Brevibacillus brevis hncs-1 and application thereof

Abstract

The application discloses a strain of Brevibacillus sp. HNCS-1, relates to the technical field of tea tree planting, and has a preservation number of CCTCC NO: M2022713. The strain is classified and named as Brevibacillus sp., and is preserved in the China Center for Type Culture Collection on May 25, 2022, and is located at Wuhan University, Wuhan, China, and has a postal code of 430072. The strain shows significant inhibitory effects on common pathogenic bacteria in tea gardens, such as tea anthracnose bacteria, tea leaf penicillium, tea fusarium, tea white star bacteria, tea round brown spot disease, and tea wheel spot disease, is environment-friendly, and is non-toxic and non-harmful.

Description

Technical Field

[0001] This invention relates to the field of tea tree cultivation technology, and more specifically to a strain of Bacillus shortiflorus HNCS-1 and its application in the preparation of biocontrol agents for tea tree diseases. Background Technology

[0002] The tea plant (Camellia sinensis) is a perennial woody or shrubby plant, an important economic crop with tea leaves as the harvested part. Tea plant diseases are common in tea gardens, especially major leaf diseases, such as tea anthracnose fungus, tea leaf mold, tea Fusarium, tea white spot fungus, tea round red spot, and tea ring spot fungus, which generally lead to a yield reduction of 10-50% and reduce the quality of tea.

[0003] Currently, tea tree diseases are mostly controlled using chemical agents. For tea production where leaves are directly harvested and processed for consumption, this increases the risk of excessive pesticide residues and increased drug resistance in pathogens. Biological control is an important direction for the development of tea tree disease control, but research on biological control of tea tree diseases is currently limited. The key lies in the screening, modification, and application of highly effective antagonistic microorganisms.

[0004] Therefore, how to achieve biological control of tea tree diseases is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a strain of Bacillus shortiflorus HNCS-1 and its application in the preparation of biocontrol agents for tea tree diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A strain of Bacillus brevicaulis HNCS-1, with accession number CCTCC NO: M2022713, was deposited on May 25, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China.

[0008] The Bacillus shortbread HNCS-1 of this invention shows significant inhibitory effects on common pathogens in tea gardens, including tea anthracnose fungus, tea mold, tea Fusarium, tea white spot fungus, tea round red spot fungus, and tea ring spot fungus. It is environmentally friendly and non-toxic.

[0009] Another object of the present invention is to provide an engineered strain, which is constructed from the above-mentioned Bacillus shortbacterium HNCS-1 as the starting strain.

[0010] Another object of the present invention is to provide a microbial inoculant comprising the above-mentioned Bacillus shortbread HNCS-1 and / or the above-mentioned engineered strain.

[0011] Preferably, the bacterial agent is fermentation supernatant, bacterial suspension, or bacterial powder.

[0012] Another object of the present invention is to provide the application of the above-mentioned Bacillus shortbreadenus HNCS-1 or the above-mentioned engineered strain or the above-mentioned microbial agent.

[0013] Preferred application in the prevention and control of tea tree diseases.

[0014] Preferably, its application in the preparation of biocontrol agents for tea tree diseases.

[0015] Another object of the present invention is to provide a biocontrol agent for tea tree diseases, comprising the above-mentioned Bacillus shortbread HNCS-1 or the above-mentioned engineered strain or the above-mentioned microbial agent.

[0016] Another objective of this invention is to provide a method for preparing a biocontrol agent for tea tree diseases, wherein the Bacillus shortbread HNCS-1 of claim 1, the engineered strain of claim 2, or the microbial agent of claim 3 / 4 is fermented on NB medium / LB medium as substrate, at a culture temperature of 24-28℃, a pH value of 6.0-8.0, and a fermentation time of 72h.

[0017] Preferably, the preparation method of the tea tree disease biocontrol agent uses NB medium as the culture medium, the culture temperature is 26℃, and the pH value is 7.0.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a strain of Bacillus shortbread HNCS-1, which shows significant inhibitory effects on common pathogens in tea gardens such as tea anthracnose fungus, tea mold, tea Fusarium, tea white spot fungus, tea round red spot fungus, and tea ring spot fungus. It is environmentally friendly and non-toxic. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 The inhibitory effects of five biocontrol bacteria obtained from the initial screening on tea anthracnose pathogens were studied, where e is Bacillus shortbread HNCS-1.

[0021] Figure 2 The inhibitory effect of Bacillus shortbread HNCS-1 fermentation supernatant on tea anthracnose bacteria was determined by re-screening.

