Method for improving response of plants to pests and pathogens

Abstract

The present invention provides methods for increasing the response of a plant to a pest and / or pathogen by increasing the production of the plant by one or more plant defense compounds derived from anthranilic acid. Also provided are methods for identifying and selecting microbial strains useful for plant treatment to increase plant response to attack by pests and / or pathogens.

Description

[0001] Citation of priority claims

[0002] This patent application claims the benefits of: U.S. Provisional Patent Application Serial No. 63 / 561,055, filed March 4, 2024; U.S. Provisional Patent Application Serial No. 63 / 606,485, filed December 5, 2023; and U.S. Provisional Patent Application Serial No. 63 / 504,299, filed May 25, 2023.

[0003] Sequence list declaration

[0004] This application contains a sequence list, which has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML file, created on May 16, 2024, is named P14472WO00.xml and has a size of 251,036 bytes. Background Technology

[0005] Plants have developed a variety of defense mechanisms to resist attacks from various organisms, including fungi, bacteria, viruses, nematodes, and insects. These mechanisms include structural barriers, the production of chemicals toxic to invading organisms, the production of chemicals that attract natural enemies of target pests or pathogens, and hypersensitive responses characterized by rapid cell death upon infection. Despite these natural protective mechanisms, crop losses due to damage caused by plant pathogens and pests incur significant costs to the global economy each year. New approaches are needed to reduce the impact of pest and pathogen attacks on plants. Summary of the Invention

[0006] This document provides methods for improving plant responses to pathogens or pests by increasing the levels of one or more plant defense compounds produced in the plant. Such methods include treating a plant, plant part, or seed with a strain of microorganism that is nonpathogenic to the plant, wherein the treatment increases the expression of one or more gene transcripts involved in the production of plant defense compounds in the plant; and growing the plant in the presence of a pathogen or pest, thereby increasing the levels of one or more plant defense compounds in the plant compared to a control plant, and improving the plant's response to the pathogen or pest compared to a control plant, wherein the control plant is not genetically modified or treated with the microbial strain. In some embodiments, the microbial strain used in the methods provided herein expresses one or more genes in pathways for producing siderophores, and / or one or more genes in pathways for producing polyketide compounds. In some embodiments, such microbial strains will enhance induced systematic resistance (ISR) plant defense responses in the treated plant or plants grown from treated seeds, plant parts, or plants grown in treated soil. In some embodiments, the plant defense response provides the production of metabolites that repel or otherwise reduce the effects of plant pests or pathogens on the plant. In some embodiments, the plant pest is an insect. In some embodiments, the protein in the pathway for generating a polyketide compound that induces a plant response to a pathogen or pest is present on a plasmid. In some embodiments, the protein in the pathway for generating the polyketide compound is encoded by a gene on SEQ ID NO:87 or a variant thereof. In some embodiments, the protein in the pathway for generating a polyketide compound that induces a plant response to a pathogen or pest is encoded by a gene on SEQ ID NO:86 or a variant thereof. In some embodiments, the protein in the pathway for generating a polyketide compound that induces a plant response to a pathogen or pest is encoded by a polynucleotide having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:36-50. In some embodiments, the protein in the pathway for generating a polyketide compound that induces a plant response to a pathogen or pest comprises a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO:21-35. In some embodiments, the protein in the microorganism that induces a plant response to a pathogen or pest is a homolog or ortholog of any of SEQ ID NO: 21-35. In some embodiments, the gene in the pathway for producing the polyketide compound is... bfmBAB_ 2 genes. In some embodiments, bfmBAB_2 comprises a polynucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:36. In some embodiments, bfmBAB_ Gene 2 encodes a protein having the sequence SEQ ID NO:21. In some embodiments, bfmBAB_ The 2 gene has at least 70% identity with SEQ ID NO:36, and / or encodes a protein having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21. In some embodiments, the bacterial strain is a genus *Methylobacterium* (…). Methylobacterium ) or methyl erythrobacter ( Methylorubrum The bacterial strain is NLS0042 (NRRL B-50932). In some embodiments, the bacterial strain is not NLS0042 (NRRL B-50932). In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots. In some embodiments, the insect larvae are repelled by plant roots. In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots compared to a control plant. In some embodiments, the plant is maize, and the insect pest is the maize rootworm. In some embodiments, root regrowth is increased after exposure to the pathogen or pest compared to a control plant.

[0007] In some embodiments provided herein, methods for improving plant responses to pathogen or pest attacks increase the levels of one or more plant defense compounds derived from anthranilic acid produced in the plant. Such methods include the steps of: modifying a plant genome to increase the expression of one or more gene transcripts involved in the production of one or more plant defense compounds derived from anthranilic acid in the plant, and / or treating the plant, plant parts, or seeds with a non-pathogenic microbial strain, wherein the treatment increases the expression of one or more gene transcripts involved in the production of one or more plant defense compounds derived from anthranilic acid in the plant; and growing the plant in the presence of a pathogen or pest, thereby increasing the levels of one or more plant defense compounds in the plant compared to a control plant, and improving the plant's response to the pathogen or pest compared to a control plant, wherein the control plant is not genetically modified or treated with the microbial strain. In some embodiments, the expression of one or more gene transcripts associated with the production of anthranilic acid and / or the conversion of anthranilic acid to plant defense compounds is increased compared to a control plant. In some embodiments, the transcription of genes encoding components of anthranilic acid synthase protein is increased compared to a control plant. In some embodiments, transcription of the α or β subunit component gene of anthranilic acid synthase is increased compared to control plants. In some embodiments, transcription of the gene encoding anthranilic acid N-benzoyltransferase is increased compared to control plants. In some embodiments, the plant defense compound is anthranilic ester. In some embodiments, the anthranilic ester is selected from the group consisting of methyl anthranilate, dimethyl anthranilate, ethyl anthranilate, phenethyl anthranilate, and menthyl anthranilate. In some embodiments, the plant defense compound is an anthranilic acid-derived phytoalexin. In some embodiments, the microbial strain that enhances the plant's response to pathogens or pests is a bacterial strain. In some embodiments, the bacterial strain is *Methylobacterium* (…). Methylobacterium ) or methyl erythrobacter ( MethylorubrumThe bacterial strain is NLS0042 (NRRL B-50932). In some embodiments, the bacterial strain is not NLS0042 (NRRL B-50932). In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots. In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots compared to a control plant. In some embodiments, the plant is maize, and the insect pest is the maize rootworm. In some embodiments, root regrowth is increased after exposure to the pathogen or pest compared to a control plant. In some embodiments, the expression of the anthranilate synthase protein component having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or the protein having the amino acid sequence of SEQ ID NO:4 is increased compared to a control plant. In some embodiments, compared with control plants, the expression of anthranilic acid synthase protein or protein components comprising amino acid sequences having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9, 10, 12, 14, 15, 17, 18, or 20 is increased. In some embodiments, compared with control plants, the expression of the gene encoding anthranilic acid N-benzoyltransferase of SEQ ID NO: 6 or SEQ ID NO: 7 is increased. In some embodiments, compared with control plants, the expression of the gene encoding anthranilic acid N-benzoyltransferase having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 6 or SEQ ID NO: 7 is increased. In some embodiments of the methods provided herein, the plant defense compound is not an indole derivative.

[0008] In some embodiments of the methods provided herein, the insect pest is a thrips, and the treated plant is corn, soybean, cotton, peanut, potato, tomato, or strawberry. In some embodiments, the insect pest is an aphid, and the plant is soybean, cotton, wheat, potato, tomato, strawberry, or pepper. In some embodiments, the insect pest is a fall armyworm, and the plant is soybean, cotton, wheat, rice, or strawberry. In some embodiments, the insect pest is a leafhopper, and the plant is rice, potato, tomato, or bean. In some embodiments, the insect pest is a lepidopteran insect, and the plant is soybean, tomato, pepper, or bean. In some embodiments, the insect pest is a nematode, and the plant is soybean, cotton, potato, or tomato. In some embodiments, the insect pest is a wireworm, and the plant is corn, soybean, or potato. In some embodiments, the insect pest is a mirid bug, and the plant is cotton, tomato, or strawberry. In some embodiments, the insect pest is a cutworm, and the plant is corn or strawberry. In some embodiments, the insect pest is a flea beetle, and the plant is brassica. In some embodiments, the insect pest is a gall midge, and the plant is a soybean plant. In some embodiments, the insect pest is a stink bug, and the plant is a tomato plant. In some embodiments, the insect pest is a potato beetle, and the plant is a potato plant. In some embodiments, the insect pest is a water weevil, and the plant is a rice plant. In some embodiments, the insect pest is a wheat stem sawfly, a cereal leaf beetle, or a wheat mite, and the plant is a wheat plant. In some embodiments, the insect pest is an alfalfa planthopper, a corn borer, or a burrowing stink bug, and the plant is a peanut plant. In some embodiments, the insect pest is a grub, and the plant is a soybean plant. In some embodiments, the insect pest is a corn rootworm, and the plant is a corn plant.

[0009] A method is provided for reducing or repelling pathogens or pests by increasing the level of one or more plant defense compounds, wherein such microbial strains protect plant roots from attack by pathogens or pests. In some embodiments, proteins in the pathway for producing polyketide compounds that induce a plant response to pathogens or pests are present on a mobile plasmid, optionally wherein the mobile plasmid is heterologous to a bacterial strain containing the mobile plasmid. In some embodiments, the proteins in the pathway for producing the polyketide compounds are encoded by a gene on SEQ ID NO:87 or a variant thereof. A variant of SEQ ID NO:87 comprises: (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:87, and / or (ii) a DNA sequence encoding the protein encoded by SEQ ID NO:87. In some embodiments, proteins in the pathway for producing polyketide compounds that induce a plant response to pathogens or pests are encoded by a gene on SEQ ID NO:86 or a variant thereof. Variants of SEQ ID NO: 86 comprise: (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 86, and / or (ii) a DNA sequence encoding a protein encoded by SEQ ID NO: 86. In some embodiments, the protein in the pathway for generating a polyketide compound that induces a plant response to a pathogen or pest is encoded by a polynucleotide SEQ ID NO: 36-50. In some embodiments, the protein in the pathway for generating a polyketide compound that induces a plant response to a pathogen or pest comprises a polypeptide having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 21-35. In some embodiments, the protein in a microorganism that induces a plant response to a pathogen or pest is a homolog or ortholog of any of SEQ ID NO: 21-35. In some embodiments, the gene in the pathway for generating the polyketide compound is... bfmBAB_ 2 genes. In some embodiments, bfmBAB_ 2 has a sequence having SEQ ID NO:36. In some embodiments, bfmBAB_ Gene 2 encodes a protein having the sequence SEQ ID NO:21. In some embodiments, bfmBAB_ The 2 gene shares at least 70% identity with SEQ ID NO:36 and / or encodes a protein sharing at least 80% identity with SEQ ID NO:21. In some embodiments, the bacterial strain is a genus *Methylobacterium* (…). Methylobacterium ) or methyl erythrobacter ( MethylorubrumIn some embodiments, the bacterial strain is NLS0042 (NRRL B-50932). In some embodiments, the bacterial strain is not NLS0042 (NRRL B-50932). In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots. In some embodiments, the insect larvae are repelled by plant roots. In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots compared to a control plant. In some embodiments, the plant is corn, and the insect pest is corn rootworm. In some embodiments, root regrowth is increased after exposure to the pathogen or pest compared to a control plant. In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots. In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots compared to a control plant. In some embodiments, the plant is corn, and the insect pest is corn rootworm. In some embodiments, the microbial strain is... Methylobacterium or Methylrubia Strain. In some embodiments, the microbial strain is NLS0042 (NRRL B-50932) or a derivative thereof. In some embodiments, the microbial strain is not NLS0042 (NRRL B-50932). In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots. In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots compared to a control plant. In some embodiments, the plant is maize, and the insect pest is the maize rootworm.

[0010] Methods for reducing or repelling pathogens or pests by treating soil, plants, plant parts, or seeds with microbial strains to produce metabolites derived from such microbial strains, wherein such metabolites enhance the defense mechanisms of the plant, plant parts, or seeds, and such defense mechanisms protect the plant roots from attack by pathogens or pests. In some embodiments, methods for reducing or repelling pathogens or pests include treating soil, plants, plant parts, or seeds with microbial strains expressing metabolites; and growing plants in the presence of pathogens or pests, whereby the treated plants, plant parts, or seeds repel more pathogens or pests or reduce pathogen or pest ingestion compared to control plants, wherein control plants are not genetically modified or treated with said microbial strains. Methods for reducing or repelling pathogens or pests by treating soil, plants, plant parts, or seeds with microbial strains to produce one or more metabolites and / or peptides derived from such microbial strains are also provided, wherein such one or more metabolites and / or peptides enhance the defense mechanisms of the plant, plant parts, or seeds, and such defense mechanisms protect the plant roots from attack by pathogens or pests. In some embodiments, methods for reducing or repelling pathogens or pests include treating soil, plants, plant parts, or seeds with a microbial strain, wherein the microbial strain expresses one, several, or gene pathways involved in metabolite biosynthesis, wherein one or more metabolites enhance the plant's response to pathogens and / or pests; and growing the plants in the presence of pathogens or pests, thereby repelling or reducing pathogen or pest ingestion by the treated plants, plant parts, or seeds compared to control plants, wherein the control plants are not the microbial strain or have been treated with the microbial strain. In some embodiments, the microbial strain produces siderophores or polyketides. In some embodiments, the polyketides produced by the microbial strain are antimicrobial compounds. In some embodiments, proteins in pathways for producing polyketides that induce plant responses to pathogens or pests are present on mobile plasmids. In some embodiments, proteins in pathways for producing polyketides are encoded by genes in SEQ ID NO: 87 or variants thereof. Variants of SEQ ID NO: 87 comprise: (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 87, and / or (ii) a DNA sequence encoding a protein encoded by SEQ ID NO: 87. In some embodiments, the protein in the pathway for generating a polyketide compound that induces a plant response to a pathogen or pest is encoded by a gene on SEQ ID NO: 86 or a variant thereof.Variants of SEQ ID NO: 86 comprise: (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 86, and / or (ii) a DNA sequence encoding a protein encoded by SEQ ID NO: 86. In some embodiments, the protein in the pathway for generating a polyketide compound that induces a plant response to a pathogen or pest is encoded by SEQ ID NO: 36-50. In some embodiments, the protein in the pathway for generating a polyketide compound that induces a plant response to a pathogen or pest has a sequence of any one of SEQ ID NO: 21-35. In some embodiments, the protein in a microorganism that induces a plant response to a pathogen or pest is a homolog or ortholog of any one of SEQ ID NO: 21-35. In some embodiments, the gene in the pathway for generating the polyketide compound is... bfmBAB_ 2 genes. In some embodiments, bfmBAB_ 2. A sequence having SEQ ID NO:36. In some embodiments, bfmBAB_ Gene 2 encodes a protein having the sequence SEQ ID NO:21. In some embodiments, bfmBAB_ 2. The gene has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:36, and / or encodes a protein with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:21. In some embodiments, the plant is a cereal crop, such as corn, rice, wheat, rye, oats, barley, and millet, or the plant is turfgrass, soybean, strawberry, or cotton, and the insect pest is an armyworm. In some embodiments, the armyworm is the fall armyworm. In some embodiments, the armyworm is the beet armyworm (Spodoptera litura). Spodoptera ) species, including but not limited to fall armyworm ( S. frugiperda ) and beet armyworm ( S. exiqua In some embodiments, the plant is a nightshade plant, such as tomato, tobacco, eggplant, pepper, and potato, and the insect pest is a hawk moth. In some embodiments, the hawk moth is a hawk moth (…). Manduca ) species, including the tomato hawk moth ( M. quinquemaculata ) and tobacco hawk moth ( M. sexta In some embodiments, the plant is soybean, and the insect pest is the soybean looper. In some embodiments, the plant is tobacco or cotton, and the pest is a thrips. In some embodiments, the thrips is the western flower thrips. In some embodiments, the microbial strain is... Methylobacterium or MethylrubiaStrain. In some embodiments, the microbial strain is NLS0042 (NRRLB-50932) or a derivative thereof. In some embodiments, the microbial strain is not NLS0042 (NRRLB-50932). In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots. In some embodiments, the plant defense compound reduces insect larvae feeding on plant roots compared to a control plant. In some embodiments, the plant is maize, and the insect pest is the maize rootworm.

[0011] This document provides a method for identifying microbial strains that enhance a plant's response to a pathogen or pest, wherein the microbial strain is non-pathogenic to the plant. Such a method includes the steps of: (i) treating a plant, plant part, or plant seed with at least a first microbial strain that is not a pathogen of the plant to obtain treated seeds and / or treated plants; (ii) growing the treated plant in the presence of the pathogen or pest, or growing a plant from a treated plant part or treated seed; (iii) collecting one or more tissue samples from the plant and an untreated control plant, wherein the tissue samples are collected during a growth phase during which the pest or pathogen is attacking the tissue samples; and (iv) measuring the samples to identify an increase in the production of one or more plant defense compounds derived from anthranilic acid in the treated plant compared to the control plant, thereby identifying a microbial strain that enhances the plant's response to the pathogen or pest. In some embodiments, the control plant is an untreated plant. In some embodiments, the control plant is treated with a different microorganism. In some embodiments, such a method further includes the step of selecting samples for analysis from treated plants that exhibit reduced damage from the pathogen or pest compared to the control plant. In some embodiments, samples are measured to determine the levels of one or more gene transcripts associated with the production of anthranilic acid and / or the conversion of anthranilic acid into plant defense compounds. In some embodiments, samples are measured to determine the levels of one or more plant defense compounds derived from anthranilic acid. In some embodiments, the pathogen or pest is a fungus, bacteria, nematode, insect, or virus. In some embodiments, the treated plant parts are selected from the group consisting of: leaves, stems, buds, flowers, fruits, shoots, roots, tubers, rhizomes, stolons, bulbs, and corms. In some embodiments, the collected tissue samples are selected from the group consisting of: leaf, stem, bud, flower, fruit, shoot, root, tuber, rhizome, stolons, bulb, and corm tissue samples. In some embodiments, plant tissue samples are analyzed to determine the levels of one or more gene transcripts encoding anthranilic acid synthase protein components or the levels of anthranilic acid synthase protein components (…). For example (by enzyme assay or immunoassay). In some embodiments, the anthranilate synthase protein component is an α or β subunit. In some embodiments, plant tissue samples are analyzed to determine the level of the gene transcript encoding anthranilate N-benzoyltransferase or the level of anthranilate N-benzoyltransferase (...). For example (by enzyme assay or immunoassay). In some embodiments, the plant defense compound is an anthranilate. In some embodiments, the anthranilate is selected from the group consisting of methyl anthranilate, dimethyl anthranilate, ethyl anthranilate, phenethyl anthranilate, and menthyl anthranilate. In some embodiments, the plant defense compound is an anthranilic acid-derived phytoalexin.

