Composition for preventing and controlling sclerotiniose and / or gibberellic disease as well as preparation method and application thereof

Abstract

The invention relates to the technical field of plant disease control. The invention provides a composition for preventing and controlling sclerotiniose and / or gibberellic disease as well as a preparation method and application thereof, and the preparation method comprises the following steps: (1) carrying out fermentation culture on bacillus velezensis and bacillus subtilis to obtain fermentation liquor; (2) carrying out solid-liquid separation on the fermentation liquid to obtain thalli and bacterial liquid; (3) carrying out ammonium sulfate precipitation on the bacterial liquid, and centrifuging to obtain supernate and precipitate; and (4) dissolving the precipitate to obtain the biocontrol pesticide. And (5) jointly preparing the composite organic bacterial fertilizer by using the thalli obtained in the step (2) and the supernate obtained in the step (3). The composite organic bacterial fertilizer prepared by the invention is simple in use method, and can be directly used as a seed fertilizer for crops such as soybeans and wheat by combining crop straws, manure and the like. The biopesticide mainly comprises macrolide and biosurfactant antibacterial cyclic lipopeptide substances, and due to the anti-adhesion, antibacterial and immunoregulation characteristics of the substances, the disease resistance of crops can be enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of plant disease control, and particularly to a composition for preventing and controlling Sclerotinia sclerotiorum and / or Fusarium head blight, a preparation method thereof, and an application thereof. Background Art

[0002] Fusarium head blight of wheat is one of the important diseases in wheat production and is known as the "cancer" of wheat. It not only causes wheat yield reduction, but also causes human poisoning when eating diseased wheat. Fusarium head blight of wheat is an explosive, epidemic and climate-type disease, which can be prevented but not cured. It mainly relies on technical measures such as "one spray, three defenses" of wheat to achieve the prevention and control goal. However, due to factors such as manpower, financial resources and climate, every spring, the prevention and control of Fusarium head blight of wheat is difficult, with heavy tasks, tight time and low efficiency. Continuous rainfall and continuous high temperature are the main factors inducing the pathogen of Fusarium head blight of wheat, and the synthetic chemical pesticides will more or less reduce the efficacy when used in the rainy season, and the effective biocontrol strains under laboratory conditions affect the field effect due to low colonization efficiency.

[0003] Sclerotinia sclerotiorum of oil crops (such as soybean, rapeseed and sunflower) is a soil-borne disease. The primary infection source is mainly sclerotia in the soil. In prevention and control, it should mainly focus on preventing the primary infection, and reducing the number of sclerotia in the field is the key to disease control. This disease mainly appears in the seedling stage, flowering stage and flower-pod stage of oil crops, and mainly harms crop leaves, stems or pod parts, etc. Crops can be infected throughout the growth period. It mainly causes stem rot, the diseased part is pale, the stem is hollow inside and there are black sclerotia, it is easy to break, and the number of pods formed is small or no pods are formed. In epidemic years, the yield reduction is 20-30%, and in severely affected fields, the yield reduction can reach 50-90%, or even a complete crop failure. The number of sclerotia in the field and environmental factors are important factors affecting the severity of this disease.

[0004] Through the joint prevention and control of the underground part and the above-ground part, the epidemic of the disease is fundamentally inhibited. Summary of the Invention

[0005] The purpose of the present invention is to provide a composition for preventing and controlling Sclerotinia sclerotiorum and / or Fusarium head blight, a preparation method thereof, and an application thereof, which maximally realizes the comprehensive utilization of the fermentation products of composite Bacillus.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a preparation method of a composition for preventing and controlling Sclerotinia sclerotiorum and / or Fusarium head blight, comprising the following steps:

[0008] (1) Ferment and culture Bacillus velezensis and Bacillus subtilis to obtain a fermentation broth;

[0009] (2) Perform solid-liquid separation on the fermentation broth to obtain bacterial cells and bacterial liquid;

[0010] (3) Perform ammonium sulfate precipitation on the bacterial solution, and obtain the supernatant and precipitate after centrifugation;

[0011] (4) Dissolve the precipitate to obtain the biocontrol pesticide;

[0012] (5) Jointly prepare a compound organic bacterial fertilizer with the bacterial cells obtained in step (2) and the supernatant obtained in step (3).

[0013] Preferably, the Bacillus velezensis described in step (1) is Bacillus velezensis YJ0-1, and the ratio of the effective viable bacteria numbers of Bacillus velezensis and Bacillus subtilis used in the fermentation culture is 1:1.5 to 2.5;

[0014] The PDA medium is used in the fermentation culture, the inoculation amounts of Bacillus velezensis and Bacillus subtilis are 0.8 to 1.2% of the weight of the medium, the fermentation culture time is 5 to 9 d, and the fermentation culture conditions are 25 to 29 °C and 120 to 140 rpm.

[0015] Preferably, centrifugation is used for the solid-liquid separation in step (2), the centrifugation conditions are 2 to 6 °C and the rotation speed is 5000 to 6000 rpm, and the centrifugation time is 15 to 25 min.

