Antagonistic strain D86 and antibiological inoculant and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to an antagonistic strain D86 and a biocontrol inoculant and application thereof, the antagonistic strain D86 is bacillus subtilis D86 and is preserved in the China Center for Type Culture Collection on November 11, 2024, the preservation number is CCTCC NO: M 20242514, and the antagonistic strain D86 is classified and named as Bacillus subtilis D86. The bacillus subtilis is separated from saline-alkali soil, has a remarkable antagonistic effect on walnut tree rot pathogenic bacteria cyst aureocladium and cryptosphere, and has a good prevention and treatment effect on walnut branch rot under an in-vitro condition.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an antagonistic strain D86 and a biocontrol agent and application thereof. Background Art

[0002] Walnut is a deciduous tree of the genus Juglandia in the family Juglandaceae. It is the first of the four famous dried fruits in the world and one of the important economic trees in my country. With the expansion of walnut planting and the monoculture of fine varieties, diseases mainly caused by rot have occurred on a large scale, seriously affecting the healthy development of the walnut industry. Rot mainly occurs on the branches and bark of walnut trees. Symptoms include bark cracking, discoloration, rotting, and branch death. Walnut rot not only affects the normal growth of walnut trees, but also leads to a decrease in fruit yield and quality. In severe cases, it may even lead to the death of the tree. Therefore, the prevention and control of walnut rot is particularly important.

[0003] At present, the prevention and control measures for walnut rot disease include chemical control, physical control and biological control. Although chemical control is more effective, the use of chemical pesticides will cause environmental pollution and pesticide residue problems. Physical control mainly uses tools such as scrapers to scrape off rot lesions, which can reduce the harm of rot disease to walnut trees, but it cannot be cured and is prone to recurrence. In contrast, biological control has gradually become the preferred option for its green, efficient, pollution-free, and harmless to humans and animals. This method uses beneficial microorganisms or their metabolites to inhibit or eliminate harmful microorganisms, thereby achieving disease control. In particular, antagonistic strains, due to their high specificity and selectivity, can prevent and control specific pathogens while avoiding damage to beneficial microorganisms, and have become a hot topic of current research.

[0004] Therefore, finding an antagonistic strain is of great significance to ensure the healthy and stable development of the walnut industry. Summary of the invention

[0005] In order to enrich the biological control methods of walnut rot disease, the present invention provides an antagonistic strain D86 and its biocontrol agent and application. The antagonistic strain D86 is Bacillus subtilis D86. The Bacillus subtilis D86 bacterial liquid prepared by Bacillus subtilis D86 has a significant antagonistic effect on Cystosporium chrysotrichum and Cryptococcus, and has a good control effect on walnut rot disease.

[0006] The technical solution of the present invention is as follows:

[0007] An antagonistic strain (Bacillus subtilis) D86 was deposited in the China Center for Type Culture Collection on November 11, 2024, with the deposit number: CCTCC NO: M 20242514, and the classification name is Bacillus subtilis D86

[0008] The antagonistic strain D86 provided by the present invention is Bacillus subtilis D86, which is a strain isolated from saline-alkali soil. The strain has a significant antagonistic effect on walnut tree rot pathogens Cystosporium aureum and Cryptosphaeria spp., and has a good control effect on walnut branch rot under in vitro conditions. It provides a new and effective way for the control of walnut rot and has good application prospects.

[0009] The invention provides a biocontrol fungicide, wherein the only active ingredient of the biocontrol fungicide is Bacillus subtilis D86.

[0010] Furthermore, the biocontrol agent is one of a liquid agent, a powder agent, and a granular agent.

[0011] Furthermore, the biocontrol agent also includes auxiliary materials; the auxiliary materials are fulvic acid, xanthan gum, sorbitol or sodium alginate.

[0012] Furthermore, the biocontrol agent is D86 bacterial liquid.

[0013] Furthermore, the OD value of the D86 bacterial solution is 0.8-1.4.

[0014] The invention provides a method for preparing a biocontrol agent. The D86 bacterial solution is obtained by inoculating the antagonistic strain D86 into an LB liquid culture medium and shaking the culture medium.

[0015] Furthermore, the shaking culture condition is to culture at 30°C to 40°C at a rotation speed of 110r / min to 130r / min for 10h to 14h.

[0016] The present invention also provides an application of the antagonistic strain D86 or the biocontrol agent in preventing and treating walnut tree rot.

