CN112852663B - Bacillus amyloliquefaciens for preventing and treating brown spot of peanut and application thereof - Google Patents
Bacillus amyloliquefaciens for preventing and treating brown spot of peanut and application thereof Download PDFInfo
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Abstract
The invention relates to bacillus amyloliquefaciens for preventing and treating peanut brown spot and application thereof, wherein the bacillus amyloliquefaciens is preserved in China general microbiological culture collection center with the preservation address: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no.1, no. 3, north Chen West Lu, the Korean region of Beijing; the preservation number is CGMCC No.18748, and the preservation date is 2019, 10 months and 28 days, and the classification is named as bacillus amyloliquefaciens. The bacillus amyloliquefaciens and the microbial inoculum are safe to plants, environment, people and livestock, do not generate harmful substances in the processes of fermentation, production, processing and use, and have no toxic or side effect; the bacillus amyloliquefaciens and fish protein mixture is used for slowing down the generation of drug resistance of pathogenic bacteria of peanut brown spot to carbendazim, tebuconazole and mancozeb.
Description
Technical Field
The invention relates to a biocontrol strain, a preparation and application thereof, in particular to bacillus amyloliquefaciens for preventing and treating peanut brown spot and application thereof, and belongs to the technical field of biocontrol preparations prepared from beneficial biocontrol strains.
Background
Peanut (Arachis hypogaea l.), an annual herb of the genus Arachis of the subfamily paphiopediforme of the order rosaceous. As an important oil crop, its range of cultivation has been over 100 or more countries and regions worldwide. The peanut planting history in China is long, and the peanut planting method is one of countries with the largest planting area and yield in the world. The first large country of world peanut planting has a planting area of 7500 mu or more and a yield of 1700 ten thousand tons and accounts for about 40 percent of the world yield, and the yield value reaches 1200 hundred million yuan and occupies the fourth place of the national crops. Liaoning is a great province for peanut planting, and in recent years, the annual average sowing area of Liaoning peanuts is about 530 ten thousand mu, and the Liaoning peanuts are in the third place of the whole country; the total annual average yield is 114 ten thousand tons, and the total annual average yield is arranged in the fourth place of China. Liaoning is a major province in peanut planting, and has become the third largest crop following corn and rice at present. Liaoning peanut has good quality and taste, does not contain aflatoxin, and is enjoyed at home and abroad.
Brown spot (Cercospora arachidicola Hori), also known as early spot, is one of the most serious foliar diseases occurring and harming in all peanut producing areas worldwide. The asexual generation of the pathogenic bacteria of the peanut brown spot is Cercospora arachidicola Hori, which belongs to the phylum of the semi-known mycotina, the order of the arbuscular mycorrhizal fungi and the genus of the cercospora; the sexual generation is Mycosphaerella arachidis (Hori) Jenkins, belonging to the genus Ascomycotina, the order Ascomycetes, the genus Stropharia. The disease mainly occurs in early and middle stages of peanut growth, and mainly damages the leaves. Disease-infected peanuts have leaf spots, the photosynthesis area is reduced sharply, chlorophyll is destroyed, photosynthesis efficiency is reduced, and plant biological yield is reduced greatly. Early leaf fall is caused with the generation of a large number of disease spots, dry matter accumulation, pod plumpness and maturity are seriously affected, and the rate of empty husks is increased. The yield of the damaged peanut field is generally reduced by 10% -20%, and the yield can reach more than 40% when serious, which is a problem to be solved in production.
At present, the most main measure for preventing and treating the disease is to use chemical pesticides, but the long-term frequent use of the bactericides causes pathogenic bacteria to generate drug resistance, and a series of serious problems such as excessive pesticide residues in agricultural products and environmental pollution reduce the quality of peanuts, influence the health of human beings and restrict the sustainable development of the peanut industry. Therefore, people need to have a biocontrol preparation capable of preventing and treating peanut brown spot.
