Vigna unguiculata endophytic non-pathogenic fusarium oxysporum fo-sy20 and application thereof

Abstract

A kind of Vigna sinensis endophytic non-pathogenic fusarium oxysporum Fo-SY20 and its application belong to the field of agricultural microorganism and biological control technology.The present application provides a kind of Vigna sinensis endophytic non-pathogenic fusarium oxysporum Fo-SY20 to solve the problems of pesticide residue,environmental pollution,pathogen resistance and soil micro-ecological destruction caused by chemical control of wilt in the prior art,with preservation number CGMCC No.42441.The biocontrol effect of the strain on Vigna sinensis wilt reaches 42.9%,and can effectively maintain rhizosphere bacterial diversity,relieve subsequent pathogen stress,and enrich growth-promoting nitrogen-fixing groups to actively construct Vigna sinensis rhizosphere microbial environment.The Vigna sinensis endophytic non-pathogenic fusarium oxysporum Fo-SY20 provided by the present application has great application potential for solving Vigna sinensis wilt hazards in Vigna sinensis production and Vigna sinensis disease resistance cultivation,rhizosphere micro-ecological regulation.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology and biological control technology, and in particular relates to a non-pathogenic Fusarium oxysporum endophyte in cowpea and its application. Background Technology

[0002] During vegetable cultivation, Fusarium oxysporum (… Fusarium oxysporum Fusarium wilt, caused by [unspecified disease], is a typical soil-borne disease and has become a major bottleneck restricting the sustainable development of the melon and vegetable industry. In recent years, with the rapid development of tropical high-efficiency agriculture, the scale of melon and vegetable planting has continued to expand. Long-term continuous cropping has led to the accumulation and spread of pathogens in the soil, causing the disease to show a trend of increasing severity year by year. Especially in winter cowpea planting areas where continuous cropping is practiced, the damage caused by Fusarium wilt is particularly significant, and the direct economic losses caused each year can reach hundreds of millions of yuan.

[0003] Currently, the control of Fusarium wilt still heavily relies on chemical agents, with benzimidazole fungicides (such as carbendazim and thiophanate-methyl) being the most widely used. Although these chemical control methods are characterized by rapid effectiveness and ease of use, their negative effects are becoming increasingly prominent: First, pesticide residues directly affect the safety and quality of vegetable products; second, the accumulation of chemical agents in the soil not only causes environmental pollution but also disrupts the soil's microecological balance; more importantly, long-term use of chemical fungicides alone can easily lead to drug resistance in pathogens, which in turn exacerbates continuous cropping obstacles and creates a vicious cycle of "drug use - drug resistance - increased dosage".

[0004] With the deepening of green and high-quality agricultural development in my country, the use of highly toxic and persistent chemical pesticides in vegetable production has been strictly limited. Against this backdrop, seeking safe, efficient, and environmentally friendly alternative biological control technologies has become a research hotspot. Among these, biological control systems targeting soil-borne diseases, particularly green control technologies centered on biocontrol agents and plant-derived antimicrobial substances, are showing broad application prospects. These technologies can not only effectively control disease occurrence but also improve the soil environment, providing new solutions for the sustainable development of the vegetable industry.

[0005] The rhizosphere refers to the micro-domain of soil surrounding plant roots, rich in microorganisms recruited by root exudates, including nitrogen-fixing bacteria, mycorrhizal fungi, and biocontrol bacteria. These microorganisms interact with plants, promoting nutrient absorption (e.g., alleviating iron deficiency) and enhancing stress resistance (e.g., drought resistance). Plant endophytes are bacteria, fungi, etc., that colonize within healthy plant tissues, living in a mutually beneficial symbiotic relationship with the host. For example, endophytes can synthesize siderophores, increasing iron absorption; they can also promote the absorption of potassium, calcium, and magnesium under salt stress, enhancing plant resistance. When plants are infected by pathogens, they release signals to recruit beneficial bacteria (such as Bacillus and Pseudomonas), forming disease-suppressing soil. These beneficial bacteria can inhibit pathogens, enhance plant immunity, and improve disease resistance. This mechanism provides an important strategy for green agriculture, and this hypothesis is known as the "cry for help" hypothesis.

