Bacillus atrophaeus YL84, microbial agent and application

Abstract

The invention discloses bacillus atrophaeus YL84, a microbial agent and application, and belongs to the technical field of microorganisms. The strain is preserved in the China Center for Type Culture Collection on February 20, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2025267. Sterile fermentation filtrate of the strain YL84 can effectively inhibit germination of conidia and growth of mycelia of valsa ceratosperma of Korla pear trees, can play a good role in preventing in-vitro branches and potted pear seedlings, in addition, the strain can secrete protease and cellulase and synthesize siderophores, has the capacity of dissolving inorganic phosphorus and producing IAA at the same time, and has a good application prospect. The strain has a remarkable antagonistic effect on five common plant pathogenic fungi in the Xinjiang Uygur autonomous region and is wide in antibacterial spectrum.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a strain of Bacillus atrophaeus YL84, a microbial inoculum and its application. Background Art

[0002] Korla fragrant pear is a characteristic pear variety in Xinjiang Uygur Autonomous Region. Due to its excellent quality and high nutritional value, it has become an important export fruit. Its unique taste, delicate fragrance and rich nutritional components have made it highly favored in domestic and international markets and become one of the representative brands of the characteristic agriculture in Xinjiang Uygur Autonomous Region. At the same time, the rapid development of the fragrant pear industry has driven the rise of related industries such as fruit processing, logistics transportation and export trade.

[0003] The trunk canker of Korla fragrant pear tree is an important disease in the production of fragrant pears. In recent years, with the increase in planting area and the change of climate environment, the trunk canker of Korla fragrant pear tree has occurred and spread rapidly in the Korla fragrant pear production area of Xinjiang Uygur Autonomous Region, seriously affecting the quality and yield of fragrant pears and greatly hitting the confidence of fruit farmers. It occurs in the main fragrant pear production areas in China. 90% of the death of Korla fragrant pear trees in the Alar Korla fragrant pear planting area of the First Division of Xinjiang Production and Construction Corps is caused by this disease. The incidence rate of Korla fragrant pear trees was 44.00% - 84.44% in 2014 and 48.00% - 92.00% in 2018. At present, the prevention and control of the trunk canker of Korla fragrant pear tree mainly adopt prevention and control measures such as cultivating disease-resistant varieties, scraping diseased spots, strengthening cultivation management, using chemical agents, etc. However, due to the small number of disease-resistant varieties of fragrant pear trees and the very limited related research at present, and the long effective period of scraping diseased spots and strengthening cultivation management, the prevention and control effect is not obvious, a series of problems such as excessive use of chemical agents, etc., have led to the break of the ecological balance, the damage of biodiversity, and the aggravation of soil and water pollution, thus threatening the sustainable development of agriculture. Therefore, finding environmentally friendly, highly efficient and safe prevention and control measures has attracted more and more attention.

[0004] Biological control is a method that uses beneficial microorganisms in nature to inhibit and control diseases. It is not only environmentally friendly but also in line with the concept of "green plant protection". Currently, certain research has been conducted on the biological control of apple canker at home and abroad. For example, a strain of actinomycete Streptomyces tauricus that antagonizes apple canker was found in the soil. The inhibition rate of the original fermentation broth of the strain against the mycelia of apple canker pathogen was 78.14%, and the relative control effect on detached apple tree branches was 76.37%. Another research report isolated a strain of Stenotrophomonas acidaminiphila from the rhizosphere soil. The fermentation filtrate of the strain significantly reduced the germination of conidia of the canker pathogen, and the control effect was still up to 81.06% after the fermentation broth was diluted 50 times. In addition, a strain of Bacillus velezensis was isolated from the bark of Korla fragrant pear trees. The sterile fermentation filtrate of the strain could induce the activity of defensive enzymes in Korla fragrant pear trees, and the inhibition effect on apple canker of Korla fragrant pear trees reached 82%. Although the current research on exploring the use of biological means to combat apple canker of Korla fragrant pear trees is increasing, these studies still cannot meet the needs of controlling apple canker in production. In view of this, screening and obtaining more antagonistic microbial strains that can efficiently inhibit apple canker of fragrant pear trees is of great significance for promoting the green control strategy of this disease, reducing the dependence on chemical pesticides, and promoting the sustainable development of agriculture. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain of Bacillus atrophaeus YL84, a microbial inoculum and its application to solve the problems existing in the above-mentioned prior art. The present invention uses V. pyri, the pathogen of apple canker of Korla fragrant pear trees, as the target, screens out antagonistic microbial strains that can efficiently inhibit V. pyri from the branches, bark and leaves of Korla fragrant pear trees, measures their antibacterial activities, antibacterial spectra and biological characteristics, and evaluates their biocontrol potential, in order to provide alternative biocontrol resources for the biological control of apple canker of Korla fragrant pear trees.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a strain of Bacillus atrophaeus YL84, which was deposited at the China Center for Type Culture Collection on February 20, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025267.

