Quercus acutissima leaf blight pathogen antagonistic bacterium and application thereof

Abstract

The invention discloses a german oak leaf blight pathogen antagonistic bacterium and application thereof, and belongs to the technical field of crop biocontrol strains. A biocontrol strain ML-1 with an antagonistic effect on Pestalotiopsis oryzae is separated from healthy bark of quercus acutissima by adopting a dilution and separation combined growth rate method, a phylogenetic tree is constructed through morphological characteristics and physiological and biochemical characteristics, analysis is carried out, and the strain ML-1 is identified as bacillus velezensis. Pot experiment results show that the strain can effectively prevent and treat the german oak leaf blight, and the prevention and treatment effect exceeds 57%. The strain has the biocontrol potential of preventing and treating related plant diseases caused by Pestalotiopsis oryzae, and can be used for preventing and treating the related plant diseases caused by Pestalotiopsis oryzae. Therefore, the ML-1 strain can effectively control infection of german oak leaf blight pathogenic bacteria on german oak leaves, achieves a good effect of preventing and controlling german oak leaf blight, and has great potential to be developed into a commercial viable biocontrol preparation.

Description

Technical Field

[0001] This invention relates to the field of biocontrol strains for crops, specifically to an antagonistic bacterium against the pathogen causing leaf blight in oak trees and its application. Background Technology

[0002] *Quercus acutissima*, a deciduous tree belonging to the genus *Quercus* in the family Fagaceae, is one of the most widely distributed oak species in my country, and a major dominant and community-building species. It has a well-developed root system, is drought-tolerant and thrives in poor soil, and produces high-quality wood, playing an important role in ecological protection, water conservation, and timber processing. Leaf blight of *Quercus acutissima* affects seedlings and mature trees, primarily affecting the leaves, and is mainly caused by *Pestalotiopsis oryzae*.

[0003] Currently, the control of Quercus acutissima leaf blight in production mainly relies on chemical control, combined with silvicultural measures. Chemical control is fast-acting, low-cost, and has a mature industrial development, making it the most common method for controlling forest diseases. However, it has unavoidable drawbacks, such as toxic residues and the development of drug resistance, which are detrimental to sustainable forestry development and ecological maintenance. The use of biological control methods to treat forest diseases is the current trend, as biological control has advantages such as low residues, low risk, and high specificity. The use of bacteria for biological control is already widely applied to forest disease control, but there is currently very little research on the biological control of Quercus acutissima leaf blight. Summary of the Invention

[0004] In order to overcome the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide an antagonistic bacterium against the pathogen of Quercus acutissima leaf blight and its application, so as to achieve the purpose of preventing and controlling Quercus acutissima leaf blight.

[0005] The technical solution of this invention to solve the above-mentioned technical problems is as follows: An antagonistic bacterium against the pathogen of Quercus acutissima leaf blight is provided. The antagonistic bacterium is Bacillus velezensis, named Bacillus velezensis ML-1, and deposited on January 20, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, with accession number GDMCC NO: 65831.

[0006] This invention provides an application of the above-mentioned antagonistic bacteria against the pathogen of Quercus acutissima leaf blight in the prevention and control of Quercus acutissima leaf blight.

[0007] Furthermore, the control of oak leaf blight is achieved by using Bacillus velezensis ML-1 to inhibit the pathogen Pestalotiopsis oryza.

[0008] Furthermore, to control Quercus acutissima leaf blight, treating Quercus acutissima leaves with Bacillus velezensis ML-1 bacterial solution can reduce the disease index and incidence of Quercus acutissima leaf blight.

[0009] Furthermore, treating Quercus acutissima leaf blight with Bacillus velezensis ML-1 bacterial solution can improve the control effect of Quercus acutissima leaf blight.

[0010] The present invention provides a microbial inoculant, which includes at least one of the above-mentioned antagonistic bacteria of the Quercus acutissima leaf blight pathogen, spores and fermentation broth.

