, sugar cane, or hemp or softwoods such as poplars, willows; and, biomass crops used in the production of biochar;Crops producing natural products useful for the pharmaceutical, agricultural nutraceutical and cosmcccutical industries

[0136] Crops producing pharmaceutical precursors or compounds or nutraceutical and cosmeceutical compounds and materials for example, star anise (shikimic acid), Japanese knotweed (resveratrol), kiwifruit (soluble fiber, proteolytic enzymes);Floricultural, Ornamental and Amenity plants grown for their aesthetic or environmental properties

[0137] Flowers such as roses, tulips, chrysanthemums;

[0138] Ornamental shrubs such as Buxus, Hebe, Rosa, Rhododendron, Hedera

[0139] Amenity plants such as Platanus, Choisya, Escallonia, Euphorbia, Carex

[0140] Mosses such as sphagnum mossPlants grown for bioremediation

[0141] In certain aspects, the microbes of the present disclosure are applied to hybrid plants to increase beneficial traits of said hybrids. In other aspects, the microbes of the present disclosure are applied to genetically modified plants to increase beneficial traits of said GM plants. The microbes taught herein are able to be applied to hybrids and GM plants and thus maximize the elite genetics and trait technologies of these plants.

[0142] It should be appreciated that a plant may be provided in the form of a seed, seedling, cutting, propagule, or any other plant material or tissue capable of growing. In one embodiment the seed may be surface- sterilised with a material such as sodium hypochlorite or mercuric chloride to remove surface-contaminating microorganisms. In one embodiment, the propagule is grown in axenic culture before being placed in the plant growth medium, for example as sterile plantlets in tissue culture.Methods of Application

[0143] The microorganisms may be applied to a plant, seed, seedling, cutting, propagule, or the like and / or the growth medium containing said plant, using any appropriate technique known in the art.

[0144] However, by way of example, an isolated microbe, consortia, or composition comprising the same, and / or a composition produced therefrom, may be applied to a plant, seed, seedling, cutting, propagule, or the like, by spraying, coating, dusting, or any other method known in the art.

[0145] In another embodiment, the isolated microbe, consortia, or composition comprising the same may be applied directly to a plant seed prior to sowing.

[0146] In another embodiment, the isolated microbe, consortia, or composition comprising the same may applied directly to a plant seed, as a seed coating.

[0147] In one embodiment of the present disclosure, the isolated microbe, consortia, or composition comprising the same is supplied in the form of granules, or plug, or soil drench that is applied to the plant growth media.

[0148] In other embodiments, the isolated microbe, consortia, or composition comprising the same are supplied in the form of a foliar application, such as a foliar spray or liquid composition. The foliar spray or liquid application may be applied to a growing plant or to a growth media, e.g., soil.

[0149] In some embodiments, the isolated microbe, consortia, or composition comprising the same are supplied in a form selected from: a soil drench, a foliar spray, a dip treatment, an in furrow treatment, a soil amendment, granules, a broadcast treatment, a post-harvest disease control treatment, or a seed treatment. In some embodiments, the agricultural compositions may be applied alone in or in rotation programs.

[0150] In some embodiments, the isolated microbe, consortia, or composition comprising the same may be compatible with tank mixing. In some embodiments, the agricultural compositions may be compatible with tank mixing with other agricultural products. In some embodiments, the agricultural compositions may be compatible with equipment used for ground, aerial, and irrigation applications.

[0151] In another embodiment, the isolated microbe, consortia, or composition comprising the same may be formulated into granules and applied alongside seeds during planting. Or the granules may be applied after planting. Or the granules may be applied before planting.

[0152] In some embodiments, the isolated microbe, consortia, or composition comprising the same are administered to a plant or growth media as a topical application and / or drench application to improve crop growth, yield, and quality. The topical application may be via utilization of a dry mix or powder or dusting composition or may be a liquid based formulation.

[0153] In embodiments, the isolated microbe, consortia, or composition comprising the same can be formulated as: (1) solutions; (2) wettable powders; (3) dusting powders; (4) soluble powders; (5) emulsions or suspension concentrates; (6) seed dressings or coatings, (7) tablets; (8) water-dispersible granules; (9) water soluble granules (slow or fast release); (10) microencapsulated granules or suspensions; (11) as irrigation components, and (12) a component of fertilizers, pesticides, and other compatible amendments, among others. In certain aspects, the compositions may be diluted in an aqueous medium prior to conventional spray application. The compositions of the present disclosure can be applied to the soil, plant, seed, rhizosphere, rhizosheath, or other area to which it would be beneficial to apply the microbial compositions. Further still, ballistic methods can be utilized as a means for introducing endophytic microbes.

[0154] In aspects, the compositions are applied to the foliage of plants. The compositions may be applied to the foliage of plants in the form of an emulsion or suspension concentrate, liquid solution, or foliar spray. The application of the compositions may occur in a laboratory, growth chamber, greenhouse, or in the field.

[0155] In another embodiment, microorganisms may be inoculated into a plant by cutting the roots or stems and exposing the plant surface to the microorganisms by spraying, dipping, or otherwise applying a liquid microbial suspension, or gel, or powder.

[0156] In another embodiment, the microorganisms may be injected directly into foliar, root, shoot, and / or vascular tissue, or otherwise inoculated directly into or onto a foliar or root cut, or else into an excised embryo, or radicle, or coleoptile. These inoculated plants may then be further exposed to a growth media containing further microorganisms; however, this is not necessary.

[0157] In other embodiments, particularly where the microorganisms are unculturable, the microorganisms may be transferred to a plant by any one or a combination of grafting, insertion of explants, injection into a plant for example into the cambium or xylem, aspiration, electroporation, wounding, root pruning, induction of stomatai opening, or any physical, chemical or biological treatment that provides the opportunity for microbes to enter plant cells or the intercellular space. Persons of skill in the art may readily appreciate a number of alternative techniques that may be used.

