Bacillus velezensis ZF520 and control effect of bacillus velezensis ZF520 on cowpea root rot

Abstract

The invention discloses bacillus velezensis ZF520 and a control effect of the bacillus velezensis ZF520 on cowpea root rot. The invention belongs to the field of microorganisms, and particularly relates to bacillus velezensis ZF520 and a control effect of the bacillus velezensis ZF520 on cowpea root rot. The bacillus velezensis disclosed by the invention is bacillus velezensis ZF520, and the preservation number of the bacillus velezensis is CGMCC (China General Microbiological Culture Collection Center) No.31235 in the China General Microbiological Culture Collection Center. The strain ZF520 has an inhibition effect on a plurality of pathogenic fungi, has broad-spectrum bacteriostatic activity, has a cowpea root rot potting control effect of 83.59%, and has a cowpea root rot control effect of 73.99% by seed coating. The research provides an efficient and safe biocontrol resource for biological control of cowpea root rot.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to Bacillus velezensis ZF520 and its control effect on cowpea root rot. Background Art

[0002] Cowpea ( Vigna unguiculata (L.) Walp.) is a food rich in nutrition and delicious taste. Its seeds contain 24% crude protein, 53% carbohydrates, and 2% fat. The leaves and flowers can also be eaten. Cowpea is planted worldwide. Hainan is the main production area of cowpea in China. At present, the commercial planting scale of cowpea in Hainan Province is 73,400 mu, and the total output is about 165,000 tons, making it the traditional main production area of winter and spring cowpea in China.

[0003] Cowpea root rot is mainly a soil-borne disease caused by the infection of Fusarium solani f.sp. phaseoli ( Fusarium solani (Burkh.) Snyder et Hansen.). At the initial stage of the disease, the main root base is slightly browned, the lateral roots are normal, and the vascular bundles of the longitudinally dissected stem are normal. As the disease progresses, the main root base turns brown and red, the lateral roots are slightly browned, the vascular bundles of the longitudinally dissected stem are browned, then the main and lateral roots are significantly browned, the red area spreads, the lateral roots fall off, the vascular bundles of the longitudinally dissected stem are significantly browned, and finally the plant wilts and dies, or the stem base and roots are severely browned or even rotted, and the vascular bundles of the longitudinally dissected stem are severely browned.

[0004] Bacillus ( Bacillus spp.) can produce a variety of antibacterial substances to inhibit the growth of plant pathogens. Lipopeptides are secondary metabolites of Bacillus, which can inhibit the growth of fungi and bacteria, induce the systemic resistance of plants, and compete for ecological niches with plant pathogens to exhibit their biological control ability. B. velezensis There are many secondary metabolite gene clusters on the genome related to the synthesis of antibacterial compounds, and Bacillus is also the main component of most registered microbial pesticides at present. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to inhibit the invasion of cowpea root rot pathogens on crops and / or control cowpea root rot.

[0006] To solve the above technical problem, the present invention first provides a strain of Bacillus velezensis.

[0007] The Bacillus velezensis provided by the present invention is Bacillus velezensis ( Bacillus velezensis ) ZF520, and its preservation number in the China General Microbiological Culture Collection Center is CGMCC No. 31235.

[0008] The present invention also provides a bacterial agent, which contains the above-mentioned Bacillus velezensis and / or the metabolites of the above-mentioned Bacillus velezensis.

[0009] The above-mentioned bacterial agent can be a pathogen inhibitor or a disease inhibitor.

[0010] In the above-mentioned pathogen inhibitor, the bacterial agent is a pathogen inhibitor or a disease inhibitor, and the pathogen can be a pathogenic fungus or / and a pathogenic bacterium.

[0011] In the above-mentioned application, the pathogenic fungus can be Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum ), Corynespora cassiicola ( Corynespora cassiicola ), Ascochyta citrullina ( Ascochyta citrullina ), Rhizoctonia solani ( Rhizoctonia solani ), Botrytis cinerea ( Botrytis cinerea ).

[0012] In the above-mentioned application, the pathogenic bacterium can be Acidovorax citrulli ( Acidovorax citrulli ), Pectobacterium brasiliense ( Pectobacterium brasiliense ), Ralstonia solanacearum ( Ralstonia solanacearum ), Pseudomonas syringae pv. lachrymans ( Pseudomonas syringae pv. lachrymans ), Corynebacterium michiganense subsp. michiganense ( Clavibacter michiganensis subsp. michiganensis ), Xanthomonas campestris pv. campestris ( Xanthomonas campestris pv. campestris ).

[0013] The disease is cowpea root rot.

[0014] The application of the above-mentioned Bacillus velezensis in the preparation of any one of the following products 1)-3) also belongs to the protection scope of the present invention: 1) A bacterial agent for controlling cowpea root rot and / or increasing the yield of cowpea; 2) A pathogen inhibitor, the bacterial agent is a pathogen inhibitor or a disease inhibitor, and the pathogenic bacterium can be Acidovorax citrulli ( Acidovorax citrulli ), Pectobacterium brasiliense ( Pectobacterium brasiliense ), Ralstonia solanacearum ( Ralstonia solanacearum ), Pseudomonas syringae pv. lachrymans ( Pseudomonas syringae pv. lachrymans ), Corynebacterium michiganense subsp. michiganense ( Clavibacter michiganensis subsp. michiganensis ), Xanthomonas campestris pv. campestris ( Xanthomonas campestris pv. campestris); The pathogenic fungus can be Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum ), Corynespora cassiicola ( Corynespora cassiicola ), Ascochyta citrullina ( Ascochyta citrullina ), Rhizoctonia solani ( Rhizoctonia solani ), Botrytis cinerea ( Botrytis cinerea ); 3) The disease is cowpea root rot.

