Compound microbial agent for preventing and treating muskmelon fusarium wilt and application thereof

Abstract

The application discloses a compound microbial agent for preventing and treating muskmelon fusarium wilt and application thereof and belongs to the technical field of biotechnology.The active ingredients of the compound microbial agent are composed of Streptomyces microflavus and Trichoderma harzianum.The compound microbial agent provided by the application can prevent and control diseases by inhibiting the colonization and propagation of pathogenic bacteria in the root system and can promote the growth of plants.The compound of Streptomyces microflavus and Trichoderma harzianum is an optimal scheme for green prevention and control of muskmelon fusarium wilt and can provide technical support for reducing the dependence of the industry on chemical fungicides.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a compound microbial agent for the prevention and control of wilt disease in melons and its application. Background Technology

[0002] Melon is a widely cultivated horticultural crop with high economic value, but melon wilt (caused by Fusarium oxysporum melonensis) is a serious disease. It is widespread, has a long incubation period, and is difficult to control, which can reduce melon yield by more than 50% or even cause crop failure, thus restricting the development of the industry.

[0003] Traditional control methods rely on chemical fungicides, which easily lead to drug resistance in pathogens and leave residues that pollute the environment and harm health, contradicting the principles of green agriculture. While there has been progress in microbial inoculants, most are single-agent formulations, limiting the stability and persistence of their field efficacy. Research on compound inoculants is also insufficient. Therefore, this study selected four potential biocontrol microbial inoculants and combined them in pairs to explore the optimal formulation, providing support for the green control of Fusarium wilt in melons. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a compound microbial agent for the prevention and control of Fusarium wilt in melons. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides a compound microbial agent for controlling Fusarium wilt in melons, wherein the active ingredient of the compound microbial agent is any one of the following: A1) is composed of Streptomyces flavus and Trichoderma harzianum; A2) Composed of Bacillus subtilis and Streptomyces flavus; A3) is composed of Trichoderma longifolia and Streptomyces flavus; A4) is composed of Trichoderma longifolia and Trichoderma harzianum; A5) is composed of Bacillus subtilis and Trichoderma longifolia; A6) is composed of Bacillus subtilis and Trichoderma harzianum.

[0006] In this invention, the term "active ingredient" refers to one or more substances in the compound microbial agent (composition) that can impart resistance to melon wilt disease to plants.

[0007] In some embodiments of the present invention, the compound microbial agent is composed of *Streptomyces flavus* and *Trichoderma harzianum* agents. The active ingredient of the *Streptomyces flavus* agent is *Streptomyces flavus*. The active ingredient of the *Trichoderma harzianum* agent is *Trichoderma harzianum*.

[0008] In some embodiments of the present invention, the compound microbial agent is composed of Bacillus subtilis agent and Streptomyces flavus agent. The active ingredient of the Bacillus subtilis agent is Bacillus subtilis. The active ingredient of the Streptomyces flavus agent is Streptomyces flavus.

[0009] In some embodiments of the present invention, the compound microbial agent is composed of *Trichoderma longicornis* and *Streptomyces flavus* agents. The active ingredient of the *Trichoderma longicornis* agent is *Trichoderma longicornis*. The active ingredient of the *Streptomyces flavus* agent is *Streptomyces flavus*.

[0010] In some embodiments of the present invention, the compound microbial agent is composed of *Trichoderma longicornis* and *Trichoderma harzianum* agents. The active ingredient of the *Trichoderma longicornis* agent is *Trichoderma longicornis*. The active ingredient of the *Trichoderma harzianum* agent is *Trichoderma harzianum*.

[0011] In some embodiments of the present invention, the compound microbial agent is composed of Bacillus subtilis inoculant and Trichoderma longifolia inoculant. The active ingredient of the Bacillus subtilis inoculant is Bacillus subtilis. The active ingredient of the Trichoderma longifolia inoculant is Trichoderma longifolia.

[0012] In some embodiments of the present invention, the compound microbial agent is composed of Bacillus subtilis and Trichoderma harzianum. The active ingredient of the Bacillus subtilis agent is Bacillus subtilis. The active ingredient of the Trichoderma harzianum agent is Trichoderma harzianum.

[0013] In some specific embodiments of the present invention, the Streptomyces flavus inoculant is a product of Henan Haomeite Biotechnology Co., Ltd. with product number 10160135850895.

[0014] In some specific embodiments of the present invention, the Trichoderma harzianum inoculant is a product of BioVolkswagen Inc. of the United States with product number 632009830503.

[0015] In some specific embodiments of the present invention, the long-branched Trichoderma inoculant is a product with product number 207106 from Beijing Leili Marine Biological New Industry Co., Ltd.

[0016] In some specific embodiments of the present invention, the Bacillus subtilis inoculant is a product with product number 207505 from Beijing Leili Marine Biological New Industry Co., Ltd.

[0017] The melon wilt disease is caused by Fusarium oxysporum melon-specific strain.

[0018] The mass ratio of the inoculum containing *Streptomyces flavus* to the inoculum containing *Trichoderma harzianum* is 1:1.

[0019] The present invention also provides a method for preventing and controlling wilt disease in melons, comprising the following steps: applying the aforementioned compound microbial agent to the recipient plant.