[0158] In one embodiment, the microorganisms infiltrate parts of the plant such as the roots, stems, leaves and / or reproductive plant pails (become endophytic), and / or grow upon the surface of roots, stems, leaves and / or reproductive plant parts (become epiphytic) and / or grow in the plant rhizosphere. In one embodiment, the microorganisms form a symbiotic relationship with the plant.

[0159] In some aspects, the invention includes:

[0160] Aspect 1 : A synthetic composition, comprising: a) a Bacillus cell, exudate therefrom, culture broth therefrom, or spore therefrom, wherein the cell comprises a heterologous polynucleotide sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 5, 13, and 15; and b) at least one composition selected from the group consisting of: a plant element, a formulation component, an agricultural composition, and any plurality and / or combination of the preceding.

[0161] Aspect 2: A synthetic composition, comprising: c) a Bacillus thuringiensis cell, exudate therefrom, culture broth therefrom, or spore therefrom, wherein the cell comprises a heterologous polynucleotide sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 5, 13, and 15; wherein the microbial cell has reduced or abolished expression of at least one gene in an endotoxin biosynthetic pathway; and

[0162] d) at least one composition selected from the group consisting of: a plant element, a formulation component, an agricultural composition, and any plurality and / or combination of the preceding.

[0163] Aspect 3: The synthetic composition of Aspect 1 or Aspect 2, wherein the heterologous polynucleotide of a) is inserted into the genome of the cell in a coding sequence located in a thuringiensin biosynthetic gene cluster.

[0164] Aspect 4: The synthetic composition of Aspect 2, wherein the endotoxin is beta endotoxin.

[0165] Aspect 5: The synthetic composition of Aspect 3, wherein said coding sequence shares at least 90% identity with at least 100 contiguous nucleotides of SEQID NO: 17, 18, 20, or 11.

[0166] Aspect 6: The synthetic composition of Aspect 3, wherein the Bacillus thuringiensis cell is an isolate of Strain PM39400, deposited as NRRL B-68090.

[0167] Aspect 7: The synthetic composition of Aspect 1, comprising at least two polynucleotide sequences selected from the group consisting of: SEQID NOs: 1-50.

[0168] Aspect 8: The synthetic composition of Aspect 1, comprising at least three polynucleotide sequences selected from the group consisting of: SEQID NOs: 1-50.

[0169] Aspect 9: The synthetic composition of Aspect 1 or Aspect 2, further comprising at least one additional microbe.

[0170] Aspect 10: The synthetic composition of Aspect 1 or Aspect 2, wherein the plant element is a seed.

[0171] Aspect 11: The synthetic composition of Aspect 10, wherein the seed comprises a transgene.

[0172] Aspect 12: The synthetic composition of Aspect 1 or Aspect 2, wherein the plant element is a leaf.

[0173] Aspect 13: The synthetic composition of Aspect 1 or Aspect 2, wherein the plant element is a root.

[0174] Aspect 14: The synthetic composition of Aspect 1 or Aspect 2, wherein the plant element is a whole plant.

[0175] Aspect 15: The synthetic composition of Aspect 1 or Aspect 2, wherein the microbe is present at a concentration of at least about 10A2 CFU / mL in a liquid formulation, or at least about 10A2 Colony Forming Units per gram in a non-liquid formulation.

[0176] Aspect 16: The synthetic composition of Aspect 1 or Aspect 2, wherein the formulation component is selected from the group consisting of: a compound that improves the stability of the microbe, a preservative, a carrier, a surfactant, an anticomplex agent, and any combination thereof.

[0177] Aspect 17: The synthetic composition of Aspect 1 or Aspect 2, wherein the agricultural composition comprises a fungicide, a nematicide, a bactericide, an insecticide, an herbicide, or any combination thereof.

[0178] Aspect 18: A plurality of synthetic compositions of Aspect 1 or Aspect 2, wherein said synthetic compositions are substantially confined within an object selected from the group consisting of: a tube, a bottle, ajar, an ampule, a package, a vessel, a bag, a box, a bin, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and a case.

[0179] Aspect 19: The plurality of synthetic compositions of Aspect 1 or Aspect 2, wherein the synthetic compositions are at a temperature below thirty degrees Celsius.

[0180] Aspect 20: The plurality of synthetic compositions of Aspect 1 or Aspect 2, wherein the synthetic compositions are at a temperature below zero degrees Celsius.

[0181] Aspect 21: The synthetic composition of Aspect 1 or Aspect 2, wherein the plant element is obtained from a plant selected from the group consisting of: maize, soybean, wheat, cotton,cucumber, tomato, pepper, potato, strawberry, orange, lemon, lime, apple, snap beans, zucchini, pea, lettuce, broccoli, celery, cauliflower, sorghum, canola, rapeseed, and okra.

[0182] Aspect 22: The synthetic composition of Aspect 1 or Aspect 2, wherein the agricultural composition comprises a growth medium.

[0183] Aspect 23: The synthetic composition of Aspect 22, wherein the growth medium comprises soil.

[0184] Aspect 24: A plurality of synthetic compositions of Aspect 23, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern with substantially equal spacing between each of the synthetic compositions.

[0185] Aspect 25: A method of conferring nematode pest tolerance to a plant, the method comprising treating a plant element of said plant with a formulation comprising at least one microbial cell, exudate therefrom, or culture broth therefrom, wherein the microbial cell is a Bacillus cell comprising a heterologous polynucleotide sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 5, 13, and 15.

[0186] Aspect 26: A method of conferring nematode pest tolerance to a plant, the method comprising treating a plant element of said plant with a formulation comprising at least one microbial cell, exudate therefrom, or culture broth therefrom, wherein the microbial cell is a Bacillus thuringiensis cell comprising a heterologous polynucleotide sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 5, 13, and 15; wherein the microbial cell has reduced or abolished expression of at least one gene in an endotoxin biosynthetic pathway.