[0017] Furthermore, the walnut tree rot disease is caused by Cytospora chrysosperma or Cryptococcus Neoformans.

[0018] The culture medium formula used in the present invention is as follows:

[0019] PDA solid culture medium (g / L): potato 200 g / L, glucose 20 g / L, agar 15 g / L, water 1 L.

[0020] LB liquid medium (g / L): tryptone 10 g / L, yeast extract 5 g / L, agar powder 15 g / L, sodium chloride 10 g / L, water 1 L.

[0021] Description of biological material deposit:

[0022] Bacillus subtilis D86, referred to as Bacillus subtilis D86 in the present invention, has been deposited in China Center for Type Culture Collection on November 11, 2024, with a deposit number of CCTCC NO: M20242514. The depository address is Wuhan University, Wuhan, China, zip code: 430072, and the classification name is: Bacillus subtilis D86 。

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The antagonistic strain D86 provided by the present invention is Bacillus subtilis D86, which is a strain isolated from saline-alkali soil. The strain has a significant antagonistic effect on walnut tree rot pathogens Cystosporium aureum and Cryptosphaeria spp., and has a good control effect on walnut branch rot under in vitro conditions. It provides a new and effective way for the control of walnut rot and has good application prospects.

[0025] The antagonistic strain D86 provided by the present invention is qualitatively analyzed for its growth-promoting indexes, and it is found that the antagonistic strain D86 has growth-promoting index effects on two indicators, nitrogen fixation and ACC deaminase; at the same time, the antibacterial rate of the strain on Chrysocytosis reaches 85%, and the antibacterial rate on Cryptococcus reaches 66%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The figure shows the antagonistic results of different strains against Cystosporum flavum. In the figure, A is the antagonistic strain D86; B is the strain D88; C is the strain D47; and D is the strain D41.

[0028] Figure 2 The figure is a result diagram of the antagonistic effect of different strains on Cryptococcus; in the figure, A is the antagonistic strain D86; B is the strain D41; C is the strain D47; and D is the strain D88.

[0029] Figure 3 The figure is a diagram of the effect of antagonistic strain D86 on the pathogenicity of Cryptococcus branches; in the figure, A is the LB control group; B is the Cryptococcus + antagonistic strain D86 group; C is the antagonistic strain D86 group alone; D is the Cryptococcus group. Each group has 4 parallel experimental results.

[0030] Figure 4The figure is a diagram showing the effect of antagonistic strain D86 on the pathogenicity of cysticercus chrysogenum branches; in the figure, A is the LB control group; B is the cysticercus chrysogenum + antagonistic strain D86 group; C is the antagonistic strain D86 group alone; and D is the cysticercus chrysogenum group. Each group has 4 parallel experimental results. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0032] Example 1: Isolation of strains

[0033] Bacterial isolation: In June 2023, soil samples were collected from the saline-alkali land of the 10th Regiment in Aral City (81°17′50.04E, 40°32′40.91N) and placed in sterile plastic bags. 10 g of each sample was weighed and placed in a 250 mL conical flask (the conical flask was pre-filled with sterile glass beads and 90 mL of sterile deionized water), and the soil suspension was obtained by shaking at 35°C and 180 r / min for 30 min. Each soil suspension was then diluted to 10 -3 , 10 -4 , 10 -5 , 100 μL was spread on LB plates respectively, and incubated at 37°C for 24 h. The single colonies that appeared were purified by streaking on LB plates three times and placed in 30% sterile glycerol tubes respectively, numbered and stored at -80°C for later use.

[0034] Antibacterial assay: Bacterial liquid of antagonistic strain D86: Use a 200 μL pipette to dip the antagonistic strain D86 into the LB liquid shaking tube and then shake it at 37°C constant temperature shaker at 120 r / min for 12 h for standby use.

[0035] The plate confrontation method was used to determine the antagonistic activity of the strain against walnut rot pathogens. First, the purified cultured golden cysts and Cryptococcus were punched out with a sterilized puncher to make 5 mm cakes and inoculated into the center of the PDA culture medium. Then, 2.5 μL of the shaken bacterial solution was taken with a pipette and inoculated with bacteria of different numbers 2.5 cm above, below, left and right from the cakes made of golden cysts and Cryptococcus. The culture was carried out in a fungal incubator at 26°C for 7 days. PDA inoculated with only golden cysts and Cryptococcus was used as a control, and each group was repeated three times. After the control pathogens have grown all over the plate (such as Figure 1 Middle A and Figure 2 The diameter of the colony on the plate was measured by the cross measurement method, and the inhibition rate of the strain was calculated. The inhibition rate calculation formula is:

[0036] Inhibition rate (%) = (control pathogen colony diameter - treated pathogen colony diameter) / control pathogen colony diameter × 100.

