Method for preventing and controlling apple continuous cropping obstacles by long-villous vicia sativa driven mycorrhizal symbiosis

Abstract

The application discloses a method for preventing and controlling apple continuous cropping obstacles by long pubescent vetch driving mycorrhizal symbiosis, and relates to the technical field of fruit tree continuous cropping obstacle prevention and control. The application realizes prevention and control of apple continuous cropping obstacles by applying AMF fungicide to apple continuous cropping soil and uniformly sowing long pubescent vetch seeds on the roots of apple plants. The application proves through tests that application of AMF fungicide and mixed cultivation of long pubescent vetch can improve the biomass of Pingyitiancha seedlings, the soil nutrient content and the soil enzyme activity, increase the number of soil cultivable microorganisms, reduce the gene copy number of soil fusarium, increase the globosin related protein content, the spore density and the mycorrhizal infection rate, improve the continuous cropping soil environment, reduce the root system infection of soil harmful fungi and the harm of toxins secreted by the soil harmful fungi to plants, and improve the colonization ability of AMF in soil, so that the apple continuous cropping obstacles are effectively prevented and controlled.

Description

Technical Field

[0001] This invention relates to the field of fruit tree continuous cropping obstacle control technology, and in particular to a method for controlling apple continuous cropping obstacle control by driving mycorrhizal symbiosis with Vitex acutissima. Background Technology

[0002] With the continuous development of the apple industry over the years, about 70% of apple orchards in my country have been established for more than 20 years, and most of the fruit trees have entered their aging stage, urgently requiring renewal and reconstruction. However, due to limited land resources and restrictions imposed by farmland protection policies, the renewal and reconstruction of old orchards can only be carried out in situ, which easily leads to apple continuous cropping obstacles. Apple continuous cropping obstacles are mainly manifested in slow plant growth, root damage, poor fruit quality, low yield, and even tree death, causing serious economic losses to fruit farmers and severely hindering the healthy and sustainable development of the apple industry.

[0003] Currently, methods to alleviate apple continuous cropping obstacles include intercropping, soil disinfection, selection of resistant rootstocks, and biological control. Intercropping, while increasing crop yield, improving soil nutrient utilization, and mitigating the promoting effect of autotoxins on pathogen proliferation, thus alleviating continuous cropping obstacles, is time-consuming and difficult to meet the production needs of simplified orchards requiring rapid renewal. Soil disinfection refers to killing or inhibiting pathogenic microorganisms in the soil through physical or chemical means to improve soil health and reduce the occurrence of continuous cropping obstacles; however, physical disinfection suffers from high costs, limited disinfection depth, and unstable effectiveness. Chemical fumigants, while fast-acting and broad-spectrum, easily cause ecological pollution and pesticide residues. Selecting resistant rootstocks can effectively alleviate apple continuous cropping obstacles, but it has drawbacks such as long breeding cycles, high costs, and limited range of resistance adaptations. Biological control refers to using beneficial bacteria to inhibit the number of pathogens in the soil or interfere with pathogen infection of host plants. Compared to intercropping, soil disinfection, and the breeding of resistant rootstocks, biological control has the advantages of being green and safe, eco-friendly, low-cost, and easy to integrate with existing orchard management practices.

[0004] Arbuscular mycorrhizal fungi ( Arbuscular mycorrhizal fungi AMF (Amyotrophic Lateral Fiber Fungus) is an important group of beneficial fungi in soil. Previous studies have shown that inoculating apple-continuously cropped soil with AMF can increase the activity of root antioxidant enzymes and the content of resistance substances in M9T337 seedlings, significantly promoting seedling growth (Wang). et al (2021). However, 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 apple plant growth and reduces fruit yield and quality, but also affects the colonization process of AMF in apple roots, resulting in a long-term low colonization rate of AMF in apple continuous cropping soil, thus limiting the application of AMF in the control of apple continuous cropping obstacles.

[0005] long-haired wild pea ( Vicia villosaAs a legume, *Vicia crassifolia* possesses advantages such as strong nitrogen fixation capacity, large biomass, good coverage, strong adaptability, and significant soil improvement effects. CN105580687A discloses that sowing *Vicia crassifolia* between rows of apple trees can increase the organic matter content, nitrogen, phosphorus, and potassium content, soil enzyme activity (phosphatase, urease, and sucrase), microbial respiration and activity, and active microbial biomass in orchard soil. However, there are no reports on the combined use of arbuscular mycorrhizal fungi and *Vicia crassifolia* for controlling continuous cropping obstacles in apple orchards. Summary of the Invention