[0022] Figure 3The colony morphology of Bacillus shortbread HNCS-1;

[0023] Figure 4 Gram-stained micrograph of Bacillus shortbread HNCS-1 (scale bar 5 μm);

[0024] Figure 5 Scanning electron microscope image of Bacillus shortbreadenus HNCS-1 (scale bar 1 μm);

[0025] Figure 6 The results of molecular biological identification of Bacillus shortbread HNCS-1 are shown in Figure a, where a is an electrophoresis image of 16S rDNA amplification products and b is a phylogenetic tree.

[0026] Figure 7 The effects of different culture media on the antibacterial activity of Bacillus shortbread HNCS-1 fermentation broth;

[0027] Figure 8 The effect of different temperatures on the antibacterial activity of Bacillus shortbread HNCS-1 fermentation broth;

[0028] Figure 9 The effect of different pH values ​​on the antibacterial activity of Bacillus shortbread HNCS-1 fermentation broth;

[0029] Figure 10 To investigate the effect of different fermentation times on the antibacterial activity of Bacillus shortbread HNCS-1 fermentation broth;

[0030] Figure 11 The inhibition rate of Bacillus shortbread HNCS-1 fermentation broth on the mycelial growth of different pathogens. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] Test materials

[0034] 1. Microorganisms

[0035] The fungi causing tea anthracnose (Gloeosporium theae-sinensis), tea white spot (Elsinoeleucospila), tea spot mold (Phyllosticta theae), tea Fusarium sp., and tea round red spot (Cercospora theae) were all isolated and preserved by the Cultivation and Plant Protection Laboratory of the Tea Research Institute of Hunan Academy of Agricultural Sciences.

[0036] 2. Soil samples

[0037] It was collected from the rhizosphere soil of perennial tea trees in Changsha City, Hengyang County, Baojing County and other places in Hunan Province.

[0038] 3. Solid culture medium

[0039] PDA medium: 6.0g potato extract powder, 20.0g glucose, 20.0g agar, 1000mL distilled water.

[0040] Gao's No. 1 culture medium: potassium nitrate 1.0g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.5g, ferrous sulfate 0.01g, sodium chloride 0.5g, soluble starch 20.0g, distilled water 1000mL.

[0041] NA medium: 10.0g peptone, 5.0g sodium chloride, 15.0g agar, 3.0g beef extract powder, 1000mL distilled water.

[0042] 4. Liquid culture medium

[0043] PDB medium: 4g potato extract powder, 20.0g glucose, 1000mL distilled water.

[0044] Landy medium: 20.0g glucose, 1.0g yeast extract, 5.0g L-glutamic acid, 2mg L-phenylalanine, 1.0g L-tryptophan, 0.5g potassium chloride, 1.0g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 5mg manganese sulfate tetrahydrate, 0.16mg copper sulfate heptahydrate, 0.15mg ferrous sulfate heptahydrate, 1000mL distilled water.

[0045] LB medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1000 mL distilled water.

[0046] NB medium: 10.0g peptone, 3.0g beef meal, 5.0g sodium chloride, 1.0g glucose, 1000mL distilled water.

[0047] Example 2

[0048] Isolation, screening and identification of strains

[0049] 1. Separation

[0050] Take 10g of tea tree rhizosphere soil, add it to an Erlenmeyer flask containing 90ml of sterile water and an appropriate amount of grinding beads, shake at 20℃ and 200rpm for 30 minutes, and then... -1 10 -2 10 -3 Serial dilutions were performed, with 100 μL of each concentration evenly spread onto PDA, NA, and Gao's No. 1 medium. After incubation in the dark at 26°C for 5 days, the colonies in the culture dishes were purified.

[0051] 2. Filtering

[0052] Initial screening: 5mm diameter mycelial cakes were collected from colonies after 5 days of purification and culture. The mycelial surface was inoculated at equal intervals on a 25mm radius circle, with a 5mm diameter *Anthracnose spp.* mycelial cake inoculated at the center. The control group was inoculated only with *Anthracnose spp.* mycelial cakes. After 7 days of dark incubation at 26℃, the colony diameter for each treatment was measured using the cross-hatching method, and the inhibition rate was calculated.

[0053] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100

[0054] The antibacterial effects of the five biocontrol strains obtained from the initial screening are shown in the appendix. Figure 1 , where e is HNCS-1.

[0055] Secondary screening: Single colonies of the purified biocontrol bacteria were inoculated into LB medium and cultured at 37°C and 200 rpm for 12 h with shaking to obtain the seed culture. The same applies below. 2% of the biocontrol bacteria seed culture was inoculated into 500 mL Erlenmeyer flasks containing 200 mL of LB liquid medium. After 7 days of shaking culture at 26°C and 180 rpm in the dark, the fermentation broth was centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was collected after removing the precipitate.