[0006] Fusarium oxysporum can be divided into two main categories based on its pathogenicity: pathogenic strains and non-pathogenic strains. Pathogenic strains can infect more than 120 plant species, causing wilt disease and resulting in huge losses to agricultural production. Non-pathogenic Fusarium oxysporum strains can be used as biocontrol materials and have shown good application potential in the green control of plant diseases. Interestingly, many non-pathogenic Fusarium oxysporum strains... Fusarium oxysporum Not only can NPFo colonize the roots of plants, but it can also enter the plant to become an endophyte, forming a stable symbiotic relationship with the host. Most importantly, it does not cause any diseases; instead, it forms a mutually beneficial symbiotic relationship with plants through various pathways, enhancing the plants' stress resistance and disease resistance, making it a research hotspot in the field of biological control. Summary of the Invention

[0007] To address the problems of pesticide residues, environmental pollution, pathogen resistance, and soil microecological damage caused by chemical control of Fusarium wilt in existing technologies, this invention provides a cowpea endophytic non-pathogenic Fusarium oxysporum Fo-SY20 and its application.

[0008] One of the objectives of this invention is to provide a non-pathogenic Fusarium oxysporum endophyte in cowpeas (… Fusarium oxysporum Fo-SY20, the non-pathogenic Fusarium oxysporum endophyte of cowpea, is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42441.

[0009] In a preferred embodiment of the present invention, the cowpea endophytic non-pathogenic Fusarium oxysporum Fo-SY20 trpB The gene sequence is shown in SEQ ID NO.1.

[0010] A second objective of this invention is to provide a microbial agent containing the aforementioned non-pathogenic Fusarium oxysporum Fo-SY20 endophytic fungus from cowpea.

[0011] In a preferred embodiment of the present invention, the preparation method of the microbial agent is as follows: the above-mentioned cowpea endophytic non-pathogenic Fusarium oxysporum Fo-SY20 is inoculated into PL medium and cultured.

[0012] In a preferred embodiment of the present invention, the culture conditions are: 180 rpm, 28°C shaker culture for 72 h.

[0013] In a preferred embodiment of the present invention, the concentration of *Fusarium oxysporum* Fo-SY20, an endophytic non-pathogenic bacterium of cowpea, in the inoculum is 1 × 10⁻⁶. 8 CFU / mL.

[0014] The third objective of this invention is to provide the application of the above-mentioned non-pathogenic Fusarium oxysporum Fo-SY20 endophytic fungus of cowpea or its inoculant in the prevention and control of cowpea wilt disease.

[0015] The beneficial effects of this invention are: This invention provides a non-pathogenic Fusarium oxysporum strain Fo-SY20 from cowpea endophytes with immune-stimulating activity against Fusarium oxysporum wilt, which is specifically targeted at the vascular-specific Fusarium oxysporum var. oxysporum, the pathogen of cowpea wilt. Fusarium oxysporum f.sp. tracheiphilum (Fj-fy) exhibits excellent specialized control effects, with the following specific beneficial effects: 1) The prevention and control effect has been significantly improved. Existing technologies using non-pathogenic Fusarium oxysporum strains for controlling cowpea wilt typically only provide qualitative descriptions of control potential, lacking specific quantitative data on control efficacy. Furthermore, their applicability to different varieties is unclear, and their control efficacy is unstable. In contrast, the Fo-SY20 strain of this invention, at a spore suspension concentration of 1.0 × 10⁻⁶, [achieved control]. 8 Under CFU / mL conditions, the average control rate against cowpea wilt reached 42.9%. Significant control efficacy was verified in both the resistant variety X480 and the susceptible variety G136: 10 days after pathogen inoculation, the disease incidence indices of X480 and G136 were only 0.37% and 0.74%, respectively, achieving near-complete early-stage disease control; 21 days after pathogen inoculation, the control efficacy of X480 was >50% and that of G136 was >50%, indicating that Fo-SY20 has stable and quantifiable excellent control efficacy in cowpea varieties with different resistance levels.

