Strain SF5 for resisting diseases and promoting growth of cotton as well as preparation method and application of strain SF5

Abstract

The invention relates to a disease-resistant and growth-promoting strain SF5 for cotton as well as a preparation method and application of the strain SF5. The strain SF5 for disease resistance and growth promotion of cotton is classified and named as bacillus methylotrophicus, the preservation date is December 3, 2020, the preservation address is China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.21291. The strain SF5 for disease resistance and growth promotion of cotton has the advantages that the preservation number is CGMCC NO.21291; the disease-resistant and growth-promoting strain SF5 for cotton can be well applied to prevention and treatment of cotton verticillium wilt, sunflower sclerotinia rot and bergamot pear rot.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and in particular to a cotton disease-resistant and growth-promoting strain SF5, a preparation method and an application thereof. Background Art

[0002] According to statistics, Xinjiang accounted for over 90% of China's total cotton production in 2023, making cotton cultivation a pillar of Xinjiang's agriculture. Verticillium wilt is one of the two major global cotton diseases, impacting cotton production globally and in China to varying degrees. Verticillium wilt is caused by Verticillium dahliae Kleb. and Verticillium alboatrum Reinkeet Berthold, with the former being the predominant disease in China. Symptoms begin to appear when cotton has three to five true leaves, with widespread disease in the field after bud formation in the middle and late stages of growth. Symptoms include pale yellow patches on the margins and between the veins of the lower leaves, which gradually spread and lose their green color, while the main vein and surrounding areas remain green. Affected leaves develop palmate mottles, thicken the mesophyll, curl downward at the leaf margins, and gradually fall off from the bottom to the top, leaving only a few leaflets at the top. Buds are sparse and crack prematurely. Grade II or higher can cause a 20%-30% or more reduction in cotton yield, and in severe cases, a 80% or more reduction in cotton yield. Therefore, the prevention and control of cotton Verticillium wilt is one of the important ways to increase cotton yield.

[0003] Current methods for controlling Verticillium wilt in cotton, in addition to selecting disease-resistant varieties, primarily involve scientific field management and chemical control, each with its own advantages, disadvantages, and limitations. Scientific field management includes deep tillage, timely cultivation, balanced nitrogen, phosphorus, and potassium fertilization, and controlled field humidity. Deep tillage after cotton harvest can reduce the number of Verticillium wilt pathogens in the tillage layer, reducing the incidence and severity of the disease. Timely cultivation improves the ecological environment of the cotton field, preventing overwatering and enhancing the cotton's nutrient transport capacity, thus reducing the disease. Balanced nitrogen, phosphorus, and potassium fertilization, particularly reducing nitrogen and increasing phosphorus and potassium, can prevent excessive growth of cotton, improve ventilation, and facilitate field control. Drip irrigation, which controls the amount of water used in the field, can effectively reduce humidity in the cotton field. A relative humidity below 80% effectively controls the disease.

[0004] Chemical control primarily involves the use of fungicides or nutrients combined with growth-promoting regulators to promote growth and seedling vigor, effectively controlling cotton disease. Immunogenic inducers such as amino oligosaccharides and brassinolide are commonly used for seed coating; ethyl allicin, 70% sodium disulfone, 3% polyoxin, 36% cotton broom, and 70% benomyl are also used to control cotton field outbreaks; nutrient agents such as potassium dihydrogen phosphate and potassium fulvic acid combined with growth-promoting regulators such as brassinolide and sodium nitrophenolate are also used to regulate cotton growth and reduce the incidence of disease. While chemical control offers strong long-term effectiveness and high stability, excessive use of chemical agents can accumulate in the food chain, degrading soil quality and harming human health. Long-term use of chemical agents can kill beneficial bacteria and induce drug resistance in pathogens, posing potential safety risks.

[0005] Therefore, developing environmentally friendly, low-cost, highly effective, risk-free, and sustainable microbial control methods for cotton Verticillium wilt is an urgent challenge. Currently, although some biocontrol agents for cotton Verticillium wilt (primarily Bacillus subtilis) have been studied and applied, their effectiveness in field control varies due to the unique biological characteristics of cotton Verticillium wilt. Summary of the Invention

[0006] In view of this, the main purpose of the present invention is to provide a cotton disease-resistant and growth-promoting strain SF5 and its preparation method and application, which can be well applied to the prevention and treatment of cotton wilt, sunflower sclerotinia wilt, and fragrant pear rot.

[0007] The purpose of the present invention and the solution of its technical problems are achieved by adopting the following technical solutions. A cotton disease-resistant and growth-promoting strain SF5 proposed in the present invention is classified and named as methylotrophic Bacillus methylotrophicus, with a preservation date of December 3, 2020, and a preservation address of the General Microbiology Center of the China Culture Collection of Microorganisms (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with a preservation number of CGMCC NO.21291.

