Bacillus velezensis, complex microbial inoculant and application of bacillus velezensis in promoting colonization of arbuscular mycorrhizal fungi

Abstract

The invention discloses bacillus velezensis, a complex microbial inoculant and application of the complex microbial inoculant in promoting colonization of arbuscular mycorrhizal fungi, and belongs to the technical field of agricultural microorganisms. The bacillus velezensis MHB4 is separated from glomus-like fungal spores, and the bacillus velezensis MHB4 has the dual functions that firstly, germination of Paraglomus sp.SW1 spores can be promoted, and colonization of the Paraglomus sp.SW1 spores in plant root systems and colonization of the Paraglomus sp.SW1 spores in soil can be promoted; and 2, 10 plant pathogenic fungi are antagonized. The MHB4 and the AMF are jointly applied to the apple continuous cropping soil, the number of AMF spores in the continuous cropping soil can be increased, colonization of the AMF in continuous cropping plant root systems is promoted, plant growth is promoted, plant photosynthesis is improved, an excellent effect is achieved in the aspect of relieving the apple tree continuous cropping obstacles, and a theoretical basis is provided for biological prevention and control of the apple tree continuous cropping obstacles.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a strain of Bacillus belye, a compound inoculant, and its application in promoting the colonization of arbuscular mycorrhizal fungi. Background Technology

[0002] Continuous cropping obstacles refer to the phenomenon that causes abnormal growth and development of crops when the same crop or crop of the same family is continuously cultivated in the same soil. Due to the limitation of land resources, the renovation and renewal of old orchards can only be carried out through continuous cropping, which leads to the widespread occurrence of continuous cropping obstacles in apples and seriously threatens the healthy and sustainable development of the apple industry.

[0003] Arbuscular mycorrhizal fungi (AMFs), as the fungi with the largest biomass in soil, can form symbiotic relationships with most terrestrial plants, constructing a root-mycelium-soil continuum through ectomycophyte networks, promoting nutrient cycling and dynamically regulating the interactions of the rhizosphere microbiome. However, long-term continuous cropping leads to soil microecological imbalance, deterioration of physicochemical properties, accumulation of autotoxic substances, and enrichment of pathogens. This not only severely inhibits apple plant growth and reduces fruit yield and quality, but also significantly affects the colonization process of AMFs in apple roots, resulting in a persistently low colonization rate of AMFs in continuously cropped apple soils. Even with artificial inoculation of highly efficient AMF strains, the colonization effect is difficult to guarantee, thus limiting the application effect of AMFs in the control of continuous cropping obstacles. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a strain of Bacillus belyssus, a compound inoculant, and its application in promoting the colonization of arbuscular mycorrhizal fungi.

[0005] Specifically, the present invention relates to the following technical solutions: In a first aspect, the present invention provides a strain of Bacillus belye ( Bacillus velezensis The strain MHB4 was deposited on January 26, 2026, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 37520; its classification name is *Bacillus belyes*. Bacillus velezensis .

[0006] The Bacillus belyssus MHB4 of the present invention has the following characteristics: (1) It can promote the germination of AMF spores and promote the colonization of AMF in the root system of plants and in the soil of continuous apple cropping. (2) It has broad-spectrum antibacterial activity against Fusarium moniliformes (Fusarium moniliformes) Fusarium proliferatum Fusarium oxysporum ( Fusarium oxysporum Fusarium solani ( ), Fusarium solani Fusarium moniliforme (Fusarium moniliforme ), apple continuous cropping obstacle specialized Fusarium moniliforme MR5 ( Fusarium proliferatum f. sp. malus domestica Rhizoctonia solani ( ) Rhizoctonia solani Alternaria ( Alternaria alternata ), *Lysimachia verrucae* ( Myrothecium verrucaria ), Pythium spp. ( Pythium aphanidermatum ), Phytophthora ( Phytophthora cactorum It has good antibacterial effects on 10 kinds of plant pathogens, including 10 kinds of plant pathogens.

[0007] (3) Bacillus belye MHB4 was combined with Glomerulosa mycorrhizal fungi ( Paraglomus sp. When used in combination with SW1, it can synergistically increase the number of AMF spores in continuously cropped soil, promote the colonization of AMF in the root system of continuously cropped plants, promote plant growth, enhance plant photosynthesis, and reduce the obstacles of continuous cropping of apples.

[0008] A second aspect of the present invention provides a microbial agent containing the aforementioned Bacillus belye ( Bacillus velezensis )MHB4.

[0009] Preferably, the bacterial agent contains Bacillus belye (B. belye). Bacillus velezensis MHB4 exists in the form of cultured live bacteria, fermentation broth, or bacterial suspension.

