Burkholderia as well as composition, application and use method thereof

Abstract

The invention provides burkholderia as well as a composition, application and a using method thereof. Specifically, the invention provides burkholderia, and a composition containing the strain and / or metabolites, cultures, fermentation broth and / or extracts thereof. The burkholderia or the composition thereof can generate siderophores, induce plants or seeds thereof to generate systemic resistance to plant pathogens and reduce the chemotaxis of the plant pathogens to the plants, so that the prevention and treatment of plant diseases and insect pests are realized; in addition, plant root development can be promoted, a nitrogen fixation effect is provided, a phosphate solubilizing effect is provided, indoleacetic acid is generated, weeds are removed, and therefore plant growth is promoted.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Burkholderia strain, a composition comprising the strain and / or its metabolites, culture, fermentation broth or extract, and uses and methods of use thereof. Background Art

[0002] In fields like agriculture and forestry, chemical pesticides are routinely used to control pests and diseases in crops and other plants, while chemical fertilizers are used to promote their growth. However, these pesticides and fertilizers often suffer from drawbacks such as residual toxicity, environmental pollution, the induction of resistance, soil damage, threats to human health, and a short duration of action. Consequently, a growing number of researchers are focusing on biopesticides and biofertilizers that can overcome these drawbacks.

[0003] Biopesticides and biofertilizers utilize pre-existing living organisms or their metabolites to control plant pests and diseases or promote plant growth. They are naturally degradable, thus overcoming the environmental pollution and toxic residues associated with chemical pesticides and fertilizers. Research has shown that many microorganisms can enhance crop resistance to pests and diseases and effectively improve the soil environment. These include strains of Bacillus subtilis, Bacillus thuringiensis, Bacillus cereus, Brevibacillus laterosporus, Paenibacillus gelatinosa, Bacillus licheniformis, Bacillus megaterium, Bacillus amyloliquefaciens, Pseudomonas putida, Pseudomonas fluorescens, Serratia marcescens, Saccharomyces cerevisiae, and Streptomyces tenuifolius.

[0004] The genus Burkholderia is a β-branch of the class Proteobacteria, consisting of more than 60 species. They are a class of Gram-negative bacteria that are widely found in water, soil, plants, and humans. Some species of Burkholderia can colonize plant roots and the rhizosphere, forming nodules in symbiosis with the plant host, and play a role in promoting plant growth, repairing soil or groundwater, and biological control. However, the functions and effects of different Burkholderia species vary greatly, and some Burkholderia species, such as Burkholderia cepacia, are pathogenic bacteria. Currently, there is no way to eliminate their pathogenicity through modification. Therefore, they cannot be used in agriculture for safety reasons.

[0005] Currently, few Burkholderia species have been isolated that possess high plant disease and insect pest control activity, particularly those that possess both plant disease and insect pest control activity and plant growth promotion activity. Studies have also shown that these species can kill a limited number of plant pathogens, primarily targeting pathogens such as nematodes and mites. Furthermore, research on their mechanisms of action remains inadequate. Therefore, there remains a need to screen and isolate more Burkholderia species with strong plant disease and insect pest control activity, the ability to kill a wider range of plant pathogens, and / or the ability to promote plant growth, and to further explore their mechanisms of action in biocontrol and plant growth promotion, thereby providing more effective, widely applicable, and environmentally friendly biocontrol and plant growth promotion products. Summary of the Invention

[0006] The above-mentioned objects can be achieved by the following aspects of the present invention. In addition, the present invention can also solve other problems that can be clearly seen from the exemplary embodiments.

[0007] In a first aspect, the present invention provides a Burkholderia sp. In some embodiments, the 16s rRNA sequence of the Burkholderia is at least 99%, 99.5%, 99.8%, 99.9% or 100% identical to the sequence shown in SEQ ID NO: 1. Preferably, the Burkholderia comprises one or more strains selected from the following: 1) Burkholderia rinojensis strain; 2) a strain having at least 99.8% or 99.9% identity to the 16s rRNA sequence of Burkholderia rinojensis; 3) a strain having at least 99.8% or 99.9% identity to the 16s rRNA sequence of Burkholderia rinojensis; rinojensis) having an average nucleotide identity of ≥86%, ≥90%, ≥95%, ≥95.5%, ≥96%, ≥96.5%, ≥97%, ≥97.5%, ≥98%, ≥98.5%, ≥99%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9%, ≥99.99% or ≥100% to the genome of R. rinojensis and / or an alignment score of ≥55%, ≥60%, ≥65%, ≥70%, ≥78%, ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%.

[0008] In a further preferred embodiment, the Burkholderia of the present invention has a 16s rRNA sequence as shown in SEQ ID NO: 1.

[0009] In a specific embodiment, the Burkholderia described in the present invention is Burkholderia sp. M222 deposited by the applicant in the Guangdong Provincial Microbiological Culture Collection Center on May 10, 2022, and its deposit number is GDMCC: 62460.

[0010] In other embodiments, the average nucleotide identity between the genome of the Burkholderia of the present invention and the genome of the strain with the deposit number GDMCC: 62460 is ≥86%, ≥90%, ≥95%, ≥95.5%, ≥96%, ≥96.5%, ≥97%, ≥97.5%, ≥98%, ≥98.5%, ≥99%, ≥99.5%, ≥99.6%, ≥99 .7%, ≥99.8%, ≥99.9%, ≥99.99% or ≥100%, and / or the alignment score between the genome of the Burkholderia sp. and the genome of the strain with the deposit number GDMCC: 62460 is ≥55%, ≥60%, ≥65%, ≥70%, ≥78%, ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%. Preferably, the 16s rRNA of the Burkholderia sp. described in the present invention is at least 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the 16s rRNA of the strain with the deposit number GDMCC: 62460. Further preferably, the 16s rRNA of the Burkholderia is at least 99.9%, 99.99% or 100% identical to the 16s rRNA of the strain with the deposit number GDMCC:62460.

[0011] In a second aspect, the present invention provides a composition comprising the Burkholderia described in the first aspect of the present invention and / or its metabolites, culture, fermentation broth, or extract. In some embodiments, the composition comprises the Burkholderia described in the first aspect of the present invention. In preferred embodiments, the Burkholderia comprises an inactivated Burkholderia described in the first aspect of the present invention. In some specific embodiments, the composition of the present invention is a culture or fermentation broth of the Burkholderia described in the first aspect of the present invention.

[0012] In some embodiments, the compositions of the present invention further comprise additional active agents and / or excipients.

[0013] In some embodiments, the additional active agent is selected from one or more additional biological control agents, one or more chemical agents, one or more fertilizers, one or more herbicides, one or more growth promoters, or any combination thereof. In preferred embodiments, the biological control agent is selected from bacteria, fungi, viral biological control agents, insect biological control agents, nematode biological control agents, or any combination thereof. In a further preferred embodiment, the biological control agent is at least one selected from Trichoderma harzianum, Purpureocillium lilacinum, Penicillium bilaiae, Bacillus subtilis, Burkholderia rinojensis, Bacillus pumilus, Bacillus velezensis, Methylobacterium rhodesianum, Methylobacterium extorquens, and Paenibacillus peoriae. Preferably, the Trichoderma harzianum is Trichoderma harzianum with a deposit number of CGMCC No. 15679. Preferably, the Purpureocillium lilacinum is Purpureocillium lilacinum with a deposit number of CGMCC No. 12773. Preferably, the Penicillium bilaiae is Penicillium bilaiae with a deposit number of CGMCC NO.12767. Preferably, the Bacillus subtilis is Bacillus subtilis with a deposit number of CGMCC NO.12908. Preferably, the Burkholderia rennogii is Burkholderia rennogii with a deposit number of GDMCC NO.61156. Preferably, the Bacillus pumilus is Bacillus pumilus with a deposit number of GDMCC NO.61962. Preferably, the Bacillus velez is Bacillus velez with a deposit number of GDMCC NO.61434. Preferably, the Methylobacterium rothei is Methylobacterium rothei with a deposit number of GDMCC NO.60729. Preferably, the Methylobacterium contortus is Methylobacterium contortus with a deposit number of GDMCC NO.62943. Preferably, the Paenibacillus pierreuil is the Paenibacillus pierreuil with a deposit number of GDMCC NO.60482.

[0014] In some embodiments, the excipients include agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film formers, spontaneity promoters, emulsifiers, dispersants, preservatives, antifreeze agents, thickeners, adjuvants, or any combination thereof.

[0015] In a preferred embodiment, the composition of the second aspect of the present invention is in solid form, liquid form, powder form, or any combination thereof. Preferably, the composition is in a form selected from the group consisting of a solution, a powder, a granule, an emulsion, an emulsion, a suspension, a tablet, a microgranule, and a wettable powder. Preferably, the composition is in the form of a microbial agent for use as a biofertilizer, biopesticide, or bioherbicide.

[0016] In a third aspect, the present invention provides use of the Burkholderia or its metabolite, culture, fermentation broth or extract as described in the first aspect of the present invention or the composition as described in the second aspect of the present invention in the preparation of a microbial agent, a biofertilizer, a biopesticide or a bioherbicide.

[0017] In a fourth aspect, the present invention provides use of the Burkholderia species described in the first aspect of the present invention, or its metabolite, culture, fermentation broth, or extract, or the composition described in the second aspect of the present invention, for controlling plant diseases and insect pests and / or weed control. Preferably, the plant diseases and insect pests are selected from plant revegetation diseases, plant bacterial diseases, plant fungal diseases, plant viral diseases, soil-borne plant diseases, plant insect pests, and / or plant oomycete diseases.

[0018] In some embodiments, the plant pests and diseases are caused by one or more of plant pathogenic bacteria, fungi, viruses, insects, insect eggs, and nematodes. Preferably, the plant diseases and insect pests are caused by at least one of the following pathogens: bacteria, such as Streptomyces scabies, Ralstonia solanacearum, Bacillus subtilis, Pseudomonas syringae pv. actinidiae, Xanthomonas campestris pv. Oryzae, Erwinia aroideae, Xanthomonas arboricola pv. juglandis, Erwinia carotovora, Staphylococcus aureus; fungi, such as Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum; graminearum), Athelia rolfsii, Sclerotinia sclerotiorum, Botrytis cirerea, Fusarium oxysporum f.sp.cucumerinum, Gaeumannomyces critici, Fusarium graminearum, Valsamali, Glomerella cingulata, Rhizoctonia solan, Pyricularia grisea, Alternaria solani, Botrytis cirerea, Phytophthora infestans, Exserohilum turcicum, Bipolaria maydis, Fusarium oxysporum f.sp.niveum), Verticillium dahliae, Fusarium oxysporum f.sp.vasinfectum, Phytophthora capsici, and Phytophthora nicotianae.

[0019] In a preferred embodiment, the plant pest is caused by a nematode or its eggs. More preferably, the nematode is selected from one or more of Meloidogyne spp. and Caenorhabditis elegans; further preferably, the root-knot nematode is selected from one or more of M. incognita, M. hapla, M. arenaria, and M. javanica.