[0008] The purpose of the present invention and the solution to its technical problems can also be achieved by adopting the following technical solutions. The present invention proposes a method for preparing a cotton disease-resistant growth-promoting strain SF5, comprising the following steps:

[0009] Step 1: In a cotton Verticillium wilt mixed disease field, 5 to 10 uninfected cotton plants are selected for isolation;

[0010] Step 2: For isolation, cotton hypocotyls are sterilized with 75% (v / v) alcohol, peeled, and the peeled stems are cut into 0.5 cm segments using sterilized pruning shears. The segments are cultured on potato agar for 7 to 10 days to obtain a culture mixture of fungi and bacteria. Bacillus is streaked onto a solid conidia medium using a sterilized pick, cultured overnight, and a single colony is picked and cultured on a new solid conidia medium plate to isolate the Bacillus strain.

[0011] Step 3: inoculate the isolated Bacillus strain into a liquid spore-forming culture medium, culture at 180-220 rpm and 26-30° C. for 12-24 hours to obtain a Bacillus bacterial solution; add glycerol to a portion and store at -80° C.; and adjust the concentration of a portion to an OD value of 0.8-1.0 for later use;

[0012] Step 4: Activate the plant wilt pathogen stored in a refrigerator at -80°C on a PDA plate and culture at 26-30°C for 48-72 hours; inoculate the activated pathogen into Czapek medium and shake at 180-220 rpm and 26-30°C for 48-72 hours to obtain a pathogenic fungal spore suspension with a concentration of 1×10 6 ~1×10 8 spores / mL;

[0013] Step 5: Pipette 10 μl of the pathogenic fungal spore suspension obtained in step 4 and drop it in the center of the PDA plate culture medium. Then, prick 10 μl of the Bacillus culture with an OD value of 0.8 to 1.0 obtained in step 3 and drop it on the edge of the plate. Drop three points on each plate to form an equilateral triangle. After the culture liquid is dried, culture it at a constant temperature of 26 to 30°C for 48 to 72 hours, observe and measure the size of the inhibition zone. Repeat the experiment three times and select the strain with the largest inhibition zone. Pick a single colony and culture it on a new spore-forming culture medium plate to isolate and obtain a purified methylotrophic Bacillus, namely the cotton disease-resistant and growth-promoting strain SF5.

[0014] The purpose of the present invention and the solution to its technical problems can be further achieved by adopting the following technical measures.

[0015] Preferably, in the aforementioned method for preparing the disease-resistant and growth-promoting cotton strain SF5, in step 2, the length of the hypocotyl is 10 cm.

[0016] Preferably, in the preparation method of the aforementioned cotton disease-resistant and growth-promoting strain SF5, in step 2, the formula of the spore-forming culture medium is as follows: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, 15.0 g agar, pH 7.0.

[0017] Preferably, in the preparation method of the aforementioned cotton disease-resistant and growth-promoting strain SF5, in step three, the formula of the liquid spore-forming culture medium is as follows: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, pH 7.0.

[0018] Preferably, in the aforementioned method for preparing the disease-resistant and growth-promoting cotton strain SF5, in step three, the volume ratio of the glycerol to the Bacillus bacterial liquid is 2-3:7-8.

[0019] Preferably, in the preparation method of the aforementioned cotton disease-resistant and growth-promoting strain SF5, in step four, the formula of the Cha's medium is as follows: 3 g sodium nitrate, 1 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.5 g potassium chloride, 0.01 g ferrous sulfate, 30 g sucrose, 20 g agar, and 1000 mL distilled water.

[0020] Preferably, in the preparation method of the aforementioned cotton disease-resistant and growth-promoting strain SF5, in step five, the formula of the PDA plate culture medium is as follows: 200g potato, 20g glucose, 3g KH2PO4, 1.5g MgSO4·7H2O, 15g agar powder, and 1L distilled water.

[0021] Preferably, in the aforementioned method for preparing the disease-resistant and growth-promoting cotton strain SF5, in step five, the three points are all circles with a diameter of 9 mm.

[0022] The purpose of the present invention and the solution of its technical problems can also be achieved by adopting the following technical solutions. A microbial biocontrol agent proposed in the present invention contains a cotton disease-resistant and growth-promoting strain SF5. The cotton disease-resistant and growth-promoting strain SF5 is classified and named as methylotrophic Bacillus methylotrophicus, with a deposit date of December 3, 2020, and a deposit address of the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCCNO.21291.

[0023] The purpose of the present invention and the solution to the technical problem can also be achieved by adopting the following technical solutions. The present invention proposes a method for preparing a microbial biocontrol agent, comprising the following steps:

[0024] The cotton disease-resistant and growth-promoting strain SF5 stored in the refrigerator was activated on a solid conidia medium and cultured at 26-30°C for 12-24 hours. A single colony was picked and shaken in a liquid conidia medium at 180-220 rpm and 26-30°C for 48-72 hours. The OD value was adjusted to 0.8-1.0 to obtain a seed solution. The seed solution was inoculated into the liquid conidia medium at an inoculum size of 1-3% (v / v), and cultured in a shaker at 26-30°C and 180-220 rpm to obtain a total viable bacterial concentration of 1×109 ~1×10 10 CFU / mL of microbial biocontrol agents.

