Bacillus subtilis and application thereof

Abstract

The invention discloses bacillus subtilis and application thereof, and belongs to the field of microorganisms. The technical problem to be solved by the invention is how to inhibit pathogenic bacteria by a biological control method. Therefore, the invention provides the bacillus subtilis, the bacillus subtilis is bacillus subtilis, the strain number of the bacillus subtilis is WR4, and the preservation number of the bacillus subtilis in the China General Microbiological Culture Collection Center is CGMCC No.29148. The invention further provides a preparation method of the bacillus subtilis. The bacillus subtilis CGMCC29148 provided by the invention is separated from rice rhizosphere soil, and the bacillus subtilis CGMCC29148 and the rice rhizosphere soil coexist in a micro-ecological harmonious manner; the strain has certain salt tolerance, can be applied to a wider range of soil, and can be used for widely preventing and treating pathogenic fungi such as fusarium, verticillium dahliae, magnaporthe oryzae and gibberella zeylanica and pathogenic bacteria such as potato scab.

Description

Technical Field

[0001] The present invention relates to a Bacillus subtilis strain in the field of microorganisms and its applications. Background Art

[0002] During the growth process of crops, they are subject to various biotic stresses, such as pathogen infections, which seriously affect the yield, quality and value of crops. In the control of crop diseases in the field, the current method is still to combine field management and chemical control to inhibit the occurrence and impact of diseases. However, field management is difficult to completely eliminate the source of diseases, and at the same time, chemical control also increases the pressure on the ecological environment and the risk of increased pathogen drug resistance. At present, the concepts of biological control and green prevention and control have initially emerged, and it has been found that they have a good effect on controlling the occurrence of diseases in the field and are more friendly to the ecology, which is one of the main trends in future development.

[0003] Taking cotton, the main cash crop in China, as an example, during the cotton production process, cotton verticillium wilt caused by Verticillium dahliae has seriously affected the cotton yield. In severely affected fields, cotton almost fails, and the quality of cotton in non-severely affected areas also significantly decreases, affecting its economic value and causing economic losses to cotton farmers.

[0004] Similarly, as the most important food crop in China - rice, it is also subject to various pathogen hazards during its production process, resulting in reduced yields. Among them, fungal pathogens causing fungal diseases have become a fatal threat to high-yield rice. Rice blast caused by Magnaporthe oryzae occurs throughout the growth period of rice. And the conidia and mycelia of the pathogenic fungi can attach to diseased rice to overwinter. When the humidity is high during the rice planting period in the following year, such as during continuous rainy weather, the spores will be spread to healthy rice by wind, rain and dew, or cause continuous diseases for many years by sowing infected rice seeds. Rice blast has a high incidence frequency and a wide range, and it will cause repeated diseases and spread in the field. Over the years, it has become the most serious fungal disease affecting rice worldwide, known as the "cancer" of rice, causing irreparable losses to the rice yield. Therefore, prevention and control measures are urgently needed. In addition, Fusarium, Gibberella fujikuroi, etc. are also the main pathogens of rice wilt, rice bakanae disease, and Solanaceae wilt. They are widely prevalent in many places in China. Among them, the widespread epidemic of Solanaceae wilt has also had a serious impact on the yield and quality of tomatoes.

[0005] Due to its drought tolerance characteristics, potatoes are widely planted in China and are also the main food crops in many regions of the world. However, potato common scab caused by the soil-borne pathogen Streptomyces causes the surface of potato tubers to be rough, forming scab lesions, affecting the appearance and quality of the crops. More importantly, infected potatoes are extremely likely to cause rapid spread. This disease has also become one of the important factors restricting the development of the potato industry.

[0006] To address the occurrence of diseases, many solutions or products have proposed biocontrol agents with significant inhibitory effects against specific pathogens. However, in the complex field environment, it does not contain only a single pathogen. Therefore, biocontrol bacteria with broad control effects against multiple diseases obviously have higher application value and economic benefits. At the same time, in biological control, due to the biological characteristics of biocontrol bacteria, it is also necessary to consider their survival activity in complex ecological conditions such as special ecological environments like saline-alkali land. Therefore, finding effective biocontrol bacteria from the recruited population of crops themselves will have a wider application environment. Summary of the Invention

[0007] The technical problem to be solved by this application is: how to inhibit pathogenic bacteria by biological control methods.

[0008] To solve the above technical problem, this application provides a strain of Bacillus subtilis, and the Bacillus subtilis is Bacillus subtilis ( Bacillus subtilis ), its strain number is WR4, and its deposit number in the China General Microbiological Culture Collection Center is CGMCC No. 29148.

[0009] This application also provides a bactericide, and the bactericide contains the above-mentioned Bacillus subtilis or / and a culture containing the above-mentioned Bacillus subtilis.

[0010] The active ingredient of the above-mentioned bactericide can be the above-mentioned Bacillus subtilis or / and a culture of the above-mentioned Bacillus subtilis (such as fermentation products). The active ingredient of the above-mentioned bactericide can also contain other biological components or non-biological components, and those skilled in the art can determine other active ingredients of the bactericide according to the effect of the bactericide.

[0011] The bactericide can be a fungal and bacterial inhibitor.

[0012] The fungi are selected from Magnaporthe oryzae ( Magnaporthe oryzae ), Verticillium dahliae ( Verticillium dahliae ), Fusarium oxysporum ( Fusarium oxysporum ), Gibberella fujikuroi ( Fusarium fujikuroi ). The bacteria are selected from Streptomyces scabies ( Streptomyces scabies ).

[0013] The term "culture" refers to the general term for liquid or solid products (all substances in the culture container) with a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biological pure culture of microorganisms, or can contain a certain amount of culture medium, metabolites or other components generated during the culture process. It can also be a mixture containing a certain amount of culture medium, microbial cell metabolites and removing microbial cells.

[0014] Further, the preparation method of the microbial agent includes culturing the above-mentioned Bacillus subtilis with a culture medium, collecting the culture (or fermentation product), and using the culture (or fermentation product) as a component of the microbial agent.

