A strain of Streptomyces sanguinii and its application in the prevention and treatment of walnut rot

Abstract

The present invention relates to the field of agricultural microbial technology, and specifically, to a strain of Streptomyces sampsonii and its application in the prevention and treatment of walnut rot disease. The Streptomyces sampsonii HT1 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC NO.30555 and a deposit time of May 11, 2024. The Streptomyces sampsonii HT1 is isolated from walnut bleeding sap collected in the Xinjiang Uygur Autonomous Region, and can effectively inhibit the mycelial growth of Cytospora chrysosperma, with an inhibition rate of up to 80%, and can effectively reduce the disease index of walnut rot disease.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and in particular to a strain of Streptomyces sanguinis and application thereof in the prevention and treatment of walnut rot disease. Background Art

[0002] Valsa canker is a plant branch and trunk disease caused by the fungus Cytospora chrysosperma, which is widespread and serious. The pathogenic fungus is a latent infectious disease that can infect more than 80 species of plants and cause rot. It mainly invades through wounds, but can also invade from the lenticels and buds of branches and trunks. The symptoms of infection vary depending on the age of the tree and the site of infection. The pathogenic fungus can reproduce between dead tissues, release toxic substances, kill surrounding living cells, produce propagules, and continue to infect. Rot is a devastating disease that causes incomplete branches and trunks in severe cases, and even causes the death of the entire tree or the destruction of the garden.

[0003] Walnut tree rot is one of the most serious and common diseases in walnut planting areas. It affects the yield and quality of walnuts, hinders the development of the walnut industry, and has become a major chronic disease for the safe production and healthy development of walnuts, causing huge economic losses to my country's walnut industry. At present, agricultural control and chemical control methods are traditionally used to prevent and control walnut rot. Simple physical scraping of rot spots can only reduce the impact of rot on walnut trees, and it is time-consuming and labor-intensive, and cannot cure the rot. Chemical control is a commonly used control method because of its simple operation, quick effect and high benefits. However, the problem of excessive and indiscriminate use of chemical pesticides is prominent, which often leads to a series of problems such as increased resistance of pathogens, excessive pesticide residues in products and environmental pollution.

[0004] Biological control of pathogenic fungi by producing antibacterial metabolites or survival competition has the advantages of little harm to humans and animals, strong control effect on pathogens, and no pollution. Therefore, the development of biocontrol microbial agents for the efficient control of walnut rot has attracted widespread attention.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The present invention aims to provide a strain of Streptomyces sampsonii and its application in the prevention and treatment of walnut rot disease. The Streptomyces sampsonii HT1 can effectively inhibit the mycelial growth of Cytosporachrysosperma with an inhibition rate of 80% and can effectively reduce the disease index of walnut infected with rot disease.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] One aspect of the present invention relates to a strain of Streptomyces sampsonii HT1, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.30555 and a deposit date of May 11, 2024.

[0009] The Streptomyces sanguinis is isolated from walnut bleeding fluid collected in Wushi County, Aksu Prefecture, Xinjiang Uygur Autonomous Region, and can effectively inhibit the mycelial growth of Cystosporum flavum with an inhibition rate of 80%, and can also effectively reduce the disease index of walnut rot infection.

[0010] The present invention measures the morphological characteristics and physiological and biochemical characteristics of the biocontrol strain HT1, and performs 16SrRNA gene sequencing to determine the taxonomic status of the strain, identifying it as Streptomyces sampsonii. Through dual verification of a plate antagonism experiment and an in vitro branch antagonism experiment, it is proved that the Streptomyces has the effect of inhibiting the pathogenic bacteria of rot disease.

[0011] Another aspect of the present invention also relates to a biocontrol microbial preparation, wherein the active ingredient of the microbial preparation comprises the Streptomyces sanguinis HT1.