[0020] In some other preferred embodiments, the plant disease is caused by insects. More preferably, the insects are selected from the group consisting of Spodoptera frugiperda (Smith), Locusta migratoria Linnaeus (migratory locusts and other migratory locusts), Loxostege sticticalis Linnaeus, armyworms (such as Mythimna separate (Walker) and Leucania loryi Duponchel), rice planthoppers (such as Nilaparvata lugens), and brown planthoppers. and white-backed planthopper Sogatella furcifera (Horváth)), rice leaf roller Cnaphalocrocis medinalis (Guenée), striped stem borer Chilo suppressalis (Walker), wheat aphids (such as Sitobion avenae (Fabricius), Rhopalosiphum padi (Linnaeus), Schizaphis graminum (Rondani)), Asian corn borer Ostrinia furnacalis (Guenée), vegetable thrips (such as Megalurothrips usitatus (Bagnall), Thrips palmi Karny, western flower thrips Frankliniella occidentalis (Pergande), flower thrips Frankliniella intonsa (Trybom)).

[0021] In a preferred embodiment, the control of plant diseases and insect pests is achieved by producing siderophores, inducing systemic resistance of plants or their seeds to plant pathogens, and / or reducing the chemotaxis of plant pathogens to plants.

[0022] In a fifth aspect, the present invention provides use of the Burkholderia species described in the first aspect of the present invention, or its metabolite, culture, fermentation broth, or extract, or the composition described in the second aspect of the present invention, for promoting plant growth. In a preferred embodiment, the plant growth promotion is achieved by at least one of promoting plant root development, providing nitrogen fixation, providing phosphate solubilization, producing indoleacetic acid, and removing weeds.

[0023] In an embodiment of the present invention, the plant is selected from food crops, cash crops, or root crops. Preferably, the food crops are selected from at least one of cereal crops, tuber crops, and legume crops. Preferably, the cash crops are selected from at least one of the Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Cruciferae, Orchidaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passiflora, Bromeliaceae, Araliaceae, Leguminosae, and Cactaceae. Preferably, the root crops are selected from potatoes, carrots, leafy vegetables, nightshade vegetables, strawberries, grapes, citrus, bananas, kiwis, pitaya, tomatoes, peppers, beans, ginger, Panax notoginseng, ginseng, and the like.

[0024] In a sixth aspect, the present invention provides a method for preparing a fermentation broth of the Burkholderia according to the first aspect of the present invention, comprising the following steps: first activating and culturing the Burkholderia, and then fermenting and culturing the Burkholderia.

[0025] In a seventh aspect, the present invention provides a method for controlling plant diseases and pests and / or promoting plant growth, comprising applying to plants or seeds the Burkholderia or its metabolites, cultures, fermentation broth or extracts as described in the first aspect of the present invention, or the composition as described in the second aspect of the present invention; preferably, applying a fermentation broth prepared by the method as described in the sixth aspect of the present invention.

[0026] Compared to the prior art, the present invention provides a newly discovered Burkholderia strain and utilizes its metabolites, culture, fermentation broth, and extracts to prepare the compositions described herein. In applications in agriculture, forestry, and other fields, the strain, its metabolites, culture, fermentation broth, extract, and composition can produce siderophores, induce systemic resistance in plants or their seeds to plant pathogens, and reduce the chemotaxis of plant pathogens toward plants, thereby killing or inhibiting a wider variety of plant pathogens and thus achieving control over a wide range of plant diseases and insect pests. Furthermore, they can promote plant root development, provide nitrogen fixation, solubilize phosphate, produce indoleacetic acid, and remove weeds, thereby more effectively promoting plant development and growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The objects, features and advantages of the present invention will be better understood by referring to the following detailed description and accompanying drawings which illustrate illustrative embodiments utilizing the principles of the invention. In these drawings:

[0028] Figure 1 The figure shows the colony morphology characteristics of Burkholderia sp. M222 after culture on LB solid medium plates.

[0029] Figure 2 The phylogenetic tree of Burkholderia sp. M222 is shown.

[0030] Figure 3 Shown are the results of testing the ability of Burkholderia sp. M222 to produce siderophores by the CAS plate overlay method.

[0031] Figure 4 Shown are staining images of tomato roots 7 days after root dip treatment in a nematode chemotaxis assay. The left image shows a sterile water control; the right image shows an M222-10× treatment.

[0032] Figure 5 Shown are staining images of cucumber roots 7 days after treatment with M222 inoculant gel in a nematode chemotaxis test. The left image shows a sterile water control; the right image shows an M222-5× treatment.

[0033] Figure 6 The results of the test of the pathogen antagonism of Burkholderia sp. M222 are shown. The upper row is the pathogen blank control, and the lower row is the confrontation culture of pathogen and M222. Figure 6 In the table, Rs represents Ralstonia solanacearum, Ss represents Streptomyces scabies, Fo represents Fusarium oxysporum, Fg represents Fusarium graminearum, Ar represents Sclerotium candidum, and Cc represents Colletotrichum anthracis.

[0034] Figure 7 Shown are the results of a qualitative test of nitrogen fixation by Burkholderia sp. M222.

[0035] Figure 8 The results of a qualitative test of phosphate solubilization of Burkholderia sp. M222 are shown.

[0036] Figure 9 Shown are the growth vigor of potted tomatoes treated with fermentation broth of Burkholderia sp. M222.

[0037] Figure 10 Shows the growth of potted cucumbers treated with the fermentation liquid of Burkholderia sp. M222.

[0038] Figure 11 The results of qualitative testing of the IAA-producing ability of Burkholderia sp. M222 are shown. DETAILED DESCRIPTION

[0039] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0040] Unless otherwise indicated, all scientific and technological terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art. Unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology and pharmacology known to those skilled in the art are employed in the practice of the present invention.

[0041] It should be noted that all headings and subheadings used herein are for convenience only and should not be construed as limiting the invention in any way.

[0042] The use of any and all examples, or exemplary language (eg, "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0043] The terms “include” and “comprising” used in the description and claims of this application refer to the presence of the described features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0044] It should also be understood that the terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the present invention. Unless the context clearly indicates otherwise, "a," "an," "the," or "said" used in the specification and claims of this application may refer to one or more than one. Unless the context clearly indicates otherwise, terms presented in the singular also include the plural.

[0045] It should be further understood that the term "and / or" used in this specification and claims refers to any combination of one or more associated listed items and all possible combinations, and includes these combinations. In addition, in some embodiments of the present invention, the features or combinations of features set forth herein may also be excluded or omitted.

[0046] In the following, various aspects and specific embodiments of the present invention will be described in more detail by way of non-limiting embodiments and examples.

[0047] In one embodiment, the present invention relates to Burkholderia deposited with the deposit number GDMCC: 62460. The Burkholderia was named M222 and was deposited on May 10, 2022, at the Guangdong Provincial Microbial Culture Collection Center, with the deposit address being: 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with the deposit number GDMCC: 62460, the test result was survival, and the taxonomic name is Burkholderia sp.

[0048] The Burkholderia sp. M222 of the present invention is a non-spore-forming Gram-negative bacterium. After being cultured in LB solid medium at 30° C. for one or several days, the colonies are white with rough surfaces and edges and are sticky.

[0049] It is well known in the art that in addition to traditional taxonomic methods, including but not limited to cell morphology observation, Gram staining, flagellar staining, and various metabolic experiments, bacterial species can also be classified and identified using molecular biological methods. Molecular biological methods include ribosomal RNA sequencing and whole genome sequencing-based methods.

[0050] The sequencing results of the 16s rRNA sequence of Burkholderia sp. M222 showed that its 16s RNA sequence is shown in SEQ ID NO: 1. Further, by comparing the 16s rRNA sequences of bacteria, a biological evolutionary tree was drawn according to the sequence difference bases and their evolutionary distances, as shown in FIG. Figure 2 shown.

[0051] In other embodiments, the present invention also relates to progeny strains, subcloned strains or genetically modified strains of Burkholderia sp. M222. Preferably, the strains retain the biological activities of M222, such as pest control and plant growth promotion effects.

[0052] In other embodiments, the present invention relates to a variant of Burkholderia sp. M222 as described herein. Preferably, the genome of the Burkholderia variant has an average nucleotide identity of ≥86%, ≥90%, ≥95%, ≥95.5%, ≥96%, ≥96.5%, ≥97%, ≥97.5%, ≥98%, ≥98.5%, ≥99%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9%, ≥99.99% or ≥100% to the genome of the above-mentioned Burkholderia sp. M222 strain. sp.) M222 strain. Preferably, the 16s rRNA of the Burkholderia variant is at least 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the 16s rRNA of the Burkholderia sp. M222 strain. Preferably, the Burkholderia variant does not include Burkholderia ambifaria, Burkholderia multivorans or Burkholderia stabilis. Further preferably, the 16s rRNA of the Burkholderia variant is at least 99.9%, 99.99% or 100% identical to the 16s rRNA of the Burkholderia sp. M222 strain.

[0053] In other embodiments, the present invention also relates to Burkholderia having at least 99%, 99.5%, 99.8%, 99.9% or 100% identity to the sequence shown in SEQ ID NO: 1. Preferably, the Burkholderia does not include Burkholderia ambifaria, Burkholderia multivorans or Burkholderia stabilis. Further preferably, the Burkholderia includes one or more strains selected from the following: 1) Burkholderia rinojensis strain; 2) strains having at least 99.8% or 99.9% identity to the 16s rRNA sequence of Burkholderia rinojensis; 3) strains having at least 99.8% or 99.9% identity to the 16s rRNA sequence of Burkholderia rinojensis; rinojensis) having an average nucleotide identity of ≥86%, ≥90%, ≥95%, ≥95.5%, ≥96%, ≥96.5%, ≥97%, ≥97.5%, ≥98%, ≥98.5%, ≥99%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9%, ≥99.99% or ≥100% to the genome of R. rinojensis and / or an alignment score of ≥55%, ≥60%, ≥65%, ≥70%, ≥78%, ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%.

[0054] As used herein, "identity" with respect to nucleic acid sequences refers to the degree to which two nucleic acid sequences have identical nucleotide residues at the same position when compared to achieve maximum identity, expressed as a percentage. Identity can be determined using computer algorithms known in the art, including but not limited to GCG, BLASTN, FASTA, etc.

[0055] As used herein, the term "variant" includes any strain derived from the Burkholderia sp. strains described herein, such as strains produced by natural mutation or recombination, or by mutation or recombination caused by resistance selection, radiation, viruses, transposons, or mutagenic chemicals, etc. In addition, variants as described herein also include other strains isolated from the natural environment that have the same or similar taxonomic characteristics and properties as the Burkholderia sp. strains described herein.

[0056] In some embodiments, the present invention relates to metabolites, cultures, fermentation broths, or extracts of Burkholderia (e.g., M222) described herein. "Metabolites," "cultures," "fermentation broths," and "extracts" as used herein include any components, compounds, substances, or byproducts (including but not limited to small molecule secondary metabolites) produced by the Burkholderia described herein that have any beneficial effects described herein. The beneficial effects include resistance to plant replantation diseases, plant bacterial diseases, plant fungal diseases, plant viral diseases, plant soil-borne diseases, plant insect pests, and / or plant oomycete diseases, as well as promoting plant growth.

[0057] In some embodiments, the present invention relates to a composition comprising the Burkholderia described herein. In preferred embodiments, the composition comprises inactivated Burkholderia described herein, such as inactivated M222. In some embodiments, the Burkholderia is cultured under suitable conditions for approximately 48 hours. Preferably, the Burkholderia is cultured with shaking at approximately 28°C to 30°C for approximately 48 hours. Alternatively, the Burkholderia can be treated at approximately 60°C for approximately 2 hours. Experiments have demonstrated that compositions comprising inactivated Burkholderia have excellent pest and disease control and plant growth promotion effects.