[0187] Aspect 27: The method of Aspect 25 or Aspect 26, wherein the trait of agronomic importance is resistance to a nematode pest.

[0188] Aspect 28: The method of Aspect 25 or Aspect 26, further comprising at least one additional microbe.

[0189] Aspect 29: The method of Aspect 25 or Aspect 26, wherein the plant further has an improved agronomic characteristic is selected from the group consisting of: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance,pathogen resistance, increase in yield, increase in yield under water-limited conditions, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increase in biomass, increase in shoot length, increase in root length, improved root architecture, increase in seed weight, altered seed carbohydrate composition, altered seed oil composition, increase in radical length, number of pods, delayed senescence, stay-green, altered seed protein composition, increase in dry weight of mature plant reproductive elements, increase in fresh weight of mature plant reproductive elements, increase in number of mature plant reproductive elements per plant, increase in chlorophyll content, increase in number of pods per plant, increase in length of pods per plant, increase in number of seeds per plant, increase in seed weight per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, increase in number of non- wilted leaves per plant, or improved plant visual appearance.

[0190] Aspect 30: The method of Aspect 25 or Aspect 26, wherein the microbial cell, exudate therefrom, or culture broth therefrom, is present in an amount capable of providing a benefit to a plant derived from the plant element, as compared to a plant derived from a plant element not treated with said microbial cell, spore, or exudate therefrom.

[0191] Aspect 31: A method of cultivating a plant, comprising:

[0192] a) introducing to a plant element of said plant a microbial cell, exudate therefrom, or culture broth therefrom, wherein the microbial cell comprises a heterologous polynucleotide sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 5, 13, and 15; and wherein said microbial cell is heterologous to the plant element;

[0193] b) placing the plant element of a) in a growth medium under conditions suitable for growth;

[0194] wherein a plant grown from the plant element of b) has improved plant health characteristics in the presence of a nematode as compared to a plant grown from a plant element not comprising the microbial cell of a).

[0195] Aspect 32: The method of Aspect 31, wherein the microbial cell has reduced or abolished expression of at least one gene in an endotoxin biosynthetic pathway.

[0196] Aspect 33: The method of Aspect 31, further comprising at least one additional microbe.

[0197] Aspect 34: The method of Aspect 31 , wherein said introducing to the plant element is accomplished by an indirect method selected from the group consisting of: in-furrow application, soil drench application, and side-dress application.

[0198] Aspect 35: The method of Aspect 31, wherein said introducing to the plant element is accomplished by coating said plant element with a liquid formulation of the microbe or exudate therefrom.

[0199] Aspect 36: The method of Aspect 31, wherein said introducing to the plant element is accomplished by coating said plant element with a substantially non-liquid formulation of the microbe or exudate therefrom.

[0200] Aspect 37: The method of Aspect 31, wherein said plant element is a seed.

[0201] Aspect 38: The method of Aspect 31, wherein said plant element is a leaf.

[0202] Aspect 39: The method of Aspect 31, wherein said plant element is a root.

[0203] Aspect 40: The method of Aspect 31, wherein said plant element is a whole plant.

[0204] Aspect 41: A method of reducing the number of pests in a composition, wherein the pests comprise nematodes, nematode eggs, nematode juveniles, or any plurality or combination of the preceding, the method comprising: introducing to the composition at least one microbial cell, spore, exudate therefrom, or culture broth therefrom, wherein the microbial cell comprises a heterologous polynucleotide sequence sharing at least 97 % identity with at least one sequence selected from the group consisting of: SEQID NOs: 5, 13, and 15.

[0205] Aspect 42: The method of Aspect 41, wherein the microbial cell has reduced or abolished expression of at least one gene in an endotoxin biosynthetic pathway.

[0206] Aspect 43: The method of Aspect 41, wherein the composition is a growth medium for a plant.

[0207] Aspect 44: The method of Aspect 43, wherein the growth medium is soil.

[0208] Aspect 45: A method of increasing the yield of a harvested product, comprising introducing to a plant or to a growth medium in which the plant is placed a microbial cell, spores, exudate therefrom, or culture broth therefrom, wherein the microbial cell comprises a heterologous polynucleotide sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 5, 13, and 15;

[0209] Aspect 46: The method of Aspect 45, wherein the microbial cell has reduced or abolished expression of at least one gene in an endotoxin biosynthetic pathway.

[0210] Aspect 47: The method of Aspect 41 or Aspect 45, further comprising at least one additional microbe.

[0211] Aspect 48: The method of Aspect 41 or Aspect 45, wherein the harvested product is a fruit.

[0212] Aspect 49: The method of Aspect 41 or Aspect 45, wherein the harvested product is a vegetable.

[0213] Aspect 50: The method of Aspect 41 or Aspect 45, wherein the harvested product is a seed.

[0214] Aspect 51: The method of Aspect 41 or Aspect 45, wherein the harvested product is a fiber.

[0215] Aspect 52: A substantially cell-free preparation obtained or derived from a culture of a microbe, wherein the microbe comprises a heterologous polynucleotide sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 5, 13, and 15.

[0216] Aspect 53: The substantially cell-free preparation of Aspect 52, wherein the microbial cell has reduced or abolished expression of at least one gene in an endotoxin biosynthetic pathway.

[0217] Aspect 54: A purified composition prepared from the substantially cell-free preparation of Aspect 52, wherein the purified composition comprises SEQID NO: 25, 33, and / or 35.

[0218] Aspect 55: Any of the methods or compositions of above, comprising a genetically modified variant of Bacillus thuringiensis deposited as NRRL Accession No. B-68090 or an isolated bacterial strain having substantially similar morphological and physiological characteristics, substantially similar or substantially identical genetic characteristics, progeny, mutants, or genetically edited, altered, or modified variants thereof.

[0219] Aspect 56: An isolated bacterial strain comprising a heterologous polynucleotide sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 1-50.

[0220] Aspect 57: The isolated bacterial strain of Aspect 56, wherein the microbial cell has reduced or abolished expression of at least one gene in an endotoxin biosynthetic pathway.

