Brevibacillus laterosporus for inhibiting verticillium dahliae of cotton and application of brevibacillus laterosporus

Abstract

The invention provides a brevibacillus laterosporus new strain M1906, the preservation number of the brevibacillus laterosporus is CGMCC (China General Microbiological Culture Collection Center) NO: 28281, the brevibacillus laterosporus M1906 has a very strong inhibition effect on cotton verticillium wilt, and the inhibition rate of sterile fermentation filtrate on the cotton verticillium wilt reaches 91.37%. The bacterial strain can produce chitinase, and the inhibition rate of sterile fermentation filtrate of the bacterial strain on sclerotia of cotton verticillium wilt reaches 100%; pot experiment results show that before inoculation of cotton verticillium wilt bacteria, spraying of the bacterial strain aqueous solution has a cotton verticillium wilt control effect of 82.52%; after inoculation, a bacterial strain aqueous solution is sprayed, and the control effect on cotton verticillium wilt is 82.52%. The bacterial strain M1906 is wide in antibacterial spectrum, sterile fermentation filtrate has a certain inhibition effect on phytopathogens such as cucumber fusarium wilt, tomato botrytis cinerea, tomato alternaria solani, jujube fruit alternaria, phytophthora capsici, corn stalk rot, astragalus verticillium wilt and the like, and the bacterial strain M1906 has potential development value on the biocontrol effect of plant diseases.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular, to the technical field of Brevibacillus laterosporus and applications thereof. Technical Background

[0002] Verticillium wilt is one of the most serious and difficult fungal diseases to control at home and abroad. It is widely distributed and causes serious damage, causing huge economic losses to agricultural production every year. Due to the strong saprophytic ability of Verticillium wilt and its wide host range, no highly resistant varieties to the disease have been found at home and abroad. At present, methyl bromide and some chemical pesticides are mainly used to prevent and control Verticillium wilt in cotton production in my country. The long-term and repeated use of methyl bromide and chemical agents has caused the pathogen to develop obvious drug resistance, resulting in a decrease or even loss of control effect, and the amount of drugs used has been increasing. The long-term and large-scale use of pesticides has caused natural ecological environment pollution and increased rice pesticide residues. The problem is becoming more and more prominent. Since Verticillium wilt is a soil-borne disease, the pathogen is not parasitic on the host, and there is little or no difference in specific resistance between crop varieties. Using beneficial microorganisms for biological control is not only economical and effective, but also has no side effects. Therefore, using antagonistic microorganisms for biological control is a new approach worth exploring. In recent years, major cotton-growing areas around the world have generally carried out biological control research using antagonistic microorganisms to control the damage caused by cotton Verticillium wilt.

[0003] The biological control research on cotton Verticillium wilt has made great progress. It has been reported that Bacillus subtilis NCD-2, Bacillus SHZ-24, SHT-15, SMT-24, BHZ-29, Bacillus cereus YUPP-10,

[0004] Bacillus amyloliquefaciens, methylotrophic Bacillus, Trichoderma, Chaetomium globosum, Streptomyces TD3-2-1, Streptomyces alba KF-43-1 and other strains have a certain antagonistic effect on cotton Verticillium wilt. The most widely used is Bacillus subtilis. The Bacillus subtilis NCD-2 developed by Ma Ping's team at Hebei Institute of Plant Protection has a prevention effect of more than 70.00% on cotton Verticillium wilt. It has been developed into a microbial pesticide and officially registered in 2015. However, at present, the main products developed are Bacillus subtilis, and most other biocontrol strains are in the laboratory research stage. Therefore, there is still a lack of new microbial pesticides for the prevention and control of cotton Verticillium wilt in production.

[0005] Bacillus laterosporus has the functions of insecticide, nematode, antibacterial, antitumor, phosphate solubilization, degradation of organic pollutants, production of lysine and various enzyme substances. The research on Bacillus laterosporus has a history of nearly 100 years, and foreign research has been very active, especially in Russia, the United States, the United Kingdom, Japan, Belgium and other countries. Early research focused on the insecticidal activity of Bacillus laterosporus, which is toxic to mosquitoes, beetle larvae, nematodes and mollusks and other invertebrates. Their insecticidal activity is mainly due to the toxicity of proteins contained in their spore inclusions or crystals to many kinds of insects. The small molecule bacterial toxins produced by Bacillus laterosporus can inhibit the hatching of nematode eggs or the development of larvae, and can prevent and control parasitic nematodes of animals and plants. The United States has made Bacillus laterosporus into powder for the prevention and control of animal nematode diseases. my country has also paid more and more attention to this field, and the nematicidal results of Bacillus laterosporus of Yunnan University have also been applied. India has completed the sequencing of the whole genome of three strains of Bacillus laterosporus. NovoTM microbial agent produced by Denmark's Novozymes, a global enzyme giant, also contains Brevibacillus laterosporus.

