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

Abstract

The present application discloses a bacillus velezensis, a compound microbial inoculant and application of the bacillus velezensis and the compound microbial inoculant in promoting colonization of arbuscular mycorrhizal fungi, and belongs to the technical field of agricultural microorganisms. Paraglomus The present application isolates a bacillus velezensis MHB4 from Glomus sp. spores, which has a dual function: one is to promote spore germination of sp.SW1 and promote colonization of the sp.SW1 in plant roots and soil; and the other is to antagonize 10 kinds of plant pathogenic fungi. The MHB4 and AMF are applied to apple continuous cropping soil, which can increase the number of AMF spores in the continuous cropping soil, promote colonization of AMF in the roots of continuous cropping plants, promote plant growth, improve photosynthesis of the plants, has excellent effect in reducing apple tree continuous cropping obstacles, and provides a theoretical basis for biological control of apple 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:

[0006] 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 .

[0007] The Bacillus belyssus MHB4 of the present invention has the following characteristics:

[0008] (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.

[0009] (2) It has broad-spectrum antibacterial activity against Fusarium moniliformes (Fusarium moniliformes) Fusarium proliferatum Fusarium oxysporum ( Fusarium oxysporumFusarium 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 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.

[0010] (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.

[0011] A second aspect of the present invention provides a microbial agent containing the aforementioned Bacillus belye ( Bacillus velezensis )MHB4.

[0012] 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.

[0013] In some preferred embodiments of the present invention, the fermentation broth is prepared by the following method:

[0014] Bacillus berberis ( Bacillus velezensis MHB4 was inoculated into LB liquid medium and fermented at 35-40℃ for 20-30 hours.

[0015] In some preferred embodiments of the present invention, the bacterial suspension is prepared by the following method:

[0016] 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.

[0017] 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.

[0018] 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):

[0019] (1) Inhibit the growth of plant pathogens;

[0020] (2) Prepare products for inhibiting plant pathogens;

[0021] (3) Prevention and control of diseases caused by plant pathogens;

[0022] (4) Prepare products for the prevention and control of diseases caused by plant pathogens.

[0023] In the above applications, the plant pathogens are one or more of the following: Fusarium oxysporum, Fusarium moniliforme, Fusarium solani, Fusarium rotundum, Fusarium moniliforme MR5 (a specialized type of Fusarium moniliforme that causes continuous cropping obstacles in apples), Rhizoctonia solani, Alternaria alternata, Lactospora verrucosum, Pythium tumefaciens, and Phytophthora spp.

[0024] 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):

[0025] (1) Promotes the germination of arbuscular mycorrhizal fungal spores;

[0026] (2) Promote the colonization of arbuscular mycorrhizal fungi in continuously cropped soils or plant roots.

[0027] 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;

[0028] The AMF is a genus of mycorrhizal fungi with accession number CGMCC NO.20744. (Paraglomus sp.) .

[0029] In some preferred embodiments of the present invention, the AMF inoculant is prepared by the following method:

[0030] 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.

[0031] In some preferred embodiments of the present invention, Bacillus belesia ( Bacillus velezensis The inoculum for MHB4 is prepared by the following method:

[0032] 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.

[0033] The Bacillus belesi ( Bacillus velezensis The viable count in the MHB4 bacterial agent is greater than or equal to 10. 9 CFU g -1 .

[0034] 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):

[0035] (1) Improve the photosynthetic capacity of apples in continuously cropped soil;

[0036] (2) Increase the amount of AMF in soil where apples are continuously cropped;

[0037] (3) Increase the AMF mycorrhizal infection rate of the underground root system of apple seedlings;

[0038] (4) Alleviate the problem of continuous cropping of apples.

[0039] The beneficial effects of this invention are:

[0040] 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

[0041] 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.

[0042] Figure 2 : Developmental phylogenetic tree of strain MHB4 constructed based on 16S rDNA.

[0043] Figure 3 : Evolutionary tree of strain MHB4 constructed based on gyrA.

[0044] Figure 4Effect of fermentation broth of strain MHB4 on spore germination of Paraglomus sp. SW1; "0" in the figure represents the control medium.

[0045] Figure 5 Figure: Strain MHB4 confronting 12 pathogens on PDA medium.

[0046] Figure 6 Phenotypic growth of Pingyi sweet tea seedlings after AMF and MHB4 inoculation.

[0047] Figure 7 Effects of AMF and MHB4 inoculation on the biomass of Pingyi sweet tea seedlings; In the figure, (a) shows the plant height measurement results; (b) shows the stem diameter measurement results; (c) shows the fresh weight measurement results; and (d) shows the dry weight measurement results.