[0008] Another technical solution of the present invention is a microbial inoculum, which includes the above-mentioned Bacillus atrophaeus YL84.

[0009] The third technical solution of the present invention is the application of the Bacillus atrophaeus YL84 or the microbial inoculum in preventing and / or treating plant diseases caused by Valsa pyri, the pathogen of pear tree canker in Korla.

[0010] The fourth technical solution of the present invention is the application of the Bacillus atrophaeus YL84 or the microbial inoculum in the production of protease.

[0011] The fifth technical solution of the present invention is the application of the Bacillus atrophaeus YL84 or the microbial inoculum in the production of cellulase.

[0012] The sixth technical solution of the present invention is the application of the Bacillus atrophaeus YL84 or the microbial inoculum in the production of siderophore.

[0013] The seventh technical solution of the present invention is the application of the Bacillus atrophaeus YL84 or the microbial inoculum in the decomposition of inorganic phosphorus.

[0014] The eighth technical solution of the present invention is the application of the Bacillus atrophaeus YL84 or the microbial inoculum in the production of IAA.

[0015] The ninth technical solution of the present invention is the application of the Bacillus atrophaeus YL84 or the microbial inoculum in the preparation of products for inhibiting plant diseases caused by pathogens.

[0016] Based on the above technical solutions, the present invention has the following technical effects:

[0017] (1) Compared with the existing biocontrol resources for pear tree canker in Korla, YL84 was identified as Bacillus atrophaeus, which is a newly identified biocontrol strain for pear tree canker in Korla.

[0018] (2) In addition to having an antagonistic effect on Valsa pyri, the pathogen of pear tree canker in Korla, this strain also has a significant antagonistic effect on 5 other common plant pathogenic fungi in Xinjiang Uygur Autonomous Region, with a wide antibacterial spectrum and a wider application range. Compared with the existing biocontrol resources for pear tree canker in Korla, YL84 has both disease resistance and growth promotion effects. Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1The strain YL84 has an antagonistic effect on the Valsa pyri causing pear tree canker in Korla. Among them, A is the colony of V. pyri growing normally; B is the colony of V. pyri after being antagonized by the strain YL84.

[0021] Figure 2 It is the identification diagram of the colony morphology of the strain YL84. Among them, A is the cultivation morphology of the strain YL84 on the LB plate, and B is the colony morphology of the strain YL84.

[0022] Figure 3 It is the phylogenetic tree of the strain YL84 based on 16S rDNA.

[0023] Figure 4 It is the inhibitory effect of the sterile fermentation filtrate of the strain YL84 on the conidial germination of the Valsa pyri causing pear tree canker.

[0024] Figure 5 It is the effect of the sterile fermentation filtrate of YL84 on the colony diameter and inhibition rate of V. pyri.

[0025] Figure 6 It is the control effect of the sterile fermentation filtrate of YL84 on the detached branches of Korla pear trees. Among them, 1 represents the protective and therapeutic effects of the strain YL84 on the detached branches of Korla pear trees infected with pear tree canker, and 2 represents the lesion length of the protective and therapeutic effects of the strain YL84 on the detached branches of Korla pear trees infected with pear tree canker. In 1, A is the protective effect of first adding the sterile fermentation filtrate of the strain YL84 and then inoculating V. pyri; B is the therapeutic effect of first inoculating V. pyri and then adding the sterile fermentation filtrate of the strain YL84; C is the control of only inoculating V. pyri without adding the sterile fermentation filtrate of the strain YL84; D is the blank control of not inoculating V. pyri and the sterile fermentation filtrate of the strain YL84