[0006] In recent years, the research on the antibacterial activity of Brevibacillus laterosporus has attracted more and more attention. The United States, Japan and other countries have reported that Brevibacillus laterosporus can produce a variety of antibacterial substances, including non-peptide spore amines, polyketide compounds, peptide antibiotics and chitinase, etc. The antibacterial substances produced by Brevibacillus laterosporus are mainly used to prevent and treat Gram-positive bacteria and Candida albicans that have developed drug resistance in clinical practice, but there are very few reports on the inhibitory effect on plant pathogens.

[0007] In recent years, the inhibitory effect of Bacillus laterosporus on plant pathogens has attracted more and more attention. my country has successively reported the antibacterial activity of Bacillus laterosporus YMF3.100003, B8, 1·864, BL-21, 2-Q-9, S62-9, X10, A60 and other strains against plant pathogenic fungi and bacteria. It can be seen that although China has carried out preliminary exploration of the biocontrol effect of Bacillus laterosporus on plant diseases in recent years, related research is still in the early stage, and there is no report on microbial fungicide with Bacillus laterosporus as the main ingredient, nor on the inhibitory effect of Bacillus laterosporus on cotton Verticillium dahliae. Summary of the invention

[0008] The purpose of the present invention is to provide a new strain with a strong inhibitory effect on cotton wilt pathogen and a broad antibacterial spectrum. The present invention has carried out the screening of biocontrol bacteria for cotton Verticillium wilt pathogen, and the strain obtained by screening is identified as Brevibacillus laterosporus through morphological characteristics, physiological and biochemical and 16S rDNA analysis. The strain has a strong inhibitory effect on cotton Verticillium wilt pathogen, strong stress resistance, stable antibacterial activity, a broad antibacterial spectrum, and obvious biocontrol potential. It is a very promising biocontrol bacteria. The present invention provides a microbial strain and its pesticide product that are non-toxic to humans and animals, friendly to the ecological environment, and have good preventive effect on cotton Verticillium wilt, and hereby apply for an invention patent for the strain. In order to achieve the purpose of the present invention, the technical scheme of the present invention is as follows:

[0009] The invention provides a Brevibacillus laterosporus M1906. The preservation number of the Brevibacillus laterosporus M1906 is CGMCC NO:28281.

[0010] The Brevibacillus laterosporus M1906 provided by the present invention was isolated and screened from rhizosphere soil samples of plants in Tianshan, Xinjiang in 2021. The strain number is M1906. After microbial taxonomy detection and identification, it was determined to be a potential new strain of Brevibacillus laterosporus. At present, the strain has been deposited in the Budapest Treaty International Microbiological Depository: China General Microbiological Culture Collection Center (CGMCC) before the application date. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is August 29, 2023, and the deposit number is CGMCC NO: 28281.

[0011] The Brevibacillus laterosporus M1906 specifically provided by the present invention belongs to a potential new strain of Brevibacillus laterosporus. After the strain is cultured on a beef extract peptone agar plate at 28° C. for 2 days, the colonies are round, milky white, flat, with relatively neat edges and a slight protrusion in the center, growing in a ring pattern, moist and smooth, and not producing pigment ( Figure 1). The morphological characteristics of the strain are: the bacteria are rod-shaped, with a size of (0.4-0.6)×(2.0-2.5)μm, with multiple flagella around the body, and can move. Gram staining is positive. The spores are oval, lateral, and the cysts are swollen and canoe-shaped. The physiological and biochemical characteristics are: anaerobic, catalase positive, oxidase negative, no amylase, nitrate reductase and L-phenylalanine deaminase are produced, and citrate cannot be utilized. .