[0056] Take 1 mL of sterile supernatant and add it to 9 mL of sterile PDA medium. After shaking, pour the mixture into a 9 cm sterile Petri dish. After the medium solidifies, inoculate the center of the medium with a 5 mm diameter anthracnose fungal cake. Repeat three times. After incubating at 26°C in the dark for 7 days, measure the colony diameter of each treatment using the cross-hatching method and calculate the inhibition rate.

[0057] The supernatant from the HNCS-1 fermentation, after being diluted 10 times, completely inhibited the growth of tea anthracnose pathogens. See attached image for the results. Figure 2 .

[0058] 3. Identification

[0059] (1) Morphological and physiological-biochemical characteristics

[0060] Strawberry strain HNCS-1 was streaked onto LB agar plates and incubated at 37°C for 12 hours. The color and morphology of the colonies were observed: On LB solid medium, the colonies of strain HNCS-1 had irregular edges, were yellowish-white, had a smooth, moist surface, were opaque, and did not produce soluble pigments (see Appendix). Figure 3 ).

[0061] Gram staining was performed on strain HNCS-1, and its basic morphological characteristics were observed under an optical microscope: strain HNCS-1 was Gram-positive, with rod-shaped cells, 2-4 μm in length, and containing spores. Preliminary identification suggests that this bacterium belongs to the family Bacillaceae (see appendix). Figure 4 ,5).

[0062] Physiological and biochemical characteristics of the biocontrol strain were determined with reference to Berger's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria: strain HNCS-1 was Gram-positive, gelatin liquefaction positive, catalase positive, and methyl red positive; negative for VP test, citrate, anaerobic growth, and starch hydrolysis; it could grow in 2% NaCl but could not grow in 5% NaCl or 7% NaCl; it produced acid by fermentation with glucose and mannitol but not by fermentation with arabinose and xylose; it is speculated that this strain may be Brevibacillus sp. (Table 1).

[0063] Table 1

[0064]

[0065] Note: + indicates positive; - indicates negative.

[0066] (2) Molecular biological identification

[0067] DNA from biocontrol bacteria was extracted according to the operating procedures of the bacterial genome extraction kit (Shangbao Biotechnology), and its 16S rDNA was sequenced.

[0068] PCR amplification reaction system (20 μL): 10 μL 2×SanTaq PCR Mix, 1 μL DNA, 0.5 μL upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) (10 mmol / L), 0.5 μL downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2) (10 mmol / L), 8 μL dd H2O. PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles; 72℃ extension for 10 min.

[0069] After the amplified PCR products passed the electrophoresis test, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for base determination.

[0070] The PCR product of the 16S rDNA gene of strain HNCS-1 was found to be approximately 1500 bp in length. Figure 6 a) The sequencing results were compared with the NCBI database for homology. The sequence information of the 12 strains with the highest similarity was selected, and a phylogenetic tree was constructed using the NJ method (neighbor-joining method). The results showed that strain HNCS-1 was more closely related to Bacillus brevis and Bacillus formosus (see Appendix). Figure 6 b) Based on the colony morphology and physiological and biochemical characteristics, strain HNCS-1 was identified as Bacillus shorthair.

[0071] The strain of this invention, *Brevibacillus sp.*, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China. The information for the strain is: *Brevibacillus sp.*, accession number CCTCC NO.M 2022713, deposit date May 25, 2022.

[0072] Example 3

[0073] Optimization of fermentation conditions for strain HNCS-1

[0074] (1) Different culture media

[0075] The HNCS-1 seed culture was inoculated at a 2% inoculum into 200 mL Erlenmeyer flasks containing one of four liquid media (LB, PDB, NB, or Landy). The pH was 7.0, the fermentation temperature was 26℃, the fermentation time was 60 h, and the rotation speed was 180 rpm. The fermentation broth was centrifuged at 10,000 rpm for 10 min at 4℃, the precipitate was removed, and the supernatant was stored at 4℃ for later use.

[0076] Antibacterial activity was assessed using the agar diffusion method. Four 5mm diameter wells were evenly punched around the circumference of a PDA medium coated with 100μL of *Anthracis chinensis* spores, 2.5cm from the center. 50μL of each of the four sterile supernatants (filtered through a 0.22μm filter) was added to each well, with sterile water used as a control. After incubation at 26℃ in the dark for 3 days, the diameter of the inhibition zone was measured using the cross-hatching method. Each treatment was repeated three times.

