Burkholderia and application thereof

Abstract

The invention discloses a Burkholderia sp. Strain and an application of the Burkholderia sp. Strain. The Burkholderia sp. Strain is Burkholderia sp., the strain number of the Burkholderia sp. Strain is B58, and the registration number of the Burkholderia sp. Strain in the China General Microbiological Culture Collection Center is CGMCC No. 26956. The Burkholderia sp. B58 provided by the invention has a prevention and treatment effect on various pathogenic bacteria, and extracellular metabolites of the Burkholderia sp. B58 have a remarkable prevention and treatment effect on the sugarcane smut disease and have potential biocontrol application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms and plant disease biocontrol, and specifically relates to a Burkholderia strain and application thereof. Background Art

[0002] Rhizosphere microorganisms participate in the whole process of plant growth and development, and play an important role in plant nutrition absorption, disease occurrence, and interaction between plants and the environment. Therefore, they are called "the second genome of plants". Plant growth promoting rhizobacteria (PGPR) mainly improve the plant's root nutrient absorption capacity and growth through biological nitrogen fixation, hormone secretion, iron chelation carrier production, and phosphorus solubilization. Some bacteria such as Bacillus, Burkholderia, Pseudomonas, Flavobacterium, and Sinorhizobium have obvious effects on promoting plant growth and improving plant stress resistance, and are considered to be typical representatives of rhizosphere growth promoting bacteria (PGPR).

[0003] The genus Burkholderia is a widely distributed and multifunctional group of bacteria with complex classification, and its members occupy various ecological niches including soil, water, plants and animals. It is reported that Burkholderia can colonize the roots of at least 30 plants in large numbers, and has a variety of growth-promoting functions such as nitrogen fixation, phosphorus solubilization, growth promotion, and disease treatment. For example, Burkholderia australi, which colonizes sugarcane roots, can promote the growth of sugarcane under low nitrogen conditions through biological nitrogen fixation. Inoculation of Burkholderia sp. MTCC 8369 effectively improved the absorption of phosphorus by rice, thereby promoting rice growth and increasing grain yield; Burkholderia fungorum significantly promoted the growth of kidney beans in nutrient-poor oxidized soil; Burkholderia pyrrocinia could inhibit the growth of the poplar canker pathogen Cystosporium chrysanthemi; and Burkholderia ambifaria BC-F could inhibit a variety of plant pathogenic fungi and nematodes. It can be seen that Burkholderia has great research value and application potential in promoting crop growth.

[0004] Fungal diseases are one of the main factors in ecological and food security. Continuous cropping and crop rotation of a single crop can lead to a large accumulation of pathogens. For a long time, chemical pesticides such as benomyl and carbendazim have been used in agriculture to prevent and control plant pathogenic fungi. However, continuous and long-term use of pesticides not only leads to drug resistance in pathogens, but also reduces the prevention effect year by year. Pesticide residues also bring many negative impacts to the environment and society. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a Burkholderia sp. B58 with biocontrol effect. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The invention provides a Burkholderia sp. with biocontrol effect, the strain number of which is B58 and the registration number of which in the General Microbiological Center of China Microbiological Culture Collection Committee is CGMCC No. 26956.

[0008] The present invention also provides a culture of the aforementioned Burkholderia, which is a substance obtained by culturing the aforementioned Burkholderia in a microbial culture medium.

[0009] The term "culture" refers to a general term for liquid or solid products (all substances in the culture container, i.e., fermentation products) that have a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying the microorganisms, which may be a biologically pure culture of the microorganisms, or may contain a certain amount of culture medium, metabolites, and / or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing the microorganisms, which may be a culture of a certain generation, or a mixture of several generations.

[0010] The culture of the present invention is obtained by the following method: strain B58 is cultured in LB medium at 180 rpm for 72 h until OD 600nm The value is 4, centrifuge at 12000rpm for 8-15min, take the supernatant, and sterilize it through a 0.22μm filter membrane to obtain sterile extracellular metabolites, which are the extracellular metabolites of strain B58.

[0011] The present invention also provides a bacterial agent, which contains the aforementioned Burkholderia and / or the metabolites of the aforementioned Burkholderia and / or the aforementioned culture.