[0048] Figure 8 Effects of AMF and MHB4 inoculation on photosynthetic capacity of leaves of Pingyi sweet tea seedlings; In the figure, (a) shows the results of intercellular CO2 concentration measurement; (b) shows the results of stomatal conductance measurement; (c) shows the results of net photosynthetic rate measurement; and (d) shows the results of transpiration rate measurement.

[0049] Figure 9 Effects of AMF and MHB4 inoculation on root growth of Pingyi sweet tea seedlings; In the figure, (a) shows the results of root length measurement; (b) shows the results of root surface area measurement; (c) shows the results of root volume measurement; and (d) shows the results of root tip number measurement.

[0050] Figure 10 Effects of AMF and MHB4 inoculation on mycorrhizal infection rate of Pingyi sweet tea seedlings.

[0051] Figure 11 Effects of AMF and MHB4 inoculation on AMF quantity and glomerulonephrine-associated protein in soil; In the figure, (a) shows the spore density measurement results; (b) shows the AMF quantity measurement results; (c) shows the easily extractable glomerulonephrine-associated protein measurement results; (d) shows the total extracted glomerulonephrine-associated protein measurement results.

[0052] Figure 12 Real-time fluorescence quantitative analysis of four pathogenic Fusarium species in soil after AMF and MHB4 inoculation treatment; In the figure, (a) is the result of Fusarium oxysporum detection; (b) is the result of Fusarium effusum detection; (c) is the result of Fusarium solani detection; (d) is the result of Fusarium moniliforme detection. Detailed Implementation

[0053] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, 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 pertains.

[0054] As mentioned earlier, long-term continuous cropping leads to soil microecological imbalance, deterioration of physical and chemical properties, accumulation of autotoxic substances and enrichment of pathogens. This not only severely inhibits the growth of apple plants and reduces fruit yield and quality, but also significantly affects the colonization process of AMF in apple roots, resulting in a long-term low colonization rate of AMF in apple-continuously cropped soil, which restricts the application effect of AMF in the control of continuous cropping obstacles.

[0055] In view of this, the present invention utilizes fungi of the genus *Glomus*. Paraglomus A 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.

[0056] 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:

[0057] 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.

[0058] The sterilization substrate used in the preparation of AMF inoculant is a common commercially available seedling substrate, which can be obtained through commercial channels.

[0059] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0060] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0061] Example 1: Isolation and Identification of Strains

[0062] 1. Isolation and purification of the strain:

[0063] from Paraglomus Several SW1 spores were extracted using the wet sieving method from the sp. SW1 inoculum. The surface of the mycelium was rinsed with sterile PBS solution to remove small soil particles, followed by multiple rinses with sterile water. The mycelium and spores were then suspended in the solution. Different gradient solutions were diluted and directly spread onto LB solid medium. The medium was incubated at 37°C for 1-2 days. After single colonies appeared, typical single colonies were selected and further purified using the streak plating method. The purified strain was stored at 4°C for later use.

[0064] 2. Screening of strains:

[0065] All isolated bacteria were subjected to plate confrontation experiments with 12 pathogenic Fusarium species (Fusarium oxysporum, Fusarium moniliforme, Fusarium flocculation, Fusarium rot, Fusarium moniliforme MR5, Rhizoctonia solani, Alternaria alternata, Lactospora verrucosum, Pythium mellea, Phytophthora hygroscopicum, Penicillium brasiliensis, and Aspergillus flavus). By statistically analyzing the growth rate and final hyphal morphology of the pathogens, the strain that inhibited the most pathogens and had the best overall inhibitory effect was selected as the subsequent experimental strain and named MHB4.

[0066] 3. Identification of the strain:

[0067] (1) Morphological and physiological / biochemical identification:

[0068] The isolated strain MHB4 was streaked onto LB agar and incubated at 37°C for 24 hours. Single colonies were observed for morphological characteristics. Gram staining was performed using crystal violet, and the morphology and size of the bacteria were observed using an optical microscope. Physiological and biochemical characteristics were analyzed according to the methods described in Bergey's Manual of Bacteriological Identification (Second Edition) and the Manual of Systematic Identification of Common Bacteria. Each indicator was tested three times, and the experiment was repeated twice.

