CN120484999A

CN120484999A - Burkholderia as well as composition, application and use method thereof

Info

Publication number: CN120484999A
Application number: CN202410178363.8A
Authority: CN
Inventors: 王科晶; 孙玉婷; 刘仁燕; 汪志翔; 谢小梅
Original assignee: Moon Guangzhou Biotech Co Ltd
Current assignee: Moon Guangzhou Biotech Co Ltd
Priority date: 2024-02-08
Filing date: 2024-02-08
Publication date: 2025-08-15
Also published as: WO2025167836A1

Abstract

The invention provides Burkholderia as well as a composition, application and a using method thereof. In particular, the invention provides a burkholderia strain and a composition comprising the strain and/or a metabolite, a culture, a fermentation broth and/or an extract thereof. The Burkholderia or the composition thereof can generate siderophores, induce systemic resistance of plants or seeds thereof to plant pathogens and reduce chemotaxis of the plant pathogens to plants, thereby realizing control of plant diseases and insect pests, and can promote root system development of the plants, provide nitrogen fixation, provide phosphate dissolution, generate indoleacetic acid and remove weeds, thereby promoting growth of the plants.

Description

Burkholderia as well as composition, application and use method thereof

Technical Field

The invention relates to the technical field of microorganisms. In particular, the invention relates to a burkholderia, a composition comprising the strain and/or a metabolite, culture, fermentation broth or extract thereof, as well as uses and methods of use thereof.

Background

In the fields of agriculture, forestry and the like, chemical pesticides and the like are conventionally used for controlling plant diseases and insect pests of crops and the like, and chemical fertilizers and the like are used for promoting the growth of the plants of the crops and the like. However, chemical pesticides and fertilizers often have the disadvantages of residual toxicity, environmental pollution, induced resistance, soil quality impairment, threat to human health, short duration of action, etc. Accordingly, more and more researches are beginning to focus on biopesticides and biofertilizers capable of overcoming the above drawbacks.

Biopesticides and biofertilizers control plant diseases and insect pests or promote plant growth by utilizing living organisms or metabolites of living organisms existing in nature, and can be degraded under natural conditions, so that the problems of environmental pollution, toxic residues and the like of chemical pesticides and fertilizers are overcome. Research shows that many microorganisms can improve the disease and pest resistance of crops and effectively improve the soil environment, and the main related microorganisms are strains of bacillus subtilis, bacillus thuringiensis, bacillus cereus, bacillus laterosporus, paenibacillus mucilaginosus, bacillus licheniformis, bacillus megaterium, bacillus amyloliquefaciens, pseudomonas putida, pseudomonas fluorescens, serratia mutans, saccharomyces cerevisiae, streptomyces microflavus and the like.

Burkholderia (Burkholderia) is a beta branch of the class Proteus, consisting of more than 60 species, which are a class of gram-negative bacteria that are widely found in water, soil, plants and humans. Some species of Burkholderia can be planted at the root and the rhizosphere of a plant, and form symbiotic nodulation with a plant host, so that the functions of promoting plant growth, restoring soil or groundwater, biologically preventing and treating and the like are exerted. However, the differences in function and effect between different burkholderia are large, and some burkholderia, such as burkholderia cepacia (Burkholderia cepacia), belong to pathogenic bacteria, and the pathogenicity of which cannot be removed by improved means at present, so that the burkholderia cepacia cannot be used for agriculture due to safety.

At present, the separated Burkholderia has higher plant disease and pest control activity, especially has the plant disease and pest control activity and plant growth promoting activity, the species of the plant pathogen which can be killed by the Burkholderia is less, the species of the pathogen mainly concentrate on the pathogens such as nematodes, mites and the like, and the research on the action mechanism of the pathogen is not deep enough. Thus, there remains a need in the art to screen and isolate more burkholderia plantarii that have stronger plant pest control activity, kill a greater variety of plant pathogens, and/or promote plant growth, and further explore their mechanisms of action in biocontrol and plant growth promotion, thereby providing more effective, more widely applied, and environmentally friendly biocontrol and plant growth promotion products.

Disclosure of Invention

The above object can be achieved by the following aspects of the present invention. In addition, the present invention may solve other problems that are apparent from the exemplary embodiments.

In a first aspect, the invention provides Burkholderia sp. In some embodiments, the 16s rRNA sequence of Burkholderia has at least 99%, 99.5%, 99.8%, 99.9%, or 100% identity with the sequence shown as SEQ ID NO. 1. Preferably, the Burkholderia includes one or more strains selected from 1) a Rennokurosis (Burkholderia rinojensis) strain, 2) a strain having at least 99.8% or 99.9% identity to the 16s rRNA sequence of Rennokurosis (Burkholderia rinojensis), 3) 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 more than 100% to the genome of Rennokurosis (Burkholderia rinojensis), and/or a strain having a genome alignment score of 55% or more than 60%, 65% or more than 70%, 78% or more than 80%, 85% or more than 90%, 95% or more than 96%, 97% or more than 98% or more than 99%.

In a further preferred embodiment, burkholderia according to the present invention has a 16s rRNA sequence as shown in SEQ ID NO. 1.

In a specific embodiment, burkholderia of the present application is Burkholderia (Burkholderia sp.) M222 deposited by the inventors at 2022, 5/10, and under accession number GDMCC:62460.

In still other embodiments, the genome of Burkholderia according to the invention 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 greater than 100% with the genome of the strain deposited under accession number GDMCC:62460, and/or the genome of Burkholderia has an alignment score of greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 78%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, or greater than or equal to 99% with the genome of the strain deposited under accession number GDMCC: 62460. Preferably, the 16s rRNA of Burkholderia according to the invention has at least 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identity with the 16s rRNA of the strain deposited under accession number GDMCC: 62460. Further preferably, the 16s rRNA of Burkholderia has at least 99.9%, 99.99% or 100% identity with the 16s rRNA of the strain deposited under accession number GDMCC: 62460.

In a second aspect, the present invention provides a composition comprising burkholderia and/or a metabolite, culture, fermentation broth or extract thereof according to the first aspect of the invention. In some embodiments, the composition comprises burkholderia as described in the first aspect of the invention. In a preferred embodiment, the burkholderia comprises an inactivated burkholderia according to the first aspect of the invention. In some specific embodiments, the composition of the invention is a culture or broth of burkholderia of the first aspect of the invention.

In some embodiments, the compositions of the present invention further comprise additional active agents and/or adjuvants.

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 a preferred embodiment, the biocontrol agent is selected from the group consisting of a bacterial, fungal, viral biocontrol agent, an insect biocontrol agent, a nematode biocontrol agent, or any combination thereof. In a further preferred embodiment, the biocontrol agent is selected from at least one of trichoderma harzianum (Trichoderma harzianum), rhodosporidium lilacinum (Purpureocillium lilacinum), penicillium beijerinum (Penicillium bilaiae), bacillus subtilis (Bacillus subtilis), renieratia holdele (Burkholderia rinojensis), bacillus pumilus (Bacillus pumilus), bacillus belgium (Bacillus velezensis), methylobacterium rosei (Methylobacterium rhodesianum), methylobacterium torvum (Methylobacterium extorquens), paenibacillus pierce (Paenibacillus peoriae). Preferably, the trichoderma harzianum is trichoderma harzianum with the preservation number of CGMCC NO. 15679. Preferably, the lilyturf root is a lilyturf root with a preservation number of CGMCC NO. 12773. Preferably, the penicillium beijerinum is penicillium beijerinum with the preservation number of CGMCC NO. 12767. Preferably, the bacillus subtilis is a bacillus subtilis with a preservation number of CGMCC NO. 12908. Preferably, the method comprises the steps of, the RenOegbeck-Hendelia is RenOegbeck-Hendelia with the preservation number of GDMCC NO. 61156. Preferably, the Bacillus pumilus is Bacillus pumilus with accession number GDMCC No. 61962. Preferably, the bacillus beleiensis is bacillus beleiensis with accession number GDMCC No. 61434. Preferably, the methylobacterium rosenbergii is methylobacterium rosenbergii having accession number GDMCC No. 60729. Preferably, the methylobacterium torvum is methylobacterium torvum having accession number GDMCC No. 62943. Preferably, the paenibacillus pierce is paenibacillus pierce deposit No. GDMCC No. 60482.

In some embodiments, the adjuvant comprises an agriculturally or horticulturally acceptable carrier, diluent, stabilizer, filler, humectant, colorant, solvent, co-solvent, film former, autonomy promoter, emulsifier, dispersant, preservative, antifreeze, thickener, adjuvant, or any combination thereof.

In a preferred embodiment, the composition according to the second aspect of the 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 solutions, powders, granules, emulsions, suspensions, tablets, microparticles and wettable powders. Preferably, the composition is in the form of a microbial inoculant for use as a biofertilizer, biopesticide or bioherbicide.

In a third aspect, the present invention provides the use of Burkholderia or a metabolite, culture, fermentation broth or extract thereof according to the first aspect of the invention or a composition according to the second aspect of the invention for the preparation of a microbial inoculant, biofertilizer, biopesticide or bioherbicide.

In a fourth aspect, the present invention provides the use of Burkholderia or a metabolite, culture, fermentation broth or extract thereof according to the first aspect of the invention or a composition according to the second aspect of the invention for controlling plant diseases and insect pests and/or for weeding. Preferably, the plant disease or pest is selected from the group consisting of plant re-planting disease, plant bacterial disease, plant fungal disease, plant viral disease, plant soil borne disease, plant pest and/or plant oomycete disease.

In some embodiments, the plant pest is caused by one or more of a plant pathogenic bacterium, fungus, virus, insect, egg, and nematode. Preferably, the plant pest is caused by at least one pathogenic bacterium selected from the group consisting of: bacteria such as Streptomyces scab (Streptomyces scabies), laurella viridis R.s (Ralstonia solanacearum), bacillus subtilis (Bacillus subtilis), actinidia canker (Pseudomonas syringae pv. Actinidiae), rhizoctonia solani (Xanthomonas campestris pv. Oryzae), rhizoctonia solani, Cabbage soft rot fungus (Erwinia aroideae), walnut black spot fungus (Xanthomonas arboricola pv. Juglandis), konjak soft rot fungus (Erwinia carotovora), staphylococcus aureus (Staphylococcus aureus), fungi such as anthrax fungus (Colletotrichum capsici), rhizoctonia solani (Rhizoctonia solani), fusarium oxysporum (Fusarium oxysporum), fusarium graminearum (Fusarium graminearum), fusarium graminearum (Athelia rolfsii), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), botrytis cinerea (Botrytis cirerea), fusarium oxysporum (Fusarium oxysporum f.sp.cuumerinum), fusarium graminearum (Gaeumannomyces critici), gibberella graminearum (Fusarium graminearum), Apple tree canker (VALSAMALI), apple anthracnose (Glomerella cingulata), rhizoctonia solani (Rhizoctonia solan), pyricularia oryzae (Pyricularia grisea), tomato early blight (ALTERNARIA SOLANI), strawberry gray mold (Botrytis cirerea), potato late blight (Phytophthora infestans), corn big spot (Exserohilum turcicum), The plant species are selected from the group consisting of Sclerotinia maydis (Bipolaria maydis), sclerotinia citrulli (Fusarium oxysporum f.sp.niveum), verticillium eggplant verticillium (Verticillium dahliae), sclerotinia gossypii (Fusarium oxysporum f.sp.gasingfectum), phytophthora capsici (Phytophthora capsici), phytophthora nicotianae (Phytophthora nicotianae).