[0012] In the above-mentioned bacterial agent, the bacterial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0013] The solid carrier may be a mineral material or a biological material; the mineral material may be at least one of peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the biological material may be at least one of various crop straws, pine husks, straw, peanut shells, corn flour, soybean flour, starch, peat and animal feces; the liquid carrier may be water; in the bacterial agent, the metabolites of Burkholderia sp. may exist in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate. The formulation of the bacterial agent may be a variety of formulations, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0014] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0015] The present invention also provides a pathogen inhibitor, which contains the aforementioned Burkholderia or / and the aforementioned metabolite of Burkholderia or / and the aforementioned culture or / and the aforementioned bacterial agent.

[0016] The pathogen inhibitor inhibits at least one of Fusarium oxysporum, Fusarium graminearum, Fusarium solani, Rhizoctonia solani, Verticillium dahliae, Ustilago officinalis, endophytic spores of plants, Alternaria alternata, Botrytis cinerea, potato black mole, pear brown rot, tomato early blight and spider web pathogen.

[0017] The present invention also provides any of the following uses of the aforementioned Burkholderia, the aforementioned metabolite of Burkholderia, the aforementioned culture, the aforementioned bacterial agent, and the aforementioned pathogen inhibitor:

[0018] N1. Application in inhibiting plant pathogens;

[0019] N2. Application in the preparation of products for inhibiting plant pathogens;

[0020] N3. Application in the prevention and treatment of diseases caused by plant pathogens;

[0021] N4. Use in the preparation of products for preventing and controlling diseases caused by plant pathogens;

[0022] The plant pathogens in the aforementioned applications include at least one of Fusarium oxysporum, Fusarium graminearum, Fusarium solani, Rhizoctonia solani, Verticillium dahliae, Ustilago officinalis, Endophytic spores of plants, Alternaria alternata, Botrytis cinerea, Potato black mole, Pear brown rot, Tomato early blight and Arachnophora spp.

[0023] The oxysporum fungus mentioned above includes strawberry root rot fungus (Fusarium oxysporum f.sp.fragariae) and banana root rot fungus (Fusarium oxysporum f.sp.cubense), the fusarium solani is specifically Fusariumsolani, the solani is specifically Rhizoctonia solani, the dahliae verticillium is specifically Verticillium dahliae, the sugarcane smut fungus is specifically Sporisorium scitamineum, the plant endophytic thorn disc spore is specifically Colletotrichum endophyticum, the chain spore is specifically Alternaria alternata, the grape seat cavity fungus is specifically Botryosphaeria dothidea, the gray grape spore is specifically Botrytiscinerea, the potato black spot fungus is specifically Rhizoctonia solani, and the pear brown rot fungus is specifically pear brown rot fungus-fruit chain sclerotinia Monilinia fructigena, the tomato early blight pathogen is specifically the tomato early blight pathogen - Alternaria solani, and the spider web pathogen is specifically the spider web pathogen - fungus parasitic Hypomyces amaniticola.

[0024] The disease in the aforementioned application is sugarcane smut.

[0025] The present invention also provides a microecological preparation containing the aforementioned bacterial agent, wherein the microecological preparation contains the aforementioned Burkholderia or the aforementioned bacterial agent.

[0026] The present invention provides a strain of Burkholderia B58 isolated from rhizosphere soil of Curcuma zedoaria, which has good inhibitory application in plant resistance to fungal diseases. Potted plant experiments show that the Burkholderia B58 provided by the present invention has a 95% control effect on sugarcane smut caused by Sporisorium scitamineum. It can be seen that Burkholderia B58 has a high application value in disease control and has the potential to be developed and applied as a new type of microbial strain resource.

[0027] Collection Instructions

[0028] Species name: Burkholderia

[0029] Latin name: Burkholderia sp.

[0030] Strain ID: B58

[0031] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0032] Abbreviation of depository institution: CGMCC

[0033] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0034] Deposit date: March 28, 2023

[0035] The registration number of the Collection Center is: CGMCC No.26956. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The morphological characteristics of the strain B58 provided by the present invention, wherein Figure A is the colony characteristics of B58 on the LB plate, and Figure B is the scanning electron microscopy characteristics of B58;

[0037] Figure 2 The phylogenetic tree of the genome of strain B58 and bacteria of the same genus provided by the present invention;

[0038] Figure 3 The genomic characteristics of the strain B58 provided by the present invention;

[0039] Figure 4Inhibitory effect of Burkholderia sp. B58 on plant pathogens. A. Fusarium oxysporum f.sp.fragariae, B. Fusarium oxysporum f.sp.cubense, C. Fusarium graminearum, D. Fusarium solani, E. Fusarium incarnatum-equiseti, F. Rhizoctonia solani, G. Verticillium dahliae, H. Sporisorium scitamineum, I. Colletotrichum endophyticum, J. Alternaria alternata, K. Botryosphaeria dothidea, L. Botrytis cinerea, M. Rhizoctonia solani, G. Verticillium dahliae, H. Sporisorium scitamineum, I. Colletotrichum endophyticum, J. Alternaria alternata, K. Botryosphaeria dothidea, L. Botrytis cinerea, M. solani), N. pear brown rot fungus-fruit chain sclerotinia (Monilinia fructigena), O. tomato early blight fungus-Solanum chain spore (Alternaria solani), P. spider web fungus-fungus parasitic (Hypomyces amaniticola).