The biocontrol bacteria are used for preventing and controlling plant diseases, are more and more important in production, are safe to people, livestock and crops, have no harm to natural enemies and beneficial organisms and are environment-friendly, but different biocontrol bacteria have different colonization capacities on plant surfaces, especially peanut leaves have smooth surfaces so that the biological bacteria are difficult to attach, and meanwhile, peanut true leaves have the characteristic of 'night motion', namely, the peanut leaves can be closed after sunset or cloudy days, petioles droop, sunlight obliquely shines in the morning and evening, and the leaves are erected, so that the biological bacteria need to be quickly attached to the leaves and exert the prevention and control effect on the bacteria in the normal growth state of the peanut leaves.
The existing biological microbial inoculum for preventing and treating peanut brown spot has poor adhesion on peanut leaves, slow colonization, large influence by movement of the peanut leaves or rain wash, unstable prevention and treatment effect and low prevention and treatment effect. Therefore, how to obtain the biocontrol microbial inoculum with strong leaf adhesion and quick colonization by preparing microbial inoculum and adjusting application method is the problem to be solved in the current peanut planting.
Disclosure of Invention
In order to remedy the differences of the prior art, the invention aims to provide the bacillus amyloliquefaciens for preventing and treating the brown spot of the peanut and the application thereof, wherein the bacillus amyloliquefaciens and the microbial inoculum are safe to plants, the environment, people and livestock, do not generate harmful substances in the processes of fermentation, production, processing and use, have no toxic or side effects, and meanwhile, the formula and the application method of the microbial inoculum improve the adhesion and the colonization speed of the bacillus amyloliquefaciens on the peanut leaves, and obviously improve the preventing and treating effect.
The invention provides bacillus amyloliquefaciens for preventing and treating peanut brown spot, which is preserved in China general microbiological culture Collection center with the preservation address: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no.1, no. 3, north Chen West Lu, the Korean region of Beijing; the preservation number is CGMCC No.18748, and the preservation date is 2019, 10 month and 28 days, and the classification is named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
The culture characteristics of the bacillus amyloliquefaciens are as follows: on NA culture medium plate, colony is milky white round or nearly round, colony is flat, surface is dry and rough, has folds, edge is saw-tooth, and gram staining is positive. In the physiological and biochemical reaction, the tests such as starch hydrolysis test, gelatin liquefaction test, oxidase reaction, V-P reaction, contact enzyme reaction, nitrate reduction reaction and the like are positive; methyl red test, H 2 S reaction and indole test are negative; has a certain salt tolerance, and can normally grow in NaCl culture medium with concentration less than or equal to 10%.
The invention also provides a preparation method of the biocontrol microbial inoculum of the bacillus amyloliquefaciens for preventing and treating peanut brown spot, which is technically characterized by comprising the following steps:
(1) Activating Bacillus amyloliquefaciens strains
Performing activation culture on bacillus amyloliquefaciens strain by adopting an LB (LB) culture medium, wherein the culture temperature is 25-30 ℃, and culturing for 3d; the formula of the LB culture medium is as follows: 10g/L peptone, 3g/L beef extract, 5g/L sodium chloride, 12-15g/L, pH 7.2.2-7.4 agar powder;
(2) Preparation of stock solution of biocontrol microbial agent