[0037] The experimental results showed that the antagonistic strain D86 had a significant inhibitory effect on walnut rot pathogens, with an inhibition rate of 85% against golden cysts and 66% against Cryptococcus.

[0038] Example 2: Determination of growth-promoting indexes of antagonistic strain D86

[0039] In this example, the growth-promoting index of the antagonistic strain D86 was determined, and the growth-promoting ability of the antagonistic strain D86 on various indexes was determined by measuring the antagonistic strain D86's organic phosphorus solution, inorganic phosphorus solution, siderophore (CAS), nitrogen fixation, potassium solution, indoleacetic acid (IAA), and ACC deaminase. The specific method is as follows:

[0040] (1) Nitrogen fixation: 1 μL of activated D86 bacterial solution was spotted in the center of an Aspergillus medium plate. After incubation at 28°C for 7 days, the plate was removed and the size and morphology of the colonies were observed. Bacteria with nitrogen fixation ability will grow sticky, translucent, white or brown colonies.

[0041] (2) Potassium solubilization: Take 1 μL of the activated D86 bacterial solution and plant it in the center of the potassium solubilizing bacteria screening culture medium plate. The culture and observation process is the same as the qualitative test of bacterial phosphate solubilization ability.

[0042] (3) Hydrolysis of organic and inorganic phosphorus: Take 1 μL of the activated D86 bacterial solution and spot it in the center of the organic and inorganic phosphorus hydrolysis bacterial culture medium plate. Incubate the plate upside down at 28°C for 7 days, then take out the plate and observe whether there is a hydrolysis zone.

[0043] (4) Indoleacetic acid (IAA): The isolated and purified D86 was inoculated into 20 mL of IAA liquid medium (wherein the concentration of L-tryptophan was 200 mg / L), with 3 replicates for each strain. The culture was shaken at 30°C and 180 r / min for 2 days. 100 μL of the bacterial suspension was dropped onto a white ceramic plate, and an equal volume of Salkowski colorimetric solution was added. A mixed solution of 100 μL of uninoculated LB liquid medium and an equal volume of colorimetric solution was used as a control. The white ceramic plate was placed at room temperature and away from light for 30 minutes and then observed. A pink color change was positive, indicating that IAA could be secreted. The darker the color, the greater the secretion intensity. No color change was negative, indicating that IAA could not be secreted.

[0044] (5) Siderophore production ability: The antagonistic strain D86 was inoculated on CAS solid culture medium for three times for each strain. The strain was cultured at 28°C for 5 days to observe whether an orange siderophore halo appeared around the antagonistic strain D86. The strain with an orange halo was a siderophore-producing strain.

[0045] (6) Determination of ACC deaminase: The antagonistic strain D86 was inoculated onto DF and ADF medium respectively by the spot seeding method, cultured at 30°C for 4 days, and the colony growth was observed every day. During the three consecutive subcultures, if the colony growth on the ADF medium was significantly better than that on the DF medium, it means that the antagonistic strain D86 can grow with ACC as the sole nitrogen source, that is, it can synthesize ACC deaminase.

[0046] The results are shown in Table 1:

[0047] Table 1 Qualitative determination of growth-promoting indexes of antagonistic strain D86

[0048] Growth promoting indicators Organic phosphorus solution Solution of inorganic phosphorus Siderophore Nitrogen fixation Potassium IAA ACC deaminase result - - - + - - +

[0049] Note: “-” in the table means that the indicator has no growth-promoting effect; “+” means that the indicator has a growth-promoting effect.

[0050] As shown in Table 1, the antagonistic strain D86 has a growth-promoting effect on both nitrogen fixation and ACC deaminase.

[0051] Example 3: Molecular identification and preservation of antagonistic strain D86

[0052] (1) Molecular identification of the antagonistic strain D86: First, a single colony of the antagonistic strain D86 was picked and inoculated into LB liquid culture medium, and cultured in a shaker at 28°C and 180 r / min for 24 h. The DNA of the antagonistic strain D86 was extracted using the instructions of the bacterial genomic DNA extraction kit and stored in a refrigerator at 4°C. The sample was sent to Novogene Technology Co., Ltd. (Beijing) for bacterial complete map sequencing analysis.