[0016] 2) Preventive control mechanism to overcome the limitations of passive competition Existing non-pathogenic strains often employ a "competitive suppression" strategy, typically requiring a very high bacterial density (e.g., more than 10 times higher than the pathogen) to be effective. Furthermore, intervention usually occurs only after disease has developed, representing a passive response and failing to achieve early intervention. The core advantage of this invention, Fo-SY20, lies in prevention rather than treatment. By adding it to the soil before sowing or soaking the roots during transplanting, pre-inoculation effectively blocks the early occurrence of disease. In unsterilized soil, the disease incidence rate is reduced to below 1% on the 10th day after inoculation, achieving an active preventative effect of "early intervention" and overcoming the efficiency bottleneck of traditional competitive mechanisms.

[0017] 3) Inducing systemic resistance and expanding the mechanism of action Current understanding of the disease control mechanisms of non-pathogenic Fusarium oxysporum is largely limited to direct competition or antagonism, without addressing research on inducing plant autoimmunity. This invention is the first to demonstrate that Fo-SY20, through pre-inoculation, can effectively induce systemic resistance in cowpeas, exhibiting immune-stimulating activity. This enables plants to mobilize their own defense systems when facing pathogen infection, thus enriching the disease control mechanisms of non-pathogenic bacteria and providing a new approach for the control of Fusarium wilt.

[0018] 4) Synergistic effect with beneficial soil microorganisms In existing technologies, many exogenous non-pathogenic strains often fail to colonize effectively in the field due to competition with native microorganisms, resulting in reduced efficacy and difficulty in achieving a stabilizing effect. This invention reveals that Fo-SY20 exhibits significantly better efficacy in unsterilized soil than in sterilized soil (a 33.33 percentage point difference in disease incidence index after 21 days). This indicates that Fo-SY20 can synergistically work with beneficial soil microbial communities to enhance plant disease resistance, overcoming the common problem of incompatibility between exogenous strains and soil microorganisms, and is more suitable for the complex soil environment of the field.

[0019] 5) Proactively build a healthy rhizosphere micro-ecology Existing technologies generally lack in-depth research on the regulation of rhizosphere microbiota by biocontrol bacteria, mostly focusing on the direct interaction between strains and pathogens, without paying attention to the improvement of rhizosphere community structure. This invention, through volcano plot and LEfSe analysis, confirms that Fo-SY20 and the pathogen Fj-fy have fundamentally different regulatory modes on rhizosphere bacteria: Fo-SY20 tends towards "active construction," enriching growth-promoting nitrogen-fixing groups such as Massilia and Rhizobium, as well as stress-responsive groups such as γ-Proteobacteria, forming a unique and stable beneficial community; while the pathogen Fj-fy tends towards "passive destruction," inhibiting sensitive groups. Pre-inoculation with Fo-SY20 effectively maintains rhizosphere bacterial diversity and alleviates subsequent pathogen stress, indicating that the strain of this invention not only prevents disease but also actively shapes a healthy rhizosphere microecology.

[0020] 6) Adaptable to routine agricultural operations, facilitating widespread application. Most existing biocontrol technologies rely on mixed inoculation during the seedling stage or complex treatment processes, increasing planting costs and operational difficulty, making them difficult to popularize in direct seeding or transplanting in large-scale field applications. The Fo-SY20 of this invention is applied by "adding it to the soil before sowing or transplanting, or soaking the roots during transplanting," directly matching the key agricultural milestones in cowpea cultivation. It eliminates the need for additional seedling stages, is easy to operate, and is more readily accepted by farmers and widely promoted, demonstrating outstanding practical value.