Preparing mother solution: the activated bacillus amyloliquefaciens is inoculated into NB culture solution and shake-cultured for 2d at 25 ℃ and 150rpm to prepare the bacillus amyloliquefaciens with the concentration of 1-9 multiplied by 10 10 The spore suspension of cfu/mL is mother liquor; the NB culture solution comprises the following formula: 10g/L peptone, 3g/L beef extract, 5g/L sodium chloride and pH 7.2-7.4;
(3) Fermentation culture
Adding the mother liquor into a fermentation culture medium for fermentation culture;
the formula of the fermentation medium is as follows: 20g/L of peanut powder, 10g/L of peanut leaf (dry) powder, 7.5g/L of sucrose, 3g/L of beef extract, 5g/L of dipotassium hydrogen phosphate and 7.2-7.4 of water pH;
the mother solution and 100mL (V: V=1:19) of fermentation medium are placed in a 250mL triangular flask, and the fermentation temperature is 25 ℃ and the vibration speed is 150rpmSwinging, fermenting and culturing for 5d to obtain a stock solution of the biocontrol microbial agent; the spore concentration of the stock solution of the biocontrol microbial agent is 1-9 multiplied by 10 10 cfu/mL；
(4) Preparation of biocontrol microbial agent
Diluting the stock solution by 100-200 times to obtain a biocontrol microbial agent, wherein the concentration of bacillus amyloliquefaciens spores of the biocontrol microbial agent is 10 8 ～10 9 cfu/mL。
The invention also provides an application method of the biocontrol microbial inoculum of the bacillus amyloliquefaciens, which has the technical key points that: the method comprises a prevention application method and a treatment application method, wherein the prevention application method comprises the following steps: spraying spore concentration of 10 before peanut brown spot is developed 8 ～10 9 cfu/mL of biocontrol microbial agent; the application method of the treatment comprises the following steps: the spore concentration sprayed in the early stage of peanut disease is 10 8 -10 9 cfu/mL of biocontrol microbial agent.
Further, the biocontrol microbial inoculum of the bacillus amyloliquefaciens is mixed with carbendazim to prepare a bacillus amyloliquefaciens-carbendazim mixture, and the spore concentration of the bacillus amyloliquefaciens in the bacillus amyloliquefaciens-carbendazim mixture is 10 8 -10 9 cfu/mL, the effective component amount of the carbendazim is 400-600mg/kg.
Further, the biocontrol microbial agent of the bacillus amyloliquefaciens is mixed with the fish protein organic fertilizer to prepare the bacillus amyloliquefaciens-fish protein mixture, and the spore concentration of the bacillus amyloliquefaciens in the bacillus amyloliquefaciens-fish protein mixture is 10 8 -10 9 cfu/mL, wherein the effective concentration of the fish protein organic fertilizer is as follows: 1-1.5g/L.
Further, the preparation method of the peanut leaf (dry) powder comprises the following steps: picking up peanut leaves, washing with clear water, drying, pulverizing the dried peanut leaves with a pulverizer, sieving with a 100-mesh sieve, bagging the peanut leaf dry powder, and storing in a dryer for later use.
Furthermore, the bacillus amyloliquefaciens-fish protein mixture is used for reducing the drug resistance of pathogenic bacteria of peanut brown spot to carbendazim, tebuconazole and mancozeb.
Compared with the prior art, the invention has the beneficial effects that:
(1) The biocontrol strain bacillus amyloliquefaciens is derived from peanut leaves, is safe to plants, environment, people and livestock, does not have extremely toxic property like a part of pesticides, does not generate harmful substances in the processes of fermentation, production and processing and use, and has no toxic or side effect.
(2) The biocontrol microbial inoculum after fermentation of the biocontrol strain enhances and knows the adhesion and colonization of the bacillus amyloliquefaciens on the leaves, and has good control effect on peanut brown spot;
(3) The bacillus amyloliquefaciens-carbendazim mixture and bacillus amyloliquefaciens-fish protein mixture have excellent control effects on controlling peanut brown spot in fields and have wide application prospects.