[0053] Then the antagonistic strain D86 was cultured on LB plates at 30°C for 24 hours and the morphological characteristics of single colonies were observed. After the strain was cultured in LB liquid medium at 30°C and 200rpm for 16 hours, the strain DNA was extracted using a bacterial genomic DNA extraction kit to amplify the 16S rRNA sequence (primer sequence 27F: 5'-AGA GTTTGATCCTGGCTCAG-3' recorded as SEQ ID NO.1, 1492R: 5'-CGGCTACCT TGTTACGAC-3' recorded as SEQ ID NO.2) and gyrA gene (primer sequence 42F: 5'-CAGTCAGGAAATGCGTACGTCCTT-3' recorded as SEQ ID NO.3, 1066R: 5'-CAAGGTAA TGCTCCAGGCATTGCT-3' recorded as SEQ ID NO.4), and the PCR reaction system was: 2×Taq PCR Mix 12.5μL, 27F / 7T237F (10μmol / L) 1.0μL, 1492R / 8261R (10μmol / L) 1.0μL, DNA template 1.0μL, ddH2O 9.5μL. PCR reaction conditions are: 95℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 2min, 30 cycles; 72℃ final extension for 5min. PCR amplification products were detected by electrophoresis in 1% agarose gel. The products after successful amplification were sent to Sangon Biotech (Xi'an) Co., Ltd. for purification and sequencing, and the 16S rRNA sequence of the antagonistic strain D86 shown in SEQ ID NO.5 and the gyrA gene of the antagonistic strain D86 shown in SEQ ID NO.6 were obtained. The sequences of SEQ ID NO.5 and SEQ ID NO.6 were compared by BLAST in the NCB I database and submitted to GenBank for acquisition. The phylogenetic tree was constructed by neighbor-joining method using Mega9.0 software and repeated 1000 times to determine the taxonomic status of the strains. The antagonistic strain D86 was found to be Bacillus subtilis.

[0054] SEQ ID NO.5:

[0055] AGGAATGGGCGGCGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGACCGTGAA。

[0056] SEQ ID NO.6:

[0057] GCCCTGGGCAATCCCCTGGGCCGCTTCCGGATGTGCGTGACGGTCTGAAGCCGGTTCACAGACGGATTTTGTACGCAATGAATGATTTAGGCATGACCAGTGACAAACCATATAAAAAATCTGCCCGTATCGTCGGTGAAGTTATCGGTAAGTACCACCCGCACGGTGACTCAGCGGTTTACGAATCAATGGTCAGAATGGCGCAGGATTTTAACTACCGCTACATGCTTGTTGACGGACACGGCAACTTCGGTTCGGTTGACGGCGACTCAGCGGCCGCGATGCGTTACACAGAAGCGAGAATGTCAAAAATCGCAATGGAAATTCTGCGTGACATTACGAAAGACACGATTGACTATCAAGATAACTATGACGGTTCAGAAAGAGAGCCTGCCGTCATGCCTTCGAGATTTCCGAATCTGCTCGTAAACGGGGCTGCCGGTATTGCGGTCGGAATGGCGACAAACATTCCCCCGCATCAGCTTGGGGAAGTCATTGAAGGCGTGCTTGCCGTAAGTGAGAATCCTGAGATTACAAACCAGGAGCTGATGGAATACATCCCGGGCCCGGATTTTCCGACTGCAGGTCAGATTTTGGGCCGGAGCGGCATCCGCAAGGCATATGAATCCGGACGGGGATCAATCACGATCCGGGCTAAGGCTGAAATCGAAGAGACTTCATCGGGAAAAGAAAGAATTATTGTCACGGAACTTCCTTATCAGGTGAACAAAGCGAGATTAATTGAAAAAATCGCGGATCTTGTCCGGGACAAAAAAATCGAAGGAATTACCGATCTGCGAGACGAATCCGACCGTAACGGAATGAGAATCGTCATTGAGATCCGCCGTG ACGCCAATGCTCACGTCATTTTGAATAACCTGTACAAACAAACGGCCCTGCAGACGTCTTTCGGAATCAACCTGCAGGCGCCTCGCTGGCCGGACGGTCCGTGCAATT。

[0058] Example 4: Pathogenic effect of antagonistic strain D86 on walnut rot.