[0020] In some specific embodiments of the present invention, the application method is root irrigation.

[0021] This invention also provides the application of compound microbial agents in the prevention and control of melon wilt or in the preparation of products for the prevention and control of melon wilt, wherein the melon wilt is caused by Fusarium oxysporum melon-specific strain.

[0022] In some embodiments of the present invention, the product is a microbial agent.

[0023] The present invention also provides the application of compound microbial agents in increasing the leaf area of ​​recipient melons or in the preparation of products that increase the leaf area of ​​recipient melons, wherein the recipient melons are melons that have developed wilt disease.

[0024] The present invention also provides the application of compound microbial agents in increasing the plant height of recipient melons or in the preparation of products that increase the plant height of recipient melons, wherein the recipient melons are melons that have developed wilt disease.

[0025] The present invention also provides the application of compound microbial agents in increasing the chlorophyll content of recipient melons or in preparing products that increase the chlorophyll content of recipient melons, wherein the recipient melons are melons that have developed wilt disease.

[0026] The present invention also provides the application of compound microbial agents in improving the antioxidant capacity of recipient melons or in preparing products that improve the antioxidant capacity of recipient melons, wherein the recipient melons are melons that have developed wilt disease.

[0027] This invention was verified through two sets of experiments: a pre-experiment with a single microbial agent and a system experiment with a compound microbial agent. While single microbial agents possess basic antibacterial and disease-preventing capabilities, their efficacy is relatively low and their growth-promoting effects are limited. In contrast, all binary compound microbial agents showed significantly better overall control and growth-promoting effects than any single agent, with the combination of *Streptomyces flavus* and *Trichoderma harzianum* exhibiting the best overall effect. This invention systematically measured plant growth and antioxidant physiological indicators only in the compound treatment group, confirming that the compound microbial agents can control Fusarium wilt through a triple mechanism: inhibiting pathogen colonization in the roots and stems, simultaneously activating the melon's antioxidant system, and promoting plant growth. This is a preferred green control solution for melon Fusarium wilt and can provide comprehensive technical support for the industry to reduce its reliance on chemical fungicides. Attached Figure Description

[0028] Figure 1 This image shows the confrontation culture between different microbial inoculants and Fusarium. Note: The left side of image AD shows the pathogen control, and the right side shows the confrontation growth of biocontrol bacteria and pathogens. The biocontrol bacteria are, in order, Bacillus subtilis, Trichoderma longifolia, Trichoderma harzianum, and Streptomyces flavus.

[0029] Figure 2 The study investigated the effects of different single microbial inoculants on the growth of melon plants; where A represents the maximum leaf area, B represents the plant height, and C represents the stem diameter; CK represents the pathogen stress control, NC represents the healthy water control, and BS, TL, SF, and TH represent single-strain treatments with Bacillus subtilis, Trichoderma longifolia, Streptomyces flavus, and Trichoderma harzianum, respectively.

[0030] Figure 3 The study compares the control effects of different microbial inoculants combined with each other on Fusarium wilt in melons. Note: A represents inoculation with the pathogen only; B represents irrigation with sterile water only; C represents inoculation with the pathogen and *Bacillus subtilis* + *Trichoderma longifolia*; D represents inoculation with the pathogen and *Bacillus subtilis* + *Streptomyces flavus*; E represents inoculation with the pathogen and *Bacillus subtilis* + *Trichoderma harzianum*; F represents inoculation with the pathogen and *Trichoderma longifolia* + *Streptomyces flavus*; G represents inoculation with the pathogen and *Trichoderma longifolia* + *Trichoderma harzianum*; H represents inoculation with the pathogen and *Streptomyces flavus* + *Trichoderma harzianum*.

[0031] Figure 4 The effects of different microbial inoculants on the colonization of pathogens in the root and stem regions of melon were investigated. A: Irrigation with sterile water only; B: Inoculation with pathogens only; C: Inoculation with pathogens and *Bacillus subtilis* + *Trichoderma longifolia*; D: Inoculation with pathogens and *Bacillus subtilis* + *Streptomyces flavus*; E: Inoculation with pathogens and *Bacillus subtilis* + *Trichoderma harzianum*; F: Inoculation with pathogens and *Trichoderma longifolia* + *Streptomyces flavus*; G: Inoculation with pathogens and *Trichoderma longifolia* + *Trichoderma harzianum*; H: Inoculation with pathogens and *Streptomyces flavus* + *Trichoderma harzianum*.

[0032] Figure 5 This study investigated the effects of combined bacterial agents on the growth and chlorophyll content of melon plants. Note: CK was the stress control group inoculated only with pathogens, and NC was the healthy control group irrigated only with water. BS + TL, BS + SF, BS + TH, TL + SF, TL + TH, and SF + TH represented the treatments of Bacillus subtilis + Trichoderma longifolia, Bacillus subtilis + Streptomyces flavus, Bacillus subtilis + Trichoderma harzianum, Trichoderma longifolia + Streptomyces flavus, Trichoderma longifolia + Trichoderma harzianum, and Streptomyces flavus + Trichoderma harzianum, respectively. All groups were irrigated with a suspension of pathogen spores. In the figure, A to D represent the maximum leaf area, plant height, stem diameter, and chlorophyll content of melon plants under different pathogen stress treatments, respectively; different uppercase and lowercase letters indicate significant differences at the P<0.01 and P<0.05 levels, respectively.