Detailed Description
Example 1
(1) Activation of biocontrol bacteria
The biocontrol strain is bacillus amyloliquefaciens, and is preserved in China center for type culture collection with a preservation number of CGMCC No.18748 and a preservation date of 2019, 10 months and 28 days, and is classified and named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
In the embodiment, the bacillus amyloliquefaciens is marked as TL-6, the TL-6 is separated from peanut leaves, and bacterial colonies are in a milky white round shape or a nearly round shape, flat, dry and rough in surface, provided with folds, serrated in edges and positive in gram staining on a flat plate of a TL-6 bacterial strain NA culture medium. In the physiological and biochemical reaction, the tests such as starch hydrolysis test, gelatin liquefaction test, oxidase reaction, V-P reaction, contact enzyme reaction, nitrate reduction reaction and the like are positive; methyl red test, H 2 S reaction and indole test are negative; has a certain salt tolerance, and can normally grow in NaCl culture medium with concentration less than or equal to 10%. The similarity with the bacillus amyloliquefaciens is 100% after morphological characteristic observation and physiological biochemical reaction and determination of the 16S rDNA sequence, and the bacillus amyloliquefaciens is named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
Activating culture with LB culture medium at 25-30deg.C for 3d; the formula of the LB culture medium is as follows: 10g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 12-15g/L of agar powder and pH7.2-7.4.
(2) Preparation of stock solution of biocontrol bacteria agent
Scraping activated Bacillus amyloliquefaciens TL6, inoculating into 250mL triangular flask containing 100mL NB culture solution (peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, pH 7.2-7.4), shake culturing at 25deg.C and 150rpm for 2d to obtain 1-9X10 concentration 10 cfu/mL spore suspension is mother liquor.
(3) Fermentation culture
The formula of the fermentation medium of the biocontrol microbial inoculum is as follows: 20g/L of peanut powder, 10g/L of peanut leaf (dry) powder, 7.5g/L of sucrose, 3g/L of beef extract, 5g/L of dipotassium hydrogen phosphate and pH of 7.2-7.4, and placing the mother liquor and 100mL (V: V=1:19) of fermentation medium in a 250mL triangular flask, and carrying out shaking fermentation culture at 150rpm for 5d at the fermentation temperature of 25 ℃ to obtain the stock solution of the biocontrol microbial inoculum.
The preparation method of the peanut leaf (dry) powder comprises the following steps: picking up peanut leaves, washing with clear water, drying, pulverizing the dried peanut leaves with a pulverizer, sieving with a 100-mesh sieve, bagging the peanut leaf dry powder, and storing in a dryer for later use.
(3) Preparation of biocontrol microbial agent
Diluting the stock solution to 100-200 times to obtain a biocontrol microbial agent, wherein the concentration of bacillus amyloliquefaciens spores of the biocontrol microbial agent is 10 8 -10 9 cfu/mL。
Example 2 evaluation of indoor in vitro peanut brown spot control Effect
(1) Test target
Performing an indoor anti-efficiency test by using the in-vitro blade;
(2) Test treatment
5 different treatments were designed, each treatment being repeated 4 times.
Treatment 1: spraying spore suspension of brown spot germ of peanut, spraying biological agent containing Bacillus amyloliquefaciens TL6 after 24 hr (for therapeutic application), and controlling spore concentration of the biological agent to 1-9×10 10 cfu/mL；
Treatment 2: spraying biological agent containing Bacillus amyloliquefaciens TL6 for 24 hr, and spraying spore suspension of brown spot germ (preventing)Application), the spore concentration of the biocontrol microbial agent is 1-9 multiplied by 10 10 cfu/mL；
Treatment 3: only the sporangium suspension of brown spot germ of peanut is sprayed.
30 leaves were treated each, 3 replicates were inoculated with 10% brown spot germ spores 5 And each mL. Test reference department of agriculture drug test institute field efficacy test guidelines (two), part 85: the classification standard of the bactericide for preventing and controlling the leaf spot disease of the peanut is investigated, the disease index is calculated, and the disease condition is investigated after 7 days.
(3) Test results
The results are shown in Table 1, the control effect of treatment 1 is 83.71%, the control effect of treatment 2 is 88.12%, and the prevention effect is remarkably higher than the treatment effect. The disease indexes of the treatment 1 and the treatment 2 are obviously lower than that of a control, and the biological microbial inoculum of the bacillus amyloliquefaciens TL6 has excellent prevention and treatment effects on peanut brown spots.
