Genetically modified bacteria for improved pest control of plants

Abstract

The present disclosure relates to edited (genetically modified) microorganisms comprising consortia of microorganisms, and to agricultural compositions comprising said microorganisms, intended to increase the tolerance of plants to nematode pests and / or to directly reduce nematode populations in soil or root systems. In addition, the present disclosure teaches methods of utilizing the described microorganisms, microbial consortia, and agricultural compositions comprising the same in methods of conferring beneficial properties to target plant species. In particular aspects, the present disclosure provides methods for improving desired plant traits and reducing undesired pests that affect crop performance in agronomically important crop species.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 581,192, filed September 7, 2023, which is incorporated herein by reference in its entirety. References to sequence lists submitted electronically An official copy of the sequence list is submitted electronically as a WIPO ST26 compliant XML sequence list, named 23072_SeqListing.xml, created on July 29, 2024, and 134,714 bytes in size, and is submitted with this specification. The sequence list contained in this file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure generally relates to the field of biology, and more specifically to genetically engineered microorganisms and microbial compositions for improving plant health. Background Technology

[0003] According to the United Nations World Food Program, nearly 900 million people worldwide suffer from malnutrition. The problem is particularly acute in developing countries, where one in six children is underweight. The lack of accessible food can be attributed to a variety of socioeconomic factors; however, regardless of the underlying causes, the reality remains that food shortages are insufficient to feed the growing world population, projected to reach 9 billion by 2050. The United Nations estimates that agricultural output must increase by 70%–100% to feed the projected global population by 2050.

[0004] These staggering figures on the world's population and malnutrition underscore the critical importance of agricultural efficiency and productivity for sustaining a growing global population. Modern field crop agriculture has achieved unprecedented crop yields, thanks to remarkable technological advancements. However, despite progress made through technological innovations such as genetically engineered crops and novel pesticide and herbicide compounds, improvements in crop performance are still needed to meet the demands of an exponentially growing global population.

[0005] Scientists estimate that if the global agricultural yield gap (i.e., the difference between the best observed yield and the results in other regions) could be eliminated, global crop production would increase by 45%-70%. In other words, if all farmers (regardless of where they are in the world) could achieve the highest yields expected in their respective regions, most of the world's food production shortages would be solved. However, addressing the challenge of achieving higher yields across the heterogeneous landscape of the world presents a significant challenge.

[0006] Typically, yield disparities can be explained by insufficient water supply, outdated farming practices, inadequate fertilizers, and lack of access to herbicides and pesticides. However, a large-scale increase in the use of water, fertilizers, herbicides, and pesticides worldwide would not only be economically unfeasible in most parts of the world but would also have negative environmental consequences.

[0007] Nematodes are among the most destructive pests of economic crops, causing an estimated $173 billion in economic losses globally each year.

[0008] Therefore, there is an urgent need in the field for improved methods to enhance crop performance and endow both plants and microorganisms with beneficial traits to improve crop health. Summary of the Invention

[0009] This encompasses genetically engineered microorganisms, particularly for use in agriculture. The disclosed microorganisms can be used in their isolated and biologically pure state, or formulated into agriculturally acceptable compositions. Agriculturally beneficial microbial aggregates comprising at least two members of the disclosed microorganisms are also provided; along with methods for utilizing said aggregates in agricultural applications. In some aspects, genomic modifications of microorganisms (individuals, aggregates, and / or communities) are envisioned to improve microbial traits and enhance microbial-associated plants.

[0010] The solutions provided by this disclosure for improving crop performance and increasing yield are harmless to Earth's resources because they do not rely on increasing water consumption or the input of synthetic chemicals into the system. Instead, this disclosure utilizes microorganisms to confer beneficial properties on desired plants, including increased yield.

[0011] Therefore, this disclosure provides an environmentally sustainable solution that allows farmers to increase the yield of important crops without relying on increased use of synthetics and pesticides.

[0012] In its implementation, this disclosure provides an effective and widely applicable agricultural platform that utilizes microorganisms and microbial aggregates (multiple microorganisms, in some respects, for improving the health of the associated plant or desired phenotypes, such as agronomic traits) that promote one or more desired plant properties.

[0013] The microorganisms disclosed herein improve the performance of plants (such as crop plants) through both direct and indirect mechanisms. In some ways, the microorganisms form a symbiotic relationship with the plant. In some ways, the microorganisms produce compounds (e.g., metabolites) that confer benefits on the plant or can be used by the plant to improve its properties. In some ways, the microorganisms increase the solubility of one or more compositions (such as nutrients), thereby benefiting the plant. In some ways, the microorganisms confer tolerance to exogenous substances (such as herbicides or pesticides). In some ways, the microorganisms produce compositions that are harmful to plant pests (such as nematodes).

[0014] In some aspects, the present invention includes engineered bacterial cells of the genus *Bacillus*, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in a biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA); wherein the engineered bacterial cells exhibit improved toxicity against nematodes compared to unengineered control bacterial cells with the same genetic background.

[0015] In some aspects, the present invention includes engineered bacterial cells of the genus *Bacillus*, wherein the engineered bacterial cells comprise at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA); wherein the engineered bacterial cells exhibit improved toxicity against nematodes compared to unengineered control bacterial cells with the same genetic background; wherein at least one gene in the toxin biosynthetic pathway comprises at least one gene selected from SEQ ID NO. NO: A sequence consisting of 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 more than 200 nucleotides 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 more than 99% identity.

[0016] In some aspects, the present invention includes engineered bacterial strains wherein heterologous polynucleotides have been inserted into the genome of a cell in coding sequences located in gene clusters. In some embodiments, the gene clusters include one or more genes encoding toxins (such as Bacillus thuringiensis), for example, but not limited to, thuE. In some embodiments, the gene clusters include one or more genes encoding proteases, such as, but not limited to, serine alkaline proteases (e.g., subtilisin E, aprE) or neutral protease A (nprA). In some embodiments, more than one gene is modified such that the expression of more than one toxin and / or protease is reduced.

[0017] In some aspects, the present invention includes engineered bacterial cells of the genus *Bacillus*, wherein the engineered bacterial cells comprise at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cr y1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in a biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA); wherein the engineered bacterial cells exhibit improved toxicity against nematodes compared to unengineered control bacterial cells with the same genetic background; wherein at least one heteropolynucleotide of (a) or (b) comprises at least one selected from SEQ ID NO: A sequence consisting of 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 more than 200 nucleotides having 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 higher than 99% identity.

[0018] In some aspects, the present invention includes engineered bacterial cells of the genus *Bacillus*, wherein the engineered bacterial cells comprise at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cr y1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA); wherein the engineered bacterial cells exhibit improved toxicity against nematodes compared to unengineered control bacterial cells with the same genetic background; wherein at least one heteropolynucleotide of (a) or (b) encodes a gene selected from SEQ ID NO. NO: A polypeptide consisting of 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 more than 200 amino acids 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 more than 99% identity.

[0019] In some aspects, the present invention includes engineered bacterial cells of the genus *Bacillus*, wherein the engineered bacterial cells comprise at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba (a) cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reduce or eliminate the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA); wherein the engineered bacterial cells exhibit improved toxicity against nematodes compared to unengineered control bacterial cells with the same genetic background; wherein the heteropolynucleotide of (a) or (b) is operatively linked to a heteropromoter.

[0020] A synthetic composition comprising engineered bacteria, or their secretions, or their culture medium, or their spores, or any combination thereof; further comprising at least one composition selected from the group consisting of: plant components, formulation components, agricultural compositions, and any combination thereof; wherein the plant component is selected from the group consisting of: seeds, leaves, roots, whole plants, and / or any combination thereof; wherein the formulation component is selected from the group consisting of: compounds that enhance the stability of microorganisms, preservatives, carriers, surfactants, anticomplex agents, and any combination thereof; and wherein the agricultural composition is selected from the group consisting of: fungicides, nematicides, bactericides, insecticides, herbicides, growth media, and any combination thereof; and wherein the engineered bacterial cells are present in a liquid formulation at a concentration of at least about 10^2. The engineered bacterial cells are present at a concentration of CFU / mL, or in a non-liquid formulation at a concentration of at least about 10^2 colony-forming units / gram; and the engineered bacterial cells belong to the genus Bacillus, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA).

[0021] A synthetic composition comprising engineered bacteria, or their secretions, or their culture medium, or their spores, or any combination thereof; further comprising at least one composition selected from the group consisting of: plant components, formulation components, agricultural compositions, and any combination thereof; and wherein the engineered bacterial cells belong to the genus Bacillus, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one heterologous polynucleotide encoding Cry toxin; (b) insertion into The absence of one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reduction or elimination of the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA).

[0022] A synthetic composition comprising engineered bacteria, or their secretions, or their culture medium, or their spores, or any combination thereof; further comprising at least one composition selected from the group consisting of: plant components, formulation components, agricultural compositions, and any combination thereof; and wherein the plant component is selected from the group consisting of: seeds, leaves, roots, whole plants, and / or any combination thereof; and wherein the engineered bacterial cells belong to the genus Bacillus, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) insertion (a) inserting at least one heteropolynucleotide encoding Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA).

[0023] A synthetic composition comprising engineered bacteria, or their secretions, or their culture medium, or their spores, or any combination thereof; further comprising at least one composition selected from the group consisting of: plant components, formulation components, agricultural compositions, and any combination thereof; wherein the formulation component is selected from the group consisting of: compounds that enhance the stability of microorganisms, preservatives, carriers, surfactants, anti-complex agents, and any combination thereof; and wherein the engineered bacterial cells belong to the genus Bacillus, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: The group includes: (a) inserting at least one heteropolynucleotide encoding Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA).

[0024] A synthetic composition comprising engineered bacteria, or their secretions, or their culture medium, or their spores, or any combination thereof; further comprising at least one composition selected from the group consisting of: plant components, formulation components, agricultural compositions, and any combination thereof; and wherein the agricultural composition is selected from the group consisting of: fungicides, nematicides, bactericides, insecticides, herbicides, growth media, and any combination thereof; and wherein the engineered bacterial cells belong to the genus Bacillus, and wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: The group consisting of: (a) inserting at least one heteropolynucleotide encoding Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA).

[0025] A synthetic composition comprising engineered bacteria, or their secretions, or their culture medium, or their spores, or any combination thereof; further comprising at least one composition selected from the group consisting of: plant components, formulation components, agricultural compositions, and any combination thereof; and wherein the plant component is selected from the group consisting of: seeds, leaves, roots, whole plants, and / or any combination thereof; and wherein the engineered bacterial cells belong to the genus Bacillus, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) insertion of at least one coding (a) a heteropolynucleotide of Cry toxin; (b) insertion of at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba and cry3Bb1; and / or (c) reduction or elimination of the expression of at least one gene in a biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA); wherein the plant component contains a transgene.

[0026] Multiple synthetic compositions, each comprising a genetically modified bacterium of the genus Bacillus heterologously treated on a plant component, 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) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in a biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA).

[0027] Multiple synthetic compositions, each comprising a genetically modified bacterium of the genus *Bacillus* heterologously treated on a plant component, 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) inserting at least one heterologous polynucleotide encoding a Cry toxin; (b) inserting at least one heterologous polynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1 Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA); wherein the synthetic composition is substantially enclosed within an object selected from the group consisting of: tubes, bottles, wide-mouth bottles, ampoules, packaging, containers, bags, boxes, silos, envelopes, cartons, containers, silos, shipping containers, truck beds, and boxes.

[0028] Multiple synthetic compositions, each comprising a genetically modified bacterium of the genus *Bacillus* heterologously treated as a plant component, 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) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reducing or eliminating the biosynthetic pathway. The expression of at least one gene (e.g., but not limited to one or more of the following: thuE, aprE, nprA); wherein the plant component is obtained from plants selected from the group consisting of: alfalfa, apple, avocado, banana, barley, legumes, broccoli, rapeseed, carrot, carrots, cassava, cauliflower, celery, chickpea, citrus, coconut, coffee, cereals, cotton, cruciferous plants, cucumber, gourd, broad bean, forest trees, garlic, ginger, grape, grass, lemon, lettuce, lime, corn, oat, oat, okra, onion, orange, fruit trees, ornamental plants, palm, pea, peanut, pepper, potato, rapeseed, kidney beans, sorghum, soybean, soybean, strawberry, strawberry, beet, sugarcane, tea, tomato, trees, vegetables, wheat, and zucchini.

[0029] Multiple synthetic compositions, each comprising a genetically modified bacterium of the genus *Bacillus* heterologously treated on a plant component, 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) inserting at least one heterologous polynucleotide encoding a Cry toxin; (b) inserting at least one heterologous polynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry5 5a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reduce or eliminate the expression of at least one gene in a biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA); wherein the plant component is selected from the group consisting of: seeds, leaves, stems, roots, buds, bulbs, offspring, branches, stolons, whole plants, and / or any and / or combinations of the foregoing.

[0030] Multiple synthetic compositions, each comprising a genetically modified bacterium of the genus *Bacillus* heterologously treated on a plant component, 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) inserting at least one heterologous polynucleotide encoding a Cry toxin; (b) inserting at least one heterologous polynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry 55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and / or (c) reducing or eliminating the expression of at least one gene in the biosynthetic pathway (e.g., but not limited to one or more of the following: thuE, aprE, nprA); it also comprises a growth medium, wherein the growth medium includes soil, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern, the spacing between each of the synthetic compositions being substantially equal.

[0031] A method for conferring plant tolerance to nematode pests, the method comprising: applying to a plant component of the plant an agent comprising engineered bacterial cells of the genus Bacillus, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and (c) reducing or eliminating the expression of at least one gene in a biosynthetic pathway.

[0032] A method for conferring plant tolerance to nematode pests, the method comprising: applying to a plant component of the plant an agent comprising engineered bacterial cells of the genus Bacillus, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and (c) reducing or eliminating the expression of at least one gene in a biosynthetic pathway; wherein the application to the plant component is performed by an indirect method selected from the group consisting of: furrow application, soil irrigation application, and lateral fertilization application.

[0033] A method for conferring tolerance to nematode pests on a plant, the method comprising: applying to a plant component of the plant a formulation comprising engineered bacterial cells of the genus Bacillus, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and (c) reducing or eliminating the expression of at least one gene in a biosynthetic pathway; wherein the application to the plant component is accomplished by coating the plant component with a liquid formulation of a microorganism or its secretions.

[0034] A method for conferring tolerance to a plant nematode pest, the method comprising: applying to a plant component of the plant a formulation comprising engineered bacterial cells of the genus Bacillus, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin; (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and (c) reducing or eliminating the expression of at least one gene in a biosynthetic pathway; wherein the application to the plant component is accomplished by coating the plant component with a substantially non-liquid formulation of the microorganism or its secretions.

[0035] A method for reducing the number of harmful organisms in or on a composition, wherein the harmful organisms include nematodes, nematode eggs, nematode larvae, or any and / or combinations thereof, the method comprising: introducing engineered bacterial cells of the genus Bacillus into the composition, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin, and (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1.

[0036] A method for reducing the number of pests in or on a composition, wherein the pests include nematodes, nematode eggs, nematode larvae, or any and / or combinations thereof, the method comprising: introducing engineered bacterial cells of the genus Bacillus into the composition, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin, and (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; wherein the composition is a plant growth medium.

[0037] A method for reducing the number of pests in or on a composition, wherein the pests include nematodes, nematode eggs, nematode larvae, or any and / or combinations thereof, the method comprising: introducing engineered bacterial cells of the genus Bacillus into the composition, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin, and (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; wherein the composition is a harvested product.

[0038] A method for reducing the number of pests in or on a composition, wherein the pests include nematodes, nematode eggs, nematode larvae, or any and / or combinations thereof, the method comprising: introducing engineered bacterial cells of the genus Bacillus into the composition, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) inserting at least one heteropolynucleotide encoding a Cry toxin, and (b) inserting at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; wherein the composition is a harvested product; wherein the harvested product is fruit, vegetable, seed, and / or fiber.

[0039] In any respect of the compositions and methods described herein, the compositions relating to engineered bacterial cells are plants or plant components.

[0040] In any respect of the compositions and methods described herein, the engineered bacterial cells belong to the genus Bacillus.

[0041] In any respect of the compositions and methods described herein, the engineered bacterial cells are the species Bacillus thuringiensis.

[0042] In any aspect of the compositions and methods described herein, the engineered bacterial cells comprise one or more sequences selected from SEQ ID NO:37-50.

[0043] In any respect of the compositions and methods described herein, the engineered bacterial cells are obtained from or derived from Bacillus thuringiensis strain PM39400, which is deposited as NRRL B-68090.

[0044] In any aspect of the compositions and methods described herein, at least one additional bacterium may be included together with engineered bacterial cells; wherein the at least one additional bacterium may be wild-type or engineered.

[0045] In any respect of the compositions and methods described herein, the taxonomic classification of nematodes is selected from the following genera: Meloidogyne, Heterodera, Pratylenchus, Radopholus, Xiphenema, Ditylenchus, and any and / or combinations thereof.

[0046] Figure descriptions and sequence lists This disclosure will be more fully understood through the following detailed description and the accompanying drawings and sequence listings that form part of this application.

[0047] Figure 1 The study showed the mortality rates of *C. elegans* under different treatments with gene-edited *Bacillus* strains on days 1, 2, and 3.

[0048] Figure 2 The study showed the mortality rates of *C. elegans* under different treatments with edited *Bacillus* strains on days 1, 3, and 4.

[0049] Figure 3 The study showed a reduction in the number of RKN egg masses per gram of dry root under different treatments with edited Bacillus strains.

[0050] Figure 4 The results showed a reduction in the number of lesions under different treatments with edited Bacillus strains.

[0051] Figure 5 The relative fluorescence (column) of different promoters in vectors containing GFP reporter gene fusions is shown, as well as spore formation observed at specified time points for each transformed strain (plus and minus signs represent spore formation and non-spore formation, respectively).

[0052] The sequence description and accompanying sequence listing conform to the rules governing the disclosure of nucleotide and amino acid sequences in regulatory patent applications as set forth in 37 CFR §§ 1.821 and 1.825. The sequence description includes the three-letter codes of the amino acids as defined in 37 CFR §§ 1.821 and 1.825, which are incorporated herein by reference.

[0053] SEQ ID NO: 1 is the app6Ba1 DNA sequence from Bacillus subtilis.

[0054] SEQ ID NO: 2 is the cry2Ab35 DNA sequence from Bacillus bombysepticus.

[0055] SEQ ID NO: 3 is a cry5B DNA sequence from Bacillus thuringiensis.

[0056] SEQ ID NO: 4 is a cry12A DNA sequence from Bacillus thuringiensis.

[0057] SEQ ID NO: 5 is a cry21Aa DNA sequence from Bacillus thuringiensis.

[0058] SEQ ID NO: 6 is a cry5Aa DNA sequence from Bacillus thuringiensis.

[0059] SEQ ID NO: 7 is a cry55a1 DNA sequence from Bacillus thuringiensis.

[0060] SEQ ID NO: 8 is the cry1Aa DNA sequence from Bacillus thuringiensis strain PM39400.

[0061] SEQ ID NO: 9 is a cry1Ba DNA sequence from Bacillus thuringiensis strain PM39400.

[0062] SEQ ID NO: 10 is the cry1Ia DNA sequence from Bacillus thuringiensis strain PM39400.

[0063] SEQ ID NO: 11 is the cry2Aa DNA sequence from Bacillus thuringiensis strain PM39400.

[0064] SEQ ID NO: 12 is a cry2Ab DNA sequence from Bacillus thuringiensis strain PM39400.

[0065] SEQ ID NO: 13 is a cry6Aa DNA sequence from Bacillus thuringiensis.

[0066] SEQ ID NO: 14 is the app4a1 DNA sequence from Bacillus thuringiensis.

[0067] SEQ ID NO: 15 is a cry5Ba DNA sequence from Bacillus thuringiensis.

[0068] SEQ ID NO: 16 is a cry3Bb1 DNA sequence from Bacillus thuringiensis.

[0069] SEQ ID NO: 17 is the dltA DNA sequence from Bacillus thuringiensis strain PM39400.

[0070] SEQ ID NO: 18 is the ugd DNA sequence from Bacillus thuringiensis strain PM39400.

[0071] SEQ ID NO: 19 is the xerC DNA sequence from Bacillus thuringiensis strain PM39400.

[0072] SEQ ID NO: 20 is the thuE DNA sequence from Bacillus thuringiensis strain PM39400.

[0073] SEQ ID NO: 21 is an App6Ba1 PRT sequence from Bacillus subtilis.

[0074] SEQ ID NO: 22 is a Cry2Ab35 PRT sequence from Bacillus spp. molluscioides.

[0075] SEQ ID NO: 23 is a Cry5B PRT sequence from Bacillus thuringiensis.

[0076] SEQ ID NO: 24 is a Cry12A PRT sequence from Bacillus thuringiensis.

[0077] SEQ ID NO: 25 is a Cry21Aa PRT sequence from Bacillus thuringiensis.

[0078] SEQ ID NO: 26 is a Cry5Aa PRT sequence from Bacillus thuringiensis.

[0079] SEQ ID NO: 27 is a Cry55a1 PRT sequence from Bacillus thuringiensis.

[0080] SEQ ID NO: 28 is the Cry1Aa PRT sequence from Bacillus thuringiensis strain PM39400.

[0081] SEQ ID NO: 29 is the Cry1Ba PRT sequence from Bacillus thuringiensis strain PM39400.

[0082] SEQ ID NO: 30 is a Cry1Ia PRT sequence from Bacillus thuringiensis strain PM39400.

[0083] SEQ ID NO: 31 is the Cry2Aa PRT sequence from Bacillus thuringiensis strain PM39400.