[0033] Figure 6This study investigated the effects of combined microbial agents on the antioxidant system of melon leaves. Note: CK was the stress control group inoculated only with pathogens, and NC was the healthy control group irrigated only with water. BS + TL, BS + SF, BS + TH, TL + SF, TL + TH, and SF + TH represented the combined treatments of *Bacillus subtilis* + *Trichoderma longifolia*, *Bacillus subtilis* + *Streptomyces flavus*, *Bacillus subtilis* + *Trichoderma harzianum*, *Trichoderma longifolia* + *Streptomyces flavus*, *Trichoderma longifolia* + *Trichoderma harzianum*, and *Streptomyces flavus* + *Trichoderma harzianum*, respectively. All groups were irrigated with a suspension of pathogen spores. In the figure, A–D represent the maximum leaf area, plant height, stem diameter, and chlorophyll content of melon plants under different pathogen stress treatments, respectively; different uppercase and lowercase letters indicate significant differences at the P<0.01 and P<0.05 levels, respectively. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] Example 1 1.1 Test Methods 1.1.1 Source of Fusarium wilt pathogen and tested microbial inoculants The fungus used in the experiment to cause wilt of melon was Fusarium oxysporum melon-specific strain (Fusarium oxysporum). Fusarium oxysporum f.sp. melonis Thin-skinned melon (item number TG611) (the test melon material and melon seeds in the following examples): Donated to the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and described in the non-patent literature "Study on the Targeted Control of Melon Wilt Disease by Bacillus vesicularis ZF2 and Its Mechanism of Action [D]. Chinese Academy of Agricultural Sciences, 2023-06-01," available to the public from Beijing University of Agriculture. This biological material is only used to replicate the relevant experiments of this invention and cannot be used for other purposes. Fusarium oxysporum is numbered TG1605240301 in the aforementioned literature.

[0037] Potato Dextrose Agar (PDA) medium is a commercially available product from Cooler Master.

[0038] The microbial agents used in the experiment were Bacillus subtilis (microbial agent, effective viable count ≥ 20 billion / g) (product number: 207505; registration certificate number: Microbial Fertilizer (2015) Approval No. (1567)) and Trichoderma longifolia ( Trichoderma longibrachiatum (Microbial inoculant, effective live bacteria count ≥ 1 billion / g) produced by Beijing Leili Marine Biological New Industry Co., Ltd. (Item No.: 207106; Registration Certificate No.: Microbial Fertilizer (2026) Approval No. (16583)), Trichoderma harzianum ( Trichoderma harzianum (Item No.: 632009830503; Registration Certificate No.: PD20140319) (Microbial agent, effective ingredient content: 300 million CFU / g) is produced by BioVol Corporation, USA. Streptomyces flavus (Item No.: 10160135850895; Registration Certificate No.: Microbial Fertilizer (2018) Approval No. (3262)) (Microbial agent, effective live bacteria count ≥ 200 million / g) is produced by Henan Haomeite Biotechnology Co., Ltd.

[0039] 1.1.2 Confrontation Cultivation Method Take 0.5g of the bacterial agent, dilute it with 50ml of sterile water, and take 0.1ml of the diluted solution. Spread it evenly on PDA medium. After the bacteria have grown, use a microbial punch to collect the mycelial cake and transfer it to a new PDA medium for further cultivation. The inhibitory effect of biocontrol bacteria (Bacillus subtilis, Trichoderma longifolia, Trichoderma harzianum, and Streptomyces flavus) on the pathogen (Fusarium wilt of melon) was tested using a confrontation culture method. The pathogen mycelial cake was placed in the center of the PDA plate, and the biocontrol mycelial cake was inoculated at the four opposite corners of the plate. A PDA plate inoculated only with pathogen mycelial cake served as a control. Each treatment was repeated three times. The plates were incubated upside down at 28 ℃. After 10 days, the diameter of the pathogen colonies in the control and each treatment was measured, and the inhibition rate was calculated.

[0040] Method for measuring the diameter of pathogens: The diameter of pathogen colonies is measured using the cross-multiplication method. The measurement is repeated three times and the average value is taken to reduce errors.

[0041] .

[0042] 1.1.3 Single-strain treatment setup Six treatment groups were used: CK (inoculation with pathogen only, stress control), NC (water only, healthy control), BS (Bacillus subtilis monotherapy), TL (Trichoderma longifolia monotherapy), SF (Streptomyces flavus monotherapy), and TH (Trichoderma harzianum monotherapy). The single-strain agent was diluted 800 times with sterile water and applied by root irrigation.