[0006] To address the aforementioned limitations of existing technologies, the present invention aims to provide a method for controlling apple continuous cropping obstacles through mycorrhizal symbiosis driven by *Vicia crassifolia*. The specific steps are as follows: applying an AMF (Amyotrophic Lateral Fiber Fungus) inoculant to the soil where apples are continuously cropped, and sowing *Vicia crassifolia* seeds at the base of the apple plants. Through field water and fertilizer management, *Vicia crassifolia*-driven mycorrhizal symbiosis is achieved to control apple continuous cropping obstacles. Experiments have demonstrated that simultaneously applying an AMF inoculant and intercropping *Vicia crassifolia* can increase the biomass of *Pingyi sweet tea* seedlings, soil nutrient content, and soil enzyme activity; increase the number of culturable microorganisms in the soil; reduce the copy number of *Fusarium* genes in the soil; increase the content of glomerulimycin-related proteins, spore density, and mycorrhizal infection rate. By improving the continuous cropping soil environment, reducing the infection of roots by harmful soil fungi and mitigating the harm of their secreted toxins to plants, and enhancing the colonization ability of AMF in the soil, effective control of apple continuous cropping obstacles is achieved. Furthermore, the method of the present invention can also improve the quality of apple fruits.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for controlling apple replanting obstacles by driving mycorrhizal symbiosis with *Vicia crassa*, comprising the following steps: Apply AMF inoculant to the soil in which apple trees are continuously cropped, and sow Vitex longifolia seeds evenly around the base of the apple trees. Then, manage water and fertilizer in the field to allow Vitex longifolia to drive mycorrhizal symbiosis and control apple continuous cropping obstacles.

[0008] Preferably, the AMF is a mycorrhizal fungus of the genus *Gloydius* ( Paraglomus (sp.), its accession number is CGMCCNO.2074, and it is recorded in patent CN113337405A.

[0009] Preferably, the AMF inoculum is prepared by the following method: The AMF strain was inoculated into the roots of clover that had been growing for one week, and preliminarily propagated by single-spore culture in a greenhouse. After the strain had preliminarily propagated, the clover roots were crushed to obtain a powder containing AMF. The sterilized seedling substrate was mixed with the AMF-containing powder, and sterilized clover seeds were sown. Propagation continued under room temperature pot conditions. After 3 months, the AMF infection rate of the clover roots was tested. When the infection rate was greater than 50%, the above-ground parts were cut off, and the clover roots were crushed together with the seedling substrate to obtain the AMF inoculum.

[0010] Furthermore, the single-spore culture time is 1 month; the mass ratio of sterilized seedling substrate to AMF-containing powder is 3:1.

[0011] Preferably, the amount of AMF inoculant applied is 400-600g per tree.

[0012] As a preferred option, the amount of long-haired vitex seeds applied is 6-10g per tree.

[0013] The beneficial effects of this invention are: This invention controls apple continuous cropping obstacles by applying AMF (ammonia pilosa) inoculant to the soil in which apples are continuously cropped and then evenly sowing Vitex trifolia seeds around the roots of apple plants. Experiments have demonstrated that the combined use of AMF and Vitex trifolia effectively promotes the growth and development of Pingyi sweet tea seedlings and apple saplings, improves the soil environment in continuous cropping, reduces the abundance of Fusarium spores in the soil, regulates the composition of the soil microbial community, and increases the mycorrhizal infection rate of Pingyi sweet tea seedling roots, soil AMF spore density, and soil glomerulonephrine-related protein content, thereby effectively alleviating apple continuous cropping obstacles. Attached Figure Description

[0014] Figure 1 Figure 1 shows the effect of AMF inoculant application on the biomass of different leguminous plants mixed with Pingyi sweet tea seedlings. Figure 2 In Experiment 1, the effect of intercropping different leguminous plants with AMF inoculant on soil enzyme activity was investigated. Figure 3 In Experiment 1, the effects of intercropping different legumes after applying AMF inoculant on the number of culturable microorganisms in the soil and the copy number of Fusarium genes were investigated. Figure 4 Figure 1 shows the effect of AMF inoculant application on the copy number of Fusarium gene after intercropping with different leguminous plants. Figure 5 In Experiment 1, the effect of intercropping different leguminous plants with AMF inoculant on the content of globulin-related proteins was investigated. Figure 6In Experiment 1, the effects of applying AMF inoculant on spore density and mycorrhizal infection rate of different leguminous plants were investigated. Figure 7 Figure 1 shows the effect of applying AMF inoculant and intercropping different leguminous plants on the two-year potted effect of Pingyi sweet tea seedlings. Figure 8 Figure 2 shows the effect of intercropping different leguminous plants with AMF inoculant on the growth of apple saplings. Figure 9 Figure 2 shows the effect of intercropping different legumes with AMF inoculant on the growth of apple saplings in the second year. Figure 10 In Experiment 2, the fruit phenotype of the Haiyang (HY2) continuous cropping apple orchard was observed. Detailed Implementation

[0015] 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.