[0012] Further methods for identifying microbial strains that enhance plant responses to pathogens or pests, wherein the microbial strains are non-pathogenic to the plant, include screening samples containing one or more microbial strains for the presence of genes encoding one or more proteins that produce compounds that enhance plant responses to pathogens or pests. In some embodiments, the microbial strain is identified by the presence of one or more genes in a siderophore biosynthesis pathway. In some embodiments, the microbial strain is identified by the presence of one or more genes in a polyketide biosynthesis pathway. In some embodiments, the polyketide is an antimicrobial compound. In some embodiments, the protein in the pathway for producing the polyketide that induces a plant response to a pathogen or pest is present on a mobile plasmid. In some embodiments, the protein in the pathway for producing the polyketide is encoded by the gene at SEQ ID NO: 87 or a variant of such a gene comprising a DNA sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein encoding the sequence of SEQ ID NO: 87. In some embodiments, the protein in the pathway for generating the polyketide compound that induces a plant response to a pathogen or pest is encoded by a gene of SEQ ID NO: 86 or a variant of such a gene comprising a DNA sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein encoding the sequence of SEQ ID NO: 86. In some embodiments, the protein in the pathway for generating the polyketide compound that induces a plant response to a pathogen or pest is encoded by a polynucleotide comprising a DNA sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 36-50. In some embodiments, the protein in the pathway for generating the polyketide compound that induces a plant response to a pathogen or pest comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 21-35. In some embodiments, the protein in the microorganism that induces a plant response to a pathogen or harmful organism is a homolog or ortholog of any of SEQ ID NO:21-35. In some embodiments, the gene in the pathway for producing the polyketide compound is... bfmBAB_ 2 genes. In some embodiments, bfmBAB_ 2 comprises a polynucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:36. In some embodiments, bfmBAB_2. Encoding a protein having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21. In some embodiments, bfmBAB_ Gene 2 has at least 70%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 36, and / or encodes a protein having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21. In some embodiments of any of the above methods and compositions, the microbial strain is a bacterial strain or a fungal strain. In some embodiments, the bacterial strain is... methyl genus Bacillus ( Methylobacterium )or Methylrubia ( Methylorubrum ) strain. In some embodiments, the plant is selected from the group consisting of: maize, wheat, rye, rice, alfalfa, barley, oats, rye, sorghum, millet, soybean, Brassica ( ) Brassica ) species, Cannabis genus ( Cannabis The species include tobacco, potatoes, peanuts, carrots, cotton, coffee, coconuts, beets, oats, barley, tomatoes, pumpkins, cucumbers, gourds, lettuce, peppers, peas, onions, green beans, kidney beans, sunflowers, safflowers, sweet potatoes, cassava, coffee, coconuts, conifers, turfgrass, leafy green vegetables, microvegetables, herbs, fruit plants (such as strawberries), and fruit trees (including but not limited to apple trees, nut trees, and ornamental plants). In some embodiments, the plant is a corn plant. In some embodiments, the pest is an insect. In some embodiments, the tissue sample is a root sample. In some embodiments, the pest is a corn rootworm.

[0013] This document also provides methods and compositions for treating plants with microorganisms identified using the methods described herein to enhance plant responses to one or more pathogens or pests. In some embodiments, the microbial compositions provided herein will comprise microbial strains that enhance plant defense responses to pathogens and / or pests, as well as additional components that enhance the long-term storage of the microbial strains, promote treatment of plants and / or plant parts, and / or serve as plant growth regulators. In some embodiments, the microbial strains provided herein will be in a solid composition, for example as a substantially dry product having a water content of about 5% or less. In some embodiments, the solid composition is in powder or granule form. In some embodiments, the compositions provided herein will be liquid cultures containing water, oil, and / or polymers. In some embodiments, the microorganisms provided herein are provided in a flowable liquid form. In some embodiments, the microorganisms are stabilized in a suspension concentrate in which the continuous phase is miscible with water but is not water. For example, the continuous phase in such a suspension concentrate may be a polymer, such as a polyether. In some embodiments, the microbial composition is provided in the form of an oil dispersion. In some embodiments, the microorganisms are encapsulated in a protective carrier. In some embodiments, the compositions provided herein comprise in the form of a dry powder. Methylobacterium Strain NLS0042 (NRRL B-50932) or a derivative thereof. In some embodiments, it contains... Methylobacterium Compositions containing strain NLS0042 (NRRL B-50932) or its derivatives include additional components to promote long-term storage in the form of a dried composition. In some embodiments, compositions containing dried powder and / or treated seeds, compared to undried compositions or compositions lacking additional components that enhance the long-term storage of microbial strains and / or promote the treatment of plants and / or plant parts, are more suitable for long-term storage. Methylobacterium The long-term stability of strain NLS0042 (NRRL B-50932) or its derivatives is enhanced. In some embodiments, it contains... Methylobacterium Compositions of strain NLS0042 (NRRL B-50932) or its derivatives contain one or more oligosaccharides or polysaccharides. In some embodiments, they contain... Methylobacterium Compositions of strain NLS0042 (NRRL B-50932) or its derivatives comprise one or more polysaccharides selected from the group consisting of dextrin, maltodextrin, disaccharides, starch, chitosan, alginate, and gums, including but not limited to ark gum, jaguar gum, xanthan gum, glucomannan, tragacanth gum, konjac gum, polysaccharide gum, mucilage, gum arabic, and other natural gums. In some embodiments, compositions exhibit enhanced stability compared to other compositions. MethylobacteriumThe dried composition of strain NLS0042 (NRRL B-50932) or its derivatives further comprises maltodextrin, trehalose and / or glucomannan. Attached Figure Description

[0014] Figure 1 Feeding selection assay results when choosing between NLS0042-treated and untreated maize roots. The vast majority of larvae chose the untreated roots over the NLS0042-treated roots. Circles indicate the percentage of larvae making a given choice in each of the 12 replicates of this experiment.

[0015] Figure 2 Results of feeding selection assays when choosing between two maize roots treated with NLS0042. In this case, most larvae did not make a choice and remained in the middle petri dish where they started. Circles indicate the percentage of larvae that made a given choice in each of the 12 replicates of this experiment.

[0016] Figure 3 Feeding selection assay results when choosing between two untreated maize roots. Circles indicate the percentage of larvae making a given choice in each of the 12 replicates of the experiment. Detailed Implementation

[0017] definition

[0018] The term “and / or” as used herein is to be considered a specific disclosure of each of two or more particular features or components having or not having the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to encompass “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0019] As used herein, the terms “include, include, and including” should be interpreted as having at least the features they refer to or covering the items they refer to, without excluding any additional unspecified features or items.

[0020] As used herein, the term "biologic" refers to a component of a composition for treating a plant or plant part composed of or derived from microorganisms. Biologics include biocontrol agents, other beneficial microorganisms, microbial extracts, natural products, plant growth activators, or plant defense agents. Non-limiting examples of biocontrol agents include bacteria, fungi, beneficial nematodes, and viruses. In some compositions, a biologic may contain... Methylobacterium A single culture or co-culture, or a culture that has been cultured separately. Methylobacterium A combination of strains or isolates.

[0021] As used in this article, the term " Methylobacterium "" refers to the genera and species within the family Methylbacteriaceae, including First Bacillus genus and recommendations Methylrubia Bacterial species in the genus (Green and Ardley (2018)). Methylobacterium This includes pink facultative methyltrophic bacteria (PPFM), and also covers non-pink ones. Methylobacterium nodosa ,as well as methyl genus Bacillus A colorless mutant of the isolate. For example, but not limited to, " Methylobacterium "" refers to the following species of bacteria, as well as any new bacteria that have not yet been reported or described. Methylobacterium Genus and species, which can be characterized based on phylogenetic analysis. Methylobacterium or First spp. of Red Bacillus Methylobacterium tumefaciens ( Methylobacterium adhaesivum ); Methylobacterium mimosinate Methylobacterium oryzae; Methylobacterium aerolatum; Methyl oxalate Methylobacterium oxalidis; Methylobacterium aquaticum; Persian beetle Methylobacterium persicinum; Methylobacterium (brachiatum); Methylobacterium phyllosphaerae; Short-chain Methylobacterium (Methylobacterium brachythecii); Methylobacterium Phyllostachyos; Methylobacterium bullatum; Methylobacterium sylvestris (Methylobacterium platani); Methylobacterium cerastii; Pseudofusiform methyl Methylobacterium pseudosasicola; Methylobacterium currus; [unclear text - possibly related to bacteria or bacteria] Methylobacterium radiotolerans; Methylobacterium dansii dankookense); Methylobacterium soli; Methylobacterium refluxum frigidaeris); Methylobacterium specialis; Methylobacterium fussawa Methylobacterium fujisawaense; Methylobacterium tardum; Methylobacterium tardum Methylobacterium gnaphalii; Methylobacterium tarhaniae; Methylobacterium goesingense; Methylobacterium thuringiensis thuringiense); Methylobacterium gossipiicola; Methylobacterium trifoliatum Methylobacterium trifolii; Methylobacterium gregans; variant methyl Methylobacterium variabile; Methylobacterium haplocladii; Methylobacterium aminovorans (Methylorubrum) aminovorans) ) Methylobacterium hispanicum; Methylobacterium truncatum (Methylobacterium extorquens); India Methylobacterium indicum; Methylobacterium podarium (foot) Methylorubrum podarium; Methylobacterium iners; group Methylobacterium populi (populi of methyltrophic bacteria); [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Methylobacterium isbilie; Methylobacterium pseudosasae (Methylorubrum pseudosasae); Methylorubrum pyrolyticus (Methylobacterium jeotgali); Methylobacterium rhodesianum (Rhodesian Methylobacterium) Methylorubrum rhodesianum; Methylobacterium Komagatae); Methylobacterium rhodinum (Methylobacterium rhodinum) Methylorubrum rhodinum; Methylobacterium longum; Methylobacterium tumefaciens (Methylobacterium salsuginis) (Methylorubrum salsuginis, a methyltrophic bacterium found in oilfield water); Horse Methylobacterium marchantiae; Methylobacterium suomiense (Methylorubrum suomiense); Mesophilic methyl bacillus (Methylobacterium mesophilicum); Methylobacterium thiocyanatum (Methylorubrum thiocyanatum); Methylobacterium nodulans); Methylobacterium zatmanii (Methylobacterium zatmanii) (Methylorubrum zatmanii)); or organic methylophilic bacteria ( Methylobacterium organophilum ).

[0022] As used herein, “mineral nutrients” (sometimes simply referred to as “nutrients”) are micronutrients or macronutrients that are required or useful for plants or plant parts, such as, but not limited to, nitrogen (N), potassium (K), calcium (Ca), magnesium (Mg), phosphorus (P) and sulfur (S), as well as micronutrients chlorine (Cl), iron (Fe), boron (B), manganese (Mn), zinc (Z), cobalt (Co), copper (Cu), molybdenum (Mo) and nickel (Ni).

[0023] As used in this article, “vitamin” refers to small amounts of organic compounds required for normal growth and metabolism. Vitamins are important for the growth of humans and / or animals, and some vitamins have been reported to be beneficial to plants. Vitamins include, but are not limited to, vitamin A (including, but not limited to, all-trans retinol and all-trans retinyl esters, as well as all-trans beta-carotene and other provitamin A carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid or folate), vitamin B12 (cobalamin), vitamin C (ascorbic acid), vitamin D (calciferol), vitamin E (tocopherol and tocotrienols), and vitamin K (quinone).

[0024] As used herein, the term “strain” should include all isolates of such strains.

[0025] As used herein, the phrase "mobile plasmid" refers to a plasmid that can be transferred from a donor strain to a recipient strain. A mobile plasmid, as defined herein, contains the cis-acting DNA element (i.e., oriT) required for conjugation and has the ability to... First spp. The origin of replication that functions in the DNA. Other elements required for conjugation may also be encoded on a mobile plasmid. Conjugated or autonomously transferable plasmids contain the cis-acting DNA required for conjugation and encode all the genes required to transfer the DNA to the recipient cell / strain or isolate, and are also considered mobile plasmids for use in the methods defined herein. As used herein, when used for Methylobacterium In the context of isolates, "variant" refers to a variant that has the same characteristics as those provided herein. Methyl rod Fungi Separates (such as, for example, sediments) MethylobacteriumAny isolate of chromosomal genomic DNA having at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity. Variants of the isolate can be obtained from a variety of sources, including soil, plants or plant material, and water, particularly water associated with plants and / or agriculture. Variants may also include derivatives obtained from preserved isolates. Sequence analysis tools such as BLAST (e.g., those developed by Altschul) can be used. et al. (As taught in 1990) or clustalw (www.ebi.ac.uk / Tools / msa / clustalw2 / ) for Methylobacterium Sequencing of isolates or strains (e.g., as by Sanger) et al. (1977), Bentley et al. (2008) or Caporaso et al. (as taught in 2012), and performed genome-wide sequence comparisons (Konstantinidis) et al. (2005). For example, variants can be identified by the presence of the 16S sequence of a reference strain, where the variant also exhibits the plant production enhancement trait of the reference strain.

[0026] As used in this article, when used for Methylobacterium In the context of the isolate, "derivative" refers to the sediment from which the present article is provided. Methylobacterium Any of the separated materials Methylobacterium . Methylobacterium Derivatives of the isolates include, but are not limited to, derivatives obtained by selection, derivatives selected by mutagenesis and selection, and derivatives derived from... Methylobacterium Genetic transformation obtained from isolates Methylobacterium "Derivatives" can be identified, for example, based on the genetic identity of the strain or isolate from which they are obtained, and will generally be chromosomal genomic DNA that exhibits at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA from which they are derived.

[0027] As used in this article, when used to evaluate a specific Methylobacterium Is the strain provided in this article the same? Methyl rod FungiWhen referring to strain variants or derivatives, "sequence identity" or "identity percentage" is a measure of nucleotide-level genomic similarity between coding regions of two genomes. Sequence identity between coding regions of bacterial genomes can be calculated, for example, using the mean nucleotide identity (ANI) score determined by FastANI (Jain et al., "High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries", Nat Communications 9, 5114 (2018)) and Han et al. ("ANI tools web: a web tool for fast genome comparison within multiplebacterial strains"; Database, 2016, 1–5).

[0028] As used herein, “leafy plants” refers to vegetable crops with edible leaves and includes, but is not limited to, spinach, kale, lettuce (including but not limited to romaine lettuce, iceberg lettuce, head lettuce, and loose-leaf lettuce), kale, cabbage, beetroot, watercress, Swiss chard, arugula, lettuce, chicory, bok choy, and turnip leaves. As used herein, leafy plants also refer to plants cultivated for the harvest of miniature leafy vegetables and / or herbs, including but not limited to: lettuce, cauliflower, broccoli, cabbage, watercress, arugula, garlic, onion, leeks, amaranth, Swiss chard, beets, spinach, melon, cucumber, squash, basil, celery, coriander, radish, red chicory, sow thistle, dill, rosemary, tarragon, basil, pennisetum, carrot, fennel, beans, peas, chickpeas, and lentils. Leafy greens also refer to mixtures of various leafy greens, such as mixed lettuce (mesclun) or other mixed salad leafy greens or mixed miniature leafy greens. As used in this article, “leafy greens” also includes other Brassica or Brassicaceae field leafy greens not specifically named herein.

[0029] As used herein, “fruit” or “fruit-bearing plant” can refer to succulent fruit-bearing plants, including but not limited to melons (including watermelons and cantaloupes), berries (including strawberries, blueberries, blackberries, and raspberries), grapes, kiwifruit, mangoes, papayas, pineapples, bananas, peppers, tomatoes, squash, and cucumbers. As used herein, “fruit” or “fruit-bearing plant” can also refer to fruit trees or fruit-bearing trees, including but not limited to apples, peaches, pears, lemons, limes, oranges (and other citrus fruits), cherries, plums, apricots, nectarines, elderberries, pomegranates, persimmons, papayas, figs, avocados, and guavas.

[0030] As used herein, “ornamental” plants refer to plants cultivated primarily for display purposes rather than for functional purposes, including but not limited to perennial plants and woody shrubs, including but not limited to azaleas, hydrangeas, forsythias, hibiscus, roses and native plants; ornamental grasses; potted plants, cut flowers and bulbous plants, including but not limited to tulips, hyacinths, daffodils, petunias and carnations; and potted flowers produced from asexually propagated cuttings, including but not limited to poinsettias and chrysanthemums.

[0031] As used herein, a “genetic element” refers to an element in a DNA or RNA molecule comprising a series of adjacent nucleotides having a length of at least 20 nucleotides and a length of up to 50, 100, 1000, or 10000 or more nucleic acids. Genetic elements may comprise different sets of adjacent nucleic acids; for example, the genome of a plant-associated microorganism may contain introns and exons. Genetic elements may be located on chromosomes or on extrachromosomal elements (such as plasmids). In eukaryotic plant-associated microorganisms, genetic elements may be located in the nucleus or mitochondria. In some embodiments, a genetic element is a functional genetic element encoding a peptide or protein. For example ,Gene).

[0032] As used herein, the terms “homologous,” “homology,” or “orthologous” refer to related genetic elements or proteins encoded by genetic elements, determined based on the degree of sequence identity. These terms describe the relationship between a genetic element or encoded protein found in one isolate, species, or strain and a corresponding or equivalent genetic element or protein in another isolate, species, or strain. As used herein, a particular genetic element in a first isolate, species, or strain is considered equivalent to a genetic element in a second isolate, species, or strain when the protein encoded by a genetic element in the isolate, species, or strain has at least 50% identity. The percentage of identity can be determined using many software programs available in the art, including BLASTP, ClustalW, ALLALIGN, DNASTAR, SIM, SEQALN, NEEDLE, SSEARCH, etc.

[0033] As used herein, the term "metabolite" refers to substances produced during the metabolism of a microbial strain, including but not limited to siderophores, nonribosomal peptides, polyketides, or combinations thereof.

[0034] Where the term is provided in singular form, other embodiments described by the plural form of the term are also provided.

[0035] It should be understood that the foregoing definitions will be used in this document when they are inconsistent with those provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference listed herein, or any patent or non-patent reference found elsewhere.

[0036] Further description

[0037] The methods and compositions provided herein can be used to improve plant responses to pests and / or pathogens by increasing the levels of one or more plant defense compounds in the plant. In some embodiments of the methods provided herein, after treatment of soil, plants, plant parts, or seeds with a microbial strain that produces the metabolites, the plants, plant parts, or seeds reduce or repel pathogens or pests, wherein such metabolites induce a defense response in the plant and protect the plant from pathogen or pest attack. In some embodiments, the plant response is improved by increasing the levels of one or more plant defense compounds derived from anthranilic acid in the plant. Anthranilic acid, the conjugate base of anthranilic acid, is synthesized in plants from branched acid by the action of anthranilic acid synthase (EC 4.1.3.27), an enzyme that is a heterotetrameric enzyme composed of two α and two β subunits. This reaction is a branch point from the aromatic amino acid pathway to tryptophan biosynthesis, and the tryptophan-binding site involved in the feedback inhibition of tryptophan synthesis is located in the α subunit. Overexpression of the tryptophan-insensitive anthranilic acid synthase (AS) α subunit has been reported to increase tryptophan levels in transgenic plants and is presumably associated with increased levels of insect-resistant indole-related compounds in the biosynthetic pathway branches involved in the tryptophan pathway. Anthranilic acid is also involved in the production of other plant secondary metabolites, including anthranilic esters and anthraquinones in various plants. Methyl anthranilate (MA) is naturally found in many plants and has been used as a repellent for birds and insects. MA is present in very small amounts in maize roots and has been reported to induce a strong repellent response in neomycete larvae in laboratory bioassays (Bernklau). et al. (2016) J. Economic Entomology 109:1683-1690).

[0038] In some embodiments, the anthranilic acid-derived plant defense compound is not derived from indole. In some embodiments, the anthranilic acid-derived plant defense compound is anthranilic ester. In some embodiments, the anthranilic ester is selected from the group consisting of methyl anthranilate, dimethyl anthranilate, ethyl anthranilate, phenethyl anthranilate, and menthyl anthranilate. In some embodiments, the anthranilic acid-derived plant defense compound is a phytoalexin. In some embodiments, the phytoalexin is an amide biosynthesized following the conversion of anthranilic acid to N-benzoyl anthranilic ester catalyzed by anthranilic acid N-benzoyltransferase (EC 2.3.1.44).