[0221] Aspect 58: An agricultural composition, comprising: a) the isolated bacterial strain of Aspect 55 or Aspect 56, and b) an agriculturally acceptable carrier; wherein the bacterialstrain is present in the agricultural composition in an amount effective for producing an improved phenotype of a plant with which it is associated.

[0222] Aspect 59: The agricultural composition of Aspect 58, wherein the agricultural composition is formulated as a seed coating, a foliar spray, a soil drench, a dip treatment, an in-furrow treatment, a soil amendment, granules, a broadcast treatment, or a post-harvest disease control treatment.

[0223] Aspect 60: A microbial cell comprising a heterologous polynucleotide sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 5, 13, and 15; wherein the microbial cell is heterologously disposed to the plant element.

[0224] Aspect 61: The microbial cell of Aspect 60, wherein the microbial cell has reduced or abolished expression of at least one gene in an endotoxin biosynthetic pathway, and a plant element.

[0225] Aspect 62: A composition comprising any of the preceding, wherein the composition confers a detrimental effect on a nematode.

[0226] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. For instance, while the particular examples below may illustrate the methods and embodiments described herein using a specific plant, the principles in these examples may be applied to any plant. Therefore, it will be appreciated that the scope of this invention is encompassed by the embodiments recited herein rather than solely by the specific examples that are exemplified below.

[0227] The present disclosure enables one of skill in the relevant art to make and use the inventions provided herein in accordance with multiple and varied embodiments. Various alterations, modifications, and improvements of the present disclosure that readily occur to those skilled in the ait, including certain alterations, modifications, substitutions, and improvements are also part of this disclosure. Accordingly, the foregoing description are by way of example to illustrate the discoveries provided herein. Furthermore, the foregoing Description and Examples are exemplary of the present invention and not limiting thereof.

[0228] All cited patents and publications referred to in this application are herein incorporated by reference in their entirety, for all purposes, to the same extent as if each were individually and specifically incorporated by reference.EXAMPLESGenetically-modified microbial strains were developed, which comprised nematode-controlling proteins not naturally occurring in a single bacterium. Nematocidal efficacy of each engineered strain was improved as compared to the wild-type parental strain, and each additional gene improved activity relative to both the parental strain and other modified strains lacking said additional gene.Example 1: Strain Selection

[0176] Exemplary Strain PM39400, a Bacillus thuringiensis isolate with previously-identified nematocidal, lepidopteran, and coleopteran activity (deposited as NRRL B-68090 on 26 January 2022; described in International Application No. PCT / US2023 / 060963) (16S RNA DNA sequences given as SEQID NOs. 37-50), was selected for genetic modification for improvement of nematocidal activity. Bacillus-sourced nematocidal toxin proteins were identified from various strains, and ranked based on factors including source strain, sequence diversity, sporeassociation, and potential nematocidal efficacy.

[0177] Other Bacillus species and strains are contemplated.Example 2: Toxin Cloning and Expression

[0178] Toxin genes were either synthesized by a third-party vendor or amplified out of genomic DNA preparations of in-house strains. Prepared toxin genes were cloned by Gibson assembly into the replicative plasmid, pBACOV, immediately downstream of the strong constitutive promoter, PaprE, which was previously amplified and cloned into pBACOV by Gibson assembly. Quality control was performed on the new plasmids by PCR amplification and subsequent amplicon size comparison to a DNA ladder using primers specific to the construction of each plasmid as well as Sanger sequencing from the upstream (5') edge to the downstream (3') edge of the construction cassette to confirm the predicted DNA sequence. Completed expression plasmids containing toxin genes were conjugated into a surrogate Gram-positive host (Bacillus velezensis) using techniques known in the art.Example 3: Insertion Locus Identification

[0179] Cassettes for insertion were designed to insert into the host bacterial genome in a manner that also resulted in removal of thuringiensin expression. Thuringiensin (beta-exotoxin)biosynthesis is reported to occur from the expression of a gene cluster (Liu et al., Toxins (Basel) 6(8):2229-2238; 2014), which comprises various components such as the gene, thuE. The thuE gene (SEQID NO:20) is one gene of the hypothetical thuringiensin biosynthetic gene cluster.

[0180] The predicted thuringiensin gene cluster in the target bacterial strain described herein was selected by identifying the thuE gene through genomic sequence analysis, which was hypothesized to be associated with thuringiensin production. A thuE::kan mutant was created to ascertain gene function by disrupting thuringiensis production through the introduction of a kanamycin antibiotic resistance gene. HPLC analysis confirmed that the thuE::kan mutation disrupted thuringiensin production.

[0181] Surrounding genes showed close operonic synteny and were hypothesized to function in the thuringiensin biosynthetic pathway. Genes including ugd (hypothesized thuD; UDP-glucose- 6-dehydrogenase) (SEQID NO: 18) and dllA (hypothesized thu2’, non-ribosomal peptide synthetase) (SEQID NO: 17) were selected as targets for insertion of toxins, as described herein.Example 4: Single Toxin Nematocidal Activity in Bacillus velezensis

[0182] Single toxin genes from Strain PM39400 were cloned into Bacillus velezensis and assessed for activity in bioassays against C. elegans and RKN (Root Knot Nematode). Results are shown in Table 1.Table 1: Single toxin bioactivity of various native genes from Strain PM39400

[0183] Cry proteins that were not natively present in Strain PM39400 were cloned into a surrogate Gram-positive Bacillus velezensis host and assessed for activity in bioassays against C. elegans and RKN (Root Knot Nematode), Results are shown in Table 2.Table 2: Single toxin bioactivity of various native genes heterologous to Strain PM39400 (B. thuringiensis) and tested in B. velezensisExample 5: Stacked Toxin Nematocidal Activity in Bacillus velezensis

[0184] cry gene expression cassettes for transformation into Bacillus velezensis were created and tested in a cucumber Root Knot Nematode (RKN) bioassay according to the protocol described herein. Results are shown in Table 3.Table 3: Stacked toxin bioactivity in Bacillus velezensisExample 6: Toxin Stacking into Bacillus thuringiensis

[0185] cry gene expression cassettes for transformation into Bacillus thuringiensis were created to integrate (and therefore disrupt) the next gene in the hypothesized thuringiensin operon in an attempt to disrupt thuringiensin production and integrate the cry toxin gene for expression.