In a preferred embodiment, the plant pest is caused by nematodes or eggs. More preferably, the nematodes are selected from one or more of the group consisting of Meloidogyne spp and caenorhabditis elegans Caenorhabditis elegans, and even more preferably, the Meloidogyne is selected from one or more of the group consisting of Meloidogyne incognita, meloidogyne arachnia (M.aremia) and Meloidogyne javanica (M.javanica).

In other preferred embodiments, the plant disease is caused by insects. More preferably, the insect is selected from Spodoptera frugiperda Spodoptera frugiperda (Smith), migratory locust Locusta migratoria Linnaeus (migratory locust and other migratory grasshoppers), meadow moth Loxostege sticticalis Linnaeus, armyworms (e.g., oriental armyworm MYTHIMNA SEPARATE (Walker) and Lawsonia inermis Leucania loryi Duponchel), rice planthoppers (e.g., brown planthopper NILAPARVATA LUGENSAnd one or more of the following species of liriope virgata Sogatella furcifera (Horv a th)), cnaphalocrocis medinalis Cnaphalocrocis medinalis (Guen e), chilo suppressalis Chilo suppressalis (Walker), wheat aphids (such as Sitobion avena (Fabricius), myzus persicae Gu Yiguan aphid Rhopalosiphum padi (Linnaeus), myzus persicae Schizaphis graminum (Rondani)), asian corn borer Ostrinia furnacalis (Guen e), vegetable thrips (such as soybean thrips Megalurothrips usitatus (bagall), melon thrips THRIPS PALMI KARNY, frankliniella occidentalis FRANKLINIELLA OCCIDENTALIS (Pergande), flower thrips FRANKLINIELLA INTONSA (Trybom)).

In preferred embodiments, the control of plant diseases and insect pests is achieved by producing siderophores, inducing systemic resistance of the plant or its seeds to plant pathogens, and/or reducing chemotaxis of plant pathogens to plants.

In a fifth aspect, the present invention provides the use of burkholderia or a metabolite, culture, fermentation broth or extract thereof according to the first aspect of the invention or a composition according to the second aspect of the invention for promoting plant growth. In a preferred embodiment, the promotion of plant growth is achieved by at least one of promoting plant root development, providing nitrogen fixation, providing phosphate solubilizing effect, producing indoleacetic acid, removing weeds.

In an embodiment of the invention, the plant is selected from the group consisting of food crops, cash crops or rhizome crops. Preferably, the food crop is selected from at least one of cereal crops, tuber crops and bean crops. Preferably, the cash crop is selected from at least one of the group consisting of plants of the Solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, brassicaceae, orchidaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, pineapple, araliaceae, leguminosae, and Cactaceae. Preferably, the rhizome crop is selected from potato, carrot, leaf vegetable, solanaceous vegetable, strawberry, grape, citrus, banana, kiwi, dragon fruit, tomato, capsicum, beans, ginger, pseudo-ginseng, etc.

In a sixth aspect, the present invention provides a method for preparing a fermentation broth of Burkholderia according to the first aspect of the present invention, comprising the steps of first subjecting said Burkholderia to an activation culture and then subjecting it to a fermentation culture.

In a seventh aspect, the present invention provides a method of controlling plant diseases and insect pests and/or promoting plant growth comprising applying to a plant or seed a burkholderia or a metabolite, culture, fermentation broth or extract thereof according to the first aspect of the invention, or a composition according to the second aspect of the invention, preferably a fermentation broth prepared by a method according to the sixth aspect of the invention.

Compared with the prior art, the invention provides a newly discovered Burkholderia, and the metabolite, the culture, the fermentation liquor and the extract of the strain are utilized to prepare the composition. In agricultural, forestry and other fields of application, the strains, metabolites, cultures, fermentation broths, extracts and compositions are capable of producing siderophores, inducing systemic resistance of plants or seeds thereof to plant pathogens and reducing chemotaxis of plant pathogens to plants, thus being capable of killing or inhibiting more kinds of plant pathogens to achieve control of various plant diseases and insect pests, and in addition, they are also capable of promoting plant root system development, providing nitrogen fixation, providing phosphate solubilizing effect, producing indoleacetic acid and removing weeds, thereby more effectively promoting plant development and growth.

Drawings

The objects, features and advantages of the present invention will be better understood by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments in which the principles of the invention are utilized. In these figures:

FIG. 1 shows colony morphology characteristics of Burkholderia (Burkholderia sp.) M222 after cultivation on LB solid medium plates.

FIG. 2 shows a phylogenetic tree of Burkholderia M222.

FIG. 3 shows the results of testing the ability of Burkholderia (Burkholderia sp.) M222 to produce siderophores by the CAS plate coverage method.

Fig. 4 shows a staining pattern of root systems after 7 days of tomato root dipping treatment in nematode chemotaxis test. The left panel shows the sterile water control and the right panel shows the M222-10X treatment.

Fig. 5 shows the staining pattern of root systems after 7 days of cucumber treatment with M222 microbial agent gel in the insect chemotaxis test. The left panel shows the sterile water control and the right panel shows the M222-5X treatment.

FIG. 6 shows the results of a test for the pathogenic bacteria antagonistic ability of Burkholderia M222. The upper row is a pathogen blank control, and the lower row is a culture diagram of the pathogen and M222 in opposition. In fig. 6 R.s represents lactobacillus subtilis, S.s represents streptomyces scab, F.o represents fusarium oxysporum, F.g represents fusarium graminearum, A.r represents southern blight, C.c represents anthrax.

FIG. 7 shows the results of a nitrogen fixation qualitative test of Burkholderia (Burkholderia sp.) M222.

FIG. 8 shows the results of a qualitative test for phosphorus dissolution of Burkholderia (Burkholderia sp.) M222.

FIG. 9 shows the growth vigor of potted tomatoes treated with Burkholderia (Burkholderia sp.) M222 fermentation broth.

FIG. 10 shows the growth vigor of potted cucumber treated with Burkholderia (Burkholderia sp.) M222 fermentation broth.

FIG. 11 shows the results of a qualitative test of the IAA-producing ability of Burkholderia M222.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, based on the embodiments of the invention, which a person skilled in the art would obtain without making any inventive effort, are within the scope of the invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. 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 unless otherwise indicated.

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

The use of any and all examples, or exemplary language (e.g., "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.

The terms "comprises" and "comprising," when used in the specification and claims of the present application, specify the presence of stated 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 groups thereof.

It is also to be understood that the terminology used in the description of the application is for the purpose of describing particular embodiments only, and is not intended to be limiting of the application. As used in the specification and claims of the present application, the terms "a," "an," "the," or "said" may mean one or more than one unless the context clearly indicates otherwise. Terms that are presented in the singular also include the plural unless the context clearly indicates otherwise.

It should be further understood that the term "and/or" as used in the present specification and claims refers to any and all possible combinations of one or more of the associated listed items, and includes such combinations. Furthermore, in some embodiments of the application, features or combinations of features set forth herein may also be excluded or omitted.

Various aspects and specific embodiments of the invention will now be described in more detail by way of non-limiting embodiments and examples.

In one embodiment, the invention relates to Burkholderia deposited under accession number GDMCC: 62460. The Burkholderia is named M222 and is deposited at the microorganism strain collection center of Guangdong province at 5/10 of 2022, the deposit address is building 5, no. 59, of the university of Guangzhou, martyr, the institute of microorganisms, the university of Guangdong province, deposit number GDMCC:62460, and the detection result is survival, and the classification name is Burkholderia sp.

Burkholderia (Burkholderia sp.) M222 of the present invention is a spore-free gram-negative bacterium that appears white in colony, rough in surface and edge, and sticky after 1 or several days of culture in LB solid medium at 30 ℃.

It is well known in the art that bacterial species can be identified by molecular biology methods in addition to traditional taxonomic methods including, but not limited to, cell morphology observation, gram staining, flagella staining, various metabolic experiments. Molecular biological methods include ribosomal RNA sequencing, sequencing based on whole genome sequencing, and the like.

Sequencing of the 16s rRNA sequence of Yu Bake Hall bacterium (Burkholderia sp.) M222 showed that the 16s RNA sequence was as shown in SEQ ID NO. 1. Further by comparing the 16s rRNA sequences of bacteria, a biological evolutionary tree was drawn according to the base of sequence differences according to the evolutionary distance thereof, as shown in FIG. 2.

In other embodiments, the invention also relates to progeny, subcloned or genetically modified strains of Burkholderia (Burkholderia sp.) M222. Preferably, the strain retains the biological activity of M222, such as pest control and plant growth promoting effects.

In other embodiments, the invention relates to variants of Burkholderia (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 more than 100% to the genome of the above-described Burkholderia M222 strain, and/or the genome of the Burkholderia variant is compared with the genome of the above Burkholderia (Burkholderia sp.) M222 strain by a fraction of no less than 55%,. Gtoreq.60%,. Gtoreq.65%,. Gtoreq.70%,. Gtoreq.78%,. Gtoreq.80%,. Gtoreq.85%,. Gtoreq.90%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98% or. Gtoreq.99%. Preferably, the 16s rRNA of the Burkholderia variant has at least 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identity with the 16s rRNA of the Burkholderia M222 strain described above, preferably the Burkholderia variant does not comprise Burkholderia ambifaria, burkholderia multivorans or Burkholderia stabilis. Further preferably, the 16s rRNA of the Burkholderia variant has at least 99.9%, 99.99% or 100% identity with the 16s rRNA of the Burkholderia (Burkholderia sp.) M222 strain described above.

In still other embodiments, the invention also relates to Burkholderia having at least 99%, 99.5%, 99.8%, 99.9% or 100% identity to the sequence as set forth 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 1) a Rennokurosis (Burkholderia rinojensis) strain, 2) a strain having at least 99.8% or 99.9% identity to the 16s rRNA sequence of Rennokurosis (Burkholderia rinojensis), 3) 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% or more to the genome of Rennokurosis (Burkholderia rinojensis), and/or a genome alignment score of 55% or more, 60% or more, 65% or more, 70% or more, 78% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or 99% or more.

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, expressed as a percentage, when aligned to achieve a maximum level of identity. Identity may be determined using computer algorithms known in the art, including but not limited to GCG, BLASTN, FASTA, etc.

The term "variant" as used herein includes any strain derived from a Burkholderia sp. Strain as described herein, such as a strain that has undergone natural mutation or recombination, or has been generated by resistance screening and mutation or recombination by any other mechanism such as radiation, viruses, transposons or mutagenic chemicals. 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 Burkholderia sp strains described herein.