[0016] Continuous cropping obstacles are prevalent in the renewal and reconstruction of old orchards, severely hindering the healthy and sustainable development of the apple industry. Currently, biological control methods using microbial agents or fertilizers have become a research hotspot for alleviating continuous cropping obstacles. Among them, arbuscular mycorrhizal fungi (AMF), as a beneficial fungal group in the soil, can promote the activation and absorption of soil mineral nutrients through hyphal networks, regulate the rhizosphere microecological balance to inhibit pathogen colonization, and secrete specific enzymes to degrade phenolic allelochemicals. However, long-term continuous cropping leads to poor colonization of AMF, limiting its application in improving continuous cropping obstacles in apples.

[0017] Based on this, the present invention provides a method for controlling apple continuous cropping obstacles by using Vitex acutissima to drive mycorrhizal symbiosis. The specific steps are as follows: applying AMF inoculant to apple continuous cropping soil in which apple plants are planted, and evenly sowing Vitex acutissima seeds at the base of the apple plants.

[0018] This invention, through experiments, found that intercropping *Vicia crassifolia* with the application of AMF (Amphioxus spp.) promotes the growth and development of *Vicia crassifolia* seedlings and apple saplings, increases soil nutrient (nitrogen, phosphorus, potassium, and organic matter) content, enhances soil enzyme (sucrase, urease, phosphatase, and catalase) activity, increases the number of culturable bacteria and actinomycetes in the soil while reducing the number of fungi, decreases the copy number of key pathogenic *Fusarium* genes in the soil, and increases the mycorrhizal infection rate of *Vicia crassifolia* seedling roots, soil spore density, and soil glomerulimycin-associated proteins (EE-GRSP and T-GRSP), thereby achieving the effect of controlling soil continuous cropping obstacles. Furthermore, field trials also verified that intercropping *Vicia crassifolia* with the application of AMF significantly increased the soluble sugar and titratable acid content of fruits, improving fruit quality. This invention promotes the colonization of *Vicia crassa* (long-haired pea) in the root system of *Vicia crassa* seedlings in Pingyi sweet tea by intercropping them with mycorrhizal symbiosis. This, in turn, enhances seedling nutrient absorption from the soil through mycorrhizal symbiosis, promoting seedling growth and effectively controlling continuous cropping obstacles. Specifically: As a legume, *Vicia crassifolia* has a well-developed fibrous root system that forms a dense root network in the soil, increasing soil porosity and loosening compacted soil. This change in soil structure provides a loose and well-aerated soil environment for AMF colonization. Furthermore, *Vicia crassifolia* can form a symbiotic nitrogen-fixing system with rhizobia in the soil, compensating for the nitrogen deficiency caused by long-term continuous cropping and providing the nitrogen nutrition needed for AMF proliferation. Its decomposed organic matter slowly releases nutrients such as phosphorus, potassium, calcium, magnesium, and zinc, improving soil fertility and providing sufficient nutrients for AMF proliferation. Its root exudates not only produce organic acids and amino acids, providing the carbon source needed for plant growth and AMF colonization, but also produce flavonoids, such as apigenin, which promote AMF colonization in the roots, stimulating AMF to infect the roots, promoting spore germination, and enhancing AMF colonization ability in the soil. Furthermore, when harmful fungi infect plant roots, they compete with AMF (a type of fungus) for ecological sites and nutrients, thus inhibiting AMF infection of the roots. Intercropping Vitex acutum (long-haired pea) can increase the abundance of beneficial soil bacteria and reduce the abundance of harmful fungi, directly creating a favorable microbial environment for AMF infection of the roots. To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0019] The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0020] In this invention, clover ( Trifolium repens ), milkvetch ( Astragalus sinicus ), long-haired wild pea ( Vicia villosa All seeds were purchased from the Shengyixuan flagship store on Taobao.