[0015] The present application also provides applications, and any one of the following A1)-A6) is applicable: A1) Application of the above-mentioned Bacillus subtilis in the preparation of a plant pathogen inhibitor; A2) Application of the above-mentioned Bacillus subtilis or the above-mentioned microbial agent in inhibiting plant pathogens; A3) Application of the above-mentioned Bacillus subtilis in the preparation of a product for preventing and / or treating plant diseases; A4) Application of the above-mentioned Bacillus subtilis or the above-mentioned microbial agent in preventing and / or treating plant diseases; A5) Application of the above-mentioned Bacillus subtilis in the preparation of a product for promoting plant growth; A6) Application of the above-mentioned Bacillus subtilis or the above-mentioned microbial agent in promoting plant growth.

[0016] Further, in the above-mentioned applications, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0017] Further, in the above-mentioned applications, the pathogen in A1) or A2) is a fungus or a bacterium; the fungus is selected from Magnaporthe oryzae ( Magnaporthe oryzae ), Verticillium dahliae ( Verticillium dahliae ), Fusarium oxysporum ( Fusarium oxysporum ), Gibberella fujikuroi ( Fusarium fujikuroi ); the bacterium is selected from Streptomyces scabies ( Streptomyces scabies ).

[0018] Further, in the above-mentioned applications, the plant disease in A3) or A4) can be a soil-borne disease or a seed-borne disease.

[0019] Further, in the above-mentioned applications, the plant disease in A3) or A4) can be rice blight caused by Fusarium oxysporum ( Fusarium oxysporum ).

[0020] Further, in the above-mentioned applications, the plant disease in A3) or A4) can be cotton verticillium wilt caused by Verticillium dahliae ( Verticillium dahliae ).

[0021] Further, in the above-mentioned applications, the plant disease in A3) or A4) can be potato scab caused by Streptomyces scabies ( Streptomyces scabies ).

[0022] Furthermore, in the application, the plant disease in A3) or A4) can be Gibberella fuscata ( Fusarium fujikuroi ) caused by rice seedling disease.

[0023] Furthermore, in the application, the plant disease in A3) or A4) can be Fusarium oxysporum ( Fusarium oxysporum ) caused by tomato wilt.

[0024] Furthermore, the promotion of plant growth described in A5) and A6) may be the promotion of plant height growth, accumulation of aboveground and root biomass (eg, plant fresh weight).

[0025] The present application also provides a method for improving plant disease resistance and / or promoting plant growth, the method comprising B1) or B2): B1), culturing the above-mentioned Bacillus subtilis in a microbial culture medium, collecting the bacteria to prepare a bacterial suspension, and using the bacterial suspension to treat the plant or the culture medium of the plant; B2) Treating plants or plant culture media with the above-mentioned microbial agents.

[0026] Furthermore, in the method, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0027] The present application also provides a method for preventing and / or treating plant diseases, which comprises C1) or C2): C1), culturing the above-mentioned Bacillus subtilis in a microbial culture medium, collecting the bacteria to prepare a bacterial suspension, and using the bacterial suspension to treat the culture medium of the plant; C2) Treat the culture medium of the plant with the above-mentioned microbial agent.

[0028] Furthermore, in the method described above, the plant disease is a soil-borne disease or a seed-borne disease.

[0029] Furthermore, in the method, the soil-borne disease may be Fusarium oxysporum ( Fusarium oxysporum ) caused by crop wilt, Fusarium fusarium ( Fusarium fujikuroi ) caused by rice seedling disease, Verticillium dahliae ( Verticillium dahliae ) caused by Verticillium wilt in cotton and / or Streptomyces scab in potato ( Streptomyces scabies ) caused by potato scab.

[0030] Furthermore, in the method, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0031] In the present application, the monocotyledonous plant may be a plant of the order Poaceae.

[0032] In this application, the gramineous plants may be plants of the family Gramineae.

[0033] In this application, the plants of the family Gramineae may be plants of the genus Oryza.

[0034] In this application, the plants of the genus Oryza may be rice ( Oryza.Sativa L.).

[0035] In this application, the dicotyledonous plants may be plants of the order Malvales, the order Tubiflorae or the order Solanales.

[0036] In this application, the plants of the order Malvales may be plants of the family Malvaceae.

[0037] In this application, the plants of the family Malvaceae may be plants of the genus Gossypium.

[0038] In this application, the plants of the genus Gossypium may be cotton (Gossypium spp).

[0039] In this application, the plants of the order Tubiflorae may be plants of the family Solanaceae.

[0040] In this application, the plants of the family Solanaceae may be plants of the genus Solanum.

[0041] In this application, the plants of the genus Solanum may be potato ( Solanum tuberosum L.).

[0042] In this application, the plants of the order Solanales may be plants of the family Solanaceae.

[0043] In this application, the plants of the family Solanaceae may be plants of the genus Solanum (formerly the genus Lycopersicon).

[0044] In this application, the plants of the genus Solanum may be tomato ( Solanum Lycopersicum ).

[0045] The beneficial technical effects obtained in this application are as follows: 1) The biocontrol Bacillus subtilis CGMCC29148 described in this application is isolated from the rhizosphere soil of rice and coexists harmoniously with the rhizosphere soil microecology of rice, and can give full play to the ecological advantages of the strain to a high degree.

[0046] 2) The microbial inoculant of this application is a pure biological preparation, which is harmless to humans and does not pollute the environment, and can solve the environmental problems caused by chemical pesticide pollution; at the same time, after the microbial inoculant is applied to the soil, it can improve the soil quality and play a positive role in reducing the occurrence of diseases.

[0047] 3) The microbial inoculant of this application can widely control pathogenic fungi such as Fusarium, Verticillium dahliae, Magnaporthe oryzae, Gibberella fujikuroi, and pathogenic bacteria such as Streptomyces scabies of potato.

[0048] 4) The microbial agent of the present application has a certain salt tolerance and can be applied to a wider range of soils.

[0049] 5) The preparation method of the biocontrol agent Bacillus subtilis CGMCC29148 described in this application is simple, easy to be produced on a large scale industrially, and has good application prospects.

