Bacillus atrophaeus strain YL84, microbial inoculant and application

Abstract

This invention discloses a strain of Bacillus atrophus YL84, a microbial inoculant, and its applications, belonging to the field of microbial technology. This strain was deposited on February 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025267. The aseptic fermentation filtrate of strain YL84 effectively inhibits the germination of conidia and mycelial growth of the pathogen causing canker in Korla fragrant pear trees, and provides good preventative effects on detached branches and potted pear seedlings. Furthermore, this strain can secrete proteases and cellulases, synthesize siderophores, and possesses the ability to dissolve inorganic phosphorus and produce IAA. This strain exhibits significant antagonistic effects against five common plant pathogenic fungi in Xinjiang Uygur Autonomous Region, demonstrating a broad antibacterial spectrum.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Bacillus atrophus YL84, microbial agents, and their applications. Background Technology

[0002] Korla fragrant pears are a specialty pear variety of Xinjiang Uygur Autonomous Region, and their superior quality and high nutritional value have made them an important export fruit. Their unique taste, fragrant aroma, and rich nutritional components have made them highly sought after in domestic and international markets, establishing them as one of the representative brands of Xinjiang Uygur Autonomous Region's specialty agriculture. Simultaneously, the rapid development of the fragrant pear industry has spurred the rise of related industries such as fruit processing, logistics, and export trade.

[0003] Korla fragrant pear tree rot is a significant disease affecting fragrant pear production. In recent years, with the increase in planting area and changes in climate, Korla fragrant pear tree rot has occurred and spread rapidly in all Korla fragrant pear producing areas of Xinjiang Uygur Autonomous Region, seriously affecting the quality and yield of fragrant pears and greatly undermining the confidence of fruit growers. It has occurred in all major fragrant pear producing areas in my country. In the Korla fragrant pear planting area of ​​the First Division of Xinjiang Production and Construction Corps, 90% of the pear tree deaths were caused by this disease. In 2014, the incidence rate of Korla fragrant pear trees was 44.00%–84.44%, and in 2018, it was 48.00%–92.00%. Currently, the main control measures for Korla fragrant pear tree rot include cultivating disease-resistant varieties, scraping off lesions, strengthening cultivation management, and using chemical agents. However, due to the limited number of disease-resistant varieties of fragrant pear trees and the very limited related research, coupled with the long time required for the effects of scraping off lesions and strengthening cultivation management, the lack of significant control efficacy, and the excessive use of chemical agents, a series of problems have led to the disruption of ecological balance, damage to biodiversity, and aggravated soil and water pollution, thereby threatening sustainable agricultural development. Therefore, the search for environmentally friendly, efficient, and safe control measures is receiving increasing attention.

[0004] Biological control utilizes beneficial microorganisms found in nature to inhibit and control diseases, making it environmentally friendly and aligned with the concept of "green plant protection." Current research on biological control of canker disease has yielded some results both domestically and internationally. For example, one study discovered an actinomycete *Streptomycestauricus* antagonistic to apple tree canker disease in soil. The original fermentation broth of this strain showed a 78.14% inhibition rate against apple tree canker mycelia and a relative control efficacy of 76.37% against detached apple tree branches. Another study reported the isolation of a microacidophilic oligoanaerobic bacterium, *Stenotrophomonas acrida miniphila*, from rhizosphere soil. The fermentation filtrate of this strain significantly reduced the germination of canker fungal conidia, and even after a 50-fold dilution, the control efficacy remained at 81.06%. Furthermore, a strain of *Bacillus velezensis* was isolated from the bark of Korla fragrant pear trees. The aseptic fermentation filtrate of this strain induced the activity of defensive enzymes in the fragrant pear trees and exhibited an 82% inhibitory effect on Korla fragrant pear tree canker. Although research exploring the use of biological methods to combat Korla fragrant pear tree canker is increasing, these studies still cannot meet the needs of canker control in production. Therefore, screening and obtaining more antagonistic microbial strains that can effectively inhibit fragrant pear tree canker is of paramount importance for promoting green control strategies for this disease, reducing reliance on chemical pesticides, and promoting sustainable agricultural development. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus atrophus YL84, a microbial agent, and its application to solve the problems existing in the prior art. This invention targets the antagonistic microbial strain *V. pyri*, the pathogen causing canker in Korla fragrant pear trees, and screens highly effective antagonistic microbial strains that inhibit *V. pyri* from the branches, bark, and leaves of Korla fragrant pear trees. The antibacterial activity, antibacterial spectrum, and biological characteristics of these strains are measured to evaluate their biocontrol potential, aiming to provide alternative biocontrol resources for the biological control of canker disease in Korla fragrant pear trees.