[0010] In some preferred embodiments of the present invention, the fermentation broth is prepared by the following method: Bacillus berberis ( Bacillus velezensis MHB4 was inoculated into LB liquid medium and fermented at 35-40℃ for 20-30 hours.

[0011] In some preferred embodiments of the present invention, the bacterial suspension is prepared by the following method: Bacillus berberis ( Bacillus velezensis Centrifuge the fermentation broth of MHB4 and collect the cell precipitate; resuspend the cell precipitate in physiological saline to obtain a cell suspension.

[0012] Furthermore, the bacterial agent contains Bacillus belye ( Bacillus velezensis In addition to MHB4, it may also include auxiliary materials or carrier substrates, such as cow dung, crop straw, etc.

[0013] A third aspect of the present invention provides the use of the above-mentioned Bacillus belye MHB4 or its agent in at least one of the following (1)-(4): (1) Inhibit the growth of plant pathogens; (2) Prepare products for inhibiting plant pathogens; (3) Prevention and control of diseases caused by plant pathogens; (4) Prepare products for the prevention and control of diseases caused by plant pathogens.

[0014] In the above applications, the plant pathogens are one or more of the following: Fusarium oxysporum, Fusarium moniliforme, Fusarium flocculation, Fusarium rot, Fusarium MR5 (a specialized type of Fusarium moniliforme that causes continuous cropping obstacles in apples), Rhizoctonia solani, Alternaria alternata, Lactospora verrucosum, Pythium mellea, and Phytophthora spp.

[0015] In a fourth aspect, the present invention provides the above-mentioned Bacillus belye ( Bacillus velezensis The application of MHB4 or the inoculant in the following (1) or (2): (1) Promotes the germination of arbuscular mycorrhizal fungal spores; (2) Promote the colonization of arbuscular mycorrhizal fungi in continuously cropped soils or plant roots.

[0016] In a fifth aspect, the present invention provides a compound bacterial agent comprising the above-mentioned Bacillus belye ( Bacillus velezensis It is a compound of MHB4 inoculant and AMF inoculant of equal quality; The AMF is a genus of mycorrhizal fungi with accession number CGMCC NO.20744. (Paraglomus sp.) .

[0017] In some preferred embodiments of the present invention, the AMF inoculant is prepared by the following method: First, the AMF strain was inoculated into the roots of clover that had been growing for one week, and single-spore culture was carried out in a greenhouse for one month to further propagate the bacteria. The clover roots were then crushed to obtain powder containing AMF. The sterilized substrate and the powder containing AMF were mixed at a mass ratio of 3:1, and sterilized clover seeds were sown to expand the propagation under greenhouse conditions. After three months, the infection rate was tested. When the infection rate was greater than 50%, the above-ground parts were removed, and the clover roots and substrate were crushed together to obtain the AMF inoculum.

[0018] In some preferred embodiments of the present invention, Bacillus belesia ( Bacillus velezensis The inoculum for MHB4 is prepared by the following method: Bacillus berberis ( Bacillus velezensis MHB4 was inoculated into a mixture of cow manure compost and wheat straw and fermented aerobically at 35-40℃ for 6-8 days. The Bacillus belesi ( Bacillus velezensis The viable count in the MHB4 bacterial agent is greater than or equal to 10. 9 CFU g -1 .

[0019] In a sixth aspect of the present invention, the above-mentioned compound microbial agent is provided for use in at least one of the following (1)-(4): (1) Improve the photosynthetic capacity of apples in continuously cropped soil; (2) Increase the amount of AMF in soil where apples are continuously cropped; (3) Increase the AMF mycorrhizal infection rate of the underground root system of apple seedlings; (4) Alleviate the problem of continuous cropping of apples.

[0020] The beneficial effects of this invention are: This invention is the first to utilize fungi of the genus *Glomus*. Paraglomus A strain of Bacillus belyi was isolated from sp. SW1 spores. Bacillus velezensis MHB4 promotes spore germination of SW1 and enhances its colonization in apple-grown soil and roots; moreover, this Bacillus belye ( Bacillus velezensis MHB4 targets *Rhizopus argentea*, a specialized form of *Fusarium solani*, the main pathogen causing continuous cropping obstacles in apples around the Bohai Bay. Fusarium proliferatum MR5 exhibits significant antagonistic effects and also has antibacterial activity against pathogens such as Fusarium oxysporum, Fusarium solani, and Pythium moniliforme; thereby promoting the quality of SW1 seedlings and enhancing their resistance to continuous cropping obstacles, providing a theoretical basis for the biological control of apple continuous cropping obstacles. Attached Figure Description

[0021] Figure 1 Morphological image and Gram staining image of strain MHB4 on LB plate; In the figure, A is the morphological image of strain MHB4 and B is the Gram staining image.