[0015] The active ingredient of the above microbial agent can be the above Bacillus velezensis and / or the metabolite of the above Bacillus velezensis. The active ingredient of the above microbial agent can also contain other biological components or non-biological components. Those skilled in the art can determine other active ingredients of the above microbial agent according to the inhibitory effect on the disease.

[0016] In the above text, the metabolite can be obtained from the fermentation broth of Bacillus velezensis. The metabolite can be the sterile metabolite of Bacillus velezensis or the bacteria-containing metabolite of Bacillus velezensis. The sterile metabolite (sterile fermentation filtrate) of Bacillus velezensis can be specifically prepared as follows: Bacillus velezensis is cultured in a liquid medium, and the Bacillus velezensis in the liquid culture (fermentation broth) is filtered off to obtain the sterile metabolite of Bacillus velezensis. The bacteria-containing metabolite of Bacillus velezensis can be specifically prepared as follows: Bacillus velezensis is cultured in a liquid fermentation medium, and the fermentation broth is collected, and this fermentation broth is the bacteria-containing metabolite of Bacillus velezensis.

[0017] In the above microbial agent, in addition to the active ingredient, a carrier is also contained. The carrier can be a carrier commonly used in the pesticide field and biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material can be at least one of corn flour, bean flour and starch; the polymer compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, a vegetable oil, a mineral oil or water; the organic solvent can be decane and / or dodecane.

[0018] The above microbial agent can be in various dosage forms, such as a liquid agent, an emulsion, a suspension, a powder, a granule, a wettable powder or a water dispersible granule.

[0019] According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the above microbial agent.

[0020] The present invention also provides a method for culturing the above-mentioned Bacillus velezensis, which includes the step of culturing the Bacillus velezensis in a medium for culturing bacillus.

[0021] In the above method, the medium can be LB medium.

[0022] The LB medium can be prepared from the following raw materials (1L): 10.0 g of tryptone, 5.0 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water; autoclaved at 121 °C for 20 min. Among them, the LB solid medium: add 20 g of agar to the LB liquid medium.

[0023] The present invention also provides a method for preparing the above-mentioned microbial agent, which may include the following steps: using the above-mentioned Bacillus velezensis as an active ingredient to obtain the microbial agent.

[0024] The application of the above-mentioned Bacillus velezensis or the microbial agent containing the above-mentioned Bacillus velezensis and / or the metabolite of the above-mentioned Bacillus velezensis in cultivating cowpea also belongs to the protection scope of the present invention.

[0025] The present invention also provides a biological organic fertilizer containing the above-mentioned Bacillus velezensis or a microbial agent containing the above-mentioned Bacillus velezensis and / or the metabolite of the above-mentioned Bacillus velezensis.

[0026] The present invention also provides a method for cultivating cowpea, which includes irrigating the roots of the cultivated cowpea with the above-mentioned Bacillus velezensis or the microbial agent containing the above-mentioned Bacillus velezensis and / or the metabolite of the above-mentioned Bacillus velezensis.

[0027] In the above method, the concentration of the Bacillus velezensis can be 1×10 8 cfu / mL. After diluting 1000 times, 20 ml of the bacterial liquid is applied to each plant. The Bacillus velezensis ( Bacillus velezensis ) is CGMCC No.31235.

[0028] The application of the above-mentioned Bacillus velezensis in preventing and treating cowpea root rot and / or increasing the yield of cowpea also belongs to the protection scope of the present invention.

[0029] The present invention provides Bacillus velezensis ZF520 and its application in preventing and treating cowpea root rot. It can inhibit plant pathogens, prepare products for inhibiting plant pathogens, prevent and treat diseases caused by plant pathogens, and prepare products for preventing and treating diseases caused by plant pathogens. And the control effect of the fermentation broth of this bacterium can reach 83.59%, and the control effect of seed coating can reach 73.99%, which can effectively control cowpea root rot from multiple aspects.

[0030] In this study, a strain ZF520 with good antagonistic effect against cowpea root rot pathogen was obtained from the rhizosphere soil of cowpea in Chengmai, Hainan. Its taxonomic status was determined by morphological observation, physiological and biochemical property determination, and construction of a multi-gene phylogenetic tree. Meanwhile, its biocontrol effect was determined through pot experiments and seed coating, and its antibacterial mechanism was further explored by amplifying antibacterial substance synthesis genes and measuring metabolic secretions, laying a theoretical foundation for the development of biological agents against cowpea root rot.

[0031] Deposit description Species name: Bacillus velezensis Latin name: Bacillus velezensis Strain number: ZF520 Depository institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms Abbreviation of depository institution: CGMCC Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Date of deposit: July 9, 2024 Registration number in the depository center: CGMCC No. 31235. Description of the drawings

[0032] Figure 1 Shows the inhibitory effect of biocontrol bacteria on cowpea root rot. Among them, A: ZF520; B: ZF521; C: ZF522; D: ZF523; E: ZF524; F: ZF525; G: ZF525; H: ZF526. The scale bar is 10 mm.

[0033] Figure 2 Shows the morphological characteristics observation of strain ZF520. Among them, A: colony morphology of strain ZF520; B: reverse colony morphology of strain ZF520; C: scanning electron micrograph of strain ZF520. The scale bars are 10 mm and 1.0 μm respectively.

[0034] Figure 3 Shows the phylogenetic tree of strain ZF520 based on 16S rRNA, rpo A and rho gene sequences.