[0016] Preferably, the method for ammonium sulfate precipitation in step (3) is: precipitate the bacterial solution with an ammonium sulfate solution with a saturation of 70 to 80% under the condition of a pH value of 1.5 to 2.0, the centrifugation conditions are 2 to 6 °C and the rotation speed is 8000 to 10000 rpm, and the centrifugation time is 15 to 25 min.

[0017] Preferably, water is used for dissolution in step (4), and the mass ratio of the precipitate to water is 1:100 to 150.

[0018] Preferably, the method for preparing the compound organic bacterial fertilizer in step (5) includes the following steps:

[0019] A. Mix according to the mass ratio of 30 to 40 parts of crop straw, 35 to 45 parts of livestock and poultry manure, 8 to 12 parts of peat soil, 0.8 to 1.2 parts of the bacterial cells obtained in step (2), and 1.8 to 31.2 parts of volcanic ash to obtain a mixed material;

[0020] B. Dilute the supernatant obtained in step (3) with water to obtain a dilution, and mix the dilution with the mixed material to obtain a fermentation material;

[0021] C. Perform aerobic fermentation on the fermentation material, and obtain the compound organic bacterial fertilizer after drying.

[0022] Preferably, in step B, the volume ratio of the supernatant to water is 1:2800 - 3200, and the mass ratio of the diluent to the mixture is 1:1 - 1.2

[0023] In step C, the temperature of the aerobic fermentation is 20 - 40°C, and the ventilation volume during the aerobic fermentation is 0.05 - 0.2 cubic meters per minute per square meter.

[0024] The present invention also provides a composition prepared by the above preparation method, and the composition includes a biocontrol pesticide and a compound organic bacterial fertilizer.

[0025] The present invention also provides the application of the above composition in preventing and controlling Sclerotinia sclerotiorum and / or Fusarium graminearum.

[0026] The present invention also provides a method for using the above composition, including the following steps: applying the compound organic bacterial fertilizer into the soil as a seed fertilizer or top dressing, and spraying the biocontrol pesticide on the plants.

[0027] The present invention provides a composition for preventing and controlling Sclerotinia sclerotiorum and / or Fusarium graminearum, its preparation method and its application, including the following steps: (1) fermenting and culturing Bacillus velezensis and Bacillus subtilis to obtain a fermentation broth; (2) performing solid-liquid separation on the fermentation broth to obtain bacterial cells and a bacterial liquid; (3) performing ammonium sulfate precipitation on the bacterial liquid, and centrifuging to obtain a supernatant and a precipitate; (4) dissolving the precipitate to obtain a biocontrol pesticide; (5) jointly preparing a compound organic bacterial fertilizer with the bacterial cells obtained in step (2) and the supernatant obtained in step (3).

[0028] The composition of the present invention maximally realizes the comprehensive utilization of the fermentation products of compound bacteria. After centrifugation, the harvested bacterial agents are directly used as seed fertilizers or top dressings after being made into organic fertilizers. When used as a seed fertilizer, the organic bacterial fertilizer should be evenly spread on the soil surface before sowing or transplanting of the crops, and then plowed into the soil. When used as a top dressing, the organic fertilizer can be applied by furrow application or hole application at the rhizosphere of the crops during the growth period of the crops, so as to facilitate the colonization of compound Bacillus in the soil rhizosphere. The supernatant of the fermentation is directly treated by a combination of ammonium sulfate and acid precipitation to enrich antibacterial cyclic lipopeptide substances and macrolide inner disease-resistant products, and the microbial metabolites are directly sprayed at the initial stage of crop diseases, with a stable and high control effect.

[0029] The intermediate products obtained in step (3) include: 30% of the macrolide antibiotic Difficidin, about 40% of antibacterial cyclic lipopeptides (20% of Surfactin type, 6.5% of Fengycin type, 7.5% of Gageostatin type, 7.0% of Bacillomycin), 2% of phytotripeptide - as a direct precursor of jasmonic acid OPDA, and the rest are inorganic salts, foam stabilizers, etc. This component is used to control crop diseases, especially fungal diseases. It can control scab, sclerotinia rot, sheath blight, powdery mildew, fusarium wilt, rust, anthracnose, sooty mold, etc., with good control effect, no toxicity, no residues, harmless to humans and animals, no environmental pollution, and repeated use will not cause plants to produce specific disease resistance, providing a low-cost, efficient, non-toxic pollution-free biological control pesticide for agricultural production, with significant economic and social benefits.

[0030] The compound organic bacterial fertilizer prepared by the present invention and its use method are simple. Combined with crop straws, manure, etc., it is directly used as seed fertilizer for crops such as soybeans and wheat. It enables the Bacillus strains to colonize the rhizosphere of plants more directly and quickly. The release of secondary metabolites during the colonization process of rhizosphere microorganisms has a lasting effect of promoting growth and improving the disease resistance of crops, benefiting plants throughout their lives.

[0031] The biological pesticide involved in the present invention mainly includes macrolide and biosurfactant antibacterial cyclic lipopeptide substances. Because of their anti-adhesion, antibacterial and immunomodulatory properties, they are beneficial to enhancing the disease resistance of crops.