[0021] In summary, the Fo-SY20 strain provided by this invention, through a multi-mechanism approach of "specialized targeting + early inoculation at sowing / transplanting points + prevention and blocking + induction of systemic resistance + active construction of rhizosphere microbiome + synergistic effect with beneficial soil microorganisms," significantly outperforms conventional non-pathogenic Fusarium oxysporum strains in existing technologies in terms of control efficacy, mechanism of action, soil compatibility, and agricultural suitability. This achieves highly efficient and green control of cowpea wilt and has enormous application potential for addressing the harm caused by cowpea wilt in cowpea production.

[0022] [Biological Preservation Information]: The preservation number of the endophytic non-pathogenic Fusarium oxysporum f. sp. Fo-SY20 of cowpea is CGMCC No. 42441, and its classification name is Fusarium oxysporum. Fusarium oxysporum It is deposited at the China General Microbiological Culture Collection Center, with a deposit date of January 26, 2026. Attached Figure Description

[0023] Figure 1 This image shows the colony morphology of Fo-SY20, a non-pathogenic endophytic Fusarium oxysporum fungus found in cowpeas. Figure 2 Phylogenetic tree of Fo-SY20, a non-pathogenic endophytic Fusarium oxysporum fungus in cowpea; Figure 3 Comparative graphs showing the efficacy of spore suspensions of the non-pathogenic Fusarium oxysporum strain Fo-SY20 in controlling Fusarium wilt in potted cowpeas; A: X480 without inoculation with Fo-SY20 and without inoculation with Fj-fy; B: X480 inoculated with Fo-SY20 but without inoculation with Fj-fy; C: X480 without inoculation with Fo-SY20 but with inoculation with Fj-fy; D: X480 inoculated with both Fo-SY20 and inoculation with Fj-fy; E: X480 inoculated with Fj-fy first and then with Fo-SY20. Figure 4Figures show the results of Alpha and Beta diversity analyses for different treatment groups; A is the Alpha diversity analysis of rhizosphere bacteria, B is the Beta diversity analysis of rhizosphere bacteria, C is the Alpha diversity analysis of rhizosphere fungi, and D is the Beta diversity analysis of rhizosphere fungi; among them, A3 is inoculated with Fo-SY20 but not Fj-fy, A4 is inoculated with Fj-fy but not Fo-SY20, A5 is the water control, and F1 is the natural soil. Figure 5 Volcano plots showing bacterial differences between different treatment groups: A is the bacterial difference volcano plot between treatment groups A3 and A4; B is the bacterial difference volcano plot between treatment groups A3 and A5; C is the bacterial difference volcano plot between treatment groups A3 and F1; D is the bacterial difference volcano plot between treatment groups A4 and A5; E is the bacterial difference volcano plot between treatment groups A4 and F1; F is the bacterial difference volcano plot between treatment groups A5 and F1. Wherein, A3 is treated with Fo-SY20 but not Fj-fy, A4 is treated with Fj-fy but not Fo-SY20, A5 is the water control, and F1 is the natural soil. Figure 6 The bar charts are for multi-level species difference judgment analysis and multi-level species difference test; the left side is the evolutionary branching diagram, and the right side is the quantitative diagram of the degree of influence of species differences; among them, A3 is the inoculation with Fo-SY20 but not Fj-fy, A4 is the inoculation with Fj-fy but not Fo-SY20, A5 is the water control, and F1 is the natural soil. Detailed Implementation

[0024] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0026] Example 1: Screening and identification of non-pathogenic Fusarium oxysporum Fo-SY20 endophytic fungus in cowpea 1. Isolation and purification of bacterial strains Diseased cowpea stem tissue was discovered and collected from the Guolingwen perennial vegetable base (109.33°E, 18.35°N) of the Tropical Agricultural Sciences Academy in Sanya City, Hainan Province. The bacterial strain was isolated, cultured, and morphologically identified using Koch's postulates. The specific steps were as follows: small pieces at the boundary of plant diseases were selected, disinfected in 5% sodium hypochlorite for 15 seconds, then disinfected in 75% alcohol for 15 seconds, washed twice with clean water, and allowed to dry. The samples were then placed on PDA medium (composed of 200 g potato, 20 g glucose, 15-20 g agar, and 1000 mL tap water) containing three antibiotics (chloramphenicol, streptomycin, and lactic acid). Bacterial isolation was performed on antibiotic-free PDA medium plates. Newly germinated hyphae were observed, and the samples were immediately transferred to new PDA medium plates to avoid mixing multiple bacteria and hindering isolation. The selected strain was then screened and named Fo-SY20.