[0012] The 16S rDNA sequence of Brevibacillus laterosporus M1906 was amplified using the total genomic DNA of the strain as a template. After PCR amplification and sequencing, the length of the 16S rDNA sequence of strain M1906 was 1427bp. The 16SrDNA sequencing results were compared with the sequences in GenBank. The results showed that the gene sequence homology between strain M1906 and Brevibacillus laterosporus BL-2 (ID: DQ371289.2) was 98.59%, further indicating that Brevibacillus laterosporus M1906 is a new species different from the existing Brevibacillus laterosporus.

[0013] Furthermore, the present invention also provides a method for culturing Brevibacillus laterosporus M1906, wherein Brevibacillus laterosporus M1906 is inoculated into a NA solid culture medium for culturing.

[0014] The invention provides a method for culturing Brevibacillus laterosporus M1906. The culturing temperature is 25-28°C and the pH value is 7.0-7.2.

[0015] Furthermore, the present invention also provides an aqueous solution for preventing and treating cotton Verticillium wilt, wherein the aqueous solution comprises a fermentation product of Brevibacillus laterosporus M1906.

[0016] Furthermore, the present invention also provides a method for preparing the aqueous solution for preventing and treating cotton wilt, which is prepared by the following method: Brevibacillus laterosporus M1906 is transferred to a fresh slant culture medium, cultured at 28°C for 1 day, inoculated into a 500ml triangular flask with a liquid volume of 100ml, and cultured on a rotary shaker at 28°C and a rotation speed of 180r / min for 20h to prepare seed liquid, which is inoculated into a liquid fermentation culture medium at a volume ratio of 3%, cultured at 28°C and a rotation speed of 180r / min for 48 to 72h, and 95% of the bacteria in the fermented liquid form spores, and the number of live spores is 1×108 to 1×109cfu / mL, so as to obtain an aqueous solution.

[0017] Furthermore, the present invention provides the use of Brevibacillus late rosporus M1906 in inhibiting the activity of cotton Verticillium dahliae.

[0018] Furthermore, the present invention provides the use of the aqueous preparation for preventing and treating Verticillium wilt in cotton in inhibiting the activity of Verticillium wilt fungi.

[0019] Furthermore, the present invention provides the use of Brevibacillus late rosporus M1906 in inhibiting the activity of cotton Verticillium dahliae.

[0020] Furthermore, the present invention provides the use of the aqueous preparation for preventing and treating Verticillium wilt in preventing and treating Verticillium wilt in cotton.

[0021] Furthermore, the present invention provides the use of Brevibacillus late rosporus M1906 in preventing and treating cotton Verticillium wilt.

[0022] Furthermore, the present invention provides the use of Brevibacillus late rosporus M1906 in inhibiting plant pathogenic activity.

[0023] The plant pathogens include but are not limited to one or more of cucumber wilt pathogen (Fusarium oxyspo rum), tomato gray mold pathogen (Botrytis cinerea), tomato early blight pathogen (Alternaria solani), jujube black spot pathogen (Alternaria alternata), pepper phytophthora capsici, corn stem rot pathogen (Fusarium graminearum) and astragalus wilt pathogen (Verticillium dahliae).

[0024] By adopting the technical solution provided above, the present invention obtains the following beneficial effects:

[0025] (1) The Brevibacillus laterosporus M1906 of the present invention is non-toxic to humans and animals, friendly to the ecological environment, and can be prepared into a microbial strain with good prevention effect on cotton Verticillium wilt and its pesticide product, and has a strong inhibitory effect on cotton Verticillium wilt. The inhibition rate of the sterile fermentation filtrate on cotton Verticillium wilt is 91.37%, which can cause the mycelium of cotton Verticillium wilt to be deformed, broken, cell membrane changed, protoplast exudation, mycelium ablation, etc. The strain M1906 can produce chitinase, and the inhibition rate of its sterile fermentation filtrate on the production of sclerotia of cotton Verticillium wilt is 100%; the inhibition rate of sclerotium germination is 49%, and the sclerotium germination can be delayed. The mycelium growth amount of the treated group after sclerotium germination is significantly less than that of the control group, and the mycelium growth rate is slow. The results of the potted plant test showed that spraying M1906 solution before inoculating cotton Verticillium wilt had a control effect of 82.52% on cotton Verticillium wilt; spraying M1906 solution after inoculating cotton Verticillium wilt had a control effect of 82.52% on cotton Verticillium wilt.