[0069] After culturing MHB4 strain on LB solid medium for 24 hours, single colonies were milky white, with a rough, opaque surface, exhibiting a typical velvety appearance, irregular colony edges, and an overall dry, dull appearance. On agar medium, they diffused in a cloud-like pattern. In LB liquid medium, after static incubation, a biofilm formed, indicating aerobic activity. Under a fluorescence microscope (100× / 1.30 oil immersion), the spores appeared as short rods and were Gram-positive. Figure 1 ).

[0070] Ten physiological and biochemical indicators of strain MHB4 were tested, and the results are shown in Table 1.

[0071] Table 1: Physiological and biochemical characteristics of strain MHB4

[0072]

[0073] Note: "+" indicates a positive reaction or is usable; "-" indicates a negative reaction.

[0074] Physiological and biochemical characteristics of strain MHB4 indicate that the strain is a facultative anaerobe with activities of catalase, amylase, cellulase and protease. It can reduce nitrate and dissolve phosphorus and potassium, but its citric acid utilization is negative.

[0075] (2) Molecular biological identification:

[0076] Genomic DNA of strain MHB4 was extracted according to the manufacturer's instructions, and its 16S rRNA was amplified according to the method of Duan et al. (2021). gyr Sequence A was used to construct a phylogenetic tree.

[0077] Phylogenetic analysis showed that the 16S rRNA gene sequence of strain MHB4 was 99% similar to that of Bacillus belyssioides OM535935.1. Figure 2 ), gyr Sequence A is 100% homologous to Bacillus belysium ON221478.1. Figure 3 ).

[0078] Based on comprehensive morphological observation, physiological and biochemical characteristics, and multi-gene phylogenetic analysis, strain MHB4 was ultimately identified as Bacillus belyssus (B. belyssus). Bacillus velezensisThe strain was biopreserved, and the preservation information is as follows:

[0079] Reference biological material: MHB4

[0080] Classification and nomenclature: Bacillus belesiensis Bacillus velezensis

[0081] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0082] Collection institution abbreviation: CGMCC

[0083] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0084] Deposit date: January 26, 2026

[0085] Registration number at the Preservation Center: CGMCC No. 37520.

[0086] Example 2: Functional identification of strain MHB4

[0087] 1. Test of ability to promote spore germination:

[0088] Propagation and treatment of AMF inoculant: First, AMF strains were inoculated into the roots of clover plants that had grown for one week. Single-spore culture was then carried out in a greenhouse for one month for further propagation. The clover roots were then crushed to obtain AMF-containing powder. Sterilized substrate and the AMF-containing powder were mixed at a mass ratio of 3:1. Sterilized clover seeds were sown, and propagation was expanded under greenhouse and pot conditions. After three months, the infection rate was tested. If 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 inoculant for subsequent experiments. The inoculant was air-dried in a cool place and stored at 4℃.

[0089] Spore disinfection and preservation: AMF bacterial agent obtained after separation and propagation by wet sieving was used. Each spore was cleaned with sterile water, rinsed twice with sterile water, sterilized with disinfectant (1% chloramine T + streptomycin 200 mg / L + gentamicin 100 mg / L), and stored at 4℃ for later use.

[0090] Preparation of fermentation supernatant for MHB4: Strawberry strain MHB4 was streaked onto LB solid medium, and then single colonies were transferred to Erlenmeyer flasks containing 50 mL of LB liquid medium and incubated at 37°C and 150 rpm until OD500 was reached. 600=0.8. Subsequently, the culture was transferred to new LB liquid medium to a final concentration of 1% (volume fraction), and shaken at 37°C and 150 rpm for 48 h to prepare the fermentation broth. The MHB4 fermentation broth was centrifuged at 12000 rpm for 5 min, and the operation was repeated twice. The broth was filtered through a 0.22 μm filter membrane three times, and the filtrate was collected to obtain the fermentation supernatant.

[0091] Culture medium preparation and SW1 spore inoculation: Fermentation supernatant from MHB4 was added to agar medium at approximately 50°C at volume ratios of 1%, 5%, and 10%, respectively. The mixture was quickly mixed and immediately poured into 9 cm diameter petri dishes, allowing it to cool and solidify. Simultaneously, a control culture medium was prepared using sterile water instead of the fermentation supernatant. Clean and sterilized SW1 spores were evenly inoculated onto the different treated culture media, with 10 spores inoculated onto each medium. The culture dishes were sealed with sealing film wrapped around them once and placed in a 28°C incubator. Spore germination was observed every 7 days for a total of 3 observations, and the germination rate was calculated.