[0035] Figure 4 Shows the plate confrontation results of Bacillus velezensis ZF520 against different pathogens. Among them, A, Fusarium oxysporum ( Fusarium oxysporum ); B, Ascochyta citrullina ( Ascochyta citrullina ); C, Rhizoctonia solani ( Rhizoctonia solani ); D, Fusarium solani ( Fusarium solani ); E, Corynespora cassiicola ( Corynespora cassiicola); F, Botrytis cinerea ( Botrytis cinerea ); G, Acidovorax citrulli ( Acidovorax citrulli ); H, Pectobacterium brasiliense ( Pectobacterium brasiliense ); I, Ralstonia solanacearum ( Ralstonia solanacearum ); J, Pseudomonas syringae pv. lachrymans ( Pseudomonas syringae pv. lachrymans ); K, Corynebacterium michiganense subsp. michiganense ( Clavibacter michiganensis subsp. michiganensis ); L, Xanthomonas campestris pv. campestris ( Xanthomonas campestris pv. campestris ). Where the scale bar: 10 mm.

[0036] Figure 5 For the determination of siderophore, protease and phosphate solubilizing ability. Among them, A, siderophore; B, protease; C, phosphate solubilizing ability.

[0037] Figure 6 For the PCR amplification results of antibacterial lipopeptides and resistance-related genes of strain ZF520. Among them, M: DNA Marker 5000 bp; CK: FZB42; Lanes 1-8 are respectively: sfr AA, pks 2C, fen D, dhb F, dif A, bae C, bac B, itu C gene amplification products.

[0038] Figure 7 For the control effect of strain ZF520 on living potted cowpea root rot. Among them, A, treatment with ZF520 bacterial suspension; B, treatment with ZF522 bacterial suspension; C, treatment with ZF525 bacterial suspension; D, treatment with CMZF524 bacterial suspension; E: treatment with ZF526 bacterial suspension; F, treatment with carbendazim wettable powder diluted 500 times; G, treatment with Fusarium solani; H, healthy cowpea control treatment.

[0039] Figure 8 For the control effect of ZF520 seed coating on cowpea root rot. Among them, A, treatment with ZF520 bacterial suspension; B, treatment with ZF522 bacterial suspension; C, treatment with 23% imidacloprid·fludioxonil·benzovindiflupyr suspension seed coating agent; D, treatment with 29% thiamethoxam·fludioxonil·metalaxyl-M suspension seed coating agent; E, treatment with Fusarium solani; F, healthy cowpea control treatment. Detailed implementation methods