[0032] Deposition description

[0033] Bacillus velezensis YJ0-1, with the deposition name of YJ0-1, Latin name of Bacillus sp., deposition date of August 7, 2020, deposition location of the General Microbiology Center of the China Microbial Culture Collection Center, deposition address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and deposition number of CGMCC No. 20507. Description of the drawings

[0034] Figure 1 It is the confrontation experiment of Bacillus velezensis YJ0-1 and Sclerotinia sclerotiorum on PDA plate;

[0035] Figure 2 It is the inhibitory effect of Bacillus velezensis cells and extracellular products on the growth of Sclerotinia sclerotiorum;

[0036] Figure 3 It is the determination of the antibacterial activity of the crude protein solution;

[0037] Figure 4 It is the determination of the antibacterial activity of the crude lipopeptide solution;

[0038] Figure 5 It is the antibacterial effect test of lipopeptide solution against Exserohilum turcicum;

[0039] Figure 6 It is the antibacterial effect test of lipopeptide solution against Sclerotinia sclerotiorum;

[0040] Figure 7 It is the antibacterial effect test of lipopeptide solution against Fusarium graminearum;

[0041] Figure 8 It is the antibacterial effect test of lipopeptide solution against Fusarium verticillioides. Specific implementation mode

[0042] The present invention provides a preparation method of a composition for preventing and controlling sclerotinia rot and / or scab, comprising the following steps:

[0043] (1) Ferment and culture Bacillus velezensis and Bacillus subtilis to obtain a fermentation broth;

[0044] (2) Perform solid-liquid separation on the fermentation broth to obtain bacterial cells and a bacterial solution;

[0045] (3) Perform ammonium sulfate precipitation on the bacterial solution, and after centrifugation, obtain a supernatant and a precipitate;

[0046] (4) Dissolve the precipitate to obtain a biocontrol pesticide;

[0047] (5) Jointly prepare a compound organic bacterial fertilizer with the bacterial cells obtained in step (2) and the supernatant obtained in step (3).

[0048] In the present invention, the Bacillus velezensis described in step (1) is preferably Bacillus velezensis YJ0-1, and the ratio of the effective viable count of Bacillus velezensis and Bacillus subtilis used in the fermentation culture is preferably 1:1.5-2.5, and more preferably 1:2;

[0049] Preferably, PDA medium is used in the fermentation culture, and the inoculation amounts of Bacillus velezensis and Bacillus subtilis are preferably 0.8-1.2% of the weight of the medium, more preferably 1%, the fermentation culture time is preferably 5-9 d, more preferably 7 d, and the fermentation culture conditions are preferably 25-29 °C, 120-140 rpm, and more preferably 27 °C, 130 rpm.

[0050] In the present invention, preferably centrifugation is used for the solid-liquid separation in step (2), the centrifugation conditions are preferably 2-6 °C, a rotation speed of 5000-6000 rpm, more preferably 4 °C, a rotation speed of 6000 rpm, and the centrifugation time is preferably 15-25 min, more preferably 20 min.

[0051] In the present invention, the method of ammonium sulfate precipitation in step (3) is preferably as follows: the bacterial solution is precipitated with an ammonium sulfate solution having a saturation of 70-80% under the condition that the pH value is 1.5-2.0. Further preferably, the bacterial solution is precipitated with an ammonium sulfate solution having a saturation of 75% under the condition that the pH value is 1.7-1.8. The conditions for centrifugation are preferably 2-6 °C and a rotation speed of 8000-10000 rpm. Further preferably, the temperature is 4 °C and the rotation speed is 10000 rpm. The centrifugation time is preferably 15-25 min, and further preferably 20 min.

[0052] In the present invention, during dissolution in step (4), water is preferably used for dissolution. The mass ratio of the precipitate to water is preferably 1:100-150, and further preferably 1:100.

[0053] In the present invention, the method for preparing the compound organic bacterial fertilizer in step (5) includes the following steps:

[0054] A. Mixing according to the mass ratio of 30-40 parts of crop straw, 35-45 parts of livestock and poultry manure, 8-12 parts of peat soil, 0.8-1.2 parts of the bacterial cells obtained in step (2), and 1.8-31.2 parts of volcanic ash to obtain a mixed material;

[0055] B. Diluting the supernatant obtained in step (3) with water to obtain a dilution, and mixing the dilution with the mixed material to obtain a fermentation material;

[0056] C. Aerobically fermenting the fermentation material and drying to obtain the compound organic bacterial fertilizer.

[0057] In the present invention, the mass ratio in step A is preferably 35 parts of straw, 40 parts of livestock and poultry manure, 10 parts of peat soil, 1 part of the bacterial cells obtained in step (2), and 14 parts of volcanic ash.

[0058] In the present invention, the volume ratio of the supernatant to water in step B is preferably 1:2800-3200, and further preferably 1:3000. The mass ratio of the dilution to the mixed material is preferably 1:1-1.2, and further preferably 1:1.1.