[0025] The purpose of the present invention and the technical problems solved therein can also be achieved by adopting the following technical solutions: The present invention proposes the use of a microbial biocontrol agent in cotton disease resistance.

[0026] The purpose of the present invention and the solution to its technical problems can be further achieved by adopting the following technical measures.

[0027] Preferably, the application of the aforementioned microbial biocontrol agent in cotton disease resistance comprises the following steps: before use, diluting the microbial biocontrol agent to 1×10 8 CFU / mL~1×10 9 CFU / mL, and then applied to the cotton field with water drops for biocontrol treatment.

[0028] By means of the above technical solution, the cotton disease-resistant and growth-promoting strain SF5 provided by the present invention and its preparation method and application have at least the following advantages:

[0029] (1) The microbial biocontrol agent provided by the present invention contains a specific strain SF5 that inhibits the growth of cotton Verticillium wilt pathogens. Its antibacterial effect is very obvious. In indoor flat plate confrontation culture, after 18 days of culture, the antibacterial rate against cotton Verticillium dahliae can reach 88.5%. Under potted conditions, the antibacterial rate against cotton Verticillium dahliae can reach 78.2%, which can significantly inhibit the production of sclerotia.

[0030] (2) The microbial biocontrol agent provided by the present invention has a simple process, short culture time and low cost. The microbial biocontrol agent for preventing and controlling Verticillium dahliae in cotton can be obtained through a simple, fast and low-cost preparation method.

[0031] (3) The microbial biocontrol agent provided by the present invention does not cause environmental pollution, is green and harmless, can reduce the application of other chemical pesticides, and is an environmentally friendly product.

[0032] (4) The microbial biocontrol agent provided by the present invention also has the disease resistance effect of preventing and controlling sunflower sclerotinia disease and fragrant pear rot; enhances plant stress resistance, increases yield and improves quality; and thus achieves the functions of promoting growth and increasing production, saving costs and increasing efficiency.

[0033] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is one of the diagrams for the separation and purification of the SF5 strain in Example 1 of the present invention;

[0035] Figure 2 This is the second diagram of the inhibitory effect of SF5 on dahlia verticillium disease in Example 2 of the present invention.

[0036] Figure 3 This is the third diagram of the inhibitory effect of SF5 of Example 2 of the present invention on sunflower sclerotinia.

[0037] Figure 4 This is the fourth diagram of the inhibitory effect of SF5 on fragrant pear rot in Example 2 of the present invention.

[0038] Figure 5 This is the fifth diagram of the phosphorus solubilization effect of SF5 on soil organic phosphorus in Example 4 of the present invention. DETAILED DESCRIPTION

[0039] To further illustrate the technical means and efficacy employed by the present invention to achieve its intended objectives, the following, in conjunction with preferred embodiments, describes in detail the specific embodiments, structure, features, and efficacy of the cotton disease-resistant and growth-promoting strain SF5, its preparation method, and its application. In the following description, different references to "one embodiment" or "embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.

[0040] The following materials or reagents, unless otherwise specified, were commercially available.

[0041] Some embodiments of the present invention provide a cotton disease-resistant and growth-promoting strain SF5, which is classified and named as Bacillus methylotrophicus, the preservation date is December 3, 2020, the preservation address is the General Microbiology Center of China Culture Collection of Microorganisms (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and the preservation number is CGMCC NO.21291.

[0042] The above-mentioned cotton disease-resistant and growth-promoting strain SF5 was obtained by screening, identification and propagation in a mixed disease nursery of cotton fusarium wilt in Xinjiang in 2020. It is a plant endophyte.

[0043] like Figure 1 As shown, some embodiments of the present invention also provide a method for preparing a cotton disease-resistant and growth-promoting strain SF5, comprising the following steps:

[0044] Step 1: In a mixed cotton blight and verticillium wilt disease field, select 5 to 10 uninfected (vascular bundles not infected by Verticillium dahliae) and resistant cotton plants, immediately bring them back to the laboratory, and separate them from the hypocotyl tissue of the cotton plants in a clean bench, i.e., step 2.

[0045] Step 2: When separating, cotton hypocotyls (about 10 cm) were taken, surface-sterilized with 75% (v / v) alcohol, peeled, and the peeled stems were cut into 0.5 cm segments using sterilized (120° C., 20 min) pruning shears. The segments were cultured on potato agar (PDA) for 7 to 10 days to obtain a culture mixture of fungi and bacteria. The culture mixture of fungi and bacteria was streaked onto a solid conidia medium (formula: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, 15.0 g agar, pH 7.0) using a sterile pick needle, cultured overnight at 30°C to 37°C, and a single colony was picked and cultured on a new solid conidia medium (formula: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, 15.0 g agar, pH 7.0) plate at 30°C to 37°C to isolate a Bacillus strain.