[0022] Figure 2 : Developmental phylogenetic tree of strain MHB4 constructed based on 16S rDNA.

[0023] Figure 3 : Evolutionary tree of strain MHB4 constructed based on gyrA.

[0024] Figure 4 Effect of fermentation broth of strain MHB4 on spore germination of Paraglomus sp. SW1; "0" in the figure represents the control medium.

[0025] Figure 5 Figure: Strain MHB4 confronting 12 pathogens on PDA medium.

[0026] Figure 6 Phenotypic growth of Pingyi sweet tea seedlings after AMF and MHB4 inoculation.

[0027] Figure 7: Effects of AMF and MHB4 inoculation on biomass of Malus hupehensis seedlings; in the figure, (a) is the determination result of plant height; (b) is the determination result of stem diameter; (c) is the determination result of fresh weight; (d) is the determination result of dry weight.

[0028] Figure 8 : Effects of AMF and MHB4 inoculation on photosynthetic capacity of leaves of Malus hupehensis seedlings; in the figure, (a) is the determination result of intercellular CO2 concentration; (b) is the determination result of stomatal conductance; (c) is the determination result of net photosynthetic rate; (d) is the determination result of transpiration rate.

[0029] Figure 9 : Effects of AMF and MHB4 inoculation on root growth of Malus hupehensis seedlings; in the figure, (a) is the determination result of root length; (b) is the determination result of root surface area; (c) is the determination result of root volume; (d) is the determination result of root tip number.

[0030] Figure 10 : Effects of AMF and MHB4 inoculation on mycorrhizal infection rate of roots of Malus hupehensis seedlings.

[0031] Figure 11 : Effects of AMF and MHB4 inoculation on AMF quantity and glomalin-related protein in soil; in the figure, (a) is the determination result of spore density; (b) is the determination result of AMF quantity; (c) is the determination result of easily-extracted glomalin-related protein; (d) is the determination result of total-extracted glomalin-related protein.

[0032] Figure 12 : Real-time fluorescence quantitative statistics of four pathogenic Fusarium in soil after AMF and MHB4 inoculation treatment; in the figure, (a) is the determination result of Fusarium oxysporum; (b) is the determination result of Fusarium proliferatum; (c) is the determination result of Fusarium solani; (d) is the determination result of Fusarium moniliforme. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0034] As described previously, long-term continuous cropping will cause soil micro-ecological imbalance, deterioration of physical and chemical properties, accumulation of autotoxic substances and enrichment of pathogenic bacteria, which not only seriously inhibits the growth of apple plants and reduces the yield and quality of fruits, but also significantly affects the colonization process of AMF in apple root systems, resulting in that the colonization rate of AMF in apple continuous cropping soil is at a low level for a long time, and the application effect of AMF in the prevention and control of continuous cropping obstacles is restricted.

[0035] In view of this, the application discloses a method for improving the colonization rate of AMF in apple continuous cropping soil. ParaglomusA bacterium, named MHB4, was isolated from sp. SW1 spores. Based on morphological, physiological and biochemical identification and multigene phylogenetic analysis, this bacterium was identified as *Bacillus belyssae*. Bacillus velezensis The *Bacillus belyssus* MHB4 strain of this invention can promote AMF spore germination and AMF colonization in the root system. Under pot cultivation conditions, strain MHB4 of this invention can be applied synergistically with SW1 in apple continuous cropping soil to promote plant growth, increase root respiration rate, and enhance chlorophyll content in the above-ground leaves; simultaneously, it promotes AMF colonization in continuous cropping soil and plant roots, and can synergistically alleviate apple continuous cropping obstacles with SW1.

[0036] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein: The pathogen used in this invention is *Fusarium moniliforme* MR5, a species specializing in apple cropping disorders. Fusarium proliferatum f. sp. malus domestica The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22426, deposited on May 17, 2021, and is described in patent CN113881573B. The *Fusarium oxysporum*, *Fusarium moniliforme*, *Fusarium solani*, *Rhizoctonia solani*, *Alternaria alternata*, *Lactobacillus verrucosum*, *Pythium spp.*, *Phytophthora infestans*, *Penicillium brasiliensis*, and *Aspergillus flavus* used are from the Shandong Agricultural Microbiological Culture Collection Center of Shandong Agricultural University. These are all pathogens already known in the prior art, and the public can obtain these pathogens from the applicant to replicate this experiment. The AMF strain used in this invention is a *Gloydiomycorrhizal* species (…). Paraglomus sp. SW1, this strain was deposited on October 16, 2020 at the China General Microbiological Culture Collection Center, with the biological accession number: CGMCC NO.20744, and is recorded in patent CN113337405B.