[0092] Spore germination rate = (Number of germinated spores / Total number of inoculated spores) × 100%

[0093] The results showed that adding an appropriate amount of MHB4 to the fermentation supernatant could promote fermentation. Paraglomus The spore germination of sp. SW1, wherein 5% (v / v) of fermentation supernatant of MHB4 was added, Paraglomus sp. SW1 had the highest spore germination rate, reaching 73.3% ( Figure 4 ).

[0094] 2. Determination of broad-spectrum antibacterial effect:

[0095] On PDA medium, strain MHB4 was separately mixed with 12 pathogens (Fusarium oxysporum). F. oxysporum Fusarium moniliforme F. moniliforme Fusarium latifolium F. proliferatum Fusarium solani F. solani Apple continuous cropping obstacle specialized Fusarium MR5, Rhizoctonia solani R. solani Alternaria 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.

[0096] Inhibition rate = [(Control colony diameter - Treated colony diameter) / Control colony diameter] × 100%

[0097] The results are shown in Table 2 and Figure 5 As shown.

[0098] Table 2: Inhibitory effects of strain MHB4 against different pathogens

[0099]

[0100] 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 ).

[0101] Example 3: Pot Experiment

[0102] 1. Experimental Design:

[0103] 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.

[0104] 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 onto 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 .

[0105] The AMF inoculant used for potted plants is the "AMF inoculant" in Example 2.

[0106] 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.

[0107] The pot experiment included the following four treatments:

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 2. Measurement Indicators

[0114] 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.

[0115] 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℃.

[0116] Root growth measurement: After washing the roots of seedlings under different treatments with clean water, they were gently placed on a transparent plastic sheet for scanning. WinRHIZO software (2007 version) was used to analyze the root length, surface area, volume and root tip images of the seedlings.

[0117] Mycorrhizal infection rate determination: Fresh root segments were selected, sterilized and decolorized by high-temperature boiling in 10% potassium hydroxide solution, acidified with 2% hydrochloric acid, stained with 0.05% tribenzyl blue lactic acid glycerol solution at high-temperature temperature, decolorized overnight with lactic acid glycerol solution, and observed under a microscope at least 100 root segments. The colonization rate of each root segment was assessed by the number of mycorrhizal fungal structures in each segment and expressed as 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%. The mycorrhizal infection rate (%) was calculated as follows:

[0118] Mycorrhizal infection rate = ∑ (0 × number of root segments + 10% × number of root segments + 20% × number of root segments + ... + 100% × number of root segments) / total number of root segments.

[0119] Spore density determination: Spore density was calculated using the wet soil sieving method. Soil samples from different treatments were sieved through 20-mesh and 400-mesh sieves. All residues on the 400-mesh sieve were transferred to centrifuge tubes, centrifuged, and the supernatant was removed. A 60% sucrose solution was added, stirred thoroughly, and centrifuged again. The supernatant was transferred to a petri dish, and the soil spore density was calculated under a stereomicroscope.

[0120] Determination of globulin-related proteins: Following the method of Li Yang (2023), the GRSP content was determined using the Coomassie Brilliant Blue method. Air-dried soil that had passed through a 100-mesh sieve was mixed with sodium citrate solution and thoroughly shaken. Extraction was performed at 121℃ and 103 kPa for 60 min, followed by centrifugation to collect the supernatant. The supernatant was collected, and Coomassie Brilliant Blue G-250 staining was added. The content of easily extractable globulin-related proteins was determined by colorimetry at 595 nm using a UV spectrophotometer. The above operation was repeated until the supernatant became clear. Finally, all supernatants were mixed thoroughly. The mixed supernatant was collected, and Coomassie Brilliant Blue G-250 staining was added. The total extracted globulin-related soil proteins were determined by colorimetry at 595 nm using a UV spectrophotometer.

[0121] Quantitative analysis of soil AMF and Fusarium: The gene copy numbers of arbuscular mycorrhizal fungi (AMF), Fusarium oxysporum, Fusarium solani, Fusarium moniliforme, and Fusarium moniliforme in the pot experiment soil were determined using the methods described in "Wang Mei. Isolation, Identification and Mechanism of Arbuscular Mycorrhizal Fungi in Controlling Apple Continuous Cropping Obstacles [D]. Doctoral Dissertation, Shandong Agricultural University, 2022." and "Wang Gongshuai. Analysis of Fungal Community Structure in Continuous Cropping Soil in the Bohai Rim and Study on the Reduction of Apple Continuous Cropping Obstacles by Intercropping with Onion [D]. Doctoral Dissertation, Shandong Agricultural University, 2018."