[0058] In other embodiments, the present invention also relates to compositions comprising metabolites, cultures, fermentation broths, or extracts of the Burkholderia species described herein (eg, M222).

[0059] In further embodiments, the compositions of the present invention further comprise an additional active agent. The additional active agents described herein are biological and / or chemical agents having specific activities other than the Burkholderia species contained in the compositions of the present invention. The additional active agents described herein are additional active agents that are compatible with metabolites, cultures, fermentation broths, or extracts of the Burkholderia species (e.g., M222).

[0060] In some embodiments, the additional active agent can be a plant nutrient, a plant growth promoter, a biostimulant (trace elements), a fertilizer, a seed coating agent, and the like.

[0061] Preferably, the fertilizer includes, but is not limited to, at least one of amino acid fertilizers, fulvic acid fertilizers, seaweed fertilizers, humic acid fertilizers, fertilizers containing pollen polysaccharides, polypeptide fertilizers, and microbial fertilizers. As used herein, the term "microbial fertilizer" refers to a product containing specific microorganisms that is applied to agricultural production and can achieve a specific fertilizer effect. Such effects include not only the supply of nutrients to the soil, environment, and plants, but also the beneficial effects of the metabolites produced by the microorganisms on plants.

[0062] In some embodiments, the additional active agent may include one or more chemical agents. In some embodiments, the chemical agent includes but is not limited to insecticides, bactericides, fungicides, virucides, nematocides, insecticides, acaricides, gastropodicides, herbicides, etc., or a combination thereof. In a preferred embodiment, the chemical agent includes one or more of bactericides, fungicides, and nematicides. Preferably, the bactericide includes but is not limited to one or more of chlorpyrifos, chloramphenicol, thiophanate-methyl, thiophanate-methyl, ethyl thiophanate, copper hydroxide, kasugamycin, chlorpyrifos, copper oxychloride, chlorobromoisocyanuric acid, trichloroisocyanuric acid, copper acetate, or copper succinate. Preferably, the fungicide includes but is not limited to one or more of carbendazim, mancozeb, fosetyl-aluminum, metalaxyl, cymoxanil, dimethomorph, flumorph, silver farinaceous, benzamidine, myclobutanil, tebuconazole, or propiconazole. Preferably, the nematicides include, but are not limited to, one or more of halogenated hydrocarbons, methyl thioisothiocyanate, organophosphorus, carbamates or avermectin.

[0063] In some embodiments, the additional active agent may include one or more additional biological control agents. Preferably, the biological control agent includes, but is not limited to, bacteria, fungi (e.g., yeast), viral biological control agents, insect biological control agents, nematode biological control agents, or any combination thereof. Preferably, the bacteria include bacteria selected from the genus Bacillus, Burkholderia, or any combination thereof.

[0064] Further preferably, the biological control agent is selected from at least one of Trichoderma harzianum, Purpureocillium lilacinum, Penicillium bilaiae, Bacillus subtilis, Burkholderia rinojensis, Bacillus pumilus, Bacillus velezensis, Bacillus coagulans, Methylobacterium rhodesianum, Methylobacterium extorquens, Paenibacillus peoriae, and Bacillus licheniformis.

[0065] Preferably, the Trichoderma harzianum includes Trichoderma harzianum with a deposit number of CGMCC NO.15679.

[0066] Preferably, the Pseudomonas lilacinus includes the Pseudomonas lilacinus with a deposit number of CGMCC NO.12773.

[0067] Preferably, the Penicillium bilaiae includes the Penicillium bilaiae with a deposit number of CGMCC NO.12767.

[0068] Preferably, the Bacillus subtilis includes the Bacillus subtilis with a deposit number of CGMCC NO.12908.

[0069] Preferably, the Burkholderia rennogii includes the Burkholderia rennogii with a deposit number of GDMCC NO.61156.

[0070] Preferably, the Bacillus pumilus includes the Bacillus pumilus with a deposit number of GDMCC NO.61962.

[0071] Preferably, the Bacillus Velez subtilis includes the Bacillus Velez subtilis with a deposit number of GDMCC NO.61434.

[0072] Preferably, the Methylobacterium rhodesi includes Methylobacterium rhodesi with a deposit number of GDMCC NO.60729.

[0073] Preferably, the Methylobacterium contortus includes Methylobacterium contortus with a deposit number of GDMCC NO.62943.

[0074] Preferably, the Paenibacillus pierreuil includes the Paenibacillus pierreuil with a deposit number of GDMCC NO.60482.

[0075] In preferred embodiments, the additional active agents described herein may include a combination of two or more of the above described chemical and biological agents.

[0076] In some embodiments, the composition of the present invention further comprises one or more excipients. Preferably, the excipients include agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film formers, spontaneity promoters, emulsifiers, dispersants, preservatives, antifreeze agents, thickeners, adjuvants, or any combination thereof. Preferably, the excipients improve the spreadability of the inoculant on plants and seeds.

[0077] In some embodiments, the composition of the present invention can be provided in the form of a microbial agent. Preferably, the composition is used as a biofertilizer, biopesticide, or bioherbicide. Additionally, the composition of the present invention can be used to prepare a microbial agent, biofertilizer, biopesticide, or bioherbicide.

[0078] The terms "biofertilizer" and "plant growth promoter" used in this specification are used interchangeably, and refer to preparations with organisms or their derivatives as the main active ingredients and the effect of promoting plant growth (promoting growth). The terms "biopesticide" and "bioinsecticide" used in this specification are used interchangeably, and refer to preparations that use organisms or their derivatives to kill or inhibit agricultural pests such as plant pathogens that cause plant diseases and insect pests. The term "bioherbicide" used in this specification refers to a preparation that uses organisms or their derivatives to remove weeds. The biofertilizers, biopesticides, and bioherbicides described herein may also be referred to as microbial fertilizers, microbial pesticides, or microbial herbicides. Specifically, the organisms used therein include Burkholderia as described above.

[0079] Preferably, the composition of the present invention has one of the activities of killing or inhibiting plant pathogens, promoting plant growth, producing indoleacetic acid, removing weeds, etc., and more preferably, has a combination of two or more of the above activities.

[0080] The Burkholderia strain of the present invention or its variant or progeny thereof or the composition of the present invention can be particularly used in at least one of the following aspects:

[0081] 1) Prevent and control plant pathogenic fungal diseases;

[0082] 2) Prevent and control plant pathogenic bacterial diseases;

[0083] 3) Preparation of products for preventing and controlling plant pathogenic fungal diseases;

[0084] 4) Preparation of products for preventing and treating plant pathogenic bacterial diseases;

[0085] 5) Resistance to plant diseases and insect pests;

[0086] 6) Preparation of products for resisting plant diseases and insect pests;

[0087] 7) Promote plant growth;

[0088] 8) Preparation of products for promoting plant growth;

[0089] 9) Prevent and control plant nematode diseases;

[0090] 10) Preparing products for preventing and controlling plant nematode diseases.

[0091] Preferably, the promoting plant growth includes promoting the emergence rate, increasing plant height and / or increasing yield.

[0092] The composition of the present invention can be provided in the form of solid, liquid, powder or any combination thereof. For example, the composition of the present invention can be formulated into dosage forms such as solution, powder, granule, emulsion, emulsion, suspension, tablet, microgranule and wettable powder.

[0093] The Burkholderia species, metabolites, cultures, fermentation broths, extracts, or compositions of the present invention can be used to control plant diseases and insect pests. Preferably, the plant diseases and insect pests are selected from plant revegetation diseases, plant bacterial diseases, plant fungal diseases, plant viral diseases, plant soil-borne diseases, plant insect pests, and / or plant oomycete diseases. Preferably, the plant diseases and insect pests are caused by one or more of plant bacterial pathogens, plant fungal pathogens, viruses, insects, insect eggs, and nematodes.

[0094] Preferably, the plant bacterial pathogens include but are not limited to at least one of Streptomyces cesscabies, Ralstonia solanacearum, Bacillus subtilis, Pseudomonas syringae pv. actinidiae, Xanthomonas campestris pv. Oryzae, Erwinia aroideae, Xanthomonas arboricola pv. juglandis, Erwinia carotovora and Staphylococcus aureus.

[0095] Preferably, the plant fungal pathogens include but are not limited to Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum, Athelia rolfsii, Sclerotiniasclerotiorum, Botrytis cirerea, Fusarium oxysporumf.sp.cucumerinum, Gaeumannomyces critici, Fusarium graminearum, Valsamali, Glomerella cingulata, Rhizoctonia solanum, Pyricularia grisea, Alternaria solani, Botrytis cirerea, Fusarium oxysporumf.sp.cucumerinum, Gaeumannomyces critici, Fusarium graminearum, Valsamali, Glomerella cingulata, Rhizoctonia solanum, Pyricularia grisea, Alternaria solani, Botrytis cirerea, cirerea), potato late blight (Phytophthora infestans), maize large blight (Exserohilum turcicum), maize small blight (Bipolaria maydis), watermelon wilt (Fusarium oxysporum f.sp. niveum), eggplant wilt (Verticillium dahliae), cotton wilt (Fusarium oxysporum f.sp. vasinfectum), pepper wilt (Phytophthora capsici) and tobacco wilt (Phytophthora nicotianae).

[0096] Preferably, the plant diseases and insect pests include but are not limited to at least one of scab, bacterial wilt, damping-off, black shank disease, and black mole disease.

[0097] Preferably, the nematodes include, but are not limited to, one or more of Meloidogyne spp. and Caenorhabditis elegans. Preferably, the root-knot nematodes include, but are not limited to, one or more of M. incognita, M. hapla, M. arenaria, and M. javanica.

[0098] Preferably, the insects include but are not limited to insects of the subclass Pterygota, such as Orthoptera, Isoptera (e.g., termites), Hemiptera (e.g., stink bugs), Homoptera, Thysanoptera (e.g., thrips), Coleoptera (e.g., beetles), Lepidoptera (e.g., moths or butterflies), Diptera and Hymenoptera (e.g., bees).

[0099] In addition, the plant diseases and insect pests also include plant diseases and insect pests caused by mites, including but not limited to the Tetranychidae, Acaridae, Leaf-flying Mites, and Acaridae of the order Acarina.

[0100] The Burkholderia of the present invention can produce a variety of siderophores that assist in survival or resistance to toxic metals, such as ornibactin, malleobactin, pyochelin, cepabactin, and cepaciachelin. Siderophores not only provide plants with iron nutrition, but also can achieve the effect of biological control by competing with plant pathogens for iron nutrition. Most pathogens do not have the ability to secrete siderophores or have a low ability to secrete siderophores, and their ability to compete with other siderophore-producing microorganisms for iron nutrition is weak. Therefore, the Burkholderia of the present invention can not only antagonize a variety of plant bacterial pathogens and plant fungal pathogens, but can also be widely used in the prevention and treatment of more plant diseases and insect pests.

[0101] In addition, the Burkholderia, its metabolites, cultures, fermentation broths, extracts or compositions of the present invention can also induce plants or their seeds to produce systemic resistance to plant pathogens, thereby further enhancing their effectiveness in preventing and controlling plant diseases and insect pests.

