Streptomyces griseoaurantiacus and application thereof

Abstract

The present application relates to a strain of Streptomyces griseoaurantiacus and its application, belonging to the technical field of biological control of plant diseases. The Streptomyces griseoaurantiacus of the present application has been preserved in Guangdong Microbial Culture Collection Center on July 28, 2023, with the preservation number of GDMCC No: 63691 and the preservation address of No. 59 Building, 5th Floor, 100, Martyrs' Road, Guangzhou, Guangdong Province. The strain can inhibit the formation of Ralstonia solanacearum biofilm and the occurrence of Ralstonia solanacearum caused by bacterial wilt, has good prevention and control effect on bacterial wilt, and can be used as a biocontrol agent and biological preparation for the prevention and control of bacterial wilt.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant disease biological control, and particularly relates to a Streptomyces griseoaurantiacus and application thereof. BACKGROUND

[0002] Tomato is a fruit and vegetable that is widely loved by people, and ranks first in the yield of facility vegetables in China. Facility tomatoes often have sharp yield reduction and soil-borne disease outbreak. For example, bacterial wilt is a common soil-borne disease of tomatoes, and the pathogenic bacteria thereof are Ralstonia solanacearum. The bacteria can lurk in the soil and irrigation water for several years, then invade the rhizosphere of tomatoes, enter the inside of the plant through the natural aperture or mechanical damage of the root, and proliferate in large quantities in the plant vessel while releasing toxic factors, and finally cause the wilting and death of the plant. Previous studies have shown that actinomycetes have a broad-spectrum inhibitory effect on plant pathogenic fungi, and recent studies have found that actinomycetes also have a good effect on the prevention and control of bacterial wilt, but only some actinomycetes have a prevention and control effect on bacterial wilt.

[0003] Streptomyces griseoaurantiacus also belongs to actinomycetes, and can produce volatile gases to inhibit the growth of Rhizoctonia solani, in addition to having a significant inhibitory effect on Pyricularia grisea, Septoria tritici, Fusarium, Botrytis cinerea, Sclerotinia sclerotiorum, Valsa mali and Coniella diplodiella, and can also prevent and control tomato verticillium wilt. However, there is almost no report on the research of Streptomyces griseoaurantiacus in the prevention and control of tomato bacterial wilt.

[0004] As a pure culture, the individual specificity of the strain is relatively strong and is greatly affected by the environment. For the biological control of bacterial wilt, it is necessary to continuously explore, screen and select new biocontrol bacteria with better prevention and control effect, so as to effectively prevent bacterial wilt. The biocontrol bacteria library is enriched to provide new resources for disease control. SUMMARY

[0005] The present application aims at overcoming the deficiencies of the prior art and providing a Streptomyces griseoaurantiacus with inhibition of bacterial wilt and application thereof.

[0006] The Streptomyces griseoaurantiacus is screened from the rhizosphere soil of healthy tomatoes, and it is found through the determination of the amount of biofilm formation that the bacteria can significantly inhibit the formation of the biofilm of the pathogenic bacteria of tomato bacterial wilt.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a strain of Streptomyces griseoaurantiacus, which has been preserved in the Guangdong Microbial Culture Collection Center on July 28, 2023, with a preservation number of GDMCC No: 63691 and a preservation address of No. 59 Building, 5th Floor, 100 Middle Liujie Road, Guangzhou, Guangdong Province.

[0009] The strain is isolated and purified from healthy rhizosphere soil collected from Zhongluotan Town, Baiyun District, Guangzhou. The nucleotide sequence length of the 16S rRNA of the strain is 1194 bp, and the homology of the strain with Streptomyces griseoaurantiacus is 100% after homology comparison. According to the phylogenetic tree constructed based on the 16S rRNA sequence, it is identified as Streptomyces griseoaurantiacus, named Streptomyces griseoaurantiacus.

[0010] The test results of the present application show that the Streptomyces griseoaurantiacus 30223 can significantly inhibit the formation of biofilm of Ralstonia solanacearum RS1; at the same time, the strain can inhibit the occurrence of tomato plant bacterial wilt caused by Ralstonia solanacearum, and the prevention and control effect on bacterial wilt can reach 70%. The strain has good prevention and control effect on Ralstonia solanacearum, and has great application value and potential in preventing and controlling bacterial wilt caused by Ralstonia solanacearum.