In some embodiments, the invention relates to a metabolite, culture, fermentation broth or extract of burkholderia (e.g., M222) as described herein. As used herein, "metabolite," "culture," "fermentation broth," and "extract" include any component, compound, substance, or by-product (including but not limited to small molecule secondary metabolites) produced by burkholderia as described herein that has any of the beneficial effects described in the present invention. The beneficial effects include resistance to plant re-planting disease, plant bacterial disease, plant fungal disease, plant viral disease, plant soil-borne disease, plant pest and/or plant oomycete disease, promotion of plant growth, and the like.

In some embodiments, the invention relates to compositions comprising burkholderia as described herein. In a preferred embodiment, the composition comprises an inactivated burkholderia as described herein, such as inactivated M222. In some embodiments, the burkholderia is cultured under suitable conditions for about 48 hours. Preferably, the Burkholderia is shake cultivated at about 28-30 ℃ for about 48 hours. Or the burkholderia may be treated at about 60 ℃ for about 2 hours. Experiments prove that the composition containing the inactivated Burkholderia has excellent effects of controlling diseases and insect pests and promoting plant growth.

In still other embodiments, the invention is directed to compositions comprising a metabolite, culture, fermentation broth or extract of Burkholderia described herein (e.g., M222).

In a further embodiment, the composition of the present invention further comprises an additional active agent. The additional active agents described herein refer to other biological and/or chemical agents having a particular activity than burkholderia contained in the compositions of the present invention. The additional active agents described herein refer to additional active agents that are compatible with the metabolite, culture, fermentation broth or extract of burkholderia (e.g., M222).

In some embodiments, the additional active agent may be a plant nutrient, a plant growth promoter, a biostimulant (medium trace element), a fertilizer, a seed coating agent, or the like.

Preferably, the fertilizer comprises at least one of amino acid fertilizer, fulvic acid fertilizer, seaweed fertilizer, humic acid fertilizer, fertilizer containing pollen polysaccharide, polypeptide fertilizer and microbial fertilizer. The term "microbial fertilizer" as used herein refers to a product containing a specific microorganism that is capable of achieving a specific fertilizer effect when applied in agricultural production. Such effects include not only the supply of soil, environment and plant nutrients, but also the beneficial effects of the metabolites produced by them on the plant.

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, an insecticide, a bactericide, a fungicide, a virucide, a nematicide, an insecticide, an acaricide, a gastropodide, a herbicide, and the like, or a combination thereof. In preferred embodiments, the chemical agent comprises one or more of a bactericide, fungicide, nematicide. Preferably, the bactericides include, but are not limited to, one or more of fluxapyroxad, mesogenic, thiabendazole, copper hydroxide, kasugamycin, bactam, copper king, chlorothalonil, 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, resina, cymoxanil, dimethomorph, flumorph, silver fali, benzamidine cyproconazole, myclobutanil, tebuconazole, or propiconazole. Preferably, the nematicide includes, but is not limited to, one or more of halogenated hydrocarbons, methyl thioisothiocyanates, organic phosphorus, carbamates, or avermectins.

In some embodiments, the additional active agent may include one or more additional biological control agents. Preferably, the biocontrol agent includes, but is not limited to, a bacterium, a fungus (e.g., yeast), a viral biocontrol agent, an insect biocontrol agent, a nematode biocontrol agent, or any combination thereof. Preferably, the bacteria include bacteria selected from the group consisting of Bacillus, burkholderia, or any combination thereof.

Further preferably, the biological control agent is selected from at least one of trichoderma harzianum (Trichoderma harzianum), rhodosporidium lilacinum (Purpureocillium lilacinum), penicillium beijerinum (Penicillium bilaiae), bacillus subtilis (Bacillus subtilis), renieratene holly (Burkholderia rinojensis), bacillus pumilus (Bacillus pumilus), bacillus bailii (Bacillus velezensis), bacillus coagulans (Bacillus coagulans), methylobacterium rosei (Methylobacterium rhodesianum), methylobacterium torvum (Methylobacterium extorquens), paenibacillus piri (Paenibacillus peoriae), and Bacillus licheniformis.

Preferably, the trichoderma harzianum comprises trichoderma harzianum with the preservation number of CGMCC NO. 15679.

Preferably, the lilyturf root comprises lilyturf root with a preservation number of CGMCC NO. 12773.

Preferably, the penicillium beijerinum comprises penicillium beijerinum with a preservation number of CGMCC No. 12767.

Preferably, the bacillus subtilis comprises bacillus subtilis with a preservation number of CGMCC NO. 12908.

Preferably, the method comprises the steps of, the RenOilybek's bacteria include RenOilybek's bacteria with a preservation number of GDMCC NO. 61156.

Preferably, the Bacillus pumilus includes Bacillus pumilus having accession number GDMCC NO. 61962.

Preferably, the bacillus belgium comprises bacillus belgium with a deposit number of GDMCC No. 61434.

Preferably, the methylobacterium rosenbergii comprises methylobacterium rosenbergii having accession number GDMCC No. 60729.

Preferably, the methylobacterium torvum comprises methylobacterium torvum having accession number GDMCC No. 62943.

Preferably, the paenibacillus pierce comprises paenibacillus pierce deposit No. GDMCC No. 60482.

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

In some embodiments, the compositions of the present invention further comprise one or more excipients. Preferably, the adjuvant comprises an agriculturally or horticulturally acceptable carrier, diluent, stabilizer, filler, wetting agent, colorant, solvent, co-solvent, film former, spontaneous emission promoter, emulsifier, dispersant, preservative, antifreeze, thickener, adjuvant, or any combination thereof. Preferably, the adjuvants improve the spreadability of the microbial agents on plants and seeds.

In some embodiments, the compositions of the present invention may be provided in the form of a microbial agent. Preferably, the composition is used as a biofertilizer, biopesticide or bioherbicide. In addition, the composition of the invention can be used for preparing microbial agents, biofertilizers, biopesticides or biological herbicides.

The term "biofertilizer" and "plant growth promoter" as used herein are used interchangeably and refer to a preparation containing a biological body or a derivative thereof as a main active ingredient and having an effect of promoting plant growth (growth promoting). The term "biopesticide" as used herein is used interchangeably with "biopesticide" and refers to a formulation that utilizes organisms or derivatives thereof to kill or inhibit agricultural pests such as plant pathogens that cause plant pests. The term "bioherbicide" as used in this specification refers to a formulation that utilizes organisms or derivatives thereof to remove weeds. The biofertilizer, biopesticide, bioherbicide described herein may also be referred to as microbial fertilizer, microbial pesticide or microbial herbicide, in particular wherein the organisms used comprise burkholderia as described above.

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., more preferably, has a combination of two or more of the above activities.

The burkholderia strain of the invention or variants thereof or the composition according to the invention is particularly applicable in at least one of the following:

1) Preventing and treating plant pathogenic fungi diseases;

2) Preventing and treating plant pathogenic bacterial diseases;

3) Preparing a product for preventing and treating plant pathogenic fungi diseases;

4) Preparing a product for preventing and treating plant pathogenic bacterial diseases;

5) The plant diseases and insect pests are resisted;

6) Preparing a product resistant to plant diseases and insect pests;

7) Promoting plant growth;

8) Preparing a product for promoting plant growth;

9) Preventing and controlling plant nematode diseases;

10A product for preventing and controlling plant nematode diseases is prepared.

Preferably, the promotion of plant growth includes promotion of emergence rate, improvement of plant height and/or improvement of yield.

The compositions of the present invention may be provided in the form of a solid, liquid, powder, or any combination thereof, and the like. For example, the compositions of the present invention may be formulated into solutions, powders, granules, emulsions, suspensions, tablets, microparticles, wettable powders and the like.

The burkholderia, the metabolite, the culture, the fermentation liquor, the extract or the composition of the invention can be used for preventing and controlling plant diseases and insect pests. Preferably, the plant disease or pest is selected from the group consisting of plant re-planting disease, plant bacterial disease, plant fungal disease, plant viral disease, plant soil borne disease, plant pest and/or plant oomycete disease. Preferably, the plant pest is caused by one or more of a plant bacterial pathogen, a plant fungal pathogen, a virus, an insect, an egg and a nematode.

Preferably, the plant bacterial pathogens include, but are not limited to, at least one of streptomyces scab (Streptomyces scabies), lactobacillus subtilis R.s (Ralstonia solanacearum), bacillus subtilis (Bacillus subtilis), kiwi fruit canker (Pseudomonas syringae pv.actinidiae), rice bacterial blight (Xanthomonas campestris pv.oryzae), cabbage soft rot (Erwinia aroideae), walnut black spot (Xanthomonas arboricola pv.juglandis), konjak soft rot (Erwinia carotovora), and staphylococcus aureus (Staphylococcus aureus).

Preferably, the plant fungal pathogens include, but are not limited to, at least one of anthrax (Colletotrichum capsici), rhizoctonia solani (Rhizoctonia solani), fusarium oxysporum (Fusarium oxysporum), fusarium graminearum (Fusarium graminearum), fusarium graminearum (Exserohilum turcicum), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), botrytis cinerea (Botrytis cirerea), fusarium cucumber (Fusarium oxysporum f.sp.curmerinum), wheat take-all (Gaeumannomyces critici), gibberella wheat (Fusarium graminearum), apple tree rot (VALSAMALI), apple anthracnose (Glomerella cingulata), sheath blight of rice (Rhizoctonia solan), rice blast (Pyricularia grisea), tomato early blight (ALTERNARIA SOLANI), strawberry gray mold (Botrytis cirerea), potato late blight (Phytophthora infestans), corn big spot (Exserohilum turcicum), corn small spot (Bipolaria maydis), watermelon fusarium wilt (Fusarium oxysporum f.sp.niveum), eggplant fusarium wilt (Verticillium dahliae), fusarium wilt (Fusarium oxysporum f.vannamese), capsicum (Phytophthora capsici), and tobacco blight (Phytophthora nicotianae).

Preferably, the plant disease and pest includes, but is not limited to, at least one of scab, bacterial wilt, damping off, black shank, black mole.

Preferably, the nematodes include, but are not limited to, one or more of root knot nematodes (Meloidogyne spp.) and caenorhabditis elegans (Caenorhabditis elegans). Preferably, the root-knot nematodes include, but are not limited to, one or more of the group consisting of meloidogyne incognita (m.incognita), meloidogyne northern (m.hapla), meloidogyne arachidis (m.areylaria) and meloidogyne javanica (m.javanica).

Preferably, the insects include, but are not limited to, orthoptera, isoptera (e.g., termites), hemiptera (e.g., stinkbugs), homoptera, thysanoptera (e.g., thrips), coleoptera (e.g., beetles), lepidoptera (e.g., moths or butterflies), diptera, and hymenoptera (e.g., bees) insects.

In addition, the plant diseases and insect pests also include plant diseases and insect pests caused by mites. The mites include, but are not limited to, the mites of the order Tetranychidae, the family Caesalpidae, the family She Yingman, the family Pinaceae, and the like.