[0186] The three heterologous toxin genes (cry21Aa SEQID NO:5, cry6Aa SEQID NO: 13, and cry5Ba SEQ ID NO: 15) that demonstrated activity were cloned into host wild-type Strain PM39400, a Bacillus thuringiensis isolate. The three toxin genes were designed to be integrated one after the other into Strain PM39400 such that after the third round of integration and excision, all three toxin genes would be located within the genome of Strain PM39400.cry6Aa

[0187] The first toxin gene, cry6Aa, was designed to integrate into the dltA gene locus of parent strain Strain PM39400 (Wild Type) such that 42% of the dltA open reading frame (from residue 335_lsoleucine to residue 761_Serine) would be removed after excision of the temperaturesensitive origin of replication. Integration of cry6Aa was facilitated by 1000 bp homologous recombination regions flanking either side of the dltA integration locus. DNA sequence 200 bp upstream of the PM39400 cry] Ba gene was cloned upstream of cry6Aa in order to utilize the strain's native cry toxin gene expression to control transcription of the incoming cry Aa toxin gene. The resultant strain was named PE39400-G15 and comprised the cry6Aa toxin expression cassette integrated at dltA, resulting in a genotype of dltA: :PCrylBa-cry6Aa (single stacked toxin strain). cry6Aa + cry5Ba

[0188] The second toxin gene, crySBa, was designed to integrate into the ugd gene of parent strain Strain PE39400-G15 such that 70% of the ugd open reading frame from position -16 to position +811 of the transcription start position) would be removed after excision of the temperature sensitive origin of replication. Integration of cry5Ba was facilitated by 1000 bp homologous recombination regions flanking either side of the ugd integration locus. The homologous recombination regions were designed to avoid capturing the downstream gene, xerC, which might have inhibited plasmid assembly in E. coli. DNA sequence 200 bp upstream of the B. thuringiensis PM39400 crylBa gene was cloned upstream of cry6Aa in order to utilize the strain's native cry toxin gene expression to control transcription of the incoming cry6Aa toxin gene. The resultant strain was named PE39400-G27 and comprised the cry6Aa toxin expression cassette integrated at dltA and the cry5Ba toxin expression cassette integrated at ugd (genotype dltA: :PcryiBa-cry6Aa + ugd: :PCrylBa-cry5Ba') (double stacked toxin strain). cry6Aa + cry5Ba + cry21Aa

[0189] The third toxin gene, cry21Aa, was designed to integrate into the cry2Aa locus such that the entire cry2Aa open reading frame (from the cry2Aa_ATG start codon to the cry2Aa_TAA stop codon) would be removed after excision of the temperature sensitive origin of replication. Additionally, the integration was designed such that the native cry2Aa promoter would drivetranscription of the incoming cry2 lAa gene. The integration would utilize homologous recombination regions 1000 bp in length flanking either side of the cry2Aa integration site. The resultant strain was named PE39400-G27 and comprised the cry6Aa toxin expression cassette integrated at dltA and the cry21Aa toxin expression cassette integrated at cry2Aa. The resultant strain was named PE39400-G30 and comprised the cry6Aa toxin expression cassette integrated at dltA, the cry21Aa toxin expression cassette integrated at cry2Aa, and the cry5Ba toxin expression cassette integrated at ngd (genotype dltA: :PCrylBa-cry6Aa ugd::PCrylBa-cry5Ba cry2Aa: :cry21 Aa) (triple stacked toxin strain).Promoter Exchange

[0190] Strain PE39400-G30 was modified as follows: the dlta locus was targeted for promoter exchange, to replace the native promoter PcrylBa (SEQ ID NO: 53). The resultant strain (CE39400-G80) comprised a new promoter driving expression of cry6Aa at the dltA locus within the PE39400-G80 chromosome. To generate plasmid pAP69 used to conduct the promoter exchange in PE39400-G30, PCR products approximately 1000 bp containing the region upstream of PcrylBa and the region downstream of PcrylBa were amplified using Q5 high- fidelity polymerase from CE39400-G30 using appropriate primer pairs. The exogenous PaprE promoter (SEQID NO: 54) approximately 200 bp in length was amplified from the Bioconsortia plasmid pBACOV using appropriate primer pairs. All primer pairs contained appropriate Gibson assembly overhangs to facilitate isothermal assembly of the fragments to each other and to the linearized cloning vector, pMMDmob, such that the final construct comprised the region upstream PcrylBa directly ligated to the PaprE promoter which in turn was directly ligated to the region downstream of PcrylBa. the cloning vector pMMDmob was linearized using the EcoRl and BamHl endonuclease enzymes and gel purified. DNA fragments consisting of the above regions were gel purified. Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragments in a 1:3:5 :3 backbone:insert:insert:insert molar ratio in a 10 ul volume, then added to 10 ul 2x Gibson master mix reagent. The reaction was incubated at 50 C for 60 minutes then used for transformation into E. coll DH5a. Ampicillin-resistant E. coli colonies were clonally isolated, and presumptive assembled plasmids were purified and confirmed using nanopore whole plasmid sequencing.The resulting correctly assembled plasmid was transformed into dam- E. coli, subsequentlypurified, and then transformed into electrocompetent PE39400-G30. PE39400-G30 colonies resistant to the antibiotic resistance marker on pAP69 (MLS) were selected for integration into and subsequent excision of the plasmid from the host chromosome. The resulting strain was confirmed to contain PaprE in place of PcrylBa at the dll A locus driving transcription of cry6Aa. Subsequent Sanger and illumina sequencing confirmed no other mutations were generated through this process. The resultant strain was named PE39400-G80 and comprised the genotype dltA: :PaprE-cry6Aa ugd: :PcrylBa-cry5Ba cry2Aa: : cry2 lAa.Deletion of aprE