[0011] In a second aspect, the present application provides the application of the above-mentioned Streptomyces griseoaurantiacus in preventing and controlling bacterial wilt.

[0012] The present application uses crystal violet staining method to quantitatively detect the amount of biofilm formation, and finds that the Streptomyces griseoaurantiacus 30223 can significantly inhibit the formation of biofilm of Ralstonia solanacearum RS1.

[0013] The present application finds through pot experiment that the Streptomyces griseoaurantiacus 30223 can significantly inhibit the incidence of tomato bacterial wilt, and the prevention and control effect on bacterial wilt reaches 70%, which has good prevention and control effect.

[0014] As a preferred embodiment of the application, the bacterial wilt is Ralstonia solanacearum disease.

[0015] As a preferred embodiment of the application, the bacterial wilt is tomato bacterial wilt.

[0016] In a third aspect, the application provides use of the Streptomyces griseoaurantiacus in the preparation of a biological agent for preventing and treating bacterial wilt.

[0017] The Streptomyces griseoaurantiacus 30223 of the application can inhibit the formation of biofilm of Ralstonia solanacearum and the incidence of bacterial wilt, and can be prepared into a biological agent for biological prevention and treatment of bacterial wilt.

[0018] As a preferred embodiment of the application, the bacterial wilt is Ralstonia solanacearum disease.

[0019] As a preferred embodiment of the application, the bacterial wilt is tomato bacterial wilt.

[0020] In a fourth aspect, the application provides a biological agent containing the Streptomyces griseoaurantiacus or a fermentation liquor thereof.

[0021] The Streptomyces griseoaurantiacus 30223 of the application can inhibit the formation of biofilm of Ralstonia solanacearum and the incidence of bacterial wilt, and the biological agent prepared from the strain can be used for biological prevention and treatment of bacterial wilt.

[0022] As a preferred embodiment of the biological agent of the application, the biological agent contains a fermentation liquor of the Streptomyces griseoaurantiacus.

[0023] As a preferred embodiment of the biological agent of the application, the fermentation liquor is a filtrate obtained by culturing the Streptomyces griseoaurantiacus in a liquid medium, centrifuging and filtering.

[0024] As a preferred embodiment of the biological agent of the application, the fermentation liquor is a filtrate obtained by culturing the Streptomyces griseoaurantiacus in Gao's liquid medium at 28°C and 180 r / min, centrifuging at 10,000 r / min, and filtering through a sterile 0.22 μm microporous filter.

[0025] In a fifth aspect, the present application provides a method for preventing and treating bacterial wilt, the method being for preventing and treating bacterial wilt by using the biological preparation.

[0026] As a preferred embodiment of the method of the present application, the method is for preventing and treating bacterial wilt by using the biological preparation containing the Streptomyces griseoaurantiacus or the fermentation liquor thereof.

[0027] As a preferred embodiment of the method of the present application, the method is for preventing and treating bacterial wilt by using the biological preparation containing the fermentation liquor of the Streptomyces griseoaurantiacus.

[0028] As a preferred embodiment of the method of the present application, the fermentation liquor is the filtrate obtained by culturing the Streptomyces griseoaurantiacus in a liquid medium, centrifuging and filtering.

[0029] As a preferred embodiment of the method of the present application, the fermentation liquor is the filtrate obtained by culturing the Streptomyces griseoaurantiacus in a Gao's liquid medium at 28°C and 180 r / min, centrifuging at 10,000 r / min and filtering through a sterile 0.22-um microporous filter.

[0030] As a preferred embodiment of the method of the present application, the bacterial wilt is Ralstonia solanacearum disease.