Burkholderia of the present invention is capable of producing a variety of siderophores (siderophores) that assist in survival or protection against toxic metals, such as ornibactin, malleobactin, pyochelin, cepabactin and CEPACIACHELIN. Siderophores not only supply plant iron nutrition, but also achieve biological control by competing with plant pathogens for iron nutrition. Most pathogens have no or low capacity to secrete siderophores and have poor ability to compete with other siderophore-producing microorganisms for iron nutrition. Therefore, the Burkholderia disclosed by the invention not only can antagonize various plant bacterial pathogenic bacteria and plant fungal pathogenic bacteria, but also can be widely used for preventing and controlling more plant diseases and insect pests.

In addition, the burkholderia, the metabolite, the culture, the fermentation liquor, the extract or the composition of the invention can induce the plant or the seed thereof to generate systemic resistance to plant pathogens, thereby further enhancing the effect of the burkholderia on controlling plant diseases and insect pests.

In addition, burkholderia, metabolites, cultures, fermentation broths, extracts or compositions of the invention can also reduce chemotaxis of plant pathogens to plants. In the case of nematodes, the nematodes are able to sense chemical signals released by plant or rhizosphere microorganisms during early interactions with plants to find hosts. By reducing chemotaxis of nematodes to plants, infection and damage of the nematodes to the plants can be prevented or reduced, so that the effect of preventing and controlling plant diseases and insect pests is achieved.

In some embodiments, the burkholderia, metabolites thereof, cultures, fermentation broths, extracts, or compositions of the invention achieve control of plant diseases and insect pests by one or more mechanisms of action that produce siderophores, induce systemic resistance of plants or seeds thereof to plant pathogens, and reduce chemotaxis of plant pathogens to plants.

The inventor compares the pest control effect of the pesticide avermectin commonly used in the field with the Burkholderia of the invention through experiments. The result shows that the abamectin has good control effect under the controllable greenhouse condition, but the effect is not ideal when being used in the field. This is probably due to the fact that 1) resistance to avermectin is produced by plant pathogens due to the problem of field abuse of avermectin, 2) the variety of plant pathogens is single in a controlled greenhouse environment, so that avermectin can still show ideal results, but there are many different plant pathogens in the field, and the killing effect of avermectin on some of the pathogens is poor, so that the control effect is poor. Compared with the prior art, the Burkholderia has good control effect in a greenhouse and a field environment, and is suggested to act through multiple mechanisms such as a pathogen killing promoting mechanism, a pathogen infection inhibiting mechanism, a crop induction resistance effect and the like, so that the Burkholderia has a broad disease and pest control spectrum, and can be used for controlling plant diseases and pests in a field complex environment.

In addition, burkholderia, metabolites, cultures, fermentation broths, extracts or compositions of the invention may also be used to promote plant growth.

Compared with other Burkholderia, the metabolite, the culture, the fermentation liquor, the extract or the composition of the Burkholderia have the capabilities of fixing nitrogen and dissolving phosphorus, can provide nutrient components required by plant growth, can generate indoleacetic acid, and further promote the development and the growth of plants.

In addition, the burkholderia, the metabolites, the cultures, the fermentation broths, the extracts or the compositions of the invention have the effect of removing weeds. Weeds as described herein refer primarily to weeds known in the agricultural arts. Preferably, the weeds include, but are not limited to, amaranthus retroflexus (Amaranthus retroflexus l.), setaria (Sedum sarmentosum), and the like.

In some embodiments, the burkholderia, metabolites thereof, cultures, fermentation broths, extracts, or compositions of the invention promote plant development and growth by one or more mechanisms of action in promoting plant root development, nitrogen fixation, phosphate dissolution, production of indoleacetic acid, and weed removal.

Plants mentioned in the description of the present application include plants in the agricultural, forestry and like fields. Preferably, the plants include, but are not limited to, food crops, cash crops or rhizome crops.

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

Preferably, the cereal crops include, but are not limited to, at least one of rice, wheat, corn, barley, oat, rye, sorghum, millet, barnyard grass, buckwheat. Preferably, the tuber crops include, but are not limited to, at least one of sweet potato, yam, potato. Preferably, the legume crops include, but are not limited to, at least one of soybeans, broad beans, peas, mung beans, peanuts.

Preferably, the cash crop includes, but is not limited to, at least one cash crop of the Solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, brassicaceae, orchidaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, pineapple, araliaceae, and Cactaceae families.

Preferably, the solanaceous plants include, but are not limited to, tomatoes, peppers, potatoes, eggplants, and the like. Preferably, the rosaceous plant includes, but is not limited to, strawberry, papaya, and the like. Preferably, the rutaceae plant includes, but is not limited to citrus and the like. Preferably, the canna plant includes, but is not limited to, banana and the like. Preferably, the cucurbitaceae plants include, but are not limited to, cucumber, wax gourd, pumpkin, bitter gourd, luffa, watermelon, grosvenor momordica fruit, and the like. Preferably, the cruciferous plants include, but are not limited to, cabbage, rape, cabbage, radish, broccoli, and the like. Preferably, the orchid includes, but is not limited to, orchid and the like. Preferably, the papilionaceous plant includes, but is not limited to, soybean and the like. Preferably, the asteraceae plants include, but are not limited to lettuce and the like. Preferably, the liliaceae plant includes, but is not limited to, garlic and the like. Preferably, the zingiberaceous plant includes, but is not limited to, ginger and the like. Preferably, the passionflower family plants include, but are not limited to passion fruit and the like. Preferably, the pineapple family plants include, but are not limited to, golden pineapple and the like. Preferably, the araliaceae plants include, but are not limited to, notoginseng, and the like. Preferably, the Cactaceae plants include, but are not limited to, dragon fruits and the like.

Preferably, the rhizome crops include, but are not limited to, potatoes, carrots, leafy vegetables, solanaceous vegetables, strawberries, grapes, citrus, bananas, kiwi fruits, dragon fruits, tomatoes, peppers, beans, gingers, pseudo-ginseng, ginseng and the like.

Plants as described herein include plants at various stages, including plants in seed germination, seedling growth, seedling development, seedling flowering, and seed formation, and thus include both seedlings and post-growth plants. In addition, plants described herein also include plant tissues and plant organs. Preferably, the plant tissue includes, but is not limited to, meristem, protective tissue, basal tissue, guide tissue, and the like. Preferably, the plant organ includes, but is not limited to, roots, stems, leaves, flowers, fruits and seeds.

In one embodiment, the present invention relates to a method of preparing a fermentation broth of Burkholderia of the present invention comprising the steps of first subjecting said Burkholderia to an activation culture and then subjecting it to a fermentation culture. Preferably, the activation culture is performed on a solid medium. Preferably, the fermentation culture is performed in a liquid medium. In a preferred embodiment, the medium is an LB medium, preferably a modified LB medium. In a specific embodiment, the LB medium comprises yeast extract, peptone, sodium chloride and water. In a more specific embodiment, the LB medium comprises 5-10 g of yeast extract powder, 8-12 g of peptone, 5-15 g of sodium chloride and water, and the pH value is 7-7.5. In a preferred embodiment, the activation culture is performed at about 30 ℃ for about 2 days. In a preferred embodiment, the fermentation culture is conducted at about 30 ℃ for about 48 hours.

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

Preferably, the application means includes root dipping, leaf spraying, composting, seed soaking, coating, field flooding, drip irrigation of the plant or plant organ, painting the plant or plant organ, drip irrigation of the plant or plant organ, and the like.

Examples

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

Example 1: isolation and identification of Burkholderia (Burkholderia sp.) M222

Isolation of M222 Strain

Burkholderia (Burkholderia sp.) M222 of the present invention was isolated from Gastrodia elata rhizosphere soil in Yunnan province. The separation method comprises the steps of weighing 5g of soil, adding 45mL of sterile water, shaking for 5min by a vortex shaker, carrying out gradient dilution on the soil to obtain 10 ^-1～10^-6, coating bacterial solutions of three dilutions of 10 ^-4、10^-5 and 10 ^-6 on a modified Luria-Bertani (LB) solid culture medium (5-10 g of yeast extract, 8-12 g of peptone, 5-15 g of sodium chloride, 15g of agar and 1L of water, and pH=7-7.5) flat plate, carrying out three repetitions on each dilution, carrying out inversion culture in a 30 ℃ constant temperature incubator for 4 days, then picking out single colony bacterial strains, streaking and inoculating the bacterial strains on a new LB flat plate, carrying out culture at 30 ℃ for 1 day, and then storing the bacterial strains in a-80 ℃ refrigerator by a glycerol tube.

Identification of M222 Strain

The isolated strain was inoculated in a modified LB solid medium and cultured at 30℃for 1 day. The strain morphology is shown in FIG. 1. The identification shows that the strain is a gram-negative bacterium which does not produce spores, and the colony of the strain is white, has rough surface and edge and is sticky (see figure 1).

Subsequently, 16s rRNA sequencing was performed on the isolated strain using the amplification primers with the sequencing primers 27F:5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2) and 1492R:5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3). The measurement result shows that the 16s rRNA sequence of the strain is as follows:

AGTCGAACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGATGAAAGCGGGGGATCTTCGGACCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGAAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTTTGGGTTAATACCTCGGAGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGACAGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGTGACTGGCAAGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATT GACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAATCCTGAAGAGATTTGGGAGTGCTCGAAAGAGAACCGATACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTGGCTAGTCTAACCGC(SEQ ID NO:1)

Then, the sequence was aligned with the sequence in NCBI database by BLAST, and the result showed that the 16s rRNA sequence of the above isolated strain had a similarity of 99% or more with the 16s rRNA sequences of a plurality of strains of Burkholderia (Burkholderia), for example, 99.78% with Burkholderia ambifaria strain LD111-1 16s rRNA sequence, 99.71% with Burkholderia rinojensis strain A396 16s rRNA sequence, 99.71% with Burkholderia multivorans strain QYGXJ-1 16s rRNA sequence, and 99.64% with Burkholderia stabilis strain LD119 16s rRNA sequence. Preliminary determination the above isolated strain belongs to Burkholderia (Burkholderia). Phylogenetic tree was constructed using MEGA 5 (see fig. 2).

To further determine the whole genome similarity of the isolated burkholderia strain to other members of the burkholderia genus, the original strain obtained by the above isolation was further subjected to genome preparation, sequencing, assembly and analysis. The genome of the original strain was sequence fragmented by ultrasound and then an Illumina sequencing library was constructed using a standard DNA banking kit (NEB Ultra ^TM). The constructed sequencing library was subjected to double-ended sequencing using NovaSeq (Illumina).

Genomic raw sequencing data was data filtered using fastp (version: 0.20.0) with filter parameters of "- -poly_g_min_len10- -poly_x_min_len10-q 15-u 40-n 5-l 50". The filtered raw data was genome assembled using SPAdes (version: v3.14.0) with the assembly parameters "- -isolate- -cov-cutoff 10". Genomic genes were subjected to genomic gene prediction analysis using a prokaryotic analysis software genome annotation procedure prokka (version: 1.14.5) with parameters "- -gcode 11- -evalue e-09".