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

[0007] One of the technical solutions of this invention is a strain of Bacillus atrophaeus YL84, which was deposited at the China Center for Type Culture Collection (CCTCC) on February 20, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025267.

[0008] The second technical solution of the present invention is a microbial inoculant, comprising the aforementioned Bacillus atrophus YL84.

[0009] The third technical solution of the present invention is the application of the Bacillus atrophus YL84 or the microbial agent in the prevention and / or treatment of plant diseases caused by V. pyri, the causal agent of canker in Korla fragrant pear trees.

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

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

[0012] The sixth technical solution of the present invention is the application of the Bacillus atrophus YL84 or the microbial agent in the production of iron carriers.

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

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

[0015] The ninth technical solution of the present invention is the application of Bacillus atrophus YL84 or the microbial agent in the preparation of products that inhibit plant diseases caused by pathogens.

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

[0017] (i) Compared with existing biocontrol resources for canker of Korla fragrant pear, YL84 was identified as Bacillusatrophaeus, a newly identified biocontrol strain for canker of Korla fragrant pear.

[0018] (II) In addition to its antagonistic effect against the pathogen causing canker in Korla fragrant pear, this strain also exhibits significant antagonistic effects against five other common plant pathogenic fungi in Xinjiang Uygur Autonomous Region, demonstrating a broad spectrum of inhibition and wider applicability. Compared with existing biocontrol resources for canker in Korla fragrant pear, YL84 possesses both disease resistance and growth-promoting effects. 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 embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1The strain YL84 exhibits antagonistic activity against *V. pyri*, the pathogen causing canker in Korla fragrant pear trees. In the diagram, A represents normally growing *V. pyri* colonies; B represents *V. pyri* colonies antagonized by strain YL84.

[0021] Figure 2 The images show the colony morphology of strain YL84. In the images, A represents the culture morphology of strain YL84 on LB agar plates, and B represents the colony morphology of strain YL84.

[0022] Figure 3 Phylogenetic tree of strain YL84 based on 16S rDNA.

[0023] Figure 4 The study investigated the inhibitory effect of sterile fermentation filtrate of strain YL84 on the germination of conidia of the rot pathogen of Pear Tree.

[0024] Figure 5 The effect of YL84 aseptic fermentation filtrate on V. pyri colony diameter and inhibition rate.

[0025] Figure 6 This study investigated the efficacy of sterile fermentation filtrate of YL84 on detached branches of Korla fragrant pear trees. In this study, 1 represents the protective and curative effects of strain YL84 on detached branches of Korla fragrant pear trees with canker disease, and 2 represents the length of lesions on these branches. Within 1, A represents the protective effect of inoculating with sterile fermentation filtrate of strain YL84 before inoculating with V. pyri; B represents the curative effect of inoculating with V. pyri first followed by sterile fermentation filtrate of strain YL84; C represents the control group (inoculated with V. pyri only, without sterile fermentation filtrate of strain YL84); and D represents the blank control group (without inoculation with either V. pyri or sterile fermentation filtrate of strain YL84).

[0026] Figure 7 This study investigated the efficacy of aseptic fermentation filtrate of YL84 against canker disease in potted Korla pear seedlings. In this study, 1 represents the protective and curative effects of strain YL84 on living branches of potted Korla pear seedlings with canker disease, and 2 represents the inhibition rate of these effects. Within 1, A represents the protective effect of spraying with aseptic fermentation filtrate of strain YL84 before inoculation with V. pyri; B represents the curative effect of inoculating with V. pyri before spraying with aseptic fermentation filtrate of strain YL84; and C represents the control group, which was inoculated with V. pyri without spraying with aseptic fermentation filtrate of strain YL84.

[0027] Figure 8 The growth-promoting characteristics of strain YL84 are shown. A represents protease, B represents hematophilic acid, C represents inorganic phosphate, and D represents cellulase.

[0028] Figure 9 This is the equation for the IAA standard curve.