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can serve as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0041] In the following experimental methods of the embodiments, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0042] Unless otherwise specified, in the following embodiments, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0043] Tested pathogenic bacteria Acidovorax citrulli ( Acidovorax citrulli ), Pectobacterium brasiliense ( Pectobacterium brasiliense ), Ralstonia solanacearum ( Ralstonia solanacearum ), Pseudomonas syringae pv. lachrymans ( Pseudomonas syringae pv. lachrymans ), Corynebacterium michiganense subsp. michiganense ( Clavibacter michiganensis subsp. michiganensis ), Xanthomonas campestris pv. campestris ( Xanthomonas campestris pv. campestris ); Tested pathogenic fungi Fusarium solani f. sp. cucurbitae ( Fusarium solani ), Fusarium oxysporum ( 、 oxysporum Fusarium ), Corynespora cassiicola ( 、 ), Ascochyta citrullina ( Corynespora cassiicola Ascochyta citrullina 、 ), Stemphylium solani ( Ascochyta citrullina ), Rhizoctonia solani ( 、 ), Botrytis cinerea ( Stemphylium solani ), Botrytis cinerea ( 、 ), Rhizoctonia solani ( Rhizoctonia solani ), Botrytis cinerea ( 、 ), Botrytis cinerea ( Botrytis cinerea ). The above strains were all isolated and preserved by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0044] Acidovorax citrulli ( Acidovorax citrulli ), has been recorded in "Zhao Zixuan, Li Junhui, Du Gongfu, et al. Isolation and identification of Pseudomonas protegens ZF509 and its control effect on bacterial fruit blotch of melon. Chinese Journal of Biological Control, 2024, 40(03): 690 - 700.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0045] Pectobacterium brasiliense Pectobacterium brasiliense has been recorded in "Qin Shiyang, Lu Lingchen, Xie Xuewen, et al. Isolation and identification of biocontrol bacterium ZF510 and its control effect on Chinese cabbage bacterial soft rot. Chinese Journal of Biological Control, 2024, 40(01).", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0046] Ralstonia solanacearum Ralstonia solanacearum has been recorded in " Kang Huajun, Chai Ali, Shi Yanxia, et al. Quadruplex PCR detection method for tomato bacterial speck, bacterial canker, bacterial wilt and bacterial scab pathogens. Acta Horticulturae Sinica, 2018, 45(11): 2254 - 2264.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0047] Pseudomonas syringae pv. lachrymans Pseudomonas syringae pv. lachrymans has been recorded in "Yuan Baojie, Li Lei, Zhang Hongjie, Shi Yanxia, Chai Ali, Xie Xuewen, Li Baoju. Screening of antagonistic bacteria against cucumber bacterial angular leaf spot and its control effect. Chinese Journal of Biological Control, 2022, 38(02): 421 - 427.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0048] Clavibacter michiganensis subsp. michiganensis Clavibater michiganensis subsp. michiganensis has been recorded in "Li Huanling, Shi Yanxia, Xie Xuewen, Li Baoju. Occurrence regularity and control techniques of tomato bacterial canker. China Vegetables 2011, 23: 24 - 27.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0049] Xanthomonas campestris pv. campestris Xanthomonas campestris pv. campestris has been recorded in "Zhang Ziyu, Xie Xuewen, Shi Yanxia, Chai Ali, Li Lei, Li Baoju. Isolation, identification and biocontrol effect of antagonistic bacterium Lysobacter enzymogenes CX03 against Chinese cabbage black rot. Chinese Journal of Biological Control, 2021, 37(06): 1221 - 1230.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0050] Rhizoctonia solaniRhizoctonia solani ) It has been recorded in "Li Lei, Chen Lida, Huang Yishuo, Xie Xuewen, Shi Yanxia, Chai Ali, Li Baoju. Establishment and application of a real-time fluorescence quantitative PCR detection system for Rhizoctonia solani Kühn. Journal of Agricultural Biotechnology, 2021, 29(07): 1417-1425", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0051] Botrytis cinerea Botrytis cinerea ) It has been recorded in "Shi Yanxia, Tang Ming, Jin Zhiwen, Xie Xuewen, Chai Ali, Li Baoju. Evaluation of the resistance of Botrytis cinerea on vegetable crops to different types of fungicides. China Vegetables, 2016(03): 60-65", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0052] Corynespora cassiicola Corynespora cassiicola ) It has been recorded in "Gao Wei, Li Baoju, Shi Yanxia, Xie Xuewen. Differentiation of pathogenicity of Corynespora cassiicola on cucumber, tomato and eggplant hosts. Acta Horticulturae Sinica 2011, 38(3): 465-470", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0053] Fusarium solani Fusarium solani ) It has been recorded in "Yan Wenxue, Shi Yanxia, Chai Ali, etc. Identification of the pathogen causing cowpea root rot [J]. Acta Phytophylacica Sinica, 2019, 46(03): 717-718.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0054] Fusarium oxysporum Fusarium oxysporum ) It has been recorded in "Chai Ali, Yang Hongmin, Li Xin, etc. Identification of the pathogen causing common bean fusarium wilt in Inner Mongolia [J]. Acta Phytopathologica Sinica, 2023, 53(01): 133-136.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0055] Ascochyta citrullina Ascochyta citrullina ) It has been recorded in "Zhao Yanjie, Li Baoju, Shi Yanxia, etc. Occurrence and control of gummy stem blight of cucurbits [J]. China Vegetables, 2008,(02): 56-57+70.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0056] Bacillus velezensis in the following examples ( Bacillus velezensis ) strain FZB42 has been described in "Wu L M, Wu H J, Chen L N, Yu X F, Borriss R, Gao X W. 2015. Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens. Scientific Reports, 5: 12975.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0057] Fusarium solani in the following examples ( Fusarium solani ) has been described in "Yan Wenxue, Shi Yanxia, Chai Ali, et al. Identification of the pathogen causing cowpea root rot [J]. Acta Phytophylacica Sinica, 2019, 46(03): 717 - 718.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0058] The preparation methods of the test media in the following examples are as follows: 1) LA (Luria Agar) medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 13 g of agar in 1 L of deionized water. After stirring evenly, adjust the pH to 7.0 ± 0.2 with NaOH and sterilize at 121 °C for 20 min; 2) NA (Nutrient Agar) medium: 10 g of tryptone, 3 g of beef powder, 5 g of sodium chloride, and 13 g of agar in 1 L of deionized water. After stirring evenly, adjust the pH to 7.0 ± 0.2 with NaOH and sterilize at 121 °C for 20 min; 3) PDA (Potato Dextrose Agar) medium: 200 g of potato, 20 g of D + anhydrous glucose, and 13 g of agar in 1 L of deionized water; 4) WA (Water Agar) medium: 4.5 g of agar in 1 L of deionized water; 5) PVK (inorganic phosphorus) solid medium: 0.50 g of yeast extract, 10.00 g of glucose, Ca 3 (PO 4 ) 25.00 g, (NH 4 ) 2 SO 4 0.50 g, KCl 0.20 g, MgCl 2 0.10 g, MnSO 4 0.10 mg, FeSO 4 0.10 mg, and 15 g of agar in every 1 L of deionized water; 6) Medium for protease detection: 5.00 g of tryptone, 3.00 g of yeast extract, 1.00 g of glucose, and 15 g of agar in every 1 L of deionized water; adjust the pH to about 7.0, autoclave at 121 °C for 20 min, and add 10% (v / v) skim milk when cooled to about 50 °C; 7) CAS medium: Solution a: Dissolve 15 g of agar, 9 g of glucose, 4.5 g of peptone, 2.7 g of beef extract, and 4.5 g of NaCl in 1 L of sterile water; Solution b: 1 mmol / L FeCl3·6H 2 O, 10 mmol / L HCl; Dissolve 121 mg of chrome azurol in 50 mL of Solution b, mix well, add 145.8 mg of HDTMA, adjust the pH to 7.0, make up the volume to 100 ml, and finally sterilize and mix with 900 mL of Solution a.

[0059] The cowpea variety "Yujing" (variety registration number: D (Jinghai) Nongzhongxuzi (2019) No. 0010) in the following examples was purchased from Zhongshu Seed Industry Technology (Beijing) Co., Ltd.

[0060] The 50% carbendazim wettable powder in the following examples was purchased from Sichuan Guoguang Agrochemical Co., Ltd. (registration number: PD85150-35), and the 23% imidacloprid·fludioxonil·benzovindiflupyr suspension seed coating agent (registration number: PD20183730) and the 29% thiamethoxam·fludioxonil·metalaxyl-M suspension seed coating agent (registration number: PD20183904) were both purchased from Dezhou Xianglong Biochemical Co., Ltd.