[0012] The active ingredient of the biocontrol microbial preparation contains Streptomyces thunbergii HT1, which is separated from walnut bleeding sap. The biocontrol microbial preparation has the advantages of being environmentally friendly and highly safe, and is quickly effective in preventing and controlling forest rot diseases. The biocontrol microbial preparation has the characteristics of low cost, no pollution, and sustainable application.

[0013] The present invention does not specifically limit the dosage form of the biocontrol microorganism preparation, and any conventional dosage form in the art can be used to implement the present invention. In some specific embodiments, it includes but is not limited to solid preparations or liquid preparations.

[0014] The biocontrol microbial preparation can also be used together with conventional pesticides and other agents in the art to improve its control effect.

[0015] The biocontrol microbial preparation also includes an adjuvant acceptable in the field of microbial pesticides. In some specific embodiments, the adjuvant includes but is not limited to at least one of a solvent, a dispersant or a carrier.

[0016] Furthermore, the active ingredient of the microbial preparation includes the fermentation broth of the Streptomyces sanguinis HT1.

[0017] The fermentation broth mentioned in the present invention is a bacterial broth of Streptomyces sansevieria HT1 obtained by culturing activated Streptomyces sansevieria HT1 in a liquid culture medium.

[0018] The present invention does not specifically limit the type of liquid culture medium, and any liquid culture medium that can be used to culture activated Streptomyces in the art can be used to implement the technical solution of the present invention. In some specific embodiments, the liquid culture medium includes but is not limited to actinomycete liquid culture medium.

[0019] Furthermore, the active ingredient of the microbial preparation includes the fermentation filtrate of the Streptomyces sanguinis HT1.

[0020] The fermentation filtrate mentioned in the present invention is obtained by shaking culture, centrifugation and filtration sterilization of spores of Streptomyces sansevieria HT1 in an actinomycete liquid culture medium (ingredients: 1 g / L potassium nitrate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.01 g / L ferrous sulfate, 0.5 g / L sodium chloride, 20 g / L soluble starch, pH value 7.2-7.4).

[0021] Another aspect of the present invention also relates to the use of the Streptomyces sanguinis HT1 or the biocontrol microbial preparation in preventing and controlling forest rot diseases.

[0022] Furthermore, the pathogenic bacteria of the forest rot disease include but are not limited to: Chrysosporium chrysotrichum.

[0023] Furthermore, the forest tree rot disease includes but is not limited to walnut rot disease.

[0024] Another aspect of the present invention also relates to a method for preventing and controlling walnut rot, which comprises applying the Streptomyces sansevieria HT1 or the biocontrol microbial preparation to walnuts.

[0025] The method has a good control effect on walnut rot by applying Streptomyces sanguinis HT1 or a biocontrol microbial preparation containing Streptomyces sanguinis HT1 to walnuts.

[0026] Furthermore, the Streptomyces sanguinis HT1 or the biocontrol microbial preparation is applied to the diseased part of the walnut.

[0027] Another aspect of the present invention also relates to a growth inhibitor of Cystosporum chrysotrichum, comprising the Streptomyces sanguinis HT1 or the biocontrol microbial preparation.

[0028] Biological sample preservation information: Streptomyces sampsonii HT1, the strain has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on May 11, 2024, with the deposit number: CGMCCNO.30555; deposit address: Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101. It was detected as a viable strain by the collection center on May 11, 2024.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The Streptomyces sampsonii HT1 provided by the present invention is isolated from walnut bleeding fluid collected in Wushi County, Aksu Prefecture, Xinjiang Uygur Autonomous Region. It can effectively inhibit the mycelial growth of Cytosporachrysosperma with an inhibition rate of 80% and can effectively reduce the disease index of walnut rot infection.

[0031] (2) The Streptomyces sampsonii HT1 and its fermentation liquid and fermentation filtrate provided by the present invention also have the effect of inhibiting the growth of walnut rot pathogens. As a biocontrol actinomycete resource, the strain has a good application prospect and can be used for safe, effective and environmentally friendly biological control of walnut rot.