[0046] Step 3: The isolated Bacillus strain is inoculated into a liquid conidia culture medium (formula: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, pH 7.0), and cultured at 180-220 rpm and 26-30° C. for 12-24 hours to obtain a Bacillus culture solution. A portion is added with glycerol (the volume ratio of glycerol to culture solution is 2-3:7-8) and stored at -80° C.; a portion is adjusted to an OD value of 0.8-1.0 for later use.

[0047] Step 4: Activate the plant wilt pathogen stored in a refrigerator at -80°C on a PDA plate and culture at 26-30°C for 48-72 hours. The activated pathogen is inoculated into Czapek medium (whose formula is: 3g sodium nitrate, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate (MgSO4·7H2O), 0.5g potassium chloride, 0.01g ferrous sulfate, 30g sucrose, 20g agar, and 1000mL distilled water) and shake-cultured at 180-220rpm and 26-30°C for 48-72 hours to obtain a pathogenic fungal spore suspension with a concentration adjusted to 1×10 6 ~1×10 8 Spores / mL are all acceptable.

[0048] Step 5: Use a 10 μl pipette to draw 10 μl of the fungal spore suspension and drop it onto the center of a PDA plate (the formula is: 200g potatoes, 20g glucose, 3g KH2PO4, 1.5g MgSO4·7H2O, 15g agar powder, 1L distilled water; the preparation method is: peel the potatoes, cut into slices, boil for 10 minutes, then filter through gauze, and add glucose and agar). Then, draw 10 μl of Bacillus culture with an OD value of 0.8-1.0 and drop it on the edge of the plate. Drop three points on each plate to form an equilateral triangle. After the culture liquid is dried, it is incubated at a constant temperature of 26°C to 30°C for 48-72 hours. The size of the inhibition zone is observed and measured. The experiment is repeated three times, and the strain with the largest inhibition zone is selected. A single colony is cultured on a new spore-forming medium plate to isolate and obtain a purified methylotrophic Bacillus, namely the cotton disease-resistant and growth-promoting strain SF5.

[0049] During specific implementation, in step 5, the existing method for identifying the antibacterial effect of fungal pathogens requires beating the bacteria cake, and then placing the bacteria cake in the center of the culture dish. It is required to punch the bacteria cake in a place where the growth is uniform and consistent to prevent errors in the experimental results. A large number of fungal plates need to be cultured in advance, and when using a punch to punch the bacteria cake, the bacteria cake is likely to remain in the punch, making it difficult to achieve rapid operation of large quantities of samples. In order to solve the above problems, for spore fungi produced by shaking culture, the method of the present invention adopts the suspension titration method, using a 10μl pipette to draw 10μl of fungal spore suspension, and dripping it on the plate just to form a dot with a diameter of 9mm, without the need for beating the bacteria cake; the biocontrol bacteria inoculation also adopts the titration method, which is convenient to operate and saves time; the concentration of the spore suspension is consistent, and the dots are uniform and consistent.

[0050] Some embodiments of the present invention further provide a microbial biocontrol agent, which contains the above-mentioned cotton disease-resistant and growth-promoting strain SF5.

[0051] Some embodiments of the present invention also provide a method for preparing a microbial biocontrol agent, comprising the following steps:

[0052] The microbial biocontrol preparation is obtained after the strain SF5 is activated, seed cultured and expanded cultured.

[0053] In some optional embodiments, the preparation method specifically includes the following steps: activating the SF5 strain stored in a refrigerator on a solid conidia medium (whose formula is: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, 15.0 g agar, pH 7.0), culturing at 26-30° C. for 12-24 h, picking a single colony and shaking it in a liquid conidia medium (whose formula is: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, pH 7.0), shaking it at 180-220 rpm, 26-30° C. for 48-72 h, adjusting the OD value to 0.8-1.0, and obtaining a seed solution; inoculating the seed solution into the liquid conidia medium at an inoculum size of 1-3% (v / v), and culturing it in a shaking table at 26-30° C. and 180-220 rpm to obtain a total viable bacteria concentration of 1×10 9 ~1×10 10 CFU / mL of finished microbial biocontrol preparations.

[0054] Some embodiments of the present invention also provide a use of the above-mentioned microbial biocontrol agent in cotton disease resistance.

[0055] The specific method is: before use, dilute the prepared microbial biocontrol agent to 1×10 8 CFU / mL~1×10 9 CFU / mL, and then applied to the cotton field with water drops for biocontrol treatment.

[0056] In the above technical scheme, the present invention screens microbial strains suitable for the prevention and control of Xinjiang cotton fields in cotton fields with mixed verticillium wilt and fusarium wilt in Xinjiang, in order to provide technical support for the prevention and control of Xinjiang cotton fields and develop effective microbial biocontrol products.

[0057] The specific implementation methods of the present invention are further described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by technicians in this field based on the above-mentioned contents of the present invention still fall within the scope of protection of the present invention.