[0031] As a preferred embodiment of the method of the present application, the bacterial wilt is tomato bacterial wilt.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] The Streptomyces griseoaurantiacus of the present application can inhibit the formation of a biofilm of Ralstonia solanacearum and the occurrence of bacterial wilt caused by Ralstonia solanacearum, has a good effect on the prevention and treatment of bacterial wilt, can be used as a biocontrol agent and a biological preparation for preventing and treating bacterial wilt, and has great application value and potential in preventing and treating bacterial wilt and other diseases caused by Ralstonia solanacearum. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Relative abundance of Streptomyces in the rhizosphere soil of healthy and diseased tomatoes in Example 1 of the present application;

[0035] Figure 2Phylogenetic tree constructed based on 16S rDNA gene sequence of Ralstonia solanacearum RS1 in Example 2 of the present application;

[0036] Figure 3 Biofilm formation amount of strain numbered 30223 in Example 2 of the present application and control treatment;

[0037] Figure 4 Phylogenetic tree results in Example 3 of the present application. DETAILED DESCRIPTION

[0038] For the purpose of better illustrating the object, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples.

[0039] Gao's solid medium: NaCl 0.5g, FeSO4 0.01g, KNO3 1.0g, K2HPO4 0.5g, MgSO4 0.5g, agar 20g, H2O 1L, pH 7.3±0.2.

[0040] Gao's liquid medium: NaCl 0.5, FeSO4 0.01g, KNO3 1.0g, K2HPO4 0.5g, MgSO4 0.5g, H2O 1L, pH 7.3±0.2.

[0041] CPG solid medium: hydrolyzed tyrosine amino acid 1g, tryptone 10g, glucose 5g, agar powder 20g, H2O 1L, pH 7.0.

[0042] CPG liquid medium: hydrolyzed tyrosine amino acid 1g, tryptone 10g, glucose 5g, H2O 1L, pH 7.0.

[0043] Other materials, reagents, etc. used in the examples can be obtained from commercial channels if not otherwise specified.

[0044] The Streptomyces griseoaurantiacus of the present application was preserved in the Guangdong Microbial Culture Collection Center on July 28, 2023, with the preservation number of GDMCC No: 63691 and the preservation address of No. 59 Building, 5th Floor, Guangzhou Institute, 100 Middle Martyrs Road, Guangzhou, Guangdong Province.

[0045] Example 1 Difference in distribution of Streptomyces in the rhizosphere soil of healthy and diseased tomatoes

[0046] Take 10 g of healthy tomato rhizosphere soil and 10 g of tomato rhizosphere soil with green and rot disease (disease) collected from Zhongluotan Town, Baiyun District, Guangzhou, according to the operation steps of soil DNA extraction kit E.Z.N.A. soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) instruction manual to extract soil DNA, with universal primer 338F and 806R for bacterial 16S rDNA V3-V4 region as primer, with extracted tomato rhizosphere soil total DNA as template, bacterial 16S rRNA high-throughput amplification was carried out. Gel recovery was performed using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), library construction was performed using NEXTFLEX Rapid DNA-Seq Kit, and sequencing was performed using Miseq PE300 platform of Illumina Company. R language (3.3.5) was used for community difference species analysis; Wilcoxon rank sum test was used for difference analysis, and the significance level was set as P<0.05.

[0047] The relative abundance of Streptomyces in healthy and diseased tomato rhizosphere soil is shown in Figure 1 , wherein ZLTH represents healthy tomato rhizosphere soil sample, and ZLTS represents diseased tomato rhizosphere soil sample; the results show that the relative abundance of Streptomyces in healthy tomato rhizosphere soil is significantly higher than that in diseased rhizosphere soil.

[0048] Example 2 Isolation and screening of strains

[0049] 1. Single colony isolation

[0050] 10 g of healthy tomato rhizosphere soil collected from Zhongluotan Town, Baiyun District, Guangzhou, was accurately weighed, added into 90 mL of sterile water with glass beads, oscillated at 30°C and 150 r / min for 30 min, and then stood for 20-30 s. The supernatant was taken, 10 times continuous dilution was performed, 10 3 , 10 4 dilution, 100 ul was respectively taken and uniformly coated on Gao's solid culture medium, and cultured at 30°C for 5-7 d. Then single colonies with different morphological characteristics were picked and numbered, and cultured in an inverted incubator at 28°C for 5-7 d.