[0037] The sterilization substrate used in the preparation of AMF inoculant is a common commercially available seedling substrate, which can be obtained through commercial channels.

[0038] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0039] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0040] Example 1: Isolation and identification of the strain 1. Isolation and purification of the strain: From Paraglomus The wet screening method was used to extract several spores of SW1 from the Bacillus subtilis SW1 inoculant. The soil particles attached to the surface of the mycelium were washed off with sterile PBS solution, and then the mycelium and spore suspension was washed several times with sterile water. The mycelium and spore suspension was then diluted to different concentrations and directly plated on LB solid medium. The plates were incubated at 37°C for 1-2 days, and when single colonies appeared, typical single colonies were selected and further purified by streaking method. The purified strain was stored in a 4°C refrigerator for future use.

[0041] 2. Screening of the strain: Plate confrontation tests were conducted between all the isolated bacteria and 12 pathogenic Fusarium species (Fusarium oxysporum, F. semitectum, F. proliferatum, F. solani, F. solani specialis apple replant disorder MR5, Rhizoctonia solani, Alternaria alternata, Myrothecium verrucaria, Pythium aphanidermatum, Phytophthora cactorum, Penicillium brasilianum, and Aspergillus flavus). The growth rate and final mycelial morphology of the pathogenic fungi were observed, and the strain that inhibited the most pathogenic fungi and had the best overall inhibition effect was selected as the subsequent experimental strain and named MHB4.

[0042] 3. Identification of the strain: (1) Morphological and physiological and biochemical identification: The isolated strain MHB4 was inoculated on LB solid medium and incubated at 37°C for 24 hours. When single colonies appeared, the colony morphology was observed. Gram staining was performed using crystal violet, and the morphology and size of the bacteria were observed under an optical microscope. Physiological and biochemical characteristics were analyzed according to the methods described in the "Berger's Bacterial Identification Manual (Second Edition)" and the "Common Bacterial System Identification Manual". Each index was tested three times, and the experiment was repeated twice.

[0043] After 24 hours of incubation on LB solid medium, the single colony of MHB4 strain was milky white, rough and opaque, with a typical velvety appearance. The colony edge was irregular, and the overall appearance was dry and matte, with a cloud-like spread on the agar medium. After static culture in LB liquid medium, a bacterial film was formed, and it was aerobic. Under a fluorescence microscope with 100x / 1.30 oil immersion lens, the spores were short rod-shaped, and the gram staining was positive. Figure 1 ).

[0044] Ten physiological and biochemical indicators of the MHB4 strain were detected, and the results are shown in Table 1.

[0045] Table 1: Physiological and biochemical characteristics of strain MHB4 Note: "+" represents positive reaction or available; "-" represents negative reaction. The physiological and biochemical characteristics of strain MHB4 showed that the strain was a facultative anaerobe with contact enzyme, amylase, cellulase and protease activity, could reduce nitrate, dissolve phosphorus and potassium, but citric acid utilization was negative.

[0046] (2) Molecular biology identification: The genomic DNA of strain MHB4 was extracted according to the manufacturer's instructions, and its 16S rRNA and gyr A sequence was amplified according to the method of Duan et al (2021), and the phylogenetic tree was constructed.

[0047] Phylogenetic analysis showed that the 16S rRNA gene sequence of strain MHB4 had 99% similarity with Bacillus velezensis OM535935.1 (E value: 0.0), Figure 2 ), gyr The A sequence was 100% homologous with Bacillus velezensis ON221478.1 (E value: 0.0). Figure 3

[0048] Combining morphological observation, physiological and biochemical characteristics and multi-gene tree results, strain MHB4 was finally identified as Bacillus velezensis (E value: 0.0). Bacillus velezensis The strain was preserved, and the preservation information is as follows: Reference biological material: MHB4 Classification name: Bacillus velezensis Bacillus velezensis Preservation agency: China General Microbiological Culture Collection Center Abbreviation of preservation agency: CGMCC Address: No. 3, Beichen West Road, Chaoyang District, Beijing Preservation date: January 26, 2026 Preservation center registration number: CGMCC No. 37520.