[0050] Preservation Instructions Bacteria name: Bacillus subtilis Latin name: Bacillus subtilis Strain ID: WR4 Depository: General Microbiology Center of China Culture Collection Administration Abbreviation of depository institution: CGMCC Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: November 27, 2023 CGMCC registration number: CGMCC No.29148 BRIEF DESCRIPTION OF THE DRAWINGS 图1 These are the test results of salt tolerance growth of Bacillus subtilis CGMCC29148.

[0051] 图2 These are the results of gene detection for antimicrobial lipopeptide synthetase.

[0052] 图3 These are the results of a four-point antibacterial test of Bacillus subtilis CGMCC29148 against Verticillium dahliae V592. The left picture is the control group, and the right picture is the experimental group. 图4 These are the results of a four-point antibacterial test of Bacillus subtilis CGMCC29148 against Fusarium oxysporum RF015. The left picture is the control group, and the right picture is the experimental group. 图5 These are the results of a four-point antibacterial test of Bacillus subtilis CGMCC29148 against Fusarium fujikuroi SS25. The left picture is the control group, and the right picture is the experimental group. 图6 These are the results of a four-point antibacterial test of Bacillus subtilis CGMCC29148 against Fusarium fujikuroi SS48. The left picture is the control group, and the right picture is the experimental group. 图7 These are the results of a four-point antibacterial test of Bacillus subtilis CGMCC29148 against Magnaporthe oryzae Guy11. The left picture is the control group, and the right picture is the experimental group.

[0053] 图8 The inhibition rate of Bacillus subtilis CGMCC29148 against pathogenic fungi.

[0054] 图9 The results of the four-point antibacterial test of Bacillus subtilis CGMCC29148 against Streptomyces scabies are shown in the figure on the left. The control group and the experimental group are shown on the right. 图10 is the inhibition rate of Bacillus subtilis CGMCC29148 against the pathogen Streptomyces scabies of potato.

[0055] 图11 To detect the disease resistance and growth-promoting effect of Bacillus subtilis CGMCC29148 on rice.

[0056] 图12 To test the disease resistance and growth promotion effect of Bacillus subtilis CGMCC29148 on tomatoes.

[0057] 图13 To test the disease resistance and growth promotion effect of Bacillus subtilis CGMCC29148 on potatoes. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0059] 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.

[0060] The rice pathogenic fungus Pyricularia oryzae ( Magnaporthe oryzae ) Guy11 was kindly donated by Liu Jun's research group at the College of Plant Protection, China Agricultural University. Magnaporthe oryzae in rice. Yang C, Liu R, Pang J, Ren B, Zhou H, Wang G, Wang E, Liu J. Nat Commun. 2021 Apr 12;12(1):2178.” was publicly disclosed, and the cotton pathogenic fungus ( Verticillium dahlia ), V592, was kindly provided by the research group of Guo Huishan from the Institute of Microbiology, Chinese Academy of Sciences. It was publicly disclosed in the literature "Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahlia. Zhou BJ, Jia PS, Gao F, Guo HS. Mol Plant Microbe Interact. 2012 Jul;25(7):964-75.", and the potato scab-causing Streptomyces scabies ( Streptomyces scabies ) and potato tissue culture seedlings were kindly provided by the research group of Zhong Naiqin from the Institute of Microbiology, Chinese Academy of Sciences. It was publicly disclosed in the literature "Phosphorus accumulation aggravates potato common scab and to be controlled by phosphorus-solubilizing bacteria. Cao J, Wang Z, Wu J, Zhao P, Li C, Li X, Liu L, Zhao Y, Zhong N. Sci Bull (Beijing). 2023 Oct 30;68(20):2316-2320.", and the variety of the potato tissue culture seedlings is Shepody, and the variety number is Mengrenshu 2013001. The public can obtain the above biological materials from the applicant, and the obtained above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0061] The Fusarium Fusarium oxysporum RF015 and Gibberella fujikuroi Fusarium fujikuroi SS48 is preserved in this laboratory and is disclosed in the paper "Heterosis in root microbiota inhibits growth of soil-borne fungal pathogens in hybrid rice. Zhang M, Wang Y, Hu Y, Wang H, Liu Y, Zhao B, Zhang J, Fang R, Yan Y. J Integr Plant Biol. 2023 Apr;65(4):1059-1076.", in which Gibberella fujikuroi Fusarium fujikuroi SS48 is labeled as Fusarium fujikuro in the paper. The public can obtain the biological material from the applicant in accordance with the relevant regulations on national biosafety. The obtained biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0062] Nipponbare in the following examples Oryza.Sativa L. spp. japonica is preserved in this laboratory and is disclosed in the paper "Heterosis in root microbiota inhibits growth of soil-borne fungal pathogens in hybrid rice. Zhang M, Wang Y, Hu Y, Wang H, Liu Y, Zhao B, Zhang J, Fang R, Yan Y. J Integr Plant Biol. 2023 Apr;65(4):1059-1076.". The public can obtain the biological material from the applicant in accordance with the relevant regulations on national biosafety. The obtained biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0063] The tomato seedlings in the following examples are products of Qingxian Chunfeng Seed Industry Co., Ltd., and the variety name is: Potted Cherry Tomato.

[0064] Preparation of the following LB liquid medium (g / L): Tryptone (OXOID, product number: LP0042) 10.0 g, Yeast extract (OXOID, product number: LP0021) 5.0 g, Sodium chloride (Sinopharm Chemical Reagent Co., Ltd., product number: 10019318) 10.0 g, made up to 1.0 L with distilled water, pH 7.0 - 7.2, sterilized at 121 °C for 15 minutes.

[0065] The 5% NaCl LB liquid medium (g / L) used in the following examples was prepared as follows: 10.0 g of tryptone (OXOID, Catalog No. LP0042), 5.0 g of yeast extract (OXOID, Catalog No. LP0021), 50.0 g of sodium chloride (Sinopharm Chemical Reagent Co., Ltd., Catalog No. 10019318), and distilled water to 1.0 L. The pH was adjusted to 7.0-7.2 and sterilized at 121°C for 15 minutes.