[0084] SEQ ID NO: 32 is a Cry2Ab PRT sequence from Bacillus thuringiensis strain PM39400.

[0085] SEQ ID NO: 33 is a Cry6Aa PRT sequence from Bacillus thuringiensis.

[0086] SEQ ID NO: 34 is an App4a1 PRT sequence from Bacillus toyonensis.

[0087] SEQ ID NO: 35 is a Cry5Ba PRT sequence from Bacillus thuringiensis.

[0088] SEQ ID NO: 36 is a Cry3Bb1 PRT sequence from Bacillus thuringiensis.

[0089] SEQ ID NO: 37 is a 16S RNA-DNA sequence from Bacillus thuringiensis strain PM39400.

[0090] SEQ ID NO: 38 is a 16S RNA-DNA sequence from Bacillus thuringiensis strain PM39400.

[0091] SEQ ID NO: 39 is a 16S RNA 3 DNA sequence from Bacillus thuringiensis strain PM39400.

[0092] SEQ ID NO: 40 is a 16S RNA 4 DNA sequence from Bacillus thuringiensis strain PM39400.

[0093] SEQ ID NO: 41 is a 16S RNA-DNA sequence from Bacillus thuringiensis strain PM39400.

[0094] SEQ ID NO: 42 is a 16S RNA-6 DNA sequence from Bacillus thuringiensis strain PM39400.

[0095] SEQ ID NO: 43 is a 16S RNA-DNA sequence from Bacillus thuringiensis strain PM39400.

[0096] SEQ ID NO: 44 is a 16S RNA 8 DNA sequence from Bacillus thuringiensis strain PM39400.

[0097] SEQ ID NO: 45 is a 16S RNA 9 DNA sequence from Bacillus thuringiensis strain PM39400.

[0098] SEQ ID NO: 46 is the 16S RNA 10 DNA sequence from Bacillus thuringiensis strain PM39400.

[0099] SEQ ID NO: 47 is the 16S RNA 11 DNA sequence from Bacillus thuringiensis strain PM39400.

[0100] SEQ ID NO: 48 is the 16S RNA 12 DNA sequence from Bacillus thuringiensis strain PM39400.

[0101] SEQ ID NO: 49 is the 16S RNA 13 DNA sequence from Bacillus thuringiensis strain PM39400.

[0102] SEQ ID NO: 50 is the 16S RNA and 14DNA sequence of strain PM39400 from Bacillus thuringiensis strain PM39400.

[0103] SEQ ID NO: 51 is an aprE DNA sequence from Bacillus thuringiensis.

[0104] SEQ ID NO: 52 is an nprA DNA sequence from Bacillus thuringiensis.

[0105] SEQ ID NO: 53 is a Pcry1 DNA sequence from Bacillus thuringiensis.

[0106] SEQ ID NO: 54 is a PaprE DNA sequence from Bacillus thuringiensis.

[0107] The microorganisms described in this application are deposited at the Agricultural Research Service Culture Collection (NRRL), an international depository located at 1815 North University Street, Peoria, IL 61604, USA.

[0108] The deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0109] The deposit is made in accordance with and meets the standards set forth in 37 CFR §§ 1.801-1.809 and the Manual of Patent Examining Procedure §§ 2402-2411.05.

[0110] Strain PM39400 was deposited in NRRL as NRRL B-68090 on January 26, 2022. Detailed Implementation

[0111] The term "a" or "an" refers to one or more of the entities, i.e., it refers to a plural entity. Therefore, the terms "a" or "an," "one or more," and "at least one" are used interchangeably herein. Furthermore, the reference to "component" by the indefinite article "a" or "an" does not preclude the possibility of more than one of the components, unless the context explicitly requires the existence of exactly one of the components.

[0112] As used herein, the term “about” means up to 10% of the listed values. For example, the term “about” may refer to ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10% of the listed values, or a non-integer percentage thereof. As another example, the term “about” may refer to ±0.2 minutes relative to the retention time listed herein.

[0113] As used herein, the term "nucleic acid" refers to a polymer of nucleotides (ribonucleotides or deoxyribonucleotides) of any length or similar. The term refers to the primary structure of a molecule and therefore includes double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. It also includes modified nucleic acids, such as methylated and / or terminally capped nucleic acids, nucleic acids containing modified bases, nucleic acids with modified backbones, etc. The terms "nucleic acid" and "nucleotide sequence" are used interchangeably.

[0114] As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Therefore, genes include, but are not limited to, coding sequences and / or regulatory sequences required for their expression. Genes may also include unexpressed segments of DNA, such as recognition sequences that form other proteins. Genes can be obtained from a variety of sources, including cloning from a target source or synthesis from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0115] Genes and / or other polynucleotides (coding sequences, non-coding sequences, regulatory elements, etc.) are included in an organism's "genome." The term "genome" encompasses all polynucleotides naturally present within the cells of a particular organism, regardless of subcellular localization (e.g., nucleoid, cytoplasm) or constitutive superstructure (e.g., chromosome, plasmid). Polynucleotides introduced into cells may be referred to as "heterologous," as further described herein.

[0116] As used herein, a “synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is known not to exist in nature or is not naturally occurring. Generally, such a synthetic nucleotide sequence will contain at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence. In this context, the term “artificial” may be considered synonymous with “synthetic.”

[0117] As used herein, the terms “homologous,” “homology,” “homologous gene,” or “orthologous” are known in the art and refer to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms “homology,” “homology,” “substantially similar,” and “substantially corresponding” are used interchangeably herein. They refer to nucleic acid fragments in which changes of one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a particular phenotype. These terms also refer to modifications made to nucleic acid fragments of this disclosure, such as the deletion or insertion of one or more nucleotides that substantially do not alter the functional properties of the resulting nucleic acid fragment relative to an initial, unmodified fragment. Therefore, it should be understood that, as those skilled in the art will appreciate, this disclosure covers not only specific exemplary sequences. These terms describe the relationship between a gene present in one species, subspecies, variety, cultivar, or strain and a corresponding or equivalent gene in another species, subspecies, variety, cultivar, or strain. For the purposes of this disclosure, homologous sequences are compared. "Homologous sequences" or "homologous" or "orthologous" are considered, believed, or known to be functionally related. Functional relationships can be indicated in any of a variety of ways, including, but not limited to: (a) the degree of sequence identity and / or (b) identical or similar biological functions. 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 (FM Ausubel et al., eds., 1987), Supplement 30, Section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK), ALIGN Plus (Scientific and Educational Software, Pennsylvania), and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), which uses default parameters.

[0118] As used herein, the term "nucleotide change" means, for example, nucleotide substitution, deletion, insertion, chemical alteration, or any of the foregoing, as is well understood in the art.

[0119] As used herein, the term “protein modification” refers to, for example, amino acid substitution, amino acid modification, deletion and / or insertion, as well as is well understood in the art.

[0120] As used herein, the terms “at least a portion” or “fragment” for nucleic acid or polypeptide mean a portion having the smallest size characteristic of such a sequence, or any larger fragment (at most and including the full-length molecule). Fragments of polynucleotides disclosed herein may encode the biologically active portion of a gene regulatory element. The biologically active portion of a gene regulatory element may be prepared by isolating a portion of one of the polynucleotides disclosed herein that contains the gene regulatory element and evaluating its activity as described herein. Similarly, a portion of a polypeptide may be 4, 5, 6, 7, etc., up to a full-length polypeptide. The length of the portion to be used will depend on the specific application. A portion of a nucleic acid that can be used as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. A portion of a polypeptide that can be used as an epitope may be as short as 4 amino acids. The portion of a polypeptide that functions as a full-length polypeptide will typically be longer than 4 amino acids.

[0121] As used herein, the term "primer" refers to an oligonucleotide that can anneal to an amplification target to allow DNA polymerase to attach, thereby acting as the starting point for DNA synthesis when under conditions that induce primer extension product synthesis (i.e., in the presence of nucleotides and reagents for polymerization (such as DNA polymerase) and at suitable temperature and pH). Primers are preferably single-stranded to obtain maximum amplification efficiency. Preferably, primers are oligodeoxyribonucleotides. Primers must be long enough to initiate the synthesis of extension products in the presence of reagents for polymerization. The precise length of the primer will depend on many factors, including temperature and primer composition (A / T versus G / C content). A bidirectional primer pair consists of a forward primer and a reverse primer, as commonly used in the field of DNA amplification (such as PCR amplification).

[0122] The term "stringency" or "stringent hybridization conditions" refers to hybridization conditions that affect the stability of the hybrid, such as temperature, salt concentration, pH, formamide concentration, etc. These conditions are empirically optimized to maximize specific binding of primers or probes to their target nucleic acid sequences and minimize nonspecific binding. The terminology used includes references to conditions under which probes or primers will hybridize with their target sequences to a much higher degree of detectability compared to other sequences (e.g., at least twice the background). Stringency conditions are sequence-dependent and vary under different conditions. Longer sequences exhibit specific hybridization at higher temperatures. Generally, stringency conditions are selected to be approximately 5°C lower than the thermal melting point (Tm) of a particular sequence at defined ionic strengths and pH. Tm is the temperature at which 50% of the complementary target sequence hybridizes with a perfectly matched probe or primer (at defined ionic strengths and pH). Typically, stringent conditions will be those involving a salt concentration of less than about 1.0 M Na+ ions at pH 7.0 to 8.3, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts), and a temperature of 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., more than 50 nucleotides). Stringent conditions can also be achieved by adding a destabilizing agent such as formamide. An exemplary low-stringent condition, or “reduced stringency condition,” involves hybridization at 37°C with a buffer of 30% formamide, 1 M NaCl, and 1% SDS, followed by washing at 40°C in 2×SSC. An exemplary high-stringency condition involves hybridization at 37°C in 50% formamide, 1 M NaCl, and 1% SDS, followed by washing at 60°C in 0.1×SSC. Hybridization procedures are well known in the art and have been described, for example, by Ausubel et al., 1998 and Sambrook et al., 2001. In some embodiments, stringent conditions are met by hybridization at 45°C in 0.25M Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA and 0.5%–20% sodium dodecyl sulfate (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 washing at 55°C to 65°C in 5×SSC containing 0.1% (w / v) sodium dodecyl sulfate.

[0123] As used herein, the terms “microbial body” or “microorganism” should be interpreted broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains—bacteria and archaea—and the eukaryotic domains fungi and protists.

[0124] Generally, "heterologous" refers to a state conferred by the non-natural combination of one composition (e.g., chemical substance, molecule, seed, plant, gene) with another composition of the same or different types. Such non-natural combinations can also be called "synthetic combinations." For example, a gene that is not naturally present at a specific location in the bacterial genome can be "heterologous" when placed in the genomes of different bacteria, or when placed at different locations in the genomes of the same bacteria. In another example, a microorganism can be "heterologous" for a particular plant if it combines in a manner not known to exist naturally (e.g., in different spaces, times, and / or concentrations). In yet another example, "heterologous" can refer to polynucleotides that are not naturally present in a particular microbial strain but may be present in other strains of the same genus and species. Typically, "heterologous" refers to a state conferred by means of not originating from the same entity or gene locus.

[0125] As used herein, the term “bacteria” generally refers to any prokaryotic organism and may include organisms from the kingdoms Eubacteria (bacteria), Archaea (archaea), or both. In some cases, bacterial genera or other taxonomic classifications have been redesignated for other reasons (such as, but not limited to, evolutionary fields of whole-genome sequencing), and it should be understood that such redesignations fall within the scope of any taxonomic claim. For example, certain species of the genus *Erwinia* have been described in the literature as belonging to the genus *Pantoea* (Zhang, Y., Qiu, S. Examining phylogenetic relations of *Erwinia* and *Pantoea* species using whole genome sequence data. Antonie van Leeuwenhoek 108, 1037-1046 (2015)).

[0126] The term "microbial community" refers to a group of microorganisms comprising two or more genera and / or species and / or strains. Unlike microbial aggregates, microbial communities do not necessarily perform a common function, nor do they necessarily participate in or cause or relate to identifiable parameters or plant phenotypic traits. The community may contain one or more species of microorganisms, or strains of a species. In some cases, these microorganisms coexist symbiotically within the community.

[0127] The term "microbial consortia" or "microbial consortium" refers to a subset of a microbial community of individual microorganisms that can be described as performing a common function or participating in or contributing to or associated with an identifiable parameter or plant phenotypic trait. The microbial consortia identified in this paper may each provide different aspects of the desired outcome (e.g., plant biotic stress control), and / or may cooperate additively (e.g., one microorganism provides control against one biotic stress factor while another provides control against a different biotic stress factor), and / or may cooperate synergistically (e.g., two or more microorganisms provide a level of biotic stress control to the plant that is higher than the sum of the effects of any single microorganism).

[0128] The terms “accelerated microbial selection” or “AMS” are used interchangeably with the terms “directed microbial selection” or “DMS”, and refer to an iterative selection methodology used in some embodiments of this disclosure to obtain a claimed microbial species or aggregates of said species.

[0129] As used herein, the terms “isolated strain,” “isolated,” “isolated microorganism,” and similar terms are intended to mean one or more microorganisms that have been isolated from at least one of the materials with which they are associated in a particular environment, such as soil, water, or plant tissue.

[0130] Therefore, the “isolated microorganism” does not exist in its natural environment; rather, it is removed from its natural environment and placed in a non-natural state of existence by means of the various techniques described herein. Thus, the isolated strain can exist, for example, as a biologically pure culture or spore (or other form of strain) combined with an agricultural carrier.

[0131] In certain aspects of this disclosure, the isolated microorganism exists as an isolated and biologically pure culture. Those skilled in the art will understand that an isolated and biologically pure culture of a particular microorganism means that the culture is substantially free of (to a scientifically reasonable extent) other living organisms and contains only the single microorganism in question. The culture may contain varying concentrations of the microorganism. This disclosure states that isolated and biologically pure microorganisms are generally “certainly different from less pure or impure material.” See, for example, In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins), also see In re Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microorganisms), also see Parke-Davis & Co. v. HK Mulford & Co., 189 F. 95 (SDNY 1911) (LearnedHand, discussing purified adrenaline), partially maintaining and partially overturning the original judgment, 196 F.496 (2d Cir. 1912), each of which is incorporated herein by reference. Furthermore, in some aspects, this disclosure provides certain quantitative measurements of concentration or purity limits that must be achieved in isolated and biologically pure microbial cultures. In some embodiments, the presence of these purity values ​​is another property distinguishing the microorganisms disclosed in this invention from those that exist in their natural state. See, for example, Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limits for vitamin B12 produced by microorganisms), which is incorporated herein by reference.

[0132] As used herein, “isolated isolate” should be considered as a composition or culture that, after being isolated from one or more other microorganisms, primarily contains a single genus, species, or strain of microorganisms. This phrase should not be considered as an indication of the degree of isolation or purification of the microorganisms. However, “isolated isolate” may essentially contain only one genus, species, or strain of microorganisms.

[0133] As used herein, the term "growth medium" is any medium suitable for supporting plant growth. By way of example, the medium can be natural or artificial, including but not limited to: soil, potting mix, bark, vermiculite, hydroponic solutions used alone and applied to solid plant support systems, and tissue culture gels. It should be understood that the medium can be used alone or in combination with one or more other media. It can also be used with or without the addition of exogenous nutrients and physical support systems for roots and leaves.

[0134] In one embodiment, the growth medium is a naturally occurring medium, such as soil, sand, mud, clay, humus, topsoil, rock, or water. In another embodiment, the growth medium is artificial. Such an artificial growth medium can be constructed to simulate the conditions of a naturally occurring medium; however, this is not required. The artificial growth medium can be made from one or more of any number and combination of materials, including sand, minerals, glass, rock, water, metals, salts, nutrients, and water. In one embodiment, the growth medium is sterile. In another embodiment, the growth medium is not sterile.

[0135] The medium can be modified or enriched with additional compounds or components, such as by adding components that can help specific groups of microorganisms interact with and / or select the plants and each other. For example, antibiotics (such as penicillin) or sterilizing agents (such as quaternary ammonium salts and oxidants) may be present, and / or physical conditions (such as salinity, plant nutrients (such as organic and inorganic minerals (such as phosphorus, nitrogen salts, ammonia, potassium and micronutrients, cobalt and magnesium), pH and / or temperature) may be modified.

[0136] The term "plant" generally includes the whole plant, plant organs, plant tissues, seeds, plant cells, and seeds and their progeny. Plant cells include, but are not limited to, cells derived from seeds, suspension cultures, embryos, meristematic zones, callus, leaves, roots, branches, gametophytes, sporophytes, pollen, and microspores. "Plant component" is intended to refer to the whole plant or plant part, which may include differentiated and / or undifferentiated tissues, such as, but not limited to, plant tissues, parts, and cell types. In one embodiment, a plant component is one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, epidermal tissue, seeds, leaves, roots, branches, stems, flowers, fruits, stolons, bulbs, tubers, corms, daughter plants, branches, buds, nodular tissues, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, callus). The term "plant organ" refers to plant tissues or groups of tissues that constitute morphologically and functionally distinct parts of a plant. As used herein, “plant part” is synonymous with “part” of a plant and refers to any part of a plant, and may include different tissues and / or organs, and may be used interchangeably with the term “tissue” throughout the text.

[0137] "Offspring" includes any subsequent generations of an organism produced through sexual or asexual reproduction.

[0138] As used herein, the term "plant component" refers to plant cells, plant protoplasts, plant cell tissue cultures, plant callus, plant clusters, and intact plant cells in a plant or plant part (such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, grain, spike, rachis, bark, stem, root, root tip, anther, etc.), and also includes these parts themselves. "Grain" is intended to refer to mature seeds produced by commercial growers for purposes other than cultivation or propagation. Progeny, variants, and mutants of regenerated plants are also included within the scope of this invention, provided that these parts contain introduced polynucleotides.

[0139] Similarly, "plant reproductive component" is intended to refer broadly to any part of a plant that can develop into another plant through the sexual or asexual reproduction of that plant, such as, but not limited to: seeds, seedlings, roots, branches, cuttings, scions, grafts, stolons, bulbs, tubers, corms, offshoots, or buds. Plant components may be located in the plant or in plant organs, tissue cultures, or cell cultures.

[0140] The term "monocotyledonous" or "monocotyledonous plant" refers to the subclass Angiosperms, also known as the "monocotyledonous plant class," whose seeds typically contain only one embryonic leaf or cotyledon. The term includes references to the whole plant, plant components, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their offspring.

[0141] The term "dicotyledonous" or "dicotyledonous plant" refers to the subclass Angiosperms, also known as the "dicotyledonous plant class," whose seeds typically contain two embryonic leaves or cotyledons. The term includes references to the whole plant, plant components, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their offspring.

[0142] As used in this article, the term "cultivar" refers to a plant variety, strain, or lineage that has been produced through horticultural or agronomic techniques and is not typically found in wild populations.

[0143] As used herein, the terms “molecular marker,” “marker,” or “genetic marker” refer to an indicator used in methods for observing differences in the 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, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), enzyme digestion amplified polymorphic sequences (CAPS) markers, or isoenzyme markers, or combinations of markers described herein (which define specific gene and chromosomal locations). The localization of molecular markers near alleles is a procedure that can be performed by a person with general experience in molecular biotechnology.

[0144] As used herein, the term "trait" refers to a characteristic or phenotype. For example, in the context of some embodiments of this disclosure, crop yield refers to the amount of marketable biomass (e.g., fruit, fiber, cereal) produced by the plant. Desired traits may also include other plant characteristics, including but not limited to: water use efficiency, nutrient use efficiency, yield, mechanical harvestability, fruit maturity, shelf life, disease and pest resistance, early maturity, stress tolerance, etc. Traits can be inherited in a dominant or recessive manner, or in a partially or incompletely dominant manner. Traits can be monogenic (i.e., determined by a single locus) or polygenic (i.e., determined by more than one locus), or may also arise from the interaction of one or more genes with the environment. Plant traits can be altered by the presence or absence of another organism. In one example, plant health may be reduced by the presence of pathogens (e.g., nematodes, fungi, bacteria, or insects). In another example, plant health can be improved by the presence of microorganisms, such as microorganisms toxic to nematodes, which may be present on or near the plant, thus providing the plant with a tolerance mechanism to the nematodes.

[0145] As used herein, the term “phenotype” refers to the observable characteristics of an individual cell, cell culture, organism (e.g., plant), or population of organisms, which arise from the interaction between an individual’s genetic makeup (i.e., genotype) and its environment.

[0146] As used herein, “improved” should be interpreted broadly to encompass improvements in plant characteristics compared to control plants or to known averages associated with the characteristics in question. For example, “improved” plant biomass associated with the application of the beneficial microorganisms or aggregates of this disclosure can be demonstrated by comparing biomass from plants treated with the microorganisms taught herein with biomass from untreated control plants. Alternatively, biomass from plants treated with the microorganisms taught herein can be compared to average biomass typically achieved by a given plant (as expressed in scientific or agricultural publications known to those skilled in the art). In this disclosure, “improved” does not necessarily require data to be statistically significant (e.g., p < 0.05); rather, any quantifiable difference indicating that one value (e.g., average treatment value) differs from another value (e.g., average control value) can be considered “improved.”

[0147] As used herein, “suppression and inhibition” and similar terms should not be interpreted as requiring complete suppression or inhibition, but may be necessary in some implementations.

[0148] As used herein, the term “genotype” refers to the genetic composition of an individual cell, cell culture, tissue, organism (e.g., plant) or population of organisms.