[0026] (2) The aseptic fermentation filtrate of Brevibacillus laterosporus M1906 described in the present invention has a broad antibacterial spectrum and has a certain inhibitory effect on plant pathogens such as cucumber wilt fungus (Fusarium oxyspo rum), tomato gray mold fungus (Botrytiscinerea), tomato early blight fungus (Alternaria solani), jujube black spot fungus (Alternaria alternata), pepper phytophthora capsici, corn stem rot fungus (Fusarium graminearum) and astragalus wilt fungus (Verticillium dahliae), thereby providing a new idea and method for agricultural disease prevention and control, and having great potential value for promoting the development of green agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown is the colony morphology of strain M1906.

[0028] Figure 2 Shown is the phylogenetic tree of strain M1906. DETAILED DESCRIPTION

[0029] The following examples are used to further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention.

[0030] The main raw and auxiliary materials and equipment involved in the present invention are:

[0031] Main reagents: beef extract peptone agar medium (NA), fermentation medium, PDA medium, chitin agar medium, etc.

[0032] Main instruments: high pressure steam sterilizer (HIRAYAMA, HVE-50), clean bench (Zhejiang Fuxia, model SW-CJ-1F), biological microscope (Nikon, Japan, model Eclipse Ci-L), constant temperature incubator (Shanghai Yiheng, model bluepard), microplate reader (BioTek, USA, model Epoch2), centrifuge (Selo Czech, model CF1524R), TS constant temperature oscillation box (Shanghai Tiancheng, model TS-2102C).

[0033] The Brevibacillus laterosporus M1906 used in the present invention is referred to as the M1906 strain.

[0034] The culture medium configuration designed in this application is as follows:

[0035] Beef extract peptone agar medium (NA): 3g beef extract, 5g peptone, 5g sodium chloride, 20g agar, 1000ml distilled water, pH 7.0-7.2.

[0036] Fermentation medium: 3g beef extract, 5g peptone, 5g sodium chloride, 1000ml distilled water, pH 7.0-7.2

[0037] PDA culture medium: including 6 g potato powder, 20 g glucose, 20 g agar and 1000 ml distilled water, pH = 5.6 ± 0.2.

[0038] Chitin agar medium: 0.5g K2HPO4, 0.5g KH2PO4, 0.5g MgSO4·7H2O, 0.1g FeSO4·7H2O, 0.1g ZnSO4·7H2O, 500mL 1% colloidal chitin, 500mL distilled water, 17g agar, pH 7.2, sterilize at 121℃ for 20min, cool to about 50℃ and pour into plates.

[0039] The cucumber wilt pathogen (Fusarium oxysporum), tomato gray mold pathogen (Botrytis cinerea), tomato early blight pathogen (Alternaria solani), jujube black spot pathogen (Alternaria alternata), pepper phytophthora capsici, corn stem rot pathogen (Fusarium graminearum), astragalus wilt pathogen (Verticillium dahliae) and cotton wilt pathogen (Verticillium dahliae Kleb) involved in this application are owned by this laboratory.

[0040] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0041] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0042] Example 1: Screening, isolation and identification of strains

[0043] Isolation and culture of strains: The culture medium is beef extract peptone agar medium (NA): 3g beef extract, 5g peptone, 5g sodium chloride, 20g agar, 1000ml distilled water, pH 7.0-7.2. The dilution coating method was used for separation. Weigh 10g of the newly collected soil, pour it into a conical flask containing 90ml sterile water, shake for 10min to form a 1 / 10 concentration suspension, and place it in a water bath at 80℃ for 20min. Take 1ml of the supernatant and prepare 10 -7 or 10 -8The concentration of dilution is reserved. Pour the melted NA into a sterilized culture dish, wait for it to solidify, take 50μl of the diluent and drop it on the surface of the solidified culture medium, spread it evenly with a sterile coating stick, and invert the culture dish in a 28℃ constant temperature box for 1-2 days. After the colonies grow, pick the colonies that are round, milky white, flat, with neat edges, slightly raised in the center, moist and smooth, and do not produce pigments. The single colony is streaked and cultured on beef extract peptone agar medium, and the separation and purification are repeated 3 times. The single colony finally obtained is named M1906, and physiological and biochemical identification and 16S rDNA sequencing are performed. The sequence length of 16S rDNA of strain M1906 is 1427bp. The 16S rDNA sequencing results were compared with the sequences in GenBank, and the results showed that the gene sequence homology of strain M1906 and Brevibacillus laterosporus BL-2 (ID: DQ371289.2) was 98.59%. According to the colony morphological characteristics and physiological and biochemical identification of Bergey's Bacterial Identification Manual (8th Edition), M1906 was determined to be Brevibacillus laterosporus, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee with a deposit number of 28281. In order to determine the control effect of the strain of the present invention and its product on Verticillium dahliae, the following in vitro antibacterial test and indoor bioassay were performed.