[0039] In some embodiments of the methods provided herein, the plant genome is modified to provide elevated levels of anthranilic acid-derived plant defense compounds. In some embodiments, the plant genome is modified to enhance the expression of one or more gene transcripts involved in the production of such plant defense compounds. In other embodiments, the plant genome is modified to reduce the expression of genes that promote tryptophan synthesis, thereby providing an accumulation of anthranilic acid for enzymatic reactions that induce the production of anthranilic esters and anthraquinones. In some embodiments, the plant genome is modified to express heterologous transcripts of genes involved in the production of plant defense compounds. Heterologous transcripts may be derived from, for example, different plant sources, microbial sources, or may be produced synthetically. Various plant regulatory elements are employed in such methods, including, for example, tissue-specific promoters, to target increased expression in specific tissues or tissues attacked by plant pathogens or pests. In some embodiments, root, leaf, green tissue, fruit, tuber, seed, or vascular tissue-specific promoters are employed. Constitutive promoters, which provide expression in a wide range of plants, plant parts, and plant tissues, are also used in this method. In some embodiments, the expression of natural plant gene transcripts is increased.

[0040] In some embodiments, gene modification is achieved through gene editing technologies that enable specific modification of endogenous genes in the plant genome, including those involved in CRISPR / CAS (Cellular, Cascade, and Cascade). For example Such as those disclosed in U.S. Patent Application Publications 20150344912, 20160138008, 20180179547, 20200172886, and 20220282244, which are incorporated herein by reference in their entirety), broad-spectrum nucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). In some embodiments, gene editing reagents can be used to edit DNA containing transcription enhancer elements via homology-directed repair (HDR) or non-homologous end joining (NHEJ). For exampleUS Patent Application Publication US2016 / 0168584 (incorporated herein by reference in its entirety) integrates into the promoter of one or more genes of a plant involved in producing plant defense compounds derived from anthranilic acid to increase their expression. In some embodiments, the promoter or a promoter containing an enhancer insert is operatively linked to anthranilic acid synthase protein components, optionally comprising the amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, or sequences having at least 90%, 95%, 98%, or 99% sequence identity therewith. In some embodiments, the promoter or a promoter containing an enhancer insert is operatively linked to a gene encoding anthranilic acid N-benzoyltransferase; optionally comprising the polypeptide sequences of SEQ ID NO:6 and / or SEQ ID NO:7, or sequences having at least 90%, 95%, 98%, or 99% sequence identity therewith, increased compared to control plants. In some embodiments, the promoter or a promoter containing an enhancer insert is operatively linked to the maize genes provided in Table 6 and optionally located in maize or other monocotyledonous plants.

[0041] In other embodiments provided herein, the level of anthranilic acid-derived plant defense compounds is increased due to treatment of the plant or portions thereof (including seeds) with one or more strains of microorganisms that are non-pathogenic to the plant, and this treatment results in increased expression of one or more gene transcripts involved in the production of anthranilic acid-derived plant defense compounds in the plant. In some embodiments, the microbial strain is non-pathogenic to the treated plant, even if the microorganism may be a pathogen of other plants not treated in the methods described herein. In some embodiments, the microbial strain is beneficial to the treated plant.

[0042] In some embodiments of the methods provided herein, after treating soil, plants, plant parts, or seeds with a microbial strain that produces metabolites, the plants, plant parts, or seeds reduce or repel pathogens or pests, wherein such metabolites enhance the plant, plant part, or seed's defense mechanisms, and these defense mechanisms protect the plant's roots or the plant itself from attack by pathogens or pests. In some embodiments, the plant defense mechanism includes increased production of anthranilic acid and / or anthranilic acid-derived compounds. In some embodiments, the production of anthranilic acid and / or anthranilic acid-derived compounds is enhanced in the roots. In some embodiments, the plant defense response protects the plant roots from attack. In some embodiments, the plant response is improved by treating the plant, plant part, seed, or soil with a microorganism that expresses genes involved in inducing an ISR response, including, for example, genes involved in the biosynthesis of siderophores and / or polyketide compounds. In some embodiments, proteins in pathways for producing polyketide compounds that induce a plant response to pathogens or pests are present on a mobile plasmid. In some embodiments, the protein in the pathway for producing the polyketide compound is encoded by a gene on DNA having the sequence SEQ ID NO: 87 or a variant thereof, the variant comprising (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 87, and / or (ii) a DNA sequence encoding the protein encoded by SEQ ID NO: 87. In some embodiments, the protein in the pathway for producing the polyketide compound that induces a plant response to a pathogen or pest is encoded by a gene on DNA having the sequence SEQ ID NO: 86 or a variant thereof, the variant comprising (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 86, and / or (ii) a DNA sequence encoding the protein encoded by SEQ ID NO: 86. In some embodiments, the protein in the pathway for generating the polyketide compound that induces a plant response to a pathogen or pest is encoded by a polynucleotide comprising a DNA sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 36-50. In some embodiments, the protein in the pathway for generating the polyketide compound that induces a plant response to a pathogen or pest comprises a polypeptide sequence of plant response to the pathogen or pest, encoded by a polynucleotide comprising a DNA sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 21-35.In some embodiments, the protein in the microorganism that induces a plant response to a pathogen or harmful organism is a homolog or ortholog of any of SEQ ID NO: 21-35. In some embodiments, the gene in the pathway for producing the polyketide compound is [gene name missing]. bfmBAB_ 2 genes. In some embodiments, bfmBAB_ Gene 2 contains the polynucleotide sequence of SEQ ID NO:36. In some embodiments, bfmBAB_ Gene 2 encodes a protein having the sequence SEQ ID NO:21. In some embodiments, bfmBAB_ Gene 2 comprises a polynucleotide having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:36, and / or encodes a protein comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21. In some embodiments, the bacterial strain is *Methylobacterium* (…). Methylobacterium ) or methyl erythrobacter ( Methylorubrum The bacterial strain is NLS0042 (NRRL B-50932). In some embodiments, the bacterial strain is not NLS0042 (NRRL B-50932). In some embodiments, the method of reducing or repelling pathogens or pests includes treating soil, plants, plant parts, or seeds with a microbial strain expressing metabolites; and growing the plants in the presence of pathogens or pests, whereby the treated plants, plant parts, or seeds repel more pathogens or pests or reduce pathogen or pest feeding compared to control plants, wherein the control plants are not genetically modified or treated with the microbial strain. In some embodiments, plant defense compounds reduce insect larvae feeding on plant roots. In some embodiments, plant defense compounds reduce insect larvae feeding on plant roots compared to a control.

[0043] In some embodiments, plants are treated with bacterial strains to increase the expression of one or more gene transcripts involved in the production of plant defense compounds derived from anthranilic acid, or to reduce or repel plant pathogens or pests. In some embodiments, the plant defense compounds are derived from anthranilic acid.

[0044] Microbial strains containing heterologous DNA are provided, as well as methods for preparing such microbial strains, which can confer resistance, tolerance, reduced damage, foraging, reduced infection, and / or reduced invasion to plants, or act as a repellent. In some embodiments, DNA is transferred from a microbial strain that can confer resistance, tolerance, reduced damage, foraging, reduced infection, and / or reduced invasion to plants, or act as a repellent, to a different microbial strain lacking that DNA. In some embodiments, the heterologous DNA transferred to the different microbial strain encodes a protein having a sequence having any of the sequences in SEQ ID NO:21-35, or a protein sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of the sequences in SEQ ID NO:21-35. In some embodiments, the DNA is transferred onto a mobile plasmid from a microbial strain that induces a plant response to insects and / or pathogens. In some embodiments, the mobile plasmid is transferred from... Methylobacterium or MethylrubiaThe strain is transferred to a *Methylobacterium* or *Methylorbacterium* strain. In some embodiments, the mobile plasmid comprises SEQ ID NO:87 or a variant thereof, comprising (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:87; and / or (ii) a DNA sequence encoding a protein encoded by SEQ ID NO:87. In other embodiments, the mobile plasmid comprises SEQ ID NO:86 or a variant thereof, comprising (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:86; and / or (ii) a DNA sequence encoding a protein encoded by SEQ ID NO:86. In a further embodiment, a mobile plasmid transferred to a microorganism to confer the ability to induce a plant defense response encodes one or more proteins comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21-35. In some embodiments, the gene encoding a protein that enhances a plant's response to insects and / or pathogens is heterologous to the microbial host. In some embodiments, genes encoding one or more of SEQ ID NO:21-35 or their derivatives, variants, homologs, or orthologs are genetically manipulated to prepare a recombinant construct that provides expression of one or more proteins comprising an amino acid sequence having at least the amino acid sequence of SEQ ID NO:21-35. In some embodiments, such a recombinant construct includes a regulatory sequence to provide expression of said one or more proteins in a target microbial host. In some embodiments, a recombinant DNA construct for expressing proteins comprising any one or more of the amino acid sequences having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21-35 is stably integrated into the genome of a target microbial host. In some embodiments, a construct for expressing proteins comprising any one or more of the amino acid sequences having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21-35 is introduced into and maintained on a plasmid or other extrachromosomal element in the target microbial host. In some embodiments, one or more genes encoding proteins comprising the amino acid sequences having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21-35 are present in an operon and expressed by the operon.In other embodiments, one or more genes encoding proteins comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21-35 are present in a separate recombinant expression construct. A recombinant DNA construct comprising a heterologous promoter is also provided, as well as a microbial cell comprising the recombinant DNA construct, the heterologous promoter being operatively linked to one or more genes encoding proteins comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21-35. In some embodiments, the microbial strain engineered to express one or more proteins comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21-35 is a bacterial strain. In some embodiments, the aforementioned or otherwise provided DNA molecules are introduced into microbial strains other than Methylobacterium species #4 (NLS0042; NRRL B-50932). In some embodiments, the aforementioned or otherwise provided DNA molecules are introduced into microbial strains selected from the group consisting of the following items in Table 1: Methylobacterium Species #1 to #3, #5 to #53, and #54. In some embodiments, the DNA molecules described above or otherwise provided are introduced into the list in Table 1. Methylrubia Among species, including Methylrubia Species #63. In some embodiments, a microbial strain engineered to express one or more proteins comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21-35 is a fungal strain.

[0045] The bacterial strains used in the methods of the present invention include, but are not limited to, the following bacterial genera: Actinomycetes (Actinomycetes), Agrobacterium, Arthrobacter, Alcaligenes (Alcaligenes), Aureobacterium, Azobacter, Rhizobium (Azorhizobium), Azospirillum, Azotobacter, Bailynkelia (Beijerinckia), Bacillus, Brevibacillus, Burkholderia (Burkholderia), Chromobacterium, Clostridium, Corynebacterium Clavibacter, Comomonas, Corynebacterium, and Brachybacterium (Curtobacterium), Enterobacter, Flavobacterium, Staphylococcus Gluconacetobacter, Gluconobacter, Herbaspirillum, Hydrogenophile Genus (Hydrogenophage), Genus (Klebsiella), Genus (Luteibacter), Species (Lysine buds) Lysinibacillus, Mesorhizobium, Methylbacterium, and Methyl Red Bacillus Genus: Microbacterium, Ochrobactrum, Bacillus (Paenibacillus), Pantoea, Pasteuria, Sphingobacterium Phingobacterium, Photorhabdus, Phyllobacterium, Pseudomonas Genus (Pseudomonas), Genus (Rhizobium), Genus (Rhodococcus), Genus (Slow-growing Rhizobium) (Bradyrhizobium), Serratia, Sinorhizobium, Sphingosine mononucleosis Genus *Sphingomonas*, Genus *Streptomyces*, Genus *Stenotrophomonas*, Genus *Gnaphalium* Genus Variovora, Genus Xanthomonas, and Genus Xenorhadbus. In some embodiments, the bacteria are selected from the group consisting of: Bacillus amyloliquefaciens, wax Bacillus cereus, Bacillus firmus, Bacillus licheniformis (Bacillus lichenformis), Bacillus pumilus, Bacillus spheroidosa Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis thuringiensis, Chromobacterium suttsuga, Pasteuria (penetrans), useful Pasteuria usage. and Pseudomonas fluorescens fluorescens) .

[0046] In some embodiments, plants are treated with beneficial fungi, including but not limited to strains of the following genera: Acrocera (Acremonium) , Alternaria, Ampelomyces, Aspergillus (Aspergillus), Aureobasidium, Beauveria, Staphylococcus Botryosphaeria, Cladosporium, Cochliobolus, Anthrax (Colletotrichum), Coniothyrium, Embellisia (Epicoccum), Fusarium, Gigaspora, Gliocladium, Globulus Genus: Glomus, Laccaria, Metarhizium, Muscodor, and Ichthyophthirius (Nigrospora), Paecilonyces, Paraglomus, Penicillium (Penicillium), Phona, Pisolithus, Podospora, [unclear text - possibly a genus name or a genus name] Genus Rhizopogon, Genus Scleroderma, Genus Trichoderma, Genus Rhizopogon (Typhula), Ulocladium and Verticillium In a specific embodiment, the fungus is... Beauveria bassiana, Coniothyrium minitans, and Broom mold (Gliocladium vixens), white aerogenous mold (Muscodor albus), pale lilac parasitic mold (Paecilomyces) lilacinus) and Trichoderma polysporum .

[0047] In some embodiments, using Methylobacterium or Methylrubia Bacterial strains are used to treat plants. In some embodiments, Methylobacterium or Methylrubia The strains are the preserved strains disclosed in Table 1.

[0048] Table 1

[0049] In some embodiments, the microbial strains provided and used in the method that can confer resistance, repellency, tolerance, reduced damage, reduced infection, reduced foraging, and / or reduced invasion against or caused by the pests or pathogens disclosed herein are obtained through various means: transferring DNA from a microbial strain to a microbial strain that does not confer such resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion by increasing the production of one or more plant defense compounds derived from anthranilic acid in plants, the DNA of which can confer resistance, tolerance, reduced damage, foraging, reduced infection, and / or reduced invasion against pests or pathogens, acting as a repellent; and recovering or selecting new microbial strains having pest or pathogen resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion conferred by the transferred DNA. In other embodiments, DNA transferred from a microbial strain that can confer resistance, tolerance, reduced damage, foraging, reduced infection, and / or reduced invasion against pests or pathogens, acting as a repellent, encodes a protein having a sequence having any of SEQ ID NO: 21-35, or a protein having a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 21-35. In some embodiments, the DNA is transferred onto a mobile plasmid from a microbial strain that induces a plant response to insects and / or pathogens. In some embodiments, the mobile plasmid is transferred from... Methylobacterium or First spp. of Red Bacillus strain transferred to second Methylobacterium or MethylrubiaThe strain. In some embodiments, the mobile plasmid comprises SEQ ID NO:87 or a variant thereof, comprising: (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:87, and / or (ii) a DNA sequence encoding a protein encoded by SEQ ID NO:87. In other embodiments, the mobile plasmid comprises SEQ ID NO:86 or a variant thereof, comprising: (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:86; and / or (ii) a DNA sequence encoding a protein encoded by SEQ ID NO:86. In a further embodiment, the mobile plasmid transferred to a microorganism to confer the ability to induce a plant defense response encodes one or more proteins comprising a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:21-35. In some embodiments, the mobile plasmid transferred to a microorganism to confer the ability to induce a plant defense response comprises one or more sequences having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:36-50. In some embodiments, the mobile plasmid transferred to a microorganism to confer the ability to induce a plant defense response encodes one or more proteins comprising a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:51-85. In some embodiments, one or more proteins having the sequence of SEQ ID NO:51-85 or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:51-85 facilitate the transfer of the mobile plasmid.

[0050] In some embodiments, the DNA conferring resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion against pests or pathogens or caused by such pests or pathogens can be derived from NLS0042 or its derivatives by increasing the production of one or more plant defense compounds derived from anthranilic acid. For example DNA donor strains), and / or microbial strains that do not confer such resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion. For example The DNA recipient strains are those listed in Table 1, excluding NLS0042. These strains are used to transfer DNA from the donor strain (…). For exampleNLS0042) was transferred to the recipient strain ( For example The methods (for another strain in Table 1) include, but are not limited to, the DNA transfer methods disclosed in U.S. Patent Application Publication US20210171961, which is incorporated herein by reference in its entirety.

[0051] In some embodiments of the methods provided herein, gene transcripts involved in the production of one or more plant defense compounds from anthranilic acid encode ASα or β components. In some embodiments, the gene transcripts encode ASβ components. In some embodiments, the ASβ gene transcripts are maize plant transcripts. In some embodiments, expression of the ASβ subunit protein having the sequence of SEQ ID NO:2 or its homologs or orthologs is increased. Homologs and orthologs of SEQ ID NO:2 include Arabidopsis thaliana (… Arabidopsis The proteins AT1G24909, AT1G25155, AT1G24807, AT1G25083, ASB2, and ASB1; and rice proteins OASB1 (Os04g0463500) and OASB2 (Os03g0718000). In some embodiments, the gene transcripts encode the ASα component. In some embodiments, the ASα gene transcripts are maize plant transcripts. In some embodiments, the expression of ASα subunit proteins having the sequence of SEQ ID NO:4 or its homologs or orthologs is increased. Homologs and orthologs of SEQ ID NO:4 include Arabidopsis proteins AT3G55870, ASA1, and ASA2; and rice proteins OASA2 (Os03g0264400) and OASA1 (Os03g0826500). For example, other plant ASα and β subunit genes can be identified from plant genome sequences. In some embodiments, the gene encoding an ASα or β protein component is transcribed to produce multiple transcripts and translated proteins. Example 4 provides an additional example of a maize AS gene for use in the methods described herein.

[0052] In other embodiments of the methods disclosed herein, gene transcripts involved in the production of one or more plant defense compounds from anthranilic acid encode anthranilic acid N-benzoyltransferase protein, which catalyzes the production of N-benzoyl-anthranilic esters from benzoyl-CoA and anthranilic acid, i.e., the reaction involved in the production of anthraquinone phytoalexins. The enzyme, EC 2.3.1.144, is sometimes referred to as anthranilic acid N-hydroxycinnamoyl / benzoyltransferase because it is capable of using other thioesters of coenzyme A as donors in its reaction with anthranilic acid, including cinnamoyl-CoA, 4-coumaryl-CoA, and salicyl-CoA. Compounds produced by reactions with such other donor substrates may also be involved in the production of plant defense compounds. In one embodiment of the methods described herein, gene transcripts involved in the production of one or more plant defense compounds encode the maize anthranilic acid N-benzoyltransferase protein represented by SEQ ID NO:6 and / or SEQ ID NO:7. Other plant anthranilic acid N-benzoyltransferase proteins have also been studied in the methods described herein, including those derived from carnations ( Dianthus caryophyllus ) of DcHCBT2_Z84386.

[0053] In other embodiments of the methods disclosed herein, gene transcripts involved in producing proteins that enhance a plant's response to pathogens or pests encode proteins having a sequence that is at least 80% identical to that of SEQ ID NO:21. In some embodiments, the gene transcripts encode proteins comprising an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% sequence identity with SEQ ID NO:21.

[0054] The methods disclosed herein are applicable to improving plant responses to a variety of plant pests and pathogens, including bacterial and fungal pathogens, viruses, nematodes, and insects. These pathogens may attack one or more parts of a plant, including but not limited to leaves, stems, buds, flowers, fruits, shoots, roots, tubers, rhizomes, stolons, bulbs, and corms. Improved plant responses to pathogens or pests will result in a reduction of damage or other adverse effects from pathogens or pests. Adverse effects of pathogen or pest attacks on plants include, but are not limited to, any type of plant tissue damage or necrosis, any type of reduced plant yield, any reduction in the value of crop products, and / or the production of unwanted metabolites or growth products of pathogens or pests, including but not limited to fungal metabolites or fungal growth byproducts, including but not limited to mycotoxins.