[0059] The temperature of the aerobic fermentation in step C is preferably 20-40 °C, and further preferably 30 °C. The aeration volume during aerobic fermentation is preferably 0.05-0.2 cubic meters per minute per square meter, and further preferably 0.1-0.15 cubic meters per minute per square meter.

[0060] The present invention also provides a composition prepared by the preparation method, and the composition includes a biocontrol pesticide and a compound organic bacterial fertilizer.

[0061] The present invention also provides the application of the composition in preventing and controlling Sclerotinia sclerotiorum and / or Fusarium graminearum.

[0062] The present invention also provides a method for using the composition, comprising the following steps: applying the compound organic bacterial fertilizer as a seed fertilizer or top dressing into the soil, and spraying the biocontrol pesticide on the plants.

[0063] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0064] Example 1 Plate confrontation test between biocontrol bacteria and pathogenic bacteria

[0065] Plate confrontation method: Take a circular bacterial cake with a diameter of 6 mm and place it in the center of the culture medium plate. At four corners equidistant from the center pathogenic bacteria at a distance of 2 cm, Bacillus velezensis YJ0-1 is inoculated respectively. After inoculation, it is placed in a constant temperature incubator at 28 °C for 3 d, and the antagonistic effect of Bacillus velezensis YJ0-1 against Sclerotinia sclerotiorum is observed.

[0066] Agar well diffusion method: Referring to the experimental method of Hultmark D, take a circular bacterial cake with a diameter of 6 mm and place it in the center of the PDA culture medium plate. At a position 2 cm horizontally and vertically from the center bacterial cake, four round holes with a diameter of 4 mm are punched, and 50 μl of sample extract is injected into each hole. After inoculation, it is placed in a constant temperature incubator at 28 °C for 3 d, and the antagonistic effect of the fermentation broth extract of Bacillus velezensis YJ0-1 against Sclerotinia sclerotiorum is observed.

[0067] Mixed plate method: The solution or bacterial cells to be mixed are placed in PDB solution, agar powder is added, and after autoclaving, a mixed PDA plate is prepared. Take a circular bacterial cake with a diameter of 6 mm and place it in the center of the culture medium plate, and place it in a constant temperature incubator at 28 °C for 3 d, and observe the growth status of the pathogenic bacteria.

[0068] The Sclerotinia sclerotiorum preserved on the slant medium at 4 °C is placed in the PDA medium for activation culture at 28 °C for 4 d; 100 μl of Bacillus velezensis YJ0-1 preserved at -80 °C is taken and activated in the LB liquid medium at 28 °C, 200 r / min, for 1 d. The antagonistic effect of the biocontrol bacteria against the pathogenic bacteria is measured in the PDA medium with the activated Bacillus velezensis YJ0-1 and Sclerotinia sclerotiorum. Take a 6-mm circular bacterial cake from the activated Sclerotinia sclerotiorum PDA medium, and use the plate confrontation method to inoculate Bacillus velezensis YJ0-1 at four positions 2 cm perpendicular to the bacterial cake, with an inoculation amount of 10 μl and a concentration of 1×10 9 CFU / ml. It is placed in a constant temperature incubator at 28 °C for culture, and the antagonistic effect of Bacillus velezensis YJ0-1 on the growth of Sclerotinia sclerotiorum is observed. The experiment includes three biological replicates, with three culture dishes for each replicate.

[0069] Example 2 Determination of Intracellular and Extracellular Antibacterial Active Substances of Biocontrol Strains

[0070] Take three bottles of 80 ml PDB medium, sterilize it by high-temperature and high-pressure, and reserve for later use. Take one bottle of PDB medium, pick up Bacillus velezensis YJ0-1 with a toothpick in a laminar flow hood and put it into a triangular flask. The culture conditions are 28 °C and 200 r / min for 2 days. The completed fermentation broth is centrifuged at 4 °C and 12,000 r / min for 5 min using a high-speed refrigerated centrifuge. The cells and supernatant are collected respectively. The cells are added to a new PDB liquid medium and mixed evenly. Agar is added to the 80 ml PDB liquid medium with cells added, 80 ml supernatant, and 80 ml PDA liquid medium respectively. After sterilization by high-temperature and high-pressure, three kinds of mixed PDA solid media are prepared. A Sclerotinia sclerotiorum cake (6 mm) is inoculated in the center of each of the three media and cultured in an incubator at 28 °C. Observe the growth of Sclerotinia sclerotiorum. Each treatment includes 3 biological replicates.

[0071] Example 3 Cultivation of Biocontrol Strains and Preparation of Fermentation Broth

[0072] The cryopreserved Bacillus velezensis YJ0-1 is inoculated on an LB solid medium by the streak plate method and cultured at 28 °C for 2 days. After single colonies grow out, pick a single colony and place it in an LB liquid medium for enrichment culture. The culture conditions are 30 °C and 200 r / min for 24 h with shaking. The enriched culture broth is inoculated into a fermentation medium at an inoculation amount of 5% (1000 ml volumetric flask, loading volume of 500 ml), and cultured at 28 °C and 200 r / min with shaking for 48 h to prepare a cell fermentation broth. The fermentation broth is placed in a high-speed centrifuge and centrifuged at 4 °C and 12,000 / min for 20 min at high speed to remove the cell precipitate, and the supernatant is collected to complete the preparation of the fermentation broth.