[0044] Example 2: Determination of the inhibitory effect of Brevibacillus lateros porus M1906 on cotton Verticillium wilt pathogen by confrontation culture

[0045] Take a 6mm diameter cotton Verticillium wilt fungus plate and place it in the center of a culture dish containing PDA. At 3cm above and below the plate, draw two parallel straight lines with an inoculation loop stained with strain M1906. Culture at 28℃ for 3 days, measure the width of the inhibition zone, and observe the growth of cotton Verticillium wilt fungus hyphae in the inhibition zone under a microscope. Continue to culture for 30 days until the cotton Verticillium wilt fungus produces sclerotia, and observe the persistence of the inhibitory effect. The experimental results show that strain M1906 can strongly inhibit the growth of cotton Verticillium wilt fungus. Microscopic observations show that the hyphae of cotton Verticillium wilt fungus in the inhibition zone are deformed and broken, the cell membrane changes, the protoplast exudation, and the hyphae ablation. When the cotton Verticillium wilt fungus produces sclerotia after 30 days of culture, the hyphae in the inhibition zone no longer grows, indicating that the antibacterial effect of strain M1906 is persistent and stable.

[0046] Example 3: Determination of antibacterial activity of Brevibacillus laterosporus M1906 fermentation broth

[0047] Preparation of sterile fermentation filtrate: Transfer Brevibacillus laterospo rus M1906 to fresh slant medium, culture at 28℃ for 1 day, inoculate into 250ml Erlenmeyer flask with 50ml liquid, shake and culture on a rotary shaker at 28℃ and 150r / min for 40h to obtain fermentation stock solution, centrifuge the fermentation stock solution at 10000r / min for 20min, filter it in a bacterial filter, obtain sterile fermentation filtrate and determine its antibacterial activity. Fermentation medium: 3g beef extract, 5g peptone, 5g sodium chloride, 1000ml distilled water, pH 7.0-7.2.

[0048] The antibacterial activity of the sterile fermentation filtrate of Brevibacillus laterosporus M1906 against Verticillium wilt of cotton was determined by the cup-disc method: a 6 mm diameter cotton Verticillium wilt bacterial disc was inverted in the center of a PDA plate, and a sterilized Oxford cup was placed around it. 100 μl of the sterile fermentation filtrate was added to the Oxford cup, and the mycelium was cultured in a 28°C incubator until the dish was full. The diameter of the inhibition zone around the Oxford cup was measured, and sterile water was used as a control. Inhibition rate (%) = colony diameter (CK) - colony diameter (treatment group) / colony diameter (CK).

[0049] The test results show that the aseptic fermentation filtrate of Brevibacillus laterosporus M1906 has a strong inhibitory effect on cotton Verticillium wilt pathogen, with an inhibition rate of 91.37%.

[0050] Example 4: Potted plant experiment to determine the control effect of the aqueous solution prepared by Brevibacillus laterosporus M1906 on cotton Verticillium wilt

[0051] Preparation of Brevibacillus laterosporus M1906 aqueous solution: Brevibacillus laterosporus M1906 was transferred to a fresh slant medium, cultured at 28°C for 1 day, inoculated into a 500ml Erlenmeyer flask with a liquid volume of 100ml, and cultured on a rotary shaker at 28°C and a speed of 180r / min for 20h to prepare a seed solution, which was inoculated into a liquid fermentation medium at a volume ratio of 3%, cultured at 28°C and a speed of 180r / min for 48 to 72h, until 95% of the bacteria in the fermentation liquid formed spores, and the number of live spores was 1×10 8 ~1×10 9 cfu / mL, and the biocontrol agent is obtained for use.