[0149] Compared to syngenetic plants that do not contain modifications derived from the methods or compositions described herein, the compositions and methods described herein can provide plants with improved “agronomical traits” or “traits of agronomic importance” or “traits of agronomic significance,” which may include, but are not limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen use, enhanced nitrogen fixation, resistance to pests, resistance to insects, resistance to nematodes, resistance to fungi, resistance to bacteria, resistance to herbivores, resistance to pathogens, increased yield, enhanced health, increased vitality, improved growth, enhanced photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased branch length, increased root length, improved root structure, metabolite regulation, proteome regulation, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, and altered seed nutrient composition. In some respects, traits of agronomic importance are achieved through the combination (physical proximity) between plants and microorganisms that provide benefits to the plants.

[0150] "Agronomic trait potential" is intended to refer to the ability of a plant component to exhibit a phenotype (preferably an improved agronomic trait) at some point in its life cycle, or to transfer the phenotype to another plant component in the same plant that is associated with it.

[0151] In some implementations, the cell or organism has at least one heterologous trait. As used herein, the term "heterologous trait" refers to a phenotype of a cell or organism conferred by a foreign molecule or other organism (e.g., a microorganism), a fragment of DNA, a heterologous polynucleotide, or a heterologous nucleic acid.

[0152] Phenotypic changes are of interest in this disclosure, including but not limited to regulating plant fatty acid composition, altering plant amino acid content, modifying plant pathogen defense mechanisms, and increasing plant yields in economically important traits (e.g., cereal yield, forage yield, etc.). These results can be achieved by using the methods and compositions of this disclosure to provide expression of heterologous products or increase the expression of endogenous products in plants. "Synthetic assemblies" may include combinations of plants and microorganisms disclosed herein, or combinations of plants and compositions. Such assemblies may be achieved, for example, by coating the seed surface of a plant (such as an agricultural plant) or host plant tissue (roots, stems, leaves, etc.) with the microorganisms disclosed herein. Furthermore, "synthetic assemblies" may include combinations of microorganisms of various strains or species. A synthetic assembly has at least one variable that distinguishes it from any assembly existing in nature. This variable may in particular be the concentration of microorganisms on seeds or plant tissues that do not exist naturally, or a combination of microorganisms and plants that do not exist naturally, or a combination of microorganisms or strains that do not coexist naturally. In each of these cases, the synthetic assembly exhibits human intervention and possesses structural and / or functional properties that do not exist when the individual elements of the assembly are considered individually.

[0153] In some embodiments, the microorganism may be "endogenous" to the seed or plant. As used herein, a microorganism is considered "endogenous" to the plant or seed if it originates from a plant sample from which it is derived. That is, if the microorganism is found to be associated with the plant in nature. In embodiments where endogenous microorganisms are applied to plants, the endogenous microorganisms are applied in amounts different from those found on plants in nature. Thus, if an endogenous microorganism for a given plant is present on the plant at a level not found in nature, the microorganism can still form a synthetic combination with the plant.

[0154] In some embodiments, a composition (such as a microorganism) may be "heterogeneous" (also called "exogenous") to another composition (such as a seed or plant), and in some respects, is referred to herein as a "heterogeneous composition." As used herein, a microorganism is considered "heterogeneous" to a plant or seed if it is not derived from a plant sample from which it is derived. That is, a situation where the microorganism is found not to be associated with said plant in nature. For example, a microorganism typically associated with leaf tissue of a corn plant is considered exogenous to leaf tissue of another corn plant in which said microorganism is not found in nature. In another example, a microorganism typically associated with a corn plant is considered exogenous to a wheat plant in which said microorganism is not found in nature.

[0155] When a composition is applied mechanically or manually, artificially inoculated, associated with a plant component, seedling, or plant, or placed on or inside a plant component, seedling, or plant, or on or inside a plant growth medium, or on or inside a treatment formulation, such that the treatment is present on or inside the plant component, seedling, plant, plant growth medium, or formulation in a manner not found in nature prior to the application of the treatment, the composition is "heterogeneously treated." For example, the composition is not present in the plant variety, at that stage of plant development, in the plant tissue, at that abundance level, or in that growth environment (e.g., drought conditions) under natural conditions. In some embodiments, this is envisioned as being selected from the group consisting of: the presence of microorganisms; the presence of microorganisms at different cell numbers, concentrations, or amounts; the presence of microorganisms in different plant components, tissues, cell types, or other physical locations within or on the plant; and the presence of microorganisms at different time periods (e.g., developmental stages of the plant or plant component, time of day, time of season, and combinations thereof). In some embodiments, "heterogeneous treatment" means applying a microorganism to a tissue or cell type of a plant component that is not naturally present in the same location as the microorganism. In some embodiments, "heterogeneous treatment" means applying a microorganism to a developmental stage of a plant component, seedling, or plant that is not associated with the microorganism in nature at that stage but may be associated at other stages. For example, if a microorganism is typically found during the flowering stage of a plant but not at other stages, then application of the microorganism to a seedling stage may be considered a heterogeneous treatment. In some embodiments, if a microorganism is typically found in the root tissue of a plant component but not in the leaf tissue, and the microorganism is applied to a leaf, then the microorganism is considered a heterogeneous treatment. In another non-limiting example, if a microorganism is naturally present in the mesophyll layer of a leaf tissue but is applied to the epidermis, then the microorganism will be considered a heterogeneous treatment. In some embodiments, "heterogeneous treatment" means that the natural plant component, seedling, or plant does not contain detectable levels of the microorganism in that same plant component, seedling, or plant. In some implementations, "heterogeneous treatment" means that microorganisms are applied to the plant component, seedling, or plant at a higher concentration, greater quantity, or greater quantity than they would be present in nature. For example, the microorganisms are heterogeneously treated when they are present at a quantity, amount, or concentration that is at least 1.5 times, between 1.5 times and 2 times, 2 times, between 2 times and 3 times, 3 times, between 3 times and 5 times, 5 times, between 5 times and 7 times, 7 times, between 7 times and 10 times, 10 times, or even more than 10 times the concentration they were present before placement.In another non-limiting example, microorganisms naturally present in the tissues of cypress trees would be considered heterologous to the tissues of maize, wheat, cotton, and soybean plants. In yet another example, microorganisms naturally present in the leaf tissues of maize, spring wheat, cotton, and soybean plants would be considered heterologous to the leaf tissues of another maize, spring wheat, cotton, or soybean plant that does not contain said microorganisms or contains varying amounts of said microorganisms in nature.

[0156] Microorganisms can also be "heterogeneously treated" on a given plant tissue. This means that the microorganism is placed on plant tissue that it has not been found in nature. For example, if a given microorganism is naturally present only on the roots of a given plant, then the microorganism can be exogenously applied to the aboveground tissue of the plant, and thus "heterogeneously treated" on said plant tissue. Therefore, when applied to a plant on which a microorganism does not naturally exist or does not naturally possess a microorganism present in the amount applied, the microorganism is considered a heterogeneous treatment.

[0157] Compared to syngenea grown from seeds without the aforementioned seed treatment agents, the compositions and methods described herein can provide host plants with "modified" "agronomical traits" or "traits of agronomic importance," which may include, but are not 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, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, enhanced nitrogen fixation, improved nitrogen use, improved root structure, increased water use efficiency, increased biomass, increased root length, increased seed weight, and increased branching. Increased length, improved yield, increased yield under water-limited conditions, grain quality, grain moisture content, metal tolerance, number of ears, number of grains per ear, number of pods, enhanced nutrition, pathogen resistance, resistance to harmful organisms, enhanced photosynthetic capacity, salt tolerance, greenness retention, improved vigor, 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 pod length per plant, decreased number of wilted leaves per plant, decreased number of severely wilted leaves per plant and increased number of non-wilted leaves per plant, detectable regulation of metabolite levels, detectable regulation of transcript levels, and detectable regulation of the proteome. The term "regulatory" is intended to refer to changes in characteristics (such as agronomic traits) altered by means of the presence of microorganisms, secretions, culture media, metabolites, etc. In some respects, this regulation provides the conferment of traits (such as traits of agronomic importance).

[0158] Bacillus thuringiensis Aspects of this disclosure include the insertion and regulation of certain polynucleotides that result in changes in the phenotype of the host cell and / or an organism associated with or potentially associated with that cell.

[0159] The cells of the bacterium Bacillus thuringiensis typically contain genomic DNA in the form of a major chromosome, as well as several plasmids, each containing various genes that contribute to the function and use of the bacteria.

[0160] Cry toxins, encoded by the cry gene, are a class of crystalline proteins of the delta-endotoxin type produced by strains of the bacterium Bacillus thuringiensis. Their mode of action is believed to be the production of pores that disrupt the intestinal epithelial membrane of pests such as insects and nematodes. Genes encoding a range of cry toxins, including cry mutants, cry chimeras, and other cry derivatives, are commercially used to enhance pest resistance, for example, in genetically modified (GM) crops. Cry toxins tend to be insect-specific; for example, a particular cry toxin may target one insect species rather than another.

[0161] Another type of toxin found in strains of the bacterium Bacillus thuringiensis is β-endotoxin, also known as thuringin. Threonine is a small oligosaccharide composed of adenosine, glucose, phosphate, and gluconic acid in a molecular ratio of 1:1:1:1. Also known as β-endotoxin, thuringin is a non-specific toxin that can affect various organisms and is prohibited from public use according to the recommendations of the World Health Organization (WHO, Guidelines for Specifications of Bacterial Larvicides for Public Health Use, WHO; Geneva, Switzerland: 1999, Publication No. WHO / CDS / CPC / WHOPES / 99.2, WHO Informal Consultation Report).

[0162] Plant traits conferred by microorganisms This disclosure utilizes genetically engineered microorganisms to confer beneficial properties (or traits) on desired plant species, such as target agronomic species. In this disclosure, the terms "beneficial property" or "beneficial trait" are used interchangeably and refer to the regulation of a desired plant phenotype or genetic property by applying microorganisms or microbial aggregates as described herein. As mentioned above, in some respects, it may be highly desirable that metabolites produced by a given microorganism ultimately play a role in regulating or conferring beneficial traits on a given plant.

[0163] There are many beneficial traits that can be modulated by applying the microorganisms disclosed herein. For example, microorganisms may have the ability to confer one or more beneficial properties on plant species, such as increased growth, increased yield, improved nitrogen use efficiency, enhanced stress tolerance, enhanced drought tolerance, increased photosynthetic rate, improved water use efficiency, enhanced pathogen resistance, and modification of plant structure (which may not necessarily affect plant yield, but rather optimize plant functionality and promote increased production of target metabolites).

[0164] In all respects, the microorganisms taught in this paper offer a wide range of agricultural applications, including: increasing the yield of grains, fruits and flowers; increasing the growth of plant parts; enhancing the ability to utilize nutrients (e.g., nitrogen, phosphate, etc.); enhancing disease resistance; biopesticide effects (including enhanced resistance to fungi, insects and / or nematodes); improving survival rates in extreme climates; and improving other desired plant phenotypic traits.

[0165] In some respects, the isolated microorganisms, aggregates, and / or agricultural compositions of this disclosure can be applied to plants to modulate or alter plant characteristics, such as, relative to a reference plant, changes in oil content, protein content, seed carbohydrate composition, seed oil composition, seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, enhanced nitrogen fixation, improved nitrogen use, improved nutrient (e.g., phosphate, potassium, etc.) use, improved root structure, increased water use efficiency, increased biomass, increased root length, increased seed weight, increased branch length, increased yield, and increased yield under water-limited conditions. High, grain quality, grain moisture content, metal tolerance, number of spikes, number of grains per spike, number of pods, enhanced nutrition, pathogen resistance, reduced pathogen levels (e.g., through the secretion of metabolites that weaken pathogen survival), resistance to pests, enhanced photosynthetic capacity, salt tolerance, greenness retention, increased vigor, 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 pod length 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, detectable regulation of metabolite levels, detectable regulation of transcript levels, and detectable regulation of the proteome.

[0166] In some aspects, the isolated microorganisms, aggregates, and / or agricultural compositions of this disclosure can be applied to plants to negatively regulate specific plant traits. For example, in some aspects, the microorganisms of this disclosure can reduce target phenotypic traits, as such functionality may be desired in some applications. For instance, the microorganisms of this disclosure may possess the ability to inhibit root growth or shorten root length. Or the microorganisms may possess the ability to inhibit branch growth or reduce plant growth rate, as such regulation of plant traits may be desired in some applications.

[0167] In some embodiments, the isolated microorganisms, aggregates and / or agricultural compositions of this disclosure may be applied to plants to confer tolerance to nematode stress.

[0168] In some embodiments, the isolated microorganisms, aggregates, and / or agricultural compositions of this disclosure may be applied to plants to provide biostimulation (biostimulant effect). In some embodiments, the isolated microorganisms, aggregates, and / or agricultural compositions of this disclosure may be applied to plants to provide disease resistance.

[0169] In some embodiments, the isolated microorganisms, aggregates and / or agricultural compositions of this disclosure may be applied to plants to directly affect the life cycle, number or population of pests in or around the plant.

[0170] Nematode control Plant-parasitic nematodes are widely recognized as one of the greatest threats to crops worldwide. These are microscopic, round worms that live in soil and plant tissues, feeding inside or on roots. Several species may coexist in the same field. Some nematode species can infect multiple crops and other plants, while others feed only on a few closely related plants, impairing their ability to absorb water and nutrients. Globally, nematodes cause billions of dollars in agricultural losses.

[0171] The engineered (genetically modified) bacteria described herein can be used to control nematode pests by conferring resistance or tolerance to another organism (e.g., a plant), pests, and / or by directly inhibiting or delaying nematode development, or by directly controlling nematode populations, or both.

[0172] Examples of nematodes to which the compositions and methods disclosed herein may be used include the following major species: *H. glycines*, such as *H. avenae*, *H. schachtii*, *H. trifolii*, *H. gottingiana*, *H. cajani*, and *H. zeae*; *Globodera*, such as *G. rostochiensis*, *G. pallida*, and *G. tabacum*; and *M. arenaria*, such as *M. incognita*, *M. javanica*, and *M. javanica*. *M. chitwoodi*, a nematode of the root-knot nematode *Hhapla*; *D. destructor*, *D. dipsaci*, and *D. angustus*; *Anguina* (grain gall nematodes), such as *A. tritici*, *A. agrostis*, and *Afrina / Anguinawevelli*; and *P. penetrans*, *P. brachyurus*, *P. coffeeeae*, *P. zeae*, *P. goodeyi*, *P. thornei*, and *P. wrathi*. *Vulnus*; *Perforator Nematodes*, such as *R. similis*, which has two host species; *Hirschmanniella* (root-seeking nematodes), such as *H. oryzae*, *H. mucronata*, and *H. spinicauda*; *Hoplolaimus* (spear-shaped nematodes), such as *H. columbus*, *H. seinhorsti*, and *H. indicus*; *Rotylenchulus* (kidney-shaped nematodes), such as *R. reniformis*; *Tylenchulus* (citrus nematodes), such as *T. citrus hemipiercing nematode*.semipenetrans); *Helicotylenchus* (spiral nematodes), such as *H. multicinctus*, *H. mucronatus*, *H. dihystera*, and *H. pseudorobustus*; *Criconemella* (ring nematodes), such as *C. xenoplax*, *C. axestis*, and *C. spharocephalum*; *Paratrichodorus* (dragon nematodes, needle nematodes, and short-rooted nematodes), such as *X. americanum*, *X. elongatum*, and *L. arachnoides*. *Africanus*, *P. minor*, and *A. fragariae* (bud and leaf nematodes and pine wood nematodes), such as *A. fragariae*, *A. besseyi* f, *A. ritzemabosi*, and *Bursaphelenchus xylophilus*.

[0173] Specifically, crop-specific nematode pests include, but are not limited to, the following: citrus nematode (citrus semi-piercing nematode); perforating nematode (similar perforating nematode); red ring nematode (coconut red ring nematode (Rhadinaphelenchuscocophilus)); palm weevil (Rhynchophorus palmarum); root rot nematode; maize cyst nematode (Heterodera zeae); root-knot nematode; kidney-shaped nematode (kidney-shaped kidney nematode); root rot nematode (short-tailed short-bodied nematode); stinging nematode (Belonolaimus longicaudatus); root-knot nematode, cyst nematode, and kidney-shaped nematode; pea cyst nematode (Heterodera goettingiana); pigeon cyst nematode on pigeon pea (Cajanus cajan); peanut root-knot nematode (M. areenaria); Javan root-knot nematode (M. javanica); northern root-knot nematode (M. hapla); root rot nematode (short-tailed short-bodied nematode); potato cyst nematode, potato golden nematode ("golden" nematode); potato cyst nematode, potato white nematode; root-knot nematode (Meloidogyne), pseudo-root-knot nematode (Nacobbus), bulb stem nematode (Ditylenchus dipsaci), potato rot nematode (rot stem nematode) and root rot nematode (short-bodied nematode); Colombian root-knot nematode (Chisley root-knot nematode); white-tipped nematode (Bessie's smooth blade nematode); rice stem nematode (Ditylenchus angustus); root-knot nematodes, genus and species of root-knot nematodes and rice cyst nematode (Heterodera oryzae); root-knot nematode (grass root-knot nematode (M. graminicola)); cyst nematode species infecting rice roots (rice cyst nematode (H. oryzae), rice cyst nematode (H. *H. elachista* and *H. sacchari* cyst nematodes; soybean cyst nematodes; beet cyst nematodes; tobacco cyst nematodes; tobacco root-knot nematodes; tobacco root-rot nematodes; tobacco stem nematodes; tobacco bulb nematodes; tobacco dwarf nematodes; tobacco spiral nematodes; tobacco kidney-shaped nematodes; vegetable root-knot nematodes; vegetable kidney-shaped nematodes, short and thick root nematodes, beet cyst nematodes, pseudo-root-knot nematodes, stinging nematodes, and dwarf nematodes; vegetable stem nematodes; vegetable kidney-shaped nematodes; potato cyst nematodes; cereal cyst nematodes; oat cyst nematodes; grain gall or spike gall nematodes; wheat grain nematodes, root-knot nematodes, dwarf nematodes, and rot nematodes; wheat gall nematodes; potato cyst nematodes (potato white nematodes), mainly infecting tuberous Solanum species; *G. sacchari* cyst nematodes.*Heteroderaoryzae*, an infecting plant of the fuchsia family (Fuchsia excorticata) and other plants in the Potato family; *Heteroderaoryzae*, a rice cyst nematode; *H. cajani*, a pigeon cyst nematode on the roots of *Cajanus cajani*; *H. mothi*, a sedge cyst nematode on the roots of *Cyprus rotundus*; *H. medicaginis*, an alfalfa cyst nematode on the roots of *Medicago sativa* in the former Soviet Union; *H. mediterranea*, a Mediterranean cyst nematode on the roots of the woody plant *Pistacia lentiscus*; *H. pakistanensis*, a Pakistani cyst nematode on the roots of wheat (*Triticum aestivum*); *H. graminis*, a grass cyst nematode from the grass (*Cynodon dactylon*); and *H. sorghum vulgare*, a sorghum cyst nematode on the roots of sorghum (*Sorghum vulgare*). sorghi); H. raskii from the roots of Cyperus bulbosus; H. spinicauda from the rhizosphere soil of Phragnutis australis; Meloidogyne coffeicola from the roots of Coffea arabica; M. salasi from the roots of Oryza sativa; M. suginamiensis from the roots of Morus alba; M. sewelli from the roots of Eleocharis acicularis; M. microcephala from the roots of Nicotiana tabacum; M. arabicida from the roots of Coffea arabica; and M. Lin's root-knot nematode from the roots of rice. lini); Kongi root-knot nematode from citrus roots; Jianyang root-knot nematode from mandarin orange roots; vandervegtei root-knot nematode from woody plants; Spanish root-knot nematode from peach (wild peach (Prunus persica silvestris)) rootstock roots; short-tailed root-knot nematode from tea (Camellia sinensis) roots.brevicauda; rice stem nematode (Ditylenchus angustus) in rice-growing areas; grain gall nematode (Vibrio villieris) from the galls of Eragrostis curvulva grains; African abrasive nematode (Afenestrata africana), a parasite of millet (Panicum maximum); and Andean sheath nematode (Thecavermiculatus andinus) from the roots of Oxalis tuberosa. Agricultural Composition In some embodiments, the microorganisms of this disclosure are combined with agricultural compositions. Agricultural compositions generally refer to organic and inorganic compounds that may include: compositions that promote the cultivation of microorganisms and / or plant components; compositions involved in the formulation of microorganisms to be applied to plant components (e.g., but not limited to: wetting agents, compatibilizers (also called "compatibilizers"), defoamers, detergents, chelating agents, drift reducers, neutralizers and buffers, corrosion inhibitors, dyes, flavorings, spreading agents (also called "dispersants"), penetration aids (also called "penetrating agents"), adhesives (also called "binders" or "binding agents"), dispersants, thickeners (also called "thickeners"), stabilizers, emulsifiers, freezing point inhibitors, antimicrobial agents, etc.); compositions involved in imparting protective effects to plant components (e.g., but not limited to: pesticides, nematicides, fungicides, bactericides, herbicides, etc.); and other compositions that may be of interest for a particular application.

[0174] In some embodiments, the agricultural compositions of this disclosure are solid. When using solid compositions, it may be desirable to include one or more carrier materials with isolated active microorganisms or aggregates. In some embodiments, this disclosure teaches the use of carriers, including but not limited to: mineral soils such as silica, silica gel, silicates, talc, kaolin, activated clay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, and magnesium oxide; pulverized synthetic materials; fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea, and urea; plant-derived products such as grain flour, bark powder, wood flour, and nutmeg powder; cellulose powder; palygorskite, montmorillonite, mica, vermiculite, synthetic silica, and synthetic calcium silicate, or combinations thereof.