[0055] In some embodiments, the methods provided herein improve plant responses to fungal pathogens selected from the group consisting of Alternaria species (Alternaria). Alternaria ) species, genus Diplosporum ( Ascochyta ) species, Aspergillus genus ( Aspergillus ) species 、 genus *Isomonium* Bipolaris ) species, Botrytis genus ( Botrytis ) species, genus *Plasmodium* ( Bremia ) species, Cercospora ( Cercospora ) species, genus Cyclospora ( Cochliobolus ) species, genus Colletotrichum ( Colletotrichum ) species, genus Chlorodiclofenac ( Diplodia ) species, powdery mildew genus ( Erysiphe ) species, Helicobacter genus (large spot disease) Exserohilum ) species, Fusarium genus ( Fusarium ) species, genus *Cephalotaxus* ( Gaeumanomyces ) species, *Coccidioides genus* ( Macrophomina ) species, genus *Oryza sativa* ( Magnaporthe ) species, genus *Cryptostoma* ( Nectria ) species, *Pseudomonas* genus ( Peronospora ) species, *Pyracantha* genus ( Phakopsora ) species, genus *Phyllostachys* Phialophora ) species, genus Stemdrops ( Phoma ) species, Texas root rot fungus ( Phymatotrichum ) species, Phytophthora ( Phytophthora ) species, genus *Peronospora* ( Plasmopara ) species, genus *Stiperus* ( Puccinia ) species, genus *Synthetium* (white-forked filamentous scaly pods) Podosphaera ) species, genus *Sclerotium* ( Pyrenophora ) species, Pyreosporium genus ( Pyricularia ) species, Pythium genus ( Pythium ) species, Rhizoctonia genus ( Rhizoctonia ) species, genus *Sclerotium* ( Sclerotium ) species, genus Sclerotium ( Sclerotinia ) species, genus *Syngonium* ( Septoria ) species, genus Polysporus ( Stagonospora ) species, Rhizospheres ( Thielaviopsis ) species, genus Uncaria ( Uncinul a) Species, Ustilago genus ( Ustilago ) species, genus *Nepeta* ( Venturia ) species and Verticillium genus ( Verticillium ) species.

[0056] In some embodiments, the methods provided herein improve plant responses to bacterial pathogens selected from the group consisting of: *Pseudomonas* spp. Pseudomonas ) species, genus Rollstonella ( Ralstonia ) species, genus *Agrobacterium* ( Agrobacterium ) species, Xanthomonas genus ( Xanthomonas ) species, Erwinia genus ( Erwinia ) species, genus *Xylobacteria* ( Xylella ), genus Dictyotis ( Dickeya ) species, genus Pectinobacter ( Pectobacterium ) species, genus *Corynebacterium* ( Clavibacter ) species and provisional candidate genera ( Candidatus ) species.

[0057] In some embodiments of plant attack by insects, an improved response can reduce damage directly caused by insects and / or reduce damage caused by insect-transmitted plant pathogens. Some viruses affecting crops are topoviruses and gemini viruses. Some common viruses that can seriously affect plants include Tomato Spotted Wilt Virus (TBV), Beet Top Curl Virus (BTVV), Tomato Yellow Leaf Curl Virus (TBV), Cucumber Mosaic Virus (CCUV), Potato Virus Y (PVY), Potato Virus X (PVX), Cauliflower Mosaic Virus (CMV), African Cassava Mosaic Virus (ACMV), Plum Pox Virus (PPV), Broccoli Mosaic Virus (BPV), Potato Virus (PVX), Tobacco Mosaic Virus (TBV), Tomato Spotted Wilt Virus (BTVV), Tomato Yellow Leaf Curl Virus (PVAV), Cucumber Mosaic Virus (CCUV), Cauliflower Mosaic Virus (CCUV), African Cassava Mosaic Virus (BPV), Plum Pox Virus (PPV), and Broccoli Mosaic Virus (BPV).

[0058] For example, in addition to the effects caused by vector-borne pathogens, piercing-sucking insects can also cause damage such as spots or streaks on branches and leaves, leaf curling, and poor or deformed fruit development. Piercing-sucking insects include leafhoppers, thrips, and aphids, and they attack plant vascular tissues, such as those found in roots, stems, leaves, and other plant organs.

[0059] In some embodiments of the methods and compositions described herein, improved plant responses to insects with chewing mouthparts (chewing insects) have been obtained. Damage caused by chewing insects can take many forms. In some cases, branches, leaves, or flowers are completely eaten by some insects, or the plant or plant parts appear jagged, with edges or centers chewed. In some cases, only the upper or lower surface is eaten, and a brown, withered appearance or openings between leaf veins can be observed. Chewing damage inside the plant is sometimes referred to as leafminer or borer. Chewing and biting pests may bite into and chew the leaves, stems, buds, flowers, and / or roots of a plant. Damage caused by such pests may include leaf drop due to excessive feeding; tunneling, such as insects (e.g., leafminers) digging holes in plant leaves; clitellating by beetles that feed on living wood; and root damage caused by insect feeding, leading to lodging. Common chewing pests include snails, slugs, caterpillars, borers, cutworms, hawk moths, and beetles. Non-limiting examples of crops and target pests to which the methods, microbial strains, and compositions provided herein are applicable include: pepper – aphids and lepidopteran insects; tomato – stink bugs, aphids, whiteflies, beet leafhoppers (BCTV vector), tobacco hawk moth; common bean – potato leafhoppers, Mexican bean beetles, lepidopteran insects; Brassica – flea beetles; soybean – armyworms, soybean loopers, nematodes; cotton – western flower thrips, fall armyworms, nematodes, aphids; rice – armyworms, water weevils; and maize – maize rootworms, cutworms, beetles, maize leaf and root aphids, white grubs, mites, armyworms, and wireworms.

[0060] In some embodiments of the methods described herein, the insect pest is the corn rootworm (CRW), which is a broad genus of beetles, namely the root leaf beetle (Cercophorus spp.). Diabrotica Members of ). In some embodiments, the methods described herein enhance the response of maize plants to western maize rootworm (WCR) attack, resulting in reduced lodging and / or increased yield compared to control plants. In some embodiments of the methods and compositions described herein, an improved response of maize plants to CRW feeding is obtained by treating maize plants, portions, or seeds with beneficial microorganisms. In some embodiments, maize plants are treated with beneficial bacteria. In some embodiments, with Methylobacterium or Methylrubia Species treatment of maize plants. In some embodiments, the preserved species listed in Table 1 are used. Methylobacterium or Methylrubia Species treatment of maize plants. In some embodiments, maize plants are treated with NLS0042 (NRRL B-50932). In some embodiments, maize plants are treated with a species other than NLS0042 (NRRL B-50932). Methylobacterium or MethylrubiaSpecies treatment of maize plants. In some embodiments, an improved response of maize plants to CRW feeding is obtained by modifying the maize plant genome to increase the expression of one or more gene transcripts involved in the production of one or more plant defense compounds. In some embodiments, the maize plant genome is modified to increase the expression of native maize gene transcripts. In some embodiments, the maize plant genome is modified to increase the expression of heterologous gene transcripts, such as those from microbial sources or from plants other than maize. In some embodiments, the plant is modified to increase the expression of anthranilic acid synthase subunit transcripts. In some embodiments, the expression of α and / or β subunit transcripts is increased. In some embodiments, the expression of anthranilic acid N-benzoyltransferase is increased. In some embodiments, the expression of anthranilic acid synthase β subunit transcripts and anthranilic acid N-benzoyltransferase transcripts is increased. In some embodiments, the expression of one or more gene transcripts encoding proteins having the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7 is increased.

[0061] Plants that can be treated and / or genetically modified using the methods provided herein include a wide variety of plants, including but not limited to field crops, leafy green vegetables, fruits or fruit trees, ornamental plants, turfgrass, and trees grown in commercial production. Such plant species include, but are not limited to, corn, soybeans, cruciferous plants, or... Brassica genus Species of vegetables (e.g., Brassica napus (B.) turnip (B. rapa), mustard greens (B. juncea) ), alfalfa, rice, rye, wheat, barley, oats, sorghum, millet (e.g., pearl millet) Pennisetum glaucum ), millet ( Panicum miliaceum ), millet ( Setaria italica ) and Dragon Claw Millet ( Eleusine coracana Sunflower, safflower, carrot, pepper, tomato, pumpkin, cucumber, melon and other melons, beans, peas, chickpeas, lentils, tobacco, potato, peanut, cotton, berries, grape, kiwi, mango, papaya, pineapple, banana, cannabis ( Cannabis ) genus species (including but not limited to cannabis) Cannabis sativa ) and industrial hemp varieties), sweet potato ( Ipomoea batatusCassava, coffee, coconut, ornamental plants (including but not limited to azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, morning glories, carnations, poinsettias, and chrysanthemums), conifers (including but not limited to pine trees such as loblolly pine, slash pine, yellow pine, black pine, and Monterey pine; Douglas fir; hemlock; western cypress; redwood; true fir trees such as silver fir and balsam fir; and cedar trees such as western red cedar and Alaskan yellow cedar) and turfgrass (including but not limited to annual Kentucky bluegrass, annual ryegrass, Canadian bluegrass, fescue, creeping bentgrass, wheatgrass, Kentucky bluegrass, orchard grass, ryegrass, red-capped grass, Bermuda grass, St. Augustine grass, and Zoysia grass).

[0062] Identification and selection of microorganisms that enhance plant responses to pests or pathogens.

[0063] This document also provides a method for identifying microorganisms that enhance a plant's response to one or more pathogens or pests, wherein the microorganisms are not pathogens of the plant, and wherein the plant response is enhanced by increasing the production of one or more plant defense compounds derived from anthranilic acid in the plant. In some embodiments, the method for identifying microorganisms that enhance a plant's response to one or more pathogens or pests includes the steps of: treating a plant, plant part, or plant seed with at least a first microbial strain that is not a pathogen of the plant to obtain treated seeds and / or treated plants; growing the treated plant in the presence of the pathogen or pest, or growing a plant from the treated plant part or treated seed; collecting one or more tissue samples from the plant and an untreated control plant, wherein the tissue samples are collected during a growth phase during which the pest or pathogen is attacking the tissue samples; and measuring the samples to identify microorganisms that increase the production of one or more plant defense compounds derived from anthranilic acid. In some embodiments, such a method includes the additional step of selecting samples from treated plants to analyze the levels of one or more plant defense compounds, wherein the treated plants exhibit reduced damage from the pathogen or pest compared to untreated control plants, or reduced damage from the plant pathogen or pest compared to other plants treated with the microorganism.

[0064] In some embodiments, a method for selecting a microbial strain that enhances a plant’s response to a pathogen or pest includes the steps of: (i) measuring one or more tissue samples from a plant treated with at least a first microbial strain that is not a pathogen of the plant, or from a plant grown from a part of a plant or seed treated with the first microbial strain, to determine an increase in the level of one or more plant defense compounds derived from anthranilic acid compared to one or more control tissue samples from an untreated control plant, wherein the tissue samples were collected from the treated and untreated plants during or after an attack on the plant tissue by the pest or pathogen; and (ii) selecting a microbial strain that is not a pathogen of the plant and provides an increased level of the one or more plant defense compounds, thereby selecting a microbial strain that enhances a plant’s response to the pathogen or pest.

[0065] In some embodiments, plant tissue extracts or plant parts, such as germinating seedlings, are treated and measured in vitro (e.g., in petri dishes or test tubes). In some embodiments, a method of selecting a microbial strain that enhances a plant's response to a pathogen or pest includes the steps of: (i) measuring one or more plant tissue samples treated with at least a first microbial strain that is not a pathogen of the plant to determine an increase in the level of one or more plant defense compounds derived from anthranilic acid compared with one or more untreated control plant tissue samples, wherein the tissue samples were exposed to the pest or pathogen during or after treatment with the first microbial strain; and (ii) selecting a microbial strain that provides an increased level of the one or more plant defense compounds, thereby selecting a microbial strain that enhances a plant's response to the pathogen or pest.

[0066] Other methods may be used to identify microorganisms that enhance plant responses to one or more pathogens or pests, wherein the microorganism is not a pathogen of the plant, and wherein the plant response is enhanced by expressing a gene in the microorganism. In some embodiments, such methods may include nucleic acid analysis of a sample and determining that the sample contains nucleic acids expressing one or more proteins involved in producing proteins that enhance plant responses to pathogens or pests. In some embodiments, the microorganism is identified as having one or more genes encoding proteins involved in the biosynthesis of polyketide compounds. In some embodiments, the gene encoding one or more polyketide compound synthesis proteins is encoded by a sequence present in SEQ ID NO:86, SEQ ID NO:87, or a variant thereof. A variant of SEQ ID NO:87 comprises: (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:87, and / or (ii) a DNA sequence encoding the protein encoded by SEQ ID NO:87. Variants of SEQ ID NO: 86 include: (i) a DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 86; and / or (ii) a DNA sequence encoding a protein encoded by SEQ ID NO: 86. In some embodiments, the gene encoding a polyketide biosynthetic protein is a homolog or ortholog of a polyketide biosynthetic protein expression sequence present in SEQ ID NO: 86 and / or SEQ ID NO: 87. In some embodiments, a polyketide biosynthetic protein encoding sequence identified in a microorganism encodes a protein having at least 80% identity with a protein having a sequence having SEQ ID NO: 21-35. In some embodiments, such polyketide biosynthetic protein encoding sequences have at least 70% identity with polyketide biosynthetic protein encoding sequences of SEQ ID NO: 36-50. In some embodiments, the polyketide biosynthetic protein has the sequence of SEQ ID NO:21 or has about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:21. Nucleic acid analysis for identifying such sequences includes, but is not limited to, techniques based on: sequencing, sequence comparison using BLAST, nucleic acid hybridization, polymerase chain reaction (PCR), mass spectrometry, nanopore-based detection, branched DNA analysis, and combinations thereof. In some embodiments, nucleic acid analysis may be used to detect at a concentration of 10 μg / g sample. 3 10 4 10 5 10 6Microbial strains present at concentrations of 100 or higher. Samples of interest for identifying microbial strains that enhance plant responses to pathogens or pests include soil samples, plants, plant parts, residual plant material, and various water sources, including water from paddy fields or crop irrigation.

[0067] Various methods can be employed to treat plants with microorganisms to identify or select one or more microorganisms that enhance the plant's response to target pathogens or pests. Such methods may include, but are not limited to, spraying, coating, partially coating, immersing, and / or imbibizing plants, plant parts, or seeds with one or more microorganisms or compositions containing such microorganisms. The microbial-containing compositions used for application to plants can be, but are not limited to, aqueous or non-aqueous liquids, dry compositions, or emulsions. In some embodiments, plant seeds or cuttings may be immersed and / or imbibized with a composition containing microbial strains. In some embodiments, seed percolation and / or immersion may be performed under gentle agitation. Seed treatment may be carried out using continuous and / or intermittent seed treatment machines. In some embodiments, coated seeds may be prepared by seed pulping with a coating composition containing microbial strains. Alternatively... ， Microbial strains can be applied to soil or other growth media for plant growth. Soil treatment or application may include, but is not limited to, application within furrows. For example Before, during, and / or after seed deposition), soil irrigation, and the distribution of granules or other dry formulations to the soil ( For example Treatments for plants grown in a hydroponic system may include pre-germination seed treatment, foliar application to germinating plants or parts thereof, and application to liquid solutions used in the hydroponic system.

[0068] The plant, plant part, or seed used in screening methods for identifying microorganisms that enhance plant responses to target pathogens or pests can be any plant that has been attacked and damaged by the target pest or pathogen, or a plant part or seed from such a plant. Without limitation, the plant can be an agricultural field crop, leafy plant, fruiting plant or tree, ornamental plant, turfgrass, or a tree grown for commercial production, such as a conifer or nut tree. Such plant species include, but are not limited to, corn, soybean, cruciferous plants, or... Brassica species Vegetables (e.g.) Brassica napus (B.) turnip (B. rapa), mustard greens (B. juncea) ), alfalfa, rice, rye, wheat, barley, oats, sorghum, millet (e.g., pearl millet) Pennisetum glaucum ), millet ( Panicum miliaceum ), millet ( Setaria italica ) and Dragon Claw Millet ( Eleusine coracanaSunflower, safflower, carrot, pepper, tomato, pumpkin, cucumber, melon and other melons, beans, peas, chickpeas, lentils, tobacco, potato, peanut, cotton, berries, grape, kiwi, mango, papaya, pineapple, banana, cannabis ( Cannabis ) genus species (including but not limited to cannabis) Cannabis sativa ) and industrial hemp varieties), sweet potato ( Ipomoea batatus Cassava, coffee, coconut, ornamental plants (including but not limited to azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, morning glories, carnations, poinsettias, and chrysanthemums), conifers (including but not limited to pine trees such as loblolly pine, slash pine, yellow pine, black pine, and Monterey pine; Douglas fir; hemlock; western cypress; redwood; true fir trees such as silver fir and balsam fir; and cedar trees such as western red cedar and Alaskan yellow cedar) and turfgrass (including but not limited to annual Kentucky bluegrass, annual ryegrass, Canadian bluegrass, fescue, creeping bentgrass, wheatgrass, Kentucky bluegrass, orchard grass, ryegrass, red-capped grass, Bermuda grass, St. Augustine grass, and Zoysia grass).

[0069] In some embodiments, the microorganisms used to treat the plant, plant parts, or plant seeds in an assay for identifying microorganisms that enhance a plant's response to a target pathogen or pest are beneficial microorganisms that provide additional benefits to the treated plant. In some embodiments, the beneficial microorganisms are bacterial strains. Bacterial strains that can be used in the assays described herein for identifying microorganisms that enhance a plant's response to a target pathogen or pest include, but are not limited to, the following non-pathogenic bacterial genera: Actinomycetes, Agrobacterium, Arthrobacterium, Alcaligenes, Chlorobacterium, Nitrogenobacterium Genus: Rhizobium, Azotospira, Azotobacter, Bacillus, Bacillus subtilis, Burkholderia *Clostridium*, *Corynebacterium*, *Trichomonas*, *Corynebacterium*, *Bruchweg*, *Enterobacter* Genus: Flavobacterium, Staphylococcus, Staphylococcus, Spirospira, Hydrophila, Klebsiella, and Strychnos nucifera Genus, Lysine Bacillus, Medium-slow Rhizobium, Methylbacterium, Methylerythrobacter, Microbacterium, Paleobacterium Bacterium genus, Bacillus spp., Pantotheca spp., Bacillus pastoris spp., Sphingobacterium spp., Bacillus spp., Leptobacterium spp., Pseudomonas spp. Genus: Rhizobium, Rhodococcus, Slow-growing Rhizobium, Serratia, Sinica Rhizobium, Sphingosomalidium, Streptococcus Molds, Oligotrophospora, Gluconobacterium, Xanthomonas, and Pathogenic Bacillus. In some embodiments, the beneficial microorganism is a fungal strain. In some embodiments, the fungal strains tested in assays used to identify an enhanced response of a plant to attack by a pathogen or harmful organism are the following non-pathogenic genera: Acrocera , Alternaria, Erysiphe, and others *Moldula*, *Beauveria bassiana*, *Botrytis*, *Cladosporium*, *Cyclospora*, *Anthracis*, *Scutellaria*, *Erythrophagus* Genus *Plasmodium*, Genus *Plasmodium*, Genus *Fusarium*, Genus *Macrocystis*, Genus *Plasmodium*, Genus *Cyclocarya*, Genus *Metarhizium*, Genus *Aeromonas* *Ichthyophthirius*, *Penicillium*, *Gymnosporium*, *Penicillium*, *Stemporus*, *Lycoperdon*, *Stemporus*, *Scleroderma*, *Scleroderma* *Puffball*, *Trichoderma*, *Sclerotium*, *Gnaphalium* or Verticillium .

[0070] In some embodiments, microorganisms that enhance plant responses to pathogens or pests will enhance induced systemic resistance (ISR) responses in plants. Some beneficial plant microorganisms, such as plant rhizosphere growth promoters (PGPRs), can induce broad-spectrum induced systemic resistance (ISR). Some PGPR strains induce responses similar to pathogen-induced acquired systemic resistance (SAR). In some embodiments, one or more genes involved in inducing ISR responses are jasmonic acid-dependent defense genes. In some embodiments, genes involved in inducing ISR responses are identified in microorganisms encoding siderophores. In some embodiments, one or more gene clusters that promote ISR production in treated plants are involved in the biosynthesis of metabolites. In some embodiments, gene clusters that promote ISR responses are polyketide synthase gene clusters, siderophore gene clusters, nonribosomal peptide synthases, or combinations thereof.