[0040] Figure 5The inhibitory effect of Burkholderia sp. B58 fermentation broth on plant pathogens. A. Fusarium oxysporum f.sp.fragariae, B. Fusariumoxysporum f.sp.cubense, C. Fusarium graminearum, D. Fusarium solani, E. Fusarium incarnatum-equiseti, F. Rhizoctonia solani, G. Verticillium dahliae, H. Sporisorium scitamineum, I. Colletotrichum endophyticum, J. Alternaria alternata, K. Botryosphaeria dothidea, L. Botrytis cinerea, M. Rhizoctonia solani, G. Verticillium dahliae, H. Sporisorium scitamineum, I. Colletotrichum endophyticum, J. Alternaria alternata, K. Botryosphaeria dothidea, L. Botrytis cinerea, M. solani), N. pear brown rot fungus-fruit chain sclerotinia (Monilinia fructigena), O. tomato early blight fungus-Alternaria solani, P. spider web fungus-fungus parasitic (Hypomyces amaniticola).

[0041] Figure 6 The control effect of Burkholderia sp. B58 on sugarcane smut. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0043] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0044] Strawberry root rot fungus (Fusarium oxysporum f.sp.fragariae); recorded in the document "Yan Chen, Yongping Xu, Tong Zhou, Mahinur S.Akkaya. Lili Wang, Shuying Li, Xiaoyu Li. Biocontrol of Fusarium wilt disease in strawberries using bioorganicfertilizer fortifed with Bacillus licheniformis X-1 and Bacillusmethylotrophicus Z-1.3 Biotech. 2020Feb; 10(2): 80..", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0045] Banana root rot fungus (Fusarium oxysporum f.sp.cubense); recorded in the document "Tingting Bai, Shengtao Xu, Franziska Rupp, Huacai Fan, Kesuo Yin, Zhixiang Guo, Lei Zhang, Baoming Yang, Yuling Huang, Yongping Li, Xundong Li, Li Zeng & Si-Jun Zheng. Temporal variations of Fusarium oxysporum f.sp.cubense tropical race 4 population in a heavily infected banana field in Southwest China. ACTAAGRICULTURAE SCANDINAVICA. 2019, VOL. 69, NO. 7, 641-648.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0046] Fusarium graminearum; recorded in the document "Ming Xu, Qinhu Wang, Guanghui Wang, Xue Zhang, Huiquan Liu and Cong Jiang. Combatting Fusarium headblight: advances in molecular interactions between Fusarium graminearum and wheat. Phytopathology Research (2022) 4: 37.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0047] Fusarium solani: recorded in the document "Chen Sijie, Du Juan, Zhang Tao, Gu Peiwen. Research on the pathogen of wolfberry root rot in Ningxia. Agricultural Science Research [J], 2021(42):7-10.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0048] Fusarium incarnatum-equiseti: recorded in the document "MM Wang, Q Chen, YZ Diao, WJ Duan, L Cai. Fusarium incarnatum-equiseti complex from China. Persoonia. 2019; 43: 70-89", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0049] Rhizoctonia solani; recorded in the document "Qun Yang, Lei Yang, Yin Wang, Yilu Chen, Keming Hu, Wei Yang, Shimin Zuo, Jiandi Xu, Zhensheng Kang, Xueqiong Xiao, and Guotian Li. A High-Quality Genome of Rhizoctonia solani, a Devastating Fungal Pathogen with a Wide Host Range. Mol Plant Microbe Interact. 2022 Oct; 35(10): 954-958.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0050] Verticillium dahliae; recorded in the document "ZHANG Yalin, ZHOU Jinglong, ZHAO Lihong, FENG Zili, WEI Feng, BAI Hongyan, FENG Hongjie and ZHU Heqin. A review of the pathogenicity mechanism of Verticillium dahliae incotton. Journal of Cotton Research, (2022) 5:3", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0051] Sugarcane smut fungus (Sporisorium scitamineum); recorded in the document "Kumaravel Nalayeni, NMR Ashwin, Leonard Barnabas, Thiyagarajan Vinodhini, VNAgisha, AmalrajRamesh Sundar1, Palaniyandi Malathi, Rasappa Viswanathan. Comparative expression analysis of potential pathogenicity-associated genes of high-and low-virulent Sporisorium scitamineum isolates during interaction with sugarcane. 3Biotech. 2021 Jul; 11(7): 353.