[0032] (3) The active ingredient of the biocontrol microbial preparation provided by the present invention comprises Streptomyces thunbergii HT1, which is isolated from walnut bleeding sap. The biocontrol microbial preparation has the advantages of being environmentally friendly and being effective in preventing and controlling forest rot diseases. The biocontrol microbial preparation has the characteristics of low cost, no pollution, and sustainable application.

[0033] (4) The method for preventing and controlling walnut rot provided by the present invention has a good prevention and control effect on walnut rot by applying Streptomyces sanguinei HT1 or a biocontrol microbial preparation containing Streptomyces sanguinei HT1 to walnuts. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The colony morphology of Streptomyces sanguinis HT1 (scale bar: 2 mm);

[0036] Figure 2 Gram staining results of Streptomyces sanguinis HT1 (scale bar: 10 μm);

[0037] Figure 3 The results of the plate confrontation between Streptomyces sansevieria HT1 and Cystosporium chrysotrichum (A is Cystosporium chrysotrichum, B is the antagonism of Streptomyces sansevieria HT1 against Cystosporium chrysotrichum, and C is the result of the inhibition rate of Streptomyces sansevieria HT1);

[0038] Figure 4 The hyphae morphology under an optical microscope (scale bar: 5 mm) (A is the hyphae morphology of Cystosporum chrysanthemi, and B is the hyphae morphology of Streptomyces sanguinis HT1 antagonizing Cystosporum chrysanthemi);

[0039] Figure 5 The Oxford cup method of Streptomyces sansevieria HT1 fermentation filtrate against the plate antibacterial effect of Cystosporum chrysotrichum (A is the plate confrontation results of the aseptic fermentation filtrate of Streptomyces sansevieria HT1 and Cystosporum chrysotrichum after inoculation for 1d, 2d, 3d and 4d respectively, the left Oxford cup contains 150μl sterile water, and the right Oxford cup contains 150μl fermentation filtrate);

[0040] Figure 6 The inhibition rate of the fermentation filtrate of Streptomyces sanguinis HT1 against Cystosporum chrysotrichum;

[0041] Figure 7 is the control effect of Streptomyces sanguinis HT1 on rot of isolated walnut branches (A is a negative control, inoculated with a small piece of PDA agar; B is a positive control, inoculated with Cystosporium chrysotrichum; C is inoculated with Cystosporium chrysotrichum + fermentation liquid of strain HT1; D is inoculated with Cystosporium chrysotrichum + fermentation filtrate of strain HT1; E is inoculated with Cystosporium chrysotrichum + spore suspension of strain HT1; F is the disease index of rot of isolated walnut branches; G is the control effect of strain HT1 on rot of isolated walnut branches);

[0042] Figure 8 This is a diagram of the disease grade of in vitro walnut branches (scale: 1 cm). DETAILED DESCRIPTION

[0043] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0044] The present invention explores the morphological characteristics and physiological and biochemical characteristics of Streptomyces sampsonii HT1, and performs 16S rRNA gene sequencing to determine the genus and species of the strain. Under the dual verification of plate antagonism experiment and in vitro walnut branch disease prevention experiment, it is proved that the Streptomyces has the effect of inhibiting rot pathogens, especially walnut rot. The specific implementation process is shown in the following examples.

[0045] Example 1 Basic information of strains

[0046] (1) Morphological identification of strains

[0047] The strain HT1 was inoculated on LB solid medium for culture. After inverted culture in a 30°C constant temperature incubator for 24 hours, the colonies were round, milky white and opaque, with a shiny surface, irregular edges, moist, and slightly convex (see Figure 1 The Gram stain of this strain is purple, indicating that it is a Gram-positive bacterium ( Figure 2 ).