[0102] Furthermore, the Burkholderia species, their metabolites, cultures, fermentation broths, extracts, or compositions of the present invention can also reduce the chemotaxis of plant pathogens toward plants. For example, during the early interactions between nematodes and plants, the nematodes can sense chemical signals released by plants or rhizosphere microorganisms to seek out hosts. By reducing the chemotaxis of nematodes toward plants, infection and damage by the nematodes can be prevented or mitigated, thereby achieving the desired effect of controlling plant diseases and insect pests.

[0103] In some embodiments, the Burkholderia, its metabolites, cultures, fermentation broths, extracts or compositions of the present invention achieve control of plant diseases and insect pests by producing siderophores, inducing systemic resistance of plants or their seeds to plant pathogens, and reducing the chemotaxis of plant pathogens to plants.

[0104] The inventors conducted experiments comparing the plant pest control effects of avermectin, a commonly used insecticide in the field, and the Burkholderia of the present invention. The results showed that while avermectin had a good control effect under controlled greenhouse conditions, it was less effective when used in the field. This may be due to the following two reasons: 1) Due to the problem of overuse of avermectin in the field, plant pathogens have developed resistance to avermectin; 2) Under controlled greenhouse conditions, the types of plant pathogens are relatively simple, so avermectin can still show good results; however, there are many different types of plant pathogens in the field, and avermectin has a poor killing effect on some of these pathogens, resulting in poor control effects. In comparison, the Burkholderia of the present invention showed good control effects in both greenhouse and field environments, suggesting that it works through multiple mechanisms such as pathogen-promoting killing mechanisms, pathogen infection inhibition mechanisms, and crop resistance induction effects, with a broad spectrum of pest control and application, making it suitable for plant pest control in complex field environments.

[0105] In addition, the Burkholderia, metabolites, cultures, fermentation broths, extracts or compositions of the present invention can also be used to promote plant growth.

[0106] Compared with other Burkholderia, the Burkholderia, its metabolites, cultures, fermentation broths, extracts or compositions of the present invention have the ability to fix nitrogen and solubilize phosphate, can provide nutrients required for plant growth, and can produce indoleacetic acid, thereby promoting plant development and growth.

[0107] In addition, the Burkholderia, metabolites, cultures, fermentation broths, extracts, or compositions of the present invention also have the effect of removing weeds. The weeds described herein primarily refer to weeds known in the agricultural field. Preferably, the weeds include, but are not limited to, Amaranthus retroflexus L., Setaria viridis (Sedum sarmentosum), and the like.

[0108] In some embodiments, the Burkholderia, metabolites, cultures, fermentation broths, extracts or compositions of the present invention promote plant development and growth by promoting one or more of the following mechanisms: root development, nitrogen fixation, phosphate solubilization, indoleacetic acid production, and weed control.

[0109] The plants mentioned in the specification of this application include plants in the fields of agriculture, forestry, etc. Preferably, the plants include but are not limited to food crops, cash crops or root crops.

[0110] Preferably, the food crops include but are not limited to at least one of cereal crops, tuber crops, and legume crops.

[0111] Preferably, the cereal crops include, but are not limited to, at least one of the following grass crops: rice, wheat, corn, barley, oats, rye, sorghum, millet, broomcorn millet, barnyard grass, and buckwheat. Preferably, the tuber crops include, but are not limited to, at least one of the following: sweet potato, yam, and potato. Preferably, the legume crops include, but are not limited to, at least one of the following: soybean, broad bean, pea, mung bean, and peanut.

[0112] Preferably, the economic crops include but are not limited to at least one of the following economic crops: Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Cruciferae, Orchidaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passiflora, Bromeliaceae, Araliaceae and Cactaceae plants.

[0113] Preferably, the Solanaceae plants include, but are not limited to, tomatoes, peppers, potatoes, and eggplants. Preferably, the Rosaceae plants include, but are not limited to, strawberries and papayas. Preferably, the Rutaceae plants include, but are not limited to, citrus fruits. Preferably, the Musaceae plants include, but are not limited to, bananas. Preferably, the Cucurbitaceae plants include, but are not limited to, cucumbers, wax gourds, pumpkins, bitter melons, loofahs, watermelons, and monk fruit. Preferably, the Cruciferae plants include, but are not limited to, cabbage, rapeseed, kale, radish, and cauliflower. Preferably, the Orchidaceae plants include, but are not limited to, orchids. Preferably, the Fabaceae plants include, but are not limited to, soybeans. Preferably, the Asteraceae plants include, but are not limited to, lettuce. Preferably, the Liliaceae plants include, but are not limited to, garlic. Preferably, the Zingiberaceae plants include, but are not limited to, ginger. Preferably, the Passiflora plants include, but are not limited to, passion fruit. Preferably, the Bromeliaceae plants include, but are not limited to, golden pineapples. Preferably, the Araliaceae plants include, but are not limited to, Panax notoginseng. Preferably, the Cactaceae plants include, but are not limited to, pitaya.

[0114] Preferably, the root crops include but are not limited to potatoes, carrots, leafy vegetables, nightshade vegetables, strawberries, grapes, citrus, bananas, kiwis, dragon fruits, tomatoes, peppers, beans, ginger, Panax notoginseng, ginseng, etc.

[0115] The plants described herein include plants at all stages, including those in the seed germination stage, seedling growth stage, seedling development stage, seedling and flowering stage, and seed formation stage. Therefore, the term "plant" includes both seedlings and plants after growth and development. Furthermore, the term "plant" as used herein also includes plant tissues and plant organs. Preferably, the plant tissues include, but are not limited to, meristematic tissue, protective tissue, cardinal tissue, and conducting tissue. Preferably, the plant organs include, but are not limited to, roots, stems, leaves, flowers, fruits, and seeds.

[0116] In one embodiment, the present invention relates to a method for preparing a fermentation broth of the Burkholderia of the present invention, comprising the steps of: first, subjecting the Burkholderia to an activation culture, and then subjecting the Burkholderia to a fermentation culture. Preferably, the activation culture is carried out on a solid culture medium. Preferably, the fermentation culture is carried out in a liquid culture medium. In a preferred embodiment, the culture medium is LB culture medium, preferably a modified LB culture medium. In a specific embodiment, the LB culture medium comprises yeast extract, peptone, sodium chloride, and water. In a more specific embodiment, the LB culture medium comprises 5-10 g of yeast extract, 8-12 g of peptone, 5-15 g of sodium chloride, and water, with a pH of 7-7.5. In a preferred embodiment, the activation culture is carried out at approximately 30° C. for approximately 2 days. In a preferred embodiment, the fermentation culture is carried out at approximately 30° C. for approximately 48 hours.

[0117] In some embodiments, the present invention relates to a method for controlling plant pests and diseases and / or promoting plant growth, comprising the step of applying to a plant, plant tissue, plant organ, or seed a Burkholderia of the present invention, or a metabolite, culture, fermentation broth, or extract thereof, or a composition of the present invention. Preferably, the method comprises applying to a plant, plant tissue, plant organ, or seed a fermentation broth of the Burkholderia of the present invention prepared as described herein.

[0118] Preferably, the application methods include root soaking, foliar spraying, spraying, composting, seed soaking, coating, field flooding, drip irrigation of plants or plant organs, smearing plants or plant organs, dripping plants or plant organs, etc.

[0119] Example

[0120] The following are non-limiting examples for practicing the present invention. The following examples are provided merely to illustrate embodiments of the present invention and should not be construed as limiting the present invention in any way.

[0121] Example 1: Isolation and identification of Burkholderia sp. M222

[0122] 1. Isolation of M222 strain

[0123] The Burkholderia sp. M222 of the present invention was isolated from the rhizosphere soil of Gastrodia elata in Yunnan Province. The isolation method is as follows: 5 g of soil was weighed, 45 mL of sterile water was added, and then vortexed for 5 minutes; the soil was gradiently diluted to 10 -1 ~10 -6 ; Take 10 -4 , 10 -5 and 10 -6Three dilutions of the bacterial solution were spread on modified Luria-Bertani (LB) solid medium (5-10 g yeast extract, 8-12 g peptone, 5-15 g sodium chloride, 15 g agar, 1 L water, pH = 7-7.5) plates, with three replicates for each dilution. The plates were incubated upside down in a 30°C constant temperature incubator for 4 days. Single colonies of the strain were then picked and streaked onto new LB plates. The plates were incubated at 30°C for 1 day to obtain pure cultures of the strains. The cultures were then stored in a -80°C freezer using glycerol tubes.

[0124] 2. Identification of M222 strain

[0125] The isolated strain was inoculated into the modified LB solid medium and cultured at 30°C for 1 day. Figure 1 Identification showed that the strain is a non-spore-forming Gram-negative bacterium, whose colonies are mostly white, with rough surfaces and edges and are sticky (see Figure 1 ).

[0126] Subsequently, the 16s rRNA sequence of the isolated strain was determined using amplification primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2) and sequencing primers 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3). The results showed that the 16s rRNA sequence of the strain was:

[0127] AGTCGAACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGATGAAAGCGGGGGATCTTCGGACCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGAAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTTTGGGTTAATACCTCGGAGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGACAGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGTGACTGGCAAGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATT(SEQ ID NO: 1)

[0128] Then, a BLAST homology comparison was performed on the sequence, and the sequence was aligned with the sequence in the NCBI database. The results showed that the 16s rRNA sequence of the isolated strain was more than 99% similar to the 16s rRNA sequence of multiple strains under the genus Burkholderia, for example: the similarity with the 16s rRNA sequence of Burkholderia ambifaria strain LD111-11 was 99.78%, the similarity with the 16s rRNA sequence of Burkholderia rinojensis strain A396 was 99.71%, the similarity with the 16s rRNA sequence of Burkholderia multivorans strain QYGXJ8-1 was 99.71%, and the similarity with the 16s rRNA sequence of Burkholderia stabilis strain LD119 was 99.64%. It was preliminarily determined that the isolated strain belonged to the genus Burkholderia. A phylogenetic tree was constructed using MEGA 5 (see Figure 2 ).

[0129] In order to further determine the genome similarity of the isolated Burkholderia strain and other members of the genus Burkholderia, the genome of the original strain isolated above was further prepared, sequenced, assembled and analyzed. The genome of the original strain was fragmented by ultrasonication and then assembled using a standard DNA library construction kit (NEB Ultra TM ) to construct an Illumina sequencing library. The constructed sequencing library was subjected to double-end sequencing using NovaSeq (Illumina).

[0130] The raw sequencing data were filtered using fastp (version 0.20.0) with the following filtering parameters: "--poly_g_min_len 10 --poly_x_min_len 10 -q 15 -u 40 -n 5 -l 50". The filtered raw data were assembled using SPAdes (version 3.14.0) with the following parameters: "--isolate --cov-cutoff 10". The genome genes were predicted and analyzed using the prokaryotic analysis software genome annotation pipeline prokka (version 1.14.5) with the following parameters: "--gcode 11 --evalue 1e-09".

[0131] The genome information of Burkholderia strains was downloaded from NCBI. The average nucleotide identity (ANI) and alignment score (AF) of the isolated strains were evaluated with these reference microorganisms and the applicant's own Burkholderia rennogii M928, as shown in Table 1. The whole genome sequence of the isolated strains was compared with other strains and species of Burkholderia to determine the degree of genome relatedness with previously identified Burkholderia species. The comparison results showed that the strain with the highest genome similarity was Burkholderia rennogii M928, with the highest average nucleotide similarity (ANI) of 85.38% and a gene coverage of 42%. Burkholderia M928 was the Burkholderia isolated by the applicant and was deposited in the Guangdong Provincial Microbial Culture Collection on August 21, 2020, with the deposit number GDMCC No: 61156. For specific information on the strain, please refer to the patent named "A composite microbial agent and its preparation method" and the publication number "CN 113980877 B".