[0058] Unless otherwise specified, the materials and reagents mentioned below are commercially available products familiar to those skilled in the art. Unless otherwise specified, the methods described are all well-known methods in the art. Unless otherwise defined, technical or scientific terms used shall have the same meanings as those commonly understood by those skilled in the art.

[0059] Example 1: Preparation of Bacillus methylotrophicusSF5

[0060] The cotton disease-resistant and growth-promoting strain SF5 of this example was obtained by screening, identification, and propagation in a cotton fusarium wilt mixed disease field in Xinjiang in 2020. It is an endophytic strain. The preparation method is as follows:

[0061] Step 1: Select 5 cotton plants that are not infected and have good resistance (the vascular bundles are not infected by Verticillium dahliae) from a mixed disease field of cotton verticillium wilt, and immediately bring them back to the laboratory for isolation.

[0062] Step 2: When separating, cotton hypocotyls (about 10 cm) were taken, surface disinfected with 75% (v / v) alcohol, peeled, and the peeled stems were cut into 0.5 cm segments with sterilized pruning shears. The segments were cultured on potato agar (PDA) for 7 days to obtain a culture mixture of fungi and bacteria. The culture mixture of fungi and bacteria was placed on a solid spore-forming medium (20 g glucose, 15.0 g peptone, The cells were streaked onto a solid conidia medium plate (5.0 g sodium chloride, 0.5 g beef extract, 15.0 g agar, pH 7.0) and cultured at 30°C for 24 h. A single colony was then streaked onto a new solid conidia medium plate (formula: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, 15.0 g agar, pH 7.0) and cultured for 24 h (30°C). The streaking process was repeated 5 times, and finally, a Bacillus strain was purified by streaking to obtain the Bacillus sporogenes strain.

[0063] Step 3: Using an inoculation needle, pick a loopful of the isolated Bacillus strain and inoculate it into a liquid conidia medium (formula: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, pH 7.0). Incubate at 220 rpm and 30°C for 12 hours to obtain a Bacillus culture solution. 1 / 10 of the Bacillus culture solution is added with glycerol (the volume ratio of glycerol to culture solution is 2 / 8) and stored at -80°C. The remaining Bacillus culture solution is adjusted to an OD value of 1.0 for later use.

[0064] Step 4: Activate the plant wilt pathogen stored in a refrigerator at -80°C on a PDA plate and culture at 26°C for 72 hours. The activated pathogen is inoculated into Czapek medium (whose formula is: 3g sodium nitrate, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate (MgSO4·7H2O), 0.5g potassium chloride, 0.01g ferrous sulfate, 30g sucrose, 20g agar, and 1000mL distilled water) and shake-cultured at 220rpm at 26°C for 72 hours to obtain a pathogenic fungal spore suspension with a concentration adjusted to 1×10 8 Spores / mL.

[0065] Step 5: Use a 10 μl pipette to draw 10 μl of the fungal spore suspension and drop it onto the center of a PDA plate medium (the formula is: 200g potatoes, 20g glucose, 3g KH2PO4, 1.5g MgSO4·7H2O, 15g agar powder, 1L distilled water; the preparation method is: peel the potatoes, cut into slices, boil for 10 minutes, then filter through gauze, and add glucose and agar). Then, draw 10 μl of Bacillus liquid with an OD value of 1.0 and drop it on the edge of the plate. Drop three points with a diameter of 9 mm on each plate to form an equilateral triangle (with a side length of 6 cm). After the liquid is dried, it is incubated at 26°C for 48 hours. The size of the inhibition zone is observed and measured. The experiment is repeated three times, and the strain with the largest inhibition zone is selected. A single colony is picked and cultured on a new spore-forming medium plate to isolate and obtain a purified methylotrophic Bacillus, namely the cotton disease-resistant growth-promoting bacterium SF5.

[0066] Based on the 16S rRNA gene systematic evolutionary analysis and biological characteristics, it was identified as Bacillusmethylotrophicus. The preservation date was December 3, 2020. The preservation address is the General Microbiology Center of China Culture Collection (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing), and the preservation number is CGMCC NO.21291.

[0067] Example 2: Preparation of cotton disease-resistant growth-promoting bacteria

[0068] The SF5 strain obtained in Example 1 is used to prepare a fungicide for preventing and treating Verticillium wilt in cotton, which specifically comprises the following steps:

[0069] A single SF5 colony was added to a liquid conidia culture medium (without agar) and cultured on a shaker at 220 rpm for 24 h at 30°C to obtain a Bacillus culture solution. The total concentration of live bacteria in the Bacillus culture solution was approximately 1 × 10 6 CFU / mL; The Bacillus liquid was inoculated again with an inoculum size of 2% (v / v) in a liquid conidia medium, filled with 5 L, and cultured in a shaking incubator at 30°C and 220 rpm for 48 h to obtain a total viable bacterial concentration of 1×10 10 Finished product of cotton disease-resistant and growth-promoting bacteria agent with CFU / mL.