Genomic information of burkholderia strains was downloaded from NCBI, and the degree of genomic correlation with previously identified burkholderia species was determined by evaluating the Average Nucleotide Identity (ANI) and alignment score (AF) of the strains obtained by the above isolation with these reference microorganisms and the inventors' autologous norkholderia M928 strain, as shown in table 1, and comparing the whole genomic sequence of the strains obtained by the above isolation with other strains and species of burkholderia. The comparison result shows that the strain with the highest genome similarity is Rennograp Hold's bacteria M928, wherein the average nucleotide similarity (ANI) is 85.38 percent, the gene coverage is 42 percent, the Burkholderia M928 is separated by the inventor and is preserved in the Guangdong province microorganism strain preservation center in 8 months 2020, the preservation number is GDMCC No:61156, and specific information of the strain can be seen in patent names of a compound microorganism microbial agent, a preparation method thereof and a patent with publication number of CN 113980877B.

Comparison with Burkholderia ambifaria showed 84.36% average nucleotide similarity (ANI) and 56% gene coverage, comparison with Burkholderia multivorans showed 84.46% average nucleotide similarity (ANI) and 62% gene coverage, and comparison with Burkholderia stabilis showed 84.51% average nucleotide similarity (ANI) and 58% gene coverage. According to the principle that ANI >95% is the same species, the genome correlation analysis based on Average Nucleotide Identity (ANI) of the application shows that the strain obtained by the separation can be obviously distinguished from the known Burkholderia species from the genotype characteristics.

Thus, the genotypic characteristics of the isolated strain are significantly different from those of the existing strains of other species of burkholderia genus, and the strain can be identified as a new species of burkholderia genus. We named it Burkholderia (Burkholderia sp.) M222.

TABLE 1M 222 Whole genome alignment results

Preservation of M222 Strain

The Burkholderia M222 isolated and identified above is sent to the Guangdong university microbiological bacterial collection center (GDMCC) for preservation, wherein the preservation number is GDMCC:62460, the preservation date is 2022, 5 months and 10 days, the preservation address is Guangzhou Mitsui martyr, no. 100, no. 59 building 5, and the Guangdong university microbiological institute.

Example 2: burkholderia (Burkholderia) sp.) preparation of M222 fermentation broths

1. Activation culture

Burkholderia (Burkholderia sp.) M222 stored after separation was transferred to a plate of modified LB solid medium (yeast extract 5-10 g, peptone 8-12 g, sodium chloride 5-15 g, agar 15g, water 1L, pH=7-7.5), and cultured at 30℃for 2 days.

2. Fermentation culture

Transferring M222 obtained by activation culture on a modified LB solid culture medium flat plate into a modified LB liquid culture medium (5-10 g of yeast extract powder, 8-12 g of peptone, 5-15 g of sodium chloride, 1L of water, pH=7-7.5 and high-temperature sterilization at 121 ℃ for 20 min), and placing the culture medium in a shaking table for shaking culture at 28-30 ℃ for 48h at 180-210 r/min to obtain a fermentation broth of M222.

EXAMPLE 3 in vitro nematicidal Activity test of M222

In this example, meloidogyne incognita (Meloidogyne incognita) was used as a pathogen.

Cleaning root systems of the water spinach for propagating the root-knot nematodes with clear water, and picking egg masses with forceps. Cutting root system after picking ovum, adding 2% sodium hypochlorite, and vortex oscillating for 1.0min. Sequentially sieving with 80 mesh, 200 mesh, 350 mesh and 500 mesh, washing for 2-3 times, collecting ovum in 500 mesh sieve, and making into ovum suspension.

The picked egg mass was surface sterilized with 0.5% naclo solution and then rinsed 3 times with sterile water. The mixture was placed in a 90mm dish and incubated in darkness for 48 hours. And then collecting the second-instar larvae of the root-knot nematode to prepare a second-instar larva suspension for standby.

The M222 strain broth prepared in example 2 was formulated as a five-fold diluted treatment broth. To a 24-well cell culture plate, 450. Mu.L of the treatment solution and 50. Mu.L of the nematode suspension (2000 bars/mL) prepared above were added, and the mixture was covered and sealed with a sealing film.

Sterile water was used as a blank (CK) in this test, and a 1500-fold dilution of 5% avermectin cream (xing Bai Kexian) was used as a positive control. Each treatment was performed in 5 replicates.

After 24h of standing in 28 ℃ dark conditions, the stiffness and death numbers of the second instar larvae were observed and recorded under a split microscope. The criteria were stiff, marked by flexural peristalsis, marked by living worms, and marked by death by nematodes that remained stiff after stimulation with 1mol/L NaOH solution. The rate of stiffness and mortality were then calculated according to the following formula:

Stiff rate (%) = (stiff nematodes number/total nematodes number) ×100%

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

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 rigidify the nematodes and cause nematode death.

TABLE 2 test results of nematicidal Activity of Burkholderia M222 fermentation broth in vitro

	Dilution factor	Rate of stiffness/%	Mortality/%
				CK	/	0.00	0.00
Avermectins	1500	100.00	-
				M222	5×	92.00	76.80

Example 4 control Effect of M222 on greenhouse centrifuge tubes and potted plant conditions and test of Protoffee

In this example, meloidogyne incognita (Meloidogyne incognita) was used as a pathogen and cucumber was used as a host. The nematode egg suspension and the second-instar larva suspension used in this example were prepared as described in example 3.

1. Test of prevention and control effect and pro-effect of M222 under centrifuge tube condition

And sowing cucumber seeds in the seedling tray. After germination, the seeds are transplanted into 50mL centrifuge tubes, 1 seed is sown in each tube, and the seeds are placed into an illumination culture chamber, and the photoperiod and the temperature are set to be 16 hours of illumination, 28 ℃ of illumination, 8 hours of darkness and 20 ℃ of illumination. After 1 week, 10mL of the treatment solution was poured into each plant of cucumber (2 mL of the M222 fermentation broth prepared in example 2 was actually poured into each plant of cucumber, and 10mL of the fermentation broth was made up with water). After 1 day, each cucumber was inoculated with 2mL of nematode egg suspension (3000/mL).

In this experiment, water was used as a blank Control (CK) and a 1500-fold dilution of 5% abamectin cream (xing Bai Kexian) was used as a positive control. Each treatment was repeated 3 times for 5 seedlings.

After 14 days, the disease grade (grade) was evaluated according to 6 grades, 0=no root knot, 1=0% -10% root knots, 2=10% -25% root knots, 3=25% -50% root knots, 4=50% -75% root knots, 5=75% -100% root knots, and the dry/fresh weight of the aerial parts was counted. The control effect and dry weight increase rate were calculated according to the following formula:

Disease index (%) = [ Sigma (number of plants at each disease stage×number of diseases stage)/(total number of plants investigated×highest stage) ] ×100

Control effect (%) = [ control group disease index-treatment group disease index)/control group disease index ] ×100

Dry weight increase (%) = [ control dry weight-treatment dry weight)/control dry weight ]

The test results are shown in table 3 below. The results show that the M222 fermentation broth can reduce the occurrence of root knots and promote the significant growth of plants.

TABLE 3 test results of control effect and promotion effect of M222 fermentation broth on centrifuge tube of meloidogyne incognita

	Index of disease condition	Prevention and control effect	Fresh weight/g of overground part	Dry weight of aerial parts/g	Rate of dry weight increase
						CK	68.00	-	0.655	0.079	-
Avermectins	0.00	100.00%	0.711	0.082	4.24%
						M222	24.00	64.71%	1.422	0.123	56.78%

2. Test of prevention and control effect and pro-effect of M222 under potting condition

And sowing cucumber seeds in the seedling tray. After cotyledons are unfolded, transplanting the cotyledons into seedling pots. When cucumber grows to 2 leaves and 1 heart, the M222 treatment liquid (20 mL of the M222 fermentation liquid prepared in example 2 is actually irrigated according to each cucumber seedling, the fermentation liquid is supplemented with water to 70 mL), and the cucumber is placed in an illumination culture chamber, and the photoperiod and the temperature are set to be 16 hours of illumination, 28 ℃ of temperature, 8 hours of darkness and 20 ℃ of temperature. After 24h, a suspension of two-instar larvae of root knot nematode (300 bars/mL) prepared as described in example 3 was inoculated, each strain was inoculated with 1mL. In the experiment, water is used as a blank Control (CK), a 1500-time diluent of 5% abamectin emulsifiable concentrate (Xing Bai Kexian) is used as a positive control, and the experiment also comprises a new dragon thread shield treatment liquid which is a commercial nematode control product and is used for comparing the control effect with a pro-effect. Each treatment of 8 seedlings was repeated 3 times.

After 15 days, the stem thickness is measured at the position 2cm below the cotyledon by using an external measuring claw of a vernier caliper perpendicular to the growth direction of the cotyledon, the first and second true leaves of the cucumber Miao Di are spread flat, and the distance between the leaf tips is measured by using a ruler to be the leaf spread opening. After 30 days, the disease grade (grade) was observed and evaluated according to the following 9 grades, 0=no root knots, 1=0% -15% root knots, 2=10% -25% root knots, 3=25% -50% root knots, 4=50% -75% root knots, 5=25% -50% root knots, or large root knots, 6=75% -100% root knots, 7=50% -75% root knots, or large root knots, 8=75% -100% root knots, or large root knots, and disease index was calculated according to the following formula:

disease index = [ Σ (number of plants per disease stage×number of disease stages)/(total number of plants investigated×highest stage) ] ×100.

The control effect of the M222 treatment fluid on the root-knot nematodes is evaluated according to the following formula:

The test results are shown in table 4 below. The result shows that the M222 fermentation liquor can reduce root knot and promote plant stem and leaf growth, and the control effect and the promotion effect of the fermentation liquor are obviously superior to those of the commercially available nematode control product Xinlong hui line shield.

TABLE 4 test of control of nematodes and of the growth of fruits by potting of M222 fermentation broth

	Stem thickness/mm	Opening/cm	Index of disease/%	Prevention and control effect
					CK	1.97	4.43	87.50	-
Avermectin 1500×	2.10	4.12	0.00	100.00%
					M222	3.19	14.10	45.24	48.30%
Xinlong Hui line shield	2.28	5.46	86.11	1.59%

Example 5 test of M222 induced plant to develop systemic resistance

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 fluid was prepared as described in example 2.

Tomato seedlings were grown in seedling trays in a 28 ℃ greenhouse. After the seedlings come out neatly, the root system of the tomato seedlings is averagely divided into two parts, the two parts are respectively planted into two adjacent basins, one basin is inoculated with the nematode eggs, the other basin is irrigated with the treatment liquid, and the treatment liquid is not in direct contact with the nematode eggs. After 7 days of tomato seedling transplanting, 100mL of M222 treatment liquid is irrigated in each pot. After 24 hours of irrigation treatment, 1 small hole with the depth of 2cm is made near the root system of the seedling, the eggs suspension of the wire worms is added, and 3000 eggs are inoculated for each plant. In this experiment, water was used as a blank Control (CK) and a 1500-fold dilution of fosthiazate was used as a positive control. 10 seedlings were treated each and repeated 3 times.