[0021] Example 1: (1) The AMF inoculum was prepared according to the method of Wang Mei (2022), as follows: The AMF strain was inoculated into the roots of clover that had been growing for one week. Single spores were cultured in a greenhouse for one month for initial propagation. After the strain had initially propagated, the clover roots were crushed to obtain powder containing AMF. Sterilized seedling substrate was mixed with the AMF-containing powder at a mass ratio of 3:1. Sterilized clover seeds were sown and propagated in pots at room temperature. After 3 months, the AMF infection rate of the clover roots was tested. When the infection rate was greater than 50%, the above-ground parts were cut off, and the clover roots were crushed together with the seedling substrate to obtain the AMF inoculum. (2) Apply AMF inoculant to the soil of apple trees planted in continuous cropping, with an application rate of 500g per tree. Sow Vitex longifolia seeds evenly at the base of the apple trees, with an application rate of 8g per tree. Then carry out field water and fertilizer management to enable Vitex longifolia to drive mycorrhizal symbiosis in order to control the obstacles of continuous cropping of apple trees.

[0022] Experiment 1: Pot Experiment The soil used in this experiment was taken from a 25-year-old apple orchard in Manzhuang Town, Tai'an City (36.04°N, 117.11°E). The soil type was loam, and its nutrient content was 5.62 mg·kg⁻¹ ammonium nitrogen. -1 Nitrate nitrogen content: 7.66 mg·kg -1 Available phosphorus content: 42.86 mg·kg -1 Available potassium content: 12.33 mg / kg -1 Organic matter content: 8.62 g·kg -1 The pH value is 6.05.

[0023] The seedlings used in the experiment were Pingyi sweet tea ( Malus hupehensi (s Rehd.) Seedlings. The cultivation process is as follows: After soaking the Pingyi sweet tea seeds in a carbendazim solution for 24 hours, they are mixed evenly with moist sand and stratified at a low temperature of 4 ℃ for about 30 days. After the seeds show white sprouts, they are sown in sterilized seedling substrate. When the seedlings grow to about 5-6 true leaves, select the strong, disease-free and uniformly growing seedlings for later use.

[0024] 1.1 Experimental Design The experiment was conducted at the Shandong Apple Technology Innovation Center from April to October 2024. The experiment consisted of nine treatments, with 12 replicates for each treatment. Details of each treatment are as follows: Continuous cropping (CK1): Blank control group, no substances were applied; Methyl bromide fumigation (CK2): Methyl bromide is used to fumigate continuously cropped soil; AMF inoculant (A): An inoculant for applying AMF only to soils that have been continuously cropped; AMF inoculant + clover (AC): Apply AMF inoculant to continuously cropped soil and sow clover seeds; Clover (C): Clover seeds are sown only in continuously cropped soils; AMF inoculant + milkvetch (AM): Apply AMF inoculant to the continuously cropped soil and sow milkvetch seeds; Milk vetch (M): Milk vetch seeds are sown only in soils where crops have been continuously grown; AMF inoculant + long-haired vitex (AV): Apply AMF inoculant to the continuously cropped soil and sow long-haired vitex seeds. Long-haired pea (V): Long-haired pea seeds are sown only in soils where crops have been continuously grown.

[0025] Each pot of soil used for potted plants weighed 10 kg. The amount of AMF inoculant applied was 1% of the potted soil mass, the amount of long-haired vitex seeds sown was 0.05% of the potted soil mass, and the amount of clover and milkvetch sown was 0.02% of the potted soil mass. The preparation method of AMF inoculant was the same as in Example 1. During the experiment, drip irrigation was used to maintain soil moisture at 60%-70% of field capacity, and no additional chemical fertilizers were applied.

[0026] Sampling was conducted in mid-July and mid-August. First, three healthy plants with uniform growth were selected, and the topsoil of the potted plants was removed. Then, rhizosphere soil was collected from the 10-25cm soil layer. The rhizosphere soil was sieved and divided into three portions: (1) stored in a refrigerator at 4 ℃ for the determination of the number of culturable microorganisms in the soil; (2) air-dried and stored at room temperature for the determination of soil physicochemical properties, soil enzyme activity, spore density and globulin-related protein content; (3) rapidly frozen in liquid nitrogen and then at -80℃ for soil DNA extraction and high-throughput sequencing analysis.

[0027] The biomass of Pingyi sweet tea seedlings, soil physicochemical properties, number of culturable microorganisms in the soil, copy number of Fusarium solani genes in the soil, soil enzyme activity, spore density, mycorrhizal infection rate, and content of glomerulonephrine-related proteins were measured. Biomass of Pingyi sweet tea seedlings: The plant height, diameter at ground level, fresh weight, and dry weight of Pingyi sweet tea seedlings were measured using a measuring tape, vernier caliper, and electronic balance, respectively.