Table 1 TL6 effects of controlling peanut brown spot on in vitro leaves
Example 3 evaluation of field peanut brown spot control Effect
(1) The test site is peanut test field of the agricultural academy of sciences of Liaoning province.
(2) Test treatment
Process 1 TL6: the concentration of the biocontrol microbial inoculum stock solution is 1-9 multiplied by 10 10 cfu/mL；
Process 2 TL6: 10-time dilution of stock solution of biocontrol bacteria;
process 3 TL6: 100 times of dilution of stock solution of biocontrol bacteria;
process 4 TL6: 200 times of dilution of stock solution of the biocontrol bacteria;
treatment 5: polyculture of polyculture fish protein organic fertilizer (polyculture of polyculture technology (Nanjing) Co., ltd.) of polyculture fish protein;
treatment 6:50% carbendazim suspension (Su Longdu Lamp chemical Co., ltd.): 1000 times of liquid.
Treatment 7: clear water control
The treatment period is as follows: is the initial stage of peanut brown spot.
(3) Test results
The results of the investigation are shown in Table 2. The bacillus amyloliquefaciens TL6 biocontrol microbial inoculum has good control effect on peanut brown spot. In the prevention and treatment of peanut brown spot, the TL6 fermentation stock solution has the prevention effect of more than 69.17 percent and has no obvious difference with the prevention and treatment effect of 1000 times of 50 percent carbendazim suspending agent.
Table 2 TL6 field control effect on peanut brown spot
EXAMPLE 4 Mixed preparation
(1) Test site
The test site is peanut test field of the agricultural academy of sciences of Liaoning province.
(2) Test treatment
Treatment 1: the polycosan protein organic fertilizer+TL 6 biocontrol microbial inoculum is prepared by 200 times of diluent of stock solution of the biocontrol microbial inoculum to 800 times of liquid of the polycosan protein organic fertilizer.
Treatment 2:50% carbendazim suspending agent+TL 6 biocontrol microbial agent, and 200 times of diluent of biocontrol microbial agent stock solution are used for preparing 1000 times of liquid of 50% carbendazim suspending agent.
Treatment 3: 200 times of dilution of stock solution of the biocontrol bacteria;
treatment 4: the polycephalum molitor protein organic fertilizer is diluted by 800 times;
treatment 5:50% carbendazim suspending agent 1000 times liquid;
treatment 6: and (5) comparing with clear water.
The treatment period is as follows: is the initial stage of peanut brown spot.
(3) Test results
The results of the investigation are shown in Table 2. The compound of 200 times liquid of bacillus amyloliquefaciens TL6 and 800 times liquid of priden fish protein organic fertilizer and the compound of 50 percent carbendazim suspending agent of bacillus amyloliquefaciens TL6 have better prevention and treatment effects on peanut brown spot. In the aspect of preventing and treating the brown spot of the peanut, the field preventing and treating effects of the two formulas on the brown spot of the peanut are 83.78% and 80.83%, respectively, a good synergistic effect is shown, and the two formulas can be alternately used when the formula is applied.
Table 3 TL6 Compound recipe for preventing and controlling peanut brown spot disease field
Example 5 Effect of Bacillus amyloliquefaciens fermentation method on controlling peanut brown spot
(1) Test site
The test site is peanut test field of the agricultural academy of sciences of Liaoning province.