After 30 days, root knot numbers were observed and recorded, and root knot reduction rate was calculated as a control effect according to the following formula to evaluate the systemic resistance of the test M222 treatment fluid to induce plant production:

Control effect (%) = [ control group root knot number-treatment group root knot number ]/control group root knot number ] x 100

The test results are shown in table 5 below. The result shows that the M222 fermentation liquor can induce plants to generate systemic resistance and reduce root knot number, and the effect is obviously better than that of the chemical drug fosthiazate.

TABLE 5 results of M222 fermentation broth induced plant resistance test

	Root knot count	Prevention and control effect
			CK	28.73	-
1500-Fold dilution of fosthiazate	14.17	50.70%
			M222	10.03	65.08%

EXAMPLE 6 determination of M222 siderophore

M222 on the plate was scraped with a sterile toothpick, inoculated into a 50mL centrifuge tube containing 10mL of MKB nonferrous medium (5 g of casamino acid, 15mL of glycerol, 2.5g of magnesium sulfate heptahydrate, and autoclaved at 1000mL,pH 7.2,121 ℃ C. For 20min; 10mL of dipotassium hydrogen phosphate solution (2.5 g of dipotassium hydrogen phosphate, 50mL of ultrapure water, and autoclaved at 121 ℃ C. For 20 min) was added to 200mL of the medium before inoculation), and cultured at 30 ℃ C., 200rpm with shaking for 24h. After the cells were produced (turbidity of the medium), the cells were centrifuged at 8000rpm for 5min, and the supernatant was discarded. The cells were washed 2 times with 5mL of sterile ultrapure water and centrifuged at 8000rpm for 5min, and diluted 10 times with sterile ultrapure water to obtain an M222 suspension. 10 mu L M of the suspension was inoculated in 3 drops on a non-ferrous MKB plate, 3 replicates per strain, and incubated in a 30℃incubator for 1 day. After a significant single colony had developed on the plate, a layer of CAS medium (Qingdao sea Bo, 400mL of ultra-pure water was added to 4.35g of medium powder, autoclaved at 116℃for 30 min) was poured onto the plate from which the colony developed, and left at 30℃for 24h. The color change of each plate was observed and the siderophore chelate halo diameter and colony diameter were measured, and siderophore solubility index was calculated according to the following formula:

siderophore solubility index = siderophore chelation halo diameter/colony diameter

The experimental results are shown in FIG. 3. As can be seen from fig. 3, M222 produced a pronounced orange siderophore chelating halo. The result shows that the solubility index of the siderophore is 3.45, and the chelating force of the siderophore on Fe ³⁺ is strong.

It will be appreciated by those skilled in the art that the nutritional competition mechanism and the point competition mechanism are two important mechanisms for the microbial strain to realize the resistance to pathogenic bacteria of soil-borne diseases in soil, and are important mechanisms for resisting fungal diseases and bacterial diseases. The complex is utilized by the ferrite-producing bacteria through the specific receptor outside the membrane, but other organisms cannot utilize the complex, the iron concentration in the environment is reduced, and the pathogenic bacteria lack iron to inhibit growth and reproduction. Experiments show that M222 can widely inhibit the growth of pathogenic bacteria in soil and has the potential of inhibiting soil-borne diseases of fungal diseases and bacterial diseases.

EXAMPLE 7 nematode chemotaxis assay

This example investigated the behavioural response of chemotaxis of root-knot nematodes to M222 using a three-dimensional transparent Pluronic gum system that mimics soil.

In this example, meloidogyne incognita was used as a pathogen, and cucumber and tomato were used as hosts. A suspension of two-instar larvae of nematodes was prepared as described in example 3.

Selecting cucumber (Guangxi 3, guangdong agricultural biotechnology) and tomato (Xinxing 101, guangdong agricultural biotechnology) seeds with basically consistent size and plumpness, carrying out surface disinfection, soaking for 30 seconds with 75% alcohol, sterilizing for 5min with 10% sodium hypochlorite after 3 times of sterile water flushing, repeatedly flushing for 5-6 times with sterile water, and finally sucking water on the surfaces of the seeds with sterile absorbent paper for later use.

Sterilized cucumber and tomato seeds were placed in sterile 1.3% water agar and uniformly aligned with the radicle direction in line. The tomatoes are placed in an illumination room with the temperature of (26+/-1) DEG C for 4 days for use. The cucumber is placed in an illumination room with the temperature of (28+/-1) DEG C for 3 days for use.

Tomato-root dipping test:

The root system of the cultivated tomato seedling is soaked in an M222 microbial inoculum (M222 fermentation broth prepared as described in the example 2, diluted 10 times (10×) as a treatment solution), sterile water is used as a blank control, and fosthiazate is used as a positive control. An electric pipette is used for sucking 23% (w/v) Pluronic glue into a square dish with a diameter of 120mm respectively, the square dish is placed in a room temperature for solidification, and after the root system of the tomato seedling is completely wrapped with M222 microbial inoculum, the square dish is placed at the top of gel Fang Min, and 5 square dishes are uniformly arranged. The plate was placed vertically in a 26/20 ℃ (day/night) light room. After 24h, 5 drops of 50. Mu.L of a suspension containing 100 2-instar larvae (J2 s) of nematodes were dropped onto the bottom edge. Roots were removed from the plates 7 days after the inoculation of the nematodes, stained with acid fuchsin (see fig. 4), counted for the total number of nematodes in 24 hours Shi Gen, the total number of nematodes in 7 days and the number of nematodes of 3-4 ages (J3/J4 s) in 7 days, and the degree of nematode infestation was evaluated.

Cucumber-bacterial agent gel test:

PF-127 (pluronic F-127 powder) was diluted to 23% (w/v) gels (5-fold (5X), 2-fold (2X), 1-fold (1X)) at different microbial concentrations using M222 microbial inoculum, with sterile water as a blank and avermectin as a positive control. 20mL of gels containing M222 bacterial agents at different concentrations were each aspirated into 120mm square dishes using an electric pipette, and after setting at room temperature, 4 3-day cucumber seedlings were transferred to the top of each square dish and the plates were placed vertically in a 28/22℃ (day/night) light room. After 24h, 4 drops of 50. Mu.L of a suspension containing 150 2-instar larvae (J2 s) of nematodes were dropped onto the bottom edge. Roots were removed from the plates 7 days after inoculation with nematodes and stained with acid fuchsin (see fig. 5), the total number of nematodes in the roots was counted for 7 days, and the degree of nematode infestation was assessed.

The above experimental data were subjected to basic calculations and statistical analysis using Excel 2019 and SPSS22.0 software, and differential significance analysis using the Duncan's new complex polar difference method, with a significance level set at P <0.05.

The test results are shown in tables 6 and 7 below. The results show that M222 can significantly reduce the attraction of crop root tips to nematodes and the nematode infestation ability (P < 0.05).

TABLE 6 test results of root dipping test

Note that the different treatments showed significant differences from the blank (P < 0.05)

TABLE 7 test results of the microbial agent gel test

EXAMPLE 8 pathogen antagonistic Capacity test of M222

In this example, streptomyces scabus, L.subtilis, fusarium oxysporum, fusarium graminearum, aphanothece, and Bacillus anthracis were used as the test pathogenic bacteria, and the antibacterial spectrum of M222 was measured.

Inhibition of pathogenic bacteria

Mu.l of bacterial pathogen (Streptomyces scab, L.subtilis) was pipetted onto LA plates, uniformly coated with glass beads and dried. Mu l M drops of fungus were pipetted onto the plates, 3 drops per plate, in a regular triangular distribution, 3 replicates per treatment. Plates inoculated with pathogen alone were used as blank.

Sealing after naturally airing the suspension on the flat plate, culturing in a constant temperature incubator at 30 ℃, taking out and observing after 1 day, measuring the radius of a bacteriostasis ring and the radius of antagonistic bacteria, and calculating an antagonistic index according to the following formula:

Antibacterial zone width = antibacterial zone radius-antagonistic bacterial cell radius

Antagonistic index = width of zone of inhibition/radius of antagonistic bacteria

The experimental results are shown in table 8 below and fig. 6.

TABLE 8 inhibition of pathogenic bacteria by M222

Inhibition of pathogenic fungi

Fungus pathogenic bacteria (fusarium oxysporum, fusarium graminearum, southern blight and anthrax) are inoculated in the center of the LA flat plate on a sterile operating table. The droplets of 5u 1M 222 bacteria were aspirated around the pathogenic bacteria. Each strain was replicated in triplicate. Plates inoculated with pathogenic fungi alone were used as blank.

Taking out and observing after culturing for 6 days in a 28 ℃ incubator, measuring the diameter of a treated colony of a treatment plate and the diameter of a control colony by adopting a crisscross method, and calculating the width of a bacteriostasis zone and the bacteriostasis rate by adopting the following formula:

Antibacterial zone width = control colony diameter-treated colony diameter

Antibacterial ratio (%) =antibacterial zone width/control colony diameter×100

The test results are shown in table 9 below and fig. 6.

TABLE 9 inhibition of pathogenic fungi by M222

As can be seen from Table 8, table 9 and FIG. 6, burkholderia (Burkholderia sp.) M222 has antagonism to both the above two pathogenic bacteria and the above four pathogenic fungi, and thus has a broad antibacterial spectrum, wherein the inhibition effect on Fusarium graminearum is strongest, and the antibacterial rate reaches 64.44%. From this, it can be concluded that Burkholderia (Burkholderia sp.) M222 of the present invention has a remarkable inhibitory effect on both bacterial diseases and fungal diseases.

Example 9 Nitrogen fixation test of M222

M222 cells on the plates were scraped off with an appropriate amount of sterile inoculating loop, resuspended in 1mL of sterile water, and inoculated evenly onto Ashby medium (0.2 g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 5.0g of calcium carbonate, 10.0g of mannitol, 0.1g of calcium sulfate, 18.0g of agar, pH 6.8-7.0) plates, each plate repeated 3 times. After 5 days of dark culture at 28 ℃, the presence of distinct colonies was observed.

Fig. 7 shows the results of this test. As can be seen from fig. 7, M222 can be grown on Ashby solid medium without N element, thus indicating that M222 has nitrogen fixation capacity. That is, the M222 strain can supplement nutrition to crops and promote the growth of the crops.

Example 10 phosphorus dissolution qualitative test of M222

M222 cells on the plates were scraped off with an appropriate amount of sterile inoculating loop, resuspended in 1mL of sterile water, and inoculated evenly onto plates of organophosphorus bacteria medium (glucose 10.0g, ammonium sulfate 0.5g, yeast extract 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, lecithin 0.2g, calcium carbonate 1.0g, agar 15.0g, water 1L, pH=7.0-7.5), each plate was repeated 3 times. After dark culture at 28 ℃ for 4-6 days, whether obvious phosphorus dissolving rings appear around the colony or not is observed.

Fig. 8 shows the results of this test. As can be seen from FIG. 8, a transparent phosphate ring appeared around the M222 colony, indicating its ability to dissolve organic phosphorus. The phosphate solubilizing circle diameter and colony diameter were then measured, and the phosphate solubilizing index was calculated according to the following formula:

phosphate solubilizing index = phosphate solubilizing circle diameter/colony diameter

The phosphorus dissolution index of M222 was calculated to be 2.84.