[0011] This invention provides a method for preventing and controlling leaf blight of Quercus acutissima caused by Pestalotiopsis oryzae, comprising the following steps: treating Quercus acutissima leaves with the above-mentioned antagonistic bacterial solution of Quercus acutissima leaf blight pathogen or the above-mentioned microbial agent.

[0012] The present invention also provides a biocontrol agent for preventing and controlling oak leaf blight caused by Pestalotiopsis oryzae, comprising the above-mentioned antagonistic bacteria against oak leaf blight pathogens or the above-mentioned microbial agents.

[0013] This invention has the following beneficial effects: The present invention uses a dilution-separation method combined with growth rate analysis to isolate a biocontrol strain, *Bacillus velezensis* ML-1 (hereinafter referred to as ML-1), which exhibits antagonistic effects against *Pestalotiopsis oryzae*, from healthy *Quercus acutissima* bark. Phylogenetic tree analysis was conducted based on morphological characteristics and physiological and biochemical properties, identifying ML-1 as *Bacillus velezensis*. Pot experiments showed that this bacterium effectively controlled the occurrence of *Quercus acutissima* leaf blight, with control efficacy exceeding 57%. This strain has biocontrol potential for controlling plant diseases caused by *Pestalotiopsis oryzae*. Therefore, the ML-1 strain of this invention can effectively control the infection of *Quercus acutissima* leaves by the pathogen causing *Quercus acutissima* leaf blight, achieving a good effect in controlling *Quercus acutissima* leaf blight, and has great potential to be developed into a commercially viable biocontrol agent. Attached Figure Description

[0014] Figure 1 This image shows the growth of strain ML-1 and the pathogen causing leaf blight of Quercus acutissima on a PDA plate. Figure 2 Morphological characteristics of strain ML-1; Figure 3 A phylogenetic tree diagram of strain ML-1 based on the gyrB gene sequence and the 16S rRNA gene sequence; Figure 4The efficacy of different dilutions of ML-1 strain against leaf blight in potted plants was investigated. Detailed Implementation

[0015] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0016] Plant samples: Quercus acutissima Carruth. samples were collected from the diseased area of ​​Quercus acutissima in Longquanshan, Chengdu, Sichuan Province.

[0017] Test strain: Pestalotiopsis oryzae, the pathogen of oak leaf blight, was isolated, identified and preserved by the Forest Protection and Forest Tree Pathology Laboratory of Sichuan Agricultural University. Preservation number: XSS202310007.

[0018] Culture media used for the test: The isolation and culture of biocontrol bacteria were conducted using beef extract peptone (NA) solid medium (g / L): beef extract 3.0, peptone 10.0, NaCl 5.0, agar 20.0, pH natural; NA liquid medium (g / L): beef extract 3.0, peptone 10.0, NaCl 5.0, pH natural; The culture of pathogens and plate confrontation experiments were conducted using potato dextrose agar (PDA) medium (g / L): potato 200.0, glucose 20.0, agar 20.0, pH natural.

[0019] Example 1: Isolation and purification of biocontrol bacteria The bacterial strain was isolated from healthy Quercus acutissima leaves using a dilution separation method. Healthy Quercus acutissima leaves were taken, washed, and ground in a sterile mortar. An appropriate amount of sterile water was added and the mixture was thoroughly ground. The homogenate was transferred to a sterile centrifuge tube and centrifuged at 12000 rpm for 2 minutes. 1 mL of the supernatant was collected and incubated at 85°C for 15 minutes. This supernatant was then added to a test tube containing 9 mL of sterile water and vortexed to prepare a 10-fold dilution separation solution. -1 10 -2 10 -3 10 -4 10 -5 Diluent. Then, take 100 μL of bacterial suspension and spread it evenly on NA solid medium. Repeat each treatment three times. Incubate in a 30°C constant temperature incubator for 48 h. Select single colonies with obvious morphological differences for purification and preservation.