(2) Test treatment
Treatment 1: TL6 biocontrol bacteria stock solution is 100-fold diluted solution, and the preparation method is shown in example 1;
treatment 2: 200 times of dilution of the stock solution of the TL6 biocontrol bacteria agent; the preparation method is shown in example 1;
treatment 3: TL6 bacteria stock solution 100 times diluted solution; the preparation of the activated strain and mother liquor of the TL6 microbial inoculum is the same as that of the example 1,
the fermentation culture is as follows: the formula of the fermentation medium of the biocontrol microbial inoculum is as follows: 200g/L of potato, 7.5g/L of sucrose, 3g/L of beef extract, 5g/L of dipotassium hydrogen phosphate and pH of 7.2-7.4, inoculating 5mL of mother liquor into a triangular flask filled with 95mL of 250mL of fermentation medium, and carrying out shaking fermentation culture at 25 ℃ and 150rpm for 5d to obtain the stock solution of the biocontrol microbial agent.
Treatment 4: TL6 bacteria stock solution 200 times diluted solution; preparing and treating the TL6 microbial inoculum stock solution in the same way as the preparation of the TL6 microbial inoculum stock solution;
treatment 5: clear water control
The treatment period is as follows: is the initial stage of peanut brown spot.
(3) Test results
The investigation results are shown in Table 2, and the test results show that the special formula of peanut powder and peanut leaf powder is adopted in the fermentation medium, and the control effect of the bacillus amyloliquefaciens TL6 on peanut brown spot after fermentation is obviously higher than that after fermentation by the conventional medium, namely, the peanut powder and the peanut leaf powder in the fermentation liquid can improve the activity of the bacillus amyloliquefaciens TL 6. The peanut leaves in the field have special movement characteristics, the leaves are always in an inclined state, and the biological bacteria act slowly, so that stable adhesion and colonization have important significance for the biological bacteria to act. The fermentation medium added with peanut powder and peanut leaf powder is beneficial to keeping the activity of bacillus amyloliquefaciens TL6 on the leaves, so that the adhesion and the colonization on the leaves are enhanced, and the peanut brown spot prevention and treatment effect is good.
TABLE 4 Effect of TL6 different fermentation Medium on peanut brown spot field control Effect
Example 6 Propritans protein organic fertilizer + TL6 biocontrol agent reduces pathogenic bacteria resistance to multiple agents.
(1) Test site
The test site is peanut test field of the agricultural academy of sciences of Liaoning province, and the pathogen source is inoculation.
(2) Test treatment
Treatment 1: the 50% carbendazim suspending agent is 1000 times liquid, and is applied once every 10 days for 4 times;
treatment 2: the 43% tebuconazole suspending agent is 3000 times, and the liquid is applied once every 10 days for 4 times;
treatment 3:80% mancozeb wettable powder 800 times of liquid, and is applied once every 10 days for 4 times;
treatment 4: the 800 times liquid of the priden fish protein organic fertilizer and the 100 times liquid of the TL6 biocontrol microbial agent are alternately used for 2 times respectively, and the 1000 times liquid of the 50% carbendazim suspending agent is used for 2 times respectively;
treatment 5: the polycosaponin protein organic fertilizer is 800 times liquid and the TL6 biocontrol microbial agent is 100 times liquid, and 43 percent tebuconazole suspending agent is 3000 times alternately used for 2 times respectively;
treatment 6: the 800 times liquid of the priden fish protein organic fertilizer and the 100 times liquid of the TL6 biocontrol microbial inoculum are alternately used for 2 times respectively by the 800 times liquid of the 80 percent mancozeb wettable powder;
treatment 7: the polycosan protein organic fertilizer is 800 times liquid and the TL6 biocontrol microbial inoculum is 100 times liquid, and the polycosan protein organic fertilizer is applied once every 10 days for 4 times;
treatment 8: and (5) comparing with clear water.
The treatment period is as follows: is the initial stage of peanut brown spot disease; collecting disease leaves after 7 days of the last application of each treatment, culturing the pathogenic bacteria spores of each treated peanut brown spot, measuring the drug resistance of the pathogenic bacteria of different treated peanut brown spots by adopting a direct disease spot conidium measuring method, and measuring the germination condition of spores under the treatment concentration of carbendazim, tebuconazole and mancozeb; concentration of carbendazim, tebuconazole, mancozeb treatment reference: liu Yong, et al, chinese oil, 1991, stage 4, 67-70; the concentration of tebuconazole and mancozeb treated is the same as that of carbendazim.