[0175] Growth composition In some embodiments, compositions that promote growth and development are provided to microorganisms and / or plant components. Exemplary compositions include liquids (such as broths, culture media) and / or solids (such as soil, nutrients). Various organic or inorganic compounds, alone or in combination with plant components, may be added to the growth composition to promote microbial health; these compounds include, but are not limited to, amino acids, vitamins, minerals, carbohydrates, monosaccharides, and lipids.

[0176] Pharmaceutical composition One or more compositions may be combined, except for microorganisms or compositions produced by microorganisms, for various applications, stability, activity and / or storage reasons. Additional compositions may be referred to as "formulation components".

[0177] In some embodiments, the agricultural compositions disclosed herein are liquids. Therefore, in some embodiments, this disclosure teaches that the agricultural compositions disclosed herein may comprise compounds or salts such as monoethanolamine salts, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium bisulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium bicarbonate, ammonium thiosulfate, ammonium diphosphate, ammonium monophosphate, sodium ammonium hydrogen phosphate, ammonium thiocyanate, ammonium aminosulfonate, or ammonium carbamate.

[0178] In some embodiments, this disclosure teaches that agricultural compositions may comprise: binders such as polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethyl cellulose, starch, ethylene pyrrolidone / vinyl acetate copolymer and polyvinyl acetate, or combinations thereof; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or combinations thereof; defoamers such as silicone emulsions, long-chain alcohols, phosphate esters, ethynyl glycol, fatty acids or organofluorine compounds; and complexing agents such as salts of ethylenediaminetetraacetic acid (EDTA), salts of triazinotriacetic acid or polyphosphate, or combinations thereof.

[0179] In some embodiments, the agricultural composition comprises a surfactant. In some embodiments, the surfactant is added to the liquid agricultural composition. In other embodiments, the surfactant is added to the solid formulation, particularly those designed to be diluted with a carrier prior to application. Thus, in some embodiments, the agricultural composition comprises a surfactant. Surfactants are sometimes used alone or in combination with other additives, such as minerals or vegetable oils, as adjuvants in spray tank mixing to improve the biocompatibility of microorganisms with their targets. The type of surfactant used for bioenhancement typically depends on the nature and mode of action of the microorganisms. Surfactants can be anionic, cationic, or nonionic and can be used as emulsifiers, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactant is a nonionic surfactant, such as alkyl ethoxylates, linear fatty alcohol ethoxylates, and fatty amine ethoxylates. Surfactants commonly used in the field of pharmaceutical formulations and also applicable to the formulations of this invention are described in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, NJ, 1998, and Encyclopedia of Surfactants, Volumes I-III, Chemical Publishing Co., New York, 1980-81. In some embodiments, this disclosure teaches the use of surfactants comprising alkali metal, alkaline earth metal, or ammonium salts of fatty acids, including aromatic sulfonic acids (e.g., lignin sulfonic acid, phenol sulfonic acid, naphthalene sulfonic acid, and dibutylnaphthalene sulfonic acid), alkyl ethers, dodecyl ethers, fatty alcohol sulfates, and fatty alcohol glycol ether sulfates; condensates of sulfonated naphthalene and its derivatives with formaldehyde; condensates of naphthalene or naphthalene sulfonic acid with phenol and formaldehyde; condensates of phenol or phenol sulfonic acid with formaldehyde; condensates of phenol with formaldehyde and sodium sulfite; polyoxyethylene octylphenyl ether; ethoxylated isooctylphenol, octylphenol, or nonylphenol; tributylphenyl polyethylene glycol ether; alkyl aryl polyether alcohol; isotretinoin; ethoxylated castor oil; ethoxylated triarylphenol; salts of phosphorylated triarylphenol ethoxylates; dodecyl alcohol polyethylene glycol ether acetate; sorbitan ester; lignin-sulfite waste liquid or methylcellulose; or combinations thereof.

[0180] In some embodiments, this disclosure teaches other suitable surfactants, including: alkyl sulfates, such as diethanolammonium dodecyl sulfate; alkyl aryl sulfonates, such as calcium dodecylbenzenesulfonate; alkylphenol-epoxide addition products, such as nonylphenol-C18 ethoxylate; ethanol-epoxide addition products, such as tridecanol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonates, such as sodium dibutylnaphthalenesulfonate; and dialkyl esters of sulfosuccinates, such as di(2-ethylhexyl)sulfonate. Sodium succinate; sorbitan esters, such as sorbitan oleate; quaternary ammonium, such as dodecyltrimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of monoalkyl and dialkyl phosphates; vegetable oils, such as soybean oil, rapeseed / canola oil, olive oil, castor oil, sunflower oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil, etc.; and esters of the above vegetable oils, especially methyl esters.

[0181] In some embodiments, the agricultural compositions include a wetting agent. A wetting agent is a substance that, when added to a liquid, improves the spreading or penetrating ability of the liquid by reducing the interfacial tension between the liquid and the surface over which it spreads. Wetting agents serve two main functions in agricultural chemical formulations: improving the wetting rate of powders in water during processing and manufacturing to prepare soluble liquids or suspension concentrates; and shortening the wetting time of wettable powders and improving the penetration rate of water into water-dispersible granules during mixing of the product with water in spray cans or other containers. Examples of wetting agents used in the agricultural compositions (including wettable powders, suspension concentrates, and water-dispersible granule formulations) of this disclosure in some embodiments are: sodium dodecyl sulfate; sodium dioctyl sulfosuccinate; alkylphenol ethoxylates; and fatty alcohol ethoxylates.

[0182] In some embodiments, the agricultural compositions of this disclosure include a dispersant. A dispersant is a substance that adsorbs onto the surface of particles and helps maintain the dispersed state of the particles and prevents them from re-aggregating. In some embodiments, a dispersant is added to the agricultural compositions of this disclosure to promote dispersion and suspension during manufacturing and to ensure that the particles are redispersed in water in a spray tank. In some embodiments, the dispersant is used in wettable powders, suspension concentrates, and water-dispersible granules. Surfactants used as dispersants have the ability to strongly adsorb onto the surface of particles and provide a charged barrier or steric barrier against particle re-aggregation. In some embodiments, the most commonly used surfactants are anionic, nonionic, or a mixture of both.

[0183] In some embodiments, sodium lignosulfonate is the most commonly used dispersant for wettable powder formulations. In some embodiments, polyelectrolytes (such as sodium naphthalenesulfonate formaldehyde condensates) are used as suspending concentrates to provide excellent adsorption and stabilization. In some embodiments, tristyrylphenol ethoxylate phosphate esters are also used. In some embodiments, alkylaryl ethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionic surfactants as dispersants for suspending concentrates.

[0184] In some embodiments, the agricultural compositions of this disclosure comprise polymeric surfactants. In some embodiments, the polymeric surfactants have extremely long hydrophobic "backbone" and numerous ethylene oxide chains that form the "teeth" of a "comb"-like surfactant. In some embodiments, these high molecular weight polymers enable the suspension concentrate to have excellent long-term stability because the hydrophobic backbone has numerous points anchored to the particle surface. Examples of dispersants used in the agricultural compositions of this disclosure in some embodiments are: sodium lignin sulfonate; sodium naphthalene sulfonate formaldehyde condensate; tristyrylphenol ethoxylate phosphate; fatty alcohol ethoxylates; alkyl ethoxylates; EO-PO block copolymers; and graft copolymers.

[0185] In some embodiments, the agricultural compositions of this disclosure include an emulsifier. An emulsifier is a substance that stabilizes a suspension of droplets in one liquid phase in another liquid phase. Without an emulsifier, the two liquids would separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends comprise alkylphenols or fatty alcohols having 12 or more ethylene oxide units and oil-soluble calcium salts of dodecylbenzenesulfonic acid. A hydrophilic-lipophilic balance (“HLB”) value in the range of 8 to 18 generally provides a well-stabilized emulsion. In some embodiments, emulsion stability can sometimes be improved by adding a small amount of EO-PO block copolymer surfactant.

[0186] In some embodiments, the agricultural compositions of this disclosure include a solubilizer. The solubilizer is a surfactant that forms micelles in water at concentrations exceeding a critical micelle concentration. The micelles then enable the dissolution or solubilization of water-insoluble materials within the hydrophobic portion of the micelles. Commonly used surfactants for dissolution are nonionic surfactants: sorbitan monooleate; sorbitan monooleate ethoxylate; and methyl oleate.

[0187] In some embodiments, the agricultural compositions of this disclosure comprise organic solvents. Organic solvents are primarily used in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent in granular formulations. Sometimes mixtures of solvents are used. In some embodiments, this disclosure teaches the use of solvents including aliphatic paraffin oils, such as kerosene or refined paraffin. In other embodiments, this disclosure teaches the use of aromatic solvents, such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons may be used as co-solvents to prevent pesticide crystallization when the formulation is emulsified in water. Sometimes alcohols are used as co-solvents to improve solubility.

[0188] In some embodiments, the agricultural composition comprises a gelling agent. Thickeners or gelling agents are primarily used in the formulation of suspension concentrates, emulsions, and suspensions to modify the rheological or flow properties of the liquid and prevent the separation and sedimentation of dispersed particles or droplets. Thickeners, gelling agents, and anti-settling agents generally fall into two categories: water-insoluble microparticles and water-soluble polymers. It is possible to use clay and silica to produce suspension concentrate formulations. In some embodiments, the agricultural composition comprises one or more thickeners, including but not limited to: montmorillonite, such as bentonite; magnesium aluminum silicate; and palygorskite. In some embodiments, this disclosure teaches the use of polysaccharides as thickeners. The most commonly used types of polysaccharides are natural extracts of seeds and seaweed or synthetic derivatives of cellulose. Some embodiments utilize xanthan gum, and some embodiments utilize cellulose. In some embodiments, this disclosure teaches the use of thickeners, including but not limited to: guar gum; locust bean gum; carrageenan; alginate; methylcellulose; sodium carboxymethyl cellulose (SCMC); and hydroxyethyl cellulose (HEC). In some embodiments, this disclosure teaches the use of other types of antisettling agents, such as modified starch, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good antisettling agent is xanthan gum.

[0189] In some implementations, a surfactant (which reduces interfacial tension) is present during production and application via spray canisters, causing foaming of the water-based formulation during mixing. Therefore, in some implementations, to reduce the tendency to foam, a defoamer is typically added during the preparation stage or before filling into the bottle / spray canister. Generally, there are two types of defoamers: silicone and non-silicone. Silicone defoamers are typically aqueous emulsions of dimethylpolysiloxane; while non-silicone defoamers are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the defoamer is to displace the surfactant from the air-water interface.

[0190] In some embodiments, the agricultural composition contains a preservative.

[0191] In some embodiments, the agricultural composition may be formulated as: soil irrigation agent, foliar spray, impregnation treatment agent, furrow treatment agent, soil conditioner, granule, broadcast treatment agent, post-harvest disease control treatment agent, or seed treatment agent. In some embodiments, the agricultural composition may be applied alone or in rotation with other agricultural products according to a spraying schedule.

[0192] In some embodiments, the agricultural composition is compatible with barrel mixing. In some embodiments, the agricultural composition is compatible with barrel mixing with other agricultural products. In some embodiments, the agricultural composition is compatible with equipment used for ground, air, and irrigation application.

[0193] In some embodiments, the agricultural composition may be applied to genetically modified seeds or plants.

[0194] Protective composition Furthermore, individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with known active agents available in the agricultural field, such as pesticides, herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, plant growth regulators, rodenticides, anti-algae agents, biocontrol agents, or beneficial agents. Additionally, microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with known fertilizers. Such combinations can exhibit synergistic properties. Furthermore, individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with inert components. Additionally, in some aspects, the disclosed microorganisms are combined with bioactive agents.

[0195] In some embodiments, individual microorganisms or microbial aggregates or communities developed according to the disclosed methods may be combined with biopesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabies mites, rodenticides, and / or antialgae agents. Such biopesticides may be, but are not limited to, macroorganisms (e.g., beneficial nematodes), microbial organisms (e.g., Serenade, QST713, Bt, etc.), plant extracts (e.g., Timorex Gold, etc.), biochemical agents (e.g., insect pheromones, etc.), and / or minerals and oils (e.g., rapeseed oil).

[0196] Pesticides and biological pesticides In some embodiments, the agricultural compositions of this disclosure comprise pesticides used in combination with the taught microorganisms. In some embodiments, the agricultural compositions of this disclosure comprise biopesticides used in combination with the taught microorganisms.

[0197] In some implementations, individual microorganisms or microbial aggregates or communities developed according to the disclosed methods can be combined with known pesticides in the agricultural field, such as pesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, rodenticides and / or antialgae agents.

[0198] In some implementations, individual microorganisms or microbial aggregates or communities developed according to the disclosed methods can be combined with known biopesticides in the agricultural field, such as biopesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, rodenticides and / or antialgae agents.

[0199] For example, in some embodiments, this disclosure teaches agricultural compositions comprising one or more of the following active ingredients: macroorganisms (e.g., beneficial nematodes, etc.), microbial organisms (e.g., Serenade, Bt, etc.), plant extracts (e.g., Timorex Gold, etc.), biochemical agents (e.g., insect pheromones, etc.), and / or minerals and oils (e.g., canola oil).

[0200] In some embodiments, the individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods may be combined with herbicides selected from the group consisting of: acetamides selected from the group consisting of: acetochlor, metolachlor, butachlor, succinyl-methyl, fenfluroxychlor, fluthiamethoxam, bensulfuron-methyl, metolachlor, pyrazosulfuron, chlorpyrifos, naphthylpropionate, clethodim, pretilachlor, thiamethoxam, and thifensulfuron-methyl; amino acid derivatives selected from the group consisting of: bisphosphonium, glufosinate, and glyphosate; aryloxyphenoxypropionates selected from the group consisting of: clodinafop-propionate, cyhalofop-butyl, quizalofop-p-ethyl, haloxyfop-P-ethyl, oxadiazon, quizalofop-P-ethyl, quizalofop-P-ethyl, and quizalofop-P-ethyl; and dichlorvos. Paraquat and glyphosate; (thiolated) carbamates selected from the following groups: chlorpyrifos, butachlor, carbaryl, betaine, piperazine, EPTC, quizalofop-p-ethyl, chlorpyrifos, chlorpyrifos, betaine, bensulfuron-methyl, barnsulfuron-methyl, quizalofop-p-ethyl, and cypermethrin; cyclohexanediones selected from the following groups: butylbenzyl, clethodim, thiamethoxam, cyclobenzyl, haloxyfop-methyl, pyrazosulfuron-methyl, and oxadiazon; dinitroanilines selected from the following groups: fluroxypyr, ethylbutadiene, sulfadiazine, pendimethalin, ambroxol, and trifluralin; diphenyl ethers selected from the following groups: trifluralin, bensulfuron-methyl, chlorpyrifos, quizalofop-p-ethyl, chlorfluazol, flusulfanilamide, quizalofop-p-ethyl, and oxyfluorfen. Hydroxybenzonitrile selected from the following groups: bromobenzonitrile, chlorpyrifos, and iodobenzonitrile; imidazolinone selected from the following groups: imazalil, methoxypromethazine, methyl imazalazine, metribuzin, metribuzin, and imazalazine; phenoxyacetic acid selected from the following groups: chlorfenapyr, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-DB, propionic acid, MCPA, MCPA-thioethyl ester, MCPB, and 2,4-methylchloropropionic acid; pyrazine selected from the following groups: chlorpyrifos, flupyridaben, cyhalofop-butyl, flumethrin, and pyrazosulfuron; pyridine selected from the following groups: chlorpyrifos, dichlorpyrifos, pyrfluthrin, flusulfuron, flupyridaben, flupyridaben, flupyridaben, flupyridaben, flupyridaben, flupyridaben, flupyridaben, flupyridaben, flupyridaben, flupyridaben, flupyridaben, flupyridaben Acetylsulfuron and thiamethoxam; sulfonylureas selected from the group consisting of: sulfadiazine, tetrazolium sulfadiazine, benzylsulfadiazine, chlorpyrifos, chlorsulfuron, ethersulfuron, cypromethazine, ethoxysulfuron, pyrimisulfuron, flupyrsulfuron, flupyrsulfuron, formamidesulfuron, chlorpyrifos, azoxysulfuron, iodosulfuron, mesosulfuron, mesosulfuron, nicosulfuron, epoxysulfuron, flupyrsulfuron, flusulfuron, pyrimisulfuron, sulfadiazine, mesosulfuron, sulfonylsulfuron, thifensulfuron, etherbensulfuron, benzylsulfuron, trifluridinesulfuron, flumethanil, trifluridinesulfuron and 14(2-chloro-6-propyl-imidazol[1,2]pyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxy-pyrimidin-2-yl)urea;Triazine compounds selected from the following groups: atrazine, atrazine, cyprodinil, isoamyl, etaziclomefone, cyclomethonium, benzoate, cyprodinil, promethazine, simazine, terbufos, decazine, and triazine fluroxypyr; urea compounds selected from the following groups: chlormequat, chlorfluazuron, diuron, fenfluroxypyr, isoproturon, linuron, methylbenzylthiazoline, and butyrazoline; acetolactate synthase inhibitors selected from the following groups: bispyribac-sodium, chlorpyrifos-methyl, dichlorvos-methyl, fluroxypyr, fluroxypyr, pyrazopyr, sulfadiazine, pyrimethanil-methyl, penoxsulam, propanil-methyl, propazine-methyl. , pyrimisulfuron, cyclopyridoxine, pyrimisulfuron, pyriproxyfen, sulfonylpyrazosulfuron, and pyrazosulfuron; and compounds selected from the group consisting of: azoxystrobin, aminotriazole, barnyardgrass, flubutyroxyfen, glyphosate, bencarbazone, benfluresate, pyrazosulfuron, bentazon, dicyclosulfuron, chlorpyrifos, brobutyroxyfen, flupropyrazosulfuron, phosmet, pyrazosulfuron-methyl, pyrazosulfuron-methyl, pyrazosulfuron-methyl, pyrazosulfuron-methyl, indole-methyl, diflubenzuron, cyclohexane, isoxaflutole, bensulfuron-methyl, propyzamide, dicamba, fenvalerate, flupyroxyfen, and Drechslera Monoceras), Herbicides, Ethoxybenzamide, Tetracycline, Fluroxypyr, Propyleneflunomide, Flumetsulam, Fluroxypyr, Fluroxypyr, Furazolidone, Indoxime, Isoxazolidone, Isoxazolidone, Cyclopyralid, Propanil, Pendimethalin, Quinolinic acid, Chlormethalin, Nitrosulfuron, Methylarsic acid, Herbicides, Propyleneoxadiazon, Oxychlor, Oxychlor, Cyclopyralid, Cyclopyralid, Bisoxazolidin, Pyrazosulfuron ... Cyclo[3.2.1]oct-3-en-2-one, (3-[2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydro-2H-pyrimidin-1-yl)-phenoxy]-pyridin-2-yloxy)-ethyl acetate, methyl 6-amino-5-chloro-2-cyclopropyl-pyrimidin-4-carboxylate, 6-chloro-3-(2-cyclopropyl-6-methyl-phenoxy)-pyridazin-4-ol, 4-amino-3-chloro-6-(4-chloro-phenyl)-5-fluoro-pyridin-2-carboxylic acid, methyl 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxy-phenyl)-pyridin-2-carboxylate, and methyl 4-amino-3-chloro-6-(4-chloro-3-dimethylamino-2-fluoro-phenyl)-pyridin-2-carboxylate.

[0201] In some embodiments, the individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods may be combined with insecticides selected from the group consisting of: organo(thio)phosphates selected from the group consisting of: acephate, methyl pyrazophos, phosmet, chlorpyrifos, methyl chlorpyrifos, chlorpyrifos, diazinon, dichlorvos, chlorpyrifos, dimethoate, phorate, ethion, fenitrothion, fenthion, isoxazophos, malathion, methamidophos, chlorpyrifos, methyl parathion, methamidophos, phosmet, phosmet, parathion, parathion, parathion, phosmet, phosmet, phosmet, phorate, phorate, phorate, phosmet, phorate, phorate, phosmet, phorate, phosmet, phosmet, triazophos, and trichlorfon; The following carbamates are included in the group consisting of: carbaryl, aldicarb, cypermethrin, carbofuran, carbaryl, carbofuran, thiocarb, fenoxycarb, furazolidone, methiocarb, methomyl, chlorpyrifos, propoxur, thiamethoxam, and pymetrozine; the following pyrethroids are included in the group consisting of: allethrin, bifenthrin, cypermethrin, deltamethrin, α-cypermethrin, β-cypermethrin, ζ-cypermethrin, deltamethrin, fenvalerate, fenvalerate, cypermethrin, fenvalerate, cypermethrin, cypermethrin, permethrin, pyrethrin I and II, benzalkonium chloride, flusilazole, deltamethrin, cypermethrin, tetrabromofenofos, tetrafluorobenzyl, and propoxur. Pyrethroids and tetrafluoromethrin; insect growth regulators selected from the group consisting of: a) chitin synthesis inhibitors, wherein the chitin synthesis inhibitors are benzoylureas selected from the group consisting of: flufenoxuron, cyramazin, diflubenzuron, flufenoxuron, flufenoxuron, flufenoxuron, lufenuron, flufenoxuron, flufenoxuron, chlorfenapyr; thiamethoxam, bensulfuron, thiamethoxam, etoxazole and tetradifon; b) ecdysone antagonists selected from the group consisting of: chlorfenapyr, methoxyfenozide, tebufenozide and azadirachtin; c) juvenile hormone analogs selected from the group consisting of: pyriproxyfen, tebufenozide and phenoxycarb; or d) lipid biosynthesis inhibitors selected from the group consisting of: spirodiclofen, spirodiclofen and spirotetramat. The following are selected from the group consisting of nicotinic receptor agonists / antagonists: thiamethoxam, fipronil, imidacloprid, thiamethoxam, acetamiprid, acetamiprid, thiamethoxam, and 1-(2-chloro-thiazo-5-ylmethyl)-2-nitromimino-3,5-dimethyl-[1,3,5]triazine; the following are selected from the group consisting of GABA antagonists: endosulfan, acetamiprid, fipronil, flupyradifurone, pyrazinoflurane, pyrazolone, and 5-amino-1-(2,6-dichloro-4-methyl-phenyl)-4-sulfinylamino-1H-pyrazole-3-thiocarbamate; the following are selected from the group consisting of macrolide insecticides: abamectin, emamectin, mibamectin, rapamectin, spinosad, and ethyl spinosad.Mitochondrial electron transport inhibitors (METI) I scabies insecticides selected from the following groups: quinclorac, pyridaben, pymetrozine, azoxystrobin, and pyrimethanil; METIs selected from the following groups Compounds II and III: acaricide, fluacyprim, and flufenoxuron; brofenoxuron; oxidative phosphorylation inhibitors selected from the group consisting of: tricyclic tin, bufenozide, fenbutatin, and chlorfenapyr; cryomazine; synergistic ethers; sodium channel blockers selected from the group consisting of: indoxacarb and cyfluthrin; and compounds selected from the group consisting of: benclothiazide, bifenazate, batan, flonicamid, acetamiprid, pymetrozine, sulfur, chlorfenapyr, flufenoxuron, chlorantraniliprole, brofenoxuron (HGW86), pyridaben, pyrimethanil, dicofol, sulfadiazine, imicyafos, diflubenzuron, and pyrifluquinazon.