[0052] Rinse the 12cm×13cm nutrient pot with clean water, wipe it with 70% alcohol, and dry it. Put 0.4kg of sterilized soil in each nutrient pot, sow 6 treated cotton seeds in each pot, and plant at a depth of 2cm-3cm. Put the nutrient pot in a 20cm×14cm plastic tray, place 3 nutrient pots in each plastic tray, add an equal amount of tap water to each plastic tray, and let the soil absorb water naturally. Put the plastic tray in an artificial climate box for cultivation, and the cultivation conditions are: 14 hours during the day, temperature 25℃, humidity 50%, light 60%; 10 hours at night, temperature 20℃, humidity 70%, light 0%. During the seedling period, water the seedlings with an equal amount of tap water every 5 days to facilitate germination. Tender buds will grow around 7 days, and the cotton seedlings will grow to two leaves and one heart around 15 days. The control effect of spraying M1906 solution before inoculating cotton Verticillium wilt was 82.52%; the control effect of spraying M1906 solution after inoculating cotton Verticillium wilt was 76.65%.

[0053] Table 1: The efficacy of M1906 aqueous solution against cotton Verticillium wilt

[0054] deal with Disease index Prevention effect (%) Apply M1906 solution before vaccination 3.18 82.52 After vaccination, M1906 aqueous solution was administered 4.25 76.65 Comparison 18.2 ——

[0055] Example 5: Effect of sterile fermentation filtrate of Brevibacillus laterosporus M1906 on sclerotial production and germination of cotton Verticillium dahliae

[0056] Experimental treatment method: Using the toxic medium method, add the sterile fermentation filtrate of Brevibacillus laterosporus M1906 in a ratio of 9:1 to the PDA medium at 50℃, mix thoroughly, make plates, and after condensation, move the cotton wilt pathogen cake with a diameter of 8mm to the center of the plate. Repeat 5 times, and use an equal amount of PDA medium without adding sterile fermentation filtrate as a control. After culturing at 25℃ for 3 days, observe the results and calculate the inhibition rate using the mycelium growth rate inhibition method.

[0057] Rinse the Verticillium wilt plate grown for about 14 days with sterile water 6 to 8 times, punch holes in the dense microsclerotia with a 6mm puncher, transfer the punched cake to a sterile mortar, grind to a homogenate, transfer to a 1.5mL centrifuge tube with a pipette, water bath at 47℃ for 5min, centrifuge at 12,000r / min for 5min, discard the supernatant, collect the precipitate, which is the microsclerotia, add 200μL of M1906 fermentation filtrate fermented for 72h on the PDA solid plate, spread it evenly with a spreader, inoculate 20 sclerotia in each culture dish, observe the germination of microsclerotia after 5d, and add an equal amount of PDA liquid as a control. Repeat each treatment 3 times. Culture at 25℃, observe and record the germination of sclerotia.

[0058] The results showed that the Verticillium wilt pathogen that was not treated with the sterile fermentation filtrate of Brevibacillus laterosporus M1906 began to form sclerotia after 20 days of constant temperature culture at 25℃, and a large number of sclerotia were produced at 23 days, while the Verticillium wilt pathogen that was treated with the sterile fermentation filtrate of Brevibacillus laterosporus M1906 did not produce sclerotia at all, and still did not produce sclerotia after one month of observation. It can be seen that the inhibition rate of the sterile fermentation filtrate of Brevibacillus laterosporus M1906 on the production of sclerotia of Verticillium wilt pathogen in cotton reached 100%. The results of the sclerotium germination test showed that the sclerotia of Verticillium wilt pathogen treated with sterile water began to germinate after 1 day of culture at 25℃, and all germinated within 7 days, and produced a large number of mycelium. However, the germination of sclerotia treated with sterile fermentation filtrate of Brevibacillus laterosporus M1906 was delayed or did not germinate. Sclerotia rarely germinated in the first two days, and only a few sclerotia germinated after 7 days of continuous culture. The inhibition rate of sclerotium germination was 42%, and sclerotium germination could be delayed. In addition, the amount of mycelium growth after sclerotium germination in the treatment group was significantly less than that in the control group, and the mycelium growth rate was slow.

[0059] Example 6: Determination of chitinase production by Brevibacillus laterosporus M1906

[0060] Chitin agar medium: 0.5g K2HPO4, 0.5g KH2PO4, 0.5g MgSO4·7H2O 0.1g, FeSO4·7H2O, 0.1g ZnSO4·7H2O, 500mL 1% colloidal chitin, 500mL distilled water, 17g agar, pH7.2, sterilized at 121℃ for 20min, cooled to about 50℃ and poured into a plate. An Oxford cup was placed on the plate, and the sterile fermentation filtrate of Brevibacillus laterosporus M1906 was added to the Oxford cup, and observed after culturing at 28℃ for 5 days. The results showed that the sterile fermentation filtrate of Brevibacillus laterosporus M1906 produced an obvious transparent circle around the Oxford cup after culturing on the chitin agar plate for 5 days, indicating that the strain can produce chitinase.