[0132] Comparison of genome similarity with Burkholderia ambifaria showed an average nucleotide similarity (ANI) of 84.36% and a gene coverage of 56%; comparison of genome similarity with Burkholderia multivorans showed an average nucleotide similarity (ANI) of 84.46% and a gene coverage of 62%; and comparison of genome similarity with Burkholderia stabilis showed an average nucleotide similarity (ANI) of 84.51% and a gene coverage of 58%. Based on the principle that ANI > 95% indicates the same species, genomic correlation analysis based on average nucleotide identity (ANI) in this application showed that the isolated strains can be clearly distinguished from known Burkholderia species based on genotypic characteristics.

[0133] Therefore, the genotypic characteristics of the isolated strain are significantly different from those of other existing Burkholderia species, and this strain can be identified as a new species of Burkholderia. We named it Burkholderia sp. M222.

[0134] Table 1. M222 whole genome alignment results

[0135]

[0136]

[0137]

[0138] 3. Deposit of M222 strain

[0139] We sent the Burkholderia sp. M222 isolated and identified above to the Guangdong Microbial Culture Collection Center (GDMCC) for preservation, with the deposit number GDMCC: 62460, the deposit date May 10, 2022, and the deposit address at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0140] Example 2: Preparation of fermentation broth of Burkholderia sp. M222

[0141] 1. Activation Culture

[0142] The isolated and preserved Burkholderia sp. M222 was transferred to a modified LB solid medium (5-10 g yeast extract, 8-12 g peptone, 5-15 g sodium chloride, 15 g agar, 1 L water, pH = 7-7.5) plate and cultured at 30°C for 2 days.

[0143] 2. Fermentation Culture

[0144] The M222 activated and cultured on a modified LB solid medium plate was transferred to a modified LB liquid medium (5-10 g yeast extract, 8-12 g peptone, 5-15 g sodium chloride, 1 L water, pH = 7-7.5, sterilized at 121°C for 20 min), and cultured in a shaker at 28°C to 30°C and 180 r / min to 210 r / min for 48 h to obtain the fermentation broth of M222.

[0145] Example 3: In vitro nematicidal activity test of M222

[0146] In this example, the southern root-knot nematode (Meloidogyne incognita) was used as a pathogen.

[0147] Wash the water spinach roots used for propagating root-knot nematodes with clean water and remove the egg masses with tweezers. Chop the roots after removing the egg masses, add 2% sodium hypochlorite, and vortex for 1.0 minute. Rinse through 80-, 200-, 350-, and 500-mesh sieves two to three times. Collect the eggs from the 500-mesh sieve to prepare a nematode egg suspension for later use.

[0148] The selected egg masses were surface-sterilized with a 0.5% NaClO solution and then rinsed three times with sterile water. The eggs were placed in a 90 mm Petri dish and incubated in the dark for 48 hours. Second-instar larvae of the root-knot nematode were then collected and prepared as a suspension for later use.

[0149] The fermentation broth of the M222 strain prepared in Example 2 was diluted fivefold to prepare a treatment solution. 450 μL of the treatment solution and 50 μL of the nematode suspension (2000 nematodes / mL) prepared above were added to a 24-well cell culture plate, which was then covered and sealed with parafilm.

[0150] In this experiment, sterile water was used as a blank control (CK), and a 1500-fold dilution of 5% avermectin emulsifiable concentrate (Xinbaike) was used as a positive control. Each treatment was repeated five times.

[0151] After 24 hours of incubation at 28°C in the dark, observe and record the number of second-instar larvae in a state of rigor mortis and mortality under a stereomicroscope. The criteria for rigor mortis were as follows: worms that were motionless were considered rigor mortis, while those that were bent and wriggling were considered alive. Worms that remained rigor mortis after stimulation with 1 mol / L NaOH solution were considered dead. Rigor mortis and mortality rates were then calculated using the following formula:

[0152] Rigor mortis rate (%) = (number of rigor mortis worms / total number of worms) × 100%

[0153] Mortality (%) = (number of dead nematodes / total number of nematodes) × 100%

[0154] The results of the in vitro nematicidal activity test are shown in Table 2. The results in Table 2 indicate that the fermentation broth of M222 can cause rigor mortis in nematodes and lead to their death.

[0155] Table 2. Results of in vitro nematicidal activity test of Burkholderia M222 fermentation liquid

[0156] Dilution multiple Rigidity rate / % mortality rate / % CK / 0.00 0.00 Abamectin 1500 100.00 - M222 5× 92.00 76.80

[0157] Example 4: Tests on the control and growth promotion effects of M222 in greenhouse centrifuge tubes and potted plants

[0158] In this example, the southern root-knot nematode (Meloidogyne incognita) was used as the pathogen and cucumber was used as the host. The nematode egg suspension and second-instar larvae suspension used in this example were prepared as described in Example 3.

[0159] 1. Test of the control and growth-promoting effects of M222 under centrifuge tube conditions

[0160] Cucumber seeds were sown in a seedling tray. After the seeds germinated, they were transplanted into 50 mL centrifuge tubes, with one seed sown per tube. They were placed in a light culture room with a photoperiod and temperature set to 16 h of light and 28 ° C; 8 h of darkness and 20 ° C. After one week, each cucumber was irrigated with 10 mL of treatment solution (according to the actual irrigating amount of 2 mL of M222 fermentation solution prepared in Example 2 per cucumber, supplemented with 10 mL of water). One day later, each cucumber was inoculated with 2 mL of nematode egg suspension (3000 eggs / mL).

[0161] In this experiment, water was used as a blank control (CK), and a 1500-fold dilution of 5% avermectin emulsifiable concentrate (Xinbaike) was used as a positive control. Each treatment consisted of 5 seedlings, and was repeated 3 times.

[0162] After 14 days, the disease level (disease grade) was evaluated according to the following 6-point scale: 0 = no root knots, 1 = 0%-10% root knots, 2 = 10%-25% root knots, 3 = 25%-50% root knots, 4 = 50%-75% root knots, and 5 = 75%-100% root knots. Aboveground dry and fresh weights were calculated. Control efficacy and dry weight growth rate were calculated using the following formula:

[0163] Disease index (%) = [∑ (number of plants at each disease level × number of disease levels) / (total number of plants surveyed × highest level)] × 100

[0164] Preventive and therapeutic effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100

[0165] Dry weight increase rate (%) = [(dry weight of control group - dry weight of treatment group) / dry weight of control group] × 100

[0166] The test results are shown in Table 3. The results show that the M222 fermentation liquid can reduce the occurrence of root knots and promote significant plant growth.

[0167] Table 3. Test results of the control and growth promotion effects of M222 fermentation liquid on southern root-knot nematode centrifuge tube

[0168] Disease Index Control effect Fresh weight of aboveground parts / g Aboveground dry weight / g Dry weight growth rate CK 68.00 - 0.655 0.079 - Abamectin 0.00 100.00％ 0.711 0.082 4.24％ M222 24.00 64.71％ 1.422 0.123 56.78％

[0169] 2. Test of the control and growth promotion effects of M222 under potted conditions

[0170] Cucumber seeds were sown in a seedling tray. After the cotyledons unfolded, they were transplanted into seedling pots. When the cucumbers grew to 2 leaves and 1 heart, they were irrigated with M222 treatment solution (20 mL of M222 fermentation solution prepared as in Example 2 was actually irrigated per cucumber seedling, and the volume was supplemented to 70 mL with water), and the seedlings were placed in a light culture room with the light cycle and temperature set to 16 h of light and 28 ° C; 8 h of darkness and 20 ° C. After 24 h, a suspension of second-instar larvae of root-knot nematodes prepared as described in Example 3 (300 larvae / mL) was inoculated, with 1 mL per plant. In this experiment, water was used as a blank control (CK), and a 1500-fold dilution of 5% avermectin emulsifiable concentrate (Xingbok Line) was used as a positive control. The commercially available nematode control product Xinlonghui Line Shield treatment solution was also included in this experiment for comparison of control and growth-promoting effects. Eight seedlings were treated in each treatment, and the results were repeated three times.

[0171] After 15 days, the stem diameter was measured 2 cm below the cotyledon using the outer measuring claws of a vernier caliper perpendicular to the growth direction of the cotyledon; the first and second true leaves of the cucumber seedlings were spread out flat, and the distance between the leaf tips was measured with a ruler as the leaf expansion degree. After 30 days, the disease level (disease grade) was observed and evaluated according to the following 9 grades: 0 = no root knot, 1 = 0%-15% root system has root knot, 2 = 10%-25% root system has root knot, 3 = 25%-50% root system has a small amount of root knot, 4 = 50%-75% root system has a small amount of root knot, 5 = 25%-50% root system has a large amount of root knot or large root knot, 6 = 75%-100% root system has a small amount of root knot, 7 = 50%-75% root system has a large amount of root knot or large root knot, 8 = 75%-100% root system has a large amount of root knot or large root knot, and the disease index was calculated according to the following formula:

[0172] Disease index = [∑(number of plants at each disease level × number of disease levels) / (total number of plants surveyed × highest level)] × 100.

[0173] The control effect of M222 treatment solution on root-knot nematodes was evaluated according to the following formula:

[0174] Preventive and therapeutic effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100

[0175] The test results are shown in Table 4. The results show that the M222 fermentation liquid can reduce the occurrence of root knots and promote the growth of plant stems and leaves. Its control and growth promotion effects are significantly better than those of the commercially available nematode control product Xinlong Huixiandun.

[0176] Table 4. Test on the effect of M222 fermentation liquid on nematode control and growth promotion in potted plants

[0177] Stem diameter / mm Depth of expansion / cm Disease index / % Control effect CK 1.97 4.43 87.50 - Abamectin 1500× 2.10 4.12 0.00 100.00％ M222 3.19 14.10 45.24 48.30％ New Longhui Line Shield 2.28 5.46 86.11 1.59％

[0178] Example 5: Test of M222-induced systemic resistance in plants

[0179] In this example, Meloidogyne incognita was used as the pathogen and tomato was used as the host. The nematode egg suspension used in this example was prepared as described in Example 3, and the M222 treatment solution was prepared as described in Example 2.

[0180] Tomato seedlings were grown in seedling trays in a 28°C greenhouse. After uniform emergence, the roots of the tomato seedlings were divided evenly into two parts and planted in two adjacent pots. One pot was inoculated with nematode eggs, and the other pot was watered with the treatment solution, without direct contact between the treatment solution and the nematode eggs. Seven days after transplanting the tomato seedlings, each pot was watered with 100 mL of the M222 treatment solution. 24 hours after the watering treatment, a 2 cm deep hole was drilled near the roots of the seedlings and the nematode egg suspension was added. 3,000 eggs were inoculated per plant. In this experiment, water was used as the blank control (CK), and a 1500-fold dilution of thiazolyl was used as the positive control. Ten seedlings were isolated for each treatment, and the experiment was repeated three times.