[0029] Figure 10 This study investigated the growth-promoting effect of sterile fermentation filtrate of strain YL84 on cotton seedlings. Group A was the control group supplemented with sterile water, and group B was the treatment group supplemented with 10 mL of the antagonistic strain YL84 sterile fermentation filtrate.

[0030] Figure 11 The study investigated the inhibitory effects of strain YL84 on six pathogenic fungi in Xinjiang Uygur Autonomous Region. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now 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, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0037] This invention provides a strain of Bacillus atrophaeus YL84, which was deposited on February 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025267.

[0038] This invention also provides a microbial inoculant, including the aforementioned Bacillus atrophus YL84.

[0039] This invention also provides the application of the Bacillus atrophus YL84 or the microbial agent in the prevention and / or treatment of plant diseases caused by V. pyri, the causal agent of canker in Korla fragrant pear trees.

[0040] This invention also provides the application of the Bacillus atrophus YL84 or the microbial agent in the production of proteases.

[0041] This invention also provides the application of the Bacillus atrophus YL84 or the microbial agent in the production of cellulase.

[0042] This invention also provides the application of the Bacillus atrophus YL84 or the microbial agent in the production of siderophores.

[0043] This invention also provides the application of the Bacillus atrophus YL84 or the microbial agent in the decomposition of inorganic phosphorus.

[0044] This invention also provides the application of the Bacillus atrophus YL84 or the microbial agent in the production of IAA.

[0045] This invention also provides the application of the Bacillus atrophus YL84 or the microbial agent in the preparation of products that inhibit plant diseases caused by pathogens.

[0046] In some specific implementations, the pathogens include *V. pyri*, *V. dahliae*, *F. oxysporum*, *V. mali*, *A. alternata*, and *V. nivea*.

[0047] This bacterium, isolated from a pear orchard in the 13th Regiment of the First Division of Xinjiang Production and Construction Corps in Alar City, exhibits significant potential for development and application of strain YL84. The aseptic fermentation filtrate effectively inhibits conidial germination and mycelial growth of the pathogen causing canker in Korla fragrant pear trees. It also provides good preventative protection against detached branches and potted pear seedlings. Furthermore, this strain secretes proteases and cellulases, synthesizes siderophores, and possesses the ability to dissolve inorganic phosphorus and produce IAA (inorganic acid). The strain also significantly increases the dry weight, plant height, and root length of cotton. It demonstrates significant antagonistic effects against five common plant pathogenic fungi in Xinjiang Uygur Autonomous Region, exhibiting a broad spectrum of inhibition and wide applicability. Compared to existing biocontrol resources for canker in Korla fragrant pear trees, strain YL84 combines disease resistance with growth promotion, indicating its significant potential for development and application in the biological control of canker in Korla fragrant pear trees.

[0048] Example 1

[0049] 1. Experimental Methods

[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. The detached branch samples 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] The dilution separation method was used. The collected samples were washed with clean water, dried, and then cut to 0.5 cm in a clean bench. 2 After thorough disinfection with 75% alcohol for 1 minute and 1% sodium hypochlorite for 3 minutes, the tissue was rinsed three times with sterile water. The final rinse was then applied to a plate to check for complete disinfection. The disinfected Korla pear tissue was ground into a paste using a sterile mortar and pestle, diluted with sterile water to obtain a tissue homogenate. This homogenate was then diluted 10 times... -1 10 -2 10 -3 10 -4 The culture was then evenly spread onto LB agar plates (LB, yeast extract 5.0 g, tryptone 10 g, NaCl 5 g, water 1000 mL; agar 15 g) using a sterile spreader, with four replicates for each concentration. The culture was incubated at 28°C for 3 days. Purification was performed by streak plating based on colony color and morphology. The purified single colonies were then stored in glycerol tubes at -20°C.

[0055] Antagonistic bacteria were screened using the plate confrontation method. Five-mm diameter *V. pyri* (provided by the Green Control Laboratory of Tarim University) were inoculated in the center of a PDA (PDA, 200g potato, 20g glucose, 1000ml water, 15g agar) plate. Purified endophytic bacteria were inoculated 2.5cm from the edge of the mycelial cake. Four endophytic bacterial cakes were inoculated on each plate. Plates inoculated only with *V. pyri* served as a control. The plates were incubated in the dark at 25℃ for 4 days. After the control colonies had completely covered the plate, the colony diameter was measured using the cross-crossing method, and the inhibition rate was calculated.