[0073] 1. Preparation of Crude Protein Solution

[0074] The fermentation broth of Bacillus velezensis YJ0-1 was stored at 4°C for later use. 500 ml of the fermentation broth was placed in a 1 L Erlenmeyer flask, and solid ammonium sulfate was slowly added until the ammonium sulfate saturation of the fermentation broth reached w = 10%. The solution was placed on a magnetic stirrer, and the rotor rotated uniformly at the bottom of the Erlenmeyer flask. It was allowed to precipitate overnight for 12 h. The next day, the solution was placed in a high-speed centrifuge and centrifuged at 4°C and 10,000 r / min for 20 min. The precipitate and the supernatant were collected separately. The precipitate was dissolved in a 25 mmol / L Tris-Hcl solution with pH = 8.0 to obtain the crude protein solution collected when the ammonium sulfate saturation of the fermentation broth was 10%, and it was stored in a 4°C refrigerator. Solid ammonium sulfate was continuously added to the supernatant with w = 10% until the ammonium sulfate saturation of the fermentation broth reached w = 20%. The above steps were repeated to obtain the crude protein solution collected when the ammonium sulfate saturation of the fermentation broth was 20%. According to this step, crude protein solutions with w = 10%, w = 20%, w = 30%, w = 40%, w = 50%, w = 60%, w = 70%, w = 80%, w = 90%, and w = 100% were finally obtained. The ten groups of crude protein solutions obtained were dialyzed using a clean dialysis bag. The dialysis solution was a 25 mmol / L Tris-Hcl solution. It was dialyzed with magnetic stirring for 2 d, and the dialysis solution was changed every 12 h to remove the ammonium sulfate salt in the crude protein solution. Then, it was filtered and sterilized with a 0.22 μm bacterial filter to obtain the sterile crude protein solutions precipitated with ammonium sulfate at different saturation concentrations. The antagonistic activity was measured against Sclerotinia sclerotiorum using the agar well diffusion method. Each concentration gradient was repeated three times, and a control group of 25 mmol / L Tris-Hcl solution was set up.

[0075] 2. Preparation of crude lipopeptide solution

[0076] The fermentation broth of Bacillus velezensis YJ0-1 was stored at 4 °C for later use. 500 ml of the fermentation broth was placed in a 1 L Erlenmeyer flask, and concentrated hydrochloric acid was slowly added until the pH of the fermentation broth was about 1.9. It was then refrigerated in a 4 °C refrigerator for 12 h. The next day, the fermentation broth was placed in a high-speed centrifuge and centrifuged at 4 °C and 12,000 / min for 20 min. The supernatant was discarded, and the precipitate was collected. The precipitate was placed in a drying oven and dried overnight at 60 °C. The crude lipopeptide solid was placed in a mortar and ground thoroughly to obtain crude lipopeptide powder. 250 mg of the crude lipopeptide powder was dissolved in 50 ml of ddH2O to prepare a crude lipopeptide solution. 30 ml of the crude lipopeptide solution was passed through a C18 solid-phase extraction column. After the C18 column was activated successively with chromatographic-grade analytical pure methanol and ddH2O (the liquid in the column must not be lower than the white sieve at the bottom of the column during activation), the lipopeptide solution was added and allowed to drip naturally. It was then washed successively with 30 ml each of ddH2O, 10% methanol, 20% methanol, 30% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol. The methanol eluates at each concentration gradient were collected. Using a rotary evaporator, the water bath temperature was set at 40 °C and rotated evenly until all the water and methanol solution were evaporated to dryness. After rotary evaporation to dryness, yellow crystals were obtained, which were then dissolved in 4 ml of 25 mmol / L Tris-Hcl and filtered through a microporous filter membrane to remove the bacteria. The agar well diffusion method was used to measure the antagonistic activity against Sclerotinia sclerotiorum, and each concentration gradient was repeated three times. The control group was set as a 25 mmol / L Tris-Hcl solution.

[0077] 4. Exploration of the broad-spectrum antibacterial effect of crude lipopeptide extract

[0078] The crude lipopeptide solution obtained by acid precipitation and 80% methanol washing was rotary evaporated to obtain yellow crystals, which were the crude lipopeptide. The crude lipopeptide crystals were dissolved in pure water to prepare a 500 mg / ml crude lipopeptide mother liquor. The crude lipopeptide mother liquor was mixed with PDB solution to prepare mixed PDA plates with crude lipopeptide concentrations of 0 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, and 250 mg / ml. Common plant disease pathogens, Sclerotinia sclerotiorum, Fusarium graminearum, Fusarium verticillioides, Setosphaeria turcica, and Fusarium oxysporum, were used for plate experiments. Sterilized punchers were used to obtain six pathogen disks (d = 4 mm) and placed in the center of the mixed PDA plates. Incubate at a constant temperature of 28 °C, and each concentration gradient was repeated three times. The control group was set as a 25 mmol / L Tris-Hcl solution, and the colony growth status was observed after 3 d.