[0020] Example 2: Screening of native pathogens controlling Quercus acutissima leaf blight Primary screening: The PDA plate confrontation growth method was used. Pathogen *Pestalotiopsis oryzae*, cultured for 5 days, was punched using a 5mm sterile punch and placed on one side of a PDA plate. Biocontrol bacteria were streaked onto the other side, 3cm away from the pathogen, using a sterile inoculation loop. Each treatment was repeated three times. A blank control was prepared by inoculating only the pathogen without biocontrol bacteria. All plates were incubated at 25℃ for 7 days. The presence or absence of an inhibition zone was observed and recorded, and the width of the inhibition zone (from the center of the bacterial colony to the edge of the pathogen hyphae) was measured. The strain with the most significant inhibitory effect was selected, purified, and prepared for secondary screening.

[0021] Secondary screening: The growth rate method was used. One loop of the biocontrol bacteria strain selected in the initial screening was inoculated into 100 mL of NA liquid medium and incubated at 30℃ and 170 rpm for 12 h to prepare a seed culture. The seed culture was then inoculated into a new 100 mL of NA liquid medium at 2% of the total volume and incubated at 30℃ and 170 rpm for 24 h. After incubation, the culture was centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm microporous membrane to prepare a sterile fermentation filtrate. 10 mL of the sterile fermentation filtrate was added to 90 mL of PDA medium, mixed well, and poured into a petri dish. Sterile water was added as a blank control. Each treatment was repeated three times. After the culture medium has cooled and solidified, use a sterile punch with a diameter of 5 mm to extract a 5-day cultured pathogenic bacteria *Pestalotiopsis oryzae* bacterial cake and inoculate it in the center of a plate. After incubation at 25℃ for 7 days, measure the colony diameter using the cross-hatching method and calculate the inhibition rate: Inhibition rate (%) = (Control pathogenic bacteria colony diameter - Treated pathogenic bacteria colony diameter) / Control pathogenic bacteria colony diameter × 100; where the colony diameter (mm) is the average of two perpendicular diameters.

[0022] Example 3: Identification of biocontrol strain ML-1 (1) Morphological observation of strain ML-1 The strain with the best biocontrol effect obtained from the secondary screening was inoculated onto NA solid medium and incubated in a 30℃ constant temperature incubator for 48 hours. The morphological characteristics of the colonies were observed, including color, smoothness or wrinkles, density or looseness, texture, and edge grooves.

[0023] (2) Determination of physiological and biochemical characteristics of strain ML-1 Physiological and biochemical characteristics were determined in accordance with the "Handbook for Systematic Identification of Common Bacteria", including Gram staining, spore staining, catalase reaction, acetylmethyl methanol test, gelatin liquefaction test, nitrate reduction reaction, hydrogen sulfide (H2S) test, citrate test, starch hydrolysis test, and sugar oxidation fermentation determination.