[0028] Soil physicochemical properties: Nitrate nitrogen was determined by ultraviolet spectrophotometry at wavelengths of 220 nm and 275 nm; ammonium nitrogen was determined by indophenol blue colorimetry at wavelength of 625 nm; available phosphorus was determined by molybdenum antimony colorimetry at wavelength of 700 nm; available potassium was determined by flame photometry, measuring the emission intensity of the filtrate at wavelength of 766 nm using a flame photometer; organic matter was determined by potassium dichromate titration method (external heating method). Soil samples were digested with potassium dichromate-sulfuric acid solution, followed by the addition of o-phenanthroline indicator, and then titrated with ferrous sulfate standard solution. The soil organic matter content was calculated by the titration difference between the blank test and the digestion test.

[0029] Number of culturable microorganisms in soil: Following the method of Yin et al. (2024), the number of culturable bacteria, fungi and actinomycetes in soil was determined by dilution plate spread method, and the ratio of bacteria to fungi was calculated.

[0030] Soil Fusarium gene copy number: Total DNA was extracted from soils treated with different methods using the EZNA@Soil DNA Kit. The gene copy number of soil Fusarium was analyzed using a CFX96™ Thermal Cycler (Bio-Rad). The primer sequences and PCR amplification reaction procedures are shown in Table 1.

[0031] Table 1 Primer sequences and amplification reaction procedures for Fusarium spores. Soil enzyme activities: Phosphatase was determined using the disodium phenyl phosphate colorimetric method, with absorbance measured at 570 nm, and activity was expressed as the amount of phenol produced; Sucrase was determined using the 3,5-dinitrosalicylic acid colorimetric method, with absorbance measured at 540 nm, and activity was expressed as the amount of glucose produced; Urease was determined using the indophenol blue colorimetric method, with absorbance measured at 625 nm, and activity was calculated as the amount of ammonium nitrogen produced; Catalase was determined using the potassium permanganate titration method, and activity was calculated as the amount of potassium permanganate consumed. The specific methods described in Duan et al. (2022a) were followed.

[0032] Spore density: Spore density was calculated using the wet soil sieving method (Wang et al (2021). Spore density = number of AM fungal spores / soil mass.

[0033] Mycorrhizal infection rate: determined according to the method of Wang et al. (2021). The infection rate of each root segment was assessed by the number of mycorrhizal structures in each slice and expressed as 0, 10, 20, ..., 100% of the root.

[0034] Mycorrhizal infection rate = Σ(0 × number of root segments + 10% × number of root segments + 20% × number of root segments + ... + (100% of the number of root segments) / total number of root segments.

[0035] Glomalin-related protein content: Following the method of Li Yang (2023), the contents of easily extractable glomalin-related soil protein (EE-GRSP) and total glomalin-related soil protein (T-GRSP) were determined using the Coomassie brilliant blue method.

[0036] 1.2 Experimental Results and Analysis: (1) Biomass of Pingyi sweet tea seedlings: Depend on Figure 1 It can be seen that intercropping with different leguminous plants significantly promotes the growth of Pingyi sweet tea seedlings. In August, compared with CK1, the plant height, ground diameter, fresh weight, and dry weight of AC, AM, and AV treatments increased by 23.24%, 18.58%, 27.72%, and 20.04%; 51.64%, 29.06%, 66.63%, and 51.90%; and 85.47%, 51.35%, 187.85%, and 149.50%, respectively. Compared with treatment A, the plant height, ground diameter, dry weight, and fresh weight of AV treatment increased by 8.33%, 9.29%, 6.97%, and 8.62%, respectively. In August, there were no significant differences in the robustness index among the treatments, while the Dickson quality index of CK2 and AV treatments showed significant differences compared with other treatments. This result indicates that CK2 and AV treatments can effectively promote seedling dry matter accumulation, promote root growth and development, and promote coordinated growth of the aboveground and underground parts, thereby improving the seedlings' resistance to continuous cropping obstacles.

[0037] (2) Soil physicochemical properties and soil enzyme activity: Table 2 Effects of AMF inoculant application on soil nutrient content after intercropping with different leguminous plants (July and August 2024) Soil nutrient content is a core indicator for measuring soil fertility. Table 2 shows that the application of AMF inoculant and intercropping with different leguminous plants significantly increased the content of soil nitrogen, phosphorus, potassium, and organic matter. In August, compared with treatment A, the AV treatment showed increases of 77.32% in nitrate nitrogen, 49.04% in ammonium nitrogen, 30.40% in available phosphorus, 26.56% in available potassium, and 30.94% in organic matter. Compared with treatment AC, except for ammonium nitrogen, the contents of other nutrients increased by 29.30%, 15.10%, 13.28%, and 19.53%, respectively. Compared with treatment AM, except for organic matter, the contents of other nutrients increased by 15.94%, 15.08%, 11.32%, and 9.85%, respectively. Therefore, the AV treatment showed the best effect in improving soil physicochemical properties and enhancing soil fertility.