[0043] 1.1.4 Single-strain test indicators 1) Plate confrontation antibacterial test (Figure 1); 2) Disease index and single-strain control efficacy of potted plants after 25 days (Table 2); 3) Maximum leaf area, plant height, and stem diameter growth indicators of melon seedlings (Figure 2); 1.1.5 Microbial inoculant compound treatment Four types of microbial agents were combined in pairs at a 1:1 mass ratio. Accurate weighing and thorough mixing were performed, followed by dilution 800 times with sterile water and thorough shaking to obtain the application solution, which was prepared and used immediately. Eight treatments were set up in the experiment: inoculation only with a suspension of *Fusarium wilt* spores (control group, CK); irrigation only with sterile water (healthy control group, NC); *Bacillus subtilis* + *Streptomyces flavus* (BS+SF); *Bacillus subtilis* + *Trichoderma longifolia* (BS+TL); *Bacillus subtilis* + *Trichoderma harzianum* (BS+TH); *Trichoderma longifolia* + *Streptomyces flavus* (TL+SF); *Trichoderma longifolia* + *Trichoderma harzianum* (TL+TH); *Streptomyces flavus* + *Trichoderma harzianum* (SF+TH).

[0044] 1.1.6 Methods for inoculating melon seedlings with Fusarium wilt pathogen, application of inoculants, and disease investigation. Soak melon seeds in 55℃ water for 30 minutes, then remove them and place them on moist filter paper. Keep them moist at 28℃ to promote germination. Once the seeds show white sprouts, sow them in 72-cell seedling trays. When the melon seedlings have fully expanded their cotyledons, transplant them into pots filled with sterilized nutrient soil, one seedling per pot. Each treatment is repeated three times, with one seedling per replicate. When the melon seedlings have two true leaves, use a root drenching method, applying 50 mL of a single or compound inoculant to each seedling (a total of three root drenches). 24 hours later, inoculate the roots of the melon seedlings with 10 mL of a 1×10⁻⁶ spore suspension of the melon wilt pathogen (the concentration of which is 1×10⁻⁶). 6 The control group (CK) used sterile water for root irrigation and inoculation with the pathogen, while the control group (NC) used sterile water for root irrigation but without pathogen inoculation. Plant growth was observed. After 25 days, the disease occurrence and colonization of the wilt pathogen in the affected parts were investigated, and the disease index, control effect, and the number of wilt pathogens in the root and stem were calculated. The wilt disease severity grading standard for melon was divided into four levels: 0: No obvious disease signs: no vascular discoloration, healthy plant, all leaves are green; 1: Yellowing of upper and lower leaves, vascular discoloration but only occurring in the roots; 2: Lower leaves die and some upper leaves wilt, roots and vascular discoloration; 3: The entire plant dies. The relative grade value refers to the disease severity level corresponding to the affected plant.

[0045] .

[0046] .

[0047] Spore count (cfu / ml) = average number of colonies on plate × dilution factor / inoculum volume.

[0048] 1.1.7 Methods for measuring plant growth-related indicators 1) Plant height measurement Measurements were taken using a ruler, starting at the base of the plant stem in contact with the soil and ending at the apical growing point of the main stem, with readings accurate to 0.1 cm. Nine plants were measured for each treatment, with three plants constituting one replicate, for a total of three replicates. The height of each plant within each group was measured, and the average value of each group was calculated. The final average value of the three replicates was taken as the plant height data for that treatment.

[0049] 2) Stem diameter measurement The stem diameter was measured using digital vernier calipers at a point 5 cm above the base of the stem, with readings accurate to 0.01 mm. Nine plants were measured for each treatment, with three plants constituting one replicate, for a total of three replicates. The mean value of each individual plant was calculated, and then the mean value of the three plants within each replicate was calculated. The final mean value of the three replicates was used as the stem diameter data for that treatment.

[0050] 3) Measurement of maximum leaf area Select the largest functional leaf on the plant and measure its length (the longest distance from the base to the tip) and width (the widest part of the leaf) using a ruler, accurate to 0.1 cm. The leaf area is calculated using the formula: S = L × W × K.

[0051] In the formula, S is the leaf area (cm²) 2 L is the leaf length (cm), and W is the maximum leaf width (cm).

[0052] Nine plants were measured for each treatment, with three plants per replicate for a total of three replicates. The maximum leaf area of ​​a single plant within each group was determined and the mean within each group was calculated. The mean of the three replicates was taken as the maximum leaf area data for that treatment.

[0053] 4) Chlorophyll content determination Sampling was conducted at the early stage of wilt disease in melons. Leaves from approximately the same location were selected, and holes were punched in the middle of the veins, avoiding the midrib. Leaves from three plants within the same replicate were mixed thoroughly, and 0.2 g of the mixed sample was weighed for identification. Chlorophyll was extracted using a slightly modified ethanol-acetone extraction method, following Arnon's (1949V) approach. The absorbance at 663 nm and 645 nm was measured using a spectrophotometer, and the total chlorophyll content was calculated using the following formula.

[0054] .

[0055] In the formula, V is the volume of the extract (ml), m is the fresh weight of the sample (g), A663 is the absorbance value at 663 nm, and A645 is the absorbance value at 645 nm.