[0049] Example 2: Functional identification of strain MHB4 1. Detection of spore germination promoting ability: AMF inoculum propagation and treatment: First inoculate AMF strain on the roots of three-leaf clover grown for one week, and carry out single spore culture in the greenhouse for one month, further propagation, crush the roots of three-leaf clover to obtain AMF-containing powder. Mix the sterilized substrate with the AMF-containing powder at a mass ratio of 3:1, sow the sterilized three-leaf clover seeds, and expand the propagation under greenhouse and potting conditions. After three months, detect the infection rate, remove the aboveground part when the infection rate is greater than 50%, and crush the roots of three-leaf clover with the substrate to obtain AMF inoculum; dry in the shade, store at 4°C for subsequent experiments.​

[0050] Spore disinfection and preservation: The AMF inoculum obtained after propagation was separated by wet screening method. Each spore was washed with sterile water, then washed twice with sterilized water, sterilized with disinfectant (1% chloramine T + streptomycin 200 mg / L + gentamicin 100 mg / L), and stored at 4°C for standby use.

[0051] Preparation of fermentation supernatant of MHB4: The strain MHB4 was streaked on LB solid medium, and then single colonies were transferred to a flask containing 50 mL of LB liquid medium, incubated at 37°C, 150 rpm until OD 600 =0.8. Subsequently, the culture was transferred to a new LB liquid medium to a final concentration of 1% (volume fraction), shaken at 37°C, 150 rpm for 48 h to prepare the fermentation broth. The fermentation broth of MHB4 was centrifuged at 12000 rpm for 5 min, repeated twice, and filtered through a 0.22 μm filter membrane three times. The filtrate was collected to obtain the fermentation supernatant.

[0052] Preparation of medium and inoculation of SW1 spores: The fermentation supernatant of MHB4 was taken and added to agar medium at about 50°C at a volume ratio of 1%, 5% and 10% respectively, mixed quickly, immediately poured into a culture dish with a diameter of 9 cm, and cooled and solidified. At the same time, a control medium was prepared by replacing the fermentation supernatant with sterile water. Clean and sterilized SW1 spores were inoculated uniformly on the medium treated in different ways, 10 spores were inoculated on each medium, and the culture dish was wrapped with a sealing film for one turn and sealed into a 28°C incubator for culture. The spore germination was observed every 7 days, a total of 3 times, and the germination rate was calculated.

[0053] Spore germination rate = number of germinated spores / total number of inoculated spores × 100% The results showed that the appropriate amount of MHB4 fermentation supernatant could promote the spore germination of Paraglomus sp. SW1, among which the spore germination rate was the highest when the fermentation supernatant of MHB4 was added at a volume ratio of 5%, reaching 73.3% (P <0.05). Paraglomus Figure 4 ).

[0054] 2. Broad-spectrum antibacterial effect determination: On PDA medium, the strain MHB4 was inoculated with 12 kinds of pathogenic fungi (Fusarium oxysporum F. oxysporum , F. moniliforme F. moniliforme , F. proliferatum F. proliferatum , F. solani F. solani , F. solani special form for apple continuous cropping obstacle MR5, Rhizoctonia solani R. solani , Alternaria alternata A. alternata ​, Varicella vulgaris M. verrucaria Fruit and vegetable mold P. aphanidermatum Phytophthora P. cactorum Penicillium brasiliensis P. brasilianum Aspergillus flavus A. flavus A plate confrontation test was conducted, with the pathogen cultured alone serving as a control. The bacteria were cultured at 28°C for 7 days to determine the direct antagonistic effect of strain MHB4 against 12 pathogens.

[0055] Inhibition rate = [(Control colony diameter - Treated colony diameter) / Control colony diameter] × 100% The results are shown in Table 2 and Figure 5 As shown.

[0056] Table 2: Inhibitory effects of strain MHB4 against different pathogens The results showed that, except for *Penicillium brasiliensis* and *Aspergillus flavus*, the mycelial growth of the other 10 pathogenic fungi was significantly inhibited, with inhibition rates ranging from 29.41% to 95.23% (Table 2). At the interface between the two pathogenic fungi, the mycelia were thinner and could not expand towards strain MHB4, resulting in an inhibition zone. Figure 5 ).

[0057] Example 3: Pot Experiment 1. Experimental Design: The pot experiment was conducted from March to October 2024 at the National Apple Engineering Experimental Center and the State Key Laboratory of Crop Biology, Shandong Agricultural University. Soil samples were taken from multiple random locations in a 37-year-old apple orchard in Manzhuang, Daiyue District, Tai'an City, Shandong Province, China. Samples were taken from 80 cm away from the tree trunk and at depths of 10–40 cm, and mixed thoroughly. The soil texture was sandy loam.