In addition, the M222 of the invention also belongs to phosphate-solubilizing bacteria, and the utilization experiment result of organic phosphorus shows that the phosphate-solubilizing bacteria can decompose solid phosphorus which is difficult to be absorbed and utilized by crops in soil, including organic phosphorus and inorganic phosphorus, and convert the solid phosphorus into a soluble phosphate form, thereby being beneficial to improving the effective utilization rate of phosphorus and promoting the growth of plants.

EXAMPLE 11 growth promoting effect of M222

1. Cultivation and treatment of tomatoes and cucumbers

Seedling raising, namely planting 2 tomatoes (New star 101, guangdong agricultural biotechnology) and cucumbers (Guangdong Xiu No.3, guangdong agricultural biotechnology) in each pot, wherein each pot is treated with 1 for 6 pots, and the total number of seedlings is 12, and the total number of seedlings is 36 after each treatment.

And (3) water and fertilizer management, namely pouring 0.01% NPK water-soluble fertilizer when drought occurs in the middle except for the on-time pouring microbial inoculum (M222 fermentation liquid).

The microbial inoculum treatment (M222) is that the 12-day seedlings are irrigated for the 1 st time and irrigated for the 2 nd time after 7 days.

Control (CK) Water was used as a control in this experiment.

2. Data collection

Culturing for 1 week after the 2 nd time of watering of the microbial inoculum, and collecting seedlings. Physiological data such as the spreading degree (the distance between the tips of two true leaves), the plant height (the height of the plant, namely the distance from the root neck to the growing point), the stem thickness (the stem diameter of the plant is measured by taking the position below the first true leaf) and the dry weight are selected and recorded for processing analysis. The biomass increase of 10% is taken as a growth promotion standard.

3. Test results

The test results are shown in Table 10, table 11, FIG. 9 and FIG. 10, respectively. Wherein Table 10 shows the effect of M222 broth treatment on tomato growth index, FIG. 9 shows the growth vigor of M222 broth treated potted tomatoes, table 11 shows the effect of M222 broth treatment on cucumber growth index, and FIG. 10 shows the growth vigor of M222 broth treated potted cucumbers.

TABLE 10 influence of M222 fermentation broth treatment on tomato growth index

TABLE 11 influence of M222 fermentation broth treatment on cucumber growth index

From the results shown in table 10, table 11, fig. 9 and fig. 10, it can be seen that the M222 fermentation broth has a significant growth promoting effect on both tomatoes and cucumbers. The dry weight of the single plant of the tomato treated by the M222 fermentation liquor is obviously improved by 89.23 percent compared with CK, and in addition, the dry weight of the single plant of the cucumber treated by the M222 fermentation liquor is obviously improved by 41.76 percent compared with CK.

Example 12 herbicidal Activity test of M222

Plate test:

The test used amaranth (Amaranthus retroflexus l.) as the weed to be tested.

Firstly, the amaranthus retroflexus seeds are placed under 70% ethanol solution for 3min, then washed 3 times with sterile water, then the seeds are washed 4min with 3% NaClO solution, and finally washed 3 times with sterile water. And selecting a rhizome inhibition method and carrying out weeding activity verification by combining a seed germination method. Specifically, 4mL of stock solution of M222 fermentation broth (prepared as described in example 2) and 10-fold dilution of M222 fermentation broth (M222-10X) were added dropwise to sterilized filter paper, respectively, and then 30 amaranthus retroflexus seeds were spread evenly over the sheet of filter paper with spore suspension. An equivalent amount of sterile water treatment was used as negative Control (CK) and a chemical, i.e. a 100 x dilution, was used as positive control. 3 plates were repeated for each treatment. Placed in a dark environment, cultured at 28 ℃ for 3 days, and then the inhibition rate and germination rate of M222 on the root length of amaranthus retroflexus seeds are measured. The inhibition rate was calculated as follows:

bud length inhibition ratio (%) = [ control group bud length-treatment group bud length ]/control group bud length ]

The test results are shown in table 12 below. The result shows that the stock solution of the M222 fermentation liquor can completely inhibit the germination of amaranthus retroflexus seeds, and the 10-time diluted M222 fermentation liquor also has a strong inhibition effect.

TABLE 12 inhibition of the germination of amaranthus retroflexus by M222 fermentation broth

Potting test:

in this experiment amaranthus retroflexus (Amaranthus retroflexus l.) and setaria (Sedum sarmentosum) were selected as weeds tested.

And (3) in the closed period, weighing equal amount of soil, putting the soil into each flowerpot, watering until water flows out from the bottom of the flowerpot, putting 30 weed seeds on the soil, and paving a layer of thin soil on the weed seeds (amaranthus retroflexus and green bristlegrass). The supernatant (stock solution) of the fermentation of M222 was sprayed on the soil with a watering can, using an equal amount of distilled water as negative Control (CK) and using a chemical B.sub.100Xdilution as positive control. Each treatment group was repeated three times. After placing the flowerpot in a lighting incubator (light-dark ratio=16:8) for culturing for about 7 days, the germination number of each weed seed (amaranthus retroflexus and green bristlegrass) is counted, and the germination rate is calculated according to the following formula:

Seed germination rate (%) = seed germination number/total number of test seeds×100%

The test results are shown in table 13 below. The result shows that the M222 fermentation liquor stock solution has an inhibiting effect on the germination of amaranthus retroflexus and setaria.

TABLE 13 inhibition of the germination of Amaranthus retroflexus and Setaria viridis seeds by M222 fermentation broth

	Amaranthus retroflexus germination rate/%	Setaria viridis germination rate/%
			M222-stock solution	8.34±8.34	26.67±10
Ethyl herbicide 100×	8.34±8.34	0±0
			CK	13.33±0	33.34±6.67

In seedling stage, weighing equal amount of soil, placing into each flowerpot, watering until water flows out from the bottom of the flowerpot, placing 30 weed seeds on the soil, and paving a layer of thin soil on the weed seeds (amaranthus retroflexus and green bristlegrass). When the flowerpots are moved to an outdoor environment and weeds grow to a 3-5 leaf period, M222 fermentation liquor is sprayed on the leaf surfaces of the weeds, a negative control group (CK) is sprayed with equal amount of clean water on the leaf surfaces, a chemical B.herbivore 100 multiplied by diluent is used as a positive control, and 3-pot repetition is set for each treatment. Continuously observing the leaf surface change of weeds and whether wilting and lodging occur or not in the outdoor growth period for 10-20 days, counting the plant height and the fresh weight of overground parts of each weed (amaranthus retroflexus and green bristlegrass), and calculating the plant height inhibition rate and the fresh weight inhibition rate according to the following formula:

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

Aboveground fresh weight inhibition ratio (%) = [ control group aboveground fresh weight-treatment group aboveground fresh weight ]/control group aboveground fresh weight ] ×100%

The test results are shown in table 14 below. The results show that when the M222 fermentation liquor is used for treating each weed, the leaf can be obviously changed from green to yellow in the growth process, and finally the weed is wilted and lodged, so that the plant height and fresh weight inhibition effect on green bristlegrass and amaranth are good.

TABLE 14 inhibition of weed growth by M222 fermentation broths

EXAMPLE 13 determination of the ability of M222 to produce IAA (indole-3-acetic acid)

1. Qualitative determination of IAA-producing ability:

50. Mu.L of the M222 strain fermentation broth was placed in a white background, and simultaneously 50. Mu.L (about one drop) of Spot color solution (color solution: 4.5g of FeCl ₃ was dissolved in 300mL of distilled water, then 98% H ₂SO₄ mL was slowly added, and the volume was set to 1000 mL after cooling). In addition, only 50. Mu.L of 5mg/L of plant growth hormone (3-indoleacetic acid) was added to the color solution as a control. At room temperature, the color change condition is recorded within 15min, the color of the IAA is changed into pink, the IAA can be secreted, the color is deeper, the IAA secretion capacity is stronger, and the IAA is not secreted as negative.

The results are shown in FIG. 11. As can be seen in FIG. 11, after addition of the colorimetric solution, the M222 fermentation broth was slightly discolored, indicating that it may be non-productive or slightly productive of IAA.

2. Quantitative determination of IAA-producing ability:

(1) A standard curve is prepared by using pure 3-indoleacetic acid, wherein IAA solutions with the following concentrations of 0mg/L, 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L, 30mg/L, 35mg/L, 40mg/L and 45mg/L are respectively prepared. The OD ₅₃₀ values of the respective concentrations were measured, and a standard curve was drawn.

(2) Centrifuging the M222 fermentation liquor for 10min at 10 000r/min and 4 ℃, taking 1mL of supernatant fluid, uniformly mixing with 1mL of colorimetric liquor, standing for 30min in a dark environment, and rapidly carrying out colorimetric comparison at a wavelength of 530nm by utilizing a spectrophotometer so as to detect the absorbance. And finally calculating the IAA content by using a standard curve according to the OD value.

The IAA standard curve linear equation is y=29.76703x+0.23426, the OD ₅₃₀ value is in the range of 0.07-1.50, and the measured OD ₅₃₀ value of the M222 treatment solution is 0.092, so that the IAA concentration generated after the M222 is fermented for 48 hours is 2.76mg/L. Thus, it is inferred that M222 is capable of producing IAA in small amounts, and thus is capable of promoting plant growth.

EXAMPLE 14 test of M222 against thrips

In the embodiment, the poisoning activity of the M222 strain fermentation liquor on the frankliniella occidentalis adults is determined by adopting a leaf dipping method.

Diluting the fermentation liquor of the M222 strain into 5 treatment liquids with preset concentration gradients by using sterile water, longitudinally cutting clean kidney beans (5 cm), immersing the clean kidney beans in each treatment liquid for 30 seconds, taking out the treated kidney beans, placing the treated kidney beans on sterilized filter paper, naturally airing the back face upwards, and placing the treated kidney beans in a 240mL tissue culture bottle for later use. Each mass concentration of the liquid medicine is 1 treatment, each treatment is provided with 3 times of repetition, and sterile water is used as a blank Control (CK).

15 Adult frankliniella occidentalis heads are connected into a tissue culture bottle through a soft brush, and a 200-mesh gauze is used for sealing. Placing the tissue culture bottle filled with the frankliniella occidentalis in a climatic chamber with the temperature of 25+/-1 ℃ and the illumination time of 16 hours per day for feeding, taking out after 24 hours, and observing and counting the survival condition of the frankliniella occidentalis adults treated by different reagents. When the insects are observed, the insects are lightly touched by a soft brush, and the death of thrips is judged if the insects are motionless for 2 times.

The test results are shown in table 15 below. The results show that the M222 fermentation liquor has stronger lethal effect on thrips in the treatment process of 5-7 days (d) at the concentration of 66.67 mu L/mL and above.

Table 15. Test results of m222 killing thrips

While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many modifications, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the invention. Accordingly, the present invention also encompasses any such alternatives, modifications, variations, or equivalents.