[0056] 5) Superoxide dismutase (SOD) activity assay: The SOD activity in plant tissues was determined using the WST-1 method based on the Superoxide Dismutase Activity Assay Kit (catalog number: BC5160) manufactured by Beijing Solarbio Science & Technology Co., Ltd. The principle is that the reaction system of xanthine and xanthine oxidase can generate superoxide anions (O2). - O2 - It can react with WST-1 reagent to form a water-soluble yellow formazan with a characteristic absorption peak at 450 nm; SOD in plant tissues can specifically scavenge O2. - This inhibits the formation of yellow formazan. The lower the absorbance of the reaction solution, the stronger the effect of SOD on O2. - The stronger the scavenging ability and the higher the enzyme activity, the SOD activity of plant tissue can be quantitatively obtained by calculating the difference in absorbance and the percentage of inhibition.

[0057] 6) Peroxidase (POD) Activity Assay: The peroxidase activity assay kit (catalog number: BC0090) manufactured by Beijing Solarbio Science & Technology Co., Ltd. was used to determine the POD activity in plant tissues by visible light spectrophotometry. The principle is as follows: In the reaction system, POD can catalyze the oxidation reaction of the substrate, and the product has a characteristic absorption peak at 470 nm. By measuring the change in absorbance (ΔA) before and after the reaction, the POD activity of the plant tissue can be calculated.

[0058] 7) Malondialdehyde (MDA) content determination: The MDA content in plant tissues was determined using a visible spectrophotometric method with a malondialdehyde content detection kit (catalog number: BC6410) manufactured by Beijing Solarbio Science & Technology Co., Ltd. The principle is as follows: MDA is the end product of lipid peroxidation in plant tissues. In an acidic and hot environment, this substance can combine with thiobarbituric acid to form a brownish-red trimethylolamine compound. This product exhibits characteristic absorption at a wavelength of 532 nm. A dual-wavelength colorimetric method at 532 nm and 600 nm was used, and the absorbance difference was calculated for analysis, thus quantitatively determining the MDA content in plant tissues.

[0059] 8) Determination of proline (Pro) activity in melon leaves: Take 0.2 g of melon leaf sample in a mortar, add 1.5 ml of 5% sulfosalicylic acid, grind thoroughly into a homogenate, place in a 10 ml centrifuge tube, seal with sealing film, boil in a water bath for 10 min, cool, and centrifuge at 11500 × g for 15 min. Mix 1 mL of the supernatant with 2 mL of acidic ninhydrin and 2 mL of glacial acetic acid in a test tube, shake well, boil in a water bath again for 30 min, cool naturally to room temperature, add 5 mL of toluene, shake thoroughly, and allow to separate into layers. Take the upper toluene extract in a cuvette and measure the absorbance at 520 nm. Calculate the proline (Pro) content using a standard curve.

[0060] 1.2 Data Analysis Each experimental treatment contained three biological replicates, with three seedlings per replicate. WPS Office 12.1.0.26375 was used for data entry, organization, and preliminary verification. GraphPad Prism 10 software was used to perform one-way ANOVA, and Duncan's multiple range test was used for multiple comparisons to determine the significance level of differences between treatments. All significance tests used P < 0.05 as the criterion for significance. Data visualization charts were also created.

[0061] 2. Results and Analysis 2.1 Inhibitory effect of biocontrol bacteria on Fusarium wilt pathogen The confrontation culture lasted for 10 days. After 10 days, the colony diameter of the pathogen in the control and treatment groups was measured, and the inhibition rate was calculated. The results showed that Bacillus subtilis inhibited the pathogen of Fusarium wilt in melon by 28.29%, Trichoderma longifolia by 60.61%, Streptomyces flavus by 69.03%, and Trichoderma harzianum by 50.00%, all of which showed significant antibacterial activity. Figure 1 (Table 1). Among them, *Streptomyces flavus* showed the best inhibitory effect on the pathogen causing wilt of melon, followed by *Trichoderma longifolia*, *Trichoderma harzianum*, and *Bacillus subtilis*.

[0062] Table 1. Antibacterial effects of different biocontrol bacteria against pathogens

[0063] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the 0.05 level.

[0064] 2.2 As shown in Table 2, although all four single bacterial agents have some disease prevention effect, the overall control efficacy is low, with the highest single-strain TL control efficacy being only 50%; combined with the single-strain growth index results in Figure 2: Leaf area and plant height in the CK pathogen-stressed group were significantly lower than those in the NC healthy group. All single-strain treatments could alleviate growth inhibition, with leaf area and plant height both lower than those in the NC healthy group. However, the growth-promoting ability of a single inoculant was limited and could not completely offset the damage caused by pathogen stress.

[0065] Table 2. Effects of single application of fungicide on disease index and control efficacy in melon

[0066] 2.3 Compound microbial inoculants reduce the severity index of Fusarium wilt in melons Different combinations of microbial inoculants showed good control effects against wilt disease in melons (Table 3). Twenty-five days after inoculation with the pathogen, the disease index of melon plants inoculated only with a suspension of spores of the wilt fungus (control treatment) was 66.67. The disease index after treatment with *Bacillus subtilis* + *Streptomyces flavus* was 27.78, with a control effect of 58.33%. The disease index after treatment with *Bacillus subtilis* + *Trichoderma longifolia* was 44.44, with a control effect of 33.34%. The disease index after treatment with *Bacillus subtilis* + *Trichoderma harzianum* was 55.56, with a control effect of 16.67%. The disease index after treatment with *Trichoderma longifolia* + *Streptomyces flavus* was 37.04, with a control effect of 44.44%. The disease index after treatment with *Trichoderma longifolia* + *Trichoderma harzianum* was 39.89, with a control effect of 41.67%. The disease index after treatment with *Streptomyces flavus* + *Trichoderma harzianum* was 16.66, with a control effect of 75.01%. The treatment groups of Streptomyces flavus + Trichoderma harzianum (SF+TH) and Bacillus subtilis + Streptomyces flavus (BS+SF) showed good control effects.