[0051] 2. Screening of strains

[0052] Biofilm formation can help pathogenic bacteria resist external adverse environment, gain advantage in tomato root surface competition, and play an important role in pathogenic bacteria colonization. Screening of microbial germplasm resources that inhibit biofilm formation of Ralstonia solanacearum is of great significance for the prevention and control of tomato bacterial wilt.

[0053] The effect of different numbered single colonies on the biofilm formation of Ralstonia solanacearum RS1 was screened, and the biofilm formation amount was quantitatively detected by crystal violet method.

[0054] (1) Preparation of Ralstonia solanacearum suspension

[0055] The Ralstonia solanacearum RS1 used in this experiment was isolated from the root system of tomato with bacterial wilt, and was identified as Ralstonia solanacearum, which was simply referred to as Ralstonia solanacearum hereinafter. The phylogenetic tree constructed based on the 16S rDNA gene sequence of Ralstonia solanacearum RS1 is shown in Figure 2 The Ralstonia solanacearum was activated by CPG solid medium, and then single colonies were picked into CPG liquid medium and cultured in a shaking incubator at 30°C to the logarithmic growth phase. After centrifugation, the supernatant was removed and resuspended with sterile water, and gradient diluted to 10 8 CFU / mL, i.e. Ralstonia solanacearum suspension was obtained.

[0056] (2) Preparation of sterile fermentation filtrate of different numbered single colonies

[0057] Different numbered single colonies were inoculated on Gao's solid medium and cultured at 30°C. When the strains grew into single colonies, 6 single colonies were picked into a 50mL Gao's liquid medium flask and cultured at 28°C, 180r / min for 3d to obtain seed culture solution. 10mL seed culture solution was inoculated into a 200mL Gao's liquid medium flask and cultured at 28°C, 180r / min for 7d. After 7d, the supernatant was obtained by centrifugation at 4°C, 10000r / min for 20min, and then filtered with sterile 0.22um micropore filter to obtain sterile fermentation filtrate.

[0058] (3) Determination of biofilm formation amount

[0059] The sterile fermentation filtrate of different numbered single colonies was mixed with Ralstonia solanacearum suspension at a volume ratio of 2:1. The mixture containing 100μL of CPG liquid medium, 66.66μL of sterile water, and 33.34μL of Ralstonia solanacearum suspension was used as control treatment, and the mixture containing 100μL of CPG liquid medium, 66.66μL of sterile fermentation filtrate, and 33.34μL of Ralstonia solanacearum suspension was used as experimental treatment, with 5 replicates for each treatment.

[0060] The control and the experimental mixture were added into 96-well plates respectively, and incubated at 30°C for 24 hours without shaking. The formation of biofilm was measured after 24 hours of incubation, and the test was repeated three times. The unformed biofilm bacteria were washed with 160 μL of ultrapure water, and 160 μL of 1% crystal violet solution was added for 10 minutes of staining. After staining, the crystal violet was gently absorbed with a gun head, and the ultrapure water was slowly washed until the water was not blue. Then, 160 μL of 30% acetic acid solution was added to dissolve the crystal violet adsorbed on the biofilm for 10 minutes, and then the absorbance value at 530 nm was measured by using a full-wavelength microplate reader. The higher the absorbance value, the lower the formation of biofilm. If the absorbance value of the experimental treatment is higher than that of the control treatment, it indicates that the formation of biofilm is promoted. If the absorbance value of the experimental treatment is lower than that of the control treatment, it indicates that the formation of biofilm is inhibited. The greater the difference between the absorbance values of the experimental treatment and the control treatment, the stronger the promotion or inhibition effect.

[0061] According to the absorbance value data, a strain capable of inhibiting the formation of biofilm of Ralstonia solanacearum was screened, and the number was 30223. The control treatment was recorded as CK group, and the sterile fermentation filtrate of the strain numbered 30223 was recorded as T1 group.

[0062] The biofilm formation of the strain numbered 30223 and the control treatment is shown in Table 1 and Figure 3 Table 1

[0063] Table 1

[0064]

[0065] Table 1 and Figure 3 The results show that the strain 30223 can significantly inhibit the formation of biofilm of Ralstonia solanacearum.

[0066] Example 3 Identification of the strain

[0067] The strain 30223 screened in Example 2 was subjected to molecular identification.