[0061] Example 7: Broad-spectrum antibacterial test of sterile fermentation filtrate of Brevibacillus laterosporus M1906

[0062] Cucumber wilt pathogen (Fusarium oxysporum), tomato gray mold pathogen (Botrytis cinerea), tomato early blight pathogen (Alternaria solani), jujube black spot pathogen (Alternaria alternata), pepper phytophthora capsici, corn stem rot pathogen (Fusarium graminearum) and astragalus wilt pathogen (Verticillium dahliae) were mixed in a PD A plate, placed on an Oxford cup, 200 μl of sterile fermentation filtrate of Brevibac illus laterosporus M1906 was added to each cup, and the plates were cultured in an incubator at 28°C until the plates were fully grown with colonies, and the diameter of the inhibition zone was measured. Each treatment was repeated 3 times and the average value was taken.

[0063] As shown in Table 2: The aseptic fermentation filtrate of Brevibacillus laterosporus M1906 has a certain inhibitory effect on a variety of plant pathogenic fungi and bacteria, among which the inhibitory effect on cotton Verticillium dahliae is the strongest, followed by tobacco target spot pathogen and pepper phytophthora.

[0064] Table 2: Inhibitory effect of Brevibacillus laterosporus M1906 on plant pathogens

[0065] Plant pathogenic fungi Diameter of inhibition zone (cm) Cucumber wilt pathogen Fusarium oxysporum 51.2 Botrytis cinerea 37.4 Tomato early blight pathogen Alternaria solani 35.5 Alternaria alternata 37.2 Phytophthora capsici 29.5 Maize stalk rot pathogen Fusarium graminearum 49.1 Verticillium dahliae 46.7

[0066] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A strain of Brevibacillus laterosporus M1906, characterized in that: The microbial preservation number of the Brevibacillus laterosporus M1906 is CGMCC NO:28281.

2. The method for culturing Brevibacillus laterosporus M1906 according to claim 1, characterized in that: The method comprises the steps of inoculating Brevibacillus laterosporus M1906 into a NA solid culture medium for culture.

3. The method for culturing Brevibacillus laterosporus M1906 according to claim 2, characterized in that: The culture conditions are: temperature 25-28°C, pH 7.0-7.

2.

4. An aqueous solution for preventing and treating cotton Verticillium wilt, characterized in that: The aqueous preparation comprises Brevibacillus laterosporus M1906 fermentation product.

5. The method for preparing an aqueous solution for preventing and treating Verticillium wilt of cotton according to claim 4, characterized in that: The preparation method is as follows: Brevibacillus laterosporus M1906 is transferred to a fresh slant medium, cultured at 28°C for 1 day, inoculated into a 500ml Erlenmeyer flask with a liquid volume of 100ml, and cultured on a rotary shaker at 28°C and a speed of 180r / min for 20h to prepare a seed solution, which is then inoculated into a liquid fermentation medium at a volume ratio of 3%, cultured at 28°C and a speed of 180r / min for 48 to 72h, until 95% of the bacteria in the fermentation liquid form spores, and the number of live spores is 1×10 8 ~1×10 9 cfu / mL, and the aqueous solution was obtained.

6. Use of Brevibacillus laterosporus M1906 according to claim 1 in inhibiting plant pathogenic activity.

7. Use of the Brevibacillus laterosporus M1906 according to claim 1 or the aqueous solution according to claim 5 in inhibiting the activity of Verticillium dahliae of cotton.

8. Use of the Brevibacillus laterosporus M1906 according to claim 1 or the aqueous solution according to claim 5 in preventing and controlling cotton Verticillium wilt.

9. The use of Brevibacillus laterosporus as claimed in claim 6 in inhibiting the activity of plant pathogens, wherein the plant pathogens include but are not limited to one or more of cucumber wilt pathogen (Fusarium oxysporum), tomato gray mold pathogen (Bo trytis cinerea), tomato early blight pathogen (Alternaria solani), jujube black spot pathogen (Alte rnaria alternata), pepper phytophthora capsici, corn stem rot pathogen (Fusarium graminearum) and Astragalus wilt pathogen (Verticillium dahliae).

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