[0061] The following examples used SPSS 11.5 statistical software to process the data. The experimental results were expressed as mean ± standard deviation, and One-way ANOVA test was used. P < 0.05 (*) indicated significant difference, P < 0.01 (**) indicated extremely significant difference, and P < 0.001 (***) indicated extremely significant difference.

[0062] Example 1: Isolation, Screening and Preservation of Strains 1. Isolation of Strains Collect the rhizosphere soil of healthy plants from the areas with frequent occurrence of cowpea root rot in Chengmai, Hainan. Mix the soil evenly to ensure no lumps, weigh 10 g of fresh soil into an Erlenmeyer flask, add 90 ml of sterile water, and shake well at 28 °C and 180 r / min for 45 min to form a uniform soil suspension. Use the gradient dilution method to isolate antagonistic bacteria. Take the dilution solutions of four gradients of 10 3 、10 4 、10 5 、10 6 respectively, spread them evenly on NA medium, repeat each gradient three times, invert and place them in an incubator at 28 °C for 48 h. Use a sterile inoculation loop to pick different-shaped single bacterial colonies for multiple purifications. After forming stable single colonies, store them for later use. A total of 385 bacterial strains were isolated and purified.

[0063] 2. Screening of antagonistic strains against cowpea root rot Using Fusarium solani ( Fusarium solani )as the target strain, the plate confrontation method was used to screen for antagonistic bacteria.

[0064] First, activate the fungus. Use a sterilized and cooled borer to punch holes on the plate covered with Fusarium solani mycelium, pick up the agar disc with a toothpick and place it on a new PDA plate, with the mycelium side facing up, and incubate it upside down at 28 °C. For the bacterial strains isolated and purified from the soil sample in step 1, use a sterile inoculation loop to pick a single colony into 3 ml of liquid LB medium, and place it in a constant temperature shaker at 28 °C for 36 h. Use a 60 mm borer to punch an agar disc on the cultured fungal medium, pick up the agar disc with a toothpick and place it in the center of a new PDA plate. Use forceps to pick up filter paper and place it on the upper, lower, left, and right sides of the plate edge, and absorb 5 μL of the bacterial suspension of different antagonistic strains and drop it on the filter paper. After the bacterial liquid is completely dry, incubate it upside down at 28 °C until the mycelium of the control group covers the plate and then investigate. Each treatment is repeated 3 times. Measure the diameter of the fungus in each repeat, and calculate the average value of the three repeats. Use the inhibition rate to represent the inhibitory ability of the screened strains against the cowpea root rot pathogen.

[0065] Inhibition rate = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0066] Through the plate confrontation method, 8 strains with obvious antagonistic effects against the cowpea root rot pathogen were screened ( Figure 1 A - H in it). Among them, strain ZF520 has good inhibitory effects on Fusarium solani, and the inhibition rate can reach more than 65% in both cases. The inhibition rates of strains ZF521, ZF522, ZF523, ZF524, and ZF525 against Fusarium solani are all above 60.00%. The inhibition rate of strain ZF526 against the cowpea root rot pathogen is the smallest, but it can reach more than 50%.

[0067] 3. Identification of Antagonistic Bacterium ZF520 1) Identification of the Morphological Characteristics of Strain ZF520 The antagonistic strain ZF520 was cultured on LB solid medium. After 36 h of incubation, the colony morphology was observed. The methods described in "Common Bacterial Systematic Identification Booklet" (Dong Xiuzhu et al., 2001) and "Bergey's Manual of Determinative Bacteriology" (Buchanan et al., 1984) were referred to. For the microscopic morphological observation of the bacteria, the samples were fixed with 2.5% glutaraldehyde, dehydrated 7 times with 20%-100% ethanol for 10 min each time, and then observed under a scanning electron microscope after the dehydration treatment of the samples was completed.

[0068] The results are as Figure 2 shown in A - C in the figure: The colonies of strain ZF520 on the LB plate were milky white, elliptical or nearly circular, with a moist and smooth surface. In the later stage of growth, the colonies turned light yellow, the surface of the bacteria became relatively dry, obvious wrinkles formed, and the inside of the bacteria remained moist ( Figure 2 A and B in the figure); Under the scanning electron microscope, the bacteria were rod-shaped, with peritrichous flagella, and the surface of the bacteria had wrinkles, with a size of (1.5 - 2.0) μm × (0.5 - 1.2) μm ( Figure 2 C in the figure).

[0069] 2) Physiological and Biochemical Determination of Strain ZF520 According to the eighth edition of "Bergey's Manual of Determinative Bacteriology" and "Common Bacterial Systematic Identification Manual", the physiological and biochemical characteristics of strain ZF520 were determined. The China Center for Agricultural Culture Collection of Microorganisms used the BIOLOG GENⅢ kit to determine the utilization of the sole carbon source by strain ZF520.

[0070] The results of the physiological and biochemical characteristics determination of strain ZF520 showed that strain ZF520 was a Gram-positive bacterium, suitable for growth under the condition of pH 6, and could grow with the NaCl content ranging from 1% to 8%. Strain ZF520 could utilize most sugars such as D-lactose, D-maltose, D-cellobiose, D-glucose, D-trehalose, dextran, D-mannose, gentiobiose, sucrose, etc., and could not utilize carbon sources such as D-trehalose, D-aspartic acid, D-arabitol, N-Acetyl-D-galactosamine, N-Acetyl mannosamine pyruvate, 3-Methyl glucose, D-serine, etc. The result of the gelatin liquefaction test was negative, and the results of the pyruvate and citrate utilization tests were positive (Table 1).