[0067] Table 3. Effects of combined microbial agents on disease index and control efficacy in melon.

[0068] 2.4 Compound formulation of microbial inoculants reduces the colonization of melon pathogens. Microbial inoculants significantly reduced the number of Fusarium wilt pathogens in the roots and stems of infected melons, thereby inhibiting the occurrence of Fusarium wilt in melons. Figure 3 The number of Fusarium wilt pathogens in the root and stem regions of melons in each treatment was detected by plate counting. The results (Table 4) showed that 25 days after inoculation with the pathogen, the number of Fusarium wilt pathogens in the root and stem regions of melons inoculated only with the pathogen was 15.87 × 10⁻⁶. 6 cfu / g ( Figure 4 (A) The number of pathogens after treatment with Bacillus subtilis + Trichoderma longifolia was 12.20 × 10⁻⁶.6 cfu / g ( Figure 4 (C) The pathogen count after treatment with Bacillus subtilis + Streptomyces flavus was 7.20 × 10⁻⁶. 6 cfu / g ( Figure 4 (D) The number of pathogens after treatment with Bacillus subtilis + Trichoderma harzianum was 12.6 × 10⁻⁶. 6 cfu / g ( Figure 4 The pathogen count after treatment with *Trichoderma longifolia* and *Streptomyces flavus* was 8.03 × 10⁻⁶. 6 cfu / g ( Figure 4 The pathogen count after treatment with *Trichoderma longifolia* and *Trichoderma harzianum* was 9.27 × 10⁻⁶. 6 cfu / g ( Figure 4 The pathogen count after treatment with *Streptomyces flavus* and *Trichoderma harzianum* was 6.40 × 10⁻⁶. 6 cfu / g ( Figure 4 (H).

[0069] Compared with the control group, the number of Fusarium wilt pathogens in the root and stem parts of melons in the treatment groups decreased by 23.13%, 54.63%, 20.60%, 49.40%, 41.59%, and 59.67%, respectively (Table 4). A certain positive correlation was found between the reduction in pathogen count and the control effect; that is, the greater the decrease in pathogen count, the higher the corresponding control effect. This further confirms that the compound microbial inoculant effectively controls the occurrence and development of Fusarium wilt in melons by inhibiting the colonization and reproduction of pathogens in the root and stem parts.

[0070] Table 4. Effects of combined microbial agents on the colonization of melon pathogens

[0071] 2.5 Compound microbial inoculants enhance the aboveground growth of melons Microbial inoculants can increase the maximum leaf area, plant height, stem diameter, and chlorophyll content of melon plants to varying degrees. Figure 5 ).

[0072] The largest leaf area of ​​melon plants in the control group (CK) treated only with the pathogen was 32.46 cm². 2 The leaf area was significantly lower than that of the blank control group (NC, 39.66 cm², P<0.01) which was only watered, indicating a significant inhibition of leaf growth. After applying the combined treatments of microbial agents, the leaf area of ​​the plants increased to varying degrees. The treatment with *Streptomyces flavus* + *Trichoderma harzianum* (SF + TH) showed the best effect, with the largest leaf area reaching 39.10 cm², close to the blank control level. The other combined treatments also effectively increased leaf area and mitigated the adverse effects of pathogens on leaf growth.

[0073] The plant height of the pathogen control group (CK) was 15.89 cm, which was significantly lower than that of the blank control (NC, 23.00 cm, P<0.01), indicating that the pathogen severely inhibited the aboveground growth of the plants. All the combined treatments of various microbial agents could significantly increase the plant height. The combination of *Streptomyces flavus* and *Trichoderma harzianum* (SF + TH) showed the best effect in alleviating pathogen damage, with a plant height of 20.61 cm. Combinations such as *Trichoderma longifolia* + *Trichoderma harzianum* and *Trichoderma longifolia* + *Streptomyces flavus* also effectively increased the plant height, and all of them showed obvious growth-promoting effects.

[0074] The stem diameter of the melon plants in the pathogen control group was 5.37 mm, lower than that of the blank control group (NC, 5.83 mm), indicating that pathogen infection significantly inhibited stem growth in melon plants. Compared with the control group, all the combined treatments of various microbial agents could increase stem diameter to varying degrees, effectively alleviating the inhibition of stem growth by the pathogen. Among them, the combination of *Trichoderma longifolia* + *Streptomyces flavus* (SF + TH) showed the best effect, with a stem diameter of 5.72 mm; combinations such as *Bacillus subtilis* + *Trichoderma harzianum* also showed some improvement, while the treatment of *Trichoderma longifolia* + *Trichoderma harzianum* was relatively weaker among the various combination treatments.