[0071] In some embodiments, a method for identifying or selecting microbial strains that enhance a plant's response to pathogens or pests described herein includes the step of growing a treated plant in the presence of the pathogen or pest. In other embodiments, the plant or plant portion is exposed to the pest or pathogen in vitro, for example, in a culture medium. The plant may be exposed to pests or pathogens in any plant growth medium, including but not limited to soil, liquids (such as hydroponic media), and nutrient media (such as nutrient media for plant tissue culture or micropropagation). Thus, in some embodiments, the treated plant portion may be a plant portion for micropropagation (such as shoot tips, anthers, petals, pollen, and other plant tissues), or may be a germinating seedling or undifferentiated plant tissue (such as callus).

[0072] The target pathogens or pests used in the assay methods described herein include bacterial and fungal pathogens as well as insect pests. Fungal pathogens include the following strains: Alternaria , genus *Cellatus* , Aspergillus, Helichrysosporus , Botrytis cinerea , genus *Panthium* , Cercospora , Cyclospora spp. , Anthrax genus Diplosporum ( Diplodia ), Powdery mildew ( Erysiphe ), Helicobacter genus of large spot disease ( Exserohilum ), Fusarium genus *Cephalopoda* ( Gaeumanomyces ), Coccidioides genus of bean shell ( Macrophomina ), genus *Oryza sativa* ( Magnaporthe ), genus *Cirsium* ( Nectria ), genus *Pseudomonas* ( Peronospora ), genus *Ipomoea* ( Phakopsora ), genus *Pyrtomyces* Phialophora ), Phytophthora spp.( Phoma ), Texas root rot fungus ( Phymatotrichum Phytophthora ( ) Phytophthora ), genus *Peronospora* ( Plasmopara ), genus *Pteris* ( Puccinia ), genus *Bryophytum* ( Podosphaera ), genus *Sclerotium* Pyrenophora ), Pyrethrum ( Pyricularisp ), Pythium ( Pythium ), Rhizoctonia spp. Rhizoctonia ), genus Sclerotium ( Sclerotium ), Sclerotium genus ( Sclerotinia ), genus *Syngonium* Septoria ), genus Polysporus ( Stagonospora ), Rhizophora genus ( Thielaviopsis ), genus *Hylocereus* ( Uncinula ), Ustilago maydis genus ( Ustilago ), Black Star Fungus ( Venturia ) and Verticillium ( Verticillium The target bacterial pathogens used in the screening methods presented herein include the following strains: Pseudomonas genus Rollston ( Ralstonia ), Agrobacterium , Xanthomonas and Erwinia (Erwinia) genus *Trichobacterium* Xylella, Dickeya, Pectobacterium, and others Clavibacter )and Candidatus (tentative candidate genus) The target insect used in the described screening methods can be any insect that causes damage to plants, including but not limited to leafhoppers, thrips, aphids, snails, slugs, caterpillars, borers, leaf miners, cutworms, hawk moths, and beetles. Most pests have four distinct growth stages: egg, larva, pupa, and adult. Piercing-sucking and chewing insects have three growth stages: egg, nymph, and adult. In some embodiments of the screening methods disclosed herein, the insect pest is applied to the plant to allow for an enhanced response of the screened plant to the pest. The pest can be applied at any growth stage, which will induce pathogen attack and damage to the plant. In some embodiments, the target pest is naturally present in the plant growth medium, such as when the plant is growing in the soil in the field. In some embodiments of the screening methods described herein, the target insect is applied to the plant, plant parts, or plant culture medium at the larval stage. In some embodiments of the screening methods described herein, the target insect pest is the corn rootworm (CRW), which is a broad genus of beetles, namely... Root Firefly Beetle Members.

[0073] The method for identifying microbial strains that enhance plant responses to pathogens or pests described herein further includes the step of collecting one or more tissue samples from the treated plant. In some embodiments, tissue samples are collected at a growth stage in which the target pest or pathogen is attacking the plant, plant part, or plant tissue. The tissues to be sampled will be selected based on the target pest and may include leaves, stems, buds, flowers, fruits, shoots, roots, tubers, rhizomes, stolons, bulbs, corms, germinating seedlings, and callus tissue.

[0074] Methods for identifying microbial strains that enhance plant responses to pathogens or pests described herein further include assaying one or more collected tissue samples to identify increased production of plant defense compounds derived from anthranilic acid and / or increased production of transcripts encoding proteins associated with anthranilic acid production and / or the conversion of anthranilic acid to plant defense compounds. In some embodiments, samples from treated plants grown in the presence of the target pathogen or pest are assayed to identify increased levels of one or more gene transcripts associated with anthranilic acid production and / or the conversion of anthranilic acid to plant defense compounds, compared to levels of such transcripts in untreated plants or control samples from plants treated with microorganisms that do not enhance the plant's response to the target pathogen or pest. Assays of gene transcript levels include RNA extraction and quantitative RNA-seq analysis, microarray analysis, high-throughput sequencing, etc. In some embodiments, samples are assayed using metabolomics methods that capture volatile and nonvolatile metabolites to identify plants with increased levels of one or more plant defense compounds. In some embodiments, microorganisms that enhance plant responses to target pathogens or pests are identified by the presence of elevated levels of such transcripts in samples from treated plants, compared to the levels of gene transcripts encoding α or β subunit protein components of anthranilic acid synthase in treated plants or from plants treated with microorganisms that do not enhance plant responses to the pathogens or pests. In some embodiments, elevated levels of transcripts encoding β subunit components indicate that the microorganism enhances plant responses to the target pathogens or pests. In some embodiments, microorganisms that enhance plant responses to target pathogens or pests are identified by the presence of elevated levels of such transcripts in samples from treated plants, compared to the levels of gene transcripts encoding proteins involved in the synthesis of one or more anthranilic acid-derived plant defense compounds in treated plants or from plants treated with microorganisms that do not enhance plant responses to the pathogens or pests. In some embodiments, the plant defense compound is an anthranilic acid-derived phytoalexin. In some embodiments, elevated levels of transcripts encoding anthranilic acid N-benzoyltransferase indicate that the microorganism improves plant responses to pathogen or pest attacks. In some embodiments, the sample is measured to determine increased expression of one or more gene transcripts encoding proteins having sequences having SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and / or SEQ ID NO:7.

[0075] The target pathogens or pests used in the assay methods described herein include bacterial and fungal pathogens as well as insect pests. Fungal pathogens include the following strains: Alternaria , genus *Cellatus* Aspergillus genus *Isomonia* , Botrytis cinerea , genus *Panthium* , Cercospora , Cyclospora spp. , Anthrax genus Diplosporum ( Diplodia ), Powdery mildew ( Erysiphe ), Helicobacter genus of large spot disease ( Exserohilum ), Fusarium genus *Cephalopoda* ( Gaeumanomyces ), Coccidioides genus of bean shell ( Macrophomina ), genus *Oryza sativa* ( Magnaporthe ), genus *Cirsium* ( Nectria ), genus *Pseudomonas* ( Peronospora ), genus *Ipomoea* ( Phakopsora ), genus *Pyrtomyces* Phialophora ), Phytophthora spp.( Phoma ), Texas root rot fungus ( Phymatotrichum Phytophthora ( ) Phytophthora ), genus *Peronospora* ( Plasmopara ), genus *Pteris* ( Puccinia ), genus *Bryophytum* ( Podosphaera ), genus *Sclerotium* Pyrenophora ), Pyricularisp genus Pythium ( Pythium ), Rhizoctonia spp. Rhizoctonia ), genus Sclerotium ( Sclerotium ), Sclerotium genus ( Sclerotinia ), genus *Syngonium* Septoria ), genus Polysporus ( Stagonospora ), Rhizophora genus ( Thielaviopsis ), genus *Hylocereus* ( Uncinula ), Ustilago maydis genus ( Ustilago ), Black Star Fungus ( Venturia ) and Verticillium ( Verticillium The target bacterial pathogens used in the screening methods presented herein include the following strains: Pseudomonas genus Rollston ( Ralstonia ), Agrobacterium , Xanthomonas and Erwinia (Erwinia) genus *Trichobacterium* Xylella, Dickeya, Pectobacterium, and others Clavibacter )and Candidatus (tentative candidate genus)The target insects used in the described screening method can be any insect that causes damage to plants, including but not limited to leafhoppers, thrips, aphids, snails, slugs, caterpillars, borers, leaf miners, cutworms, hawk moths, and beetles. In some embodiments of the screening method described herein, the plant is corn, and the target insect pest is the corn rootworm (CRW), which is a broad genus of beetles, namely… Root Firefly Beetle Members.

[0076] Composition for treating plants

[0077] In some embodiments of any of the methods identified herein, the composition for treating plants to improve plant responses to pest and / or pathogen attacks will comprise a microorganism that enhances the plant response by increasing the production of one or more plant defense compounds derived from anthranilic acid and at least one additional component. In other embodiments, the composition will comprise a microorganism that enhances the plant response by increasing the production of one or more plant defense compounds and at least one additional component. In some embodiments, the additional component may be an additional active ingredient, such as a pesticide or a second biological agent. In some embodiments, the pesticide may be an insecticide, fungicide, herbicide, nematicide, or other biocide. Non-limiting examples of insecticides and nematicides include carbamates, diamides, macrolides, neonicotinoids, organophosphates, phenylpyrazoles, pyrethroids, spinosads, synthetic pyrethroids, terfenicol, and tetramic acids. In specific embodiments, the insecticides and nematicides include abamectin, aldicarb, aldoxycarb, bifenthrin, carbofuran, chlorantraniliporle, chlothianidin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, dinotefuran, emamectin, ethiprole, fenamiphos, fipronil, and flubendiamide. The following pesticides are listed: diamide, fosthiazate, imidacloprid, ivermectin, lambda-cyhalothrin, milkemectin, nitenpyram, oxamyl, permethrin, tioxazafen, spinosad, spirodichlofen, spirotetramat, tefluthrin, thiacloprid, thiamethoxam, and thiodicarb.Non-limiting examples of useful fungicides include aromatic hydrocarbons, benzimidazoles, benzothiadiazoles, formamides, carboxylic amides, morpholines, benzamides, phosphonates, quinone external inhibitors (e.g., strobilurin), thiazolidines, thiophanates, thiophenecarboxamides, and triazoles. Specific examples of fungicides include acibenzolar-S-methyl, azoxystrobin, benalaxyl, bixafen, boscalid, carbendazim, cyproconazole, dimethomorph, epoxiconazole, fluopyram, fluoxastrobin, flutianil, flutolanil, fluxapyroxad, fosetyl-Al, ipconazole, isopyrazam, kresoxim-methyl, and metalaxyl. The fungicides listed include mefenoxam, metalaxyl, metconazole, myclobutanil, orysastrobin, penflufen, penthioopyrad, picoxystrobin, propiconazole, prothioconazole, pyraclostrobin, sedaxane, silthiofam, tebuconazole, thifluzamide, thiophanate, tolclofos-methyl, trifloxystrobin, and triticonazole.Other non-limiting examples of biocides include isothiazolinones, such as 1,2-benzothiazolin-3-one (BIT), 5-chloro-2-methyl-4-isothiazolin-3-one (CIT), 2-methyl-4-isothiazolin-3-one (MIT), octylisothiazolinone (OIT), dichlorooctylisothiazolinone (DCOIT), and butylbenzisothiazolinone (BBIT); 2-bromo-2-nitropropane-1,3-diol (Bronopol), 5-bromo-5-nitro-1,3-dioxane (Bronidox), tris(hydroxymethyl)nitromethane, 2,2-dibromo-3-nitropropamide (DBNPA), and alkyl dimethyl benzyl ammonium chloride. Non-limiting examples of herbicides include ACCase inhibitors, acetanilide, AHAS inhibitors, carotenoid biosynthesis inhibitors, EPSPS inhibitors, glutamine synthase inhibitors, PPO inhibitors, PS II inhibitors, and synthetic auxins. Specific examples of herbicides include acetochl, clethodim, dicamba, flumioxazin, fomesafen, glyphosate, glufosinate, mesotrione, quizalofop, saflufenacil, sulcotrione, and 2,4-D.

[0078] In some embodiments, the compositions or methods disclosed herein may include additional active ingredients selected from the group consisting of: clothianidin, ipconazole, imidacloprid, metalaxyl, mefenoxam, thiazoxafen, azoxystrobin, thiomethoxam, fluopyram, prothioconazole, piraclostrobin, and sedaxane.

[0079] In some embodiments, the second biological agent may be additional beneficial microorganisms, microbial extracts, plant extracts, yeast extracts, plant chitosan, natural products, plant growth activators, or plant defense agents. Non-limiting examples of the second biological agent may include bacteria, fungi, beneficial nematodes, and viruses. In some embodiments, the second biological agent is... First spp. or MethylrubiaStrains, including but not limited to those listed in Table 1. In some embodiments, plants are treated with methanogenic bacteria. Methanogenic bacteria strains that can be used in the compositions and methods described herein comprise bacterial species selected from the group consisting of: Methyloacidimicrobium, methyl acid *Methyloacidiplilum*, *Methylobacter*, *Methylwarmophyte* (Methylocaldum), Methylocapsa, Methylocella, Methylcoccus Genus (Methylococcus), Genus (Methylocystis), Genus (Methyloferula), [unclear text - possibly related to bacteria or fungi] Methylogaea, Methyloglobus, and Methylsalobacteria (Methylohalobius), Methylomagnum, Methylomarinum, Methyl Methylomicrobium, Methylomonas, and Methylparacoccus Methyloparacoccus, Methyloperedens, Methyloperedens Methyloprofundus, Methylosarcina, Methylosinus Methylosoma, Methylosphaera, Methylthermomycin (Methylothermus) and Methylovulum In some embodiments, the methanogenic bacteria are the following species: Methylbacterium, Methylcystis, Methylmicrobe, Methylmonas, Methylocytosporum or Methyl bend Aspergillus. In some embodiments, the methanogenic bacteria are the following strains: Methyl microbial lake bacteria Methylomicrobium lacus, Methylosarcina fibrata, Methylcurvularia (Methylosinus Trichosporium, Methylosinus sporium, Rosa rugosa Methylocystis rosea, Methylocystis parvus )or Fiber Methylcystis (Methylocystis hirsuta). In some embodiments, Methyl microbial lake bacteria For the preservation of strain NRRL B-68261. In some embodiments, Fiber Methylcystis For the preservation of strain NRRL B-68262. In some embodiments, species of the genus *Methylcystis* (… Methylocystis sp The strains selected are from the following: NRRL B-68282, NRRL B-68283, NRRL B-68284, NRRL B-68285, NRRL B-68286, NRRL B-68319, NRRL B-68321, NRRL B-68323, and NRRL B-68347. In some embodiments, species of the genus *Methylocyclocarya* (…) Methylosarcina sp. The strain is NRRL. B-68281. In some embodiments, species of the genus *Methylcampylobacter* (… Methylosinus sp. The strains are selected from the following preserved strains: NRRL B-68320, NRRL B-68322 and NRRL B-68348.

[0080] In some embodiments, the second biological agent may be bacteria belonging to the following genera: Actinomycetes (Actinomycetes), Agrobacterium, Arthrobacter, Alcaligenes (Alcaligenes), Aureobacterium, Azobacter, Rhizobium (Azorhizobium), Azospirillum, Azotobacter, Bailynkelia (Beijerinckia), Bacillus, Brevibacillus, Burkholderia (Burkholderia), Chromobacterium, Clostridium, Corynebacterium Clavibacter, Comomonas, Corynebacterium, and Brachybacterium (Curtobacterium), Enterobacter, Flavobacterium, Staphylococcus Gluconacetobacter, Gluconobacter, Herbaspirillum, Hydrogenophile Genus (Hydrogenophage), Genus (Klebsiella), Genus (Luteibacter), Species (Lysine buds) Lysinibacillus, Mesorhizobium, Microbacterium Microbacterium, Ochrobactrum, Paenibacillus, Pantotheca (Pantoea), Pasteuria, Phingobacterium, and other bacteria (Photorhabdus), Phyllobacterium, Pseudomonas, Rhizobium (Rhizobium), Rhodococcus, Bradyrhizobium, Serratia (Serratia), *Sinorhizobium*, *Sphingomonas*, *Streptomyces* (Streptomyces), Stenotrophomonas, Variotrophorax (Xanthomonas) and pathogenic bacteria (Xenorhadbus). In a specific embodiment, the bacteria are selected from the group consisting of: Bacillus amyloliquefaciens, Bacillus cereus Bacillus cereus, Bacillus firmus, Bacillus lichenformis, and short-lived Bacillus cereus Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis (Bacillus subtilis), Bacillus thuringiensis, and pigment bacilli (Chromobacterium suttsuga), Pasteuria penetrans, useful Pasteuria Pasteuria usage and Pseudomonas fluorescens .

[0081] In some embodiments, the second biological agent may be the following fungal genera: Acremonium , Chain Genus Alternaria, Genus Ampelomyces, Genus Aspergillus, Genus Short-stemmed Pyrrosia (Aureobasidium), Beauveria, Botryosphaeria, Cladosporium Cladosporium, Cochliobolus, Colletotrichum, and Shield-shell molds Coniothyrium, Embellisia, Epicoccum, Fusarium The genera *Gigaspora*, *Gliocladium*, *Glomus*, and *Laccaria*. Metarhizium, Muscodor, Nigrospora, and Paecilomyces (Paecilonyces), Paraglomus, Penicillium, Phona *Pisolithus*, *Podospora*, *Rhizopogon*, *Ploxera* (Scleroderma), Trichoderma, Typhula, Ulocladium and genus Verticilium In a particular embodiment, the fungus is Beauveria bassiana Coniothyrium minitans, Gliocladium vixens, and white gas-producing mold (Muscodor albus), Paecilomyces lilacinus or Trichoderma polysporum) In some embodiments, the composition comprises a variety of additional biological components, including aggregates comprising any of the above-described bacterial or fungal genera or species.

[0082] In further embodiments, the second biological agent may include, but is not limited to, various Bacillus Bacillus sp., Pseudomonas sp., Coniothyrium sp., Pantotheca. Genus: *Pantoea* sp., *Streptomyces* sp. and Trichoderma sp. Microbial biopesticides can be bacteria, fungi, viruses, or protozoa. Particularly useful biopesticides include a wide variety of... withered grass sprouts Bacillus subtilis, Bacillus thuringiensis, and Bacillus pumilus (Bacillus pumilis), Pseudomonas syringae, Trichoderma harzianum), Trichoderma virens and Streptomyces lydicus Strains. Other added microorganisms may be genetically engineered or wild-type isolates, which may be used in pure culture form. In some embodiments, the second biological agent is expected to be provided in the composition in the form of spores. In further embodiments, the second biological agent may be a biostimulant, including but not limited to seaweed extracts or humic acid, a plant growth activator or plant defense agent, including but not limited to harpin, Polygonum cuspidatum (… Reynoutria sachalinensis), jasmonate, lipochito-oligosaccharides, and isoflavones.

[0083] In some embodiments for plant treatment, the microbial inoculant is incorporated in the form of a dry powder or granules into a non-aqueous continuous phase comprising a non-aqueous solvent (e.g., a solvent immiscible with water). In some embodiments, the microbial inoculant is uniformly dispersed in the non-aqueous continuous phase. Such compositions contain additional components to enhance the mixing of the microbial inoculant with an aqueous composition comprising an agrochemical and / or to enhance the stability of the microbial inoculant in such aqueous compositions. See, for example, U.S. Patent Publication US20230337681, which is incorporated herein by reference in its entirety, for non-limiting examples of components useful in such compositions. Various methods can be used to produce dry microbial powders for use in such compositions, including but not limited to spray drying, freeze drying, air drying, fluidized bed drying, electrospray drying, or other drying methods. See, for example, U.S. Patent Publication US20220312772, which is incorporated herein by reference in its entirety, for non-limiting examples of methods for preparing dry microbial compositions.