[0202] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial aggregates disclosed herein with known pesticides in the agricultural field, such as pesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, rodenticides, and / or antialgae agents.

[0203] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial aggregates disclosed herein with known biopesticides in the agricultural field, such as biopesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, rodenticides, and / or antialgae agents.

[0204] In some embodiments, an additive effect on target plant phenotypic traits is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with pesticides. In other embodiments, a synergistic effect on target plant phenotypic traits is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with pesticides.

[0205] In some embodiments, an additive effect on target plant phenotypic traits is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with biopesticides. In other embodiments, a synergistic effect on target plant phenotypic traits is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with biopesticides.

[0206] The synergistic effect obtained through the taught method can be quantified using the Colby formula (i.e., (E) = X + Y - (X*Y / 100)). See Colby, RS, “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967 Weeds, Vol. 15, pp. 20-22, the full text of which is incorporated herein by reference. Therefore, “synergistic” means that the presence of one component enhances the desired effect beyond the sum of its components.

[0207] The isolated microorganisms and aggregates disclosed herein can synergistically enhance the effectiveness of agricultural active pesticide compounds and agricultural auxiliary pesticide compounds.

[0208] The isolated microorganisms and aggregates disclosed herein can synergistically enhance the effectiveness of agriculturally active biopesticide compounds and agricultural auxiliary biopesticide compounds.

[0209] Plant growth regulators and biostimulants In some embodiments, the agricultural compositions of this disclosure comprise plant growth regulators and / or biostimulants used in combination with the taught microorganisms.

[0210] In some implementations, individual microorganisms or microbial aggregates or communities developed according to the disclosed methods can be combined with known plant growth regulators in the agricultural field, such as auxins, gibberellins, cytokinins, ethylene generators, growth inhibitors, and growth retardants.

[0211] For example, in some embodiments, this disclosure teaches agricultural compositions comprising one or more of the following active ingredients, including: cyclopropionylpyridinol, sec-butylamine, alcohol, chlormequat chloride, cytokinin, butyrylhydrazine, ethephon, furazolidone, gibberellic acid, gibberellin mixture, indole-3-butyric acid (IBA), maleic hydrazine, mefludide, mepiquat chloride, mepiquat pentaborate, naphthaleneacetic acid (NAA), 1-naphthylacetamide (NAD), n-decyl alcohol, placlobutrazol, calcium cyclohexane, anti-buckwheat ester, uniconazole, salicylic acid, abscisic acid, ethylene, brassinolide, jasmonic acid, polyamine, nitric oxide, strigolactone, or karrikins, etc.

[0212] In some embodiments, individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with seed inoculants known in the agricultural field, such as QUICKROOTS. ®VAULT ® RHIZO-STICK ® NODULATOR ® DORMAL ® SABREX ® In some embodiments, the Bradyrhizobium inoculum is used in combination with any single microorganism or microbial aggregate disclosed herein. In certain aspects, when one of the aforementioned inoculums (e.g., QUICKROOTS) is used... ® Synergistic effects were observed when *Or slow-growing rhizobia* were combined with microorganisms or microbial aggregates as taught herein.

[0213] In some embodiments, the agricultural compositions disclosed herein comprise plant growth regulators comprising: kinetin, gibberellic acid, and indolebutyric acid, as well as copper, manganese, and zinc.

[0214] In some embodiments, this 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®, CottonQuik®, 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®, Antac®, FST-7®, Royaltac®, Bonzi®, Cambistat®, Cutdown®, Downsize®, Florazol®, Paclo®, Paczol®, Piccolo®, Profile®, Shortstop®, Trimmit®, TurfEnhancer®, Apogee®, ArmorTech®, Goldwing®, Governor®, Groom®, Legacy®, Primeraone®, Primo®, Provair®, Solace®, T-Nex®, T-Pac®, Concise® and Sumagic®.

[0215] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial aggregates disclosed herein with plant growth regulators and / or stimulants (such as plant hormones or chemicals that affect the production or destruction of plant growth regulators).

[0216] In some embodiments, the present invention teaches plant hormones that may include: auxins (e.g., indoleacetic acid IAA), gibberellins, cytokinins (e.g., kinetin), abscisic acid, ethylene (and their production, such as being regulated by ACC synthase and destroyed by ACC deaminase).

[0217] In some implementations, individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods may be combined with biostimulants. Such biostimulants may be, but are not limited to, microbial organisms, plant extracts, algae, acids, biochar, etc.

[0218] In some embodiments, individual microorganisms or microbial aggregates or communities developed according to the disclosed methods may be combined with fertilizers, which may be organic (e.g., manure, blood, fish, urine, seaweed, kelp, etc.), nitrogen-based (e.g., nitrates, ammonium, urea, etc.), phosphates, and potassium. Such fertilizers may also contain micronutrients, including but not limited to sulfur, iron, zinc, etc.

[0219] In some embodiments, the present invention teaches additional plant growth-promoting chemicals that can work synergistically with the microorganisms and microbial aggregates disclosed herein, such as humic acid, fulvic acid, amino acids, polyphenols, and protein hydrolysates.

[0220] Therefore, in some embodiments, this disclosure provides for the application of the taught microorganism in combination with Ascend® to any crop. Furthermore, this disclosure provides for the application of the taught microorganism in combination with Ascend® to any crop and for any method or application rate.

[0221] In some embodiments, this disclosure teaches agricultural compositions containing biostimulants.

[0222] As used herein, the term "biostimulant" refers to any substance used to stimulate the growth of microorganisms that may exist in soil or other plant growth media.

[0223] The level of microorganisms in the soil or growing medium is directly related to plant health. Microorganisms feed on biodegradable carbon sources, and therefore plant health is also related to the amount of organic matter in the soil. While fertilizers provide nutrients to nourish and support plant growth, in some embodiments, biostimulants provide biodegradable carbon (e.g., molasses, carbohydrates (e.g., sugars)) to nourish and support microorganisms. Unless otherwise explicitly stated, biostimulants may contain a single ingredient or a combination of several different ingredients that, due to the effects of one or more of these ingredients (acting alone or in combination), can enhance microbial activity or plant growth and development.

[0224] In some embodiments, biostimulants are compounds that produce non-nutritive plant growth responses. In some embodiments, many important benefits of biostimulants are based on their ability to influence hormone activity. Hormones in plants (plant hormones) are chemical messengers that regulate normal plant development and responses to the environment. Root and branch growth, as well as other growth responses, are regulated by plant hormones. In some embodiments, compounds in biostimulants can alter the hormonal state of plants and have a significant impact on their growth and health. Therefore, in some embodiments, this disclosure teaches kelp, humic acid, fulvic acid, and B vitamins as common components of biostimulants. In some embodiments, the biostimulants of this disclosure enhance antioxidant activity, thereby enhancing the plant's defense system. In some embodiments, vitamin C, vitamin E, and amino acids (such as glycine) are antioxidants contained in the biostimulant.

[0225] In other embodiments, biostimulants can be used to stimulate the growth of microorganisms present in soil or other plant growth media. Previous studies have shown that when certain biostimulants containing specific organic seed extracts (e.g., soybean) are used in combination with microbial inoculants, the biostimulants can stimulate the growth of microorganisms contained in the microbial inoculants. Therefore, in some embodiments, this disclosure teaches one or more biostimulants that, when used with microbial inoculants, can increase the population size of both native microorganisms and inoculum microorganisms. For a review of some mainstream uses of biostimulants, see Calvo et al., 2014, Plant Soil 383:3-41.

[0226] Combination of plant components, microorganisms and agricultural compositions In some embodiments, this disclosure teaches individual microorganisms or microbial aggregates or communities or any combination thereof, comprising, for example, any one or more microorganisms, including genetically modified Bacillus thuringiensis or any strain containing at least 95% identity with any of the polynucleotides in SEQ ID NO. 1-50, which may optionally be combined with any agricultural composition and applied to plant components to improve plant phenotype.

[0227] Isolated microorganisms, communities, or aggregates (often interchangeably referred to as "microbes" or "microbes") can be applied to heterologous plant components to form a synthetic assemblage. Microorganisms are considered heterologous to plant components if they are not normally associated with plant components in nature or are applied in quantities different from those found in nature. In some embodiments, microorganisms may be present in one part of a plant but not in another, and the introduction of microorganisms into another part of the plant is considered a heterologous association.

[0228] It is also envisioned that isolated microorganisms, or those combined with plants or plant components, could be further associated with one or more agricultural compositions (such as those described above).

[0229] The conception of synthetic combinations of microorganisms with plant components, microorganisms with agricultural compositions, and microorganisms with plant components and agricultural compositions (often referred to as "synthetic compositions," i.e., compositions containing components that are not typically found in nature) is envisioned.

[0230] Plant component treatment agents In some embodiments, this disclosure also relates to the finding that treating plant components with one or more of the microorganisms or agricultural compositions of this disclosure prior to sowing or planting can enhance desired plant traits, such as plant growth, plant health, plant yield, and plant tolerance to pests. In some aspects, plants benefit from the harmful effects exerted by the microorganisms on another organism that affects the plant.

[0231] Therefore, in some embodiments, this disclosure teaches the use of one or more microorganisms or microbial aggregates as plant component treatment agents. Plant component treatment agents may be coatings applied directly to untreated and "naked" plant components. However, plant component treatment agents may be coatings applied to plant components that have already been coated with one or more prior plant component coatings or plant component treatment agents. Prior plant component treatment agents may include one or more active compounds (chemical or biological) and one or more inert components.

[0232] The term "plant component treatment agent" generally refers to the application of material to plant components before or during planting in the soil. The advantage of plant component treatment agents containing the microbial and other agricultural compositions disclosed herein is that the treatment agent can be delivered to the area where the plant components are planted shortly before germination and emergence.

[0233] In other embodiments, this disclosure also teaches that the use of plant component treatment agents can minimize the amount of microorganisms or agricultural compositions required for successful plant treatment and further limit worker exposure to microorganisms and compositions compared to application techniques such as spraying over soil or over germinating plant components.

[0234] Furthermore, in some embodiments, this disclosure teaches that the microorganisms disclosed herein are important for enhancing the early stages of plant life (e.g., within the first thirty days after the emergence of plant components). Therefore, in some embodiments, delivering the microorganisms and / or compositions of this disclosure in the form of a plant component treatment agent allows them to be placed in the area of ​​action at a time when microbial activity is critical.

[0235] In some embodiments, the microbial compositions of this disclosure are formulated as plant component treatment agents. In some embodiments, it is envisioned that one or more layers of the microorganisms and / or agricultural compositions disclosed herein can be substantially uniformly coated onto plant components using treatment agent application equipment specially designed and manufactured to apply the plant component treatment product precisely, safely, and effectively, using conventional mixing, spraying methods, or combinations thereof. Such equipment utilizes various types of coating technologies, such as rotary coating machines, drum coating machines, fluidized bed technology, jet beds, rotary sprays, or combinations thereof. Liquid plant component treatment agents (such as those disclosed herein) can be applied via a spinning “atomizer” disc or nozzle, which distributes the plant component treatment agent uniformly onto the plant component as it moves in a spray mode. In various embodiments, the plant component is subsequently mixed or tumbled for a period of time to achieve further treatment agent distribution and drying.

[0236] Prior to coating with the microbial composition, the plant components may be induced or uninitiated to improve the uniformity of germination and emergence. In an alternative embodiment, the dry powder formulation may be metered onto the mobile plant components.

[0237] In some embodiments, at least a portion of the surface area of ​​the plant component is coated with the microbial composition according to the present disclosure. In some embodiments, a plant component coating containing the microbial composition is applied directly to a bare plant component. In some embodiments, a plant component coating containing the microbial composition is applied to a plant component that has already been coated with a plant component. In some aspects, the plant component may have a plant component coating containing, for example, thiamethoxam and / or Bacillus firmus-I-1582, on which the composition of the present invention is applied as a plant component coating. In some aspects, the taught microbial composition is applied as a plant component coating to a plant component that has been treated with PONCHO™ VOTiVO™. In some aspects, the plant component may have a plant component coating containing, for example, metalaxyl and / or thiamethoxam and / or Bacillus firmus-I-1582, on which the composition of the present invention is applied as a plant component coating. In some aspects, the taught microbial composition is applied as a plant component coating to a plant component that has been treated with ACCELERON™.

[0238] In some embodiments, the plant components treated with microorganisms have the following microbial spore concentrations or microbial cell concentrations: approximately 10^2 to 10^12, 10^2 to 10^11, 10^2 to 10^10, 10^2 to 10^9, 1^02 to 10^8, 10^2 to 10^7, 10^2 to 10^6, 10^2 to 10^5, 10^2 to 10^4, or 10^2 to 10^3 per plant component.

[0239] In some embodiments, the plant components treated with microorganisms have the following microbial spore concentrations or microbial cell concentrations: approximately 10^3 to 10^12, 10^3 to 10^11, 10^3 to 10^10, 10^3 to 10^9, 10^3 to 10^8, 10^3 to 10^7, 10^3 to 10^6, 10^3 to 10^5, or 10^3 to 10^4 per plant component.

[0240] In some embodiments, the plant components treated with microorganisms have the following microbial spore concentrations or microbial cell concentrations: approximately 10^4 to 10^12, 10^4 to 10^11, 10^4 to 10^10, 10^4 to 10^9, 10^4 to 10^8, 10^4 to 10^7, 10^4 to 10^6, or 10^4 to 10^5 per plant component.

[0241] In some embodiments, the plant components treated with microorganisms have the following microbial spore concentrations or microbial cell concentrations: about 10^5 to 10^12, 10^5 to 10^11, 10^5 to 10^10, 10^5 to 10^9, 10^5 to 10^8, 10^5 to 10^7, or 10^5 to 10^6 per plant component.

[0242] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or microbial cell concentration of approximately 10^5 to 10^9 per plant component.

[0243] In some embodiments, the plant components treated with microbial bodies have the following microbial spore concentrations or microbial cell concentrations: at least about 1×10^3 or 1×10^4 or 1×10^5 or 1×10^6 or 1×10^7 or 1×10^8 or 1×10^9 per plant component.

[0244] In some embodiments, the amount of one or more of the microorganisms and / or agricultural composition applied to the plant component depends on the final formulation and the size or type of the plant or plant component utilized. In some embodiments, one or more of the microorganisms are present at about 2% w / w to about 80% w / w of the total formulation. In some embodiments, by weight, one or more of the microorganisms used in the composition are about 5% w / w to about 65% w / w, or 10% w / w to about 60% w / w of the total formulation.

[0245] In some implementations, the plant component may also have more spores or microbial cells per plant component, such as about 10^2, 10^3, 10^4, 10^5, 10^6, 10^7, 10^8, 10^9, 10^10, 10^11, 10^12, 10^13, 10^14, 10^15, 10^16 or 10^17 spores or cells per plant component.

[0246] In some embodiments, the thickness of the disclosed plant component coating can be up to 10μm、20μm、30μm、40μm、50μm、60μm、70μm、80μm、90μm、100μm、110μm、120μm、130μm、140μm、150μm、160μm、170μm、180μm µm、190µm、200µm、210µm、220µm、230µm、240µm、250µm、260µm、270µm、280µm 、290µm、300µm、310µm、320µm、330µm、340µm、350µm、360µm、370µm、380µm、39 0µm, 400µm, 410µm, 420µm, 430µm, 440µm, 450µm, 460µm, 470µm, 480µm, 490µm, 500µm, 510µm, 520µm, 530µm, 540µm, 550µm, 560µm, 570µm, 580µm, 590µm, 6 00µm, 610µm, 620µm, 630µm, 640µm, 650µm, 660µm, 670µm, 680µm, 690µm, 700µm, 710µm, 720µm, 730µm, 740µm, 750µm, 760µm, 770µm, 780µm, 790µm, 800µm, 810µm, 820µm, 830µm, 840µm, 850µm, 860µm, 870µm, 880µm, 890µm, 900µm, 910µm, 920µm, 930µm, 940µm, 950µm, 960µm, 970µm, 980µm, 990µm, 1000µm, 101 0µm, 1020µm, 1030µm, 1040µm, 1050µm, 1060µm, 1070µm, 1080µm, 1090µm, 1100µm, 1110µm, 1120µm, 1130µm, 1140µm, 1150µm, 1160µm, 1170µm, 1180µm, 11 90µm, 1200µm, 1210µm, 1220µm, 1230µm, 1240µm, 1250µm, 1260µm, 1270µm, 1280µm, 1290µm, 1300µm, 1310µm, 1320µm, 1330µm, 1340µm, 1350µm, 1360µm, 1 370µm, 1380µm, 1390µm, 1400µm, 1410µm, 1420µm, 1430µm, 1440µm, 1450µm, 1460µm, 1470µm, 1480µm, 1490µm, 1500µm, 1510µm, 1520µm, 1530µm, 1540µm,1550µm, 1560µm, 1570µm, 1580µm, 1590µm, 1600µm, 1610µm, 1620µm, 1630µm, 1640µm, 1650µm, 1660µm, 1670µm, 1680µm, 1690µm, 1700µm, 1710µm, 1720µm ,1730µm,1740µm,1750µm,1760µm,1770µm,1780µm,1790µm,1800µm,1810µm,1820µm,1830µm,1840µm,1850µm,1860µm,1870µm,1880µm,1890µm,1900µ m, 1910µm, 1920µm, 1930µm, 1940µm, 1950µm, 1960µm, 1970µm, 1980µm, 1990µm, 2000µm, 2010µm, 2020µm, 2030µm, 2040µm, 2050µm, 2060µm, 2070µm, 2080 µm, 2090µm, 2100µm, 2110µm, 2120µm, 2130µm, 2140µm, 2150µm, 2160µm, 2170µm, 2180µm, 2190µm, 2200µm, 2210µm, 2220µm, 2230µm, 2240µm, 2250µm, 226 0µm, 2270µm, 2280µm, 2290µm, 2300µm, 2310µm, 2320µm, 2330µm, 2340µm, 2350µm, 2360µm, 2370µm, 2380µm, 2390µm, 2400µm, 2410µm, 2420µm, 2430µm, 24 40µm, 2450µm, 2460µm, 2470µm, 2480µm, 2490µm, 2500µm, 2510µm, 2520µm, 2530µm, 2540µm, 2550µm, 2560µm, 2570µm, 2580µm, 2590µm, 2600µm, 2610µm, 2 620µm, 2630µm, 2640µm, 2650µm, 2660µm, 2670µm, 2680µm, 2690µm, 2700µm, 2710µm, 2720µm, 2730µm, 2740µm, 2750µm, 2760µm, 2770µm, 2780µm, 2790µm, 2800µm, 2810µm, 2820µm, 2830µm, 2840µm, 2850µm, 2860µm, 2870µm, 2880µm, 2890µm, 2900µm, 2910µm, 2920µm, 2930µm, 2940µm, 2950µm, 2960µm, 2970µm,2980µm, 2990µm, or 3000µm.

[0247] In some embodiments, the thickness of the plant component coating disclosed herein may be 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm or 5 mm.

[0248] In some embodiments, the plant component coating of this disclosure may 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%, or 24% of the weight of the uncoated plant component. 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%.

[0249] In some embodiments, microbial spores and / or cells may be freely coated onto plant components, or may be formulated in a liquid or solid composition prior to coating onto plant components. For example, a solid composition containing microorganisms may be prepared by mixing a solid carrier with a spore suspension until the solid carrier is impregnated with a spore or cell suspension. The mixture may then be dried to obtain the desired particles.

[0250] In some other embodiments, the solid or liquid microbial compositions of this disclosure are envisioned to also contain functional agents, such as activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the product or combinations thereof.

[0251] Plant component coating methods and compositions known in the art can be particularly useful when modified by incorporating one of the embodiments of this disclosure. Such coating methods and their application devices are disclosed, for example, in U.S. Patent Nos. 5,916,029, 5,918,413, 5,554,445, 5,389,399, 4,759,945, 4,465,017 and U.S. Patent Application No. 13 / 260,310, each of which is incorporated herein by reference.