[0068] The total DNA of the strain 30223 was extracted by using a bacterial genomic DNA extraction kit (Tiangen Biotech Co., Ltd.), and the 16S rRNA gene sequence was amplified by using the bacterial universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACCTTGTTACGACTT-3' with the total DNA of the strain as the template.

[0069]

[0070] According to the sequencing results of 16S rRNA gene sequence, homology search was performed on EzBioCloud Database nucleic acid database, the sequence of the model strain with high homology was downloaded, and the phylogenetic tree was constructed by using Mega11.0 software, and the phylogenetic tree results are shown in Figure 4 .

[0071] The results show that the 30223 strain has the highest homology with Streptomyces griseoaurantiacus, which is 100%. The 30223 strain is identified as Streptomyces griseoaurantiacus, and the 30223 strain has been preserved in Guangdong Microbial Culture Collection Center on July 28, 2023, with the preservation number of GDMCC No: 63691 and the preservation address of No. 59 Building, 5th Floor, Guangzhou, Guangdong Province, China.

[0072] Example 4: Test of biocontrol effect of strain 30223

[0073] 1. Preparation of Ralstonia solanacearum suspension

[0074] Using the Ralstonia solanacearum RS1 isolated in Example 2, referred to as Ralstonia solanacearum, to prepare Ralstonia solanacearum suspension. Activate Ralstonia solanacearum with CPG solid medium, then pick single colony to CPG liquid medium, shake culture at 30℃ until logarithmic growth phase; centrifuge, remove supernatant, resuspend with sterile water, gradient dilute to 10 8 CFU / mL, and the Ralstonia solanacearum suspension is obtained.

[0075] 2. Preparation of Streptomyces griseoaurantiacus 30223 fermentation broth

[0076] The saved Streptomyces griseoaurantiacus 30223 strain screened in Example 2 was taken out from the -80℃ refrigerator, inoculated on Gao's solid culture medium, and cultured at 30℃. When the strain grew into single colonies, 6 single colonies were picked into 50 mL Gao's liquid culture medium in a flask, and cultured at 28℃ and 180 r / min for 3 days to obtain a seed culture solution. 10 mL of the seed culture solution was inoculated into 200 mL Gao's liquid culture medium in a flask, and cultured at 28℃ and 180 r / min for 7 days. After 7 days, the solution was centrifuged at 4℃ and 10,000 r / min for 20 min, and the supernatant was filtered with a sterile 0.22 um microporous filter to obtain a sterile fermentation filtrate, that is, the Streptomyces griseoaurantiacus 30223 fermentation solution.

[0077] 3. Biocontrol test

[0078] (1) Treatment

[0079] The tomato seedlings were about 4-leaf stage, and the tomato seedlings with good and uniform growth state were selected and acclimated for 2 days, and then the corresponding treatment was performed.

[0080] The CK group was treated with sterile water, and the root injury irrigation inoculation method was used. The lateral roots were cut with a high-temperature sterilized scissors, and the roots were irrigated with sterile water, and 30 mL was applied.

[0081] The RS group was treated with Ralstonia solanacearum suspension, and the root injury irrigation inoculation method was used. The lateral roots were cut with a high-temperature sterilized scissors, and the roots were irrigated with the prepared Ralstonia solanacearum suspension, and 30 mL was applied.

[0082] The R30223 group was treated with Streptomyces griseoaurantiacus 30223 fermentation solution + Ralstonia solanacearum suspension. The root irrigation method was used, the roots were irrigated with the prepared Streptomyces griseoaurantiacus 30223 fermentation solution, and 30 mL was applied. After 24 hours, the root injury irrigation inoculation method was used. The lateral roots were cut with a high-temperature sterilized scissors, and the roots were irrigated with the prepared Ralstonia solanacearum suspension, and 30 mL was applied.

[0083] The 30223 group was treated with Streptomyces griseoaurantiacus 30223 fermentation solution. The root irrigation method was used, the roots were irrigated with the prepared Streptomyces griseoaurantiacus 30223 fermentation solution, and 30 mL was applied.

[0084] Each treatment group had 6 replicates.