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0052] Colletotrichum endophyticum; recorded in the document "Liwei Liang, Shujiang Li, Shan Han, Tianmin Qiao, Shuying Li, Tianhui Zhu, First Report of Colletotrichum endophyticum, a Causal Agent of Leaf Spot of Bauhinia blakeana in Southwest China. Plant Dis. 2022 Aug 10.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0053] Alternaria alternata; recorded in the document "Kaoru Ito, Takayoshi Tanaka, Rieko Hatta, Mikihiro Yamamoto, Kazuya Akimitsu and Takashi Tsuge. Dissection of the host range of the fungal plant pathogen Alternaria alternata by modification of secondary metabolism. Molecular Microbiology (2004) 52 (2), 399-411.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0054] Botryosphaeria dothidea; recorded in the document "Yuxin Rao, Li Mei, Liqin Zhang, Hong Jiang, Liangjin Ma, Yongjun Wang. Genome Sequence Resource of Botryosphaeria dothidea CK16, a Fungal Pathogen Causing Chinese Hickory TrunkCanker Disease. Plant Dis. 2021 Oct; 105(10): 3282-3284.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0055] Botrytis cinerea; recorded in the document “BRIAN WILLIAMSON, BETTINATUDZYNSKI, PAUL TUDZYNSKI AND JAN ALVANKAN., Botrytis cinerea: the cause of grey mould disease. MOLECULAR PLANT PATHOLOGY (2007) 8(5), 561–580.”, which is publicly available from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0056] Potato black blight pathogen (Rhizoctonia solani); recorded in the document "Manoranjan Senapati, Ajit Tiwari, Neha Sharma, Priya Chandra, Bishnu Maya Bashyal, Ranjith KumarEllur, Prolay Kumar Bhowmick, Haritha Bollinedi, KK Vinod, Ashok Kumar Singh, SGopala Krishnan. Rhizoctonia solani Kühn Pathophysiology: Status and Prospects of Sheath Blight Disease Management in Rice. Front Plant Sci. 2022 May 3; 13: 881116.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0057] Pear brown rot fungus - Monilinia fructigena; recorded in the document "Lucia Landi, Rita M De Miccolis Angelini, Stefania Pollastro, Domenico Abate, Francesco Faretra, Gianfranco Romanazzi. Genome sequence of the brown rot fungal pathogen Monilinia fructigena. BMC Res Notes. 2018 Oct 23; 11 (1): 758.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0058] Tomato early blight pathogen - Alternaria solani; recorded in the document "Radha Sivarajan Sajeevan, Ingi Abdelmeguid, Ganapathi Varma Saripella, Marit Lenman1 and Erik Alexandersson. Comprehensive transcriptome analysis of different potato cultivars provides insight into early blight disease caused by Alternaria solani. BMC Plant Biol. 2023 Mar 8; 23(1): 130.", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0059] Spider web pathogen - fungal parasite (Hypomyces amaniticola): recorded in the document "Zeng Zhaoqing, Zhuang Wenying. Three new species of the genus Hypomyces and two new species recorded in China, Journal of Mycology 2016, 35(9): 1048-1055. State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0060] Example 1. Isolation, screening and identification of Burkholderia sp.

[0061] 1. Strain Isolation and Screening

[0062] Rhizosphere soil samples of the medicinal plant Curcuma zedoaria were collected from the Guangxi Botanical Garden in Nanning, Guangxi Zhuang Autonomous Region, China, and separated as follows:

[0063] Weigh 5 g of rhizosphere soil and dissolve it in 45 mL of sterile water. Shake it in a constant temperature shaker at 28°C for 30 min to mix it thoroughly. -1 Up to 10 -5 Perform gradient dilutions, pipette 100 μL of each dilution to a concentration of 10 -3 , 10 -4 , 10 -5The suspension was coated on LB plates, and 3 plates were coated for each concentration. After constant temperature culture at 28°C for 24 hours, single colonies with different morphologies were picked and streaked on sterile LB plates for purification until single colonies with the same morphology grew. The newly obtained single colony strain was further purified by streaking to obtain a pure culture strain, which was named strain B58.