[0071] Table 1. Physiological and Biochemical Reactions of Strain ZF520

[0072] Note: +: Positive reaction or can grow; -: Negative reaction or cannot grow; W: Weak positive reaction or can grow 4. Multigene Amplification and Sequence Analysis of Strain ZF520 The genomic DNA of strain ZF520 was extracted using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing). Three groups of genes, 16S rRNA and rpo A, rho were amplified. For 16S rRNA, universal primers 27F / 1492R were used, and for the target genes rpo A, rho the amplification primers: 72F / 328R, OF / OR (Table 3) were used to specifically amplify strain ZF520.

[0073] The total volume of the amplification reaction system was 50 μL. The reaction conditions were: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, with 34 cycles, and sufficient extension at 72°C for 10 min. The reaction products were sent to Beijing Bomed Biotechnology Co., Ltd. for sequencing.

[0074] The 16S rRNA and rpo A 、rho genes of strain ZF520 were compared with those of strains in the NCBI database. Software such as MEGA 7.0 was used to analyze the sequencing results. Strain ZF520 has a 16S rRNA with a nucleotide sequence that is sequence 1 in the sequence listing, a rho gene with a nucleotide sequence that is sequence 2 in the sequence listing, and a rpo A gene with a nucleotide sequence that is sequence 3 in the sequence listing.

[0075] A phylogenetic tree was constructed to analyze the genetic relationship of strain ZF520. The multi-gene phylogenetic tree constructed with the 16S rRNA and rpo A, rho gene sequences showed that strain ZF520 and Bacillus velezensis ( Bacillus velezensis ) FZB42 clustered in the same branch ( Figure 3 ).

[0076] In view of the above morphological characteristic analysis, physiological and biochemical indexes, BIOLOG sole carbon source utilization characteristics, 16S rRNA and rpo A, rho gene sequence analysis results, strain ZF520 was identified as Bacillus velezensis ( Bacillus velezensis ).

[0077] 5. Preservation of Strain ZF520 Bacillus velezensis ZF520 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 9, 2024, with the deposit number CGMCC No. 31235. Hereinafter, it is referred to as Bacillus velezensis CGMCC No. 31235 or strain ZF520.

[0078] Example 2: Determination of the antibacterial spectrum of strain ZF520 1. Determination of the antifungal spectrum of strain ZF520 The inhibitory effect of strain ZF520 on pathogenic fungi was determined by the plate confrontation method, and the method was the same as that for screening the cowpea root rot antagonistic strain in Example 1. After the control mycelium covered the plate, the investigation was carried out, the colony diameter was measured, and the inhibition rate was calculated.

[0079] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100 The results are as Figure 4 shown in A-L and Table 2: The fermentation broth of strain ZF520 of Bacillus velezensis has a good plate inhibitory effect on 6 pathogenic fungi, and the inhibition rate of most fungi can reach more than 50%, among which the inhibition rate on Fusarium solani Fusarium solani ), Botrytis cinerea Botrytis cinerea ), Rhizoctonia solani Rhizoctonia solani can all reach more than 70%.

[0080] 2. Determination of the antibacterial spectrum of strain ZF520 The double-layer culture method was adopted. Take 5 μL of the bacterial suspension of the strain to be screened and drop it onto the center of the LA medium. After natural drying, it was cultured in an incubator at 28 °C for 36 h. Then it was inverted and placed in a fume hood. 3 mL of chloroform was dropped along the lid of the glass petri dish, and after overnight fumigation, the second layer of bacterial liquid was inoculated. Take 150 μL of the pathogenic bacteria suspension with OD 600 = 1.0, add it to 5 mL of WA semi-solid medium at about 50 °C, shake well and pour it onto the upper layer of the LA medium, and culture it in an incubator at 28 °C for 36 h. If a clear antibacterial zone appears around the central biocontrol colony, it indicates that the strain has an antagonistic effect on pathogenic bacteria. Measure the size of the antibacterial zone, and use the inhibition rate to represent the antibacterial effect of the strain.

[0081] Inhibition rate (%) = (diameter of the digestion zone - diameter of the strain inoculated in the center) / diameter of the control colony × 100.

[0082] The results showed (Table 2): The fermentation broth of strain ZF520 also had a good inhibitory effect on 6 pathogenic bacteria, and its fermentation broth had no effect on most pathogenic bacteria.

[0083] Table 2. Antibacterial effect of Bacillus velezensis ZF520 on pathogenic bacteria

[0084] Note: Data are presented as mean ± standard deviation. Different lowercase letters after the data indicate significant differences (P < 0.05).

[0085] 3. Detection of biocontrol factors of strain ZF520 1) Detection of phosphate-solubilizing ability Absorb 5 μL of the ZF520 bacterial suspension (OD 600 = 0.8) and drop it in the center of the PVK medium. Place it in an incubator at 28 °C. After culturing for 48 h, observe whether a hydrolysis zone is produced.

[0086] 2) Detection of siderophore Absorb 5 μL of the ZF520 bacterial suspension (OD 600 = 0.8) and drop it in the center of the siderophore medium. After natural drying, culture it in an incubator at 28 °C and observe whether a yellow halo is produced around the colony.

[0087] 3) Detection of protease Absorb 5 μL of the ZF520 bacterial suspension (OD 600 = 0.8) and drop it in the center of the protease detection medium. Place it in an incubator at 28 °C. After culturing for 24 h, observe the production of a clear zone around the colony.