[0252] Plant component coating compositions are disclosed in, for example, U.S. Patent 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 herein by reference.

[0253] In some embodiments, a variety of additives may be added to the plant component treatment formulation comprising the composition of the present invention. A binder may be added, and the binder comprises those composed of natural or synthetic adhesive polymers that are non-phytotoxic to the plant component being coated. The binder may be selected from polyvinyl acetate; polyvinyl acetate copolymers; ethylene-vinyl acetate (EVA) copolymers; polyvinyl alcohol; polyvinyl alcohol copolymers; cellulose, including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose; polyvinylpyrrolidone; polysaccharides, including starch, modified starch, dextrin, maltodextrin, alginate, and deacetylated chitosan; fats; oils; proteins, including gelatin and corn gluten; gum arabic; shellac; vinylidene chloride and vinylidene chloride copolymers; calcium lignin sulfonate; acrylic acid copolymers; polyvinyl acrylate; polyethylene oxide; acrylamide polymers and copolymers; hydroxyethyl polyacrylate, methacrylamide monomers; and polychloroprene.

[0254] A variety of colorants can be used, including organic chromophores classified as: nitroso; nitro; azo, including monoazo, diazo, and polyazo; acridine, anthraquinone, azazine, diphenylmethane, indoleamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, and xanthracene. Other additives that can be added include trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, molybdenum, and zinc.

[0255] Polymers or other dust control agents can be applied to leave the treatment on the surface of the plant components.

[0256] In some specific embodiments, in addition to microbial cells or spores, the coating may also comprise an adhesive layer. The adhesive should be non-toxic, biodegradable, and adhesive. Examples of such materials include, but are not limited to: polyvinyl acetate; polyvinyl acetate copolymers; polyvinyl alcohol; polyvinyl alcohol copolymers; cellulose, such as methylcellulose, hydroxymethylcellulose, and hydroxymethylpropylcellulose; dextrin; alginate; sugars; molasses; polyvinylpyrrolidone; polysaccharides; proteins; fats; oils; gum arabic; gelatin; syrups; and starches. Further examples can be found, for example, in U.S. Patent No. 7,213,367, which is incorporated herein by reference.

[0257] Plant component treatment formulations may also contain various additives, such as adhesives, dispersants, surfactants, nutrients, and buffering agents. Other common plant component treatment additives include, but are not limited to, coating agents, wetting agents, buffers, and polysaccharides. At least one agriculturally acceptable carrier, such as water, solid, or dry powder, may be added to the plant component treatment formulation. Dry powders may be derived from a variety of materials, such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon, and various phosphorus compounds.

[0258] In some embodiments, the plant component coating composition may comprise at least one filler, which is an organic or inorganic, natural or synthetic component, combined with the active component to facilitate its application to the plant component. In various respects, the filler is an inert solid, such as clay, natural or synthetic silicates, silica, resin, wax, solid fertilizer (e.g., ammonium salts), natural soil minerals (such as kaolin, clay, talc, lime, quartz, palygorskite, montmorillonite, bentonite, or diatomaceous earth), or synthetic minerals (such as silica, bauxite, or silicates, particularly aluminum silicate or magnesium silicate).

[0259] In some embodiments, the plant component treatment formulation may also include one or more of the following ingredients: other pesticides, including compounds that act only below ground level; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of these materials; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroaniline, glyceryl ethers, pyridazinones, uracil, phenoxy compounds, urea, and benzoic acid; and herbicide safeners, such as benzoxazine, diphenylmethyl derivatives, and N,N-diallyl. Dichloroacetamide, various dihaloacetyl, oxazolyl and thiazolyl compounds, acetone, naphthalenecarboxylic anhydride compounds and oxime derivatives; chemical fertilizers; biofertilizers; and biocontrol agents, such as other naturally occurring or recombinant bacteria and fungi derived from Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. These components may be added as a separate layer on plant components or alternatively as part of the plant component coating compositions disclosed herein.

[0260] In some embodiments, the formulations used in this disclosure for treating plant components may be in the following forms: suspensions; emulsions; granular slurries in an aqueous medium (e.g., water); wettable powders; wettable granules (dry and flowable); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation may be from about 0.5% by weight to about 99% by weight (w / w), or 5%-40%, or otherwise formulated by those skilled in the art.

[0261] As mentioned above, other conventional inactive or inert ingredients may be incorporated into the formulation. Such inert ingredients include, but are not limited to: conventional binders; dispersants, such as methylcellulose, which acts as a combined dispersant / binder for plant component treatments; polyvinyl alcohol; lecithin; polymeric dispersants (e.g., polyvinylpyrrolidone / vinyl acetate); thickeners (e.g., clay thickeners used to increase viscosity and reduce sedimentation of particulate suspensions); emulsion stabilizers; surfactants; antifreeze compounds (e.g., urea); dyes; colorants, etc. Further inert ingredients that may be used in this disclosure can be found in McCutcheon's, Volume 1, “Emulsifiers and Detergents,” MC Publishing Company, Glen Rock, NJ, USA, 1996, which is incorporated herein by reference.

[0262] The plant component coating formulations of this disclosure can be applied to plant components by a variety of methods, including but not limited to: mixing in a container (e.g., bottle or bag), mechanical application, tumbling, spraying, and immersion. A variety of active or inert materials can be used to contact the plant components with the microbial composition according to this disclosure.

[0263] In some embodiments, the amount of microorganisms or agricultural composition used to treat the plant component will vary depending on the type of plant component and the type of active ingredient, but the treatment will involve contacting the plant component with an agriculturally effective amount of the composition of the present invention.

[0264] As discussed above, an effective amount means an amount of the composition of the present invention sufficient to affect beneficial or desired results. An effective amount may be applied in one or more applications.

[0265] In some implementations, in addition to the coating layer, the plant components may be treated with one or more of the following: other pesticides, including fungicides and herbicides; herbicide-safe agents; fertilizers and / or biocontrol agents. These components may be added as separate layers or alternatively added to the coating layer.

[0266] In some embodiments, the plant component coating formulations of this disclosure can be applied to the plant components using a variety of techniques and machines, such as fluidized bed technology, roller milling, rotary static plant component processors, and drum coating machines. Other methods, such as sprayed beds, may also be useful. The plant components can be pre-sized before coating. After coating, the plant components are typically dried and then transferred to a sizing machine for further sizing. Such procedures are known in the art.

[0267] In some embodiments, the plant components treated with microorganisms may also be coated with an external film to protect the coating. Such coatings are known in the art and can be applied using fluidized bed and cylindrical film coating techniques.

[0268] In other embodiments of this disclosure, the composition according to this disclosure can be introduced onto a plant component by using a solid matrix. For example, a certain amount of the composition of the present invention can be mixed with a solid matrix material, and then the plant component can be placed in contact with the solid matrix material for a period of time to allow the composition to be introduced into the plant component. The plant component can then optionally be separated from the solid matrix material and stored or used, or the mixture of the solid matrix material and the plant component can be stored or grown directly. Solid matrix materials that can be used in this disclosure include polyacrylamide, starch, clay, silica, bauxite, soil, sand, polyurea, polyacrylate, or any other material capable of absorbing or adsorbing the composition of the present invention for a period of time and releasing the composition into the interior or surface of the plant component. It is useful to ensure that the composition of the present invention and the solid matrix material are compatible with each other. For example, the solid matrix material should be selected such that it can release the composition at a reasonable rate (e.g., over a period of minutes, hours, or days).

[0269] In some embodiments, the present disclosure teaches that individual microorganisms or microbial aggregates or microbial communities developed according to the disclosed methods can be combined with any plant biostimulant.

[0270] In some embodiments, this 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™, PlantPlus™, Accomplish LM®, Titan®, Soil Builder™, Nutri Life, Soil Solution™, SeedCoat™, PercPlus™, Plant Power®, CropKarb®, Thrust™, Fast2Grow®, Baccarat®, and Potente®.

[0271] In some embodiments, an additive effect on the phenotypic traits of the target plant is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with active chemical agents. In other embodiments, a synergistic effect on the phenotypic traits of the target plant is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with active chemical agents.

[0272] In some embodiments, an additive effect on the target plant phenotypic traits is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with fertilizers. In other embodiments, a synergistic effect on the target plant phenotypic traits is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with fertilizers.

[0273] In some embodiments, an additive effect on the target plant phenotypic traits is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with plant growth regulators. In some embodiments, a synergistic effect is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with plant growth regulators. In some aspects, the microorganisms of this disclosure are combined with Ascend... ® The study combined these findings and observed synergistic effects on one or more target phenotypic traits.

[0274] In some embodiments, an additive effect on the target plant phenotypic traits was observed when microorganisms or microbial aggregates identified according to the taught methods were combined with biostimulants. In some embodiments, a synergistic effect was observed when microorganisms or microbial aggregates identified according to the taught methods were combined with biostimulants.

[0275] The synergistic effect obtained through the taught method can be quantified using the Colby formula (i.e., (E) = X + Y - (X*Y / 100)). See Colby, RS, “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967 Weeds, Vol. 15, pp. 20-22, the full text of which is incorporated herein by reference. Therefore, “synergistic” means that the presence of one component enhances the desired effect beyond the sum of its components.

[0276] The isolated microorganisms and aggregates disclosed herein can synergistically enhance the effectiveness of agricultural active compounds and agricultural auxiliary compounds.

[0277] In other implementations, synergistic effects were observed when microorganisms or microbial aggregates identified according to the taught methods were combined with fertilizers.

[0278] Furthermore, in some embodiments, this disclosure utilizes synergistic interactions to define microbial aggregates. That is, in some aspects, this disclosure combines certain isolated microbial species that exert synergistic effects into aggregates that confer beneficial traits to plants, or are associated with enhancing beneficial plant traits.

[0279] The agricultural compositions developed according to this disclosure can be formulated with certain adjuvants to enhance the activity of known active agricultural compounds. This has the advantage of reducing the amount of active ingredient in the formulation while maintaining the efficacy of the active compound, thus keeping costs as low as possible and complying with any official regulations. In individual cases, it is also possible to broaden the scope of action of the active compound, since plants (where treatment with a specific active ingredient without addition is not successful enough) can actually be successfully treated by adding certain adjuvants as well as the disclosed microbial isolates and aggregates. Furthermore, when environmental conditions are unfavorable, the performance of the active substance can be enhanced in individual cases by appropriate formulations.

[0280] Such adjuvants, which can be used in agricultural compositions, are typically adjuvants. Adjuvants are usually in the form of surfactants or salt-like compounds. Depending on their mode of action, they can be broadly classified as regulators, activators, fertilizers, pH buffers, etc. Regulators affect the wetting, adhesion, and spreading properties of formulations. Activators disrupt the waxy cuticle of plants and enhance the penetration of active ingredients into the cuticle (both short-term (minutes) and long-term (hours)). Fertilizers (such as ammonium sulfate, ammonium nitrate, or urea) improve the absorption and solubility of active ingredients and can reduce antagonistic behavior of active ingredients. pH buffers are often used to adjust formulations to their optimal pH.

[0281] For further embodiments of the agricultural compositions disclosed herein, see “Chemistry and Technology of Agrochemical Formulations”, ed. DA Knowles, 1998 edition, Kluwer Academic Publishers, which is incorporated herein by reference.

[0282] Plant and agricultural benefits A wide variety of plants (including those cultivated in agriculture) can benefit from the application of microorganisms (such as those described herein, including single microorganisms, aggregates, and / or compositions produced therefrom, or containing any of the foregoing). A wide variety of different plants, including mosses, lichens, and algae, can be used in the methods of this disclosure. In embodiments, the plants have economic, social, or environmental value. For example, plants may include those used as food crops, fiber crops, oil crops, for forestry, for the pulp and paper industry, as feedstock for biofuel production, and as ornamental plants.

[0283] In other implementations, the plants may be economically, socially, or environmentally undesirable, such as weeds. The following is a list of non-limiting examples of plant types to which the methods of this disclosure may be applied: Food crops Cereals, such as corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, and buckwheat; Leafy greens, such as cruciferous vegetables like cabbage, broccoli, bok choy, and arugula; salad greens like spinach, watercress, and lettuce; Fruits and flowering vegetables, such as avocados, sweet corn, feed corn, and artichokes; melons, such as zucchini, cucumbers, cantaloupes, squash, and pumpkins; and nightshade vegetables / fruits, such as tomatoes, eggplants, and peppers. Legume vegetables, such as peanuts, peas, soybeans, green beans, lentils, chickpeas, and okra; Bulbous and stem vegetables, such as asparagus, celery, and Allium crops, such as garlic, onion, and leeks; Root and tuber vegetables, such as carrots, beets, bamboo shoots, cassava, yams, ginger, Jerusalem artichokes, parsnip, radishes, potatoes, sweet potatoes, taro, turnips, and wasabi; Sugar crops, including sugar beets (Beta vulgaris) and sugarcane (Saccharum officinarum); Crop cultivation for the production of non-alcoholic beverages and stimulants, such as coffee, black tea, herbal tea and green tea, cocoa, cannabis and tobacco; Fruit crops, such as true berries (e.g., kiwifruit, grapes, currants, gooseberries, guavas, feijoas, pomegranates), citrus fruits (e.g., oranges, lemons, limes, grapefruits), fruits with superior ovaries (e.g., bananas, cranberries, blueberries), aggregate fruits (blackberries, raspberries, boysonberries), compound fruits (e.g., pineapples, figs), stone fruits (e.g., apricots, peaches, cherries, plums), pome fruits (e.g., apples, pears), and other fruits such as strawberries and sunflower seeds; Culinary and medicinal herbs, such as rosemary, basil, bay leaf, coriander, mint, dill, Hypericum, foxglove, aloe vera, rosehip, and cannabis; Spices are produced by crop plants such as black pepper, cumin, cinnamon, nutmeg, ginger, cloves, saffron, cardamom, nutmeg skin, red chili peppers, masala, and star anise. Crop crops grown for nut production, such as almonds and walnuts, Brazil nuts, cashews, coconuts, chestnuts, macadamia nuts, pistachios, peanuts, and pecans; Crop cultivation for the production of beer, wine and other alcoholic beverages, such as grapes and hops; Oilseed crops, such as soybeans, peanuts, cotton, olives, sunflowers, sesame, lupin species and brassica crops (e.g., canola / rapeseed); and edible fungi, such as white mushrooms, shiitake mushrooms and oyster mushrooms; Plants used in rural agriculture Leguminosae: Species of the genera *Trifolium*, *Medicago*, and *Lotus*; white clover (*T. repens*); red clover (*T. pratense*); Caucasian clover (*T. ambigum*); ground clover (*T. subterraneum*); alfalfa / purple alfalfa (*Medicago sativum*); annual alfalfa; caltrop alfalfa; alfalfa scabra; red clover (*Onobrychis viciifolia*); *Lotus corniculatus*; large *Lotus pedunculatus*. Seed legumes / dried beans, including peas (Pisum sativum), common beans (Phaseolus vulgaris), broad beans (Vicia faba), mung beans (Vigna radiata), cowpeas (Vigna unguiculata), chickpeas (Cicerarietum), and lupins (Lupinus species); cereals, including corn / maize (Zea mays), sorghum (Sorghum spp.), millet (Panicum miliaceum, P. sumatrense), rice (Oryza sativaindica, Oryza sativa japonica), wheat (Triticum aestivum), barley (Hordeum vulgare), rye (Secale cereale), triticale (Triticum X Secale), and oats (Avena sativa). Forage and woodland grasses: temperate grasses, such as species of *Lolium*; species of *Festuca*; species of *Agrostis*; perennial ryegrass (*Lolium perenne*); hybrid ryegrass (*Lolium hybridum*); annual ryegrass (*Lolium multiflorum*); tall fescue (*Festuca arundinacea*); cowtail grass (*Festucapratensis*); red fescue (*Festuca rubra*); fescue (*Festuca ovina*); fescue ryegrass (*Festuloliums*) (ryegrass × fescue crosses); orchardgrass (*Dactylis glomerata*); Kentucky bluegrass (*Poa pratensis*); Kentucky bluegrass (*Poa palustris*); woodland bluegrass (*Poa nemoralis*); common bluegrass (*Poa trivialis*); Kentucky bluegrass (*Poa kounii*). (compresa); Bromus species; Phalaris (Phleum species); Arrhenatherum elatius; Agropyron species; Avena strigosa; Setaria italic. Tropical grasses, such as species of the genera *Phalaris*, *Brachiaria*, *Eragrostis*, *Panicum*, *Paspalum notatum*, *Brachypodium*, and grasses used for biofuel production, such as switchgrass (*Panicum virgatum*) and miscanthus. Fiber crops Cotton, kapok, jute, coconut, sisal, flax (Linum species), New Zealand flax (Phormium species); cultivated and natural forest species harvested for paper and engineered wood fiber products, such as coniferous and broadleaf species; Tree and shrub species used in the planting of forestry and biofuel crops Pine (species of the genus *Pinus*); Fir (species of the genus *Pseudotsuga*); Spruce (species of the genus *Picea*); Cypress (species of the genus *Cupressus*); Acacia (species of the genus *Acacia*); Alder (species of the genus *Alnus*); Oak (species of the genus *Quercus*); Redwood (species of the genus *Sequoiadendron*); Willow (species of the genus *Salix*); Birch (species of the genus *Betula*); Cedar (species of the genus *Cedurus*); Ash (species of the genus *Fraxinus*); Larch (species of the genus *Larix*); Eucalyptus (species of the genus *Eucalyptus*); Bambuseae (species of the tribe Bambuseae); and Poplar (species of the genus *Populus*).

[0284] Plants are cultivated for conversion into energy, biofuels, or industrial products through extraction, biological, physical, or biochemical processes. Oil-producing plants, such as oil palm, jatropha, corn, soybean, cotton, and flaxseed; latex-producing plants, such as rubber tree (Hevea brasiliensis) and Panama tree (Castilla elastica); plants used as direct or indirect feedstocks for the production of biofuels (i.e., following chemical, physical (e.g., thermal or catalytic), biochemical (e.g., enzymatic pretreatment), or biological (e.g., microbial fermentation) conversions during the production of biofuels, industrial solvents, or chemical products (e.g., ethanol or butanol, propylene glycol, or other fuels or industrial materials), including sugar crops (e.g., sugar beets, sugarcane), starch crops (e.g., C3 and C4 cereals and tuber crops), and cellulosic crops (e.g., trees such as pine and eucalyptus). ) and cereals and grasses (such as bamboo, switchgrass, miscanthus); crops used in energy, biofuel or industrial chemical production by gasification and / or microbial or catalytic conversion into biofuels or other industrial feedstocks (such as solvents or plastics, producing or not producing biochar), such as biomass crops, such as conifers, eucalyptus, tropical or broadleaf tree species, cereals and grasses (such as bamboo, switchgrass, miscanthus, sugarcane or hemp) or cork (such as poplar, willow); and biomass crops used for biochar production; Crops that produce natural products that can be used in the pharmaceutical, agricultural nutrition, and cosmeceutical industries Crops that produce drug precursors or compounds, or nutritional and cosmeceutical compounds and materials, such as star anise (shikimic acid), Japanese knotweed (resveratrol), and kiwifruit (soluble fiber, proteolytic enzymes). Flowering, ornamental, and landscaping plants grown for their aesthetic appeal or environmental characteristics Flowers, such as roses, tulips, and chrysanthemums; Ornamental shrubs, such as those in the genera *Buxus*, *Hebe*, *Rosa*, *Rhododendron*, and *Hedera*. Beautifying plants include those from the genera *Platanus*, *Platanus*, *Escallonia*, *Euphorbia*, and *Carex*. Mosses, such as peat moss Planting plants for bioremediation In some respects, the microorganisms of this disclosure are applied to hybrid plants to enhance the beneficial traits of said hybrids. In other respects, the microorganisms of this disclosure are applied to genetically modified plants to enhance the beneficial traits of said GM plants. The microorganisms taught herein can be applied to hybrids and GM plants, and thus enable the maximization of the superior genetic and trait technologies of these plants.

[0285] It should be understood that the plant may be provided as seeds, seedlings, cuttings, propagules, or any other plant material or tissue form capable of growth. In one embodiment, the seeds may be surface-sterilized with a material such as sodium hypochlorite or mercuric chloride to remove surface contaminants of microorganisms. In one embodiment, the propagule is grown in a pure culture, such as in the form of a sterile seedling in a tissue culture, before being placed in a plant growth medium.

[0286] Application method Microorganisms can be applied to plants, seeds, seedlings, cuttings, propagules, etc., and / or growth media containing said plants, using any appropriate technique known in the art.

[0287] However, for example, isolated microorganisms, aggregates, or compositions containing them and / or compositions derived therefrom can be applied to plants, seeds, seedlings, cuttings, propagules, etc. by spraying, coating, dusting, or any other method known in the art.

[0288] In another embodiment, the isolated microorganisms, aggregates, or compositions containing them can be applied directly to plant seeds before sowing.

[0289] In another embodiment, the isolated microorganisms, aggregates, or compositions containing them can be applied directly to plant seeds in the form of seed coating.

[0290] In one embodiment of this disclosure, the isolated microorganisms, aggregates, or compositions comprising them are supplied in the form of granules or fillers applied to plant growth media or soil irrigation.

[0291] In other embodiments, the isolated microorganisms, aggregates, or compositions containing them are supplied in the form of foliar application, such as foliar sprays or liquid compositions. Foliar sprays or liquid applications may be applied to growing plants or growing media (e.g., soil).