[0064] 2. Strain Identification

[0065] 1. Morphological identification of strains

[0066] The morphology and characteristics of the bacteria were observed. The results showed that strain B58 could form colonies of 1 to 2 mm after culturing on LB medium for 24 hours. The colonies had no protrusions in the middle, were cream yellow, smooth and shiny, had neat edges, were opaque, and did not produce fluorescence ( Figure 1 Scanning electron microscopy showed that B58 bacteria were short rod-shaped and had no flagella ( Figure 1 (shown in B).

[0067] 2. Physiological and chemical classification and identification

[0068] Physiological and biochemical tests were performed on strain B58 with reference to the Bergey Bacteria Identification Manual and the Common Bacteria System Identification Manual. The results showed that strain B58 could utilize most monosaccharides, but could not utilize carbon sources such as d-maltose, dextrin, and d-cellobiose as described in Table 1, could grow at pH 5, and could grow in NaCl greater than 8%. Combined with the strain morphology and staining results, it was basically consistent with the characteristics of the genus Burkholderia (Table 1).

[0069] Table 1. Physiological and biochemical test results of strain B58 Biolog GEN III

[0070]

[0071]

[0072] Note: “+” means positive, “-” means negative.

[0073] 3. Whole genome sequencing of strain B58

[0074] The total DNA of strain B58 was extracted and purified. The genome of strain B58 was sequenced and spliced ​​using the Pacbio third-generation sequencing platform. The genome was assembled using the HGAP4 analysis process provided by the Pacbio official analysis software SMRT Link (V6.0.0.47841). The basic characteristics of the genome were analyzed using RAST. The genome size of strain B58 is 7.78M, the GC content is 66.4, and it contains 3 chromosomes, of which chromosome 1, chromosome 2, and chromosome 3 are 3734459bp, 3110240bp, and 1058508bp, respectively (without plasmid). 8015 genes were predicted, and their basic characteristics are as follows: Figure 3 shown.

[0075] The genome sequences of the type strains of Burkholderia species were downloaded from the GenBank database, and the phylogenetic tree of Burkholderia B58 and its congeners was constructed using UBCG. The phylogenetic tree is shown in Figure 2 , strain B58 and Burkholderia pyrrocinia DSM10685 T The clusters were in the same branch and identified as Burkholderia sp. In the figure, "Burkholderia sp B58" represents strain B58.

[0076] In view of the above morphological characteristics analysis, physiological and biochemical characteristics, and 16SrRNA gene analysis results, strain B58 was identified as Burkholderia sp. It was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC; address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postal Code: 100101) on March 28, 2023, with the deposit number CGMCC No. 26956, hereinafter referred to as Burkholderia B58 or strain B58 or B58. Average Nucleotide Identity (ANI) analysis showed that its similarity with Burkholderia pyrrocinia was 94.9% (less than the new species identification value of 95%), and DNA relatedness (dDDH) analysis showed that its correlation with Burkholderia pyrrocinia was 64.1% (less than the new species identification value of 70%), indicating that strain B58 and Burkholderia pyrrocinia are different species and are a new species of the genus Burkholderia.

[0077] Example 2, Determination of the antibacterial spectrum of Burkholderia B58

[0078] A single colony of Burkholderia B58 was selected and inoculated into LB medium. The culture was shaken at 28°C and 180 rpm for 48 h. The bacterial solution was adjusted to a concentration of 1×10 8 cfu·mL -1 , the Burkholderia B58 content was 1×10 8 cfu·mL -1 The culture fluid (bacterial suspension) of strain B58.

[0079] 1. Inhibitory effect of Burkholderia sp. B58 on plant pathogens

[0080] Pipette 10 μL of a 1×10 8 cfu·mL -1 The culture solution of strain B58 was inoculated on the PDA medium at a point 2 cm away from the edge of the culture dish, and the pathogenic fungi to be tested were inoculated on the extension line 2 cm away from the edge of the culture dish for confrontation culture. Each treatment was repeated three times, and then the culture dish was placed in an incubator at 25°C for 3-5 days to observe the antibacterial situation and measure the distance from the inhibited point to the B58 inoculation point. The greater the distance between the inhibition point and the B58 inoculation point, the stronger the inhibition ability. The pathogenic fungi to be tested were grouped as follows:

[0081] A. Strawberry root rot fungus (Fusarium oxysporum f.sp.fragariae);