[0066] The 10% NaCl LB liquid medium (g / L) used in the following examples was prepared as follows: 10.0 g of tryptone (OXOID, Catalog No. LP0042), 5.0 g of yeast extract (OXOID, Catalog No. LP0021), 100.0 g of sodium chloride (Sinopharm Chemical Reagent Co., Ltd., Catalog No. 10019318), and distilled water to 1.0 L. The pH was adjusted to 7.0-7.2 and sterilized at 121°C for 15 minutes.

[0067] The LB solid medium (g / L) used in the following examples was prepared as follows: 10.0 g of tryptone (OXOID, Catalog No. LP0042), 5.0 g of yeast powder (OXOID, Catalog No. LP0021), 10.0 g of sodium chloride (Sinopharm Chemical Reagent Co., Ltd., Catalog No. 10019318), 15 g of agar (Beijing Aoboxing Biotechnology Co., Ltd., Catalog No. 01-023), and distilled water to 1.0 L, with a pH of 7.0-7.2; sterilized at 121°C for 15 minutes.

[0068] Preparation of PDA (Potato Dextrose Agar) medium (g / L) in the following examples: 200 g of fresh potatoes were boiled in water, filtered, and the filtrate retained. Add 20.0 g of glucose (Sinopharm Chemical Reagent Co., Ltd., Catalog No. 10010518) and 15.0 g of agar (Beijing Aoboxing Biotechnology Co., Ltd., Catalog No. 01-023). The volume was adjusted to 1.0 L with distilled water, and the pH was adjusted to natural. Sterilize at 121°C for 15 minutes.

[0069] NA medium (Nutrient agar) in the following examples: glucose (Sinopharm Chemical Reagent Co., Ltd., catalog number: 10010518) 10 g, tryptone (OXOID, catalog number: LP0042) 5 g, beef extract 3 g, yeast powder (OXOID, catalog number: LP0021) 0.5 g, sodium chloride (Sinopharm Chemical Reagent Co., Ltd., catalog number: 10019318) 10.0 g, agar 15 g, distilled water to 1.0 L, pH 7.0-7.2. Sterilize at 121°C for 15 minutes.

[0070] The 2% CTAB extraction solution in the following examples: 2 g of cetyltrimethylammonium bromide (Sinopharm Chemical Reagent Co., Ltd., product number: 30037416), 8.18 g of sodium chloride (Sinopharm Chemical Reagent Co., Ltd., product number: 10019318), tris(hydroxymethyl)aminomethane (NOVON product number: 1600515), disodium ethylenediaminetetraacetate dihydrate (Sinopharm Chemical Reagent Co., Ltd., product number: 10009717) 7.44 g, and made up to 1.0 L with distilled water.

[0071] In the quantitative tests in the following examples, unless otherwise specified, 4 replicates were set, and the results were averaged.

[0072] In the following examples, GraphPad Prism 8.0 statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and significant differences were represented by letters. The same letter indicated p > 0.05, that is, no significant difference; different letters represented p < 0.05, that is, significant difference, and were marked in descending order of the mean.

[0073] Example 1: Isolation, identification and cultivation of strain WR4 A strain numbered WR4 was obtained by self-isolation. The colony morphology was a white wrinkled colony. Through sequence alignment analysis of the 16srRNA gene and gyrA gene, it was the same as that of Bacillus subtilis ( Bacillus subtilis ), with a similarity of 99.99%. Strain WR4 was identified as Bacillus subtilis ( Bacillus subtilis ). The nucleotide sequence of the 16srRNA gene of WR4 is SEQ ID No.1, and the nucleotide sequence of the gyrA gene is SEQ ID No.2 (the nucleotide sequences are shown in Table 1).

[0074] Strain WR4 has been deposited in the China General Microbiological Culture Collection Center (CGMCC). Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101. The deposit date was November 27, 2023. The deposit number is CGMCC No. 29148. Strain name: Bacillus subtilis; Latin name: Bacillus subtilis ; The biological material (strain) referred to: WR4. Hereinafter referred to as Bacillus subtilis CGMCC29148.

[0075] Table 1 Sequences of the 16srRNA gene and gyrA gene of Bacillus subtilis CGMCC29148

[0076] Example 2. Characteristics of Bacillus subtilis CGMCC29148 2.1 Preparation of the culture of Bacillus subtilis CGMCC29148 and extraction of its genome 1) Bacillus subtilis CGMCC29148 was activated 3 times on LB solid medium to obtain highly active Bacillus subtilis CGMCC29148 cells; 2) One inoculation loop of highly active Bacillus subtilis CGMCC29148 cells was inoculated into 3 mL of LB liquid medium and cultured in a shaker at 28 °C and 220 rpm for 12 hours to obtain a fermentation broth; 3) 200 μL of the fermentation broth was inoculated into 500 ml of LB liquid medium and cultured in a shaker at 28 °C and 220 rpm for 12 hours to obtain the culture of Bacillus subtilis CGMCC29148; 4) The fermentation broth obtained in 2) was centrifuged at 13000 rpm for 1 minute, and the culture broth was removed to obtain pure cells, and the genome was extracted by the CTAB method; 5) The cells were added with 500 μL of 2% CTAB extraction solution, mixed well by inverting up and down, and then placed in a water bath at 65 °C for 30 min; 500 μL of phenol-chloroform-isoamyl alcohol (product number: C493, Guangzhou Meiji Biotechnology Co., Ltd.) was added, shaken, and centrifuged at 13000 rpm for 3 minutes; 500 μL of isopropanol (product number: 67-63-0, Tianjin Concord Technology Co., Ltd.) was added to the obtained supernatant, and after centrifugation at 13000 rpm for 2 min, a precipitate was obtained, which was dissolved in 30 μL of water to obtain genomic DNA.