[0048] (2) Molecular biological identification of strains

[0049] The genomic DNA of strain HT1 was extracted and used as template DNA to perform PCR amplification of the 16S rRNA gene using universal primers 27F and 1492R (27F: 5'-AGAGTTTGATCMTGGCTCAG-3'; 1492R: 5'-TACGGYTACCTTGTTACGACTT-3'). PCR reaction system: template DNA 1μL, primer 27F 1μL, primer 1492R 1μL, 2×Taq Mixture 12.5μL, ddH2O 9.5μL. PCR reaction procedure: 98℃ pre-denaturation for 5min, 98℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 2min, denaturation, annealing, extension for 30 cycles, 72℃ for 5min. The amplified product was detected by 1% agarose gel electrophoresis, purified and sequenced, and the sequencing result is shown in SEQ ID No.1.

[0050] SEQ ID No.1:

[0051]

[0052] The obtained sequence was subjected to BLAST search in the National Center for Bioinformation (NCBI) of the United States. The one with the highest similarity to the 16SrRNA gene fragment sequence of strain HT1 was Streptomyces sampsonii, with a homology of up to 99.43%. It was determined to be a strain of Streptomyces sampsonii, named Streptomyces sampsonii HT1, and deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC NO.30555 and a deposit time of May 11, 2024.

[0053] (3) Physiological and biochemical properties of strains

[0054] The physiological and biochemical identification of strain HT1 mainly refers to the Manual of Identification of Common Bacterial Systems. Obvious hydrolysis circles can be observed in starch and hydrogen peroxide culture media, so the strain has the ability to secrete amylase and catalase. The hydrogen sulfide test result of the strain is positive, and the methyl red and VP reaction tests are both negative. The optimal pH value is 8.0, and it can survive in NaCl content between 2% and 10%. The test results are shown in Table 1.

[0055] Table 1 Test results of physiological and biochemical characteristics of Streptomyces sanguinii HT1

[0056]

[0057]

[0058] Example 2 Determination of the Antagonism of Streptomyces sanguinis HT1 to Cystosporum chrysotrichum on Plates

[0059] (1) Activation of walnut rot pathogens:

[0060] Use a sterile blade to pick out the walnut rot pathogen Cytospora chrysosperma from a refrigerator stored at 4°C and inoculate it onto PDA medium (potato dextrose agar medium: 200g potato, 20g glucose, 20g agar), and culture it at a constant temperature of 25°C until the fungus fills the plate.

[0061] The Streptomyces sanguinis strain HT1 stored in a -80°C ultra-low temperature freezer was activated by streaking on a lysing broth (LB) solid culture medium, placed in an incubator at 30°C for 24 h, and a single colony was picked and inoculated into an actinomycete liquid culture medium, which was then placed in a shaker at 30°C, 200 rpm, for 24 h.

[0062] (2) Strain HT1 plate antagonism experiment:

[0063] Use a 6mm diameter puncher to punch a bacterial cake on the PDA plate of walnut rot pathogen, and place the bacterial cake upside down in the center of the PDA plate. Use the cross method to inoculate Streptomyces sansevieriae (2μL) at four points equidistant from the center (2.5cm), and repeat 3 times. Use the PDA plate inoculated with only the pathogen Cystosporum chrysotrichum in the center as a control. Place it in a 25℃ constant temperature incubator and invert it for 3-5 days. When the pathogen in the control group grows all over the PDA plate, observe and take pictures to record the antagonistic effect and calculate the inhibition rate (see Figure 3 ) and observe the morphology of mycelium after antagonism (see Figure 4 ).

[0064] Calculation formula:

[0065]

[0066] The results showed that strain HT1 had a high efficiency in antagonizing the ability of Cystosporum chrysogenum ( Figure 3 B), and its plate antagonistic inhibition rate was 80% ( Figure 3 C). Observation of the mycelium of Chrysospora chrysotrichum after antagonism under an optical microscope revealed that the mycelium showed obvious swelling, deformation, shortening, and irregular curling ( Figure 4 ).