[0122] Data processing: All statistical analyses were performed using IBM SPSS 26.0 (IBM SPSS Statistics, IBM Corporation, Armonk, NY, United States). Different lowercase letters indicate significant differences between treatments (one-way ANOVA). p <0.05), based on Duncan's multiple range test. GraphPad Prism 9.0 plots the image.

[0123] 3. Experimental Results

[0124] (1) Effects of AMF and MHB4 inoculation on the biomass of Pingyi sweet tea seedlings

[0125] Depend on Figure 6 and Figure 7 It can be seen that among all treatments under continuous cropping conditions, the combined treatment of MHB4 and AMF (M4A) had the most significant promoting effect on apple seedlings. Its plant height, stem diameter, fresh weight and dry weight increased by 59.02%, 55.08%, 96.15% and 44.08% respectively compared with the CK treatment. Compared with the AMF inoculant treatment alone (A) and the MHB4 inoculant treatment alone (M4), it had a significant synergistic effect.

[0126] (2) Effects of AMF and MHB4 inoculation on photosynthetic capacity of leaves of Pingyi sweet tea seedlings

[0127] Depend on Figure 8 It can be seen that different treatments have different effects on the photosynthetic parameters of Pingyi sweet tea seedlings. Among them, the M4A treatment increased the intercellular CO2 concentration, stomatal conductance, net photosynthetic rate, and transpiration rate of apple seedling leaves to varying degrees. Compared with the control (CK), the M4A treatment increased the intercellular CO2 concentration, stomatal conductance, net photosynthetic rate, and transpiration rate by 8.1%, 46.8%, 40.2%, and 28.2%, respectively.

[0128] (3) Effects of AMF and MHB4 inoculation on root growth of Pingyi sweet tea seedlings

[0129] from Figure 9 It can be seen that under continuous cropping conditions, the M4A treatment significantly promotes the root development of apple seedlings, and the root growth of this treatment is significantly better than that of the A and M4 treatments alone. Compared with the control (CK), the root length, root surface area, root volume, and number of root tips of seedlings treated with M4A increased by 52.92%, 69.31%, 66.51%, and 29.48%, respectively.

[0130] (4) Effects of AMF and MHB4 inoculation on the mycorrhizal infection rate of Pingyi sweet tea seedlings

[0131] Depend on 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.

[0132] (5) Effects of AMF and MHB4 inoculation on AMF levels and globulin-associated proteins in soil

[0133] 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.

[0134] (6) Effects of AMF and MHB4 inoculation treatments on the number of four pathogenic Fusarium species in the soil

[0135] 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.

[0136] 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 belye ( Bacillus velezensis MHB4, characterized in that, Its accession number is CGMCC No.37520.

2. A microbial agent, characterized in that, The bacterial agent contains Bacillus belye MHB4 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, In the bacterial agent, Bacillus belyssus MHB4 exists in the form of cultured live bacteria.

4. The microbial agent according to claim 2, characterized in that, In the bacterial agent, Bacillus berreatus MHB4 exists in the form of fermentation broth or bacterial suspension.

5. The application of the fermentation supernatant of Bacillus belye MHB4 as described in claim 1 in promoting the germination of arbuscular mycorrhizal fungal spores.

6. The application of Bacillus belye MHB4 as described in claim 1 or the fungal agent as described in any one of claims 2-4 in promoting the colonization of arbuscular mycorrhizal fungi in continuously cropped soils or plant roots.

7. The use of Bacillus belyssus MHB4 according to claim 1 or the bacterial agent according to any one of claims 2-4 in at least one of the following (1)-(4): (1) Inhibits 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; The plant pathogens mentioned 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, Pythium spp., and Phytophthora.

8. A compound microbial agent, characterized in that, Bacillus belesiensis as described in claim 1 ( 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.) .

9. The compound microbial agent according to claim 8, characterized in that, Bacillus belesiensis ( 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. Bacillus belesiensis ( Bacillus velezensis The viable count in the MHB4 inoculant is greater than or equal to 10. 9 CFUg -1 .

10. The compound microbial agent according to claim 8, characterized in that, The AMF bacterial agent is prepared by the following method: First, the AMF strain was inoculated into the roots of clover that had been growing for one week. Single spores were cultured for one month for further propagation. The clover roots were then crushed to obtain powder containing AMF. The sterilized substrate and the AMF-containing powder were mixed at a mass ratio of 3:

1. Sterilized clover seeds were then sown and propagated under greenhouse conditions. After three months, the infection rate was tested. Once 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.

11. The use of the compound microbial agent according to claim 8 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.

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