[0058] The MHB4 inoculant used in potted plants is a solid inoculant, and its preparation method is as follows: Activated Bacillus belye (… Bacillus velezensis MHB4 was inoculated into a blank microbial fertilizer carrier (the blank microbial fertilizer carrier was composed of cow manure compost and wheat straw in a mass ratio of 1:2; purchased from Shandong Dezhou Chuangdi Microbial Resources Co., Ltd.), and fermented aerobicly at 37℃ for 7 days to prepare the MHB4 microbial agent. The viable count in the MHB4 microbial agent was 3.0 × 10⁻⁶. 9 CFU g -1 .

[0059] The AMF inoculant used for potted plants is the "AMF inoculant" in Example 2.

[0060] In March, Pingyi sweet tea seeds were stratified at around 4℃ for 40 days. A layer of substrate was first laid out and thoroughly watered. Some of the newly sprouted seeds were sown in uninoculated AMF (Amino Acid) seedling substrate to form non-inoculated seedlings; the other portion was sown in AMF-inoculated substrate (AMF:seedling substrate = 1:3 by weight) to form inoculated seedlings. Both types were managed normally. At the end of April, healthy, uniformly growing inoculated and non-inoculated seedlings were transplanted into continuously cropped soils with different treatments.

[0061] The pot experiment included the following four treatments: Non-acidic seedlings planted in continuous cropping soil (CK): As a blank control, the continuous cropping soil was not treated and non-acidic seedlings that were not inoculated with AMF were transplanted. Cultivating seedlings in continuous cropping soil (A): Add 80g of AMF inoculum to each pot of continuous cropping soil, mix well, and transplant seedlings inoculated with AMF inoculum. MHB4 inoculum treatment followed by non-acidic seedlings in continuous cropping soil (M4): Add 80 g of MHB4 inoculum to the continuous cropping soil, mix well, and transplant non-acidic seedlings that have not been inoculated with AMF inoculum. M4A: MHB4 and AMF inoculants were used to treat the seedlings in the continuous cropping soil. 80g of compound inoculant consisting of MHB4 and AMF in a 1:1 mass ratio was added to each pot of continuous cropping soil. The mixture was mixed evenly and then transplanted with SW1 seedlings.

[0062] Each treatment consisted of 20 pots, each containing 8 kg of continuously cropped soil. The amount of microbial agent used was 1% of the soil mass. Other management conditions remained consistent across all treatment groups. Samples were collected in August 2024.

[0063] 2. Measurement Indicators Biomass determination: Plant height and diameter at ground level were measured using a meter stick and a vernier caliper, respectively. The above-ground and underground roots of the plant were rinsed with tap water and dried. Fresh weight was measured on an electronic balance. The plant was dried to constant weight and then weighed again.

[0064] Photosynthetic capacity was measured using the CIRAS-3 portable photosynthetic fluorescence system (PP system, Hansatech, UK). P n ) and porosity ( G s The internal light intensity is 450 μmol·m. -2 ·s -1 The CO2 concentration was 360 μL·L -1 The blade chamber temperature is 25℃.

[0065] Root growth determination: The roots of seedlings treated differently were washed with clean water and placed gently on a transparent plastic sheet for scanning. The WinRHIZO software (2007 version) was used to analyze the length, surface area, volume, and root tip image of the seedling roots.

[0066] Mycorrhizal infection rate determination: Fresh root segments were selected, 10% potassium hydroxide solution was used for high-temperature decolorization, 2% hydrochloric acid was used for acidification, 0.05% curli blue lactic acid glycerol solution was used for high-temperature staining, lactic acid glycerol solution was used for decolorization overnight, and at least 100 root segments were observed under a microscope. The colonization rate of each root segment was evaluated by the number of mycorrhizal fungal structures in each segment and expressed as 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%. The mycorrhizal infection rate (%) was calculated as follows: Mycorrhizal infection rate = ∑ (0 x number of root segments + 10% x number of root segments + 20% x number of root segments + … + 100% x number of root segments) / total number of root segments.

[0067] Spore density determination: The soil wet screening method was used to calculate the spore density. The soil treated differently was passed through a 20-mesh and a 400-mesh sieve. The residue on the 400-mesh sieve was transferred to a centrifuge tube, the supernatant was removed by centrifugation, 60% sucrose solution was added, and the mixture was stirred evenly and centrifuged. The supernatant was transferred to a culture dish, and the soil spore density was calculated under a stereomicroscope.