[0067] Example 3 Determination of antibacterial ability of fermentation filtrate of Streptomyces sanguinis HT1

[0068] (1) Preparation of fermentation filtrate of strain HT1:

[0069] The Streptomyces sanseri strain HT1 stored in a -80°C ultra-low temperature freezer was activated on LB solid medium by streaking method and incubated in a 30°C constant temperature incubator for 7 days. 5 ml of sterile water was added to the LB plate of Streptomyces sanseri cultured for 7 days and fully shaken to make the spores of Streptomyces sanseri fall off in the sterile water, thus obtaining a suspension of conidia of Streptomyces sanseri. The concentration of the spore suspension was counted using a hemocytometer (concentration was 2×10 7 CFU / ml) for later use.

[0070] The spore suspension was inoculated into 100 ml of actinomycete liquid culture medium at a ratio of 2%, and cultured at 28°C and 180 rpm for 1-4 days, and the fermentation liquid was taken out in turn. The fermentation liquid was centrifuged at 8000 rpm for 5 minutes, and then filtered through a 0.22 μm filter membrane to obtain the sterile fermentation filtrate of strain HT1.

[0071] The pathogen cake was inoculated in the center of the PDA culture medium for a confrontation experiment. An Oxford cup was placed at both ends of the horizontal line 2.5 cm away from the center of the plate. 150 μl of sterile water was added to the Oxford cup of the control group, and 150 μl of the fermentation filtrate of the strain was added to the Oxford cup of the experimental group. Each treatment was repeated 5 times. When the cake of the positive control group filled the plate, the diameter of the inhibition zone could be observed and recorded, and the inhibition rate could be calculated.

[0072] The results show that the fermentation filtrate of strain HT1 has a good and stable antibacterial ability. After 2 days of shaking, the antibacterial rate of its fermentation filtrate reached 59.4%, and the antibacterial rate of the fermentation filtrate after 3 days of shaking was 46.3%, which was not significantly different from the antibacterial effect of shaking for 2 days; the antibacterial rate of the fermentation filtrate of the strain after shaking for 4 days was 31.2%, and its antibacterial effect was significantly reduced ( Figure 5 and Figure 6 ). Therefore, the fermentation filtrate of strain HT1 after two days of shaking was selected for the in vitro walnut branch disease prevention test.

[0073] Example 4 In vitro walnut branch disease prevention test

[0074] (1) Take walnut branches with a diameter of about 1-2 mm and trim them into 10 cm long, rinse them three times with sterile water, and then clean and disinfect them with 75% alcohol for later use. Scald the walnut branches to make a 6 mm × 6 mm wound;

[0075] (2) Prepared spore suspension (2.85×10 8 CFU / ml) was inoculated into 100 ml of actinomycete liquid culture medium at a ratio of 2%, and cultured at 30°C with shaking at 180 rpm for 2 days to obtain fermentation broth; the fermentation broth was centrifuged at 8000 rpm for 5 min, and then filtered through a 0.22 μm filter membrane to obtain the fermentation filtrate of strain HT1;

[0076] (3) Set up a blank control group ( Figure 7 A: only inoculated with PDA solid medium) and positive control group ( Figure 7 B: Inoculated with only the fungus cake of Chrysosporium chrysosporium), the isolated walnut branches were soaked in sterile water for 1.5 h, with 5 branches in each group, and repeated three times; treatment group 1 ( Figure 7 C: inoculated with golden cystosporium cake + fermentation liquid of strain HT1), treatment group 2 ( Figure 7 D: inoculated with golden cystosporium cake + fermentation filtrate of strain HT1), treatment group 3 ( Figure 7E: Inoculation of golden shell cystospore cake + strain HT1 spore suspension), the in vitro walnut branches were immersed in the fermentation liquid, fermentation filtrate and spore suspension of strain HT1 for 1.5 hours. After the in vitro branches of each group were air-dried, the wound sites of treatment groups 1, 2, 3 and the positive control group were inoculated with golden shell cystospore cakes with a diameter of about 6 mm, and the blank control group was inoculated with a small piece of PDA solid culture medium with the same diameter. The treated walnut branches were placed in a 25°C incubator for constant temperature culture.