[0027] The morphological identification results of the target strains obtained from the above isolation are as follows: Figure 1 As shown, the hyphae are nearly circular, the aerial hyphae are white and fluffy, the hyphae of the strain are dense and have concentric rings; the hyphae are filamentous, colorless and septate, and the small conidia are elliptical.

[0028] 2. Strain identification The Fo-SY20 strain obtained above was subjected to primer pairs with sequences shown in SEQ ID NO.2-3 (trpBF: 5′-TCATGATAGACAAGGTTT-3′, SEQ ID NO.2; trpBR: 5′-AATTTGCTGTGATAGGGTGGAT-3′, SEQ ID NO.3). trpB Gene sequence fragment amplification yielded a 765 bp FO-specific DNA fragment (SEQ ID NO. 1). Homology and phylogenetic analyses were performed on this FO-specific DNA fragment, and the results are as follows: Figure 2 As shown, the most recent species of the Fo-SY20 strain obtained from the above screening was determined to be Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum ).

[0029] Example 2: Application of endophytic non-pathogenic Fusarium oxysporum Fo-SY20 in the control of cowpea wilt disease 1. Research Materials Cowpea varieties: disease-resistant variety X480, susceptible variety G136 (provided by the Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences); Fusarium oxysporum: Fusarium oxysporum var. ... Fusarium oxysporum f . sp . tracheiphilum(From the Disease Research Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences); Fo-SY20 strain obtained by screening in Example 1.

[0030] 2. Treatment of cowpea seeds Select 200 seeds each of the disease-resistant variety X480 and the susceptible variety G136 that are uniform and plump. Disinfect them with 1% sodium hypochlorite for 3 minutes, and rinse them with distilled water to remove any residual sodium hypochlorite from the surface of the beans. Place them on a 28°C shaker and soak them for 4 hours while shaking. After the beans absorb water and swell, remove them and clean the surface mucus. Soak two layers of gauze, wring them dry (but not dripping), wrap the soaked beans in the gauze, spray them with water (but not dripping), place them in a sealed bag, and then put them in a 28°C incubator. Check daily to see if water needs to be added, and wait for the beans to sprout.

[0031] Sowing: Sterilize the nutrient soil and vermiculite in advance, and mix them thoroughly according to the ratio of vermiculite to substrate soil = 1:2. Wait for the cowpeas to sprout and then sow them in the soil.

[0032] 3. Inoculation treatment Preparation of spore suspension: Prepared PL medium in advance (PL medium: 200g potatoes boiled in distilled water, filtered and brought to a final volume of 1000mL, then filtered again and 20g lactose added, sterilized at 121℃ for 15 min). The cultured *Fusarium oxysporum* vascularophile-specific strain Fj-fy and the Fo-SY20 strain obtained in Example 1 were used to create 2-3 mycelial cakes using a 1 mL blue pipette tip. These were then placed in PL medium supplemented with three antibiotics (chloramphenicol, streptomycin, and lactic acid) and incubated at 28℃ for 72 h on a shaker. The spore suspension was obtained by filtration through four layers of gauze. Spore concentration was calculated using a hemocytometer, and the inoculation concentration was 1.0 × 10⁻⁶. 8 CFU / mL.

[0033] 4. Inoculation test The first inoculation treatment steps were as follows: 30 seedlings were treated per group. The cowpea seedlings with two true leaves were gently pulled out, the soil was shaken off, and the roots were treated to remove any damage. Inoculation was then performed according to Table 1, and the seedlings were soaked in the prepared spore solution (concentration 1.0 × 10⁻⁶). 8 After 30 minutes (CFU / mL), transplant into a new pot containing a mixture of substrate, vermiculite, and natural soil in a ratio of 2:3:1.