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

[0057] In this embodiment, 63 bacterial strains were isolated from 144 pear tree tissue samples using a dilution separation method. After plate confrontation screening, 30 strains were found to have antagonistic effects against *V. pyri*, the causal agent of canker in Korla fragrant pear. Among them, one strain was selected to have the best antagonistic effect against *V. pyri*, with an inhibition rate as high as 78.82%. Figure 1 It was named YL84.

[0058] 1.2 Identification of strain YL84

[0059] Morphological identification: Antagonistic bacteria were streaked onto LB agar and incubated at 28°C for 3 days. The morphological characteristics of the colonies on LB plates were observed according to Berger's Manual of Bacterial Identification (Buchanan Berger, 1984) and the Manual of Systematic Identification of Common Bacteria (Cai Miaoying and Dong Xiuzhu, 2001).

[0060] Physiological and biochemical characteristics determination: The physiological and biochemical characteristics of antagonistic strains were determined using a fully automated microbial biochemical analyzer (BIOLOG).

[0061] Antagonistic bacterial DNA was extracted using a bacterial DNA extraction kit. PCR amplification was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction system was: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 45 s, 55℃ annealing for 45 s, 72℃ extension for 90 s, 30 cycles of 72℃ extension for 5 min. The amplified products were detected by 1% agarose gel electrophoresis, then excised and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were subsequently compared for homology in GenBank and NCBI databases, and a phylogenetic tree was constructed using MEGA11 software.

[0062] Colony morphology characteristics of strain YL84: After culturing on LB medium for 1 day, the colonies of strain YL84 were pale yellow, translucent, 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-salicylic acid, gentiobiose, sucrose, α-D-glucose, Tween 40, 1% NaCl, 4% NaCl, 8% NaCl, gelatin, pectin, VP test, dextrin, 1% sodium lactate, citric acid, L-lactic acid, L-arginine, and L-histamine; it cannot utilize D-cellobiose, D-malinobiose, D-serine, D-aspartic acid, D-arabinol, stachyose, α-D-lactose, L-rhamnose, naphthidinone acid, and α-keto-butyric acid. Based on the results of morphological and physiological biochemical index measurements, strain YL84 conforms to the characteristics of Bacillus.

[0064] Table 2

[0065]

[0066]

[0067] Molecular biological identification of strain YL84: Sequencing results showed that the 16S rDNA of strain YL84 was 1449 bp in length. Comparison with GenBank sequences revealed that strain YL84 showed the highest homology with *Bacillus atrophaeus*, with a similarity exceeding 99%. Phylogenetic tree construction using MEGA11 showed clustering in one clade. Based on morphological and physiological / biochemical characteristics, strain YL84 was identified as *Bacillus atrophaeus*. Figure 3 ).

[0068] Bacillus atrophaeus YL84 was deposited on February 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025267.

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

[0070] The antagonistic strain YL84 was purified by streaking in LB medium in a clean bench. A small amount of the purified antagonistic bacterial solution was transferred to 100 ml of liquid LB and cultured at 37°C and 120 rpm for 2 days to obtain the fermentation broth of the antagonistic bacterial strain. The fermentation broth was transferred to a centrifuge tube and centrifuged at 4°C and 13000 rpm for 10 min. The supernatant was then filtered through a 0.22 μm pore size microporous membrane to obtain the sterile fermentation filtrate of the antagonistic bacterial strain.

[0071] 1.4 Inhibitory effect of aseptic fermentation filtrate of strain YL84 on the pathogen causing canker in Korla fragrant pear.

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

[0073] Conidia of *V. pyri*, the rot disease of Korla fragrant pear, were cultured using an in vitro branch inoculation method. One-year-old Korla fragrant pear branches of uniform growth and good condition were taken from the fragrant pear orchard of Tarim University. Branches of uniform diameter were selected and cut into 15cm sections. After washing with clean water, the branches were soaked in 75% alcohol for 1 minute, disinfected with 1% sodium hypochlorite for 3 minutes, rinsed with sterile distilled water, and air-dried. Paraffin wax was used to seal both ends of the branches. A 5mm diameter hole was punched in the center of each branch down to the xylem. *V. pyri* fungal discs cultured for 3 days were inoculated into the punched holes and cultured at a constant temperature and humidity of 25℃. Once conidial horns appeared on the branches, a conidial suspension of 1×10⁻⁶ was prepared using liquid PDA. 6 Units / mL are available for use.