[0024] (3) Molecular biological identification of strain ML-1 DNA was extracted from biocontrol bacteria using a bacterial genomic DNA kit. The 16S rRNA gene sequence was amplified using universal primers 27F and 1492R. PCR reaction conditions were: 95℃ for 4 min; 94℃ for 1 min, 50℃ for 1 min, 72℃ for 2 min, for a total of 34 cycles; 72℃ for 10 min. The gyrA gene sequence was amplified using specific primers gyrA-F and gyrA-R. PCR reaction conditions were: 95℃ for 5 min; 94℃ for 40 s, 50℃ for 40 s, 68℃ for 1 min, for a total of 30 cycles; 68℃ for 10 min. The primers for gyrB gene amplification were UP1f and UP2r (see Zhu Bichun, Gu Li, Li Zheng, et al. Isolation and identification of Antarctic soil Bacillus and its control of bacterial brown rot in maize [J]. Journal of Nanjing Agricultural University, 2017, 40(4):641-648). PCR reaction conditions: 95℃ for 54 min; 98℃ for 10 s, 62℃ for 1 min, 72℃ for 2 min, for a total of 30 cycles; 72℃ for 8 min. The PCR reaction system (50 μL) consisted of: 2×Taq PCR Master Mix 25 μL, primers (10 μmol / L) 2 μL each, DNA template (100 mg / L) 1 μL, and ddH2O 20 μL. After the PCR reaction was completed, 6 μL of the reaction product was taken, and the PCR results were detected by 1% agarose gel electrophoresis. The PCR product was then sent to Chengdu Qingke Biotechnology Co., Ltd. for sequencing. The primer sequences are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 3); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 4); gyrA-F: 5'-CAGTCAGGAAATGCGTACGTCCTT-3' (SEQ ID NO. 5); gyrA-R: 5'-CAAGGTAATGCTCCAGGCATTGCT-3' (SEQ ID NO. 6); UP1f: 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAART ARTTYGA-3' (SEQ IDNO.7); UP2r: 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTC NGTCAT-3' (SEQ ID NO.8).

[0025] (4) Constructing a phylogenetic tree The sequenced gene sequences were BLAST-aligned in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) and then submitted to GenBank. Sequences of strains with high gene similarity were downloaded, and phylogenetic trees of the 16S rRNA, gyrA, and gyrB gene sequences were constructed using MEGA 6.0 software with neighbor-joining, with 1000 replicates for the bootstrap test, to determine the phylogenetic position of the biocontrol strain.

[0026] Example 4: Pot test of biocontrol strain ML-1 Pot experiments were conducted in the greenhouse of the Fifth Teaching Building at the Chengdu Campus of Sichuan Agricultural University. The biocontrol strain ML-1 was inoculated into NA liquid medium and cultured at 30℃ with shaking at 180 rpm for 48 h to prepare a bacterial suspension for later use. The pathogen of Quercus acutissima leaf blight, cultured for 15 days in PDA plates, was rinsed with sterile water and diluted to prepare a 1×10⁻⁶ solution. 6 CFU / mL conidial suspension. Healthy two-year-old Quercus acutissima seedlings with a height of approximately 60-70 cm were selected and treated as follows: After making wounds on the main trunk of the Quercus acutissima seedlings with a needle, 5 mL of F. fujikuroi conidial suspension was evenly sprayed on each seedling. Seven days later, Bacillus amyloliquefaciens ML-1 bacterial suspension was sprayed on the same site. Four treatments were set up: 100-fold, 200-fold, 400-fold, and 800-fold dilutions, 5 mL per seedling. Sterile water was used as a blank control. Ten seedlings were treated per treatment, with three replicates. After 15 days, the disease incidence, disease index, and relative control effect of the biocontrol bacteria on Quercus acutissima leaf blight were calculated. The disease grading standards are as follows: Grade I, represented by a value of 0, no disease or almost no disease; Grade II, represented by a value of 1, leaf disease incidence less than 25%; Grade III, represented by a value of 2, leaf disease incidence 25%-50%; Grade IV, represented by a value of 3, leaf disease incidence 51%-75%; Grade V, represented by a value of 4, leaf disease incidence exceeding 75%. The calculation formulas for incidence rate, disease index, and control effect are shown below: Incidence rate (%) = (Number of infected plants / Total number of inoculated plants) × 100; Disease index = [∑(number of diseased plants × representative value) / (total number of plants × representative value of the most severe disease level)] × 100; Prevention and control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100.

[0027] Results Analysis (1) Isolation of biocontrol strains A total of 14 Bacillus isolates were obtained from healthy Quercus acutissima leaf tissue, numbered ML-1-ML-14. Single colonies were picked with a sterile inoculation loop and streaked onto NA solid medium. After purification, the isolates were transferred to NA solid slant medium and incubated at 30°C for 48 h to obtain purified strains, which were then stored at 4°C.