[0082] B. Banana root rot pathogen (Fusarium oxysporum f.sp.cubense);

[0083] C. Fusarium graminearum;

[0084] D. Fusarium solani;

[0085] E. Fusarium incarnatum-equiseti

[0086] F. Rhizoctonia solani;

[0087] G. Verticillium dahliae;

[0088] H. Sugarcane smut fungus (Sporisorium scitamineum);

[0089] I. Colletotrichum endophyticum;

[0090] J. Alternaria alternata;

[0091] K. Botryosphaeria dothidea;

[0092] L. Botrytis cinerea;

[0093] M. Potato black spot disease (Rhizoctonia solani);

[0094] N. Pear brown rot pathogen - Monilinia fructigena;

[0095] O. Tomato early blight pathogen - Alternaria solani;

[0096] P. Arachnid pathogen-fungus parasite (Hypomyces amaniticola).

[0097] Table 2. Antibacterial effect of Burkholderia B58 on pathogenic fungi

[0098]

[0099]

[0100] The results are as follows Figure 4 The results show that after inoculation with the culture solution of strain B58, strain B58 has an effective effect on strawberry root rot pathogen (Fusarium oxysporum f.sp.fragariae) in the culture dish ( Figure 4 A) Banana root rot pathogen (Fusarium oxysporumf.sp.cubense) Figure 4 B) Fusarium graminearum Figure 4 C) Fusarium solani ( Figure 4 D), Fusarium incarnatum-equiseti ( Figure 4 E), Rhizoctonia solani ( Figure 4 F), Verticillium dahliae ( Figure 4 G), Sporisorium scitamineum ( Figure 4H), Colletotrichumendophyticum ( Figure 4 I), Alternaria alternata ( Figure 4 J), Botryosphaeria dothidea ( Figure 4 K), Botrytis cinerea ( Figure 4 L), Rhizoctonia solani ( Figure 4 M), pear brown rot pathogen - Monilinia fructigena ( Figure 4 N), tomato early blight pathogen - Alternaria solani ( Figure 4 O), spider web pathogen-fungus parasite (Hypomyces mycophilus) Figure 4 P) showed different inhibitory effects, among which strain B58 had a significant inhibitory effect on D. solani (Fusarium solani) Figure 4 D), Fusarium incarnatum-equiseti ( Figure 4 E), Rhizoctonia solani ( Figure 4 F), Sporisorium scitamineum ( Figure 4 H), Colletotrichum endophyticum ( Figure 4 I), Alternaria alternata ( Figure 4 J) and pear brown rot pathogen Monilinia fructigena ( Figure 4 N) has a strong antibacterial effect, and the antibacterial distance reaches more than 20 mm.

[0101] 2. Inhibitory effect of Burkholderia sp. B58 fermentation broth on plant pathogens

[0102] The strain B58 was cultured in LB medium with shaking at 180 rpm for 72 h until OD 600nm The value is 4, centrifuge at 12000rpm for 8-15min, take the supernatant, and sterilize it through a 0.22μm filter membrane to obtain a sterile fermentation broth, which is the extracellular metabolite of strain B58.

[0103] Use a hole puncher to make a hole with a diameter of 6mm on the PDA medium, and add 50μl of the extracellular metabolites of strain B58 to each hole. Inoculate the test pathogenic fungi on their extension line 2-3cm away from the edge of the culture dish for confrontation culture. Repeat each treatment three times, then put the culture dish into the incubator and culture at 25℃ for 3-5 days, observe the antibacterial situation, and measure the distance from the inhibition point to the B58 inoculation point. The test pathogenic fungi are grouped as follows:

[0104] A. Strawberry root rot fungus (Fusarium oxysporum f.sp.fragariae);

[0105] B. Banana root rot pathogen (Fusarium oxysporum f.sp.cubense);

[0106] C. Fusarium graminearum;

[0107] D. Fusarium solani;

[0108] E. Fusarium incarnatum-equiseti

[0109] F. Rhizoctonia solani;

[0110] G. Verticillium dahliae;

[0111] H. Sugarcane smut fungus (Sporisorium scitamineum);

[0112] I. Colletotrichum endophyticum;

[0113] J. Alternaria alternata;

[0114] K. Botryosphaeria dothidea;

[0115] L. Botrytis cinerea;

[0116] M. Potato black spot disease (Rhizoctonia solani);

[0117] N pear brown rot pathogen - Monilinia fructigena;

[0118] O. Tomato early blight pathogen - Alternaria solani;

[0119] P. Arachnid pathogen-fungus parasite (Hypomyces amaniticola).