[0191] Strain PE39400-G30 was modified as follows: the aprE gene (SEQ ID NO: 51) was targeted for in frame marker-less deletion. The resultant strain (PE39400-G89) comprised an aprE gene deletion within the PE39400-G30 chromosome. To generate plasmid pAP79 used to create the in-frame marker- less deletion in PE39400-G89, PCR products approximately 1000 bp containing the region upstream of aprE and the region downstream of aprE were amplified using Q5 high-fidelity polymerase from PM39400 using appropriate primer pairs. Primer pairs contained appropriate Gibson assembly overhangs to facilitate isothermal assembly of the fragments to each other and to the linearized cloning vector, pMMDmob, such that the final construct comprised the region upstream of aprE directly ligated to the region downstream of aprE such that the reading frame was maintained from the aprE start codon to the aprE stop codon while also eliminating 2658 bp of the middle of the aprE coding sequence. pMMDmob was linearized using the EcoRl and BamHl endonuclease enzymes and gel purified. DNA fragments consisting of the above regions were gel purified. Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragments in a 1:3:3 backbone:insert:insert molar ratio in a 10 ul volume, then added to 10 ul 2x Gibson Reagent. The reaction was incubated at 50 C for 60 minutes then used for transformation into E. coli DH5 a . Ampicillin resistant E. coli colonies were clonally isolated, and presumptive assembled plasmids were purified and confirmed using nanopore whole plasmid sequencing. The resulting correctly assembled plasmid was transformed into dam- E. coli, subsequently purified, and then transformed into electrocompetent PE39400-G30. PE39400-G30 colonies resistant to the antibiotic resistance marker on pAP79 (MLS) were selected for integration into and subsequent excision of the plasmid from the host chromosome. The resulting strain was confirmed tocontain a markerless in-frame deletion of aprE. Subsequent Sanger and illumina sequencing confirmed no other mutations were generated through this process. The resultant strain was named PE39400-G89 and comprised the genotype dltA: :PcrylBa-cry6Aa ugd::PcrylBa-cry5Ba cry2Aa::cry21Aa aprE.Deletion of nprA

[0192] Strain PE39400-G30 was modified as follows: the nprA gene (SEQ ID NO: 52) was targeted for in frame marker-less deletion. The resultant strain (PE39400-G91) comprised an nprA gene deletion within the PE39400-G30 chromosome. To generate plasmid pAP80 used to create the in-frame marker- less deletion in PE39400-G91, PCR products approximately 1000 bp containing the region upstream of nprA and the region downstream of nprA were amplified using Q5 high-fidelity polymerase from PM39400 using appropriate primer pairs. Primer pairs contained appropriate Gibson assembly overhangs to facilitate isothermal assembly of the fragments to each other and to the linearized cloning vector, pMMDmob, such that the final construct comprised the region upstream of nprA directly ligated to the region downstream of nprA such that the reading frame was maintained from the nprA start codon to the nprA stop codon while also eliminating 1251 bp of the middle of the nprA coding sequence. pMMDmob was linearized using the EcoRl and BamHl endonuclease enzymes and gel purified. DNA fragments consisting of the above regions were gel purified. Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragments in a 1:3:3 backbone:insert:insert molar ratio in a 10 ul volume, then added to 10 ul 2x Gibson Reagent. The reaction was incubated at 50 C for 60 minutes then used for transformation into E. coli DH5 a . Ampicillin resistant E. coli colonies were clonally isolated, and presumptive assembled plasmids were purified and confirmed using nanopore whole plasmid sequencing. The resulting correctly assembled plasmid was transformed into dam- E. coli, subsequently purified, and then transformed into electrocompetent PE39400-G30. PE39400-G30 colonies resistant to the antibiotic resistance marker on pAP80 (MLS) were selected for integration into and subsequent excision of the plasmid from the host chromosome. The resulting strain was confirmed to contain a markerless in-frame deletion of nprA. Subsequent Sanger and illumina sequencing confirmed no other mutations were generated through this process. The resultant strain wasnamed PE39400-G91 and comprised the genotype dltA: :Pcryl Ba-cry6Aa ugd::PcrylBa-cry5Ba cry2Aa: :cry21Aa AnprA.Deletion of aprE and nprA

[0193] Strain PE39400-G89 was modified as follows: the nprA gene was targeted for in frame marker-less deletion. The resultant strain (PE39400-G94) comprised an nprA gene deletion within the PE39400-G89 chromosome that itself contained the aprE gene deletion (see above). To generate plasmid pAP80 used to create the in-frame marker-less deletion in PE39400-G94, PCR products approximately 1000 bp containing the region upstream of nprA and the region downstream of nprA were amplified using Q5 high-fidelity polymerase from PM39400 using appropriate primer pairs. Primer pairs contained appropriate Gibson assembly overhangs to facilitate isothermal assembly of the fragments to each other and to the linearized cloning vector, pMMDmob, such that the final construct comprised the region upstream of nprA directly ligated to the region downstream of nprA such that the reading frame was maintained from the nprA start codon to the nprA stop codon while also eliminating 1251 bp of the middle of the nprA coding sequence. pMMDmob was linearized using the EcoRl and BamHl endonuclease enzymes and gel purified. DNA fragments consisting of the above regions were gel purified. Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragments in a 1:3:3 backbone:insert:insert molar ratio in a 10 ul volume, then added to 10 ul 2x Gibson Reagent. The reaction was incubated at 50 C for 60 minutes then used for transformation into E. coli DH5a. Ampicillin resistant E. coli colonies were clonally isolated, and presumptive assembled plasmids were purified and confirmed using nanoporc whole plasmid sequencing. The resulting correctly assembled plasmid was transformed into dam- E. coli, subsequently purified, and then transformed into electrocompetent PE39400-G30.PE39400-G30 colonies resistant to the antibiotic resistance marker on pAP80 (MLS) were selected for integration into and subsequent excision of the plasmid from the host chromosome. The resulting strain was confirmed to contain a markerless in-frame deletion of nprA. Subsequent Sanger and illumina sequencing confirmed no other mutations were generated through this process. The previously generated aprE deletion was confirmed to remain intact after nprA deletion. The resultant strain was named PE39400-G94 and comprised the genotype dltA: :PcrylBa-cry6Aa ugd::PcrylBa-cry5Ba cry2Aa::cry21Aa AaprE AnprA.Example 7: Promoter Efficacy