[0077] 2.2 Salt tolerance growth range of Bacillus subtilis CGMCC29148 The experiment was divided into three groups according to the added amount of NaCl in the culture medium: 1% NaCl group, 5% NaCl group, and 10% NaCl group, with 10 replicates in each group. The operation steps for the 1% NaCl group were as follows: Pick the highly active Bacillus subtilis CGMCC 29148 colonies in Example 2.1 into 3 mL of LB liquid medium (with the added amount of NaCl being 10 g / L), culture overnight at 28 °C and 220 rpm. After measuring the OD600 nm using a micro-spectrophotometer (Hangzhou Allsheng Instruments Co., Ltd., model: Nano-300), adjust the OD600 nm to 0.4 with LB liquid medium, continue to dilute 100 times with LB liquid medium, and then inoculate 300 μL into a sterile microplate (Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number: P8366). Use a fully automatic growth curve analyzer (Bioscreen, model: Bioscreen C) for shaking culture and measurement. The culture temperature was 28 °C, the rotation speed was 220 rpm, and the absorbance value of the culture medium at a wavelength of 600 nm was measured every 2 h. The results were the average of 10 replicates.

[0078] The operation steps for the 5% NaCl group were referred to those of the 1% NaCl group, and the only difference was the culture medium. The culture medium for the 5% NaCl group was LB liquid medium with 5% NaCl, and the added amount of NaCl was 50 g / L.

[0079] The operation steps for the 10% NaCl group were referred to those of the 1% NaCl group, and the only difference was the culture medium. The culture medium for the 10% NaCl group was LB liquid medium with 10% NaCl, and the added amount of NaCl was 100 g / L.

[0080] The results were as 图1 shown. Under the condition of inoculating the same initial biomass, the absorbance values (i.e., OD600 nm) of the culture media with different salt contents at a wavelength of 600 nm were detected every 2 h. During the 0 - 48 h culture time of Bacillus subtilis CGMCC 29148, the OD600 nm in the 1% NaCl and 5% NaCl culture media gradually increased, indicating that with the extension of the culture time, the biomass of the bacteria increased and the bacteria had proliferative activity; within the same culture time, there was no significant change in the OD600 nm of the 10% NaCl group, indicating that with the increase of the culture time, the biomass of the bacteria did not increase and the bacteria did not have proliferative activity. 图1 The results showed that Bacillus subtilis CGMCC 29148 could tolerate an NaCl concentration of 5% and below, and still had proliferative activity at this concentration, showing a certain salt tolerance.

[0081] 2.3. Detection of the gene encoding antibacterial lipopeptide synthase Detection of genes encoding antimicrobial lipopeptide synthase produced by Bacillus subtilis CGMCC29148: sfp Encodes a transferase necessary for surfactin synthesis; AlbF It is one of the genes necessary for the synthesis of subtilosin A; fenD 、 fenB Encoding Fengycin synthase; bmyC Encoding Bacillomycin D synthase C; ituA Encodes iturin A synthase, thereby regulating the synthesis of iturin; bioA Encoding biotin synthase; pmxB Gene encoding polymycin synthase.

[0082] Sfp The reference sequence number of the gene is 56652119 (NCBI update date 2023.12.2), which encodes an essential transferase for the production of surfactin; AlbF The reference sequence number of the gene is 938515 (NCBI update date 2023.10.6), encoding subtilosin A; fenD The reference sequence number of the gene is 76982609 (NCBI update date 2023.4.13), fenB The reference sequence number of the gene is 76982606 (NCBI update date 2023.4.13), which encodes Fengycin synthase; bmyC The reference sequence number of the gene is 17137328 (NCBI update date 2015.2.4), which encodes bacillomycin D synthase C; ituA The reference sequence number of the gene is 14664694 (NCBI update date 2015.2.4), which encodes iturin A synthase; bioA The reference sequence number of the gene is 72450804 (NCBI update date 2023.11.20), which encodes biotin synthase; pmxB The reference sequence number of the gene is 17691193 (NCBI update date 2015.3.1), which encodes polymycin synthase.

[0083] The coding or regulatory genes of the above Bacillus subtilis antimicrobial substances were selected as the detection objects, and the genomic DNA of Bacillus subtilis CGMCC29148 obtained in 2.1 was used as the amplification template for PCR amplification. The amplified products were tested by 1% agarose gel electrophoresis.

[0084] Table 2 Primer sequences for antimicrobial lipopeptide detection

[0085] Reaction system: 1 μL genomic DNA; 10 μL 2× Rapid Taq Master Mix; 0.5 μL primer 1 (10 μM); 0.5 μL primer 2 (10 μM); Nuclease-free ddH2O to 20 μL (except genomic DNA and primers, all the above are from the high-fidelity amplification kit, which is a product of Nanjing Novozymes Biotech Co., Ltd., catalog number: P505-d1 / d2 / d3).

[0086] Reaction program: 95°C for 5 min; (95°C for 25 s, 55°C for 30 s, 72°C for 45 s)*30 cycles; final extension at 72°C for 10 min.

[0087] Result: The test result is as follows: 图2 As shown, the product detection after amplification using the above primers can be seen albF 、 bmyC 、 ituA and fenD Has a single, bright, correctly sized band, while sfp 、 pmxB 、 bioA and fenB No target bands were amplified. The amplified products were sequenced by Ruibo Xingke Co., Ltd., and the sequence results were compared with the NCBI database, confirming the correct sequence. These results indicate that Bacillus subtilis CGMCC29148 has the potential to synthesize subtilisin, bacillimycin, fengycin, and iturin.

[0088] Example 3, Determination of antibacterial ability of Bacillus subtilis CGMCC29148 3.1 Pathogen culture The fungi involved in the experiment are: Rice blast fungus ( Magnaporthe oryzae )Guy11, Verticillium dahliae ( Verticillium dahliae ) V592, Fusarium oxysporum ( Fusarium oxysporum ), RF015, Gibberella fujikuroi Fusarium fujikuroi ), SS48 and Gibberella fujikuroi Fusarium fujikuroi ), SS25.

[0089] The bacteria participating in the experiment included Streptomyces scabies Streptomyces scabies ).