The investigation method refers to: wei Lei, the spore germination method is adopted to detect the drug resistance of the botrytis cinerea, and 19-21 are the Beijing agricultural science.
(3) Test results
The investigation result is shown in Table 5, and the result shows that the alternate application of the Proteus-Goldfish protein organic fertilizer plus TL6 biocontrol microbial inoculum, carbendazim, tebuconazole and mancozeb obviously slows down the generation of drug resistance of peanut brown spot pathogens to the single agent, and is beneficial to the effective period of the field agent control of the peanut brown spot at present.
Table 5 TL6 Compound recipe for preventing and controlling peanut brown spot disease field
Claims (3)
1. The application of the bacillus amyloliquefaciens for preventing and treating peanut brown spot is characterized in that: the bacillus amyloliquefaciens is preserved in China general microbiological culture collection center, and the preservation address is: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no.1, no. 3, north Chen West Lu, the Korean region of Beijing; the preservation number is CGMCC No.18748, the preservation date is 2019, 10 and 28, and the classification is named as bacillus amyloliquefaciens @Bacillus amyloliquefaciens）；
The biocontrol microbial agent of the bacillus amyloliquefaciens is mixed with the fish protein organic fertilizer to prepare the bacillus amyloliquefaciens-fish protein mixture, and the spore concentration of the bacillus amyloliquefaciens in the bacillus amyloliquefaciens-fish protein mixture is 10 8 -10 9 cfu/mL, the effective concentration of the fish protein organic fertilizer is as follows: 1-1.5g/L; the bacillus amyloliquefaciens-fish protein mixture is used for slowing down the generation of drug resistance of pathogenic bacteria of peanut brown spot to carbendazim, tebuconazole and mancozeb.
2. The use of bacillus amyloliquefaciens for preventing and treating peanut brown spot according to claim 1, wherein the bacillus amyloliquefaciens is characterized in that: the preparation method of the bacillus amyloliquefaciens biocontrol microbial inoculum comprises the following steps:
(1) Activating Bacillus amyloliquefaciens strains
Performing activation culture on bacillus amyloliquefaciens strain by adopting an LB culture medium;
(2) Preparation of stock solution of biocontrol microbial agent
Preparing mother solution: the activated bacillus amyloliquefaciens is inoculated into NB culture solution, shake-cultured at 25 ℃ and 150rpm for 2d to prepare the bacillus amyloliquefaciens with the concentration of 1-9 multiplied by 10 10 The spore suspension of cfu/mL is mother liquor;
(3) Fermentation culture
Adding the mother liquor into a fermentation culture medium for fermentation culture, and carrying out shaking fermentation culture at 150rpm at the fermentation temperature of 25 ℃ to obtain a stock solution of the biocontrol microbial agent; the spore concentration of the stock solution of the biocontrol microbial agent is 1-9 multiplied by 10 10 cfu/mL；
(4) Preparation of biocontrol microbial agent
Diluting the stock solution to obtain a bacillus amyloliquefaciens biocontrol microbial agent, wherein the bacillus amyloliquefaciens spore concentration of the bacillus amyloliquefaciens biocontrol microbial agent is 10 8 -10 9 cfu/mL。
3. The use of bacillus amyloliquefaciens for preventing and treating peanut brown spot according to claim 2, wherein the bacillus amyloliquefaciens is characterized in that: the formula of the fermentation medium is as follows: 20g/L of peanut powder, 10g/L of peanut leaf (dry) powder, 7.5g/L of sucrose, 3g/L of beef extract, 5g/L of dipotassium hydrogen phosphate and pH of 7.2-7.4.
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