[0034] The second inoculation treatment procedure is the same as the first inoculation treatment procedure, with 30 strains treated per group. The first and second inoculation treatments should be spaced 7 days apart.

[0035] Table 1

[0036] 5. Observe and record the experimental results. During the disease progression in cowpeas, disease incidence was recorded at 10, 13, 15, 18, and 21 days after inoculation, based on the disease index grading standard for individual diseased cowpea plants. The disease index grading standard for each treatment was calculated as follows: Level 0: No symptoms; Grade 1: Two cotyledons are yellow, but growth is normal; Level 3: Cotyledons yellow and die, affecting growth; Level 5: Cotyledons wither and die, and a small portion of the plant wilts; Level 7: Part of the plant withers or stops growing; Level 9: The entire plant wilts, falls over, or dies.

[0037] The formula for calculating the disease index is as follows: .

[0038] like Figure 3 As shown in Tables 2-3, the Fo-SY20 strain provided by this invention demonstrates significant application potential in controlling cowpea wilt, especially as a preventative biological control agent, as detailed below: (1) Excellent preventive and protective effect (connect Fo-SY20 first, then Fj-fy): In unsterilized soil, early inoculation with Fo-SY20 almost completely blocked the early occurrence of the disease. On the 10th day after Fj-fy inoculation, the disease incidence rates of X480 and G136 varieties were only 0.37% and 0.74%, respectively, which were significantly lower than those of the treatment group directly inoculated with Fj-fy (2.59% and 3.33%).

[0039] This protective effect lasted until the end of the experiment (21 days). For the X480 variety, the morbidity index of the treatment group that was first vaccinated with Fo-SY20 was only 28.89%, while that of the control group that was directly vaccinated with Fj-fy was as high as 79.26%, with a prevention efficacy of more than 50%. The trend of the G136 variety was the same (30.37% vs 65.93%).

[0040] (2) Limited therapeutic and recovery effects (Fj-fy first, then Fo-SY20): When plants were first infected with Fj-fy and then inoculated with Fo-SY20, their morbidity index was not significantly different from that of the control group directly inoculated with Fj-fy, indicating that Fo-SY20 could not effectively reverse the established infection.

[0041] (3) Synergistic enhancement effect of soil microbial community: Comparing experimental data from unsterilized and sterilized soil revealed that the disease incidence rates were generally lower in all treatment groups in the unsterilized soil. For example, the disease incidence rate of the G136 variety treated with Fo-SY20 was 30.37% after 21 days in the unsterilized soil, but rose to 63.70% in the sterilized soil. This indicates that a healthy soil microbial community can synergistically work with Fo-SY20 to enhance the plant's disease resistance.

[0042] (4) Variety resistance differences: The disease incidence of the resistant variety X480 was generally lower than that of the susceptible variety G136 in all treatments, but Fo-SY20 showed significant and consistent preventive effects in both varieties, proving its universality.

[0043] Therefore, the *Fusarium oxysporum* strain Fo-SY20, an endophytic non-pathogenic bacterium of cowpea provided by this invention, is a highly promising biocontrol strain. Its core advantage lies in prevention rather than treatment. Through pre-inoculation, it can effectively induce systemic resistance in cowpea and possess immune-stimulating activity, significantly reducing the incidence of cowpea wilt disease by more than 50%, with sustained and stable effects. In practical applications, it is recommended to use it as a seed treatment or root soaking agent before sowing or transplanting to achieve the best preventive control effect. The efficacy of this strain can synergize with beneficial microbial communities in the soil; therefore, maintaining soil health helps improve its disease resistance.