[0090] Taking Magnaporthe oryzae Magnaporthe oryzae ), Guy11 as an example, Magnaporthe oryzae Magnaporthe oryzae ), Guy11 was inoculated on PDA solid medium and cultured at 28 °C for 14 days. Using a puncher with a 5 mm pore diameter, on the PDA solid medium that had grown for 14 days, the outermost and best-growing and newly grown mycelia were punched out as the inoculum of Magnaporthe oryzae Magnaporthe oryzae ), Guy11 for the antibacterial test.

[0091] The preparation of the inocula of the remaining fungi participating in the test was referred to that of Magnaporthe oryzae Magnaporthe oryzae ), Guy11, and the only difference was the strain.

[0092] In the antibacterial experiment of bacteria, Streptomyces scabies Streptomyces scabies ) was inoculated on PDA solid medium and cultured at 28 °C for 7 days. A single colony was picked with a sterile toothpick and resuspended in 200 μL of sterile water to form an inoculum of Streptomyces scabies Streptomyces scabies ) for the antibacterial experiment.

[0093] 3.2 Four-point antibacterial test of pathogenic fungi Taking Magnaporthe oryzae Magnaporthe oryzae ), Guy11 as an example, a 5-mm-diameter inoculum of Magnaporthe oryzae Magnaporthe oryzae ), Guy11 was placed in the center of NA solid medium (the culture dish had a specification of 90 mm). At about 3 cm in 4 directions from the mycelial cake of Magnaporthe oryzae Magnaporthe oryzae ), Guy11, 10 μL of the Bacillus subtilis CGMCC29148 culture obtained in Example 2.1 was evenly added dropwise. Using the same fungal inoculation method, LB medium was added dropwise in 4 directions as the control group. After culturing at 28 °C for 7 days, the growth of the fungi was observed, and the inhibition rate of Bacillus subtilis CGMCC29148 against Magnaporthe oryzae Magnaporthe oryzae ), Guy11 was calculated. A total of 4 groups of repeated groups with the same operation were set, and the results were averaged.

[0094] Calculation formula for the inhibition rate of pathogenic fungi: Inhibition rate = (radius of fungal growth in the control group - radius of fungal growth in the experimental group) / radius of fungal growth in the control group.

[0095] The four-point inhibition test of Bacillus subtilis CGMCC29148 against other fungi involved in the experiment was carried out with reference to the rice blast fungus ( Magnaporthe oryzae )Guy11, which differ only in the fungi tested.

[0096] The results are as follows Figures 3 - 8 As shown in Table 3: Bacillus subtilis CGMCC29148 against rice blast fungus ( Magnaporthe oryzae ) Guy11 inhibition rate is about 65%, against Verticillium dahliae ( Verticillium dahliae ) V592 inhibition rate is about 65%, against Fusarium oxysporum ( Fusarium oxysporum ) RF015 inhibition rate is about 37%, against Fusarium fuscata ( Fusarium fujikuroi ) SS48 inhibition rate is about 41%, against Fusarium fuscata ( Fusarium fujikuroi ) The SS25 inhibition rate was approximately 44%.

[0097] The results showed that Bacillus subtilis CGMCC29148 had a significant inhibitory effect on the growth of the above crop pathogenic fungi.

[0098] Table 3 Statistics of the inhibition rate of Bacillus subtilis CGMCC29148 against pathogenic fungi

[0099] 3.3 Four-point antibacterial test of pathogenic bacteria Take 100 μL of potato scab Streptomyces ( Streptomyces scabies ) inoculated solution was evenly spread on NA solid culture medium (90 mm dish size). After drying, 10 μL of the Bacillus subtilis CGMCC29148 culture obtained in Example 2.1 was evenly added dropwise in four directions at the same position. LB culture medium was added dropwise in four directions as a control group using the same inoculation method. After culturing at 28°C for 7 days, the growth of Streptomyces scabii ( Streptomyces scabies ) and the growth of Bacillus subtilis CGMCC29148, and the effect of Bacillus subtilis CGMCC29148 on Streptomyces scabii ( Streptomyces scabies ) inhibition rate. A total of 4 replicate groups with the same operation were set up, and the results were averaged.

[0100] The calculation formula of pathogen inhibition rate is: inhibition rate = (radius of the transparent zone around Bacillus subtilis - growth radius of Bacillus subtilis colony) / growth radius of Bacillus subtilis colony.

[0101] The results are as follows Figures 9 - 10 As shown in the data in the last row of Table 3, Bacillus subtilis CGMCC29148 is effective against Streptomyces scabii ( Streptomyces scabies ) has an inhibition rate of about 106%.

[0102] The results showed that Bacillus subtilis CGMCC 29148 also had an obvious effect on inhibiting the growth of pathogenic bacteria.

[0103] Example 4. Detection of the disease resistance potential of Bacillus subtilis CGMCC 29148 in rice Fusarium oxysporum Fusarium oxysporum After being cultured on PDA medium at 28 °C for 7 d, the mycelia on the surface of the medium were scraped off with a sterile inoculation loop. The fungal mycelia were suspended in 400 μL of PBS, and tissue grinding was carried out using stainless steel beads (diameter 4 mm) and an appropriate amount of quartz sand. The rotation speed was set at 800 rpm, and grinding was carried out for 10 min. An inoculum of Fusarium oxysporum Fusarium oxysporum RF015 was obtained.

[0104] Vermiculite and nutrient soil were mixed evenly at a volume ratio of 1:1, and then autoclaved at 121 °C for 30 min to obtain sterile soil.

[0105] Three groups of treatments were set: control group (Mock), fungal group (F), and antagonistic group (BF).

[0106] The operation of the antagonistic group was as follows: 400 g of sterile soil was filled in each pot, and according to the addition amount of 0.025 g of the inoculum of Fusarium oxysporum Fusarium oxysporum RF015 and 1 mL of the bacterial suspension of Bacillus subtilis CGMCC 29148 (prepared in 2.1) per 100 g of sterile soil, the inoculum of Fusarium oxysporum Fusarium oxysporum RF015 and the culture of Bacillus subtilis CGMCC 29148 were added simultaneously. After mixing evenly, they were filled into pots. 5 germinated seeds of Oryza sativa L. spp. japonica Oryza.Sativa that had germinated for 2 d were transplanted into each pot and cultured in a greenhouse for 28 days. The plant height, aboveground fresh weight, and survival rate were statistically analyzed. A total of 4 pots were set as replicates. The results were averaged.