[0075] The chlorophyll content in the pathogen control group was only 0.167 mg / g, significantly lower than that in the blank control (NC, 0.669 mg / g, P<0.01), indicating that pathogen stress can disrupt photosynthetic pigment synthesis and reduce the photosynthetic capacity of melon plants. All microbial combination treatments significantly increased chlorophyll content, with the *Streptomyces flavus* + *Trichoderma harzianum* treatment (SF + TH) showing the highest chlorophyll content at 0.559 mg / g. Combinations such as *Bacillus subtilis* + *Streptomyces flavus* and *Trichoderma longifolia* + *Streptomyces flavus* also increased chlorophyll content and enhanced the photosynthetic capacity of plants under pathogen stress.

[0076] 2.6 Microbial inoculant compound enhances leaf antioxidant system Compared with the pathogen stress group (CK), the healthy control group (NC), which was only watered with clean water, showed increased activities of superoxide dismutase (SOD) and peroxidase (POD) in the leaves of melons. This can remove reactive oxygen species, reduce membrane lipid peroxidation, and protect the integrity of leaf cell membrane structure. In contrast, the activities of SOD and POD in the leaves of the CK melons decreased, which promoted the accumulation of reactive oxygen species and increased the content of malondialdehyde (MDA), further aggravating leaf cell membrane damage. At the same time, the changes in the content of proline (Pro), as an osmotic regulator, can reflect the plant's resistance to pathogen stress. Under pathogen stress, the plant can reduce leaf damage by maintaining cell osmotic balance.

[0077] SOD scavenges superoxide anions, acting as the first line of defense against oxidation. According to... Figure 6 The results showed that, compared with the control (CK), all microbial inoculant combinations significantly increased SOD activity in melon leaves (P<0.01). Among them, the combination of *Streptomyces flavus* and *Trichoderma harzianum* (SF + TH) had the strongest effect on increasing SOD activity, with an activity increase of 62.93% compared to the CK; the combination of *Trichoderma longiflora* and *Trichoderma harzianum* (TL + TH) was the second strongest, increasing SOD activity by 48.93% compared to the CK. The combinations of *Bacillus subtilis* + *Trichoderma longiflora* (BS + TL), *Bacillus subtilis* + *Streptomyces flavus* (BS + SF), *Bacillus subtilis* + *Trichoderma harzianum* (BS + TH), and *Trichoderma longiflora* + *Streptomyces flavus* (TL + SF) also significantly increased SOD activity, by 28.68%, 44.47%, 27.09%, and 39.12%, respectively, indicating that each treatment effectively activated the plant's first line of defense against oxidation.

[0078] POD catalyzes the decomposition of H2O2, forming the second line of defense against oxidation. Compared with the control (CK), all microbial inoculant combinations significantly increased POD activity in melon leaves (P<0.01). Among them, the SF + TH combination treatment showed the greatest increase in POD activity, exceeding the CK by 103.05%. The BS + TL, BS + SF, BS + TH, TL + SH, and TL + TH combinations also significantly increased POD activity in melon roots, exceeding the CK by 68.72%, 59.57%, 26.92%, 39.73%, and 80.62%, respectively, indicating that each treatment effectively activated the plant's second line of defense against oxidation.

[0079] MDA is an end product of lipid peroxidation and can reflect the degree of oxidative damage. Compared with the control group (CK), the SF + TH combination treatment showed the best effect in alleviating oxidative damage, with MDA content significantly reduced by 43.56% compared to CK (P<0.05). The BS + SF combination treatment was similar, reducing MDA content by 42.79% compared to CK. The BS + TL, BS + TH, TL + SH, and TL + TH combination treatments also significantly reduced MDA content, by 35.07%, 31.46%, 35.84%, and 38.45% respectively compared to CK, showing a stable effect in alleviating oxidative damage.

[0080] Proline (Pro) is an important osmotic regulator, and its excessive accumulation usually reflects abiotic stress in plants. Compared with the control (CK), all microbial inoculant combinations significantly reduced the Pro content in melon leaves (P<0.01). The CK treatment had the highest proline accumulation and content, significantly higher than all other treatments; the SF + TH combination treatment had the lowest proline content, reduced by 51.88% compared to the CK; the BS + TL, BS + SF, BS + TH, TL + SH, and TL + TH combinations all significantly reduced proline accumulation, decreasing by 54.87%, 54.94%, 36.49%, 39.56%, and 37.87%, respectively, indicating that all treatments could effectively alleviate osmotic stress and physiological damage caused by continuous cropping.