[0074] The aseptic fermentation filtrate of strain YL84 was diluted 0, 10, 100, and 1000 times using liquid LB. 100 μL of each aseptic fermentation filtrate was transferred to a sterile centrifuge tube. Then, 100 μL of the prepared conidial suspension was added to centrifuge tubes containing aseptic fermentation filtrate of antagonistic bacteria at different dilutions. After incubation at 25°C for 36 h, conidial germination was observed under an optical microscope. Five fields of view were observed, with 20 conidia per field, and the germination inhibition rate was calculated. A conidial suspension without antagonistic bacteria was used as a control. Each treatment was repeated three times.

[0075] Spore germination inhibition rate = (number of germinating control spores - number of germinating treated spores) / number of germinating control spores × 100%.

[0076] The aseptic fermentation filtrate of strain YL84 at different dilutions effectively inhibited the germination of conidia of *V. pyri*, the rot disease of Korla fragrant pear trees. The inhibitory effect weakened with increasing dilution factor of the antagonistic bacteria's aseptic fermentation filtrate. The inhibition rate was highest at a dilution of 0 (91.07%), while the lowest inhibition rate was observed at a dilution of 1000 times (35.71%). Figure 4 ).

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

[0078] The aseptic fermentation filtrate of strain YL84 was poured into Erlenmeyer flasks containing 100 ml of PDA at 55°C at volume fractions of 2%, 4%, 6%, 8%, and 10%, respectively. After thorough mixing, the filtrate was transferred to plates, with a 5 mm diameter *V. pyri* mycelial cake inoculated in the center of each plate. Plates containing only *V. pyri* mycelial cakes without the addition of antagonistic bacteria aseptic fermentation filtrate served as controls. Each treatment was repeated 5 times. The plates were incubated at 25°C for 3 days. After 3 days, the colony diameter was measured and the inhibition rate was calculated.

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

[0080] Using *V. pyri*, the pathogen causing canker in Korla fragrant pear, as the target bacterium, the effect of different concentrations of aseptic fermentation filtrate from strain YL84 on *V. pyri* mycelial growth was determined. The results showed that the aseptic fermentation filtrate of strain YL84 inhibited the growth of *V. pyri* mycelia. The best inhibitory effect (88.75%) was observed when the volume fraction of the aseptic fermentation filtrate was 10%. The inhibitory effect decreased with decreasing volume fraction of the antagonistic bacteria fermentation filtrate, reaching its lowest level (56.25%) at a volume fraction of 2%. Figure 5 ).

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

[0082] One-year-old Korla fragrant pear branches with good and uniform growth were pruned from the fragrant pear orchard of Tarim University. Branches of uniform diameter were selected and cut into 15cm sections. After washing with clean water, they were soaked in 75% alcohol for 1 minute, disinfected with 1% sodium hypochlorite for 3 minutes, rinsed with sterile distilled water after surface disinfection, and then air-dried. Paraffin wax was used to seal both ends of the branches. A 5mm diameter hole was drilled in the center of each branch down to the xylem. Treatment 1 was a protective effect test: 60μL of sterile fermentation filtrate of antagonistic bacteria was pipetted into the hole, and the mixture was incubated at 25℃ for 1 day. Then, 5mm diameter *V. pyri* mycelium was added. The mixture was then incubated at 25℃ under humidity. Treatment 2 was a therapeutic effect test: 5mm diameter *V. pyri* mycelium was transferred to the hole, and the mixture was incubated at 25℃ for 1 day. After removing the mycelium, 60μL of sterile fermentation filtrate of antagonistic bacteria was pipetted into the hole. After the branch absorbed the filtrate, the mixture was incubated at 25℃ under humidity. The treatment inoculated only with V. pyri mycelium cakes served as a control, and the treatment inoculated only with PDA blocks served as a blank control. Each treatment was replicated three times. The length and size of lesions were observed on day 7, and the control efficacy was calculated.

[0083] Control efficacy = (Leg length of control group - lesion length of treatment group) / lesion length of control group × 100%.