[0292] In some embodiments, the isolated microorganisms, aggregates, or compositions comprising them are supplied in forms selected from: soil irrigation agents, foliar sprays, impregnation treatments, furrow treatments, soil conditioners, granules, broadcast treatments, post-harvest disease control treatments, or seed treatments. In some embodiments, these agricultural compositions may be applied alone or in rotational procedures.

[0293] In some embodiments, the isolated microorganisms, aggregates, or compositions containing them are compatible with barrels. In some embodiments, the agricultural composition is compatible with barrels of other agricultural products. In some embodiments, the agricultural composition is compatible with equipment used for ground, air, and irrigation application.

[0294] In another embodiment, the isolated microorganisms, aggregates, or compositions containing them may be formulated into granules and applied simultaneously with the seeds during planting. Alternatively, the granules may be applied after planting. Or, the granules may be applied before planting.

[0295] In some embodiments, isolated microorganisms, aggregates, or compositions containing them are applied to plants or growing media in the form of topical and / or irrigant applications to improve crop growth, yield, and quality. Topical application can be carried out using dry mixtures, powders, or dusting compositions, or can be a liquid-based formulation.

[0296] In embodiments, the isolated microorganisms, aggregates, or compositions containing them may be formulated as: (1) solutions; (2) wettable powders; (3) spreadable powders; (4) soluble powders; (5) emulsions or suspension concentrates; (6) seed dressings or seed coatings; (7) tablets; (8) water-dispersible granules; (9) water-soluble granules (slow-release or fast-release); (10) microencapsulated granules or suspensions; (11) as irrigation components; and (12) components of fertilizers, pesticides, and other compatibility modifiers, etc. In some respects, the compositions may be diluted in an aqueous medium prior to conventional spray application. The compositions of this disclosure may be applied to soil, plants, seeds, rhizosphere, root sheath, or other areas where application of the microbial composition will be beneficial. Furthermore, micro-bombardment may be used as a means of introducing endophytic microorganisms.

[0297] The composition is applied to the leaves of plants in various ways. It can be applied to the leaves as an emulsion or suspension concentrate, a liquid solution, or a foliar spray. Application can be carried out in a laboratory, growth chamber, greenhouse, or field.

[0298] In another embodiment, microorganisms can be inoculated into plants by pruning roots or stems and exposing the plant surface to microorganisms by spraying, dipping, or otherwise applying a liquid microbial suspension, gel, or powder.

[0299] In another embodiment, the microorganisms can be injected directly into leaves, roots, branches, and / or vascular tissues, or otherwise inoculated directly into the interior or surface of leaf or root cuts, or into excised embryos, radicles, or coleoptiles. These inoculated plants may then be further exposed to a growth medium containing additional microorganisms; however, this is not necessary.

[0300] In other embodiments, particularly where the microorganisms are not culturable, the microorganisms can be transferred into the plant by any or a combination of physical, chemical, or biological treatments that provide an opportunity for the microorganisms to enter plant cells or intercellular spaces, such as grafting, insertion of explants, injection into the plant (e.g., injection into the cambium or xylem), aspiration, electroporation, wounding, root pruning, induction of stomatal opening, or any physical, chemical, or biological treatment that provides an opportunity for the microorganisms to enter plant cells or intercellular spaces. Many alternative techniques are readily apparent to those skilled in the art.

[0301] In one embodiment, the microorganisms infiltrate parts of the plant (such as roots, stems, leaves, and / or propagating plant parts (becoming endophytic)), and / or grow on the surface of roots, stems, leaves, and / or propagating plant parts (becoming epiphytic) and / or grow in the plant rhizosphere. In one embodiment, the microorganisms form a symbiotic relationship with the plant.

[0302] In some aspects, the present invention includes: Aspect 1: A synthetic composition comprising: a) Bacillus cells, their secretions, their culture medium or spores thereof, wherein the cells comprise a heteropolynucleotide sequence having at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 5, 13 and 15; and b) a composition of at least one of the following groups: plant components, formulation components, agricultural compositions, and any and / or combinations thereof.

[0303] Aspect 2: A synthetic composition comprising: c) Bacillus thuringiensis cells, their secretions, their culture medium, or spores thereof, wherein the cells comprise a heteropolynucleotide sequence sharing at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 5, 13, and 15; wherein the microbial cells have the expression of at least one gene in a reduced or eliminated biosynthetic pathway; and d) At least one composition selected from the group consisting of: plant components, formulation components, agricultural compositions, and any and / or combinations thereof.

[0304] Aspect 3: The synthetic composition as described in aspect 1 or aspect 2, wherein the heteropolynucleotide of a) is inserted into the genome of the cell in a coding sequence located in the thallium biosynthesis gene cluster.

[0305] Aspect 4: The synthetic composition as described in aspect 2, wherein the endotoxin is a β-endotoxin.

[0306] Aspect 5: The synthetic composition as described in aspect 3, wherein the coding sequence shares at least 90% identity with at least 100 consecutive nucleotides of SEQ ID NO: 17, 18, 20 or 11.

[0307] Aspect 6: The synthetic composition as described in aspect 3, wherein the Bacillus thuringiensis cells are isolates of strain PM39400 preserved as NRLRB-68090.

[0308] Aspect 7: The synthetic composition as described in aspect 1, comprising at least two polynucleotide sequences selected from the group consisting of: SEQ ID NO: 1-50.

[0309] Aspect 8: The synthetic composition as described in aspect 1, comprising at least three polynucleotide sequences selected from the group consisting of: SEQ ID NO: 1-50.

[0310] Aspect 9: The synthetic composition as described in aspect 1 or aspect 2 further comprises at least one additional microorganism.

[0311] Aspect 10: The synthetic composition as described in aspect 1 or aspect 2, wherein the plant component is a seed.

[0312] Aspect 11: The synthetic composition as described in aspect 10, wherein the seed contains a genetically modified organism.

[0313] Aspect 12: The synthetic composition as described in aspect 1 or aspect 2, wherein the plant component is a leaf.

[0314] Aspect 13: The synthetic composition as described in aspect 1 or aspect 2, wherein the plant component is a root.

[0315] Aspect 14: The synthetic composition as described in aspect 1 or aspect 2, wherein the plant component is the whole plant.

[0316] Aspect 15: A synthetic composition as described in aspect 1 or aspect 2, wherein the microorganism is present in a liquid formulation at a concentration of at least about 10^2 CFU / mL, or in a non-liquid formulation at a concentration of at least about 10^2 colony-forming units / gram.

[0317] Aspect 16: A synthetic composition as described in aspect 1 or aspect 2, wherein the formulation component is selected from the group consisting of: compounds with microbial stability, preservatives, carriers, surfactants, anti-complexing agents, and any combination thereof.

[0318] Aspect 17: A synthetic composition as described in aspect 1 or aspect 2, wherein the agricultural composition comprises a fungicide, a nematicide, a bactericide, a pesticide, a herbicide, or any combination thereof.

[0319] Aspect 18: A variety of synthetic compositions as described in aspect 1 or aspect 2, wherein the synthetic composition is substantially enclosed within an object selected from the group consisting of: tubes, bottles, wide-mouth bottles, ampoules, packaging, utensils, bags, boxes, silos, envelopes, cartons, containers, silos, shipping containers, truck beds and boxes.

[0320] Aspect 19: Various synthetic compositions as described in aspect 1 or aspect 2, wherein the synthetic composition is at a temperature below thirty degrees Celsius.

[0321] Aspect 20: A variety of synthetic compositions as described in aspect 1 or aspect 2, wherein the synthetic composition is at a temperature below zero degrees Celsius.

[0322] Aspect 21: The synthetic composition as described in aspect 1 or aspect 2, wherein the plant component is obtained from plants selected from the group consisting of: corn, soybean, wheat, cotton, cucumber, tomato, pepper, potato, strawberry, orange, lemon, lime, apple, green beans, zucchini, pea, lettuce, broccoli, celery, cauliflower, sorghum, rapeseed, rapeseed and okra.

[0323] Aspect 22: A synthetic composition as described in aspect 1 or aspect 2, wherein the agricultural composition comprises a growth medium.

[0324] Aspect 23: The synthetic composition as described in aspect 22, wherein the growth medium comprises soil.

[0325] Aspect 24: A plurality of synthetic compositions as described in aspect 23, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern, the spacing between each of the synthetic compositions being substantially equal.

[0326] Aspect 25: A method for conferring plant tolerance to nematode pests, the method comprising treating a plant component of the plant with a formulation comprising at least one microbial cell, its secretions or a culture medium thereof, wherein the microbial cell is a Bacillus cell and comprises a heteropolynucleotide sequence having at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 5, 13 and 15.

[0327] Aspect 26: A method for conferring plant tolerance to nematode pests, the method comprising treating a plant component of the plant with a formulation comprising at least one microbial cell, its secretions or a culture medium thereof, wherein the microbial cell is a Bacillus thuringiensis cell and comprises a heteropolynucleotide sequence having at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 5, 13 and 15; wherein the microbial cell has the expression of at least one gene in a reduced or eliminated biosynthetic pathway.

[0328] Aspect 27: The method as described in aspect 25 or aspect 26, wherein the trait of agronomic importance is resistance to nematode pests.

[0329] Aspect 28: The method described in aspect 25 or aspect 26 further comprises at least one additional microorganism.

[0330] Aspect 29: The method as described in aspect 25 or aspect 26, wherein the plant further possesses improved agronomic characteristics selected from the group consisting of: disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved nitrogen use, enhanced nitrogen fixation, resistance to pests, resistance to herbivores, resistance to pathogens, increased yield, increased yield under water-limited conditions, enhanced health, increased vitality, improved growth, enhanced photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased branch length, increased root length, improved root structure, and increased seed weight. Increased quantity, altered seed carbohydrate composition, altered seed oil composition, increased root length, increased pod number, delayed senescence, increased greenness, altered seed protein composition, increased dry weight of mature plant reproductive components, increased fresh weight of mature plant reproductive components, increased number of mature plant reproductive components per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, increased number of seeds per plant, increased seed weight per plant, decreased number of withered leaves per plant, decreased number of severely withered leaves per plant, increased number of non-withered leaves per plant, or improved ornamental appearance of the plant.

[0331] Aspect 30: The method as described in aspect 25 or aspect 26, wherein the presence of the microbial cells, their secretions, or their culture medium is sufficient to provide benefit to plants derived from the plant components compared to plants derived from plant components not treated with the microbial cells, spores, or their secretions.

[0332] Aspect 31: A method of cultivating plants, comprising: a) Introducing microbial cells, their secretions, or their culture medium into a plant component of the plant, wherein the microbial cells contain a heteropolynucleotide sequence that shares at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 5, 13, and 15; and wherein the microbial cells are heterologous to the plant component; b) Place the plant components described in a) in a growth medium under suitable growth conditions; Plants grown from plant components described in b) exhibit improved plant health characteristics in the presence of nematodes, compared to plants grown from plant components that do not contain the microbial cells described in a).

[0333] Aspect 32: The method of aspect 31, wherein the microbial cell has the expression of at least one gene in a reduced or eliminated biosynthetic pathway.

[0334] Aspect 33: The method as described in aspect 31 further comprises at least one additional microorganism.

[0335] Aspect 34: The method of aspect 31, wherein the introduction into the plant component is accomplished by an indirect method selected from the group consisting of: furrow application, soil irrigation application, and lateral fertilization application.

[0336] Aspect 35: The method of aspect 31, wherein the introduction into the plant component is accomplished by coating the plant component with a liquid formulation of the microorganism or its secretions.

[0337] Aspect 36: The method of aspect 31, wherein the introduction into the plant component is accomplished by coating the plant component with a substantially non-liquid formulation of the microorganism or its secretions.

[0338] Aspect 37: The method as described in aspect 31, wherein the plant component is a seed.

[0339] Aspect 38: The method as described in aspect 31, wherein the plant component is a leaf.

[0340] Aspect 39: The method as described in aspect 31, wherein the plant component is a root.

[0341] Aspect 40: The method as described in aspect 31, wherein the plant component is the whole plant.

[0342] Aspect 41: A method for reducing the number of harmful organisms in a composition, wherein the harmful organisms include nematodes, nematode eggs, nematode larvae, or any combination thereof, the method comprising: introducing into the composition at least one microbial cell, spore, secretions thereof, or culture medium thereof, wherein the microbial cell comprises a heteropolynucleotide sequence having at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 5, 13, and 15.

[0343] Aspect 42: The method of aspect 41, wherein the microbial cell has the expression of at least one gene in a reduced or eliminated biosynthetic pathway.

[0344] Aspect 43: The method as described in aspect 41, wherein the composition is a growth medium for plants.

[0345] Aspect 44: The method as described in aspect 43, wherein the growth medium is soil.

[0346] Aspect 45: A method for increasing the yield of harvested products, comprising introducing microbial cells, spores, secretions or culture media thereof into a plant or a growth medium in which the plant is placed, wherein the microbial cells comprise a heteropolynucleotide sequence that shares at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 5, 13 and 15; Aspect 46: The method of aspect 45, wherein the microbial cell has the expression of at least one gene in a reduced or eliminated biosynthetic pathway.

[0347] Aspect 47: The method described in aspect 41 or aspect 45 further comprises at least one additional microorganism.

[0348] Aspect 48: The method as described in aspect 41 or aspect 45, wherein the harvested product is fruit.

[0349] Aspect 49: The method as described in aspect 41 or aspect 45, wherein the harvested product is vegetables.

[0350] Aspect 50: The method as described in aspect 41 or aspect 45, wherein the harvested product is a seed.

[0351] Aspect 51: The method of aspect 41 or aspect 45, wherein the harvested product is fiber.

[0352] Aspect 52: A substantially cell-free preparation obtained or derived from a culture of a microorganism, wherein the microorganism comprises a heteropolynucleotide sequence sharing at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 5, 13 and 15.

[0353] Aspect 53: A substantially cell-free preparation as described in aspect 52, wherein the microbial cells have the expression of at least one gene in a reduced or eliminated biosynthetic pathway.

[0354] Aspect 54: A purified composition prepared from a substantially cell-free formulation as described in aspect 52, wherein the purified composition comprises SEQ ID NO: 25, 33 and / or 35.

[0355] Aspect 55: Any of the methods or compositions described above, comprising a genetically modified variant of Bacillus thuringiensis deposited with NRRL accession number B-68090, or an isolated bacterial strain having substantially similar morphological and physiological characteristics, substantially similar or substantially identical genetic characteristics, its progeny, mutants or genetically edited, altered or modified variants.

[0356] Aspect 56: An isolated bacterial strain comprising a heteropolynucleotide sequence having at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 1-50.

[0357] Aspect 57: The isolated bacterial strain as described in aspect 56, wherein the microbial cell has the expression of at least one gene in a reduced or eliminated biosynthetic pathway.

[0358] Aspect 58: An agricultural composition comprising: a) an isolated bacterial strain as described in aspect 55 or aspect 56, and b) an agriculturally acceptable carrier; wherein the bacterial strain is present in the agricultural composition in an amount that effectively produces an improved phenotype of the plant associated therewith.

[0359] Aspect 59: Agricultural compositions as described in aspect 58, wherein the agricultural compositions are formulated as seed coatings, foliar sprays, soil irrigation agents, impregnation agents, furrow treatment agents, soil conditioners, granules, broadcast treatment agents, or postharvest disease control treatment agents.

[0360] Aspect 60: A microbial cell comprising a heteropolynucleotide sequence having at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 5, 13 and 15; wherein the microbial cell is heterologously treated into the plant component.

[0361] Aspect 61: Microbial cells as described in aspect 60, wherein the microbial cells have reduced or eliminated endotoxin biosynthetic pathways and the expression of at least one gene in plant components.

[0362] Aspect 62: A composition comprising any of the preceding items, wherein the composition imparts a harmful effect on nematodes.

[0363] While the invention has been particularly shown and described in conjunction with preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes to its form and details may be made without departing from the spirit and scope of the invention. For example, although specific embodiments described below use specific plants to illustrate the methods and embodiments described herein, the principles in these embodiments are applicable to any plant. Therefore, it should be understood that the scope of the invention is covered by the embodiments described herein, and not only by the specific embodiments illustrated below.

[0364] This disclosure enables those skilled in the art to make and use the invention provided herein according to numerous and varied embodiments. Various changes, modifications, and improvements (including certain alterations, modifications, substitutions, and improvements) that will readily occur to those skilled in the art are also part of this disclosure. Therefore, the foregoing detailed descriptions are by way of example illustrating the findings provided herein. Furthermore, the foregoing detailed descriptions and embodiments are illustrative of the invention and not intended to limit it.

[0365] All patents and publications cited in this application are incorporated herein by reference in their entirety for all purposes, as if each were individually and explicitly incorporated by reference.

[0366] Example Genetically modified microbial strains containing nematode-controlling proteins not naturally present in single bacteria were developed. The nematicidal efficacy of each engineered strain was enhanced compared to the wild-type parent strain, and the activity of each added gene was increased relative to the parent strain and other modified strains lacking said added genes.

[0367] Example 1: Strain Selection The exemplary strain PM39400 was selected for genetic modification to enhance nematicidal activity. This strain is a Bacillus thuringiensis isolate with previously identified nematicidal, lepidopteran, and coleopteran activities (deposited on January 26, 2022 as NRLRB-68090; described in international application PCT / US2023 / 060963) (16S RNA DNA sequence given as SEQ ID NO. 37-50). Nematicidal toxin proteins derived from Bacillus were identified from various strains and ranked based on factors including source strain, sequence diversity, spore attachment, and potential nematicidal efficacy.

[0368] Other Bacillus species and strains were considered.

[0369] Example 2: Toxin Cloning and Expression The toxin gene was synthesized by a third-party supplier or amplified from genomic DNA preparations of internal strains. The prepared toxin gene was cloned into the replicative plasmid pBACOV using the Gibson assembly method, adjacent to the strongly constitutive promoter P. aprE Downstream of the gene, the promoter was pre-amplified and cloned into pBACOV using Gibson assembly. Quality control of the new plasmids was performed, including PCR amplification using primers specific to each plasmid construction, followed by size comparison of the amplicon with DNA molecular weight standards, and Sanger sequencing from the upstream (5') edge to the downstream (3') edge of the cassette to confirm the predicted DNA sequence. Using techniques known in the art, the complete expression plasmid containing the toxin gene was conjugated into an alternative Gram-positive host (Bacillus velezensis).

[0370] Example 3: Identification of Insertion Locus The insert cassette is designed to insert into the host bacterial genome in a manner that simultaneously causes the removal of Bacillus thuringiensis expression. Bacillus thuringiensis (β-exotoxin) biosynthesis has been reported to occur via the expression of a gene cluster (Liu et al., Toxins(Basel) 6(8):2229-2238; 2014), which contains various components such as the gene thuE. The thuE gene (SEQ ID NO: 20) is one of the genes in the putative Bacillus thuringiensis biosynthesis gene cluster.

[0371] Genomic sequence analysis was used to identify the thuE gene, which was used to select the predicted Bacillus thuringiensis gene cluster in the target bacterial strains described in this paper. This gene was hypothesized to be associated with Bacillus thuringiensis production. Bacillus thuringiensis production was disrupted by introducing a kanamycin antibiotic resistance gene, and a thuE::kan mutant was created to determine gene function. HPLC analysis confirmed that the thuE::kan mutation disrupts Bacillus thuringiensis production.

[0372] Surrounding genes showed close operon linearity and were hypothesized to function in the thuringin biosynthetic pathway. As described herein, genes including ugd (plaused thuD; UDP-glucose-6-dehydrogenase) (SEQ ID NO: 18) and dltA (plaused thu2; nonribosomal peptide synthase) (SEQ ID NO: 17) were selected as targets for the insertion toxin.

[0373] Example 4: Single toxin nematicidal activity in Bacillus belesiensis A single toxin gene from strain PM39400 was cloned into Bacillus bereaves, and its activity against Caenorhabditis elegans and RKN (root-knot nematodes) was evaluated in a bioassay. The results are shown in Table 1.

[0374] Table 1: Single toxin bioactivity of various natural genes of strain PM39400 The Cry protein, which is not naturally present in strain PM39400, was cloned into an alternative Gram-positive Bacillus belyssus host, and its activity against Caenorhabditis elegans and RKN (root-knot nematode) was evaluated in a bioassay. The results are shown in Table 2.

[0375] Table 2: Single toxin bioactivity of various natural genes heterologous to strain PM39400 (Bacillus thuringiensis) and tested in Bacillus belesia spp. Example 5: Nematode-killing activity of superimposed toxins in Bacillus belyssus A cry gene expression cassette was created for transformation into Bacillus belye and tested in a cucumber root-knot nematode (RKN) bioassay according to the protocol described herein. The results are shown in Table 3.

[0376] Table 3: Nematode-killing activity of superimposed toxins in Bacillus belyssus Example 6: Toxin superposition introduction into Bacillus thuringiensis A cry gene expression cassette was created for transformation into Bacillus thuringiensis to integrate (and thus disrupt) the next gene in the putative Bacillus thuringiensis operon, intended to disrupt Bacillus thuringiensis production and integrate the cry toxin gene for expression.

[0377] Three heterologous toxin genes exhibiting activity (cry21Aa SEQ ID NO: 5, cry6Aa SEQ ID NO: 13, and cry5Ba SEQ ID NO: 15) were cloned into the host wild-type strain PM39400 (Bacillus thuringiensis isolate). The three toxin genes were designed to be integrated sequentially into strain PM39400, so that after a third round of integration and excision, all three toxin genes would be located within the genome of strain PM39400.