[0088] 4) Detection of genes related to antibiotic synthesis of strain ZF520 To detect the Surfactin, Macrolactin, Fengycin, Difficidin, Bacillibactin, Bacillaene, Bacilysin and Iturin lipopeptide antibiotic synthesis genes in the Bacillus genome (Table 3), using FZB42 as a positive control strain, and the genomic DNA of ZF520 as a template for PCR amplification.

[0089] PCR reaction system: 2×Taq PCR Mix 10 μL, 1 μL each of 10 μmol / L forward and reverse primers, 1 μL of genomic DNA (50 ng) as a template, and ddH 2 O was added to make up to 20 μL.

[0090] PCR amplification conditions were as follows: pre-denaturation at 95 °C for 5 min; denaturation at 94 °C for 45 s, annealing at 50 °C for 40 s, extension at 72 °C for 50 s, for 34 cycles; extension at 72 °C for 10 min, and stored at 4 °C. After the PCR reaction was completed, 5 μL of the amplified product was electrophoresed on a 1.5% agarose gel, observed and photographed on a BIO-RAD gel imaging system, and analyzed by comparing with the PCR amplified product of the expected size.

[0091] Table 3. Primer sequence information for strain identification and genes related to antibacterial lipopeptide synthesis

[0092] The results are as follows: Through the determination of the biocontrol factors of Bacillus velezensis ZF520, it was found that when the iron ions in the CAS detection plate were taken away by the siderophore secreted by microorganisms, the plate changed from blue to orange-yellow. Strain ZF520 could produce a large yellow transparent halo on the CAS medium. Therefore, it was indicated that strain ZF520 could produce siderophore ( Figure 5 in A); by observing the situation of strain ZF520 in the protease detection medium, it was found that it could produce a transparent hydrolysis zone, indicating that strain ZF520 could produce protease ( Figure 5 in B); on the inorganic phosphorus-containing plate, an obvious transparent hydrolysis zone could be produced ( Figure 5 in C), indicating that strain ZF520 had the ability to dissolve phosphorus.

[0093] Using the genomic DNA of Bacillus velezensis ( Bacillus velezensis ) FZB42 as a positive control, the lipopeptide substances secreted by ZF520 were determined. The test results showed that: bright and single bands could be amplified in each lane, and the sizes of the amplified bands were consistent with the expected results ( Figure 6 ). It was proved that the genome of strain ZF520 contained genes related to the synthesis of lipopeptide substances such as Surfactin, Macrolactin, Fengycin, Difficidin, Bacillibactin, Bacillaene, Bacilysin, and Iturin, indicating that strain ZF520 might inhibit bacteria by secreting lipopeptide substances.

[0094] Example 3. Determination of the biocontrol efficacy of cowpea root rot biocontrol strains in pots After Fusarium solani f. sp. phaseoli grew on the PDA plate for 3 - 5 days, 5 bacterial cakes were punched out with a sterilized puncher and placed in 1 L of PD medium prepared and shaken for 7 days, and then diluted to 10 with sterile water 6Reserve at a concentration of [[[spores / mL]]]. Activate the biocontrol strain on an LA medium plate and incubate it upside down at 28 °C for 1 day. Use a sterilized inoculation loop to pick a single colony and place it in LB medium. Incubate it on a shaker at 28 °C and 180 r / min for 36 h and then reserve. Select the root puncture method for inoculation. At the stage of 2-3 true leaves of cowpea, use an inoculation needle to puncture a small wound from the root or the base of the stem, and pour 10 mL of the fermentation broth of [[[Fusarium solani]]] for root irrigation. Each replicate has 15 seedlings, with a total of 3 replicates. Use sterile water for root irrigation inoculation as a control. After inoculation, place it in a humidity-controlled glass house and cultivate it conventionally for 24-36 h under the conditions of 30 °C and 80%-90% humidity. Use the root irrigation inoculation method for biocontrol bacteria inoculation. After inoculation, continue to cultivate it in the glass house for 5-7 d. After the pathogen control has fully developed, investigate the disease index and control efficacy. Among them, the treatment method for the disease control is to inoculate only the fermentation broth of [[[Fusarium solani]]]; the treatment method for the healthy control is to inoculate the same amount of clear water as the bacterial solution.

[0095] Disease index = ∑(number of diseased plants at each level × disease level) / (total number of plants investigated × representative value of the highest level) × 100.

[0096] Control efficacy (%) = [(disease index of the disease control - disease index of each treatment) / disease index of the disease control] × 100.

[0097] The results of the indoor pot experiment showed that after 7 d of inoculation, the pathogen control developed fully. The roots of cowpea turned reddish-brown, the epidermis cracked, the main roots and lateral roots were significantly browned, the reddened area spread, the lateral roots fell off, the vascular bundles of the longitudinally dissected stem were significantly browned, and the disease index reached 71.67. The incidence of cowpea root rot treated with the fermentation broth of strain ZF520 was significantly reduced ( Figure 7 in A-H), and the disease index was only 11.77, and the control efficacy reached 83.59%, which was better than the control efficacy of carbendazim wettable powder against cowpea root rot (Table 4).

[0098] Table 4. Pot control efficacy of strain ZF520 against cowpea root rot

[0099] Note: Data are mean ± standard error. Different lowercase letters indicate significant differences at the 0.05 level.

[0100] Example 4. Determination of pot control efficacy of cowpea seed coating against root rot Shake-culture the seed solution of the biocontrol bacillus ZF520. After 12 h, transfer it to KB medium and culture it at 28 °C and 200 r / min for 48 h to obtain the ZF520 fermentation broth, and the concentration of the microbial agent is 1×10 8 cfu / mL.