[0077] See the appendix for the results. Figure 7The antibacterial effects of strain HNCS-1 on the supernatant after 60 hours of fermentation in four media: PDB, Landy, LB, and NB showed significant differences. The inhibition zone diameter of NB medium reached 2.10 cm, which was significantly larger than that of the other media, while the inhibition zone diameter of Landy medium was the smallest, at only 1.33 cm.

[0078] (2) Different fermentation temperatures

[0079] The fermentation temperatures were 24℃, 26℃, 28℃, 30℃, 32℃, and 34℃, respectively. Other initial conditions and methods for determining antibacterial activity were the same as in (1).

[0080] See the appendix for the results. Figure 8 Within the fermentation temperature range of 24-28℃, the antibacterial effect of the fermentation supernatant of strain HNCS-1 against *Anthracnose causal agent* initially increased and then decreased with increasing temperature, but the difference was not significant. The strongest antibacterial effect was observed at 26℃, with an inhibition zone diameter of 2.29 cm. Within the temperature range of 28-34℃, the antibacterial effect of the fermentation supernatant of strain HNCS-1 decreased significantly with increasing temperature.

[0081] (3) Different pH values

[0082] The initial pH values ​​were 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. Other initial conditions and methods for determining antibacterial activity were the same as in (1).

[0083] See the appendix for the results. Figure 9 When the fermentation pH was between 3.0 and 5.0, the supernatant of strain HNCS-1 showed no antibacterial activity. Between pH 6.0 and 8.0, the supernatant exhibited the strongest antibacterial activity, significantly higher than other treatments; the best antibacterial effect was observed at pH 7.0, with an inhibition zone diameter of 2.43 cm. Between pH 9.0 and 10.0, the antibacterial activity of the supernatant decreased significantly.

[0084] (4) Different fermentation times

[0085] The biocontrol bacteria seed solution was inoculated into 500 mL Erlenmeyer flasks containing 200 mL of NB liquid culture medium at an inoculation rate of 2%. Other initial conditions and methods for determining antibacterial activity were the same as in (1).

[0086] See the appendix for the results. Figure 10 Within 12-72 hours, the antibacterial effect of the fermentation supernatant significantly increased with increasing fermentation time. After 72 hours of fermentation, the antibacterial effect of the fermentation supernatant was the strongest, with an inhibition zone diameter of 2.17 cm. Within 72-132 hours, the antibacterial effect gradually decreased with increasing fermentation time, but the decrease was not significant.

[0087] Example 4

[0088] Antibacterial spectrum of strain HNCS-1

[0089] Strain HNCS-1 was inoculated into LB medium and cultured at 37°C with shaking at 200 rpm for 12 h to obtain the seed culture. The same applies below. 2% of the biocontrol bacterial seed culture was inoculated into 500 mL Erlenmeyer flasks containing 200 mL of LB liquid medium. After 7 days of culture in the dark with shaking at 26°C and 180 rpm, the fermentation broth was centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was collected after removing the precipitate.

[0090] Take 1 mL of sterile supernatant and add it to 9 mL of sterile PDA medium. After shaking, pour the mixture into 9 cm sterile Petri dishes. After the medium solidifies, inoculate the center of each treatment with a 5 mm diameter mycelial cake of *Tea bud mold*, *Fusarium*, *Tea white spot*, *Tea round red spot*, or *Tea ring spot*, repeating the process three times. After incubating at 26°C in the dark for 7 days, measure the colony diameter of each treatment using the cross-sectional method and calculate the inhibition rate. See the appendix for results. Figure 11 When the supernatant fermentation broth of strain HNCS-1 was diluted 10 times, it could completely inhibit the growth of tea mold, Fusarium, Cercospora, and tea leaf spot fungus, and also had a high inhibition rate of 61.12% on the mycelial growth of tea white spot fungus.

[0091] Example 5

[0092] Pot experiment on the control efficacy of strain HNCS-1 against tea white spot disease.

[0093] After fermentation culture of strain HNCS-1 under the above optimal conditions, the fermentation broth was centrifuged at 4℃ and 10000rpm for 10min, and the supernatant was retained and stored at 4℃ for later use.

[0094] Twenty healthy, one-year-old tea seedlings of similar growth and normal development were selected and divided into two groups. The experimental group was sprayed with a supernatant diluted 20 times, while the control group was sprayed with sterile water. Both groups were inoculated with tea white spot pathogen 24 hours later. Twenty-four hours after inoculation, the disease severity of the first, second, and third leaves of each seedling was investigated and recorded, and the disease index and control efficacy were calculated.