[0068] Glomalin-related protein determination: According to the method of Li Yang (2023), the GRSP content was determined using the Coomassie Brilliant Blue method. Dry soil was passed through a 100-mesh sieve, sodium citrate solution was added, and the mixture was shaken well. The mixture was extracted at 121°C and 103 kPa for 60 min, and the supernatant was collected by centrifugation. The supernatant was taken and stained with Coomassie Brilliant Blue G-250, and the content of easily extractable glomalin-related protein was determined by UV spectrophotometry at 595 nm. The above operation was repeated until the supernatant was clear, and finally all the supernatants were mixed evenly. The mixed supernatant was taken and stained with Coomassie Brilliant Blue G-250, and the total extractable glomalin-related soil protein was determined by UV spectrophotometry at 595 nm.

[0069] Quantitative analysis of AMF and Fusarium in soil: The methods of Wang Mei (2022) and Wang Gongshuai (2018) were used to determine the gene copy number of arbuscular mycorrhizal fungi, Fusarium oxysporum, F. solani, F. proliferatum, and F. equiseti in the pot experiment soil.

[0070] Data processing: All data statistical analysis was performed using IBM SPSS 26.0 (IBM SPSS Statistics, IBM Corporation, Armonk, NY, United States). Different lowercase letters represent significant differences between treatments (one-way ANOVA, P < 0.05) according to Duncan's multiple range test. Pictures were drawn using GraphPad Prism 9.0. p <0.05), according to Duncan's multiple range test. Pictures were drawn using GraphPad Prism 9.0.

[0071] 3、Experimental results (1) Effects of AMF and MHB4 inoculation on the biomass of Pingyin sweet tea seedlings From Figure 6 and Figure 7 it can be seen that under the condition of continuous cropping, the MHB4 and AMF complex treatment (M4A) has the most significant promoting effect on apple seedlings, and the plant height, stem diameter, fresh weight and dry weight are increased by 59.02%, 55.08%, 96.15% and 44.08% compared with CK treatment; Compared with AMF inoculation (A) and MHB4 inoculation (M4) alone, it has significant synergistic effect.

[0072] (2) Effects of AMF and MHB4 inoculation on the photosynthetic capacity of Pingyin sweet tea seedlings From Figure 8 it can be seen that different treatments have different effects on the photosynthetic parameters of Pingyin sweet tea seedlings. Among them, the intercellular CO2 concentration, stomatal conductance, net photosynthetic rate and transpiration rate of apple seedling leaves after M4A treatment are increased to different degrees, and compared with CK, the intercellular CO2 concentration, stomatal conductance, net photosynthetic rate and transpiration rate after M4A treatment are increased by 8.1%, 46.8%, 40.2% and 28.2%, respectively.

[0073] (3) Effects of AMF and MHB4 inoculation on the root growth of Pingyin sweet tea seedlings From Figure 9 it can be seen that under the condition of continuous cropping, M4A treatment can significantly promote the development of apple seedling roots, and the growth status of the roots of this treatment is significantly better than that of A and M4 single treatment. Compared with CK, the root length, root surface area, root volume and root tip number of seedlings after M4A treatment are increased by 52.92%, 69.31%, 66.51% and 29.48%, respectively.

[0074] (4) Effects of AMF and MHB4 inoculation on the mycorrhizal infection rate of Pingyin sweet tea seedlings From Figure 10As can be seen, compared with the continuous cropping soil control (CK), the mycorrhizal infection rate of the roots of the continuously cropped Pingyi sweet tea seedlings treated with MHB4 alone increased by 25.33%, and the mycorrhizal infection rate of the AMF treatment alone increased by 30.89%; while the mycorrhizal infection rate of the combined treatment of MHB4 and AMF (M4A) increased by 57.48%. Therefore, combining MHB4 strain with AMF can synergistically improve the mycorrhizal infection effect.

[0075] (5) Effects of AMF and MHB4 inoculation on AMF levels and globulin-associated proteins in soil Depend on Figure 11 It can be seen that, compared with the continuous cropping soil control (CK), the application of MHB4 alone (M4), AMF alone (A), and both combined (M4A) all increased the AMF spore density and total AMF in the continuously cropped soil to varying degrees, with the M4A treatment showing the most significant effect. Regarding the content of glomerulonemycin-related proteins in the continuously cropped soil, there was no significant difference between the M4 treatment and the continuous cropping control, but the M4A treatment increased the readily extractable glomerulonemycin-related protein and the total extractable glomerulonemycin-related protein in the continuously cropped soil by 65.65% and 39.56%, respectively.