[0084] In some embodiments, the composition for treating plants, plant parts, or plant seeds will contain microbial strains that enhance plant defense responses to pathogens and / or harmful organisms. ， And agriculturally acceptable excipients or agricultural adjuvants. Agriculturally acceptable excipients include, but are not limited to, wood flour, clay, activated carbon, diatomaceous earth, fine-grained inorganic solids, calcium carbonate, etc. Clays and inorganic solids that can be used include, but are not limited to, calcium bentonite, kaolin, porcelain clay, talc, perlite, mica, vermiculite, silica, quartz powder, montmorillonite, and mixtures thereof. Agriculturally acceptable excipients also include various lubricants, such as talc, graphite, polyethylene wax-based powders (such as lubricants), protein powders such as soy protein powder, or combinations of protein powders and lipids such as lecithin or vegetable oils. In some embodiments, the composition includes additional components to promote or enhance long-term storage and / or stability as a dry composition. In some embodiments, with other compositions ( For example,The composition lacks components that promote long-term storage and / or stability, including stability on treated plant parts or seeds, and its long-term stability as a dry powder and / or on treated seeds is enhanced. Additional components that can promote or enhance long-term storage and / or stability may include, but are not limited to, one or more oligosaccharides or polysaccharides. In some embodiments, the polysaccharide is selected from dextrin, maltodextrin, disaccharides, starch, chitosan, alginate, and gums, including but not limited to ark gum, jaguar gum, xanthan gum, glucomannan, tragacanth gum, konjac gum, polysaccharide gum, mucilage, gum arabic, and other natural gums. In some embodiments, a combination of any of the above or other agriculturally acceptable excipients and / or agriculturally acceptable adjuvants is used. Agriculturally acceptable adjuvants that promote adhesion to seeds may include, but are not limited to, polyvinyl acetate, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, polyesters, polyvinyl methyl ether, polyvinyl methyl ether-maleic anhydride copolymers, waxes, latex polymers, cellulose (including ethyl cellulose and methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxymethylpropyl cellulose), polyvinylpyrrolidone, alginate, dextrin, maltodextrin, polysaccharides, fats, oils, proteins, guar gum, tragacanth gum, tragacanth gum, polysaccharide gum, gum paste, gum arabic, shellac, vinylidene chloride polymers and copolymers, soybean-based protein polymers and copolymers, lignosulfonates, acrylic acid copolymers, starch, polyvinyl acrylate, corn protein, gelatin, carboxymethyl cellulose, polyglucosamine, polyethylene oxide, acrylamide polymers and copolymers, hydroxyethyl polyacrylate, methacrylamide monomer, alginate, ethyl cellulose, polychloroprene, and syrups or mixtures thereof. Other suitable agriculturally acceptable adjuvants that can promote coating include, but are not limited to, polymers and copolymers of vinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymers, and water-soluble waxes. Further, agriculturally acceptable adjuvants also include various lubricants (which can provide smooth flow and separation (single-cut) of seeds) such as talc, graphite, polyethylene wax-based powders (such as flow agents), protein powders such as soy protein powder, or combinations of protein powders and lipids such as lecithin or vegetable oils. This document and U.S. Patent No. 8,181 , The various surfactants, dispersants, anti-caking agents, foam control agents, and dyes disclosed in 388 are suitable for use with the composition to treat with microbial strains that enhance the plant's response to pathogens and / or harmful organisms. In some embodiments, the dried composition comprises Methylobacterium The strain and other components selected from the group consisting of: maltodextrin, trehalose, glucomannan, soy protein, soy-based protein polymers and copolymers, talc and graphite.

[0085] Application methods for treating plants with microorganisms and additional components include spraying, coating, partial coating, immersion, watering, and / or imbibition of seeds, plants, or plant parts with the composition. In some embodiments, the composition is used to expose seeds and / or seedlings to soil or other plant growth media in which plants or seed-derived plants are grown. Examples of application methods that provide microbial strains in soil include furrow application. 、 Soil irrigation, etc. In some embodiments, the effective amount of one or more microbial strains providing enhanced plant responses to pathogens and / or harmful organisms provided in the treatment of seeds or plant parts is at least about 10 per seed or treated plant part. 3 10 4 10 5 Or 10 6 CFU.

[0086] Other embodiments

[0087] In addition, this disclosure includes the following non-limiting embodiments.

[0088] Example 1. A method for improving plant responses to pathogens or harmful organisms, wherein the method comprises increasing the level of plant defense compounds produced in the plant by: (i) Modifying a plant genome to increase the expression of one or more gene transcripts involved in the production of one or more plant defense compounds derived from anthranilic acid in the plant, and / or treating a plant, plant part, or seed with a non-pathogenic microbial strain, wherein said treatment increases the expression of one or more gene transcripts involved in the production of plant defense compounds derived from anthranilic acid in the plant; and (ii) The plant is grown in the presence of the pathogen or pest, thereby increasing the level of one or more plant defense compounds in the plant compared to a control plant, and improving the plant’s response to the pathogen or pest compared to a control plant, wherein the control plant was not modified or treated as in (i).

[0089] 2. The method according to Example 1, wherein the expression of one or more gene transcripts or polypeptides associated with the production of anthranilic acid and / or the conversion of anthranilic acid into plant defense compounds is increased.

[0090] 3. The method according to Example 2, wherein the gene transcript encodes an anthranilate synthase protein component, or the polypeptide is an anthranilate synthase protein component.

[0091] 4. The method according to Example 3, wherein the anthranilic acid synthase protein component is an α or β subunit.

[0092] 5. The method according to Example 2, wherein the gene transcript encodes anthranilic acid N-benzoyltransferase, or the polypeptide is anthranilic acid N-benzoyltransferase.

[0093] 6. The method according to Example 1, wherein the plant defense compound is anthranilate.

[0094] 7. The method according to Example 6, wherein the anthranilate is selected from the group consisting of: methyl anthranilate, dimethyl anthranilate, ethyl anthranilate, phenyl ethyl anthranilate and menthyl anthranilate.

[0095] 8. The method according to Example 1, wherein the plant defense compound is an anthranilic acid-derived phytoalexin.

[0096] 9. The method according to any one of Examples 1 to 8, wherein the microbial strain is a bacterial strain.

[0097] 10. The method according to any one of Examples 1 to 9, wherein the microbial strain contains one or more genes encoding proteins involved in the biosynthesis of polyketide compounds.

[0098] 11. The method according to Example 10, wherein the one or more genes: (i) encode one or more proteins having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% with one or more of SEQ ID NO:21-35; (ii) encode one or more proteins having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% with SEQ ID NO:21; (iii) contain a sequence of one or more of SEQ ID NO:36-50; and / or (iv) are present on a plasmid containing SEQ ID NO:86 or a variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:86.

[0099] 12. The method according to any one of Examples 9 to 11, wherein the bacterial strain is Methylobacterium or methyl genus Rhodotorula strains.

[0100] 13. The method according to any one of Examples 9 to 12, wherein the bacterial strain is a bacterial strain other than NLS0042 (NRRL B-50932) containing heterologous DNA from NLS0042 (NRRL B-50932), the heterologous DNA conferring on the bacterial strain other than NLS0042 a phenotype that increases the expression of one or more gene transcripts involved in the production of plant defense compounds in the plant, optionally wherein the heterologous DNA encodes one of a plurality of proteins having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with one or more of SEQ ID NO:21-35.

[0101] 14. The method according to Example 13, wherein the bacterial strain other than NLS0042 is one of the bacterial strains shown in Table 1.

[0102] 15. The method according to any one of Examples 1 to 14, wherein the plant defense compound reduces insect larvae feeding on plant roots compared to the control plant.

[0103] 16. The method according to Example 3 or 4, wherein the anthranilate synthase protein component has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0104] 17. The method according to Example 5, wherein, compared with the control plant, the expression of the gene encoding anthranilic acid N-benzoyltransferase comprising a polypeptide sequence of SEQ ID NO: 6 or SEQ ID NO: 7 is increased.

[0105] 18. The method according to any one of Examples 1 to 17, wherein the plant defense compound is not an indole derivative.

[0106] 19. The method according to any one of Examples 1 to 18, wherein the plant is a crop plant shown in Table 10, and / or wherein the pathogen or pest is shown in Table 10 and / or 11, or wherein the plant is a maize plant.

[0107] 20. A method for identifying microbial strains that enhance plant responses to pathogens or harmful organisms, the method comprising: (i) Treating a plant, plant part or plant seed with at least a first microbial strain that is not a pathogen of the plant to obtain treated seeds and / or treated plants; (ii) Growing the treated plant in the presence of the pathogen or harmful organism, or growing the plant from the treated plant parts or the treated seeds; (iii) Collecting one or more tissue samples from the plant and from an untreated control plant, wherein the tissue samples were collected during a growth phase during or after which the pest or pathogen is attacking or has attacked the plant and the untreated control plant; and (iv) The samples are measured to identify an increase in the production of one or more plant defense compounds derived from anthranilic acid in the treated plants compared with untreated control plants, thereby identifying microbial strains that enhance the plant’s response to the pathogen or pest.

[0108] 21. A method for selecting microbial strains that enhance plant responses to pathogens or harmful organisms, the method comprising: (i) Determining, in relation to one or more tissue samples from plants treated with at least a first microbial strain that is not a pathogen of the plant, or from plants grown from plant parts or seeds treated with the first microbial strain, an increased level of one or more plant defense compounds derived from anthranilic acid compared to one or more control tissue samples from untreated control plants, wherein the tissue samples were collected from treated and untreated plants during or after attack of the plant tissue by the pest or pathogen; and (ii) Selecting microbial strains that are not pathogens of the plant and provide elevated levels of one or more plant defense compounds, thereby selecting microbial strains that enhance the plant’s response to the pathogen or pest.

[0109] 22. The method according to Example 20 or 21, further comprising the step of selecting samples from treated plants that exhibit reduced damage from the pathogen or pest compared to the untreated control plants for analysis.

[0110] 23. The method according to Example 20 or 21, wherein the sample is measured to determine the levels of one or more gene transcripts or polypeptides associated with the production of anthranilic acid and / or the conversion of anthranilic acid into plant defense compounds.

[0111] 24. The method according to Example 20, wherein the sample is measured to determine the level of one or more plant defense compounds derived from anthranilic acid.

[0112] 25. The method according to Example 20 or 21, wherein the pathogen or harmful organism is a fungus, bacteria, nematode, insect or virus.

[0113] 26. The method according to Example 20, wherein the treated plant part is selected from the group consisting of: leaves, stems, buds, flowers, fruits, shoots, roots, tubers, rhizomes, stolons, bulbs and corms.

[0114] 27. The method according to Example 20, wherein the collected tissue samples are selected from the group consisting of: leaf, stem, bud, flower, fruit, shoot, root, tuber, rhizome, stolon, bulb and corm tissue samples.

[0115] 28. The method according to any one of Examples 20 to 27, wherein the levels of one or more gene transcripts encoding anthranilic acid synthase protein components in the treated and untreated tissues are determined.

[0116] 29. The method according to Example 28, wherein the anthranilic acid synthase protein component is an α or β subunit.

[0117] 30. The method according to Example 23, wherein the levels of gene transcripts encoding anthranilic acid N-benzoyltransferase in the treated and untreated tissues are determined.

[0118] 31. The method according to Example 21 or 24, wherein the plant defense compound is an anthranilate.

[0119] 32. The method according to Example 31, wherein the anthranilate is selected from the group consisting of: methyl anthranilate, dimethyl anthranilate, ethyl anthranilate, phenethyl anthranilate and menthyl anthranilate.

[0120] 33. The method according to Example 21 or 24, wherein the plant defense compound is an anthranilic acid-derived phytoalexin.

[0121] 34. The method according to any one of Examples 20 to 33, wherein the microbial strain is a bacterial strain or a fungal strain.

[0122] 35. The method according to Example 34, wherein the bacterial strain is Methylobacterium or Methyl red bacillus genus strains.

[0123] 36. The method according to any one of Examples 20 to 35, wherein the plant is selected from the group consisting of: corn, wheat, rye, rice, alfalfa, barley, oats, rye, sorghum, millet, soybean, Brassica genus Species, CannabisSpecies, tobacco, potatoes, peanuts, carrots, cotton, coffee, coconuts, beets, oats, barley, tomatoes, pumpkins, cucumbers, gourds, lettuce, peppers, peas, onions, green beans, sunflowers, safflowers, sweet potatoes, cassava, coffee, coconuts, conifers, lawn grass, leafy green vegetables, miniature vegetables, herbs, fruit plants including fruit trees, and ornamental plants.

[0124] 37. The method according to Example 36, wherein the plant is corn.

[0125] 38. The method according to Example 36 or 37, wherein the tissue sample is a root sample.

[0126] 39. The method according to any one of Examples 20 to 38, wherein the pest is a corn rootworm.

[0127] 40. The method according to any one of Examples 20 to 39, wherein the first microbial strain is obtained by transferring DNA from a second microbial strain to a third microbial strain, the DNA being conferred resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion against or caused by the pest or pathogen by increasing the production of one or more plant defense compounds derived from anthranilic acid in the treated plant, the third microbial strain not conferred resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion against or caused by the pest or pathogen in the plant treated with the third microbial strain; optionally wherein the second microbial strain is NLS0042, and / or optionally wherein the third microbial strain is a microbial strain other than NLS0042 provided in Table 1; optionally wherein the transferred DNA: (i) encodes one or more having the same SEQ ID Proteins containing one or more of SEQ ID NO:21-35 having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity; or (ii) proteins present on plasmids containing SEQ ID NO:86 or variants thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO:86; and / or (iii) proteins containing one or more of SEQ ID NO:37-50 or variants thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with one or more of SEQ ID NO:37-50.

[0128] 41. A method for monitoring resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion in plants, the method comprising determining, from one or more tissue samples of a plant treated with at least a first microbial strain, the first microbial strain being capable of conferring resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion on the harmful organism or pathogen and not being a pathogen of the plant or from plants grown from plant parts or seeds treated with the first microbial strain: (i) increasing the level of one or more plant defense compounds derived from anthranilic acid; and / or (ii) Increase the expression of one or more gene transcripts or polypeptides associated with the production of anthranilic acid and / or the conversion of anthranilic acid into plant defense compounds. The increased levels and / or elevated expression are compared with one or more control tissue samples from untreated control plants, wherein the tissue samples were collected from treated and untreated control plants during or after the attack of the pest or pathogen on the plant tissue, and thus the increased levels of one or more plant defense compounds, gene transcripts and / or peptides in the tissue samples from the treated plants compared with the control plants indicate increased resistance, tolerance, reduced damage, reduced infection and / or reduced invasion of the pest or pathogen in the treated plants.

[0129] 42. The method according to Example 41, wherein the treated plants do not exhibit increased levels of the one or more plant defense compounds, gene transcripts and / or peptides, and wherein the method further comprises retreatment with the first microbial strain and / or treatment with another biocontrol agent, insecticide, fungicide or pesticide.

[0130] 43. A method for selecting a microbial strain that enhances a plant’s response to a pathogen or pest, the method comprising: (i) measuring one or more tissue samples from a plant treated with at least a first microbial strain that is not a pathogen of the plant, or from a plant grown from a part of a plant or seed treated with the first microbial strain, to determine an increase in the level of one or more plant defense compounds derived from anthranilic acid compared with one or more control tissue samples from an untreated control plant, wherein the tissue samples were collected from the treated and untreated plants during or after an attack of the plant tissue by the pest or pathogen; and (ii) selecting a microbial strain that is not a pathogen of the plant and provides an increased level of the one or more plant defense compounds, thereby selecting a microbial strain that enhances a plant’s response to the pathogen or pest.

[0131] 44. A method for selecting a microbial strain that enhances a plant’s response to a pathogen or pest, the method comprising: (i) measuring one or more plant tissue samples treated with at least a first microbial strain that is not a pathogen of the plant to determine that the level of one or more plant defense compounds derived from anthranilic acid is increased compared with one or more untreated control plant tissue samples, wherein the tissue samples were exposed to the pest or pathogen during or after treatment with the first microbial strain; and (ii) selecting a microbial strain that provides an increased level of the one or more plant defense compounds, thereby selecting a microbial strain that enhances a plant’s response to the pathogen or pest.

[0132] 45. A method for improving a plant's response to a pathogen or pest, wherein the method comprises increasing the level of plant defense compounds produced in the plant by: (i) Modifying a plant genome to increase the expression of one or more gene transcripts involved in the production of one or more plant defense compounds in the plant, and / or treating a plant, plant part, or seed with a non-pathogenic microbial strain, wherein said treatment increases the expression of one or more gene transcripts involved in the production of plant defense compounds in the plant; and (ii) The plant is grown in the presence of the pathogen or pest, thereby increasing the level of one or more plant defense compounds in the plant compared to a control plant, and improving the plant’s response to the pathogen or pest compared to a control plant, wherein the control plant was not modified or treated as in (i).

[0133] 46. ​​The method according to Example 45, wherein the plant defense compound is derived from anthranilic acid.

[0134] 47. A method for repelling pathogens or harmful organisms, wherein the method includes increasing the level of plant defense compounds produced in the plant, including

[0135] (i) Treatment of soil, plants, plant parts, or seeds with a plant-nonpathogenic microbial strain, wherein said treatment increases the expression of one or more gene transcripts involved in the production of plant defense compounds in said plant; and

[0136] (ii) The plant is grown in the presence of the pathogen or pest, thereby increasing the level of one or more plant defense compounds in the plant compared to a control plant, and repelling more of the pathogen or pest compared to a control plant, wherein the control plant is not modified or treated as in (i).

[0137] 48. The method according to Example 47, wherein the plant defense compound is derived from anthranilic acid.

[0138] 49. The method according to Example 47, wherein the microbial strain produces metabolites that, compared with the control plant, increase the production of such plant defense compounds.

[0139] 50. The method according to Example 49, wherein the metabolite is selected from the group consisting of siderophores, nonribosomal peptides, polyketides, or combinations thereof.

[0140] 51. The method according to Example 50, wherein the microbial strain comprises one or more gene clusters encoding proteins involved in the biosynthesis of the metabolite.

[0141] 52. The method according to any one of Examples 47 to 51, wherein the microbial strain comprises one or more genes encoding: (i) one or more proteins having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% with one or more of SEQ ID NO:21-35; (ii) one or more proteins having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% with SEQ ID NO:21; (iii) a sequence comprising one or more of SEQ ID NO:36-50; and / or (iv) present on a plasmid comprising SEQ ID NO:86 or a variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:86; optionally wherein the plant is a crop plant shown in Table 10, and / or wherein the pathogen or pest is shown in Tables 10 and / or 11.

[0142] 53. A method for selecting a microbial strain that confers resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion to a plant pest or pathogen, the method comprising identifying in the microbial strain: (i) one or more polynucleotides encoding proteins in a polyketide biosynthesis pathway; and / or (ii) one or more proteins in a polyketide biosynthesis pathway.

[0143] 54. The method according to Example 53, wherein the polynucleotide is identified by detecting at least one polynucleotide that: (i) encodes one or more proteins having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with one or more of SEQ ID NO: 21-35; (ii) encodes one or more proteins having at least 80% sequence identity with SEQ ID NO: 21; (iii) comprises one or more sequences of one or more of SEQ ID NO: 36-50 or having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 36-50; and / or (iv) is present on a plasmid comprising SEQ ID NO: 86 or having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 86.

[0144] 55. The method according to Example 53 or 54, wherein the polynucleotide is identified using nucleic acid amplification, hybridization and / or sequencing techniques.