[0026] Figure 7 It is the control effect of the sterile fermentation filtrate of YL84 on the canker of potted Korla pear seedlings. Among them, 1 represents the protective and therapeutic effects of the strain YL84 on the living branches of potted Korla pear seedlings infected with canker, and 2 represents the inhibition rate of the protective and therapeutic effects of the strain YL84 on the living branches of potted Korla pear seedlings infected with canker. In 1, A is the protective effect of first spraying the sterile fermentation filtrate of the strain YL84 and then inoculating V. pyri; B is the therapeutic effect of first inoculating V. pyri and then spraying the sterile fermentation filtrate of the strain YL84; C is the control of only inoculating V. pyri without spraying the sterile fermentation filtrate of the strain YL84.

[0027] Figure 8 It is the growth-promoting characteristics of the strain YL84. Among them, A is protease, B is siderophore, C is inorganic phosphorus, and D is cellulase.

[0028] Figure 9 It is the equation of the IAA standard curve.

[0029] Figure 10 It is about the growth-promoting effect of the sterile fermentation filtrate of strain YL84 on cotton seedlings. Among them, A is the control group added with sterile water, and B is the treatment group added with 10 mL of the sterile fermentation filtrate of the antagonistic strain YL84.

[0030] Figure 11 It is about the inhibitory effect of strain YL84 on 6 pathogenic fungi in Xinjiang Uygur Autonomous Region. Specific embodiments

[0031] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are only exemplary.

[0035] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0036] The technical solutions of the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been made public.

[0037] An embodiment of the present invention provides a strain of Bacillus atrophaeus YL84, which was deposited at the China Center for Type Culture Collection on February 20, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025267.

[0038] An embodiment of the present invention also provides a microbial inoculant, including the Bacillus atrophaeus YL84.

[0039] An embodiment of the present invention also provides the application of the Bacillus atrophaeus YL84 or the microbial inoculant in preventing and / or treating plant diseases caused by Valsa pyri, the pathogen of pear tree canker in Korla.

[0040] An embodiment of the present invention also provides the application of the Bacillus atrophaeus YL84 or the microbial inoculant in the production of protease.

[0041] An embodiment of the present invention also provides the application of the Bacillus atrophaeus YL84 or the microbial inoculant in the production of cellulase.

[0042] An embodiment of the present invention also provides the application of the Bacillus atrophaeus YL84 or the microbial inoculant in the production of siderophore.

[0043] An embodiment of the present invention also provides the application of the Bacillus atrophaeus YL84 or the microbial inoculant in the solubilization of inorganic phosphorus.

[0044] An embodiment of the present invention also provides the application of the Bacillus atrophaeus YL84 or the microbial inoculant in the production of IAA.

[0045] An embodiment of the present invention also provides the application of the Bacillus atrophaeus YL84 or the microbial inoculant in the preparation of products for inhibiting plant diseases caused by pathogens.

[0046] In some specific embodiments, the pathogens include Valsa pyri, the pathogen of pear tree canker in Korla, Verticillium dahliae, the pathogen of cotton verticillium wilt, Fusarium oxysporum, the pathogen of cotton fusarium wilt, Valsa mali, the pathogen of apple tree canker, Alternaria alternata, the pathogen of red date black spot, and Valsa nivea, the pathogen of walnut tree canker.

[0047] This strain was isolated from the pear orchard in the 13th Regiment of Alar City, the First Division of Xinjiang Production and Construction Corps. The sterile fermentation filtrate of strain YL84 can effectively inhibit the germination of conidia and the growth of hyphae of the pathogen causing pear tree canker of Korla fragrant pear, and can play a good preventive role in detached branches and potted pear seedlings. In addition, this strain can secrete protease, cellulase, synthesize siderophores, and also has the ability to dissolve inorganic phosphorus and produce IAA. This strain can also significantly increase the dry weight, plant height and root length of cotton. This strain has a significant antagonistic effect against 5 common plant pathogenic fungi in Xinjiang Uygur Autonomous Region, has a wide antibacterial spectrum and a wide range of application. Compared with the existing biological control resources for pear tree canker of Korla fragrant pear, strain YL84 has both disease resistance and growth promotion effects, indicating that strain YL84 has significant development and application prospects in the biological control of pear tree canker of Korla fragrant pear.