[0070] The formula of liquid Bacillus sporulation medium is as follows: glucose 20g, peptone 15g, NaCl: 5g, beef extract 0.5g, distilled water to 1L, pH 7.0, and 1×10 5 Sterilize at 400 Pa for 30 min.

[0071] Application Example 1: Preparation and testing of the antibacterial effect of the bacterial agent.

[0072] The cotton disease-resistant growth-promoting bacterial agent prepared in Example 2 was diluted 100 times to obtain a total concentration of 1×10 8 CFU / mL of diluted bacterial culture.

[0073] Prepare PDA medium: Peel and slice potatoes, boil for 10 minutes, filter through cheesecloth, and add glucose and agar. The recipe is: 200g potatoes, 20g glucose, 3g KH2PO4, 1.5g MgSO4·7H2O, 15g agar powder, and 1L distilled water.

[0074] The pathogenic fungi of cotton dahliae, sunflower sclerotinia rot, and fragrant pear rot stored in a refrigerator at -80°C were activated and cultured. The three plant pathogens were activated on PDA medium and cultured at 28°C for 72 hours. The activated pathogens were inoculated into Cha's medium and shaken at 220 rpm and 28°C for 72 hours to obtain a plant pathogenic fungal spore suspension with a concentration of 1×10 8 CFU / mL is reserved.

[0075] The suspension titration method was used for detection and examination. 10 μl of the fungal spore suspension was aspirated with a 10 μl pipette and dropped in the center of the PDA plate, forming a dot with a diameter of 9 mm. Then, 10 μl of Bacillus culture with an OD value of 1.0 was aspirated and dropped on the edge of the plate. Three dots were dropped on each plate, forming an equilateral triangle (6 cm). The treated group was dried up and the control group was cultured without the Bacillus culture. The plates were cultured at 26°C. When the colonies in the control group filled the plate (diameter 90 mm), the colony diameters were measured by the cross-cross method, the mean colony diameters were calculated, and the average inhibition rate of fungal hyphae growth was calculated according to the following formula.

[0076] Average mycelial growth inhibition rate = (mean colony diameter of the control group - mean colony diameter of the treatment group) / (mean colony diameter of the control group - diameter of the inoculated cake) × 100%. Specific calculation results are shown in Tables 1 to 3.

[0077] Table 1 Inhibitory effect of Bacillus methylotrophicusSF5 on Verticillium dahliae

[0078] strain Colony diameter d / mm Inhibition rate (%) SF5 10.35 88.5 comparison 90.00 —

[0079] Table 2 Inhibitory effect of Bacillus methylotrophicusSF5 on sunflower tuberculosis

[0080] strain Colony diameter d / mm Inhibition rate (%) SF5 2.196 75.6 comparison 90.00 —

[0081] Table 3 Inhibitory effect of Bacillus methylotrophicusSF5 on fragrant pear rot

[0082] strain Colony diameter d / mm Inhibition rate (%) SF5 27.99 68.9 comparison 90.00 —

[0083] The results in Tables 1 to 3 show that the cotton disease-resistant and growth-promoting bacterial agent prepared from Bacillus methylotrophicus SF5 can inhibit the growth of cotton Verticillium dahliae, sunflower tuberculosis, and fragrant pear rot by 88.5%, 75.6%, and 68.9%, respectively.

[0084] Figure 2 The results showed that Bacillus methylotrophicus SF5 had an inhibitory effect on dahlia verticillium disease. The inhibition rate was 88.5% after 18 days of culture. No sterile sclerotia were produced after 18 days of culture, and the production of sclerotia was significantly inhibited.

[0085] Figure 3 The results showed that Bacillus methylotrophicus SF5 had an inhibitory effect on sunflower sclerotinia disease, with an inhibition rate of 75.6% after 7 days of culture. No sclerotia were produced after 18 days of culture, while the control produced 30 sclerotia.

[0086] Figure 4 The results showed that Bacillus methylotrophicus SF5 had an inhibitory effect on fragrant pear rot, with an inhibition rate of 68.9% after 4 days of culture.

[0087] Application Example 2: Determination of the potted control effect of cotton disease-resistant and growth-promoting agent prepared from Bacillus methylotrophicus SF5 on Verticillium dahliae

[0088] The Bacillus stored in a -80°C refrigerator was activated on a solid spore-forming medium and cultured at 30°C for 12 h. A single colony was picked and placed in a Bacillus spore-forming liquid medium and shaken at 220 rpm and 30°C for 72 h. The OD value was adjusted to 1.0 and the culture was stored at 4°C for later use.

[0089] The Verticillium dahliae strain was inoculated into a bran medium (100 g wheat bran, 10 g sucrose, 2 g peptone, 0.5 g potassium dihydrogen phosphate, 0.2 g MgSO4·7H2O, and 70 ml deionized water), cultured at 28°C for 7 days, air-dried at 30°C after full mycelium growth, and then crushed to 80 mesh to obtain the Verticillium dahliae inoculum.