[0038] Depend on Figure 2 It can be seen that in August, compared with AC and AM, the AV treatment increased the activities of sucrase, urease, phosphatase, and catalase by 7.22%, 14.11%, 5.84%, 6.24%, and 16.07%, 26.90%, 2.85%, and 1.43%, respectively; compared with A, the AV treatment increased by 24.14%, 17.42%, 6.09%, and 9.52%, respectively. In August, except for a decrease in phosphatase activity compared with July, the activities of the other three enzymes all increased. This result indicates that the AV treatment not only effectively activated the activities of enzymes related to soil carbon, nitrogen, and phosphorus nutrient transformation, but also enhanced the activity of catalase related to oxidative stress scavenging, significantly improving the soil environment after continuous cropping.

[0039] (3) Number of culturable microorganisms in the soil and copy number of Fusarium genes in the soil: Depend on Figure 3 It can be seen that AC, AM, and AV treatments all have a regulatory effect on the rhizosphere soil microbial community, specifically manifested as an increase in the number of culturable bacteria and actinomycetes, and a decrease in the number of fungi, with all effects being superior to those of monoculture of leguminous plants (C, M, and V treatments). In August, compared with A, there were no significant differences in soil actinomycetes and fungi in AC and AM treatments, while in AV treatment, actinomycetes increased by 14.85% and fungi decreased by 26.44%; soil bacteria increased by 16.96%, 10.87%, and 17.83% in AC, AM, and AV treatments, respectively. Therefore, it can be concluded that intercropping with Vitex acutissima (AV) has the best effect.

[0040] To further investigate the changes in the abundance of harmful fungi in the soil, we used qPCR to determine the changes in gene copy numbers of four key pathogenic Fusarium species in the soil, such as... Figure 4As shown in the figure. The results showed that, compared with continuous cropping (CK1), intercropping with different leguminous plants (AC, AM, AV) reduced the gene copy number of the four Fusarium species. In August, compared with A, there was no significant difference in the gene copy number of *Fusarium solani* in AC, AM, and AV treatments; there was no significant difference in the gene copy number of *Fusarium oxysporum* in AC and AM treatments, while the AV treatment reduced it by 36.06%; there was no significant difference in the gene copy number of *Fusarium moniliforme* in AM treatment, while AC and AV treatments reduced it by 22.23% and 48.71%, respectively; there was no significant difference in the gene copy number of *Fusarium chrysogenum* in AC treatment, while AM ​​and AV treatments reduced it by 17.81% and 35.91%, respectively. The imbalance of soil microbial community structure caused by the proliferation of harmful soil fungi is the main biological factor leading to ARD. AV treatment effectively inhibited the proliferation of Fusarium, not only reducing its infection of the roots but also reducing the damage caused to plants by the various toxins it secretes, significantly alleviating the obstacle of continuous cropping.

[0041] (4) Content of globulin-associated protein: Glomalin-related soil protein (GRSP) is a special glycoprotein produced by arbuscular mycorrhizal fungi with good thermal stability. It has significant effects on promoting soil aggregate formation, increasing soil organic carbon, and improving plant stress resistance (Gan et al., 2022). Figure 5 It can be seen that in August, compared with CK1, except for the AC treatment which showed no significant difference in total glomerulone-related protein content (T-GRSP), the AM and AV treatments showed a significant increase in T-GRSP content. Simultaneously, the content of easily extractable glomerulone-related protein (EE-GRSP) increased significantly in all three treatments. Compared with A, the AV treatment increased EE-GRSP and T-GRSP by 28.22% and 16.35%, respectively. This result indicates that there are significant differences in the regulation of different components of soil GRSP by intercropping different leguminous plants, with the AV treatment showing the best effect in increasing EE-GRSP and T-GRSP content.

[0042] (5) Spore density and mycorrhizal infection rate: The results are as follows Figure 6 As shown in the figure, in September, compared with CK1, the AC, AM, and AV treatments all significantly increased soil spore density, increasing by 27.05%, 48.36%, and 68.85%, respectively; compared with A, only the AV treatment showed a significant increase in spore density of 15.41%. The spore density of each treatment showed a trend of first increasing and then decreasing from July to October, reaching its highest value in September. This result indicates that intercropping with long-haired vitex (AV) effectively promoted the reproductive capacity of AMF in the soil, enhanced seedling stress resistance, and effectively alleviated continuous cropping obstacles.