[0081] 2.7 Pearson Correlation Analysis of Melon Wilt Disease Disease Index with Control Effects and Physiological Indicators To clarify the relationship between the control efficacy of each inoculant treatment for Fusarium wilt in melon and the disease index and plant physiological indicators, Pearson correlation analysis was performed on each indicator. The results (Table 5) showed a significant linear correlation between the Fusarium wilt disease index and each indicator; a highly significant negative correlation was found between the control efficacy of each inoculant treatment and the disease index (r=-1.000, P<0.01), meaning that the better the inoculant treatment effect, the lower the disease index of the melon plant; the disease index was also highly significant negatively correlated with SOD activity (r=-0.951, P<0.01) and POD activity (r=-0.916, P<0.01), indicating that in treatments with poor control efficacy and high disease index, the SOD and POD activities of the melon plant were significantly lower. The activity was significantly reduced, and the antioxidant system's ability to scavenge reactive oxygen species was weakened. The disease index was significantly positively correlated with MDA content (r=0.828, P<0.05) and Pro content (r=0.788, P<0.05), indicating that in the treatment with a high disease index, the plant cell membrane lipid peroxidation damage was severe, the accumulation of MDA increased, and a large amount of Pro accumulated at the same time to cope with pathogen stress and maintain cell osmotic pressure stability.

[0082] Table 5. Correlation analysis between the disease index of Fusarium wilt in melon and the control effect and physiological indicators

[0083] Note: The data in the table are Pearson correlation coefficients; * indicates a significant correlation at the P<0.05 level, and ** indicates a highly significant correlation at the P<0.01 level.

[0084] 3. Conclusion The single-agent plate confrontation experiment showed that all four tested microbial agents had an inhibitory effect on the wilt pathogen of melon, among which Streptomyces flavus had the best inhibitory effect (69.03%).

[0085] When compound microbial agents were applied to melon seedlings infected with Fusarium wilt, Streptomyces flavus + Trichoderma harzianum showed the best control effect on the disease, with a control effect of 75.01%, the number of pathogens decreased by 60% compared with the control, and it could also significantly promote the growth of melon seedlings, with significant improvement in various growth indicators; Bacillus subtilis + Streptomyces flavus also had good control effect (58.33%).

[0086] The compound microbial agents can significantly increase the activity of SOD and POD in melon leaves, reduce the content of MDA and Pro, and alleviate the oxidative damage and membrane lipid peroxidation caused by melon wilt. Among them, the treatment with the compound of Streptomyces flavus and Trichoderma harzianum is better than other treatments in all indicators and has a better mitigation effect.

[0087] After the application of different microbial agents, the disease index of wilt disease in melon was significantly negatively correlated with SOD activity and POD activity, and significantly positively correlated with MDA content and Pro content. This indicates that the higher the activity of antioxidant enzymes in the plant, the lower the level of membrane lipid peroxidation damage and accumulation of osmotic regulatory substances, the lower the disease index, and the better the control effect of the microbial agent.

[0088] In summary, the compounded microbial inoculants control diseases by inhibiting the colonization and reproduction of pathogens in the root and stem regions, and also promote plant growth. The combination of *Streptomyces flavus* and *Trichoderma harzianum* is a preferred green control solution for melon wilt, providing technical support for the industry to reduce its reliance on chemical fungicides.

[0089] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A compound microbial agent, characterized in that, The compound microbial agent is used to control wilt disease in melons, and the active ingredient of the compound microbial agent is any one of the following: A1) is composed of Streptomyces flavus and Trichoderma harzianum; A2) Composed of Bacillus subtilis and Streptomyces flavus; A3) is composed of Trichoderma longifolia and Streptomyces flavus; A4) is composed of Trichoderma longifolia and Trichoderma harzianum; A5) is composed of Bacillus subtilis and Trichoderma longifolia; A6) is composed of Bacillus subtilis and Trichoderma harzianum.

2. The compound microbial agent according to claim 1, characterized in that, The melon wilt disease is caused by Fusarium oxysporum melon-specific strain.

3. The compound microbial agent according to claims 1 or 2, characterized in that, The mass ratio of the inoculum containing *Streptomyces flavus* to the inoculum containing *Trichoderma harzianum* is 1:

1.

4. A method for controlling Fusarium wilt in melons, characterized in that, The procedure includes the following steps: applying the compound microbial agent according to any one of claims 1-3 to the tested melons to prevent and control melon wilt disease.

5. The method according to claim 4, characterized in that, The method of application is root irrigation.

6. The application of the compound microbial agent according to any one of claims 1-3 in the prevention and control of Fusarium wilt in melon or in the preparation of products for the prevention and control of Fusarium wilt in melon, wherein the Fusarium wilt is caused by Fusarium oxysporum melon-specific strain.

7. The use of the compound microbial agent according to any one of claims 1-3 in increasing the leaf area of ​​a test melon or in preparing a product that increases the leaf area of ​​a test melon, wherein the test melon is a melon infected with melon wilt disease.

8. The use of the compound microbial agent according to any one of claims 1-3 in increasing the plant height of recipient melons or in preparing products that increase the plant height of recipient melons, wherein the recipient melon is a melon that has developed wilt disease.

9. The use of the compound microbial agent according to any one of claims 1-3 in increasing the chlorophyll content of recipient melons or in preparing products that increase the chlorophyll content of recipient melons, wherein the recipient melon is a melon exhibiting wilt disease.

10. The use of the compound microbial agent according to any one of claims 1-3 in improving the antioxidant capacity of recipient melons or in preparing products that improve the antioxidant capacity of recipient melons, wherein the recipient melon is a melon that has developed wilt disease.

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