[0028] (2) Screening of biocontrol strains that antagonize Pestalotiopsis oryzae The 14 Bacillus strains obtained were re-screened using sterile fermentation broth. The results showed that strain ML-1 had the best antibacterial effect. The pathogen *Pestalotiopsis oryzae* had the smallest colony diameter (2.37 cm) and the highest inhibition rate (66.67%) (see Table 1). Compared with the control, the pathogen's colony margins showed underdeveloped, sparse, and regressive hyphae growth, indicating a significant inhibitory effect (see Table 1). Figure 1 Based on the initial screening results, strain ML-1 exhibited a strong antagonistic effect against the pathogen, demonstrating stability and significantly outperforming the other 13 strains. Therefore, strain ML-1 will be selected as the target Bacillus for subsequent experiments. Strain ML-1 showed a significant antibacterial effect, producing a 1.64 cm inhibition zone. Microscopic observation of the hyphae at the edge of the inhibition zone and those of normal hyphae revealed that the inhibited hyphae were twisted, swollen, and deformed.

[0029] Table 1. Antibacterial effect of antagonistic bacteria Note: All data are "mean ± standard deviation" of 3 replicates. Different letters after the data in the same column indicate that the difference is significant at the P<0.05 level according to the LSD(L) test.

[0030] (2) Identification of antagonistic bacteria ① Morphological identification: In NA medium, strain ML-1 colonies are opaque, milky white, nearly circular, with irregularly raised edges and a soft, sticky texture. It can produce a milky white biofilm on NA medium (see...). Figure 2 A). Microscopic observation revealed that ML-1 cells were rod-shaped (0.54-0.86) × (1.91-2.83) μm, with oval-shaped, centrally located spores (see...). Figure 2 B).

[0031] ② Physiological and biochemical characteristics identification: The results of the physiological and biochemical characteristics test of strain ML-1 showed that the strain is an aerobic Bacillus that can hydrolyze starch and gelatin. The catalase reaction and nitrate reduction reaction were both positive. It can utilize citrate and various glycogen. The acetylmethane test and hydrogen sulfide (H2S) test were both negative. The results are shown in Table 2.

[0032] Table 2 Physiological and biochemical characteristics of strain ML-1 Note: + indicates a positive reaction; - indicates a negative reaction.

[0033] ③ Molecular biological identification: The amplified 16S rRNA sequence (SEQ ID NO.1) and gyrB sequence (SEQ ID NO.2) were compared for homology using NCBI. The results showed that the 16S rRNA gene sequence of strain ML-1 had over 99% similarity to *Bacillus velezensis*. Phylogenetic analysis, combining 16S rRNA and gyrB gene sequence analysis, revealed that strain ML-1 shared over 99% sequence similarity with *Bacillus velezensis*, and both strains belonged to the same phylogenetic branch (see...). Figure 3 Therefore, based on the comprehensive morphological characteristics, physiological and biochemical properties, and the results of 16S rRNA and gyrB gene sequence alignment, strain ML-1 was ultimately identified as Bacillus velezensis. The biocontrol strain Bacillus velezensis ML-1, after morphological and molecular biological identification, has been sent to the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO: 65831.

[0034] (3) Test of potted plant efficacy The results of the pot experiment are shown in Table 3 and Figure 4 (Figure A: Control (CK), Figure B: 800x dilution, Figure C: 400x dilution, Figure D: 200x dilution, Figure E: 100x dilution) As shown. Almost all Quercus acutissima trees treated with no Bacillus leucocephala ML-1 suspension developed leaf blight, with an incidence rate as high as 100%. However, the disease was effectively controlled after treatment with ML-1 suspension. The incidence rate was significantly reduced, decreasing by 70% compared to the control, with a disease index decrease of 59.23, achieving the best control effect of 83.78%. Diluted ML-1 suspension also reduced the incidence of Quercus acutissima leaf blight, but as the dilution ratio increased, the incidence rate and disease index gradually increased, while the control effect decreased accordingly. The control effect was better at dilutions below 100x, reaching over 70%.