[0043] To investigate the effects of different leguminous plants on mycorrhizal infection rates, the mycorrhizal infection rates of *Camellia sinensis* seedlings from Pingyi during July to October were measured. In terms of root type, the mycorrhizal infection rate was highest in absorbing roots, while there was no significant difference between the mycorrhizal infection rates of woody roots and growing roots. In terms of the growing season, the mycorrhizal infection rate increased continuously from July to September, reaching its peak in September, and then began to decline in October. In September, compared with treatment A, the mycorrhizal infection rates of absorbing roots in treatments AC, AM, and AV increased by 17.55%, 7.56%, and 28.10%, respectively, while the mycorrhizal infection rates of woody roots increased by 11.05%, 3.39%, and 27.20%, respectively. The mycorrhizal infection rate of growing roots in treatment AC showed no significant difference from that in treatment A, while the rates in treatments AM and AV increased by 19.11% and 41.76%, respectively. This indicates that intercropping of clover (AC), milkvetch (AM), and long-haired vitex (AV) can all increase the mycorrhizal infection rate of Pingyi sweet tea seedlings to varying degrees, with long-haired vitex showing the best effect. Furthermore, the synergistic effect of AMF inoculant and leguminous plants on increasing the mycorrhizal infection rate varies significantly among different root types.

[0044] (6) To verify the stability of the synergistic effect between AMF and legumes, the first-year potted seedlings were kept in pots and cultured until the second year to continuously explore the subsequent effects of the synergistic effect. The results are as follows: Figure 7 As shown.

[0045] Depend on Figure 7 It can be seen that among the three different legumes, *Vicia crassifolia* (AV2) showed the best effect, significantly promoting the growth of *Pingyi sweet tea* seedlings and increasing the number of culturable bacteria and actinomycetes in the soil while reducing the number of fungi. Compared with CK-1, the plant height, diameter at breast height, fresh weight, and dry weight of the AV2 treatment increased by 20.34%, 64.92%, 70.30%, and 72.44%, respectively; compared with A2, they increased by 2.91%, 16.88%, 48.37%, and 27.70%, respectively. Meanwhile, compared with A2, the soil bacteria and actinomycetes in the AV2 treatment increased by 17.77% and 15.22%, respectively, while the fungi decreased by 32.73%.

[0046] Experimental Example 2: Field Experiment Field trials were conducted from April to October 2024 in Yiyuan County, Zibo City, Shandong Province (118.17 °N, 36.18 °E) and Haiyang City, Yantai City, Shandong Province (111.37 °N, 32.22 °E). The basic physicochemical properties of the tested soils are shown in Table 3.

[0047] Table 3 Basic physicochemical properties of the tested soils The experimental seedlings were two-year-old grafted seedlings (rootstock M9T337, scion Xin '2001'). This experiment consisted of 5 treatments, with 10 trees replicated for each treatment. The details of each treatment are as follows: Continuous cropping (T-CK): Blank control, no substances were applied; AMF agent (TA): A microbial agent for applying AMF; AMF inoculant + clover (T-AC): Apply AMF inoculant and sow clover seeds; AMF inoculant + milkvetch (T-AM): Apply AMF inoculant and sow milkvetch seeds; AMF inoculant + long-haired vitex (T-AV): Apply AMF inoculant and sow long-haired vitex seeds.

[0048] In this case, 500g of AMF inoculant was applied to each tree, 5g of clover seeds and milkvetch seeds were sown per tree, and 8g of long-haired wild vetch seeds were sown per tree. The preparation method of AMF inoculant was the same as in Example 1, and field fertilizer and water management were carried out uniformly.

[0049] The biomass of apple saplings and the number of culturable microorganisms in the soil were measured using the method described in Experiment Example 1. The results are as follows: Figures 8-10 As shown in Table 4.

[0050] Depend on Figure 8 It can be seen that intercropping with different leguminous plants can effectively promote the growth of continuously cropped apple saplings and improve the soil environment. Regarding sapling growth, the T-AV treatment showed good growth-promoting effects at both experimental sites. At the Yiyuan (YY) experimental site, the plant height, number of branches, and branch length of the AV treatment increased by 21.03%, 44.00%, and 10.10% respectively compared to the A treatment, but the ground diameter showed no significant difference. There was no significant difference in the biomass of apple saplings between the AC and AV treatments. Except for the plant height and ground diameter of the AM treatment, which showed no significant difference compared to the AV treatment, all other biomass indicators were significantly lower than those of the AV treatment. Regarding soil microbial communities, intercropping with different leguminous plants effectively optimized the soil microbial composition at each experimental site. At the Haiyang (HY) experimental site, the number of soil bacteria and actinomycetes, as well as the ratio of bacteria to fungi, increased by 23.03%, 39.78%, and 76.10% respectively compared to the A treatment, while the number of fungi decreased by 29.51%. The AC and AM treatments had similar effects on the number of soil microorganisms, but their effects were both lower than those of the AV treatment.