Claims

1. A burkholderia wherein the 16srRNA sequence of burkholderia has at least 99%, 99.5%, 99.8%, 99.9% or 100% identity with the sequence shown in SEQ ID No. 1;

Preferably, the Burkholderia comprises one or more strains selected from 1) a Rennokurosis (Burkholderia rinojensis) strain, 2) a strain having at least 99.8% or 99.9% identity to the 16s rRNA sequence of Rennokurosis (Burkholderia rinojensis), 3) 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 more than 100% to the genome of Rennokurosis (Burkholderia rinojensis), and/or a strain having a genome alignment score of 55% or more than 60%, 65% or more than 70%, 78% or more than 80%, 85% or more than 90%, 95% or more than 96%, 97% or more than 98% or more than 99% or less;

Preferably, the Burkholderia has the 16s rRNA sequence shown in SEQ ID NO. 1.

2. The burkholderia of claim 1, wherein the burkholderia is a burkholderia deposited with the collection of microorganism strains in Guangdong province under accession number GDMCC:62460 at 5/10 2022.

3. The burkholderia of claim 1, wherein the average nucleotide identity of the genome of the burkholderia to the genome of the strain with accession number 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 more than 100%, and/or the comparison score of the genome of the burkholderia to the genome of the strain with accession number GDMCC:62460 is more than or equal to 55%, > 60%, > 65%, > 70%, > 78%, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98% or more than 99%;

Preferably, the 16s rRNA of Burkholderia has at least 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identity with the 16s rRNA of the strain deposited under accession number GDMCC: 62460;

preferably, the 16s rRNA of Burkholderia has at least 99.9%, 99.99% or 100% identity with the 16s rRNA of the strain deposited under accession number GDMCC: 62460.

4. A composition comprising burkholderia and/or a metabolite, culture, fermentation broth or extract thereof according to any of claims 1-3.

5. The composition of claim 4, wherein the burkholderia comprises an inactivated burkholderia of any one of claims 1-3.

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

7. The composition of claim 6, wherein 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 of claim 7, wherein the biocontrol agent is selected from the group consisting of a bacterial, fungal, viral biocontrol agent, an insect biocontrol agent, a nematode biocontrol agent, or any combination thereof.

9. The composition of claim 8, wherein the biocontrol agent is selected from at least one of trichoderma harzianum (Trichoderma harzianum), rhodosporidium lilacinum (Purpureocillium lilacinum), penicillium beijerinum (Penicillium bilaiae), bacillus subtilis (Bacillus subtilis), renieratia holdele (Burkholderia rinojensis), bacillus pumilus (pumilus), bacillus belicus (Bacillus velezensis), methylobacterium roseum (Methylobacterium rhodesianum), methylobacterium torvum (Methylobacterium extorquens), and paenibacillus pierce (Paenibacillus peoriae);

Preferably, the trichoderma harzianum is trichoderma harzianum with the preservation number of CGMCC NO. 15679;

preferably, the lilyturf root is a lilyturf root with a preservation number of CGMCC NO. 12773;

Preferably, the penicillium beijerinum is penicillium beijerinum with the preservation number of CGMCC NO. 12767;

Preferably, the bacillus subtilis is a bacillus subtilis with a preservation number of CGMCC NO. 12908;

preferably, the method comprises the steps of, the RenOilybek's bacillus is RenOilybek's bacillus with the preservation number of GDMCC NO. 61156;

Preferably, the bacillus pumilus is bacillus pumilus with a deposit number of GDMCC No. 61962;

Preferably, the bacillus beleiensis is bacillus beleiensis with the preservation number of GDMCC No. 61434;

preferably, the methylobacterium rosei is methylobacterium rosei with accession number GDMCC No. 60729;

preferably, the methylobacterium torvum is methylobacterium torvum with deposit number GDMCC No. 62943;

Preferably, the paenibacillus pierce is paenibacillus pierce deposit No. GDMCC No. 60482.

10. The composition of claim 6, wherein the adjuvant is selected from the group consisting of agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film formers, spontaneous accelerators, emulsifiers, dispersants, preservatives, antifreeze agents, thickening agents, adjuvants, or any combination thereof.

11. The composition according to any of claims 4-10, wherein 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, suspensions, tablets, microparticles and wettable powders.

12. Use of burkholderia or a metabolite, culture, fermentation broth or extract thereof according to any of claims 1-3 or a composition according to any of claims 4-11 for the preparation of a microbial agent, a biofertilizer, a biopesticide or a bioherbicide.

13. Use of Burkholderia or a metabolite, culture, fermentation liquor or extract thereof according to any one of claims 1 to 3 or a composition according to any one of claims 4 to 11 for controlling plant diseases and/or herbicides, preferably selected from plant re-planting diseases, plant bacterial diseases, plant fungal diseases, plant viral diseases, plant soil-borne diseases, plant pests and/or plant oomycete diseases.

14. The use according to claim 13, wherein the plant pest is caused by one or more of a plant pathogenic bacterium, fungus, virus, insect, egg and nematode.

15. The use according to claim 13 or 14, wherein the plant disease and pest is caused by at least one pathogen selected from the group consisting of Streptomyces scab (Streptomyces scabies), laurella solani R.s (Ralstonia solanacearum), bacillus subtilis (Bacillus subtilis), actinidia canker (Pseudomonas syringae pv. Actinidiae), rhizoctonia solani (Xanthomonas campestris pv. Oryzae), rhizoctonia solani, Cabbage soft rot fungus (Erwinia aroideae), walnut black spot fungus (Xanthomonas arboricola pv. Juglandis), konjak soft rot fungus (Erwinia carotovora), staphylococcus aureus (Staphylococcus aureus), anthrax fungus (Colletotrichum capsici), rhizoctonia solani (Rhizoctonia solani), fusarium oxysporum (Fusarium oxysporum), fusarium graminearum (Fusarium graminearum), fusarium graminearum (Athelia rolfsii), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), botrytis cinerea (Botrytis cirerea), fusarium oxysporum (Fusarium oxysporum f.sp.cuumerinum), fusarium graminearum (Gaeumannomyces critici), gibberella graminearum (Fusarium graminearum), Apple tree canker (VALSAMALI), apple anthracnose (Glomerella cingulata), rhizoctonia solani (Rhizoctonia solan), pyricularia oryzae (Pyricularia grisea), tomato early blight (ALTERNARIA SOLANI), strawberry gray mold (Botrytis cirerea), potato late blight (Phytophthora infestans), corn big spot (Exserohilum turcicum), The plant species are selected from the group consisting of Sclerotinia maydis (Bipolaria maydis), sclerotinia citrulli (Fusarium oxysporum f.sp.niveum), verticillium eggplant verticillium (Verticillium dahliae), sclerotinia gossypii (Fusarium oxysporum f.sp.gasingfectum), phytophthora capsici (Phytophthora capsici), phytophthora nicotianae (Phytophthora nicotianae).

16. The use according to claim 13 or 14, wherein the plant pest is caused by nematodes or eggs, preferably the nematodes are selected from one or more of the group consisting of Meloidogyne spp and caenorhabditis elegans Caenorhabditis elegans, more preferably the Meloidogyne is selected from one or more of the group consisting of Meloidogyne incognita, meloidogyne northern (m.hapla), meloidogyne arachnoides (m.arenicia) and Meloidogyne javanica (m.java);

Or the plant disease and pest is caused by insects, preferably selected from Spodoptera frugiperda (Spodoptera frugiperda (Smith)), migratory locust (Locusta migratoria Linnaeus) and other migratory locust, meadow moth (Loxostege sticticalis Linnaeus), myxomycetes (such as Oriental myxoworm (MYTHIMNA SEPARATE (Walker)) and myxoworm (Leucania loryi Duponchel)), rice planthoppers (such as brown planthoppers (NILAPARVATA LUGENS) ) And one or more of the species sogatella furcifera (Sogatella furcifera (Horv a th))), cnaphalocrocis medinalis (Cnaphalocrocis medinalis (Guen e)), chilo suppressalis (Chilo suppressalis (Walker)), wheat aphids (such as Sitobion avena (Fabricius)), grass Gu Yiguan aphids (Rhopalosiphum padi (Linnaeus)), wheat binary aphids (Schizaphis graminum (Rondani)), asian corn borers (Ostrinia furnacalis (Guen e)), vegetable thrips (such as soybean thrips (Megalurothrips usitatus (Bagnall)), melon thrips (THRIPS PALMI KARNY), frankliniella occidentalis (FRANKLINIELLA OCCIDENTALIS (Pergande)), frankliniella occidentalis (FRANKLINIELLA INTONSA (Trybom))).

17. The use according to any one of claims 13 to 16, wherein control of the plant pest is achieved by producing siderophores, inducing systemic resistance of the plant or seed thereof to plant pathogens and/or reducing chemotaxis of plant pathogens to plants.

18. Use of burkholderia or a metabolite, culture, fermentation broth or extract thereof according to any of claims 1-3 or a composition according to any of claims 4-11 for promoting plant growth.

19. The use according to claim 18, wherein said promotion of plant growth is achieved by at least one of promoting plant root development, providing nitrogen fixation, providing phosphate solubilizing, producing indoleacetic acid, and removing weeds.

20. Use according to any one of claims 13 to 19, wherein the plant is selected from a food crop, an economic crop or a rhizome crop;

Preferably, the food crop is 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 rice, wheat, corn, barley, oat, rye, sorghum, millet, barnyard grass, buckwheat, preferably, the tuber crops include, but are not limited to, at least one of sweet potato, yam, potato, preferably, the legume crops include, but are not limited to, at least one of soybean, broad bean, pea, mung bean, peanut;

preferably, the cash crop includes, but is not limited to, at least one cash crop of the Solanaceae, rosaceae, rutaceae, musaceae, cucurbitaceae, brassicaceae, orchidaceae, papilionaceae, compositae, liliaceae, zingiberaceae, passifloraceae, pineapple, araliaceae, and Cactaceae plants;

Preferably, the Solanaceae plants include but are not limited to tomato, capsicum, potato, eggplant, etc., preferably the Rosaceae plants include but are not limited to strawberry, papaya, etc., preferably the Rutaceae plants include but are not limited to citrus, etc., preferably the Plantaceae plants include but are not limited to banana, etc., preferably the Cucurbitaceae plants include but are not limited to cucumber, wax gourd, pumpkin, balsam pear, luffa, watermelon, momordica grosvenori, etc., preferably the Brassicaceae plants include but are not limited to cabbage, rape, cabbage, radish, cauliflower, etc., preferably the Orchidaceae plants include but are not limited to orchid, etc., preferably the Papilionaceae plants include but are not limited to soybean, etc., preferably the Plantaceae plants include but are not limited to garlic, etc., preferably the Zingiberaceae plants include but are not limited to ginger, etc., preferably the Passifloraceae plants include but are not limited to passion fruit, etc., preferably the Canariaceae plants include but are not limited to pineapple, etc., preferably include but are not limited to being obtained;

21. A method for preparing the fermentation broth of burkholderia of any of claims 1-3, comprising the steps of first subjecting said burkholderia to an activation culture and then subjecting it to a fermentation culture.

22. A method of controlling plant diseases and insect pests and/or promoting plant growth, which method comprises applying to a plant or seed a burkholderia or a metabolite, culture, fermentation liquor or extract thereof according to any one of claims 1 to 3 or a composition according to any one of claims 4 to 11.