[0048] Example 1

[0049] 1 Test method

[0050] 1.1 Isolation and screening of antagonistic strains

[0051] Test samples: Information on the collection of branches, bark and leaves of Korla fragrant pear trees is shown in (Table 1). Samples of detached branches of Korla fragrant pear trees were collected from the fragrant pear orchard of Tarim University (81°17′23″, 40°32′53″).

[0052] Table 1

[0053]

[0054] Using the dilution isolation method, the collected samples were washed with clean water respectively, dried in the ultra-clean workbench and cut into pieces about 0.5 cm. After disinfecting with 75% alcohol for 1 min and 1% sodium hypochlorite for 3 min, they were then washed three times with sterile water, and the last sterile water was used to coat the plate to check whether the disinfection was complete. The disinfected Korla fragrant pear tree tissues were ground into pulp with a sterilized mortar, and tissue homogenates were obtained after dilution with sterile water. The tissue homogenates were diluted 10 2 , 10 -1 , 10 -2 , 10 -3 , 10 -4 and then evenly smeared on LB medium (LB, 5.0 g of yeast extract, 10 g of tryptone, 5 g of NaCl, 1000 mL of water; 15 g of agar) plates with a sterilized spreader, with 4 replicates for each concentration. The medium was placed in an incubator and incubated at a constant temperature of 28 °C for 3 d. Purification was carried out according to the colony color and morphology by the streaking method. The single colonies after the third purification were stored in glycerol tubes at -20 °C.

[0055] The antagonistic bacteria were screened by the plate confrontation method. V. pyri with a diameter of 5 mm (provided by the Green Prevention and Control Laboratory of Tarim University) was inoculated in the center of a PDA (PDA: 200 g of potato, 20 g of glucose, 1000 ml of water, 15 g of agar) plate. The purified endophytes were inoculated 2.5 cm away from the edge of the fungal disc. Four endophyte discs were inoculated on each plate, and the plate inoculated only with V. pyri was used as a control. They were placed in an incubator and cultured in the dark at 25 °C for 4 days. After the control colony covered the entire plate, the colony diameter was measured by the cross method and the inhibition rate was calculated.

[0056] Inhibition rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0057] In this example, 63 strains were isolated from 144 pear tissue samples by the dilution isolation method. After screening by the plate confrontation method, it was found that 30 strains had antagonistic effects on V. pyri, the pathogen causing pear tree canker in Korla fragrant pear. Among them, 1 strain with the best antagonistic effect on V. pyri was selected. The inhibition rate of this strain against V. pyri was as high as 78.82% ( Figure 1 ), and it was named YL84.

[0058] 1.2 Identification of strain YL84

[0059] Morphological identification: The antagonistic bacteria were streaked on an LB medium and cultured at a constant temperature of 28 °C for 3 days. According to "Bergey's Manual of Determinative Bacteriology" (Buchanan Bergey, 1984) and "Handbook for Identification of Common Bacteria" (Cai Miaoying and Dong Xiuzhu, 2001), the morphological characteristics of the colonies on the LB plate were observed.

[0060] Determination of physiological and biochemical characteristics: A fully automatic microbial biochemical analyzer (BIOLOG) was used to determine the physiological and biochemical characteristics of the antagonistic strains.

[0061] The DNA of the antagonistic bacteria was extracted using a bacterial DNA extraction kit. PCR amplification was carried out using the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). PCR reaction system: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 90 s, 30 cycles, and extension at 72 °C for 5 min. After the amplified products were detected by 1% agarose gel electrophoresis, the gel was cut and recovered and sent to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing. The sequencing results were then subjected to homology comparison in the GenBank and NCBI databases, and a phylogenetic tree was constructed using MEGA11 software.

[0062] Colony morphological characteristics of strain YL84: After culturing strain YL84 on LB medium for 1 day, the colony is light yellow, semi-transparent, with a smooth and moist surface and irregular edges( Figure 2 ).