[0051] The apple seedlings from each treatment group were further cultured, and the effects of intercropping with different leguminous plants after applying AMF inoculant on the growth of apple saplings in the second year were as follows: Figures 9-10 As shown in Table 4.

[0052] Table 4. Determination of fruit growth and quality indicators in the Haiyang (HY2) continuous cropping apple orchard (October 2025) Depend on Figure 9 It can be seen that among the three leguminous plants, *Vicia crassa* (AV) showed the best results, consistent with the results of the first year's experiment. At the Haiyang (HY2) experimental site, compared to T-A2, the T-AV2 treatment increased plant height, diameter at root, number of fruits, and branch length by 21.81%, 29.25%, 77.78%, and 22.25%, respectively. At the Yiyuan (YY) experimental site, compared to T-A2, the T-AV2 treatment increased the number of soil bacteria and actinomycetes, as well as the bacterial-to-fungal ratio, by 25.31%, 28.88%, and 141.02%, respectively, while reducing the number of fungi by 47.97%.

[0053] In the second year of the experiment, the fruit trees in the Haiyang (HY) continuous cropping orchard were bearing fruit normally. Therefore, fruits treated with T-CK2, T-A2, and T-AV2 were harvested, and fruit growth and quality indicators were measured. The results are as follows: Figure 10 As shown in Table 4. From Figure 10 As shown in Table 4, compared with the AMF (T-A2) treatment alone, the T-AV2 treatment increased the transverse and longitudinal diameters, fresh fruit weight, dry fruit weight, fruit firmness, and titratable acid content of the fruit by 7.10%, 8.17%, 12.95%, 35.00%, 18.57%, and 22.58%, respectively, but there was no significant difference in soluble sugar content. This indicates that intercropping Vitex trifolia with AMF inoculant can promote fruit growth and improve fruit quality in continuously cropped orchards to a certain extent.

[0054] 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 method for controlling apple replanting obstacles through mycorrhizal symbiosis driven by *Vicia crassifolia*, characterized in that, Includes the following steps: Apply AMF inoculant to the soil in which apple trees are continuously cropped, and sow Vitex longifolia seeds evenly around the base of the apple trees. Then, manage water and fertilizer in the field to allow Vitex longifolia to drive mycorrhizal symbiosis and control apple continuous cropping obstacles.

2. The method for controlling apple replanting obstacles by driving mycorrhizal symbiosis with *Vicia crassifolia* as described in claim 1, characterized in that, The AMF is a fungal mycorrhizal fungus of the genus *Gloydiomycota* ( Paraglomus (sp.), its accession number is CGMCC NO.2074.

3. The method for controlling apple replanting obstacles by driving mycorrhizal symbiosis with *Vicia sativa* as described in claim 1, characterized in that... The AMF inoculum is prepared by the following method: The AMF strain was inoculated into the roots of clover that had been growing for one week, and preliminarily propagated by single-spore culture in a greenhouse. After the strain had preliminarily propagated, the clover roots were crushed to obtain a powder containing AMF. The sterilized seedling substrate was mixed with the AMF-containing powder, and sterilized clover seeds were sown. Propagation continued under room temperature pot conditions. After 3 months, the AMF infection rate of the clover roots was tested. When the infection rate was greater than 50%, the above-ground parts were cut off, and the clover roots were crushed together with the seedling substrate to obtain the AMF inoculum.

4. The method for controlling apple replanting obstacles by using *Vicia sativa* (a type of pea) to drive mycorrhizal symbiosis as described in claim 3, characterized in that... The single-spore culture time is 1 month; the mass ratio of sterilized seedling substrate to powder containing AMF is 3:

1.

5. The method for controlling apple replanting obstacles by driving mycorrhizal symbiosis with *Vicia crassifolia* as described in claim 1, characterized in that, The application rate of AMF fungicide is 400-600g per tree.

6. The method for controlling apple replanting obstacles by driving mycorrhizal symbiosis with *Vicia sativa* as described in claim 1, characterized in that, The application rate of long-haired vitex seeds is 6-10g per tree.

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