[0090] Healthy soil (free of yellow wilt) was potted at a standard filling rate of 30 kg / pot. A Verticillium dahliae inoculant was evenly mixed with the soil before planting, with an inoculum rate of 0.5 g / pot. Five cotton holes were sown in each pot for a pot experiment. The inoculant was drip-applied after sowing at a rate of 0.5 ml / pot. Cotton seedlings were thinned when they reached 5 cm in height. At 15 days old, five plants were retained per pot and managed according to conventional field practices. The number of diseased plants was observed and recorded 60 days after sowing. The efficacy of the Bacillus methylotrophicus SF5 inoculant against Verticillium dahliae in cotton was recorded. Specific results are shown in Table 4.

[0091] Grading standards for cotton dahliae verticillium disease:

[0092] Level 0: healthy plants, no diseased leaves;

[0093] Level 1: Symptoms are evident on less than 1 / 4 of the leaves, with pale yellow or irregular yellow lesions appearing between the main veins of the leaves;

[0094] Level 2: Symptoms are evident on 1 / 4-1 / 2 of the leaves, with most lesions being yellow or yellow-brown, and the leaf edges slightly curled and withered;

[0095] Level 3: Symptoms are evident on more than 1 / 2 of the leaves, most of the lesions are yellow-brown, and the leaf edges are curled and withered;

[0096] Level 4: All leaves of the plant are infected, or the leaves dry up and fall off to become bare stems, and the plant dies.

[0097] The calculation formulas for disease index and relative prevention effect are as follows:

[0098] Disease index = Σ(number of diseased plants × disease level) / (total number of plants × most diseased level)

[0099] Preventive effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%

[0100] Table 4 Inhibitory effect of Bacillus methylotrophicusSF5 on Verticillium dahliae disease in potted cotton

[0101] Pot treatment Disease Index Relative prevention effect (%) SF5 16.4±2.8 78.2 comparison 75.3±5.4 —

[0102] From the results in Table 4, it can be seen that the control efficiency of Bacillus methylotrophicus SF5 on cotton dahliae verticillium disease in greenhouse potted plants reached 78.2%, and the control effect was significant.

[0103] Application Example 3: Determination of the Soil Phosphate Solubility Effect of Bacillus methylotrophicus SF5, a Cotton Disease Resistance and Growth-Promoting Bacterial Agent

[0104] The Bacillus stored in a -80°C refrigerator was activated on a solid conidia medium and cultured at 30°C overnight. A single colony was picked and placed in a liquid conidia medium. The culture was shaken at 220 rpm and 30°C for 72 h. The OD value was adjusted to 1.0 and the culture was stored at 4°C for later use.

[0105] Prepare organophosphorus culture medium: glucose 10g, ammonium sulfate 0.5g, yeast extract powder 0.5g, NaCl 0.3g, KCl 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, calcium sulfate 1.0g, lecithin 0.2g, pH 7.0, distilled water to 1L, and add 1×10 5 Pa sterilization for 30 minutes.

[0106] The suspension titration method was used for detection and examination. 10 μl of the fungal spore suspension was aspirated with a 10 μl pipette and dropped in the center of the PDA plate, forming a dot with a diameter of 9 mm. Then, 10 μl of Bacillus culture with an OD value of 1.0 was aspirated and dropped on the edge of the plate. Three dots were dropped on each plate to form an equilateral triangle (with a side length of 6 cm). The culture was dried and used as the treatment group. No Bacillus culture was dripped in the control treatment. All the samples were cultured at 26°C for 5 days. The colony diameter and the diameter of the phosphate-dissolving circle were measured by the cross-cross method, and the phosphate-dissolving amount was calculated. See Table 5.

[0107] from Figure 5 From the results in Table 5, it can be seen that the strain has the effect of dissolving organic phosphorus in soil. After culturing for 5 days, the colony diameter d3mm, the diameter of the phosphorus dissolving zone D6.5mm, D / d=2.16, and the phosphorus dissolving amount is 298.32μg / ml.

[0108] Table 5

[0109]

[0110] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0111] The numerical ranges described in the present invention include all values ​​within the range, and include range values ​​formed by any two values ​​within the range. Different numerical values ​​of the same indicator appearing in all embodiments of the present invention can be arbitrarily combined to form a range value.

[0112] The technical features in the claims and / or the specification of the present invention may be combined, and the manner of combination is not limited to the combination obtained by reference in the claims. The technical solutions obtained by combining the technical features in the claims and / or the specification are also within the scope of protection of the present invention.

[0113] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiment based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A cotton disease-resistant and growth-promoting strain SF5, characterized in that: Its classification name is Bacillus methylotrophicus, the preservation date is December 3, 2020, the preservation address is the General Microbiology Center of China Culture Collection Administration, and the preservation number is CGMCC NO.21291.