[0194] The promoters used in the triple-stacked strain (PaprE, Very] Ba. Pcry2Aa) were tested for transcriptional activity in plasmids with Green Fluorescent Protein (GFP) as a reporter. The following plasmids were constructed: PE39400-T106 (oriPUB PaprE-sfGFP kan amp), PE39400-G118 (pAP86 oriPUB traJ PcrylBa-sfGFP kan amp), PE39400-G119 (pAP87 oriPUB traJ Pcry2Aa-sfGFP kan amp), and PE39400-G120 (pAP88 oriPUB traJ PaprE-His6x kan amp). Each plasmid was transformed into parental strain PM39400.

[0195] All strains were streaked on LB plates containing 50 ug / mL kanamycin and grown overnight at 37C. The next day, strains were inoculated in LB broth containing 50 ug / ml kanamycin, 5 ml in 50 ml conical tube, and grown at 30C at 200 RPM for 5 days. Each 24 hours, 200 uL was sampled from each culture and resuspended in PBS (Phosphate Buffered Saline) to the same concentration. 100 uL of each was aliquoted into separate wells of a 96- well plate. OD600 and fluorescence was measured on a Cytation 5 imaging reader. Relative fluorescence units were calculated as the average fluorescence divided by the average OD600. Sporulation was assessed at each of the timepoints by microscopy. Gross sporulation phenotype was based on cytological examination.

[0196] Data is shown in FIG. 5. Plus and minus signs indicate presence or absence, respectively, of sporulation. PaprE (strain T106) was shown to be constitutively-active. Under these growth conditions, PcrylBa was strongly active during sporulation.Example 8: BioassayC. elegans

[0229] To determine possible modes of action of nematocidal microbes the model organism C. elegans, a free living, soil dwelling nematode, was used in several different assays. First, a developmental test was conducted to determine activity. Young larval worms were made to feed on the microbes. While doing this the development and fecundity of the worms was tracked. Based on how quickly C. elegans reaches reproductive age and the resulting population of offspring, how these microbes affect the nematodes was determined.

[0230] Second, mortality of adult worms was assessed. Adult worms were introduced to the microbes and rate of death was tracked. This demonstrated the toxicity of the microbe toward the nematodes.

[0231] C. elegans was also tested in a chemotaxis assay. Worms were introduced to two different stimuli, (e.g., microbial, chemical, metabolic, etc.). Based on the movement of the nematodes in response to these stimuli, a chemotactic index, repulsion or attraction, was calculated.RKN

[0197] Seeds of a susceptible crop were planted in pots containing soil and sand mixture at 1:1 ratio, two weeks before treatment application. An excess of seeds were planted per pot and then thinned to one seedling per pot.

[0198] Two weeks after planting, microbial treatment solution was drenched at lxlOA7 CFUs / mL per plant, at the base of the plant into the soil. Application volume was sufficient to cover the root zone in the pot.

[0199] Plants were not watered immediately before or after treatment application (no heavy watering that day - before or after treatment application). Regular watering was resumed the next day.

[0200] Forty-eight hours after treatment application, nematodes were applied at the desired concentration, for example in some cases, J2 stage was used instead of eggs, to reduce variability.

[0201] On the day of nematode application, plants were misted with water in the morning before nematode infestation. Four weeks after nematode application, shoots were harvested for dry weights and roots washed for nematode assessments.

[0202] Roots were stained for egg masses, count egg masses, galls, eggs, etc. (Galls, eggs, egg masses, cyst, females, etc.).

[0203] Root dry weights were obtained, and nematodes per gram dry root weight was calculated.

[0204] Positive and negative controls included: Untreated (no nematode inoculation, no microbial treatment, just drench with water), Inoculated control (nematode inoculation, no microbial treatment, just drench with water), Positive controls (known nematicide products against the nematodes tested - ideally have both chemical nematicide and biological nematicide as positive controls in the same experiment).Lesion

[0205] Seeds of susceptible crop (Sorghum) were planted in pots containing soil and sand mixture at 1:1 ratio, two weeks before treatment application. An excess of seeds were planted per pot and then thinned to one seedling per pot.

[0206] Two weeks after planting, microbial treatment solution was drenched at lxlOA7 CFUs / mL per plant, at the base of the plant into the soil. Application volume was sufficient to cover the root zone in the pot.

[0207] Plants were not watered immediately before or after treatment application (no heavy watering that day - before or after treatment application). Regular watering was resumed the next day.

[0208] Forty-eight hours after treatment application, Lesion nematodes were applied to the roots at the desired concentration. On the day of nematode application, plants were misted with water in the morning before nematode infestation. Six weeks after nematode application, shoots were harvested for dry weights and roots were extracted for nematode counts.

[0209] Nematodes were counted per plant.

[0210] Root dry weights were obtained, and nematodes per gram dry root weight was calculated.

[0211] Positive and negative controls included: Untreated (no nematode inoculation, no microbial treatment, just drench with water), Inoculated control (nematode inoculation, no microbial treatment, just drench with water), Positive controls (known nematicide products against the nematodes tested - ideally have both chemical nematicide and biological nematicide as positive controls in the same experiment).