[0076] (6) Effects of AMF and MHB4 inoculation treatments on the number of four pathogenic Fusarium species in the soil The antagonistic effects of different treatments on four Fusarium species in soil—*Fusarium oxysporum*, *Fusarium chrysogenum*, *Fusarium solani*, and *Fusarium moniliforme*—were investigated using real-time quantitative PCR. Figure 12 It can be seen that, compared with the continuously cropped soil control (CK), the combined application of AMF and MHB4 (M4A) significantly reduced the copy number of the four Fusarium species in the continuously cropped soil. The M4A treatment reduced the copy numbers of *Fusarium oxysporum*, *Fusarium solani*, *Fusarium solani*, and *Fusarium moniliforme* by 63.7%, 88.1%, 59.2%, and 68.8%, respectively, compared to the control (CK). Meanwhile, the individual application of MHB4 (M4) and AMF (A) also inhibited the growth of the four pathogenic Fusarium species to varying degrees. Compared with the control (CK), the M4 treatment reduced the copy numbers of *Fusarium oxysporum*, *Fusarium solani*, *Fusarium solani*, and *Fusarium moniliforme* by 23.3%, 20.1%, 25.17%, and 26.5%, respectively, while the A treatment reduced the copy numbers of *Fusarium solani*, *Fusarium solani*, and *Fusarium moniliforme* by 14.9%, 32.4%, and 39.9%, respectively.

[0077] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A strain of Bacillus velezensis (B. velezensis) MHB4, characterized in that, Bacillus velezensis ) MHB4, characterized in that, with the accession number CGMCC No. 37520.

2. An inoculant characterized in that, The bacterial agent contains the Bacillus velezensis MHB4 in claim 1.

3. The bacterial agent of claim 2, wherein In the bacterial agent, the Bacillus velezensis MHB4 exists in the form of cultured live bacteria, fermentation broth or bacterial suspension.

4. The use of the Bacillus velezensis MHB4 in claim 1 or the bacterial agent in claim 2 or 3 in at least one of the following (1) or (2): (1) promoting spore germination of arbuscular mycorrhizal fungi; (2) promoting colonization of arbuscular mycorrhizal fungi in continuous cropping soil or plant roots.

5. The use of the Bacillus velezensis MHB4 in claim 1 or the bacterial agent in claim 2 or 3 in at least one of the following (1)-(4): (1) inhibiting the growth of plant pathogenic fungi; (2) preparing a product for inhibiting plant pathogenic fungi; (3) preventing diseases caused by plant pathogenic fungi; (4) preparing a product for preventing diseases caused by plant pathogenic fungi.

6. Use according to claim 5, characterized in that, The plant pathogenic fungi are one or more of the following: Fusarium oxysporum, F. semitectum, F. proliferatum, F. solani, F. solani speciality form of apple replant disorder MR5, Rhizoctonia solani, Alternaria alternata, Mycosphaerella, Pythium aphanidermatum and Phytophthora cactorum.

7. A complex microbial agent, characterized in that, Bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) MHB4 and AMF, etc. The AMF is Glomus versiforme with the preservation number CGMCC NO. 20744 (Paraglomus sp.) . 8.The complex bacterial agent according to claim 7, characterized in that, Bacillus velezensis (Bacillus velezensis) Bacillus velezensis The bacterial agent of MHB4 is prepared by the following method: Bacillus velezensis (Bacillus velezensis) Bacillus velezensis MHB4 was inoculated into the mixture of cow manure compost and wheat straw, and aerobic fermentation was carried out at 35-40℃ for 6-8d; Bacillus velezensis (Bacillus velezensis) Bacillus velezensis The viable cell count in the bacterial agent of MHB4 is greater than or equal to 10 9 CFU / g -1 . 9.The complex bacterial agent according to claim 7, characterized in that, The AMF bacterial agent is prepared by the following method: First, inoculate the AMF strain into the roots of clover grown for one week, and single-spore culture for one month, then further propagate, crush the clover roots, and obtain the AMF-containing powder; mix the sterilized substrate with the AMF-containing powder at a mass ratio of 3:1, sow the sterilized clover seeds, and propagate in a greenhouse; after three months, detect the infection rate, remove the aboveground part when the infection rate is greater than 50%, and crush the clover roots and the substrate together to obtain the AMF bacterial agent.

10. The use of the complex bacterial agent in claim 7 in at least one of the following (1)-(4): (1) improving the photosynthetic capacity of apple in continuous cropping soil; (2) increasing the number of AMF in apple continuous cropping soil; (3) increasing the AMF mycorrhizal infection rate of the underground roots of apple seedlings; (4) alleviating apple replant disorder.

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