[0079] Results and analysis

[0080] 1. Plate confrontation experiment between Bacillus velezensis YJ0-1 and Sclerotinia sclerotiorum

[0081] On the 3rd day after inoculation of Sclerotinia sclerotiorum in the PDA plate, the mycelium of Sclerotinia sclerotiorum covered the entire plate. On the 7th day after inoculation, large sclerotia appeared, indicating that the growth rate of Sclerotinia sclerotiorum was relatively fast. In the PDA plate inoculated with Sclerotinia sclerotiorum and Bacillus velezensis YJ0-1 simultaneously, the colony diameter of Bacillus velezensis YJ0-1 on the 7th day of culture was significantly larger than that on the 3rd day of culture, indicating that the growth rate of Bacillus velezensis YJ0-1 was also relatively fast. However, in the PDA plate inoculated with both bacteria simultaneously, the colony diameter of Sclerotinia sclerotiorum on the 7th day was basically the same as that on the 3rd day, indicating that the growth of Sclerotinia sclerotiorum was inhibited by Bacillus velezensis. Based on the above observations, it is shown that Bacillus velezensis YJ0-1 has an obvious inhibitory effect on the growth of Sclerotinia sclerotiorum (see Figure 1 ).

[0082] 2. Determination of the antibacterial effect of the cells and extracellular products of Bacillus velezensis YJ0-1 by the mixed plate method

[0083] After culturing on the PDA plate for 6 days, the mycelium of Sclerotinia sclerotiorum in the PDA plate of the control group had covered the entire plate, and obvious sclerotia had grown on the 10th day; compared with the control, in the experimental group with Bacillus velezensis YJ0-1 cells in the PDA medium, the mycelium did not completely cover the entire plate on the 6th day. On the 10th day, compared with the 6th day, the mycelium slowly spread outwards but still did not cover the entire plate, but obvious large sclerotia could be seen in the center of the plate; compared with the control group, in the experimental group with the extracellular secretion of Bacillus velezensis YJ0-1 in the PDA medium, Sclerotinia sclerotiorum could not grow in the plate on either the 6th day or the 10th day (see Figure 2 ). The above results show that the antibacterial active products of Bacillus velezensis YJ0-1 that have an inhibitory effect on Sclerotinia sclerotiorum are mostly bacterial extracellular products, and the antibacterial effect is extremely significant. Moreover, these antibacterial active substances will not be inactivated under high temperature and high pressure conditions, that is, at 121 °C, 103.4 KPa, and 20 min. Therefore, the extracellular secretion of Bacillus velezensis YJ0-1 was used for the plate antibacterial experiment in the subsequent experiments and all were treated with high-pressure steam sterilization.

[0084] 3. Crude extraction of antibacterial active substances from the fermentation broth of Bacillus velezensis YJ0-1

[0085] (1) Antibacterial experiment of crude protein solution

[0086] The crude protein solutions extracted at different ammonium sulfate saturation concentrations after desalting with dialysis bags were used for the plate antibacterial experiment. Before the experiment, the extracted crude protein solutions were sterilized by high-pressure steam at 121 °C, 103.4 KPa for 20 min. The ten saturated ammonium sulfate concentrations were w = 10%, w = 20%, w = 30%, w = 40%, w = 50%, w = 60%, w = 70%, w = 80%, w = 90%, and w = 100%, and the antibacterial activities of the crude protein solutions at each concentration against Sclerotinia sclerotiorum were measured. The results showed that the crude protein solutions extracted at ammonium sulfate saturation concentrations of w = 10% and w = 100% had no antibacterial effect on Sclerotinia sclerotiorum; before the ammonium sulfate saturation concentration of the crude protein solution was 40%, the antibacterial zone around the agar well expanded with the increase in ammonium sulfate saturation, indicating that within the range of w = 10% to w = 40%, as the ammonium sulfate saturation increased, the antibacterial activity of the crude protein solution also increased. At w = 40%, the antibacterial effect of the extracted crude protein solution on Sclerotinia sclerotiorum was the best, and the diameter of the antibacterial zone reached 18.03 mm, which was the best antibacterial effect among the crude protein solutions at each concentration gradient; after the ammonium sulfate saturation was w = 40%, the antibacterial activity of the crude protein solution obtained with the increase in ammonium sulfate saturation gradually decreased, and the antibacterial zone around the agar well also decreased accordingly, and there was no inhibitory effect on the growth of Sclerotinia sclerotiorum in the crude protein solution with w = 100%. This may be because as the ammonium sulfate concentration increased, the content of impurity proteins in the obtained crude protein solution also increased (see Figure 3 ).