[0084] The aseptic fermentation filtrate of strain YL84 effectively inhibited the infection and lesion expansion of detached branches infected with canker disease in Korla fragrant pear trees. Furthermore, the protective efficacy of the treatment group inoculated with the aseptic fermentation filtrate of strain YL84 followed by inoculation with *V. pyri* was 84.72%. The therapeutic efficacy of the treatment group inoculated with *V. pyri* followed by inoculation with the aseptic fermentation filtrate of YL84 was 72.72%. This indicates that the aseptic fermentation filtrate of strain YL84 has both protective and therapeutic effects against canker disease in Korla fragrant pear trees, with the protective effect being superior to the therapeutic effect. Figure 6 ).

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

[0086] Healthy, disease-free, two-year-old pear seedlings with uniform growth were selected. Treatment 1 involved inoculating the center of the trunk with 5mm diameter *V. pyri* mycelial cakes, followed by spraying with 300ml of antagonistic bacteria sterile fermentation broth 3 days later. Treatment 2 involved spraying with 300ml of antagonistic bacteria sterile fermentation broth, followed by inoculating the center of the trunk with 5mm diameter *V. pyri* mycelial cakes 3 days later. Control 1 involved inoculating the center of the trunk with 5mm diameter *V. pyri* mycelial cakes. Control 2 involved inoculating with 300ml of water. The length of lesions was measured and the control efficacy calculated at 10 and 21 days after the first application of the antagonistic bacteria sterile fermentation filtrate. Each treatment was repeated three times.

[0087] Control efficacy = (Leg length of control group - lesion length of treatment group) / lesion length of control group × 100%.

[0088] Using Korla fragrant pear seedlings as experimental material, the aseptic fermentation filtrate of the antagonistic strain YL84 was used to treat potted Korla fragrant pear seedlings with rot disease, and its control effect was determined. The results showed that the preventive and curative effects of spraying the potted pear seedlings with the aseptic fermentation filtrate of strain YL84 were 73.50% and 51.81%, respectively. The lesion length with preventive effect was 1.19±0.071 cm, and the lesion length with curative effect was 2.16±0.029 cm. Figure 7 ).

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

[0090] 1.5.1 Determination of nitrogen fixation, phosphorus solubilization, potassium solubilization, cellulase production, and protease production capabilities of strain YL84

[0091] Strain YL84 was transferred to casein agar, Assumption medium, CAS agar, organic phosphorus bacteria medium, inorganic phosphorus bacteria medium, and sodium carboxymethyl cellulose medium. After incubation at 28°C for 7 days, the presence of a clear zone around the inoculation site and the ability of colonies to grow normally were observed.

[0092] Strain YL84 produced clear zones on casein medium, CAS agar medium, and sodium carboxymethyl cellulose medium, and grew normally on inorganic phosphorus medium, indicating that strain YL84 has the ability to secrete proteases and cellulases, synthesize siderophores, and degrade inorganic phosphorus. Figure 8 ).

[0093] 1.5.2 Determination of IAA production capacity of strain YL84

[0094] IAA secretion capacity assay: A colorimetric method was used. The strain was inoculated into King's B medium containing tryptophan and cultured in a shaker at 28°C for 7 days, with 3 replicates. 2 mL of fermentation broth was centrifuged at 12,000 rpm for 10 minutes, and 1 mL of supernatant was collected and placed in a centrifuge tube. An equal volume of colorimetric reagent was added, and the tube was incubated in the dark for 30 minutes to observe for any color change.

[0095] Prepare concentrations of 0.5, 1.0, 5.0, 10.0, 15.0, and 20.0 mg·L⁻¹. -1 The IAA standard solution was analyzed using the above-described colorimetric method, and its absorbance was measured at 530 nm using a UV spectrophotometer. A standard curve was plotted and its equation calculated. The absorbance of the chromogenic strain at OD was then determined. 530nm The value of is substituted into the standard curve equation to calculate the content of IAA produced by the strain.

[0096] After culturing the strain in King's B medium containing tryptophan for 7 days, the fermentation broth underwent a colorimetric reaction, indicating that the strain possessed the ability to secrete IAA. A standard curve (absorbance at 530 nm) was plotted using the prepared IAA standard solution, yielding the regression equation: y = 0.06954 + 0.00199x. Substituting the absorbance value of strain YL84 after colorimetric development into the standard curve equation, the final measured IAA content of strain YL84 within 7 days was 29.88 mg / L. This indicates that it has a strong IAA synthesis ability. Figure 9 ).