[0120] Table 3. Antibacterial effect of extracellular metabolites of Burkholderia B58 on pathogenic fungi

[0121]

[0122]

[0123] The results are as follows Figure 5 As shown, after inoculation with the extracellular metabolites of strain B58, the extracellular metabolites of strain B58 had an effect on the strawberry root rot pathogen (Fusarium oxysporum f.sp.fragariae) in the culture dish ( Figure 5 A), banana root rot fungus (Fusarium oxysporum f.sp.cubense) ( Figure 5 Middle B), Fusarium graminearum ( Figure 5 C), Fusarium solani ( Figure 5 D), Fusarium incarnatum-equiseti ( Figure 5 Middle E), Rhizoctonia solani ( Figure 5 Middle F), Verticillium dahliae ( Figure 5 Middle G), Sporisorium scitamineum ( Figure 5 Middle H), Colletotrichum endophyticum ( Figure 5 I), Alternaria alternata ( Figure 5 Chinese J), ​​Botryosphaeria dothidea ( Figure 5 Middle K), Botrytis cinerea ( Figure 5 Middle L), potato black mole pathogen (Rhizoctonia solani) ( Figure 5 M), pear brown rot pathogen - Monilinia fructigena ( Figure 5N), tomato early blight pathogen - Alternaria solani ( Figure 5 O), spider web pathogen-fungus parasite (Hypomyces mycophilus) ( Figure 5 The extracellular metabolites of strain B58 showed different inhibitory effects on Fusarium incarnatum-equiseti ( Figure 5 It has a good inhibitory effect on all tested pathogens except E).

[0124] Example 3: Effect of Controlling Sugarcane Smut

[0125] Preparation of B58 inoculum: Inoculate strain B58 into a 250 mL Erlenmeyer flask containing 100 mL LB liquid medium and culture at 28 °C and 180 rpm for 48 h until OD 600nm The value is 3, and then the bacterial solution is sterilized by filtering it through a 0.22μm filter membrane for later use.

[0126] Sugarcane seedlings: Sugarcane seedlings (Guangxi black sugarcane) were soaked in 0.5% potassium permanganate for 30 min, rinsed with sterile water several times, and then sown in nutrient pots (15 cm × 13 cm) containing sterile culture matrix (peat soil: vermiculite: fine sand = 2:1:1, volume ratio). Two seedlings were inoculated in each pot, covered with 2 cm thick sterile soil, and then watered and placed in a greenhouse for cultivation. After the seedlings emerged from the soil, the seedlings were fixed to 1 plant per pot. Routine daily care was carried out, watering once every 2 days (100 mL / pot), and Hoagland nutrient solution (100 mL / pot) was poured once a week (purchased from Beijing Wokai Biotechnology Co., Ltd., item number GCCM0544-5L).

[0127] Sugarcane smut MAT-1 mating type strain U1 and sugarcane smut MAT-2 mating type strain U2: recorded in the document "Meixin Yan, Guining Zhu, Shanyu Lin, Xiaoyong Xian, Changqing Chang, Pinggen Xi, Wankuan Shen, Weihua Huang, Enping Cai, Zide Jiang, Yi Zhen Deng, Lian-Hui Zhang, The mating-type locus b of the sugarcane smut Sporisorium scitamineum is essential for mating, filamentous growth and pathogenicity, Fungal Genetics and Biology, Volume 86, 2016, Pages 1-8", the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0128] Preparation of sugarcane smut fungus liquid: Sporisorium scitamineum different mating type strains U1 and U2 were inoculated into 250 mL of PDA medium, respectively, and cultured at 25 ° C and 150 r / min for 2 days to obtain sugarcane smut fungus mating type strain U1 liquid and sugarcane smut fungus mating type strain U2 liquid. The concentration of sugarcane smut fungus mating type strain U1 liquid and sugarcane smut fungus mating type strain U2 liquid was adjusted to 1×10 7 After CFU, the mixture was mixed in a 1:1 ratio to obtain the smut fungus culture. PDA medium: 200.0 g peeled potatoes, 20.0 g glucose, 20.0 g agar, dilute to 1000 mL with distilled water, and sterilize at 121°C for 15 min.

[0129] Ustilago + B58 group: The sugarcane smut fungus solution and B58 inoculant were injected into the stems of sugarcane at the 3-4 leaf stage using a sterile syringe, and then cultured until the sugarcane seedlings in the control group became diseased, and the disease of the sugarcane seedlings in this group was observed.