[0063] Physiological and biochemical characteristics of strain YL84: The physiological and biochemical characteristics of strain YL84 are shown in Table 2. Strain YL84 can utilize D-mannose, D-fructose, D-galactose, D-trehalose, D-gluconic acid, D-malic acid, D-galacturonic acid, D-glucuronic acid, D-sorbitol, D-salicin, gentiobiose, sucrose, α-D-glucose, Tween 40, 1% NaCl, 4% NaCl, 8% NaCl, gelatin, pectin, V-P test, dextrin, 1% sodium lactate, citric acid, L-lactic acid, L-arginine, L-histamine; it cannot utilize D-cellobiose, D-turanose, D-serine, D-aspartic acid, D-arabitol, stachyose, α-D-lactose, L-rhamnose, nalidixic acid, α-keto-butyric acid. According to the determination results of morphological and physiological and biochemical indexes, strain YL84 conforms to the characteristics of Bacillus.

[0064] Table 2

[0065]

[0066]

[0067] Molecular biological identification of strain YL84: The sequencing results show that the length of the 16S rDNA of strain YL84 is 1449 bp. By comparing with the GenBank sequence, it is found that strain YL84 has the highest homology with Bacillus atrophaeus, and the similarity is above 99%. Using MEGA11 to construct a phylogenetic tree, it is found that they cluster together. Combining morphological and physiological and biochemical characteristics, strain YL84 is identified as Bacillus atrophaeus( Figure 3 ).

[0068] Bacillus atrophaeus YL84 was deposited in the China Center for Type Culture Collection on February 20, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025267.

[0069] 1.3 Preparation of sterile fermentation filtrate of strain YL84

[0070] In a laminar flow hood, the antagonistic strain YL84 was streaked and purified on LB medium. A small amount of the purified antagonistic bacterial liquid was taken and transferred to 100 ml of liquid LB. After culturing at 37 °C and 120 rpm for 2 days, the fermentation broth of the antagonistic strain was obtained. The strain fermentation broth was transferred to a centrifuge tube and centrifuged at 4 °C and 13,000 rpm for 10 min. Subsequently, the supernatant was filtered through a microporous filter membrane with a pore size of 0.22 μm to obtain the sterile fermentation filtrate of the antagonistic strain.

[0071] 1.4 Inhibitory effect of the sterile fermentation filtrate of strain YL84 on the Valsa mali causing pear tree canker in Korla

[0072] 1.4.1 Inhibitory effect of the sterile fermentation filtrate of the strain on the germination of V. pyri conidia

[0073] The conidia of V. mali causing pear tree canker in Korla were cultured by the method of inoculating detached branches. One-year-old Korla pear tree branches with good growth status and consistent growth were cut from the pear orchard of Tarim University. Branches with uniform diameters were selected and cut into 15-cm segments. After washing with clean water, they were soaked in 75% alcohol for 1 min and disinfected with 1% sodium hypochlorite for 3 min. After surface disinfection, they were rinsed with sterilized distilled water and dried, and the two ends of the branches were sealed with paraffin. A hole with a diameter of 5 mm was punched in the center of the branch to reach the xylem. The V. pyri pathogen cake cultured for 3 days was inoculated at the punched site and cultured at a constant temperature of 25 °C with humidity. After conidial horns grew on the branches, the conidial suspension was prepared with liquid PDA to 1×10 6 cells / mL for standby.

[0074] The sterile fermentation filtrate of strain YL84 was diluted 0, 10, 100, and 1000 times with liquid LB. 100 μL of the sterile fermentation filtrate was respectively pipetted into sterilized centrifuge tubes. Subsequently, 100 μL of the prepared conidial suspension was respectively added to the centrifuge tubes containing the sterile fermentation filtrate of the antagonistic strain with different dilution multiples. After culturing at a constant temperature of 25 °C for 36 h, the germination of conidia was observed under an optical microscope. A total of 5 fields of view were observed, and 20 spores were observed in each field of view. The inhibition rate of spore germination was calculated. The conidial suspension without adding the sterile fermentation filtrate of the antagonistic strain was used as a control, and each treatment was repeated 3 times.

[0075] Inhibition rate of spore germination = (number of germinated spores in the control - number of germinated spores in the treatment) / number of germinated spores in the control × 100%.

[0076] The sterile fermentation filtrate of strain YL84 at different dilution multiples could effectively inhibit the germination of V. pyri conidia causing pear tree canker in Korla, and the inhibitory effect weakened with the increase in the dilution multiple of the sterile fermentation filtrate of the antagonistic strain. When the dilution multiple was 0, the inhibition rate was the highest, at 91.07%. The inhibition rate of the sterile fermentation filtrate diluted 1000 times was the lowest, at 35.71% ( Figure 4 ).