2. A method for preparing a cotton disease-resistant and growth-promoting strain SF5, characterized in that: The steps include: Step 1: In a cotton Verticillium wilt mixed disease field, 5 to 10 uninfected cotton plants are selected for isolation; Step 2: For isolation, cotton hypocotyls are obtained, surface-sterilized with 75% (v / v) alcohol, peeled, and the peeled stems are cut into 0.5 cm segments using sterilized pruning shears. The segments are cultured on potato agar for 7 to 10 days to obtain a fungal and bacterial culture mixture; the fungal and bacterial culture mixture is streaked onto a solid conidia medium using a sterilized pick, cultured overnight, and a single colony is picked and cultured on a new solid conidia medium plate to isolate a Bacillus strain; Step 3: inoculate the isolated Bacillus strain into a liquid spore-forming culture medium, culture at 180-220 rpm and 26-30° C. for 12-24 hours to obtain a Bacillus bacterial solution; add glycerol to a portion of the solution and store it at -80° C.; and adjust the concentration of a portion of the solution to an OD value of 0.8-1.0 for later use; Step 4: Activate the plant wilt pathogen stored in a refrigerator at -80°C on a PDA plate and culture at 26-30°C for 48-72 hours; inoculate the activated pathogen into Czapek medium and shake at 180-220 rpm at 26-30°C for 48-72 hours to obtain a pathogenic fungal spore suspension with a concentration of 1×10 6 ~1×10 8 spores / mL; Step 5: Pipette 10 μl of the pathogenic fungal spore suspension obtained in step 4 and drop it in the center of the PDA plate culture medium. Then, prick 10 μl of the Bacillus culture with an OD value of 0.8 to 1.0 obtained in step 3 and drop it on the edge of the plate. Drop three points on each plate to form an equilateral triangle. After the culture liquid is dried, culture it at a constant temperature of 26 to 30°C for 48 to 72 hours, observe and measure the size of the inhibition zone. Repeat the experiment three times and select the strain with the largest inhibition zone. Pick a single colony and culture it on a new spore-forming culture medium plate to isolate and obtain a purified methylotrophic Bacillus, namely the cotton disease-resistant and growth-promoting strain SF5.

3. The method for preparing the disease-resistant and growth-promoting cotton strain SF5 according to claim 2, wherein: In step 2, the length of the hypocotyl is 10 cm; the formula of the solid spore production medium is as follows: 20 g glucose, 15.0 g peptone, 5.0 g sodium chloride, 0.5 g beef extract, 15.0 g agar, pH 7.

0.

4. The method for preparing the disease-resistant and growth-promoting cotton strain SF5 according to claim 2, wherein: In step 3, the formula of the liquid spore production medium is as follows: 20g glucose, 15.0g peptone, 5.0g sodium chloride, 0.5g beef extract, pH 7.0; the volume ratio of the glycerol to the Bacillus bacterial liquid is 2-3:7-8.

5. The method for preparing the disease-resistant and growth-promoting cotton strain SF5 according to claim 2, wherein: In step 4, the formula of the Czapek medium is as follows: 3 g sodium nitrate, 1 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.5 g potassium chloride, 0.01 g ferrous sulfate, 30 g sucrose, 20 g agar, and 1000 mL distilled water; in step 5, the formula of the PDA plate medium is as follows: 200 g potato, 20 g glucose, 3 g KH2PO4, 1.5 g MgSO4·7H2O, 15 g agar powder, and 1 L distilled water.

6. The method for preparing the disease-resistant and growth-promoting cotton strain SF5 according to claim 2, wherein: In step 5, the three points are all circles with a diameter of 9 mm.

7. A microbial biocontrol agent, characterized in that: The microbial biocontrol preparation contains the cotton disease-resistant and growth-promoting strain SF5. The classification name of the cotton disease-resistant and growth-promoting strain SF5 is methylotrophic Bacillus methylotrophicus. The preservation date is December 3, 2020. The preservation address is the General Microbiology Center of the China Culture Collection Administration, and the preservation number is CGMCCNO.21291.

8. A method for preparing a microbial biocontrol agent, characterized in that: The following steps are involved: The cotton disease-resistant and growth-promoting strain SF5 stored in the refrigerator was activated on a solid conidia medium and cultured at 26-30°C for 12-24 hours. A single colony was picked and shaken in a liquid conidia medium at 180-220 rpm and 26-30°C for 48-72 hours. The OD value was adjusted to 0.8-1.0 to obtain a seed solution. The seed solution was inoculated into the liquid conidia medium at an inoculum size of 1-3% (v / v), and cultured in a shaker at 26-30°C and 180-220 rpm to obtain a total viable bacterial concentration of 1×10 9 ~1×10 10 CFU / mL of microbial biocontrol agents.

9. Use of the microbial biocontrol agent according to claim 7 in cotton disease resistance.

10. Use of the microbial biocontrol agent according to claim 9 in cotton disease resistance, characterized in that: The application comprises the following steps: before use, dilute the microbial biocontrol agent to 1×10 8 CFU / mL~1×10 9 CFU / mL, and then applied to the cotton field with water drops for biocontrol treatment.

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