[0212] Results are shown in Tables 4 and 5, and FIGs. 1-4.Table 4: Toxin Stacks Nematocidal ActivityTable 5: Toxin Stacks Nematocidal Activity

Claims

IT IS CLAIMED:

1. An engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of:(a) insertion of at least one heterologous polynucleotide encoding a Cry toxin;(b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, cry 1 Ba, cry Ha, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl and(c) reduced or abolished expression of at least one gene in a biosynthetic pathway; wherein the engineered bacterial cell confers an improved phenotype to a plant with which it is associated, as compared to a control bacterial cell of the same strain that has not been engineered; wherein the improved phenotype to the plant is selected from the group consisting of: improved toxicity activity against a nematode, improved plant growth, improved plant health, and any plurality and / or combination of the preceding.

2. The engineered bacterial cell of Claim 1 , wherein the at least one gene in a biosynthetic pathway is selected from the group consisting of: thuE, aprE, and nprA;3. The engineered bacterial cell of Claim 1, wherein the at least one gene in a toxin biosynthetic pathway comprises a sequence sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 20, 51, and 52.

4. The engineered bacterial cell of Claim 1, wherein the heterologous polynucleotide of (a) or (b) is inserted into the genome of the cell in a coding sequence located in a thuringiensin biosynthetic gene cluster.

5. The engineered bacterial cell of Claim 1 , wherein the at least one heterologous polynucleotide of (a) or (b) comprises a sequence sharing at least 97 % identity with at least one sequence selected from the group consisting of: SEQID NOs: 1-16.

6. The engineered bacterial cell of Claim 1, wherein the at least one heterologous polynucleotide of (a) or (b) encodes a polypeptide sharing at least 97% identity with at least one sequence selected from the group consisting of: SEQID NOs: 21-36.

7. The engineered bacterial cell of Claim 1, wherein the heterologous polynucleotide of (a) or (b) is operably linked to a heterologous promoter.

8. They engineered bacterial cell of Claim 1, wherein the Bacillus cell is of the species Bacillus thuringiensis.

9. A synthetic composition comprising the engineered bacterial cell of Claim 1, or an exudate therefrom, or culture broth therefrom, or spore therefrom, or any plurality or combination of the preceding; further comprising at least one composition selected from the group consisting of: a plant element, a formulation component, an agricultural composition, and any plurality and / or combination of the preceding; wherein the plant element is selected from the group consisting of: a seed, a leaf, a root, a whole plant, and / or any plurality and / or combination of the preceding; wherein the formulation component is selected from the group consisting of: a compound that improves the stability of the microbe, a preservative, a carrier, a surfactant, an anticomplex agent, and any combination and / or plurality of the preceding; and wherein the agricultural composition is selected from the group consisting of: comprises a fungicide, a nematicide, a bactericide, an insecticide, an herbicide, a growth medium, and any combination and / or plurality of the preceding;and wherein the engineered bacterial cell is present at a concentration of at least about 10A2 CFU / mL in a liquid formulation, or at least about 10A2 Colony Forming Units per gram in a non-liquid formulation.

10. The synthetic composition of Claim 9, further comprising at least one additional microbe.

11. The synthetic composition of Claim 9, wherein the plant element comprises a transgene.

12. A plurality of synthetic compositions of Claim 9, wherein said synthetic compositions are substantially confined within an object selected from the group consisting of: a tube, a bottle, ajar, an ampule, a package, a vessel, a bag, a box, a bin, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and a case.

13. The synthetic composition of Claim 9, wherein the plant element is obtained from a plant selected from the group consisting of: maize, soybean, wheat, cotton, cucumber, tomato, pepper, potato, strawberry, orange, lemon, lime, apple, snap beans, zucchini, pea, lettuce, broccoli, celery, cauliflower, sorghum, canola, rapeseed, and okra.

14. A plurality of synthetic compositions of Claim 9, wherein the growth medium comprises soil, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern with substantially equal spacing between each of the synthetic compositions.

15. A method of conferring nematode pest tolerance to a plant, the method comprising: applying to a plant element of said plant a formulation comprising an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of:(a) insertion of at least one heterologous polynucleotide encoding a Cry toxin;(b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, cry Ila, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl', and (c) reduced or abolished expression of at least one gene in a biosynthetic pathway.

16. The method of Claim 15, wherein said applying to the plant element is accomplished by an indirect method selected from the group consisting of: in-furrow application, soil drench application, and side-dress application.

17. The method of Claim 15, wherein said applying to the plant element is accomplished by coating said plant element with a liquid formulation of the microbe or exudate therefrom.

18. The method of Claim 15, wherein said applying to the plant element is accomplished by coating said plant element with a substantially non-liquid formulation of the microbe or exudate therefrom.

19. The method of Claim 15, wherein said plant element is a seed, a leaf, a root, a whole plant, and / or any plurality and / or combination of the preceding.

20. A method of reducing the number of pests in or on a composition, wherein the pests comprise nematodes, nematode eggs, nematode juveniles, or any plurality and / or combination of the preceding, the method comprising: introducing to the composition at an engineered bacterial cell of the genus Bacillus, wherein the engineered bacterial cell comprises at least one modification to its genome, wherein the at least one modification is selected from the group consisting of:(a) insertion of at least one heterologous polynucleotide encoding a Cry toxin; and(b) insertion of at least one heterologous polynucleotide selected from the group consisting of: app6Bal, cry2Ab35, cry5B, cryl2A, cry21Aa, cry5Aa, cry55al, crylAa, crylBa, cry Ila, cry2Aa, cry2Ab, cry6Aa, app4al, cry5Ba, and cry3Bbl.

21. The method of Claim 20, wherein the composition is a growth medium for a plant.

22. The method of Claim 20, wherein the composition is a harvested product.

23. The method of Claim 22, wherein the harvested product is a fruit, a vegetable, a seed, and / or a fiber.

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