[0077] 1.4.2 Inhibitory effect of the sterile fermentation filtrate of strain YL84 on the mycelial growth of V. pyri

[0078] Pour the sterile fermentation filtrate of strain YL84 into conical flasks containing 100 ml of PDA at 55°C according to volume fractions of 2%, 4%, 6%, 8%, and 10% respectively. After thorough mixing, pour onto plates. Place a V. pyri fungal disc with a diameter of 5 mm in the center of each plate. Use the plate that only contains the V. pyri fungal disc without adding the sterile fermentation filtrate of the antagonistic bacterium as the control. Each treatment is repeated 5 times. Incubate in an incubator at 25°C. After 3 days, measure the colony diameter and calculate the inhibition rate.

[0079] Inhibition rate = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0080] Using V. pyri, the causal agent of pear tree canker in Korla fragrant pear trees, as the target fungus, the effects of the sterile fermentation filtrate of strain YL84 at different concentrations on the mycelial growth of V. pyri were determined. The results showed that the sterile fermentation filtrate of strain YL84 had an inhibitory effect on the mycelial growth of V. pyri. Among them, when the volume fraction of the sterile fermentation filtrate was 10%, the inhibitory effect was the best, reaching 88.75%. The inhibitory effect decreased with the decrease in the volume fraction of the fermentation filtrate of the antagonistic bacterium. When the volume fraction was 2%, the inhibitory effect was the lowest, at 56.25%( Figure 5 ).

[0081] 1.4.3 Determination of the control effect of the sterile fermentation filtrate of strain YL84 on detached branches of Korla fragrant pear trees

[0082] Cut one-year-old Korla fragrant pear tree branches with good growth status and consistent growth from the fragrant pear orchard of Tarim University. Select branches with uniform diameters and cut them into 15-cm segments. After washing with clean water, soak them in 75% alcohol for 1 minute and disinfect them with 1% sodium hypochlorite for 3 minutes. After surface disinfection, rinse them with sterile distilled water and air-dry. Seal both ends of the branches with paraffin. Use a punch with a diameter of 5 mm to punch holes in the center of the branches until reaching the xylem. Treatment 1 is the protection test: Use a pipette to aspirate 60 μL of the sterile fermentation filtrate of the antagonistic bacterium to the punched hole. After incubating at 25°C for 1 day, add a V. pyri fungal disc with a diameter of 5 mm. Incubate under humid conditions at 25°C. Treatment 2 is the treatment test: Move a V. pyri fungal disc with a diameter of 5 mm to the punched hole. After incubating at 25°C for 1 day, remove the fungal disc and use a pipette to aspirate 60 μL of the sterile fermentation filtrate of the antagonistic bacterium to the punched hole. After the branch absorbs it, incubate under humid conditions at 25°C. Use the treatment with only the inoculation of the V. pyri fungal disc as the control and the treatment with only the inoculation of the PDA block as the blank control. Each treatment is repeated three times. Observe the length of the disease spots on the 7th day and calculate the control effect.

[0083] Prevention effect = (lesion length of control group - lesion length of treatment group) / lesion length of control group × 100%.

[0084] The aseptic fermentation filtrate of strain YL84 can effectively inhibit the infection and lesion expansion of in vitro branches of Korla fragrant pear tree rot. The protective effect of the treatment group inoculated with the aseptic fermentation filtrate of strain YL84 first and then with V.pyri was 84.72%. The protective effect of the treatment group inoculated with V.pyri first and then with the aseptic fermentation filtrate of YL84 was 72.72%. This shows that the aseptic fermentation filtrate of strain YL84 has a protective and therapeutic effect on Korla fragrant pear tree rot, and the protective effect is better than the therapeutic effect ( Figure 6 ).