[0145] 56. The method according to Example 53 or 54, wherein the protein is identified by detecting: (i) one or more proteins having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98% or 99% with one or more of SEQ ID NO:21-35; or (ii) one or more proteins having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98% or 99% with SEQ ID NO:21.

[0146] 57. The method according to Example 56, wherein the protein is identified by immunoaffinity and / or mass spectrometry.

[0147] 58. The method according to any one of Examples 53 to 57, further comprising the step of isolating and / or culturing the identified microbial stain containing the polynucleotide and / or protein.

[0148] 59. The method according to any one of Examples 53 to 58, wherein the microbial strain comprises a gene that: (i) encodes one or more proteins having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% with one or more of SEQ ID NO:21-35; (ii) encodes one or more proteins having a sequence identity of at least 80% with SEQ ID NO:21; or (iii) comprises a sequence of one or more of SEQ ID NO:36-50 or a variant thereof having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% with SEQ ID NO:36-50.

[0149] 60. The method according to any one of Examples 53 to 58, wherein the microbial strain is genetically modified with DNA: (i) encoding one or more proteins having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with one or more of SEQ ID NO: 21-35; (ii) encoding one or more proteins having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 21; (iii) comprising one or more sequences of SEQ ID NO: 36-50 or having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 36-50; and / or (iv) present on a plasmid comprising SEQ ID NO: 86 or having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 86.

[0150] 61. A method for improving plant responses to pathogens or harmful organisms, wherein the method comprises: (i) Treating plants, plant parts, or seeds with NLS0042 or a microbial stain containing DNA, wherein the DNA: (i) encodes one or more proteins having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% with one or more of SEQ ID NO:21; (ii) encodes one or more proteins having a sequence identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% with one or more of SEQ ID NO:21; (iii) contains a sequence of one or more of SEQ ID NO:37-50 or a variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with one or more of SEQ ID NO:37-50; and / or (iv) is present in a sequence containing SEQ ID NO:86 85%, 90%, 95%, 97%, 98%, or 99% or a variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:37-50; and / or (iv) is present in a sequence containing SEQ ID NO:86 85%, 90%, 95%, 97%, 98%, or 99% or a variant thereof having at least 80%, 85%, 90 ... On plasmids containing variants of NO:86 with at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity; and (ii) The plant or a plant grown from the seed is grown in the presence of the pathogen or pest, thereby improving the plant’s response to the pathogen or pest compared to a control plant; and wherein the plant is not a maize plant.

[0151] 62. The method according to Example 61, wherein the plant is selected from the group consisting of: pepper plants, tomatoes, beans, brassica plants, soybeans, cotton and rice.

[0152] 63. The method according to Example 61 or 62, wherein the level of one or more plant defense compounds in the plant is increased compared with that of a control plant.

[0153] 64. The method according to any one of Examples 61 to 63, wherein the pathogen or pest is an insect pest selected from the group consisting of: aphids, lepidopteran insects, stink bugs, whiteflies, beet leafhoppers, tobacco hawk moths, potato leafhoppers, Mexican bean beetles, flea beetles, fall armyworms, soybean loopers, western flower thrips, and water weevils.

[0154] 65. The method according to any one of Examples 61 to 64, wherein the improved response of the treated plant or the plant grown from the treated portion or seed, compared with the untreated control plant, includes improved resistance to or tolerance to pests or pathogens of the treated plant or the plant grown from the treated portion or seed, reduced damage, reduced infection, and / or reduced contamination.

[0155] 66. The method according to any one of Examples 61 to 65, wherein the plant is a crop plant shown in Table 10, and / or wherein the pathogen or pest is shown in Tables 10 and / or 11.

[0156] Example

[0157] Example 1: Analysis of Upregulated Maize Genes in Field Trials

[0158] Establish field trials by including insect-attracting crops in the field to attract maize root-worms (CRWs), or by planting maize on top of it for several years to increase CRW stress. In a fully causal design, plants are used... Methylobacterium Strain NLS0042 was used, either treated or untreated (UNT). The maize varieties used in the experiment were P1197AM and P1197AMXT, and are described in the following Pioneer 2022 Maize Hybrid-Herbicide Management Guide: AM - Optimum® AcreMax® Insect Protection System, equipped with YGCB, HX1, LL, and RR2. Contains a single-bag integrated refuge solution for ground insects. In EPA-designated cotton-growing counties, 20% of independent corn borer refuges must be planted with Optimum AcreMax products.

[0159] AMXT (Optimum® AcreMax® XTreme) - Contains a single-bag integrated refuge solution targeting both above-ground and underground insects. Key components include the Agrisure® RW trait, Bt trait, and Herculex® XTRA gene. In EPA-designated cotton-growing counties, 20% of independent corn borer refuges must be planted with Optimum AcreMax XTreme products.

[0160] Methylobacterium Apply as a spray-dried powder at planting time, with at least 1 x 10 g per seed. 6 The CFU ratio is applied to the seeds in the furrows.

[0161] Roots were collected when CRW larvae reached their peak feeding activity. Root samples from each of the four treatments (NLS0042 on P1197AM maize, UNT on P1197AM maize, NLS0042 on P1197AMXT maize, and UNT on P1197AMXT maize) were excavated from the ground, as much dirt as possible was removed, and the roots were immediately frozen on dry ice. The samples were returned to the laboratory, and the roots were ground under liquid nitrogen. RNA was extracted from the roots using the Qiagen RNeasy mini kit. The RNA was DNase-treated with DNase Max, and the quality was checked using a bioanalyzer. Quantseq (3' RNAseq) was performed by Lexogen (Greenland, NH). Using the Bluebee platform and the B73 maize genome as references, differentially expressed (DE) genes were analyzed in samples with the desired CRW phenotype (NLS0042-treated plants with lower node damage scores (NIS) than UTC under the same genetic background, and UTC plants with higher root damage scores than NLS0042-treated plants under the same genetic background). NIS (also known as the Iowa root damage score) ranged from 0 to 3, where 0 indicated no damage and 3 indicated that 3 nodes within 2 inches of the stem were eaten (Oleson). et al. (2005). For each of the four treatments, there were 3 samples for each condition.

[0162] Table 2. Node damage score (NIS) of field samples analyzed by RNAseq

[0163] Differentially expressed gene codes were matched with maize gene annotations v3 to v5 obtained from MaizeGDB. Differentially expressed genes with significant p-values ​​after multiple comparison correction were manually searched for additional annotation information.

[0164] Table 3. Differentially expressed genes upregulated in maize roots treated with NLS0042.

[0165]

[0166] Two of the nine genes upregulated by NLS0042 relative to UNT in AMXT maize were found to be associated with anthranilic acid in maize. Compared to untreated maize plants, AMXT maize plants treated with *Methylobacterium* showed significant increases in the transcripts of anthranilic acid N-benzoyltransferase protein 2 and anthranilic acid synthase homolog 1 (ASβ subunit). Smaller increases in gene expression were also observed for transcripts encoding other components of the anthranilic acid synthase protein.

[0167] Example 2: Greenhouse Measurement Results

[0168] Table 4 shows the results of treating maize plants with NLS0042 as a freeze-dried powder, applied as a seed treatment before sowing, and comparing them with untreated seeds (UTC). An additional group of untreated seeds was also included in the study of seeds infested with the western maize rootworm, also known as the maize root leaf beetle. Diabrotica virgifera Prior to larval infestation, seedlings received jasmonic acid as a positive control for ISR, applied via foliar spray (1 mM concentration, sprayed onto runoff). Two types of destructive harvesting were performed in a parallel set of pots at 3, 6, and 9 days post-infestation: 1) roots and soil were transferred to a Berlese funnel to extract live larvae, who were then enumerated, weighed, and individually measured in length; 2) roots were removed and processed for RNA extraction and gene expression analysis. A parallel set of seedlings were potted in 2-gallon pots and grown to near maturity, with root damage at the VT growth stage assessed using the Iowa Node Damage Scale (NIS).

[0169] Table 4

[0170] Table 5. For “CRW-inoculated” and “UTC” maize plants inoculated with maize rootworms, the root damage rating on the Iowa Node Damage Scale (NIS) was 0-3 at the VT stage.

[0171]

[0172] By using Methylobacterium Seed treatment with NLS0042 or seedling irrigation with jasmonic acid did not appear to have a negative impact on larval survival, biomass, and length. As measured by the Iowa Node Damage Score (NIS), both NLS0042 as a seed treatment and jasmonic acid as a foliar irrigation agent showed a trend toward reduced root damage compared to the untreated control, where 0 indicated no larval root feeding pressure and 3 indicated extremely high root feeding pressure (Table 5). Gene expression results comparing NLS0042-treated samples (NLS0042_UTC) and jasmonic acid-treated samples (UTC_JA) with the untreated control showed strong signaling in jasmonic acid-regulated genes at 9 DAI; compared to the untreated control, both NLS0042 seed-treated and jasmonic acid-irrigated plants showed ribosome-inactivating protein 2 (rip2), terpene synthase (tps23), and maize protease inhibitor (mpi).

[0173] Example 3: A method for selecting microorganisms that enhance the response of maize plants to CRW.

[0174] The following assays were performed to identify and / or select plant beneficial microorganisms that enhance the response of maize plants to maize rootworm larvae. Microorganisms that are not maize pathogens (e.g., Methylobacterium or Methylrubia Maize seeds were treated with a strain of [specific strain name], sown in a growth medium, and grown to the seedling stage V2-V4. The plants were removed from the growth medium and gently washed to remove the medium from the roots. The root system was placed in a petri dish lined with moistened cellulose filter paper and infected with early-stage maize rootworm larvae. The larvae were allowed to feed on the maize roots and then removed. Roots and / or root exudates and topspace were assessed in genetic and / or metabolomics assays to determine the levels of anthranilic acid-derived plant defense compounds and / or the levels of gene transcripts associated with the production of anthranilic acid-derived plant defense compounds. Microorganisms that increased the production of anthranilic acid-derived plant defense compounds and / or increased the levels of gene transcripts associated with the production of anthranilic acid-derived plant defense compounds compared to levels in untreated control plants were selected as microorganisms enhancing the maize plant response to maize rootworms.

[0175] Further steps for selecting samples for analysis include assessing larval feeding behavior before analyzing root samples. After feeding, the larvae are gently removed from the root using a brush and placed away from the root. Larval behavior is tracked, and the time it takes to reach the root surface is recorded. Larval behavior from control roots of untreated plants is compared to behavior from roots of treated plants. The presence of chemical feeding deterrents (such as methyl anthranilate) in root exudates will prevent larvae from returning to the corn root, or will require a significantly longer time to return to the roots of treated plants compared to untreated control roots.

[0176] The following selection assays were performed on the roots treated with NLS0042 as described above and the control roots to analyze the larval feeding behavior: 1) 10-day-old untreated plants, without WCR feeding 2) 10-day-old NLS0042-treated plants, without WCR feeding 3) Untreated 10-day-old plants have a 3-day feeding pressure from WCR larvae. 4) Plants treated with NLS0042 at 10 days old had a 3-day feeding pressure from WCR larvae.

[0177] When choosing between NLS0042-treated roots and untreated roots, the vast majority of larvae chose the untreated roots. Figure 1 This basic condition was tested in four of the six possible treatment combinations, and it was only slightly affected if the root was subjected to WCR larval feeding stress prior to the selected test.

[0178] When selection is made between roots treated with two NLS0042, most larvae do not make a selection and remain in the intermediate culture dish where they started. Figure 2 This means that these larvae are unable to feed.

[0179] When choosing between two untreated roots, the larvae selected either untreated root, with a slight preference for the untreated root that had undergone WCR larval feeding stress prior to the selection test. Figure 3 ).

[0180] These results indicate that NLS0042-treated plants are releasing volatile chemicals that inhibit feeding by WCR larvae. These results are consistent with the WCR larvae's response to methyl anthranilate.

[0181] Example 4. Analysis of metabolites produced by Methylobacterium.

[0182] The contribution of metabolites produced by *Methylobacterium* NLS0042 to reducing maize root damage caused by maize rootworms was investigated. Two gene clusters involved in the biosynthesis of metabolites were identified in the NLS0042 genome. Two genes (one from each of the clusters) were selected to create knockout mutants: asbA It is expected to participate in the generation of ferrocarriers, and bfmBAB It is expected that these genes will participate in the biosynthesis of antimicrobial compounds. Allelic exchange vectors for each of the gene targets were assembled and moved into NLS0042 via conjugation and integration at the target sites. Anti-selection and screening were performed to identify knockout mutants. PCR evidence supporting the creation of knockout mutants targeting both gene targets was obtained. Sequencing of the regions expected to contain deletions confirmed the generation of the desired knockout mutants.

[0183] asbA and bfmBAB The mutant strain was tested in the feeding selection assay described above to determine whether the mutation blocked the ability of Methylobacterium to repel CRW larvae from feeding.

[0184] Example 5. Selection determination and identification of volatiles produced by treated maize plants.

[0185] As described above, additional selection assays were performed using treated and untreated maize roots. Treatments included 1) untreated seeds, 2) seeds treated with NLS0042, 3) seeds treated with NLS0042mut1, and 4) seeds treated with NLS0042mut2. Ten newly hatched larvae were placed in the center, and their positions were recorded in the dark after 5 hours. The following treatment groups were evaluated through at least 10 replicates: Process 1 relative to Process 2 Process 1 relative to Process 3 Process 1 relative to Process 4 Process 2 relative to Process 3 Process 2 compared to Process 4 Process 3 compared to Process 4 The larval selection between analytical treatments was performed, and the volatile compounds that led to the selection were identified as follows.

[0186] Untreated non-Bt maize seeds will be rinsed and incubated overnight in distilled water at 25°C to accelerate germination. Seeds will be dried, weighed, and treated in a biosafety cabinet at a rate of 1E6 cfu / seed. The maize seeds will be planted and germinated and grown for at least ten days without larval feeding to allow the initiation activity of ISRs and the production of defensive volatile chemicals to occur. After 10 days of growth, a vacuum will be applied to adsorb the volatiles onto a solid substrate. This substrate will be used to identify known plant volatiles using GC-MS, including methyl anthranilate as a control compound.

[0187] The two treatments in this experiment will be an untreated control and the original NLS0042 powder, and the comparison will be between the volatiles produced by the untreated plants and the plants treated with NLS0042 after 10 days of growth.

[0188] Example 6: Maize genes, sequences, and homologs

[0189] Table 6. Maize o-aminobenzoic acid synthase genes

[0190] Gene accession number on the World Wide Web website "maizegdb.org / .", which is the maize genome database.

[0191] Example 7. Assess larval feeding behavior before analyzing root samples.

[0192] Larval feeding selection assays were performed largely as described in Example 5. Larval behavior from control roots of untreated plants was compared with larval behavior from roots of treated plants, and the results are provided in Table 7. The presence of methyl anthranilate in root exudates prevented larvae from returning to maize roots, or required a significantly longer time to return to roots of treated plants compared to roots of untreated control plants.

[0193] Table 7. Results of dietary choice assays using untreated corn roots or different concentrations of methyl anthranilate.

[0194] Example 8. Selective determination and identification of volatiles produced by treated maize plants.

[0195] As described above, additional selection assays were performed using treated and untreated maize roots. Treatments included 1) untreated seeds, 2) seeds treated with NLS0042, and 3) seeds treated with NLS0042mut2 (bfmBAB mutant). The NLS0042mut2 strain was obtained as described in Example 4. Ten newborn larvae were placed in the center, and their positions were recorded after incubation in the dark. The results are shown in Table 8 below. The bfmBAB mutant significantly reduced the repulsive effect of NLS0042, indicating that this gene contributes to inducing the repulsive effect of NLS0042 in maize plants. Other genes identified in the polyketide synthase pathway were evaluated in a similar manner to identify similar effects on induced insect defense responses in maize plants.

[0196] Table 8 Results of feeding selection assays using treatment combinations of untreated roots or roots treated with NLS0042 wild type or NLS0042 mut2.

[0197]

[0198] Example 9. Screening for polyketide biosynthetic sequences in microorganisms.

[0199] The methods used to identify the presence of sequences in microorganisms are as follows: Obtain the complete or partial genome sequence of the microorganism or a metagenomic sequence from an environmental sample. For isolated microorganisms or combinations of cultured microorganisms, use BLAST (Basic Local Alignment Search Tool; Altschul) to perform the following: et al. Sequences of interest can be identified using PCR (1990) or other nucleic acid analysis software. Sequences can be nucleotide or protein sequences. Sequences can also be searched using annotations of genes, gene clusters, and / or protein domains. Alternatively, standard PCR or qPCR primers specific to the sequence of interest can be used to identify the microorganisms of interest. Environmental samples containing multiple characterized or uncharacterized microorganisms are also screened in this manner, and microorganisms with positive signals are purified from aliquots of the original sample. Positive microorganisms are identified using colony PCR and / or aliquot dilution methods. The sequences used in the analysis are provided in Table 9.

[0200] Table 9. Genes in NLS0042 of the polyketide compound synthesis cluster.

[0201]

[0202] In this manner, sequences present on (i) SEQ ID NO:87, SEQ ID NO:86 and / or variants thereof; or (ii) sequences encoding one or more proteins having sequences of SEQ ID NO:21-35 or their homologs or orthologs are identified. Microorganisms identified in this manner are screened to identify microorganisms that enhance plant defense responses to pathogens and / or insect pests.

[0203] Example 10. Methylobacterium strain Field trial analysis of the effects of NLS0042 on the infection of specialty crops.

[0204] Field trials were conducted on pepper to determine the ability of NLS0042 to enhance plant defense against aphids and caterpillars.

[0205] Field trials were conducted on tomatoes to determine the ability of NLS0042 to enhance plant defense against aphids and stink bugs.

[0206] Field trials were conducted on common beans to determine the ability of NLS0042 to enhance plant defense against potato leafhoppers, Mexican bean beetles, and lepidopteran insects.

[0207] Field trials were conducted on Brassica species to determine the ability of NLS0042 to enhance plant defense against flea beetles.

[0208] Use NLS0042 via foliar spraying, seed treatment, and irrigation. For example The plants, plant parts, and / or seeds were treated in the form of soil irrigation, furrow treatment, or a combination thereof. The treated plants were grown in the presence of natural and / or artificially supplemented target insect pest infestation. Plant yield and insect damage were assessed and compared with control plants to identify the enhanced plant response to insect pests induced by NLS0042 treatment.

[0209] Example 11. Greenhouse tomato experiment on the effects of NLS0042 on whiteflies.

[0210] Tomato plants, parts, and / or seeds were treated with NLS0042 in the form of foliar spray, seed treatment, irrigation, or a combination thereof. The treated plants and untreated control plants were inoculated with whiteflies. Whitefly counts were performed weekly after inoculation and compared with counts in untreated control plants to identify enhanced plant response to whiteflies.

[0211] Example 12. Tomato experiment on the effect of NLS0042 on leafhoppers.

[0212] In a greenhouse, treated and untreated tomato plants were exposed to leafhoppers carrying beet top-turning virus (BCTV). NLS0042 was applied via foliar spray, seed treatment, irrigation, or a combination thereof. Digital PCR was used to assess the viral load in treated plants. Treated and untreated controls were transplanted into field trials to determine the effects on plant vigor (hyperspectral imaging), yield, and fruit quality. Insect presence was also determined using net scanning and / or sticky traps.

[0213] Example 13. Greenhouse experiment to assess the impact of NLS0042 on caterpillar pests in Solanaceae, soybean, cotton and rice crops.

[0214] Tomato, eggplant, pepper, soybean, cotton, and rice plants were treated with NLS0042 via foliar spraying, seed treatment, irrigation, or a combination thereof. Pre-weighed tobacco hawk moth caterpillars were allowed to feed on treated and untreated control tomato plants at different phenological stages for fixed periods. Pre-weighed soybean inchworm and fall armyworm caterpillars were allowed to feed on treated and untreated control soybean plants at different phenological stages for fixed periods. Pre-weighed fall armyworm caterpillars were allowed to feed on treated and untreated control soybean, cotton, and rice plants at different phenological stages for fixed periods. Data on caterpillar growth, mortality, volume, and developmental milestones were collected and analyzed during all treatment periods to identify the enhanced plant response to insect pests induced by NLS0042 treatment.