[0107] The treatment steps of the fungal group were referred to those of the antagonistic group, and the only difference was that the culture of Bacillus subtilis CGMCC 29148 was not added to the sterile soil of the fungal group.

[0108] The treatment steps of the control group were referred to those of the antagonistic group, and the only differences were that the culture of Bacillus subtilis CGMCC 29148 and the inoculum of Fusarium oxysporum Fusarium oxysporum RF015 were not added to the sterile soil of the control group.

[0109] The formula for calculating the survival rate: Survival rate = (Number of surviving seedlings in the control group - Number of surviving seedlings in the experimental group) / Number of surviving seedlings in the control group × 100%.

[0110] Results: The experimental results were asFigure 11 As shown, all rice seedlings in the control group survived. After 28 days of growth, the seedlings were strong, with expanded, bright green leaves. Their average aboveground height was approximately 35 cm, and their aboveground fresh weight was approximately 1.6 g. In the fungus group, only 65% of the seedlings survived after 28 days of treatment. The surviving seedlings were slender, with green, dry, and wrinkled leaves. The number of leaves was significantly reduced compared to the control group. The average aboveground height was approximately 15 cm, a 57.14% decrease compared to the control group, and the aboveground fresh weight was approximately 0.6 g, a 62.50% decrease compared to the control group. After 28 days of growth, 95% of the seedlings in the antagonistic group survived. The surviving seedlings were healthy, with upright stems and expanded green leaves. Their aboveground height was approximately 31 cm, double that of the fungus group, and their average aboveground fresh weight was approximately 1.25 g, double that of the fungus group.

[0111] The results showed that Bacillus subtilis CGMCC29148 can effectively improve the pathogenic fungus Fusarium oxysporum ( Fusarium oxysporum ) RF015 caused death and growth inhibition on plants, and Bacillus subtilis CGMCC29148 had the potential to be used as a field biocontrol agent.

[0112] Example 5: Detection of the disease resistance potential of Bacillus subtilis CGMCC29148 on tomatoes Fusarium oxysporum ( Fusarium oxysporum ) After RF015 was cultured in PDA medium at 28°C for 7 days, the mycelia on the surface of the medium were scraped with a sterile inoculating loop, the fungal mycelia were suspended in 1 ml of PBS, and filtered through two layers of gauze to obtain Fusarium oxysporum ( Fusarium oxysporum ) RF015 spore suspension was prepared and the spore concentration was adjusted by microscopic counting.

[0113] Vermiculite and nutrient soil were mixed evenly in a volume ratio of 1:1, and then sterilized in an autoclave at 121°C for 30 min to obtain sterile soil.

[0114] Four treatment groups were set up: control group (Mock), fungal group (F), bacterial group (B) and antagonistic group (BF).

[0115] The antagonistic group was prepared as follows: 1 tomato seedling (potted cherry tomato) was planted in each pot with 400 g of sterile soil. After 3 weeks, when the seedlings had two leaves and one heart, the inoculation was performed by root dredging. 4 mL of OD600 1.0 Bacillus subtilis CGMCC29148 suspension (prepared in 2.1) was inoculated into the roots of each seedling. 24 hours later, 8 mL of Fusarium oxysporum ( Fusarium oxysporum )RF015 spore suspension (10 6 After 60 days of greenhouse culture, phenotypic characteristics such as plant height and aboveground fresh weight were measured. Six replicates were set up, and the results were averaged.

[0116] The treatment steps of the fungal group refer to those of the antagonistic group, with the only difference being that Bacillus subtilis CGMCC29148 culture is not inoculated into the sterile soil of the fungal group.

[0117] The treatment steps of the bacterial group refer to those of the antagonistic group, with the only difference being that only Bacillus subtilis CGMCC29148 culture is added to the sterile soil of the bacterial group.

[0118] The treatment steps of the control group refer to those of the antagonistic group, with the only difference being that neither Bacillus subtilis CGMCC29148 culture nor Fusarium oxysporum ( Fusarium oxysporum ) RF015 inoculum is added to the sterile soil of the control group.

[0119] Results: The experimental results are as Figure 12 shown. After 60 days of growth, the tomato seedlings in the control group grew vigorously, with their leaves spreading out and being bright green. The average above-ground plant height was about 14 cm, and the above-ground fresh weight was about 2.6 g. After 60 days of treatment in the bacterial group, the seedlings grew significantly stronger. Compared with the control group, the number and area of leaves were both significantly increased, and the plants entered the flowering stage earlier. The average above-ground plant height was about 19 cm, and the above-ground fresh weight was about 6.5 g, which was about 4 times higher than that of the control group. After 60 days of treatment in the fungal group, the seedlings grew slender and weak, with their leaves being yellowish-green, dry, wrinkled, and the number of leaves significantly reduced compared with the control group, and the leaf area decreased. The average above-ground plant height was about 10 cm, which was 28.57% less than that of the control group, and the above-ground fresh weight was about 1.7 g, which was 34.62% less than that of the control group. After 60 days of growth of the seedlings in the antagonistic group, the seedlings grew significantly stronger, with straight stems, large leaf areas, many leaves, and a green and spreading shape. The above-ground plant height was about 19 cm, which was about twice that of the fungal group. At the same time, the average above-ground fresh weight was about 7 g, which was 4 times higher than that of the fungal group.

[0120] This result indicates that Bacillus subtilis CGMCC29148 culture can significantly promote the growth of tomatoes, increase biomass, and effectively improve the growth inhibition effect on plants caused by the pathogenic fungus Fusarium oxysporum ( Fusarium oxysporum ) RF015. Bacillus subtilis CGMCC29148 has the potential to be used as a biocontrol agent in the field.