[0130] Ustilago group: The difference between this group and the Ustilago + B58 group is that sterile water was used instead of B58 inoculation agent, and the rest of the operations were the same as those of the Ustilago + B58 group.

[0131] CK group: The difference between this group and the Ustilago + B58 group is that sterile water was used instead of the sugarcane Ustilago liquid and the B58 inoculation agent, and the rest of the operations were the same as those of the Ustilago + B58 group.

[0132] The experiment was repeated 3 times.

[0133] Disease classification is carried out according to the "Pesticide Field Efficacy Test Guidelines" (Pesticide Testing Laboratory, Ministry of Agriculture, 1993), and the disease index and control effect are calculated. Classification standard:

[0134] Level 0: no symptoms;

[0135] Level 1: Slight lesions appear at the inoculation point and do not expand, and the lesion area accounts for less than 10% of the total leaf area;

[0136] Level 2: The lesion area accounts for more than 10% and less than or equal to 20% of the total leaf area;

[0137] Level 3: The area of ​​necrotic spots accounts for more than 20% and less than or equal to 30% of the total leaf area, and the yellowing area of ​​the inoculation point accounts for less than 50% of the total area;

[0138] Level 4: The area of ​​necrotic spots accounts for more than 30% and less than or equal to 60% of the total leaf area;

[0139] Level 5: The area of ​​necrotic spots accounts for more than 60% of the total leaf area.

[0140] Disease index = ∑ (number of diseased leaves at each level × disease level) / (total number of leaves surveyed × highest disease level) × 100.

[0141] Control effect = (control disease index - treatment disease index) / control disease index × 100%.

[0142] The results are as follows Figure 6 As shown in the figure, 30 days after inoculation with the sugarcane smut fungus solution, there was no significant difference in the growth status of the sugarcane seedlings in the smut fungus + B58 group and the control group, indicating that the Burkholderia sp. B58 filtrate, i.e. the extracellular metabolites of strain B58, can effectively control the occurrence of sugarcane smut, while the sugarcane seedlings inoculated with smut fungus alone (CK group) died.

[0143] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Burkholderia, characterized in that The Burkholderia is Burkholderia sp., the strain number of which is B58, and the registration number of which in the General Microbiological Center of China National Microbiological Culture Collection is CGMCC No.26956.

2. The culture of Burkholderia according to claim 1, which is obtained by culturing the Burkholderia according to claim 1 in a microbial culture medium.

3. A bacterial agent, characterized in that The bacterial agent contains the Burkholderia according to claim 1 or / and the metabolite of the Burkholderia according to claim 1 or / and the culture according to claim 2.

4. A pathogen inhibitor, characterized in that: The pathogen inhibitor contains the Burkholderia according to claim 1 or / and the metabolite of the Burkholderia according to claim 1 or / and the culture according to claim 2 or / and the bacterial agent according to claim 3.

5. The pathogen inhibitor according to claim 4, characterized in that The pathogen inhibitor inhibits at least one of Fusarium oxysporum, Fusarium graminearum, Fusarium solani, Rhizoctonia solani, Verticillium dahliae, Ustilago officinalis, endophytic spores of plants, Alternaria alternata, Botrytis cinerea, potato black mole, pear brown rot, tomato early blight and spider web pathogen.

6. Any of the following uses of the Burkholderia according to claim 1, the metabolite of the Burkholderia according to claim 1, the culture according to claim 2, the bacterial agent according to claim 3, and the pathogen inhibitor according to claim 4 or 5: N1. Application in inhibiting plant pathogens; N2. Application in the preparation of products for inhibiting plant pathogens; N3. Application in the prevention and treatment of diseases caused by plant pathogens; N4. Use in the preparation of products for preventing and treating diseases caused by plant pathogens.

7. The use according to claim 6, characterized in that: The plant pathogenic bacteria include at least one of Fusarium oxysporum, Fusarium graminearum, Fusarium solani, Rhizoctonia solani, Verticillium dahliae, Ustilago officinalis, Endophytic thorny spores of plants, Alternaria alternata, Botrytis cinerea, Potato black mole, Pear brown rot, Tomato early blight and Cobweb pathogen.

8. The use according to claim 6 or 7, characterized in that: The disease is sugarcane smut.

9. A microecological preparation containing the bacterial agent according to claim 2, characterized in that: The microecological preparation contains the Burkholderia described in claim 1 or the bacterial agent described in claim 2.

Images