[0181] After 30 days, the number of root knots was observed and recorded, and the root knot reduction rate was calculated according to the following formula as the control effect to evaluate the systemic resistance induced by the test M222 treatment solution in the plants:

[0182] Control effect (%) = [(number of root knots in the control group - number of root knots in the treatment group) / number of root knots in the control group] × 100

[0183] The test results are shown in Table 5. The results show that the M222 fermentation liquid can induce systemic resistance in plants and reduce the number of root knots, and its effect is significantly better than that of the chemical drug thiazolyl.

[0184] Table 5. Results of the test on systemic resistance induced by M222 fermentation broth in plants

[0185] Number of root knots Control effect CK 28.73 - 1500-fold dilution of thiazolyl 14.17 50.70％ M222 10.03 65.08％

[0186] Example 6: Determination of siderophore production in M222

[0187] Use a sterile toothpick to scrape M222 from the plate and inoculate it into a 50mL centrifuge tube containing 10mL of MKB iron-free medium (5g of casamino acids, 15mL of glycerol, 2.5g of magnesium sulfate heptahydrate, 1000mL of ultrapure water, pH 7.2, autoclaved at 121°C for 20min; before inoculation, add 10mL of dipotassium hydrogen phosphate solution (2.5g of dipotassium hydrogen phosphate, 50mL of ultrapure water, autoclaved at 121°C for 20min) per 200mL of medium. Incubate at 30°C and 200rpm with shaking for 24h. After bacterial cells are produced (the medium becomes turbid), centrifuge at 8000rpm for 5min and discard the supernatant. Wash the cells twice with 5mL of sterile ultrapure water by centrifugation at 8000rpm for 5min, then dilute 10-fold with sterile ultrapure water to obtain an M222 suspension. Take 10 μL of the M222 suspension and inoculate three drops onto iron-free MKB plates, with three replicates for each strain; incubate in a 30°C incubator for 1 day. After a single colony clearly grows on the plate, pour a layer of CAS medium (Qingdao Haibo, add 400 mL of ultrapure water to 4.35 g of culture medium powder and autoclave at 116°C for 30 minutes) on the plate with the colony and incubate at 30°C for 24 hours. Observe the color change of each plate, measure the siderophore chelation halo diameter and colony diameter, and calculate the siderophore solubility index according to the following formula:

[0188] Siderophore solubility index = siderophore chelation halo diameter / colony diameter

[0189] The experimental results are shown in Figure 3 .from Figure 3 It can be seen that M222 produces an obvious orange-yellow siderophore chelation halo. The siderophore solubility index is calculated to be 3.45, which shows that it has a strong affinity for Fe. 3+ The chelating power is strong.

[0190] Those skilled in the art will understand that nutrient competition and site competition are two important mechanisms by which microbial strains achieve resistance to soil-borne pathogens in the soil, and are crucial for combating fungal and bacterial diseases. Siderophore-producing bacteria utilize this complex through specific extracellular receptors, while other organisms cannot utilize it. This reduces environmental iron concentrations, leading to iron deficiency and the inhibition of pathogen growth and reproduction. Experiments have shown that M222 can widely inhibit the growth of pathogens in the soil, demonstrating its potential for combating soil-borne fungal and bacterial diseases.

[0191] Example 7: Nematode chemotaxis test

[0192] In this example, a three-dimensional transparent Pluronic gel system simulating soil was used to study the behavioral response of root-knot nematodes to the chemotaxis of M222.

[0193] In this example, Meloidogyne incognita was used as the pathogen, and cucumber and tomato were used as hosts. A suspension of second-instar larvae of the nematode was prepared as described in Example 3.

[0194] Cucumber (Yuexiu No. 3, Guangdong Kenong Biotechnology) and tomato (Xingxing 101, Guangdong Kenong Biotechnology) seeds of basically the same size and plumpness were selected for surface disinfection. They were soaked in 75% alcohol for 30 seconds, rinsed with sterile water three times, and then disinfected with 10% sodium hypochlorite for 5 minutes. Then, they were rinsed repeatedly with sterile water for 5 to 6 times. Finally, the surface moisture of the seeds was absorbed with sterile absorbent paper for later use.

[0195] Sterilized cucumber and tomato seeds were placed in sterile 1.3% water agar and arranged evenly with the radicles in the same direction. Tomatoes were cultured in a light chamber at (26±1)°C for 4 days before use. Cucumbers were cultured in a light chamber at (28±1)°C for 3 days before use.

[0196] Tomato - Root Dip Test:

[0197] The cultivated tomato seedling roots were immersed in the M222 bacterial agent (M222 fermentation liquid prepared as described in Example 2, diluted 10 times (10×) as the treatment liquid), with sterile water as the blank control and thiazolylphos as the positive control. Use an electric pipette to draw 23% (w / v) Pluronic gel into each 120mm square dish, place it at room temperature to solidify, and wait until the tomato seedling roots are completely coated with the M222 bacterial agent, place it on top of the gel square dish, and evenly arrange 5 pieces. Place the plate vertically in a 26 / 20℃ (day / night) light room. After 24h, drip 5 drops of 50μL of a suspension containing 100 2nd instar larvae (J2s) of nematodes to the bottom edge. 7 days after the nematode inoculation, remove the roots from the plate and stain with acid fuchsin (see Figure 4 ), the total number of nematodes in the roots within 24 hours, the total number of nematodes in the roots within 7 days, and the number of 3rd and 4th instar nematodes (J3 / J4s) in the roots within 7 days were counted to assess the degree of nematode infestation.

[0198] Cucumber-microbial gel test:

[0199] Use M222 inoculant to dilute PF-127 (pluronic F-127 powder) into 23% (w / v) gel with different inoculant concentrations (diluted 5 times (5×), 2 times (2×), and 1 times (1×) respectively). Sterile water was used as a blank control, and avermectin was used as a positive control. Use an electric pipette to draw 20 mL of gel containing different concentrations of M222 inoculant into 120 mm square dishes. After solidifying at room temperature, transfer 4 3-day-old cucumber seedlings to the top of each square dish, and place the plate vertically in a 28 / 22°C (day / night) light room. After 24 hours, drip 4 drops of 50 μL of a suspension containing 150 2nd instar larvae (J2s) nematodes to the bottom edge. 7 days after inoculation of nematodes, remove the roots from the plate and stain with acid fuchsin (see Figure 5 ), the total number of nematodes in the roots was counted for 7 days to assess the degree of nematode infestation.

[0200] The above experimental data were calculated and statistically analyzed using Excel 2019 and SPSS22.0 software. Duncan's new multiple range method was used for significance analysis, and the significance level was set at P < 0.05.

[0201] The test results are shown in Tables 6 and 7. The results showed that M222 could significantly reduce the attractiveness of crop root tips to nematodes and the nematode infestation capacity (P<0.05).

[0202] Table 6. Test results of root dipping test

[0203]

[0204] Note: * indicates that there are significant differences between different treatments and blank control (P<0.05)

[0205] Table 7. Test results of the microbial gel test

[0206]

[0207] Note: * indicates that there are significant differences between different treatments and blank control (P<0.05)

[0208] Example 8: Pathogen Antagonism Test of M222

[0209] In this example, Streptomyces scabies and Ralstonia solanacearum were used as test pathogenic bacteria, and Fusarium oxysporum, Fusarium graminearum, Sclerotium sclerotiorum, and Colletotrichum anthracis were used as test pathogenic fungi to determine the antibacterial spectrum of M222.

[0210] Inhibition of pathogenic bacteria

[0211] On a sterile workbench, pipette 100 μl of bacterial pathogen solution (Streptomyces scabies, Ralstonia solanacearum) onto LA plates, spread evenly with glass beads, and air dry. Pipette 5 μl of M222 bacterial solution onto the same plates, 3 drops per plate, distributed in a regular triangle pattern. Repeat each treatment three times. Use a plate inoculated with only the pathogen as a blank control.

[0212] After the suspension on the plate is naturally dried, seal it and place it in a constant temperature incubator at 30°C. Take it out after 1 day for observation, measure the radius of the inhibition zone and the radius of the antagonistic bacteria, and calculate the antagonism index according to the following formula:

[0213] Inhibition zone width = inhibition zone radius - antagonistic bacteria radius

[0214] Antagonistic index = inhibition zone width / antagonistic bacteria radius

[0215] The experimental results are shown in Table 8 and Figure 6 As shown in .

[0216] Table 8. Inhibitory effect of M222 on pathogenic bacteria

[0217]

[0218] Inhibition of pathogenic fungi

[0219] On a sterile workbench, inoculate the center of an LA plate with a cake of fungal pathogens (Fusarium oxysporum, Fusarium graminearum, Sclerotium sclerotiorum, and Colletotrichum anthracis). Place 5 μl of M222 bacterial solution around the pathogens. Repeat in triplicate for each strain. Use a plate inoculated with only the pathogens as a blank control.

[0220] After culturing in a 28°C incubator for 6 days, the plates were taken out for observation. The diameters of the treated colonies and the control colonies on the treated plates were measured using the cross method. The width of the inhibition zone and the inhibition rate were calculated using the following formula:

[0221] Inhibition zone width = control colony diameter - treated colony diameter

[0222] Inhibition rate (%) = width of inhibition zone / diameter of control colony × 100

[0223] The test results are shown in Table 9 and Figure 6 As shown in .

[0224] Table 9. Inhibitory effect of M222 on pathogenic fungi

[0225]

[0226] From Table 8, Table 9 and Figure 6As can be seen, Burkholderia sp. M222 has antagonistic effects against the two aforementioned pathogenic bacteria and the four aforementioned pathogenic fungi, thus exhibiting a broad spectrum of inhibition. The strongest inhibitory effect was achieved against Fusarium graminearum, with an inhibition rate of 64.44%. This suggests that the Burkholderia sp. M222 of the present invention has significant inhibitory effects against both bacterial and fungal diseases.

[0227] Example 9: Qualitative test of nitrogen fixation of M222

[0228] Use a sterile inoculating loop to scrape an appropriate amount of M222 cells from the plate, resuspend in 1 mL of sterile water, and evenly inoculate onto Ashby medium (0.2 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 0.2 g sodium chloride, 5.0 g calcium carbonate, 10.0 g mannitol, 0.1 g calcium sulfate, 18.0 g agar per liter, pH 6.8-7.0) in triplicate. Incubate in the dark at 28°C for 5 days and observe for the appearance of distinct colonies.

[0229] Figure 7 The results of this experiment are shown. Figure 7 As can be seen, M222 can grow on Ashby solid medium without nitrogen, indicating that M222 has the ability to fix nitrogen. In other words, the M222 strain can supplement crop nutrition and promote crop growth.

[0230] Example 10: Qualitative test of phosphorus solubility of M222

[0231] Use a sterile inoculating loop to scrape an appropriate amount of M222 cells from the plate, resuspend in 1 mL of sterile water, and evenly inoculate onto an organophosphorus bacterial culture medium (10.0 g glucose, 0.5 g ammonium sulfate, 0.5 g yeast extract, 0.3 g sodium chloride, 0.3 g potassium chloride, 0.3 g magnesium sulfate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, 0.2 g lecithin, 1.0 g calcium carbonate, 15.0 g agar, 1 L water, pH = 7.0-7.5). Repeat three times for each plate. After incubation at 28°C in the dark for 4-6 days, observe whether a clear phosphate-dissolving zone appears around the colonies.