[0101] In a beaker, add 10% Filmer-17, 0.3% xanthan gum and 1% magnesium aluminum silicate as film-forming agents, then add the biocontrol bacterial solution to make up to 200 g, and stir evenly. Add warning color acid fuchsin to the mixed solution at a volume-weight ratio of 0.5%, and stir well to prepare a suspension seed coating agent containing biocontrol bacteria ZF520.

[0102] The results showed that: 7 days after inoculation, the pathogen control showed full disease development. The roots of cowpea turned reddish-brown, the epidermis cracked, the main roots and lateral roots were significantly browned, the vascular bundles of the longitudinally dissected stems were significantly browned, and the disease index reached 57.67. The incidence of cowpea root rot after inoculation of seeds coated with strain ZF520 was significantly reduced, the disease index was only 15.00, and the control effect reached 73.99%, which was better than the control effects of 23% imidacloprid·fludioxonil·benzovindiflupyr and 29% thiamethoxam·fludioxonil·metalaxyl-M on cowpea root rot ( Figure 8 in A-F, Table 5).

[0103] Table 5. Pot control effect of strain ZF520 on cowpea root rot

[0104] Note: Data are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0105] In summary: 1) A strain of Bacillus velezensis ZF520 with good antagonistic effect against cowpea root rot pathogen was obtained in this invention. It can carry out antibacterial action by secreting 8 kinds of lipopeptide substances, and has good ability to produce siderophores, playing its competitive and antibiotic effects with the pathogen. At the same time, it can also produce protease and has certain phosphorus solubilizing ability; 2) Strain ZF520 is mainly effective against a variety of pathogenic fungi and also has a consistent inhibitory effect on pathogenic bacteria, with broad-spectrum antibacterial activity. It has antibacterial activity against 6 pathogenic fungi and 6 pathogenic bacteria; 3) The control effect of the fermentation broth of strain ZF520 on potted cowpea root rot reached 83.59%, which was better than that of carbendazim wettable powder. The control effect of using ZF520 for seed coating on cowpea root rot reached 73.99%, which was better than commercial agents such as 23% imidacloprid·fludioxonil·benzovindiflupyr and 29% thiamethoxam·fludioxonil·metalaxyl-M. This study provides an efficient and safe biocontrol resource for the biological control of cowpea root rot. The antibacterial mechanism and field control effect of this strain still need to be further verified.

[0106] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Bacillus Velez, characterized in that: The Velez Bacillus is Velez Bacillus ( Bacillus velezensis ) ZF520, whose deposit number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.31235.

2. A bacterial agent, characterized in that: The bacterial agent contains the Bacillus Velezii according to claim 1 and / or a metabolite of the Bacillus Velezii; The bacterial agent is a pathogen inhibitor or a disease inhibitor, and the pathogen is a pathogenic fungus or / and a pathogenic bacteria; The pathogenic fungus is Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum )、Multiple main coryneformes( Corynespora cassiicola )、Citrullus citrinum( Ascochyta citrullina )、Rhizoctonia solani( Rhizoctonia solani )、Botrytis cinerea( Botrytis cinerea ); The pathogenic bacteria is Acidophilus citrullus ( Acidovorax citrulli )、Pectobacterium brasiliensis( Pectobacterium brasiliense ), Solanum coryllis ( Ralstonia solanacearum ), Pseudomonas syringae pv. Pseudomonas syringae pv. lachrymans ), Corynebacterium michiganensis subsp. michiganensis ( Clavibacter michiganensis subsp. michiganensis ), Xanthomonas campestris var. Xanthomonas campestris pv. campestris ); The disease is cowpea root rot.

3. Use of the Bacillus Velezii according to claim 1 in the preparation of any of the products in 1) to 3) below: 1) A microbial agent for preventing and controlling cowpea root rot and / or increasing cowpea yield; 2) Pathogen inhibitors, wherein the pathogens are pathogenic fungi and / or pathogenic bacteria; The pathogenic fungus is Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum )、Multiple main coryneformes( Corynespora cassiicola )、Citrullus citrinum( Ascochyta citrullina )、Rhizoctonia solani( Rhizoctonia solani )、Botrytis cinerea( Botrytis cinerea ); The pathogenic bacteria is Acidophilus citrullus ( Acidovorax citrulli )、Pectobacterium brasiliensis( Pectobacterium brasiliense ), Solanum coryllis ( Ralstonia solanacearum ), Pseudomonas syringae pv. Pseudomonas syringae pv. lachrymans ), Corynebacterium michiganensis subsp. michiganensis ( Clavibacter michiganensis subsp. michiganensis ), Xanthomonas campestris var. Xanthomonas campestris pv. campestris ); 3) A disease inhibitor, wherein the disease is cowpea root rot.

4. A method for culturing the Bacillus Velez according to claim 1, comprising the step of culturing the Bacillus Velez in a culture medium for culturing Bacillus.

5. A method for preparing the bacterial agent according to claim 2, comprising the following steps: using the Bacillus Velez subtilis according to claim 1 as an active ingredient to obtain the bacterial agent.

6. Use of the Bacillus Velezii described in claim 1 or the microbial agent described in claim 2 in cultivating cowpea.

7. Bio-organic fertilizer, characterized by: The biological organic fertilizer contains the Bacillus Velez subtilis described in claim 1 or the bacterial agent described in claim 2.

8. A method for cultivating cowpea, comprising inoculating the cultivated cowpea with the Bacillus Velez subtilis according to claim 1 or the bacterial agent according to claim 2 by root irrigation.

9. Use of the Bacillus Velezii according to claim 1 in preventing and controlling cowpea root rot and / or increasing cowpea yield.

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