[0097] 1.5.3 Growth-promoting effect of aseptic fermentation filtrate of strain YL84 on cotton seedlings

[0098] The experiment used "Tahe No. 2" cotton seeds. Soil and vermiculite were mixed in a 2:1 ratio and sterilized in 2L beakers. The cotton seeds were germinated at 28℃ for 24 hours until they showed signs of germination. After thorough sterilization with 75% alcohol for 1 minute and 1% sodium hypochlorite for 3 minutes, the seeds were rinsed three times with sterile water and then planted in seedling trays. In the treatment group, 10 ml of YL84 sterile fermentation filtrate 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. The seeds were grown in a greenhouse at 28℃ with a 14h / 10h light / dark cycle. Each treatment was repeated three times. Dry weight, plant height, and root length were measured after 7 days.

[0099] Analysis of the results of the seed treatment experiment on "Tahe No. 2" cotton yielded the following conclusions: Compared with the control group, the treatment group with 10 mL of aseptic fermentation filtrate of antagonistic strain YL84 added to the seed trays showed significantly increased dry weight, plant height, and root length of cotton seedlings, indicating that YL84 fermentation filtrate can effectively promote the growth of cotton seedlings (Table 3). Figure 10 ).

[0100] Table 3. Growth-promoting effect of aseptic fermentation filtrate of strain YL84 on cotton seedlings.

[0101]

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

[0103] The inhibitory effect of strain YL84 on six plant pathogens—*V. pyri* (causing Korla pear canker), *V. dahliae* (causing cotton Verticillium wilt), *F. oxysporum* (causing cotton wilt), *V. mali* (causing apple canker), *A. alternata* (causing jujube black spot), and *V. nivea* (causing walnut canker)—was determined using the plate confrontation method. Pathogens were inoculated in the center of PDA plates, with YL84 fungal cakes inoculated 2.5 cm from the edge of the cake. Plates inoculated only with pathogens served as controls. The plates were incubated in the dark at 25°C. Each treatment was replicated four times. The inhibition rate was calculated after the control group colonies had fully colonized the plates. Inhibition rate (%) = (Control group colony diameter - Treatment group colony diameter) / Control group colony diameter × 100%.

[0104] The inhibition rate of mycelial growth of antagonistic strain YL84 against six tested pathogens was determined by the plate confrontation method. The results showed that strain YL84 exhibited varying degrees of antagonistic effect on the mycelial growth of all six pathogens. The best inhibition was observed against *Verticillium wilt*, reaching 84.11%, while the inhibition against *Black Spot Disease of Jujube* was relatively poor, at 58.82%. Figure 10These results indicate that strain YL84 not only has good biocontrol effects on the main branch and trunk diseases of Korla fragrant pear trees, but also has a good inhibitory effect on diseases in farmland and shelterbelts surrounding Korla fragrant pear orchards, demonstrating a certain broad spectrum of activity.

[0105] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of Bacillus atrophus ( Bacillus atrophaeus YL84, characterized in that, This strain was deposited on February 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO: M 2025267.

2. A microbial inoculant, characterized in that, Includes Bacillus atrophus YL84 as described in claim 1.

3. The Bacillus atrophus YL84 as described in claim 1 or the microbial agent as described in claim 2 in the prevention and / or treatment of rot fungi in Korla fragrant pear trees. V. pyri Applications in plant diseases caused by it.

4. The application of Bacillus atrophus YL84 as described in claim 1 or the microbial agent as described in claim 2 in the decomposition of inorganic phosphorus.

5. The application of Bacillus atrophus YL84 as described in claim 1 or the microbial agent as described in claim 2 in the production of IAA.

6. The application of Bacillus atrophus YL84 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of products that inhibit plant diseases caused by pathogens, characterized in that, The pathogen is the pathogen causing canker in Korla fragrant pear trees. V. pyri、 cotton Verticillium wilt pathogen V. dahliae 1. Cotton wilt pathogen F. oxysporum Apple tree rot pathogen V. mali Black spot disease pathogen of jujube A.alternata Walnut tree rot pathogen V. nivea .

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