[0232] Figure 8 The results of this experiment are shown. Figure 8 It can be seen that a transparent phosphate-dissolving ring appears around the M222 colony, indicating that it has the ability to dissolve organic phosphorus. The diameter of the phosphate-dissolving ring and the colony diameter are then measured, and the phosphate-dissolving index is calculated according to the following formula:

[0233] Phosphate solubility index = phosphate solubility circle diameter / colony diameter

[0234] The calculated phosphorus solubility index of M222 is 2.84.

[0235] In addition, the M222 of the present invention is also a phosphate-solubilizing bacterium. Experimental results on the utilization of organic phosphorus show that it can decompose solid phosphorus in the soil that is difficult for crops to absorb and utilize, including organic phosphorus and inorganic phosphorus, and convert it into soluble phosphate form, which helps to improve the effective utilization rate of phosphorus and thus promote plant growth.

[0236] Example 11: Growth-promoting effect of M222

[0237] 1. Cultivation and processing of tomatoes and cucumbers

[0238] Seedling cultivation: Tomato (Xingxing 101, Guangdong Kenong Biotechnology) and cucumber (Yuexiu No. 3, Guangdong Kenong Biotechnology) were planted 2 per pot, 6 pots per tray as one treatment, with a total of 12 seedlings, 3 replicates per treatment, and a total of 36 seedlings.

[0239] Water and fertilizer management: In addition to watering the bacterial agent (M222 fermentation liquid) on time, 0.01% NPK water-soluble fertilizer should be applied when drought occurs.

[0240] Treatment with microbial agent (M222): The first watering was performed on 12-day-old seedlings; after 7 days, the second watering was performed.

[0241] Blank control (CK): Water was used as a blank control in this experiment.

[0242] 2. Data Collection

[0243] After the second inoculum application, continue culturing for one week before harvesting the seedlings. Physiological data such as spread (the distance between the tips of the two true leaves), plant height (the height of the plant, measured from the root collar to the growing point), stem diameter (stem diameter measured below the first true leaf), and dry weight were recorded for analysis. A 10% increase in biomass was used as the growth promotion standard.

[0244] 3. Test results

[0245] The test results are shown in Table 10, Table 11, Figure 9 and Figure 10 Table 10 shows the effect of M222 fermentation liquid treatment on tomato growth indicators. Figure 9 Table 11 shows the effect of M222 fermentation liquid treatment on the growth index of cucumber. Figure 10 Shows the growth of potted cucumbers treated with M222 fermentation liquid.

[0246] Table 10. Effects of M222 fermentation broth treatment on tomato growth indicators

[0247]

[0248] Table 11. Effects of M222 fermentation liquid treatment on cucumber growth indicators

[0249]

[0250] From Table 10, Table 11, Figure 9 and Figure 10 The results show that the M222 fermentation liquid has a significant growth-promoting effect on both tomatoes and cucumbers. The dry weight per plant of tomatoes treated with the M222 fermentation liquid was significantly increased compared to the CK, reaching 89.23%. In addition, the dry weight per plant of cucumbers treated with the M222 fermentation liquid was significantly increased compared to the CK, reaching 41.76%.

[0251] Example 12: Herbicidal activity test of M222

[0252] Petri dish test:

[0253] In this experiment, Amaranthus retroflexus L. was selected as the test weed.

[0254] First, the seeds of Amaranthus retroflexus were placed in a 70% ethanol solution and rinsed for 3 minutes, then rinsed 3 times with sterile water, and then the seeds were rinsed with a 3% NaClO solution for 4 minutes, and finally rinsed 3 times with sterile water. The rhizome inhibition method was selected and combined with the seed germination method to verify the herbicidal activity. Specifically, 4 mL of M222 fermentation broth (prepared as described in Example 2) and a 10-fold diluted M222 fermentation broth (M222-10×) were evenly added to sterilized filter paper, and then 30 Amaranthus retroflexus seeds were evenly sprinkled on the filter paper with spore suspension. An equal amount of sterile water was used as a negative control (CK), and a 100× dilution of the chemical ethyl ester was used as a positive control. Each treatment was repeated 3 times on a plate. Placed in a dark environment, cultured at 28°C for 3 days, and then the inhibition rate and germination rate of M222 on the root length of Amaranthus retroflexus seeds were measured. The calculation formula for the inhibition rate is as follows:

[0255] Bud growth inhibition rate (%) = [(bud length of control group - bud length of treatment group) / bud length of control group] × 100%

[0256] The test results are shown in the following Table 12. The results show that the original M222 fermentation solution can completely inhibit the germination of Amaranthus retroflexus seeds, and the 10-fold diluted M222 fermentation solution also has a strong inhibitory effect.

[0257] Table 12. Inhibitory effect of M222 fermentation liquid on the germination of Amaranthus retroflexus seeds

[0258]

[0259] Pot test:

[0260] In this experiment, Amaranthus retroflexus L. and Setaria viridis (Sedum sarmentosum) were selected as test weeds.

[0261] Closed period: Weigh an equal amount of soil and put it into each flower pot, water until water flows out from the bottom of the flower pot, then place 30 weed seeds on the soil, and spread a thin layer of soil on the weed seeds (Amaranthus retroflexus and Setaria viridis). Use a watering can to spray the M222 fermentation supernatant (stock solution) on the soil, use an equal amount of distilled water as a negative control (CK), and use a 100× dilution of the chemical ethyl benzoate as a positive control. Repeat three times for each treatment group. After the flower pots are placed in a light incubator (light-dark ratio = 16:8) for about 7 days, the number of germinations of each weed seed (Amaranthus retroflexus and Setaria viridis) is counted, and the germination rate is calculated according to the following formula:

[0262] Seed germination rate (%) = number of seeds germinated / total number of test seeds × 100%

[0263] The test results are shown in the following Table 13. The results show that the M222 fermentation broth has an inhibitory effect on the germination of Amaranthus retroflexus and Setaria viridis.

[0264] Table 13. Inhibitory effect of M222 fermentation liquid on the germination of Amaranthus retroflexus and Setaria viridis seeds

[0265] Amaranthus retroflexus germination rate / % Setaria germination rate / % M222-solution 8.34±8.34 26.67±10 B. Weed 100× 8.34±8.34 0±0 CK 13.33±0 33.34±6.67

[0266] Seedling stage: Weigh an equal amount of soil and put it into each flower pot. Water until water flows out from the bottom of the flower pot, then place 30 weed seeds on the soil, and spread a thin layer of soil on the weed seeds (Amaranthus retroflexus and Setaria viridis). Move the flower pot to an outdoor environment and wait until the weeds grow to the 3-5 leaf stage. Spray the M222 fermentation liquid on the weed leaves. The negative control group (CK) sprays an equal amount of clean water on the leaves. A 100× dilution of the chemical ethyl benzoate is used as a positive control. Each treatment is repeated in 3 pots. Grow outdoors for 10-20 days. During this period, continuously observe the changes in the weed leaves and whether wilting and lodging occur. Count the plant height and fresh weight of each weed (Amaranthus retroflexus and Setaria viridis). Calculate the plant height inhibition rate and fresh weight inhibition rate according to the following formula:

[0267] Plant height inhibition rate (%) = [(plant height of control group - plant height of treatment group) / plant height of control group] × 100%

[0268] Inhibition rate of aboveground fresh weight (%) = [(aerial fresh weight of control group - aerial fresh weight of treatment group) / aerial fresh weight of control group] × 100%

[0269] The test results are shown in Table 14. The results showed that when the M222 fermentation liquid was used to treat the various weeds, the leaves clearly turned from green to yellow during their growth, and the weeds eventually wilted and fell over. It also had a good inhibitory effect on the plant height and fresh weight of Setaria viridis and Amaranthus retroflexus.

[0270] Table 14. Inhibitory effect of M222 fermentation broth on weed growth

[0271]

[0272] Example 13: Determination of IAA (indole-3-acetic acid) production ability of M222

[0273] 1. Qualitative determination of IAA production capacity:

[0274] Place 50 μL of the fermentation broth of the M222 strain on a white background. Simultaneously, add 50 μL (approximately one drop) of Spot colorimetric solution (colorimetric solution: dissolve 4.5 g of FeCl₃ in 300 mL of distilled water, then slowly add 1 mL of 98% H₂SO₄. Cool and dilute to 1000 mL). Additionally, add only 50 μL of 5 mg / L plant growth hormone (3-indoleacetic acid) to the colorimetric solution as a control. At room temperature, record the color change within 15 minutes. A pink color indicates IAA secretion; a darker color indicates a higher IAA secretion capacity. No color change is negative, indicating no IAA secretion.

[0275] See the results Figure 11 .like Figure 11 As can be seen, after adding the colorimetric solution, the M222 fermentation broth changed color slightly, indicating that it may not produce or produce a small amount of IAA.

[0276] 2. Quantitative determination of IAA production capacity:

[0277] (1) Prepare a standard curve using pure 3-indoleacetic acid: Prepare IAA solutions at the following concentrations: 0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, and 45 mg / L. Measure the OD at each concentration. 530 The standard curve was drawn based on the values.

[0278] (2) Centrifuge the M222 fermentation broth at 10,000 rpm and 4°C for 10 min. Take 1 mL of the supernatant and mix it with 1 mL of colorimetric solution. After standing in the dark for 30 min, quickly perform colorimetry at a wavelength of 530 nm using a spectrophotometer to measure the absorbance. Calculate the IAA content based on the OD value using a standard curve.

[0279] The linear equation of the IAA standard curve is y=29.76703x+0.23426, OD 530 The value range is between 0.07-1.50, and the measured OD of M222 treatment solution 530 The value is 0.092, which means that the IAA concentration produced by M222 after 48 hours of fermentation is 2.76 mg / L. Therefore, it is inferred that M222 can produce a small amount of IAA, which can promote plant growth.

[0280] Example 14: Test of M222 against thrips

[0281] In this example, the leaf immersion method was used to determine the toxicity of the fermentation broth of the M222 strain against adults of western flower thrips.

[0282] The M222 fermentation broth was diluted with sterile water to create five treatments with a predetermined concentration gradient. Clean green beans (5 cm) were cut lengthwise and immersed in each treatment for 30 seconds. Afterwards, they were placed on sterile filter paper, air-dried with the back facing upward, and placed in 240 mL tissue culture flasks until ready for use. Each concentration constituted one treatment, with three replicates per treatment. Sterile water served as a blank control (CK).

[0283] Fifteen adult western flower thrips were placed into a tissue culture flask using a soft-bristled brush and sealed with a 200-mesh screen. The flasks were then placed in a climate chamber maintained at 25 ± 1°C with 16 hours of light per day. After 24 hours, the flasks were removed and the survival rate of the adult western flower thrips treated with the different test agents was observed and recorded. During observation, the thrips were gently touched with a soft-bristled brush; if they remained motionless twice, they were considered dead.

[0284] The test results are shown in Table 15. The results showed that the M222 fermentation broth at a concentration of 66.67 μL / mL and above exhibited a strong lethal effect on thrips during the 5-7 day (d) treatment process.

[0285] Table 15. Test results of M222 against thrips

[0286]

[0287]

[0288] Although a plurality of embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art may conceive of many changes, variations, and substitutions. It should be understood that various alternatives to the embodiments described herein may be employed in the process of implementing the present invention. Therefore, the present invention also encompasses any such substitutions, changes, variations, or equivalents.