[0121] Example 6. Detection of the disease resistance potential of Bacillus subtilis CGMCC29148 on potatoes Streptomyces scabies ( Streptomyces scabies ) After being cultured on PDA medium at 28 °C for 7 d, the mycelium on the surface of the medium was scraped with a sterile inoculation loop and inoculated into 100 ml of TSB liquid medium. After shaking culture at 28 °C and 220 rpm for 5 days, the supernatant was removed by centrifugation, and the inoculum of Streptomyces scabies ( Streptomyces scabies ) was obtained by resuspending with sterile water.

[0122] Mix vermiculite and nutrient soil evenly at a volume ratio of 1:1, and then use an autoclave for high-pressure steam sterilization at 121 °C for 30 min to obtain sterile soil.

[0123] Set up four groups of treatments: control group (Mock), fungal group (F), bacterial group (B), and antagonistic group (BF).

[0124] The operation of the antagonistic group is as follows: Each pot is filled with 400 g of sterile soil, and 3 tissue-cultured potato seedlings (cultivar: Shepody) grown for 20 days are transplanted. After 15 days of acclimation, 35 ml of Streptomyces scabies inoculum is inoculated. Streptomyces scabies ), 10 - 15 days later, 10 ml of Bacillus subtilis CGMCC29148 bacterial suspension with an OD600 of 1 (prepared in 2.1) is inoculated, and after culturing in the greenhouse for 60 days, phenotypic characteristics such as the number of potato fruits, the number of disease spots, and the fruit weight are counted. A total of 4 pots are set as replicates. The results are averaged.

[0125] The treatment steps of the fungal group refer to those of the antagonistic group, and the only difference is that: in the sterile soil of the fungal group, Bacillus subtilis CGMCC29148 culture is not inoculated.

[0126] The treatment steps of the bacterial group refer to those of the antagonistic group, and the only difference is that: in the sterile soil of the bacterial group, only Bacillus subtilis CGMCC29148 culture is inoculated.

[0127] The treatment steps of the control group refer to those of the antagonistic group, and the only differences are that: in the sterile soil of the control group, Bacillus subtilis CGMCC29148 culture and Streptomyces scabies Streptomyces scabies ) inoculum are not inoculated.

[0128] Results: The experimental results are as Figure 13 shown. After 60 days of growth, the potatoes in the control group grew vigorously, the tubers were fair and had no disease spots, and the fresh weight was about 2.0 g per tuber. After 60 days of treatment in the bacterial group, the seedlings grew significantly stronger. Compared with the control group, there were no significant changes in the number and average weight of tubers, and the tubers were clean and had no disease spots. After 60 days of treatment in the fungal group, there were no significant changes in the number and size of tubers, but there were obvious disease spots on the tubers, and the total number of disease spots was 23. After 60 days of growth of the seedlings in the antagonistic group, the size of the tubers increased, the fresh weight increased significantly, the average weight was about 5 g, which was 2 times higher than that of the control group. At the same time, the total number of disease spots was 10, which was significantly lower than that of the fungal group.

[0129] The results indicate that Bacillus subtilis CGMCC29148 culture can significantly promote the growth of potato tubers, increase the biomass, and at the same time can effectively improve the infection of pathogenic Streptomyces scabies Streptomyces scabies ) on plants and the formation of scabs. Bacillus subtilis CGMCC29148 has the potential to be used as a biocontrol agent in the field.

[0130] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a 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 general, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art.

Claims

1. A Bacillus subtilis strain, characterized in that: The Bacillus subtilis is Bacillus subtilis ( Bacillus subtilis ), its strain number is WR4, and its preservation number in the China General Microbiological Culture Collection Center is CGMCC No. 29148.

2. Bacterial agent, characterized in that: The microbial agent contains the Bacillus subtilis described in claim 1 or / and the culture of the Bacillus subtilis described in claim 1.

3. Application, characterized in that: The application is any one of the following A1)-A6): A1), The application of the Bacillus subtilis described in claim 1 in the preparation of a plant pathogen inhibitor; A2), The application of the Bacillus subtilis described in claim 1 or the microbial agent described in claim 2 in inhibiting plant pathogens; A3), The application of the Bacillus subtilis described in claim 1 in the preparation of a product for preventing and / or treating plant diseases; A4), The application of the Bacillus subtilis described in claim 1 or the microbial agent described in claim 2 in preventing and / or treating plant diseases; A5), The application of the Bacillus subtilis described in claim 1 in the preparation of a product for promoting plant growth; A6), The application of the Bacillus subtilis described in claim 1 or the microbial agent described in claim 2 in promoting plant growth.

4. The application according to claim 3, characterized in that: The plant is a monocotyledonous plant or a dicotyledonous plant.

5. The application according to claim 3 or 4, characterized in that: A1) or A2) the pathogenic bacterium is a fungus or a bacterium; the fungus is selected from Magnaporthe oryzae ( Magnaporthe oryzae ), Verticillium dahliae ( Verticillium dahliae ), Fusarium oxysporum ( Fusarium oxysporum ), Gibberella fujikuroi ( Fusarium fujikuroi ); the bacterium is selected from Streptomyces scabies ( Streptomyces scabies ).

6. A method for enhancing plant disease resistance and / or promoting plant growth, characterized in that: The method includes B1) or B2): B1), Culturing the Bacillus subtilis described in claim 1 in a microbial culture medium, collecting the bacterial cells to prepare a bacterial suspension, and treating the plant or the culture medium of the plant with the bacterial suspension; B2), Treating the plant or the culture medium of the plant with the microbial agent described in claim 2.

7. The method according to claim 6, wherein: The plant is a monocotyledonous plant or a dicotyledonous plant.

8. A method for preventing and / or treating plant diseases, characterized in that: The method includes C1) or C2): C1), Culturing the Bacillus subtilis described in claim 1 in a microbial culture medium, collecting the bacterial cells to prepare a bacterial suspension, and treating the culture medium of the plant with the bacterial suspension; C2), Treating the culture medium of the plant with the microbial agent described in claim 2.

9. The method according to claim 8, characterized in that: The plant disease is a soil-borne disease or a seed-borne disease.

10. The method according to claim 8 or 9, characterized in that: The plant is a monocotyledonous plant or a dicotyledonous plant.

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