[0077] (4) Every 3 days, 50 μl of the fermentation liquid, fermentation filtrate, spore suspension and 50 μl of sterile water of strain HT1 were dripped onto the wound of the detached walnut branches of treatment groups 1, 2, 3 and the control group. When the branches of the positive control group were fully infected by the pathogen, the length of the lesions was observed and recorded, and the disease condition of the walnut branches was graded according to the ratio of the lesions to the total length of the walnut branches, and the disease index and the control effect of strain HT1 were calculated. The disease grade classification of walnut detached branches is shown in Figure 8 and Table 2.

[0078] Table 2 Disease grade of walnut in vitro branches

[0079]

[0080]

[0081] The disease index calculation formula is:

[0082]

[0083] The formula for calculating the control effect is:

[0084]

[0085] like Figure 7 As shown, 7 days after inoculation with the pathogen, it was observed that the in vitro walnut branches of the positive control group were completely infected. After peeling off the outer skin, the branches appeared reddish brown and shriveled; while the walnut branches of the group treated with the fermentation liquid and fermentation filtrate of strain HT1 only showed slight symptoms of the disease on the surface.

[0086] The length of the walnut branches and lesions in vitro was measured 7 days after inoculation. The average lesion length of the branches in the positive control group accounted for 100% of the total branch length, and the disease index was 100; the average lesion length of the treatment groups C, D, and E accounted for 17.4%, 33.6%, and 58.6% of the total branch length, respectively, and the disease indexes were 28, 44, and 68, respectively. Figure 7 F); the fermentation liquid, fermentation filtrate and spore suspension of strain HT1 for two days had a control effect of 72%, 56% and 32% on walnut detached branches respectively ( Figure 7G), the results showed that the fermentation broth, fermentation filtrate and spore suspension of Streptomyces sansevieria HT1 all had different degrees of control effects on walnut rot, among which the fermentation broth had the best control effect.

[0087] In summary, it can be seen that the fermentation broth of Streptomyces sampsonii HT1 provided by the present invention is found for the first time to be able to be used for the prevention and treatment of forest rot diseases. This strain has a good prevention and treatment effect, especially on walnut rot disease, and has good application prospects in the prevention and treatment of rot diseases.

[0088] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. Streptomyces sanguinis ( Streptomyces sampsonii )HT1, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCCNO.30555 and the deposit date May 11, 2024.

2. A biocontrol microbial preparation, characterized in that: The active ingredient of the microbial preparation comprises the Streptomyces sanguinis HT1 described in claim 1.

3. The biocontrol microbial preparation according to claim 2, characterized in that: The active ingredients of the microbial preparation include the fermentation broth of the Streptomyces sanguinis HT1.

4. The biocontrol microbial preparation according to claim 2, characterized in that: The active ingredient of the microbial preparation includes the fermentation filtrate of the Streptomyces sanguinis HT1.

5. Use of the Streptomyces sanguinis HT1 according to claim 1 or the biocontrol microbial preparation according to any one of claims 2 to 4 in preventing and controlling forest rot caused by Cystosporium chrysotrichum.

6. The use according to claim 5, characterized in that: The forest rot disease is walnut rot.

7. A method for preventing and treating walnut rot, characterized in that: The Streptomyces sanguinis HT1 of claim 1 or the biocontrol microbial preparation of any one of claims 2 to 4 is applied to walnuts.

8. The method for preventing and treating walnut rot according to claim 7, characterized in that: Apply the Streptomyces sanguinis HT1 or the biocontrol microbial preparation to the diseased part of the walnut.

9. A growth inhibitor of Cystosporum chrysogenum, characterized in that: The method comprises the Streptomyces sanguinis HT1 according to claim 1 or the biocontrol microbial preparation according to any one of claims 2 to 4.

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