Claims

1. A Burkholderia species, characterized in that The 16s rRNA sequence of the Burkholderia is at least 99%, 99.5%, 99.8%, 99.9% or 100% identical to the sequence shown in SEQ ID NO: 1; Preferably, the Burkholderia comprises one or more strains selected from the group consisting of: 1) a Burkholderia rinojensis strain; 2) a strain having at least 99.8% or 99.9% identity to the 16s rRNA sequence of Burkholderia rinojensis; 3) a strain having at least 99.8% or 99.9% identity to the 16s rRNA sequence of Burkholderia rinojensis; rinojensis) having an average nucleotide identity of ≥86%, ≥90%, ≥95%, ≥95.5%, ≥96%, ≥96.5%, ≥97%, ≥97.5%, ≥98%, ≥98.5%, ≥99%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9%, ≥99.99% or ≥100% to the genome; and / or a genome alignment score of ≥55%, ≥60%, ≥65%, ≥70%, ≥78%, ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% to the genome; Preferably, the Burkholderia has the 16s rRNA sequence shown in SEQ ID NO:

1.

2. The Burkholderia according to claim 1, characterized in that The Burkholderia is a Burkholderia deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 10, 2022 with the deposit number GDMCC: 62460.

3. The Burkholderia according to claim 1, characterized in that the average nucleotide identity of the genome of the Burkholderia sp. to the genome of the strain deposited with GDMCC: 62460 of claim 2 is ≥86%, ≥90%, ≥95%, ≥95.5%, ≥96%, ≥96.5%, ≥97%, ≥97.5%, ≥98%, ≥98.5%, ≥99%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9%, ≥99.99% or ≥100%, and / or the alignment score of the genome of the Burkholderia sp. to the genome of the strain deposited with GDMCC: 62460 is ≥55%, ≥60%, ≥65%, ≥70%, ≥78%, ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%; Preferably, the 16s rRNA of the Burkholderia is at least 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the 16s rRNA of the strain deposited with GDMCC:62460; Preferably, the 16s rRNA of the Burkholderia is at least 99.9%, 99.99% or 100% identical to the 16s rRNA of the strain with the deposit number GDMCC:62460.

4. A composition, characterized in that The composition comprises the Burkholderia and / or its metabolite, culture, fermentation broth or extract according to any one of claims 1 to 3.

5. The composition according to claim 4, characterized in that The Burkholderia includes the inactivated Burkholderia according to any one of claims 1 to 3.

6. The composition according to claim 4 or 5, characterized in that The composition further comprises additional active agents and / or excipients.

7. The composition according to claim 6, characterized in that The additional active agent is selected from one or more additional biological control agents, one or more chemical agents, one or more fertilizers, one or more herbicides, one or more growth promoters, or any combination thereof.

8. The composition according to claim 7, characterized in that The biological control agent is selected from bacteria, fungi, viral biological control agents, insect biological control agents, nematode biological control agents, or any combination thereof.

9. The composition according to claim 8, characterized in that The biological control agent is at least one selected from the group consisting of Trichoderma harzianum, Purpureocillium lilacinum, Penicillium bilaiae, Bacillus subtilis, Burkholderia rinojensis, Bacillus pumilus, Bacillus velezensis, Methylobacterium rhodesianum, Methylobacterium extorquens, and Paenibacillus peoriae; Preferably, the Trichoderma harzianum is the Trichoderma harzianum with a deposit number of CGMCC NO.15679; Preferably, the Pseudomonas lilacinus is the Pseudomonas lilacinus with a deposit number of CGMCC NO.12773; Preferably, the Penicillium bilairum is the Penicillium bilairum with a deposit number of CGMCC NO.12767; Preferably, the Bacillus subtilis is the Bacillus subtilis with a deposit number of CGMCC NO.12908; Preferably, the Burkholderia rennogii is the Burkholderia rennogii with a deposit number of GDMCC NO.61156; Preferably, the Bacillus pumilus is the Bacillus pumilus with a deposit number of GDMCC NO.61962; Preferably, the Bacillus Velez is the Bacillus Velez with a deposit number of GDMCC NO.61434; Preferably, the Methylobacterium rhodesi is Methylobacterium rhodesi with a deposit number of GDMCC NO.60729; Preferably, the Methylobacterium contortus is Methylobacterium contortus with a deposit number of GDMCC NO.62943; Preferably, the Paenibacillus pierreuil is the Paenibacillus pierreuil with a deposit number of GDMCC NO.60482.

10. The composition according to claim 6, characterized in that The auxiliary material is selected from agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film formers, spontaneity promoters, emulsifiers, dispersants, preservatives, antifreeze agents, thickeners, adjuvants, or any combination thereof.

11. The composition according to any one of claims 4 to 10, characterized in that The composition is in solid form, liquid form, powder form or any combination thereof; preferably selected from the group consisting of solutions, powders, granules, emulsions, emulsions, suspensions, tablets, microgranules and wettable powders.

12. Use of the Burkholderia according to any one of claims 1 to 3 or its metabolite, culture, fermentation broth or extract, or the composition according to any one of claims 4 to 11 in the preparation of a microbial agent, a biofertilizer, a biopesticide or a bioherbicide.

13. Use of the Burkholderia or its metabolite, culture, fermentation broth or extract according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 11 in controlling plant diseases and insect pests and / or weeding; preferably, the plant diseases and insect pests are selected from plant replant diseases, plant bacterial diseases, plant fungal diseases, plant viral diseases, plant soil-borne diseases, plant insect pests and / or plant oomycete diseases.

14. The use according to claim 13, characterized in that The plant diseases and insect pests are caused by one or more of plant pathogenic bacteria, fungi, viruses, insects, insect eggs and nematodes.

15. The use according to claim 13 or 14, characterized in that The plant diseases and insect pests are caused by at least one of the following pathogens: Streptomyces scabies, Ralstonia solanacearum, Bacillus subtilis, Pseudomonas syringae pv. actinidiae, Xanthomonas campestris pv. Oryzae, Erwinia aroideae, Xanthomonas arboricola pv. juglandis, Erwinia carotovora, Staphylococcus aureus; Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum, and the like. graminearum), Athelia rolfsii, Sclerotinia sclerotiorum, Botrytis cirerea, Fusarium oxysporum f.sp.cucumerinum, Gaeumannomyces critici, Fusarium graminearum, Valsamali, Glomerella cingulata, Rhizoctonia solanum, Pyricularia grisea, Alternaria solani, Botrytis cirerea, Phytophthora infestans, Exserohilum turcicum, Bipolaria maydis, Fusarium oxysporum f.sp.niveum), Verticillium dahliae, Fusarium oxysporum f.sp.vasinfectum), pepper blight (Phytophthora capsici), tobacco blight (Phytophthora nicotianae).

16. The use according to claim 13 or 14, characterized in that The plant pests and diseases are caused by nematodes or nematode eggs; preferably, the nematodes are selected from one or more of Meloidogyne spp. and Caenorhabditis elegans; more preferably, the root-knot nematodes are selected from one or more of M. incognita, M. hapla, M. arenaria, and M. javanica; Alternatively, the plant pests and diseases are caused by insects; preferably, the insects are selected from the group consisting of fall armyworm (Spodoptera frugiperda (Smith)), migratory locusts (Locusta migratoria Linnaeus) and other migratory locusts, meadow moth (Loxostege sticticalis Linnaeus), armyworms (such as Mythimna separate (Walker) and Leucania loryi Duponchel), rice planthoppers (such as brown planthoppers (Nilaparvatalugens ), and one or more of: white-backed planthopper (Sogatella furcifera (Horváth)), rice leaf roller (Cnaphalocrocis medinalis (Guenée)), striped stem borer (Chilo suppressalis (Walker)), wheat aphids (such as wheat stem aphid (Sitobion avenae (Fabricius)), cereal stem aphid (Rhopalosiphum padi (Linnaeus)), wheat stem aphid (Schizaphis graminum (Rondani))), Asian corn borer (Ostriniafurnacalis (Guenée)), vegetable thrips (such as bean thrips (Megalurothrips usitatus (Bagnall)), melon thrips (Thrips palmi Karny), western flower thrips (Frankliniella occidentalis (Pergande)), and flower thrips (Frankliniella intonsa (Trybom))).

17. The use according to any one of claims 13 to 16, characterized in that The control of the plant diseases and insect pests is achieved by producing iron carriers, inducing plants or their seeds to produce systemic resistance to plant pathogens and / or reducing the chemotaxis of plant pathogens to plants.

18. Use of the Burkholderia or its metabolite, culture, fermentation broth or extract according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 11 in promoting plant growth.

19. The use according to claim 18, characterized in that The plant growth promotion is achieved by promoting at least one of the following: promoting the development of plant roots, providing nitrogen fixation, providing phosphorus solubilization, producing indoleacetic acid, and removing weeds.

20. The use according to any one of claims 13 to 19, characterized in that The plant is selected from food crops, cash crops or root crops; Preferably, the food crops are selected from at least one of cereal crops, tuber crops and legume crops; Preferably, the cereal crops include but are not limited to at least one of the following gramineous crops: rice, wheat, corn, barley, oats, rye, sorghum, millet, broomcorn millet, barnyard grass, and buckwheat; preferably, the tuber crops include but are not limited to at least one of the following: sweet potato, yam, and potato; preferably, the legume crops include but are not limited to at least one of the following: soybean, broad bean, pea, mung bean, and peanut; Preferably, the economic crops include but are not limited to at least one of the following economic crops: Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Cruciferae, Orchidaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passifloraaceae, Bromeliaceae, Araliaceae and Cactaceae plants; Preferably, the Solanaceae plants include but are not limited to tomatoes, peppers, potatoes, eggplants, etc.; preferably, the Rosaceae plants include but are not limited to strawberries, papayas, etc.; preferably, the Rutaceae plants include but are not limited to citrus, etc.; preferably, the Musaceae plants include but are not limited to bananas, etc.; preferably, the Cucurbitaceae plants include but are not limited to cucumbers, wax gourds, pumpkins, bitter melons, loofahs, watermelons, monk fruit, etc.; preferably, the Cruciferae plants include but are not limited to cabbage, rapeseed, kale, radish, cauliflower, etc.; preferably, the Orchidaceae plants include but are not limited to orchids, etc.; preferably, the Fabaceae plants include but are not limited to soybeans, etc.; preferably, the Asteraceae plants include but are not limited to lettuce, etc.; preferably, the Liliaceae plants include but are not limited to garlic, etc.; preferably, the Zingiberaceae plants include but are not limited to ginger, etc.; preferably, the Passiflora plants include but are not limited to passion fruit, etc.; preferably, the Bromeliaceae plants include but are not limited to golden pineapples, etc.; preferably, the Araliaceae plants include but are not limited to Panax notoginseng, etc.; preferably, the Cactaceae plants include but are not limited to pitaya, etc.; Preferably, the root crops include but are not limited to potatoes, carrots, leafy vegetables, nightshade vegetables, strawberries, grapes, citrus, bananas, kiwis, dragon fruits, tomatoes, peppers, beans, ginger, Panax notoginseng, ginseng, etc.

21. A method for preparing a fermentation broth of the Burkholderia according to any one of claims 1 to 3, comprising the following steps: The Burkholderia bacteria are first activated and then fermented.

22. A method for preventing and controlling plant diseases and insect pests and / or promoting plant growth, characterized in that: The method comprises applying the Burkholderia of any one of claims 1 to 3 or a metabolite, culture, fermentation broth or extract thereof, or the composition of any one of claims 4 to 11 to a plant or seed.

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