[0059] Treatment of branches: The in vitro branch method was used to detect the control effect of antagonistic bacteria on walnut rot. The experiment was divided into 4 groups, with 4 branches in each group. Two-year-old walnut branches with a length of 10 cm were selected. The surface was disinfected with 75% alcohol solution for the first time and then rinsed with distilled water. The second time, it was disinfected with 2% sodium hypochlorite for 3 minutes, rinsed with sterile water, and dried at room temperature.

[0060] Pathogenicity test:

[0061] (1) Cryptococcus branch treatment experiment

[0062] The treated branches were divided into four groups and an inoculation point was punched with a 5mm sterilized puncher, namely: LB control group, Cryptococcus + antagonistic strain D86 group, antagonistic strain D86 group alone and Cryptococcus group. LB control group: 2.5μL liquid LB was added with a pipette; Cryptococcus + antagonistic strain D86 group: 2.5μL of isolated, purified and sterilized antagonistic strain D86 bacterial solution was first added, and the antagonistic strain D86 bacterial solution was added after drying for 5min; Antagonistic strain D86 group alone: ​​2.5μL antagonistic strain D86 bacterial solution was added with a pipette; Cryptococcus group: Cryptococcus cake was inoculated. The above groups were sealed with film, placed in an incubator at 25℃ for 14d, and then peeled to observe pathogenicity.

[0063] (2) Experiment on the treatment of golden shell cyst branches

[0064] The treated branches were divided into four groups and an inoculation point was punched with a 5mm sterilized puncher, namely: LB control group, Chrysocytis chrysanthemi + antagonistic strain D86 group, antagonistic strain D86 group alone and Chrysocytis chrysanthemi group. LB control group: 2.5μL liquid LB was added with a pipette; Chrysocytis chrysanthemi + antagonistic strain D86 group: 2.5μL of isolated, purified and sterilized antagonistic strain D86 bacterial solution was first added, and the antagonistic strain D86 bacterial solution was added after drying for 5 minutes; Antagonistic strain D86 group alone: ​​2.5μL of antagonistic strain D86 bacterial solution was added with a pipette; Chrysocytis chrysanthemi group: Chrysocytis chrysanthemi cake was inoculated. The above groups were sealed with film, placed in an incubator at 25℃ for 14 days, and then peeled to observe pathogenicity.

[0065] Depend on Figure 3 It can be seen that there were local diseases on the branches and trunks in the Cryptococcus + antagonistic strain D86 group; there was no disease on the branches and trunks in the antagonistic strain D86 group alone; and there was some effect in the treatment of the branches and trunks in the Cryptococcus group.

[0066] Depend on Figure 4It can be seen that the branches and trunks in the group of Cystosporium chrysotrichum + antagonistic strain D86 did not show obvious disease; the branches and trunks in the group of antagonistic strain D86 alone showed no disease; and the branches and trunks in the group of Cystosporium chrysotrichum showed local rot.

[0067] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0068] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An antagonistic strain (Bacillus subtilis) D86, characterized in that: It was deposited in the China Center for Type Culture Collection on November 11, 2024, with the deposit number: CCTCC NO: M 20242514, and the classification name is Bacillus subtilis D86.

2. A biocontrol agent, characterized in that: The only active ingredient of the biocontrol agent is the Bacillus subtilis D86 described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that: The biocontrol agent is one of a liquid agent, a powder agent, and a granular agent.

4. The biocontrol agent according to claim 3, characterized in that: The biocontrol agent also includes auxiliary materials; the auxiliary materials are fulvic acid, xanthan gum, sorbitol or sodium alginate.

5. The biocontrol agent according to claim 3, characterized in that: The biocontrol agent is D86 bacterial liquid.

6. The biocontrol agent according to claim 5, characterized in that: The OD value of the D86 bacterial solution is 0.8-1.

4.

7. A method for preparing the biocontrol agent according to claim 6, characterized in that: The D86 bacterial liquid is obtained by inoculating the antagonistic strain D86 into LB liquid culture medium and shaking it.

8. The method for preparing the biocontrol agent according to claim 7, characterized in that: The shaking culture conditions are: culturing at 30°C to 40°C and a rotation speed of 110 r / min to 130 r / min for 10 h to 14 h.

9. Use of the antagonistic strain D86 according to claim 1 or the biocontrol agent according to any one of claims 2 to 6 in preventing and controlling rot disease of walnut trees.

10. The use according to claim 9, characterized in that: The walnut tree rot disease is caused by Cytospora chrysosperma or Cryptococcus Neoformans.

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