[0087] (2) Antibacterial experiment of crude lipopeptide solution

[0088] The crude lipopeptide solutions obtained after eluting the C18 solid-phase extraction column with different concentrations of methanol were evaporated by rotary evaporation and then dissolved with Tris-Hcl to obtain 11 groups of crude lipopeptide solutions with different antibacterial activities, and the agar well diffusion experiment was carried out. The results showed that in the agar well diffusion experiment with the crude lipopeptide solution eluted with ddH2O, an antibacterial zone with a very small visible range was observed around the agar well. In the agar well diffusion experiments with the crude lipopeptide solutions eluted with 10% methanol to 60% methanol, no antibacterial zone was shown around the agar well, indicating that the content of antibacterial active substances in the crude lipopeptide solutions eluted with 10% methanol, 20% methanol, 30% methanol, 40% methanol, 50% methanol, and 60% methanol was extremely low and had no inhibitory effect on the growth of Sclerotinia sclerotiorum; during the elution process with 70% methanol to 100% methanol, the crude lipopeptide solution eluted with 70% methanol showed an obvious antibacterial zone. The antibacterial zone of the crude lipopeptide solution eluted with 80% methanol was the largest in this experiment, reaching 25.23 mm. Subsequently, as the methanol concentration increased, the range of the antibacterial zone gradually decreased. The size of the antibacterial zone produced by the crude lipopeptide solution obtained with 100% methanol was 20.08, which was smaller than the antibacterial zone of 22.47 mm of the crude lipopeptide solution obtained with 70% methanol (seeFigure 4 ), from left to right are the antibacterial effects of the crude lipopeptide solution eluted with ddH2O, 10% methanol, 20% methanol, 30% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol. It is worth noting that the antibacterial effect of the crude lipopeptide solution eluted with ddH2O is greater than that of the crude lipopeptide solution eluted with 10% - 60% methanol. This may be because the solubility of some antibacterial active substances in the acid precipitation in ddH2O is greater than that in methanol, so they are eluted first.

[0089] (3) Broad-spectrum antibacterial effect of the crude lipopeptide solution

[0090] The colony morphology of four plant pathogens, Sclerotinia sclerotiorum, Fusarium graminearum, Fusarium verticillioides, and Exserohilum turcicum, after 3 days of separate purification culture on a mixed PDA medium was observed. The results showed that the colonies of the four plant pathogens grew to varying degrees in the control group (0 mg / ml). The growth of the Exserohilum turcicum colony was not obvious, but the mycelium had covered the agar disc with white hyphae (see Figure 5 ); the mycelium of Sclerotinia sclerotiorum had almost completely covered the bottom of the petri dish (see Figure 6 ); the colony of Fusarium graminearum had an obvious extension, and the mycelium extended outwards in a fluffy and divergent shape (see Figure 7 ); the growth of Fusarium verticillioides was relatively slow, and it was only surrounded by fluffy mycelium on the agar disc (see Figure 8 ). Among the six different concentration gradient treatments, the colony of Exserohilum turcicum did not expand. In the mixed PDA medium with a crude lipopeptide concentration of 150 mg / ml, white hyphae still grew on the agar disc, but in the mixed PDA medium with a crude lipopeptide concentration of 200 mg / ml and 250 mg / ml, the agar disc of Exserohilum turcicum had turned black and necrotic, and no white hyphae could grow (see Figure 5 ). This indicates that when the crude lipopeptide concentration reaches 200 mg / ml, its antibacterial activity is sufficient to inhibit the normal growth of Exserohilum turcicum and even cause the death of Exserohilum turcicum; in the mixed PDA plate with a crude lipopeptide concentration of 50 mg / ml for Sclerotinia sclerotiorum, the growth of the mycelium on the stipe did not spread around the bottom of the plate like in the control group, but showed a tendency to grow towards the air from the agar disc, and the newly grown mycelium did not contact the mixed PDA medium (see Figure 6) It shows that the crude lipopeptide substances contained in the culture medium have an inhibitory effect on the growth of Sclerotinia sclerotiorum hyphae. When the concentration of crude lipopeptide reaches 100 mg / ml, the growth inhibition rate of Sclerotinia sclerotiorum has reached 100%. As the concentration of crude lipopeptide increases, the inhibitory effect is the same. For Fusarium graminearum, there is no significant difference in the colony size at a crude lipopeptide concentration of 50 mg / ml compared to the control group, but the hyphal density is significantly lower than that of the control group. Among the six treatment groups with different concentration gradients, as the concentration of crude lipopeptide gradually increases, the colonies of Fusarium graminearum show a gradually decreasing trend. When the concentration of crude lipopeptide is 250 mg / ml, there is no hyphal extension around the fungal cake (see Figure 7 ). It is proved that when the concentration of crude lipopeptide is 250 mg / ml, the inhibition rate on Fusarium graminearum has reached 100%. For the growth of the colonies of Verticillium pseudotheobromae, since Verticillium pseudotheobromae grows relatively slowly, only in the control group is the fungal cake surrounded by villous hyphae and the colony does not grow outwards. However, obviously, in the mixed PDA plate with a crude lipopeptide solution concentration of 200 mg / ml, the fungal cake of Verticillium pseudotheobromae has turned white. After 3 days of constant temperature culture, the fungal cake does not grow but instead dies (see Figure 8 ). It shows that when the concentration of crude lipopeptide reaches 200 mg / ml, it can cause the death of Verticillium pseudotheobromae. In summary, the crude lipopeptide solution obtained by acid precipitation, 80% methanol elution and then dissolution by rotary evaporation has different degrees of inhibitory effects on Alternaria alternata, Sclerotinia sclerotiorum, Fusarium graminearum and Verticillium pseudotheobromae. As the concentration of crude lipopeptide increases, the inhibitory effects on the four pathogenic fungi also increase. When the concentration of crude lipopeptide reaches 250 mg / ml, the inhibition rates on the four plant pathogenic fungi have all reached 100% (see Figures 5 to 8 ).