[0044] Table 2: Disease index of cowpeas in various treatments (natural soil, unsterilized)

[0045] Note: X480fj is the disease-resistant variety X480 that was inoculated with strain Fj-fy during the first inoculation; X480 Fo-SY20fj is the disease-resistant variety X480 that was inoculated with strain Fo-SY20 during the first inoculation and strain Fj-fy during the second inoculation; X480fjFo-SY20 is the disease-resistant variety X480 that was inoculated with strain Fj-fy during the first inoculation and strain Fo-SY20 during the second inoculation. G136fj is a susceptible variety of G136 that was inoculated with strain Fj-fy during the first inoculation; G136 Fo-SY200fj is a susceptible variety of G136 that was inoculated with strain Fo-SY20 during the first inoculation and strain Fj-fy during the second inoculation; G136fjFo-SY20 is a susceptible variety of G136 that was inoculated with strain Fj-fy during the first inoculation and strain Fo-SY20 during the second inoculation.

[0046] Table 3: Disease index of cowpea in various treatments (natural soil sterilization)

[0047] Example 3: Comprehensive Analysis of Differences in the Rhizosphere Microbial Community of Cowpea 1. Experimental materials Cowpea varieties: Disease-resistant variety X480; Disease-susceptible variety G136; Fusarium oxysporum: Fusarium oxysporum var. ... Fusarium oxysporum f . sp. tracheiphilum Example 1 shows the obtained Fo-SY20 strain.

[0048] 2. Test methods Based on the experimental groups in Table 1 of Example 2 after inoculation treatment, the temperature was controlled at 26-28℃ and cultured for 30 days. Rhizosphere soil samples were collected from each treatment group of the disease-resistant variety X480 and the disease-susceptible variety G136. At the same time, blank soil samples were collected from the unplanted plants cultured at the same time.

[0049] The community structure and diversity of rhizosphere bacteria in the rhizosphere soil samples were analyzed using high-throughput sequencing technology (16S rRNA amplicon sequencing). Microbial DNA was extracted from the cowpea rhizosphere soil using a soil DNA extraction kit (MagPure Soil DNA LQKit), and DNA quantification was performed using NanoDrop. DNA quality was assessed by 1.2% agarose gel electrophoresis. PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.4) and 1492R (5'-CCGTCAATTCCTTTRAGTTT-3', SEQ ID NO.5).

[0050] Bioinformatics analysis of soil bacterial communities was performed based on sequencing data. The workflow was as follows: First, paired-end 16S rRNA amplicon sequences were assembled, and forward and reverse primers were removed from the data after quality control. Sequences were merged and quality controlled using Vsearch software. Redundancy was removed from reads (sequences with copy number > 2 were retained), and clustering was performed at 97% similarity. Chimeras were removed, and species annotation was performed based on the Greengenes database. Ineligible OTUs were eliminated, and an OTU abundance table at 97% similarity level was constructed. The community composition of samples at each taxonomic level was statistically analyzed. Then, Qiime 1.9.1 was used to generate abundance tables at each taxonomic level, and Alpha diversity indices (Chao1, Shannon, Simpson, ACE) were calculated. Diversity differences between groups were analyzed using the Wilcoxon rank-sum test (P < 0.05). Finally, differential enrichment analysis of microorganisms, plotting, and non-metric multidimensional scaling analysis were performed using R 4.0.5 packages such as edgeR, ggplot2, and vegan.

[0051] 3. Analysis of differences in rhizosphere microbiota between the Fo-SY20 strain inoculation group and the Fj-fy strain inoculation group. Alpha diversity primarily reflects the richness and evenness within a community, while Beta diversity primarily reflects the magnitude of differences in community structure among different treatments.

[0052] like Figure 4 As shown, inoculation with different pathogens significantly affected the diversity and community structure of rhizosphere bacteria and fungi in cowpea, and the patterns of change for bacteria and fungi differed. Combined Alpha and Beta diversity analyses revealed that inoculation with both Fo-SY20 and Fj-fy strains reduced rhizosphere microbial diversity, with bacteria showing a more pronounced effect. Both pathogens altered community structure, clearly distinguishable on the Beta diversity plot. The rhizosphere effect was significant, with rhizosphere soil diversity far exceeding that of natural soil, and the community structure was completely different. Overall, bacteria were more sensitive to pathogen inoculation, while fungi were relatively more stable.