[0085] 1.4.4 Determination of the effect of aseptic fermentation filtrate of strain YL84 on the rot of potted Korla pear seedlings

[0086] Select healthy pear seedlings with consistent growth, two years old, and no disease. In treatment one, a 5mm diameter V.pyri cake was inoculated into the center of the trunk, and 300ml of antagonistic bacteria aseptic fermentation liquid was sprayed after 3 days. In treatment two, 300ml of antagonistic bacteria aseptic fermentation liquid was sprayed, and 3 days later, a 5mm diameter V.pyri cake was inoculated into the center of the trunk. In control one, a 5mm diameter V.pyri cake was inoculated into the center of the trunk. In control two, 300ml of water was inoculated. The length of the lesions was measured and the prevention effect was calculated at 10d and 21d after the first application of the antagonistic bacteria aseptic fermentation filtrate, and each treatment was repeated 3 times.

[0087] Prevention effect = (lesion length of control group - lesion length of treatment group) / lesion length of control group × 100%.

[0088] The Korla fragrant pear seedlings were used as the test material, and the sterile fermentation filtrate of the antagonistic strain YL84 was used to treat the potted pear seedlings with Korla fragrant pear rot disease. The results showed that the prevention and treatment effects of potted pear seedlings sprayed with the sterile fermentation filtrate of the strain YL84 were 73.50% and 51.81%, respectively. The length of the lesion for the prevention effect was 1.19±0.071cm, and the length of the lesion for the treatment effect was 2.16±0.029cm ( Figure 7 ).

[0089] 1.5 Determination of growth-promoting properties of strain YL84

[0090] 1.5.1 Determination of the ability of strain YL84 to fix nitrogen, dissolve phosphorus, dissolve potassium, produce cellulase and protease

[0091] The strain YL84 was transferred to casein agar medium, Ashby medium, CAS agar medium, organic phosphorus bacteria medium, inorganic phosphorus bacteria medium, and sodium carboxymethyl cellulose medium, and cultured at a constant temperature of 28°C for 7 days. Then, it was observed whether a transparent circle was formed around the inoculation site or whether the colonies could grow normally.

[0092] The strain YL84 could produce a transparent circle on casein medium, CAS agar medium, and sodium carboxymethyl cellulose medium, and could grow normally on inorganic phosphorus medium, indicating that the strain YL84 has the ability to secrete protease, cellulase, synthesize siderophore, and decompose inorganic phosphorus ( Figure 8 ).

[0093] 1.5.2 Determination of the IAA-producing ability of strain YL84

[0094] Determination of the IAA-secreting ability: The colorimetric method was used. The strain was inoculated into King’s B culture medium containing tryptophan and cultured on a shaker at 28°C for 7 days, with 3 replicates set. Take 2 mL of the fermentation broth, centrifuge at 12,000 r / min for 10 minutes, collect 1 mL of the supernatant and place it in a centrifuge tube. After adding an equal volume of the colorimetric solution, let it stand in the dark for 30 min, and observe whether there is a color change.

[0095] Prepare IAA standard solutions with concentrations of 0.5, 1.0, 5.0, 10.0, 15.0, and 20.0 mg·L -1 . Using the above color development method, measure their absorbance at 530 nm using an ultraviolet spectrophotometer, and draw a standard curve and calculate its equation. Measure the OD 530nm value of the color-developed strain, and substitute it into the standard curve equation to calculate the content of IAA produced by the strain.

[0096] After the strain was cultured in King’s B culture medium containing tryptophan for 7 days, its fermentation broth showed color development after the colorimetric reaction, indicating that the strain has the ability to secrete IAA. The regression equation obtained from the standard curve (absorbance at 530 nm) drawn with the prepared IAA standard solution is: y = 0.06954 + 0.00199x. Substitute the absorbance value of the strain YL84 after color development into the standard curve equation for calculation. Finally, it was measured that the IAA content produced by the strain YL84 within 7 days was 29.88 mg / L, indicating that it has a strong IAA synthesis ability ( Figure 9 ).

[0097] 1.5.3 Growth promotion effect of the sterile fermentation filtrate of strain YL84 on cotton seedlings

[0098] The "Tahe No. 2" cotton seeds were selected for the experiment. The soil and vermiculite were filled into a 2L beaker in a ratio of 2:1 and sterilized for later use. The cotton seeds were germinated at 28°C for 24 hours until the seeds showed white tips. After being disinfected with 75% alcohol for 1 minute and 1% sodium hypochlorite for 3 minutes, they were washed three times with sterilized water and then implanted into the seedling trays. In the treatment group, 10 ml of the sterile fermentation filtrate of YL84 was added to every 100 g of soil in the seedling trays. In the control group, 10 ml of sterile water was added to every 100 g of soil in the seedling trays. They were placed in the greenhouse to grow at a temperature of 28°C with a light / dark cycle of 14 h / 10 h, and each treatment was repeated three times. After 7 days, the dry weight, plant height, and root length were measured.