[0215] In the second experiment, an artificial diet was prepared, in which 10% leaf material from both treated and untreated control plants was added. Hawk moths, fall armyworms, and soybean inchworms were allowed to feed, develop, and complete their life cycles on this diet. Data on life history traits were collected to further evaluate the effects of the NLS0042-treated plants on insect pests.

[0216] Selection assays were performed between treated and control plants to assess the effect of NLS0042 treatment on herbivore selection and to evaluate potential antibiotic effects.

[0217] Example 14. Greenhouse experiment used to evaluate the effects of NLS0042 on cotton thrips and aphid pests.

[0218] Population assays were performed using known numbers of western flower thrips or aphids. Thrips or aphids were allowed to feed and develop on NLS0042-treated and untreated control plants, and population growth was monitored over extended time periods. Electrophysiological experiments were also conducted using a technique called electroosmography, which provides information on how NLS0042-treated and untreated control plants changed in their antibial and xenobiotic properties.

[0219] Selection assays were also conducted between the treated plants and the control plants to assess the effect of the NLS0042 treatment on herbivore selection and to evaluate potential antibiotic effects.

[0220] Example 15. Evaluation of the effects of microorganisms containing genes for inducing plant defense responses to insects and / or pathogenic pests.

[0221] Microbial strains containing one or more genes are identified through genomic screening and / or selection as described herein to express one or more sequences having any of SEQ ID NO: 21-35 or their homologs or orthologs. Alternatively, such strains are generated by plasmid transfer and / or genetic transformation with recombinant constructs by transferring the gene encoding the polyketide synthesis protein identified herein. The microbial strains are used to treat target plants in greenhouse, growhouse, and / or field assays and to assess enhanced plant defense responses to target pests. Treatments include foliar application, imbibition, or irrigation, as well as seed treatment. Table 10 below shows the plants and pests assessed.

[0222] Table 10 Assessment of plant defense responses to insect pests

[0223] Table 11. Assessment of plant defense responses of crops (including but not limited to maize, soybean, peanut, and cotton) to additional pests.

Claims

1. A method for improving a plant's response to a pathogen or pest, wherein the method comprises increasing the level of plant defense compounds produced in the plant by: (i) treating a plant, plant part, or seed with a non-pathogenic microbial strain, wherein the treatment increases the expression of one or more gene transcripts involved in the production of plant defense compounds derived from anthranilic acid in the plant, and / or modifies the plant genome to increase the expression of one or more gene transcripts involved in the production of one or more plant defense compounds derived from anthranilic acid in the plant; and (ii) The plant is grown in the presence of the pathogen or pest, thereby increasing the level of one or more plant defense compounds in the plant compared to a control plant, and improving the plant’s response to the pathogen or pest compared to a control plant, wherein the control plant is not modified or treated as in (i).

2. The method of claim 1, wherein the expression of one or more gene transcripts or polypeptides associated with the production of anthranilic acid and / or the conversion of anthranilic acid into plant defense compounds is increased.

3. The method according to claim 2, wherein the gene transcript encodes an anthranilate synthase protein component, or the polypeptide is an anthranilate synthase protein component.

4. The method according to claim 3, wherein the anthranilate synthase protein component is an α or β subunit.

5. The method according to claim 2, wherein the gene transcript encodes anthranilic acid N-benzoyltransferase, or the polypeptide is anthranilic acid N-benzoyltransferase.

6. The method according to claim 1, wherein the plant defense compound is anthranilate.

7. The method of claim 6, wherein the anthranilate is selected from the group consisting of methyl anthranilate, dimethyl anthranilate, ethyl anthranilate, phenethyl anthranilate, and menthyl anthranilate.

8. The method according to claim 1, wherein the plant defense compound is a phytoalexin derived from anthranilic acid.

9. The method according to claim 1, wherein the microbial strain is a bacterial strain.

10. The method of claim 1, wherein the microbial strain comprises one or more genes encoding proteins involved in the biosynthesis of polyketide compounds.

11. The method of claim 10, wherein the one or more genes: (i) encode one or more proteins having a sequence having at least 80% sequence identity with one or more of SEQ ID NO:21-35; (ii) encode one or more proteins having at least 80% sequence identity with SEQ ID NO:21; (iii) contain a sequence of one or more of SEQ ID NO:36-50; and / or (iv) are present on a plasmid containing SEQ ID NO:86 or a variant thereof having at least 80% identity with SEQ ID NO:

86.

12. The method according to claim 11, wherein the bacterial strain is Methylobacterium or Methylrubia strains.

13. The method of claim 11, wherein the bacterial strain is a bacterial strain other than NLS0042 (NRRL B-50932) containing heterologous DNA from NLS0042 (NRRL B-50932), the heterologous DNA conferring on the bacterial strain other than NLS0042 a phenotype of increased expression of one or more gene transcripts involved in the production of plant defense compounds in the plant, optionally wherein the heterologous DNA encodes one of a plurality of proteins having at least 80% sequence identity with one or more of SEQ ID NO:21-35.

14. The method of claim 13, wherein the bacterial strain other than NLS0042 is one of the bacterial strains shown in Table 1.

15. The method of claim 1, wherein the plant defense compound reduces insect larvae feeding on plant roots compared to the control plant.

16. The method according to claim 3, wherein the anthranilate synthase protein component has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

4.

17. The method of claim 5, wherein, compared with the control plant, the expression of the gene encoding anthranilic acid N-benzoyltransferase comprising the polypeptide sequence of SEQ ID NO: 6 or SEQ ID NO: 7 is increased.

18. The method according to claim 1, wherein the plant defense compound is not an indole derivative.

19. The method according to any one of claims 1 to 18, wherein the plant is a maize plant.

20. A method for identifying microbial strains that enhance plant responses to pathogens or harmful organisms, the method comprising: (i) Treating a plant, plant part or plant seed with at least a first microbial strain that is not a pathogen of the plant to obtain treated seeds and / or treated plants; (ii) Growing the treated plant in the presence of the pathogen or harmful organism, or growing the plant from the treated plant parts or the treated seeds; (iii) Collecting one or more tissue samples from the plant and from an untreated control plant, wherein the tissue samples were collected during a growth phase during or after which the pest or pathogen is attacking or has attacked the plant and the untreated control plant; and (iv) The samples are measured to identify an increase in the production of one or more plant defense compounds derived from anthranilic acid in the treated plants compared with untreated control plants, thereby identifying microbial strains that enhance the plant’s response to the pathogen or pest.

21. A method for selecting microbial strains that enhance plant responses to pathogens or harmful organisms, the method comprising: (i) Determining, on one or more tissue samples from plants treated with at least a first microbial strain that is not a pathogen of the plant, or from plants grown from plant parts or seeds treated with the first microbial strain, to determine an increase in the level of one or more plant defense compounds derived from anthranilic acid compared with one or more control tissue samples from untreated control plants, wherein the tissue samples were collected from treated and untreated plants during or after the attack of the pest or pathogen on the plant tissue; as well as (ii) Selecting microbial strains that are not pathogens of the plant and provide elevated levels of one or more plant defense compounds, thereby selecting microbial strains that enhance the plant’s response to the pathogen or pest.

22. The method of claim 20 or 21, further comprising the step of selecting samples from treated plants that exhibit reduced damage from the pathogen or pest compared to the untreated control plants for analysis.

23. The method of claim 20 or 21, wherein the sample is measured to determine the levels of one or more gene transcripts or polypeptides associated with the production of anthranilic acid and / or the conversion of anthranilic acid into plant defense compounds.

24. The method of claim 20, wherein the sample is measured to determine the level of one or more plant defense compounds derived from anthranilic acid.

25. The method according to claim 20 or 21, wherein the pathogen or harmful organism is a fungus, bacteria, nematode, insect or virus.

26. The method of claim 20, wherein the treated plant part is selected from the group consisting of: leaves, stems, buds, flowers, fruits, shoots, roots, tubers, rhizomes, stolons, bulbs, and corms.

27. The method of claim 20, wherein the collected tissue sample is selected from the group consisting of: leaf, stem, bud, flower, fruit, shoot, root, tuber, rhizome, stolon, bulb and corm tissue samples.

28. The method of claim 23, wherein the levels of one or more gene transcripts encoding a component of the anthranilate synthase protein are determined in the treated and untreated tissues.

29. The method of claim 28, wherein the anthranilate synthase protein component is an α or β subunit.

30. The method of claim 23, wherein the level of the gene transcript encoding anthranilic acid N-benzoyltransferase in the treated and untreated tissues is determined.

31. The method according to claim 21 or 24, wherein the plant defense compound is an anthranilate.

32. The method of claim 31, wherein the anthranilate is selected from the group consisting of methyl anthranilate, dimethyl anthranilate, ethyl anthranilate, phenethyl anthranilate, and menthyl anthranilate.

33. The method according to claim 21 or 24, wherein the plant defense compound is a phytoalexin derived from anthranilic acid.

34. The method according to claim 20 or 21, wherein the microbial strain is a bacterial strain or a fungal strain.

35. The method of claim 34, wherein the bacterial strain is Methylobacterium or Methylrubia strains.

36. The method according to claim 20 or 21, wherein the plant is selected from the group consisting of: corn, wheat, rye, rice, alfalfa, barley, oats, rye, sorghum, millet, soybean, Brassica genus Species, Cannabis Species, tobacco, potatoes, peanuts, carrots, cotton, coffee, coconuts, beets, oats, barley, tomatoes, pumpkins, cucumbers, gourds, lettuce, peppers, peas, onions, green beans, sunflowers, safflowers, sweet potatoes, cassava, coffee, coconuts, conifers, lawn grass, leafy green vegetables, miniature vegetables, herbs, fruit plants including fruit trees, and ornamental plants.

37. The method of claim 36, wherein the plant is corn.

38. The method of claim 37, wherein the tissue sample is a root sample.

39. The method according to claim 20 or 21, wherein the pest is a corn rootworm.

40. The method of claim 20 or 21, wherein the first microbial strain is obtained by transferring DNA from a second microbial strain to a third microbial strain, the second microbial strain conferring resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion against or caused by the pest or pathogen by increasing the production of one or more plant defense compounds derived from anthranilic acid in the treated plant, and the third microbial strain not conferring resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion against or caused by the pest or pathogen in the plant treated with the third microbial strain; optionally wherein the second microbial strain is NLS0042, and / or optionally wherein the third microbial strain is a microbial strain other than NLS0042 provided in Table 1; optionally wherein the transferred DNA: (i) encodes one or more proteins having at least 80% sequence identity with one or more of SEQ ID NO: 21-35; or (ii) is present in a protein comprising SEQ ID NO: 86 or thereof and SEQ ID NO:

86. 86 on a plasmid having at least 80% sequence identity; and / or (iii) containing a sequence of one or more of SEQ ID NO:37-50 or a variant thereof having at least 80% sequence identity with one or more of SEQ ID NO:37-50.

41. A method for monitoring resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion in plants, the method comprising determining, from one or more tissue samples of a plant treated with at least a first microbial strain, the first microbial strain being capable of conferring resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion on the harmful organism or pathogen and not being a pathogen of the plant or from plants grown from plant parts or seeds treated with the first microbial strain: (i) increasing the level of one or more plant defense compounds derived from anthranilic acid; and / or (ii) Increase the expression of one or more gene transcripts or polypeptides associated with the production of anthranilic acid and / or the conversion of anthranilic acid into plant defense compounds. The increased levels and / or elevated expression are compared with one or more control tissue samples from untreated control plants, wherein the tissue samples were collected from treated and untreated control plants during or after the attack of the pest or pathogen on the plant tissue, and thus the increased levels of one or more plant defense compounds, gene transcripts and / or peptides in the tissue samples from the treated plants compared with the control plants indicate increased resistance, tolerance, reduced damage, reduced infection and / or reduced invasion of the pest or pathogen in the treated plants.

42. The method of claim 41, wherein the treated plants do not exhibit increased levels of the one or more plant defense compounds, gene transcripts and / or polypeptides, and wherein the method further comprises retreatment with the first microbial strain and / or treatment with another biocontrol agent, insecticide, fungicide or pesticide.

43. A method for selecting microbial strains that enhance plant responses to pathogens or harmful organisms, the method comprising: (i) Determining, on one or more tissue samples from plants treated with at least a first microbial strain that is not a pathogen of the plant, or from plants grown from plant parts or seeds treated with the first microbial strain, to determine an increase in the level of one or more plant defense compounds derived from anthranilic acid compared with one or more control tissue samples from untreated control plants, wherein the tissue samples were collected from treated and untreated plants during or after attack on the plant tissue by the pest or pathogen; and (ii) Selecting microbial strains that are not pathogens of the plant and provide increased levels of the one or more plant defense compounds, thereby selecting microbial strains that enhance the plant's response to the pathogen or pest.

44. A method for selecting microbial strains that enhance plant responses to pathogens or harmful organisms, the method comprising: (i) determining, in combination with at least a first microbial strain that is not a pathogen of the plant, the level of one or more plant defense compounds derived from anthranilic acid is increased compared with one or more untreated control plant tissue samples, wherein the tissue samples were exposed to the pest or pathogen during or after treatment with the first microbial strain; and (ii) selecting a microbial strain that provides an increased level of the one or more plant defense compounds, thereby selecting a microbial strain that enhances the plant’s response to the pathogen or pest.

45. A method for improving a plant's response to a pathogen or pest, wherein the method comprises increasing the level of plant defense compounds produced in the plant by: (i) modifying a plant genome to increase the expression of one or more gene transcripts involved in the production of one or more plant defense compounds in the plant, and / or treating a plant, plant part, or seed with a non-pathogenic microbial strain, wherein the treatment increases the expression of one or more gene transcripts involved in the production of plant defense compounds in the plant; and (ii) The plant is grown in the presence of the pathogen or pest, thereby increasing the level of one or more plant defense compounds in the plant compared to a control plant, and improving the plant’s response to the pathogen or pest compared to a control plant, wherein the control plant is not modified or treated as in (i).

46. ​​The method of claim 45, wherein the plant defense compound is derived from anthranilic acid.

47. A method for repelling pathogens or harmful organisms, wherein the method includes increasing the level of plant defense compounds produced in the plant, including (i) treating soil, plants, plant parts, or seeds with a plant-nonpathogenic microbial strain, wherein said treatment increases the expression of one or more gene transcripts involved in the production of plant defense compounds in said plant; and (ii) The plant is grown in the presence of the pathogen or pest, thereby increasing the level of one or more plant defense compounds in the plant compared to a control plant, and repelling more of the pathogen or pest compared to a control plant, wherein the control plant is not modified or treated as in (i).

48. The method of claim 47, wherein the plant defense compound is derived from anthranilic acid.

49. The method of claim 47, wherein the microbial strain produces metabolites, wherein such metabolites increase the production of such plant defense compounds compared to the control plant.

50. The method of claim 49, wherein the metabolite is selected from the group consisting of siderophores, nonribosomal peptides, polyketides, or combinations thereof.

51. The method of claim 50, wherein the microbial strain comprises one or more gene clusters encoding proteins involved in the biosynthesis of the metabolite.

52. The method of claim 47, wherein the microbial strain comprises one or more genes encoding: (i) one or more proteins having a sequence having at least 80% sequence identity with one or more of SEQ ID NO:21-35; (ii) one or more proteins having at least 80% sequence identity with SEQ ID NO:

21.

53. A method for selecting a microbial strain that confers resistance, tolerance, reduced damage, reduced infection, and / or reduced invasion to a plant pest or pathogen, the method comprising identifying in the microbial strain: (i) one or more polynucleotides encoding proteins in a polyketide biosynthesis pathway; and / or (ii) one or more proteins in a polyketide biosynthesis pathway.

54. The method of claim 53, wherein the polynucleotide is identified by detecting at least one polynucleotide, said at least one polynucleotide: (i) encodes one or more proteins having at least 80% sequence identity with one or more of SEQ ID NO:21-35; (ii) encodes one or more proteins having at least 80% sequence identity with SEQ ID NO:21; (iii) comprises a sequence of one or more of SEQ ID NO:36-50 or one or more sequences having at least 80% sequence identity with any of SEQ ID NO:36-50; and / or (iv) is present on a plasmid comprising SEQ ID NO:86 or a sequence having at least 80% sequence identity with SEQ ID NO:

86.

55. The method of claim 54, wherein the polynucleotide is identified using nucleic acid amplification, hybridization and / or sequencing techniques.

56. The method of claim 53, wherein the protein is identified by detecting: (i) one or more proteins having a sequence having at least 80% sequence identity with one or more of SEQ ID NO:21-35; or (ii) one or more proteins having at least 80% sequence identity with SEQ ID NO:

21.

57. The method of claim 56, wherein the protein is identified by immunoaffinity and / or mass spectrometry.

58. The method of claim 53, further comprising the steps of isolating and / or culturing the identified microbial stain containing the polynucleotide and / or protein.

59. The method according to any one of claims 53 to 58, wherein the microbial strain comprises a gene, said gene: (i) encoding one or more proteins having a sequence having at least 80% sequence identity with one or more of SEQ ID NO:21-35; (ii) encoding one or more proteins having at least 80% sequence identity with SEQ ID NO:21; or (iii) comprising a sequence of one or more of SEQ ID NO:36-50 or a variant thereof having at least 80% sequence identity with SEQ ID NO:36-50.

60. The method according to any one of claims 53 to 58, wherein the microbial strain is genetically modified with DNA: the DNA: (i) encodes one or more proteins having at least 80% sequence identity with one or more of SEQ ID NO: 21-35; (ii) encodes one or more proteins having at least 80% sequence identity with SEQ ID NO: 21; (iii) comprises one or more sequences of one or more of SEQ ID NO: 36-50 or having at least 80% sequence identity with any one of SEQ ID NO: 36-50; and / or (iv) is present on a plasmid comprising SEQ ID NO: 86 or a sequence having at least 80% sequence identity with SEQ ID NO:

86.

61. A method for improving a plant's response to a pathogen or harmful organism, wherein the method comprises: (i) A plant, plant part, or seed is treated with NLS0042 or a microbial stain containing DNA, wherein the DNA: (i) encodes one or more proteins having a sequence having at least 80% sequence identity with one or more of SEQ ID NO:21-35; (ii) encodes one or more proteins having at least 80% sequence identity with SEQ ID NO:21; (iii) contains a sequence of one or more of SEQ ID NO:37-50 or a variant thereof having at least 80% identity with one or more of SEQ ID NO:37-50; and / or (iv) is present on a plasmid containing SEQ ID NO:86 or a variant thereof having at least 80% identity with SEQ ID NO:86; and (ii) The plant or a plant grown from the seed is grown in the presence of the pathogen or pest, thereby improving the plant’s response to the pathogen or pest compared to a control plant; and wherein the plant is not a maize plant.

62. The method of claim 61, wherein the plant is selected from the group consisting of: pepper plants, tomatoes, beans, brassica plants, soybeans, cotton, and rice.

63. The method of claim 61, wherein the level of one or more plant defense compounds in the plant is increased compared to the control plant.

64. The method of claim 61, wherein the pathogen or pest is an insect pest selected from the group consisting of: aphids, lepidopteran insects, stink bugs, whiteflies, beet leafhoppers, tobacco hawk moths, potato leafhoppers, Mexican bean beetles, flea beetles, fall armyworms, soybean loopers, western flower thrips, and water weevils.

65. The method according to any one of claims 61 to 64, wherein the improved response of the treated plant or the plant grown from the treated portion or seed, compared to the untreated control plant, includes improved resistance to, tolerance to, damage reduction, infection reduction, and / or infestation reduction of pests or pathogens in the treated plant or the plant grown from the treated portion or seed.

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