[0053] like Figure 5 As shown, the response patterns of rhizosphere bacterial communities to disturbances caused by inoculation with different bacterial strains were revealed. The rhizosphere effect was the core driving force, and the differences between the rhizosphere environment and natural soil were far greater than the differences between different pathogen inoculations. The screening effect of root exudates significantly reshaped the bacterial community structure, inhibiting oligotrophic saprophytic bacteria and enriching eutrophic and plant-associated bacteria. Differential regulation by pathogen inoculation: The Fo-SY20 and Fj-fy strains exhibited different regulatory patterns on rhizosphere bacteria. The Fo-SY20 strain was more inclined towards "active construction," forming a unique community by enriching new taxa; the Fj-fy strain was more inclined towards "passive destruction," mainly inhibiting existing sensitive taxa, resulting in poorer community stability. High sensitivity of bacterial communities: Both changes in the rhizosphere environment and pathogen stress triggered fluctuations in the abundance of numerous bacterial species.

[0054] By using LEfSe (LDA effect size) analysis, differential species (Biomarkers) among the groups inoculated with Fo-SY20 (A3), inoculated with Fj-fy (A4), water control (A5), and natural soil (F1) were identified at the phylum to genus level, visually demonstrating the core indicator groups of rhizosphere bacteria under different treatments.

[0055] like Figure 6As shown, the cladogram on the left displays the phylogenetic hierarchy from phylum (inner layer) to genus (outer layer) in concentric circles. Color nodes mark the dominant taxa that are significantly associated with each group. A3 (following Fo-SY20, red): The core indicator taxa are Gammaproteobacteria, Enterobacterales, and Enterobacteriaceae, etc. These taxa are mostly related to environmental stress response and organic matter decomposition, reflecting the stress adaptation characteristics of rhizosphere bacteria after CS-20 inoculation.

[0056] like Figure 6 As shown, the bar chart on the right quantifies the degree of influence of different species (LDAS score). The longer the bar, the greater the contribution of that group to the differences between groups. In group A3, the LDA scores of Gamma-Proteobacteria and Enterobacteriaceae are outstanding, and they are the most discriminative biomarkers after Fo-SY20 inoculation.

[0057] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A non-pathogenic Fusarium oxysporum endophyte in cowpea ( Fusarium oxysporum Fo-SY20, characterized in that, The cowpea endophytic non-pathogenic Fusarium oxysporum Fo-SY20 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42441.

2. The cowpea endophytic non-pathogenic Fusarium oxysporum Fo-SY20 according to claim 1, characterized in that, The cowpea endophytic non-pathogenic Fusarium oxysporum Fo-SY20 trpB The gene sequence is shown in SEQ ID NO.

1.

3. A microbial agent, characterized in that, The inoculum contains the non-pathogenic Fusarium oxysporum Fo-SY20 endophytic fungus of cowpea as described in any one of claims 1 to 2.

4. The microbial agent according to claim 3, characterized in that, The preparation method of the microbial agent is as follows: the cowpea endophytic non-pathogenic Fusarium oxysporum Fo-SY20 as described in any one of claims 1 to 2 is inoculated into PL medium and cultured.

5. The microbial agent according to claim 4, characterized in that, The culture conditions were: 180 rpm, 28℃ shaker culture for 72 h.

6. The microbial agent according to claim 4, characterized in that, The concentration of the non-pathogenic Fusarium oxysporum Fo-SY20 endophytic strain in the inoculum was 1×10⁻⁶. 8 CFU / mL.

7. The application of the non-pathogenic Fusarium oxysporum Fo-SY20 endophytic fungus of cowpea as described in any one of claims 1 to 2, or the fungal agent as described in any one of claims 3 to 6, in the prevention of cowpea wilt disease, characterized in that, The application involves adding the non-pathogenic Fusarium oxysporum Fo-SY20 or the bacterial agent to the soil before sowing or transplanting cowpeas, or soaking the roots of cowpea plants with the non-pathogenic Fusarium oxysporum Fo-SY20 or the bacterial agent during cowpea transplanting.

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