[0378] cry6Aa The first toxin gene, cry6Aa, was engineered to integrate into the dltA locus of the parental strain PM39400 (wild-type), resulting in the removal of 42% of the dltA open reading frame (from residue 335_isoleucine to residue 761_serine) after excision of the temperature-sensitive origin of replication. Integration of cry6Aa was facilitated by flanking a 1000 bp homologous recombination region on either side of the dltA integration locus. A 200 bp DNA sequence upstream of the PM39400 cry1Ba gene was cloned upstream of cry6Aa to control the transcription of the introduced cry6Aa toxin gene using the expression of the strain's native cry toxin gene. The resulting strain was named PE39400-G15 and contained a cry6Aa toxin expression cassette integrated at dltA, producing dltA::P cry1Ba -cry6Aa genotype (single superimposed toxin strain).

[0379] cry6Aa + cry5Ba The second toxin gene, cry5Ba, was engineered to integrate into the ugd gene of the parental strain PE39400-G15, such that 70% of the ugd open reading frame (from position -16 to position +811) would be removed after excision of the temperature-sensitive origin of replication. Integration of cry5Ba was facilitated by flanking a 1000 bp homologous recombination region on either side of the ugd integration locus. The homologous recombination region was engineered to avoid capturing the downstream gene xerC, which may inhibit plasmid assembly in *E. coli*. A 200 bp DNA sequence upstream of the cry1Ba gene from *Bacillus thuringiensis* PM39400 was cloned upstream of cry6Aa to utilize the expression of the strain's native cry toxin gene to control the transcription of the introduced cry6Aa toxin gene. The resulting strain was named PE39400-G27 and contained a cry6Aa toxin expression cassette integrated at dltA and a cry5Ba toxin expression cassette integrated at ugd (genotype dltA::P). cry1Ba -cry6Aa + ugd::P cry1Ba -cry5Ba (double superimposed toxin strain).

[0380] cry6Aa + cry5Ba + cry21Aa The third toxin gene, cry21Aa, was designed to integrate into the cry2Aa locus, ensuring 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. Furthermore, the integration was designed so that the native cry2Aa promoter would drive the transcription of the introduced cry21Aa gene. The integration utilized a 1000 bp homologous recombination region flanking either side of the cry2Aa integration site. The resulting strain was named PE39400-G27 and contained both a cry6Aa toxin expression cassette integrated at dltA and a cry21Aa toxin expression cassette integrated at cry2Aa. The resulting strain was named PE39400-G30 and contained a cry6Aa toxin expression cassette integrated at dltA, a cry21Aa toxin expression cassette integrated at cry2Aa, and a cry5Ba toxin expression cassette integrated at ugd (genotype dltA::P). cry1Ba -cry6Aa ugd::P cry1Ba -cry5Ba cry2Aa::cry21Aa)(Triadic toxin strain).

[0381] Promoter swapping The strain PE39400-G30 was modified as follows: the dlta locus was targeted for promoter exchange to replace the natural promoter Pcry1Ba (SEQ ID NO: 53). The resulting strain (CE39400-G80) contained a new promoter driving cry6Aa expression at the dltA locus within the PE39400-G80 chromosome. To generate plasmid pAP69 for promoter exchange in PE39400-G30, a PCR product of approximately 1000 bp containing upstream and downstream regions of Pcry1Ba was amplified from CE39400-G30 using appropriate primer pairs and Q5 high-fidelity polymerase. The exogenous PaprE promoter (SEQ ID NO: 54), approximately 200 bp in length, was amplified from the Bioconsortia plasmid pBACOV using appropriate primers. All primer pairs contained appropriate Gibson assembly overhangs to facilitate isothermal assembly of fragments with each other and with the linearized cloning vector pMMDmob, resulting in a final construct containing an upstream region of Pcry1Ba directly linked to the PaprE promoter, which in turn directly links to a downstream region of Pcry1Ba. The cloning vector pMMDmob was linearized using EcoR1 and BamH1 endonucleases and purified by gel electrophoresis. The DNA fragment composed of the aforementioned regions was purified by gel electrophoresis. Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment in a 1:3:5:3 backbone:insert:insert:insert molar ratio in 10 μL, followed by addition to 10 μL of 2x Gibson master mix reagent. The reaction mixture was incubated at 50°C for 60 min and then used to transform *E. coli* DH5α. Ampicillin-resistant *E. coli* colonies were cloned and isolated, and the putatively assembled plasmid was purified and confirmed by nanopore full-length plasmid sequencing. The correctly assembled plasmid was transformed into *E. coli* dam, purified, and then transformed into electrocompetent cells PE39400-G30. PE39400-G30 colonies resistant to antibiotic resistance markers on pAP69 (MLS) were selected for integration into the host chromosome, followed by excision of the plasmid from the host chromosome. The resulting strain was confirmed to contain PaprE instead of Pcry1Ba at the dltA locus driving cry6Aa transcription. Subsequent Sanger and Illumina sequencing confirmed that no other mutations were generated during this process. The resulting strain was named PE39400-G80 and contained the genotype dltA::PaprE-cry6Aa ugd::Pcry1Ba-cry5Bacry2Aa::cry21Aa.

[0382] aprE deficiency The strain PE39400-G30 was modified as follows: the aprE gene (SEQ ID NO: 51) was targeted for in-frame label-free deletion. The resulting strain (PE39400-G89) contained the aprE gene deletion within the PE39400-G30 chromosome. To generate plasmid pAP79 for creating in-frame label-free deletion in PE39400-G89, a PCR product containing approximately 1000 bp of upstream and downstream regions of aprE was amplified from PM39400 using appropriate primer pairs and Q5 high-fidelity polymerase. The primer pairs contained appropriate Gibson assembly overhangs to promote isothermal assembly between fragments and with the linearized cloning vector pMMDmob, such that the final construct contained an upstream region of aprE directly linked to the downstream region of aprE, maintaining the reading frame from the aprE start codon to the aprE stop codon, while also eliminating the 2658 bp in the middle of the aprE coding sequence. pMMDmob was linearized using EcoR1 and BamH1 endonucleases and purified by gel electrophoresis. DNA fragments comprising the aforementioned regions were purified by gel electrophoresis. Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment at a 1:3:3 backbone:insert:insert molar ratio in 10 μL, followed by addition to 10 μL of 2x Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used to transform into *E. coli* DH5α. Ampicillin-resistant *E. coli* colonies were cloned and isolated, and the proposed assembled plasmid was purified and confirmed by nanopore full-length plasmid sequencing. The correctly assembled plasmid was transformed into *dam-E. coli*, purified, and then transformed into electrocompetent PE39400-G30 cells. Colonies of PE39400-G30, resistant to antibiotic resistance markers on pAP79 (MLS), were selected for integration into the host chromosome, followed by excision of the plasmid from the host chromosome. The resulting strain was confirmed to contain an unmarked in-frame deletion of aprE. Subsequent Sanger and Illumina sequencing confirmed that no other mutations were generated through this process. The resulting strain was named PE39400-G89 and contained the genotype dltA::Pcry1Ba-cry6Aa ugd::Pcry1Ba-cry5Ba cry2Aa::cry21Aa ΔaprE.

[0383] nprA missing The strain PE39400-G30 was modified as follows: the nprA gene (SEQ ID NO: 52) was targeted for in-frame label-free deletion. The resulting strain (PE39400-G91) contained the nprA gene deletion within the PE39400-G30 chromosome. To generate plasmid pAP80 for creating in-frame label-free deletion in PE39400-G91, a PCR product containing approximately 1000 bp of upstream and downstream regions of nprA was amplified from PM39400 using appropriate primer pairs and Q5 high-fidelity polymerase. The primer pairs contained appropriate Gibson assembly overhangs to promote isothermal assembly between fragments and with the linearized cloning vector pMMDmob, such that the final construct contained an upstream region of nprA directly linked to the downstream region of nprA, maintaining the reading frame from the nprA start codon to the nprA stop codon, while also eliminating the 1251 bp in the middle of the nprA coding sequence. pMMDmob was linearized using EcoR1 and BamH1 endonucleases and purified by gel electrophoresis. DNA fragments comprising the aforementioned regions were purified by gel electrophoresis. Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment at a 1:3:3 backbone:insert:insert molar ratio in 10 μL, followed by addition to 10 μL of 2x Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used to transform into *E. coli* DH5α. Ampicillin-resistant *E. coli* colonies were cloned and isolated, and the proposed assembled plasmid was purified and confirmed by nanopore full-length plasmid sequencing. The correctly assembled plasmid was transformed into *dam-E. coli*, purified, and then transformed into electrocompetent PE39400-G30 cells. Colonies of PE39400-G30, resistant to antibiotic resistance markers on pAP80 (MLS), were selected for integration into the host chromosome, followed by excision of the plasmid from the host chromosome. The resulting strain was confirmed to contain an unmarked in-frame deletion of nprA. Subsequent Sanger and Illumina sequencing confirmed that no other mutations were generated during this process. The resulting strain was named PE39400-G91 and contained the genotype dltA::Pcry1Ba-cry6Aa ugd::Pcry1Ba-cry5Ba cry2Aa::cry21Aa ΔnprA.

[0384] aprE and nprA deficiency The strain PE39400-G89 was modified as follows: the nprA gene was targeted for in-frame marker-free deletion. The resulting strain (PE39400-G94) contained an nprA gene deletion within the PE39400-G89 chromosome, which itself contained an aprE gene deletion (see above). To generate plasmid pAP80 for creating an in-frame marker-free deletion in PE39400-G94, a PCR product containing approximately 1000 bp of upstream and downstream regions of nprA was amplified from PM39400 using appropriate primer pairs and Q5 high-fidelity polymerase. The primer pairs contained appropriate Gibson assembly overhangs to promote isothermal assembly between fragments and with the linearized cloning vector pMMDmob, resulting in a final construct containing an upstream region of nprA directly linked to the downstream region of nprA, maintaining the reading frame from the nprA start codon to the nprA stop codon, while also eliminating the 1251 bp in the middle of the nprA coding sequence. pMMDmob was linearized using EcoR1 and BamH1 endonucleases and purified by gel electrophoresis. DNA fragments comprising the aforementioned regions were purified by gel electrophoresis. Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment at a 1:3:3 backbone:insert:insert molar ratio in 10 μL, followed by addition to 10 μL of 2x Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used to transform into *E. coli* DH5α. Ampicillin-resistant *E. coli* colonies were cloned and isolated, and the proposed assembled plasmid was purified and confirmed by nanopore full-length plasmid sequencing. The correctly assembled plasmid was transformed into *dam-E. coli*, purified, and then transformed into electrocompetent PE39400-G30 cells. Colonies of PE39400-G30, resistant to antibiotic resistance markers on pAP80 (MLS), were selected for integration into the host chromosome, followed by excision of the plasmid from the host chromosome. The resulting strain was confirmed to contain an unmarked in-frame deletion of nprA. Subsequent Sanger and Illumina sequencing confirmed that no other mutations were generated during this process. The previously generated aprE deletion was confirmed to remain intact after the nprA deletion. The resulting strain was named PE39400-G94 and contained the genotype dltA::Pcry1Ba-cry6Aa ugd::Pcry1Ba-cry5Ba cry2Aa::cry21Aa ΔaprE ΔnprA.

[0385] Example 7: Promoter Function Transcriptional activity of the promoters (PaprE, Pcry1Ba, Pcry2Aa) used in triple-addition strains was tested in plasmids containing green fluorescent protein (GFP) as a reporter gene. The following plasmids were constructed: PE39400-T106 (oriPUBPaprE-sfGFP kan amp), PE39400-G118 (pAP86 oriPUB traJ Pcry1Ba-sfGFP kan amp), PE39400-G119 (pAP87 oriPUB traJ Pcry2Aa-sfGFP kan amp), and PE39400-G120 (pAP88oriPUB traJ PaprE-His6x kan amp). Each plasmid was transformed into the parental strain PM39400.

[0386] All strains were streaked onto LB agar plates containing 50 μg / mL kanamycin and grown overnight at 37°C. The next day, the strains were inoculated into LB medium containing 50 μg / mL kanamycin, 5 mL of which was transferred to 50 mL conical tubes and grown at 30°C at 200 RPM for 5 days. Every 24 hours, 200 μL of each culture was sampled and resuspended in PBS (phosphate-buffered saline) to the same concentration. 100 μL of each culture was aliquoted into the wells of a 96-well plate. OD600 and fluorescence were measured using a Cytation 5 imaging plate reader. Relative fluorescence units were calculated as mean fluorescence divided by mean OD600. Spore formation was assessed at each time point using a microscope. Macroscopic spore formation phenotypes were based on cytological examination.

[0387] Data shown Figure 5 The plus and minus signs indicate the presence or absence of sporulation, respectively. PaprE (strain T106) shows constitutive activity. Under these growth conditions, Pcry1Ba exhibits strong activity during sporulation.

[0388] Example 8: Bioassay Caenorhabditis elegans To determine the possible modes of action of nematode-killing microorganisms, the model organism *C. elegans* (a free-living, soil-dwelling nematode) was used in several different assays. First, developmental tests were conducted to determine activity. Young larvae were fed the microorganisms. During this process, the worm's development and reproductive capacity were tracked. Based on the rate at which *C. elegans* reached the reproductive stage and the number of offspring produced, how these microorganisms affect the nematode was determined.

[0389] Secondly, the mortality rate of the adult nematodes was assessed. Adult nematodes were introduced into the microorganisms, and the mortality rate was tracked. This demonstrated the toxicity of the microorganisms to the nematodes.

[0390] The chemotaxis of *C. elegans* was also tested in a chemotaxis assay. The worm was introduced into two different stimuli (e.g., microorganisms, chemicals, metabolites, etc.). The chemotaxis index, i.e., repulsion or attraction, was calculated based on the worm's movement in response to these stimuli.

[0391] RKN Two weeks before application of the treatment, sow the seeds of the susceptible crop in pots containing a 1:1 mixture of soil and sand. Sow an excess of seeds per pot, then thin to one seedling per pot.

[0392] Two weeks after planting, apply the microbial treatment solution to the soil at the base of the plant at a rate of 1 × 10^7 CFU / mL per plant. The volume applied should be sufficient to cover the root zone in the pot.

[0393] Avoid watering the plants immediately before or after treatment (do not water heavily on the day of treatment). Resume regular watering the following day.

[0394] Forty-eight hours after treatment, apply nematodes at the desired concentration. In some cases, second-instar larvae (J2) are used instead of eggs to reduce variability.

[0395] On the morning of the nematode application, the plants were first sprayed with water via mist, followed by nematode infestation. Four weeks after nematode application, branches were harvested to determine their dry weight, and the roots were cleaned for nematode assessment.

[0396] Stain the egg masses on the roots and count the egg masses, galls, eggs, etc. (galls, eggs, egg masses, cysts, female insects, etc.).

[0397] Obtain the root dry weight and calculate the number of nematodes per gram of root dry weight.

[0398] Positive and negative controls include: untreated group (no nematode inoculation, no microbial treatment, only water irrigation), inoculated control (nematode inoculation, no microbial treatment, only water irrigation), and positive control (using a known nematicide product targeting the tested nematode, ideally with both chemical and biological nematicides as positive controls in the same experiment).

[0399] Root rot nematodes Two weeks before application of the treatment, sow the seeds of the susceptible crop (sorghum) in pots containing a 1:1 mixture of soil and sand. Sow an excess of seeds in each pot, then thin the seedlings to one seedling per pot.

[0400] Two weeks after planting, apply the microbial treatment solution to the soil at the base of the plant at a rate of 1 × 10^7 CFU / mL per plant. The volume applied should be sufficient to cover the root zone in the pot.

[0401] Avoid watering the plants immediately before or after treatment (do not water heavily on the day of treatment). Resume regular watering the following day.

[0402] Forty-eight hours after application, apply root-rot nematodes to the roots at the desired concentration. On the morning of the nematode application, first mist the plants with water, then infect them with nematodes. Six weeks after nematode application, harvest branches to determine their dry weight and extract roots for nematode counting.

[0403] Count the number of nematodes on each plant.

[0404] Obtain the root dry weight and calculate the number of nematodes per gram of root dry weight.

[0405] Positive and negative controls include: untreated group (no nematode inoculation, no microbial treatment, only water irrigation), inoculated control (nematode inoculation, no microbial treatment, only water irrigation), and positive control (using a known nematicide product targeting the tested nematode, ideally with both chemical and biological nematicides as positive controls in the same experiment).

[0406] The results are shown in Tables 4 and 5, and Figures 1-4 middle.

[0407] Table 4: Cumulative nematicidal activity of toxins Table 5: Cumulative nematicidal activity of toxins

Claims

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) Insert at least one heterologous polynucleotide encoding Cry toxin; (b) Insert at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and (c) Reduce or eliminate the expression of at least one gene in the biosynthetic pathway; The engineered bacterial cells, compared to control bacterial cells of the same strain that are not engineered, confer an improved phenotype on the associated plant; wherein the improved phenotype on the plant is selected from the group consisting of: improved toxicity against nematodes, improved plant growth, improved plant health, and any and / or combinations thereof.

2. The engineered bacterial cell of claim 1, wherein the at least one gene in the 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 the toxin biosynthesis pathway comprises a sequence that shares at least 97% identity with a sequence selected from at least one of the following groups: SEQ ID NO: 20, 51 and 52.

4. The engineered bacterial cell of claim 1, wherein the heteropolynucleotide of (a) or (b) is inserted into the genome of the cell in a coding sequence located in the Bacillus thuringiensis biosynthesis gene cluster.

5. The engineered bacterial cell of claim 1, wherein the at least one heteropolynucleotide of (a) or (b) comprises a sequence having at least 97% identity with a sequence selected from the group consisting of: SEQ ID NO: 1-16.

6. The engineered bacterial cell of claim 1, wherein the at least one heteropolynucleotide of (a) or (b) encodes a polypeptide that shares at least 97% identity with a sequence selected from the group consisting of: SEQ ID NO: 21-36.

7. The engineered bacterial cell of claim 1, wherein the heteropolynucleotide of (a) or (b) is operatively linked to a heteropromoter.

8. The engineered bacterial cell as described in claim 1, wherein the Bacillus genus cell is a Bacillus thuringiensis species.

9. A synthetic composition comprising engineered bacterial cells as described in claim 1, or their secretions, or their culture medium, or their spores, or any combination thereof; further comprising at least one composition selected from the group consisting of: plant components, formulation components, agricultural compositions, and any combination thereof. The plant component is selected from the group consisting of: seeds, leaves, roots, whole plant, and / or any of the foregoing and / or combinations thereof; The formulation component is selected from the group consisting of: compounds that improve microbial stability, preservatives, carriers, surfactants, anti-complexing agents, and any combination and / or more of the foregoing; and The agricultural composition thereof is selected from the group consisting of fungicides, nematicides, bactericides, insecticides, herbicides, growth media, and any combination and / or more of the foregoing. Furthermore, the engineered bacterial cells are present in the liquid formulation at a concentration of at least about 10^2 CFU / mL, or in the non-liquid formulation at a concentration of at least about 10^2 colony-forming units / gram.

10. The synthetic composition of claim 9, further comprising at least one additional microorganism.

11. The synthetic composition of claim 9, wherein the plant component comprises a genetically modified organism.

12. The synthetic compositions of various claims 9, wherein the synthetic compositions are substantially enclosed within an object selected from the group consisting of: tubes, bottles, wide-mouth bottles, ampoules, packaging, containers, bags, boxes, silos, envelopes, cartons, containers, silos, shipping containers, truck beds, and boxes.

13. The synthetic composition of claim 9, wherein the plant component is obtained from plants selected from the group consisting of: corn, soybean, wheat, cotton, cucumber, tomato, pepper, potato, strawberry, orange, lemon, lime, apple, kidney bean, zucchini, pea, lettuce, broccoli, celery, cauliflower, sorghum, rapeseed, rapeseed and okra.

14. The synthetic compositions of claim 9, wherein the growth medium comprises soil, wherein the synthetic compositions are arranged in a regular pattern in the soil, and the spacing between each of the synthetic compositions is substantially equal.

15. A method for conferring plant nematode tolerance, the method comprising: Applying a formulation comprising engineered bacterial cells of the genus Bacillus to a plant component of the plant, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) Insert at least one heterologous polynucleotide encoding Cry toxin; (b) Insert at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba, and cry3Bb1; and (c) Reduce or eliminate the expression of at least one gene in the biosynthetic pathway.

16. The method of claim 15, wherein the application to the plant component is performed by an indirect method selected from the group consisting of: furrow application, soil irrigation application, and lateral fertilization application.

17. The method of claim 15, wherein the application to the plant component is accomplished by coating the plant component with a liquid formulation of the microorganism or its secretions.

18. The method of claim 15, wherein the application to the plant component is accomplished by coating the plant component with a substantially non-liquid formulation of the microorganism or its secretions.

19. The method of claim 15, wherein the plant component is a seed, leaf, root, whole plant and / or any and / or combination of the foregoing.

20. A method for reducing the number of pests in or on a composition, wherein the pests include nematodes, nematode eggs, nematode larvae, or any combination thereof, the method comprising: Introducing engineered Bacillus cells into the composition, wherein the engineered bacterial cells contain at least one modification to their genome, wherein the at least one modification is selected from the group consisting of: (a) Insertion of at least one heterologous polynucleotide encoding Cry toxin; and (b) Insert at least one heteropolynucleotide selected from the group consisting of: app6Ba1, cry2Ab35, cry5B, cry12A, cry21Aa, cry5Aa, cry55a1, cry1Aa, cry1Ba, cry1Ia, cry2Aa, cry2Ab, cry6Aa, app4a1, cry5Ba and cry3Bb1.

21. The method of claim 20, wherein the composition is a growth medium for plants.

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

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

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