[0085] (2) Disease assessment

[0086] The treated potted tomato seedlings were placed in a greenhouse with a temperature of 28-30°C, a relative humidity (RH) of 75-90%, and a light period of 12h. Each treatment group was randomly placed, and watered evenly and appropriately every day to avoid loss of fertilizer due to water flowing out of the pot bottom. After the tomato plants showed symptoms of disease, the disease index of each treatment was observed every day, and the seedlings were collected after 30 days of cultivation. The grade, incidence, disease index, and control effect of the tomato plants on bacterial wilt were counted.

[0087] The grading standard for tomato bacterial wilt is as follows:

[0088] 0: healthy plants;

[0089] 1: the wilting degree of plant leaves is less than 25%;

[0090] 2: the wilting degree of plant leaves is between 25% and 50%;

[0091] 3: the wilting degree of plant leaves is between 50% and 75%;

[0092] 4: the wilting degree of plant leaves is more than 75%.

[0093] Incidence % (DI) = number of diseased plants / total number of plants x 100%.

[0094] Disease index = [Σ (disease grade x number of plants of that disease grade) / (highest disease grade x total number of plants)] x 100%.

[0095] Control effect % (BE) = [(RS group disease index - R30223 group disease index) / RS group disease index] x 100%.

[0096] The collected seedling data was statistically analyzed using SPSS21 software, and the differences between multiple groups were compared using one-way ANOVA, and multiple comparisons were performed using Duncan's method.

[0097] The incidence and control of bacterial wilt in different treatment groups are shown in Table 2.

[0098] Table 2

[0099]

[0100] The results in Table 2 show that after inoculation with bacterial wilt bacteria suspension, all tomato seedlings were diseased. However, inoculation with the fermentation broth of the Streptomyces griseoaurantiacus 30223 strain of the application in advance can significantly reduce the incidence of bacterial wilt in tomato seedlings, and the control effect reaches 70%.

[0101] Therefore, the Streptomyces griseoaurantiacus 30223 strain of the present application can inhibit the formation of the Ralstonia solanacearum biofilm, significantly inhibit the bacterial wilt caused by the Ralstonia solanacearum, and the prevention and control effect on the bacterial wilt can reach 70%. The Streptomyces griseoaurantiacus 30223 strain can be used as a biocontrol agent for the prevention and control of the bacterial wilt caused by the Ralstonia solanacearum.

[0102] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not a limitation on the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.​

Claims

1. A strain of Streptomyces glaucus (Gray Orange) Streptomyces griseoaurantiacus ), characterized in that, It has been preserved in Guangdong Microbial Culture Collection Center on July 28, 2023, with the preservation number GDMCC No: 63691, and the preservation address is No. 59, Building 5, 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

2. The use of Streptomyces griseorubens (ATCC 25937) according to claim 1 for the control of bacterial wilt, characterized in that, Streptomyces griseoaurantiacus The bacterial wilt is a Ralstonia solanacearum disease. ​ 3. The Streptomyces griseorubens (S. griseorubens) of claim 1 for use in the preparation of a biological agent for the control of bacterial wilt, characterized in that, Streptomyces griseoaurantiacus ) the use of the Streptomyces griseorubens (S. griseorubens) of claim 1 for the preparation of a biological agent for the control of bacterial wilt, characterized in that, The bacterial wilt is a Ralstonia solanacearum disease.

4. Use according to claim 2 or 3, wherein the compound is ###0002### The bacterial wilt is a tomato bacterial wilt.

5. A biological agent, characterized in that, A composition comprising Streptomyces grisorchacus of claim 1 Streptomyces griseoaurantiacus or a fermentation broth thereof.

6. The biological preparation of claim 5, wherein, Contains the Streptomyces glaucusi as described in claim 1 ( Streptomyces griseoaurantiacus The fermentation broth.

7. The biological preparation of claim 6, wherein, The fermentation broth is a filtrate obtained by culturing the Streptomyces Streptomyces griseoaurantiacus ) of claim 1 in a liquid medium, centrifuging, and filtering.

8. A method of controlling bacterial wilt, characterized by, The biological agent of any one of claims 5-7 is used for preventing and treating bacterial wilt, and the bacterial wilt is a Ralstonia solanacearum disease.

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