[0091] As can be seen from the above examples, the present invention provides a composition for preventing and controlling sclerotinia rot and / or scab, its preparation method and its application, including the following steps: (1) Fermenting and culturing Bacillus velezensis and Bacillus subtilis to obtain a fermentation broth; (2) Separating the solid and liquid of the fermentation broth to obtain bacterial cells and a bacterial solution; (3) Precipitating the bacterial solution with ammonium sulfate, and centrifuging to obtain a supernatant and a precipitate; (4) Dissolving the precipitate to obtain a biopesticide; (5) Jointly preparing a compound organic bacterial fertilizer with the bacterial cells obtained in step (2) and the supernatant obtained in step (3). The compound organic bacterial fertilizer prepared by the present invention has a simple usage method. Combined with crop straws and manure, etc., it can be directly used as a seed fertilizer for crops such as soybeans and wheat. The biopesticide mainly includes antibacterial cyclic lipopeptide substances such as macrolides and biosurfactants. Because of its anti-adhesion, antibacterial and immunomodulatory properties, it is beneficial to enhance the disease resistance of crops.

[0092] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a composition for preventing and controlling sclerotinia and / or fusarium head blight, characterized in that: The steps include: (1) fermenting Bacillus Velezii and Bacillus subtilis to obtain a fermentation liquid; (2) performing solid-liquid separation on the fermentation liquid to obtain bacterial cells and bacterial liquid; (3) subjecting the bacterial solution to ammonium sulfate precipitation, and obtaining a supernatant and a precipitate after centrifugation; (4) dissolving the precipitate to obtain the biocontrol pesticide; (5) preparing a composite organic bacterial fertilizer by combining the bacterial cells obtained in step (2) and the supernatant obtained in step (3).

2. The preparation method according to claim 1, characterized in that: The Bacillus Velez subtilis described in step (1) is Bacillus Velez subtilis YJ0-1, and the ratio of the effective viable counts of Bacillus Velez subtilis used in the fermentation culture is 1:1.5-2.5; The PDA culture medium is used in the fermentation culture, the inoculation amount of the Bacillus Velez and the Bacillus subtilis is 0.8-1.2% of the weight of the culture medium, the fermentation culture time is 5-9 days, and the fermentation culture conditions are 25-29° C. and 120-140 rpm.

3. The preparation method according to claim 2, characterized in that: The solid-liquid separation in step (2) is carried out by centrifugation, the centrifugation conditions are 2-6°C, the rotation speed is 5000-6000rpm, and the centrifugation time is 15-25min.

4. The preparation method according to claim 3, characterized in that: The method of ammonium sulfate precipitation in step (3) is: the bacterial liquid is precipitated with an ammonium sulfate solution with a saturation of 70-80% under the condition of pH value of 1.5-2.0, the centrifugation conditions are 2-6°C, the rotation speed is 8000-10000rpm, and the centrifugation time is 15-25min.

5. The preparation method according to claim 4, characterized in that: The dissolution in step (4) is carried out using water, and the mass ratio of the precipitate to water is 1:100-150.

6. The preparation method according to claim 5, characterized in that: The method for preparing the composite organic fertilizer in step (5) comprises the following steps: A. Mixing 30 to 40 parts of crop straw, 35 to 45 parts of livestock and poultry manure, 8 to 12 parts of peat soil, 0.8 to 1.2 parts of the bacterial cells obtained in step (2), and 1.8 to 31.2 parts of volcanic ash in a mass ratio to obtain a mixture; B. diluting the supernatant obtained in step (3) with water to obtain a diluent, and mixing the diluent with the mixed material to obtain a fermentation material; C. Aerobically ferment the fermented material and obtain a composite organic fertilizer after drying.

7. The preparation method according to claim 6, characterized in that: The volume ratio of the supernatant to water in step B is 1:2800-3200, the mass ratio of the diluent to the mixed material is 1:1-1.2; the temperature of the aerobic fermentation in step C is 20-40°C, and the ventilation volume during the aerobic fermentation is 0.05-0.2 cubic meters / minute·square meters.

8. The composition prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The composition comprises a biocontrol pesticide and a composite organic bacterial fertilizer.

9. Use of the composition according to claim 8 in preventing and controlling sclerotinia disease and / or fusarium head blight.

10. The method for using the composition according to claim 8, characterized in that: The method comprises the following steps: applying the composite organic bacterial fertilizer into the soil as seed fertilizer or topdressing fertilizer, and spraying the biocontrol pesticide on the plants.

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