[0095] Disease severity grading criteria: Grade 0: normal appearance, no symptoms; Grade 1: lesions cover 1%-10% of the leaf area; Grade 2: lesions cover 11%-50% of the leaf area; Grade 3: lesions cover 51%-80% of the leaf area; Grade 4: lesions cover more than 81% of the leaf area.

[0096] Disease index = [∑(number of diseased plants at each level × corresponding level value) / (total number of plants surveyed × highest level value)] × 100

[0097] Prevention and control efficacy (%) = (Disease index of control group - Disease index of experimental group) / Disease index of control group × 100

[0098] Spraying the supernatant of strain HNCS-1 fermentation diluted 20 times and then inoculating it with tea white star pathogen 24 hours later showed good control effect against tea white star pathogen (see Table 2).

[0099] Table 2

[0100]

[0101] Example 6

[0102] Pot experiment on the control efficacy of strain HNCS-1 against tea anthracnose fungus

[0103] After fermentation culture of strain HNCS-1 under the above optimal conditions, the fermentation broth was centrifuged at 4℃ and 10000rpm for 10min, and the supernatant was retained and stored at 4℃ for later use.

[0104] Three experimental groups and one control group were set up in tea gardens with uniform planting levels (25-year-old tea trees). The experimental groups were sprayed with HNCS-1 fermentation broth, HNCS-1 fermentation broth diluted 50 times, and carbendazim diluted 1000 times, respectively, while the control group was sprayed with sterile water. Each treatment was replicated 4 times, and each replicate plot was 15m². 2 The samples were randomly arranged in blocks with alternating rows between adjacent blocks. After spraying, tea anthracnose was inoculated using the wound inoculation method. At 2, 4, and 12 days after inoculation, 50 tea leaves were randomly sampled in parallel from each block, and the disease severity was investigated and recorded to calculate the incidence rate, disease index, and control efficacy.

[0105] Disease grading criteria: Grade 0, normal appearance, no symptoms; Grade 1, lesion diameter 0-1mm; Grade 2, lesion diameter 1-3mm; Grade 3, lesion diameter 3-5mm; Grade 4, lesion diameter 5mm or more.

[0106] Disease index = [∑(number of diseased plants at each level × corresponding level value) / (total number of plants surveyed × highest level value)] × 100

[0107] Prevention and control efficacy (%) = (Disease index of control group - Disease index of experimental group) / Disease index of control group × 100

[0108] The results showed that all three treatment groups (1000x dilution of carbendazim, HNCS-1 fermentation broth, and 50x dilution of HNCS-1 fermentation broth) had good control efficacy against tea anthracnose (see Table 3). Carbendazim showed good rapid action but poor sustained efficacy; while HNCS-1 fermentation broth initially showed slightly lower efficacy, its efficacy gradually improved as the biocontrol bacteria colonized in the field, resulting in better long-term control. Furthermore, while a 50x dilution of HNCS-1 fermentation broth slightly reduced its efficacy, the difference in efficacy between the diluted solution, the original fermentation broth, and carbendazim was not significant after 12 days of inoculation.

[0109] Table 3

[0110]

[0111]

[0112] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0113] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A short-lived Bacillus strain ( Brevibacillus sp.) HNCS-1, characterized in that, The preservation number of the Bacillus shortbread HNCS-1 is CCTCC NO: M2022713.

2. A microbial inoculant, characterized in that, It contains the short-lived Bacillus HNCS-1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is fermentation supernatant, bacterial suspension, or bacterial powder.

4. The application of the Bacillus shortbreadenus HNCS-1 according to claim 1 or the microbial agent according to claims 2 / 3, characterized in that, The application is for the prevention and control of tea tree diseases or the preparation of biocontrol agents for tea tree diseases; The tea tree diseases mentioned are caused by one or more of the following: tea anthracnose fungus, tea mold fungus, tea falciparum fungus, tea white spot fungus, tea round red spot fungus, and / or tea ring spot fungus.

5. A biocontrol agent for tea tree diseases, characterized in that, Includes Bacillus shortbread HNCS-1 as described in claim 1 or the microbial agent as described in claims 2 / 3.

6. A method for preparing a biocontrol agent for tea tree diseases, characterized in that, The *Bacillus shortiflora* HNCS-1 of claim 1 or the microbial agent of claim 2 / 3 were fermented using NB / LB medium as substrate at a temperature of 24-28°C and a pH of 6.0-8.0 for 72 h.

7. The method for preparing the biocontrol agent for tea tree diseases according to claim 6, characterized in that, NB medium was used as the culture medium, and the culture temperature was 26℃ with a pH of 7.0.

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