[0099] Through the analysis of the results of the treatment experiment on "Tahe No. 2" cotton seeds, the following conclusions were drawn. In the treatment group where 10 mL of the sterile fermentation filtrate of the antagonistic strain YL84 was added to the seedling trays, compared with the control group, the dry weight, plant height, and root length of the cotton all increased significantly, indicating that the fermentation filtrate of YL84 can effectively promote the growth of cotton seedlings (Table 3, Figure 10 ).

[0100] Table 3 Promoting effect of the sterile fermentation filtrate of strain YL84 on cotton seedlings

[0101]

[0102] 1.6 Determination of the antibacterial spectrum of strain YL84

[0103] The plate confrontation method was used to determine the inhibitory effect of strain YL84 on six plant pathogens, namely V. pyri causing the canker disease of Korla fragrant pear trees, V. dahliae causing cotton verticillium wilt, F. oxysporum causing cotton fusarium wilt, V. mali causing apple tree canker disease, A. alternata causing red date black spot disease, and V. nivea causing walnut tree canker disease (all six pathogens were provided by the Green Prevention and Control Laboratory of Tarim University). The pathogens were inoculated in the center of the PDA plate, and the YL84 bacterial cake was inoculated 2.5 cm away from the edge of the bacterial cake. The plate inoculated only with the pathogens was used as the control, and it was placed in the incubator for dark cultivation at 25°C. Each treatment had 4 replicates, and the inhibition rate was measured after the colonies in the control group covered the culture dish. Inhibition rate (%) = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group × 100%.

[0104] By using the plate confrontation method to determine the mycelial growth inhibition rate of the antagonistic strain YL84 against 6 tested pathogens, the results showed that strain YL84 had different degrees of antagonistic effects on the mycelial growth of the 6 tested pathogens. Among them, the inhibitory effect on the mycelial growth of cotton verticillium wilt was the best, reaching 84.11%, and the inhibitory effect on the mycelial growth of red date black spot disease was relatively poor, being 58.82% ( Figure 10)。These results indicate that strain YL84 not only has good biocontrol effects on the main trunk diseases of Korla fragrant pear trees, but also has good inhibitory effects on the diseases of farmland and shelterbelts around Korla fragrant pear orchards, showing a certain broad spectrum.

[0105] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A strain of Bacillus atrophaeus YL84, characterized in that: This strain was deposited in the China Center for Type Culture Collection on February 20, 2025, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: M 2025267.

2. A microbial agent, characterized in that: Including the Bacillus atrophaeus YL84 described in claim 1.

3. Use of the Bacillus atrophaeus YL84 according to claim 1 or the microbial agent according to claim 2 in preventing and / or treating plant diseases caused by V. pyri, a Korla fragrant pear tree rot pathogen.

4. Use of the Bacillus atrophaeus YL84 according to claim 1 or the microbial agent according to claim 2 in the production of protease.

5. Use of the Bacillus atrophaeus YL84 according to claim 1 or the microbial agent according to claim 2 in the production of cellulase.

6. Use of the Bacillus atrophaeus YL84 according to claim 1 or the microbial agent according to claim 2 in the production of siderophores.

7. Use of the Bacillus atrophaeus YL84 as described in claim 1 or the microbial agent as described in claim 2 in the decomposition of inorganic phosphorus.

8. Use of the Bacillus atrophaeus YL84 according to claim 1 or the microbial agent according to claim 2 in the production of IAA.

9. Use of the Bacillus atrophaeus YL84 according to claim 1 or the microbial agent according to claim 2 in the preparation of products for inhibiting plant diseases caused by pathogens.

10. The use according to claim 9, characterized in that: The pathogens include V. pyri, a pathogen of Korla fragrant pear tree rot, V. dahliae, a pathogen of cotton verticillium wilt, F. oxysporum, a pathogen of apple tree rot, V. mali, a pathogen of red date black spot, A. alternata, and a pathogen of walnut tree rot, V. nivea.

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