[0035] Table 3. Potted plant control efficacy of Bacillus vesalis ML-1 against Quercus acutissima leaf blight Note: Data in the same column marked with different lowercase letters indicate significant differences (P<0.05).

[0036] In summary, this invention is the first to isolate the biocontrol bacterium *Bacillus belyssiopsis ML-1*, which exhibits good inhibitory activity against *Pestalotiopsis oryzae*, the pathogen causing leaf blight in *Quercus acutissima*, from healthy leaves of *Quercus acutissima*. Simultaneously, molecular identification was performed using 16S rRNA and gyrB gene sequences. Combined with morphological characteristics and physiological and biochemical properties, strain ML-1 was rapidly and accurately identified as *Bacillus belyssiopsis*, demonstrating excellent biocontrol potential. In confrontation experiments, a 1.64 cm inhibition zone was produced, and the sterile filtrate showed an inhibition rate of 67.25% against the pathogen. In pot experiments, the 100-fold dilution of the ML-1 bacterial suspension still showed significant control effects, proving the biocontrol potential of ML-1. This invention can provide a potential biocontrol strain for the biological control of *Quercus acutissima* leaf blight and provides a reference for the rapid and accurate classification and identification of closely related *Bacillus* species.

[0037] The 16S rRNA and gyrB gene sequences of the ML-1 strain in this invention are shown below: (1) 16S rRNA: TATCTATCACTTCGGCGGCTGGCTCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCCCTGGTGTGCCTCCACATCTCTACGCATTTTACCCGCTACACGTGGGAAATCCACTCTCCCTCTTTCTGCACTCAAGATCC(SEQ ID NO.1); (2)gyrB: (SEQ ID NO.2).

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antagonistic bacterium against the pathogen causing leaf blight of Quercus acutissima, characterized in that, The antagonistic bacterium against the pathogen causing the leaf blight of Quercus acutissima is Bacillus velezensis, named Bacillus velezensis ML-1. It was deposited on January 20, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, with accession number GDMCC NO: 65831.

2. The application of the antagonistic bacterium against the pathogen of Quercus acutissima leaf blight as described in claim 1 in the prevention and control of Quercus acutissima leaf blight.

3. The application according to claim 2, characterized in that, The control of oak leaf blight is achieved by using Bacillus velezensis ML-1, which can inhibit the pathogen Pestalotiopsis oryzae.

4. The application according to claim 2, characterized in that, The control of Quercus acutissima leaf blight involves treating Quercus acutissima leaves with Bacillus velezensis ML-1 bacterial solution, which can reduce the disease index and incidence of Quercus acutissima leaf blight.

5. The application according to claim 2, characterized in that, The method of controlling Quercus acutissima leaf blight involves treating Quercus acutissima leaves with Bacillus velezensis ML-1 bacterial solution, which can improve the control effect of Quercus acutissima leaf blight.

6. A microbial inoculant, characterized in that, The microbial agent includes at least one of the cell bodies, spores, and fermentation broth of the antagonistic bacteria against the leaf blight pathogen of Quercus acutissima as described in claim 1.

7. A method for controlling leaf blight of Quercus acutissima caused by Pestalotiopsis oryzae, characterized in that, Includes the following steps: The leaves of Quercus acutissima were treated with the antagonistic bacterial solution of the Quercus acutissima leaf blight pathogen as described in claim 1 or the microbial agent as described in claim 6.

8. A biocontrol agent for controlling leaf blight of Quercus acutissima caused by Pestalotiopsis oryzae, characterized in that, The biocontrol agent comprises the antagonistic bacteria against the leaf blight pathogen of Quercus acutissima as described in claim 1 or the microbial agent as described in claim 6.

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