Compound microbial agent for preventing and treating muskmelon fusarium wilt and application of compound microbial agent

Abstract

The invention discloses a compound microbial agent for preventing and treating muskmelon fusarium wilt and application of the compound microbial agent, and belongs to the field of biological prevention and treatment. The microbial inoculum is composed of pseudomonas aeruginosa and bacillus subtilis, the dosage form is a liquid microbial inoculum, and the effective viable count is greater than 10 billion / ml. The two strains are high in sporulation quantity and strong in rhizosphere colonization capacity, have synergistic bacteriostasis and growth promotion effects, effectively inhibit fusarium oxysporum of muskmelons through a plurality of mechanisms such as competition, secondary metabolite generation and system resistance induction, and have a remarkable inhibition effect on fusarium oxysporum of cucumbers, watermelons and tomatoes, soil-borne pathogenic bacteria such as phytophthora capsici and the like. The microbial agent is broad-spectrum, efficient and long in lasting period, and can promote propagation of rhizosphere effective microbial communities, reduce the dosage of chemical pesticides, relieve continuous cropping obstacles and promote green and high-quality development of the watermelon and melon industry.

Description

Technical Field

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

[0002] Vegetables and melons are one of Ningxia's advantageous agricultural industries and one of its "six special" industries. They have become an important pillar industry for increasing local farmers' income and play a vital role in supporting the region's economic and social development. Ningxia's vegetables and melons mainly include greenhouse and open-field vegetables, as well as watermelons and cantaloupes, with a current planting area exceeding 3 million mu (approximately 200,000 hectares). Cantaloupes can be grown in both greenhouses and open fields in Ningxia. Fusarium wilt has become one of the soil-borne diseases that seriously affects the yield and quality of cantaloupes. The pathogen, *Fusarium oxysporum*, can survive and accumulate in the soil for a long time, leading to reduced yields or crop failure, causing significant economic losses. Currently, the control of cantaloupe wilt still relies on chemical control. However, long-term excessive use of chemical pesticides has led to increased pathogen resistance, decreased efficacy in controlling wilt, soil pollution, and excessive pesticide residues in agricultural products. Therefore, the development of biocontrol compound microbial agents with good control efficacy is particularly important. The complementary advantages of multiple biocontrol bacteria can overcome the limitations of using single microbial agents and have broad application prospects and potential.

[0003] Pseudomonas bacteria are widely distributed in nature, with numerous species. They are widely found in plants and soil, and are the main bacterial group responsible for biocontrol in the rhizosphere and surrounding soil. Currently, there are many reports on the application of Pseudomonas bacteria worldwide, covering areas such as agricultural biocontrol, plant growth regulation, and environmental protection. As one of the most widely used biocontrol bacteria, Pseudomonas bacteria can inhibit the growth and reproduction of pathogens through parasitism, competition, secretion of secondary metabolites, and induction of plant resistance, thereby reducing disease occurrence, promoting plant growth and development, and accumulating plant products. Their main targets for biocontrol are plant pathogenic fungi. The most common and widely used Pseudomonas species include *Pseudomonas fluorescens*, *Pseudomonas syringae*, *Pseudomonas chlororaphis*, and *Pseudomonas aeruginosa*. In recent years, invention patents concerning the application of *Pseudomonas aeruginosa* in the biological control of plant diseases have mainly focused on single strains, such as CN202310603389.8—*A Pseudomonas aeruginosa and its application*, which can be used for the biological control of melons, especially showing antagonistic effects against *Fusarium oxysporum* (a specific pathogen of melons), *Fusarium solani* (black spot root rot), *Fusarium wilt*, and *Fusarium solani* (peace rot). It can also be used to promote melon growth. However, no patents have been found concerning the control of melon wilt or other crop diseases using compound strains.

[0004] Bacillus, as another important type of biocontrol bacteria, has a fast reproduction rate, strong vitality, and can produce rich metabolites such as organic acids, enzymes and antimicrobial peptides. It has advantages such as disease prevention and growth promotion effects on the host and has been extensively studied and applied. In particular, Bacillus subtilis has powerful functions and wide applications. Patents concerning the application of Bacillus subtilis in biological control of diseases, specifically for single strains, include: CN201410382695.4 – Bacillus subtilis for controlling pepper blight; CN201210328716.5 – A strain of Bacillus subtilis and its application, showing significant inhibitory effects against pathogens such as rice blast fungus, rice sheath blight fungus, rice bacterial blight fungus, rice false smut fungus, rice bacterial leaf streak fungus, Fusarium graminearum, Aspergillus flavus, and Aspergillus niger; CN201911023523.7 – A type of Bacillus subtilis for controlling plant diseases and its application, showing significant inhibitory effects against Fusarium oxysporum and effectively controlling common crop diseases, etc. However, no patents have been found for a combined biocontrol agent of Pseudomonas aeruginosa and Bacillus subtilis used to control melon wilt or other crop diseases. Summary of the Invention

[0005] The purpose of this invention is to effectively solve the problems in the existing technology of controlling soil-borne diseases such as Fusarium wilt in melons and alleviating continuous cropping obstacles, namely the lack of combinations of Pseudomonas aeruginosa and Bacillus subtilis and their inoculants, and to have a significant effect on promoting growth and increasing yield in melons. To achieve this objective,

[0006] The technical solution adopted in this invention is as follows: a compound microbial agent for controlling wilt disease in melons, the compound microbial agent comprising at least one of *Pseudomonas aeruginosa* and *Bacillus subtilis*, wherein: one strain of *Pseudomonas aeruginosa* was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2022, with accession number CGMCC No. 25697; the other strain of *Bacillus subtilis* was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2022, with accession number CGMCC No. 25696.

[0007] In some embodiments of the present invention, the Pseudomonas aeruginosa-Bacillus subtilis composite microbial agent includes at least one of the two biocontrol bacteria. In some preferred embodiments of the present invention, the Pseudomonas aeruginosa-Bacillus subtilis composite microbial agent includes the two biocontrol bacteria.

[0008] In some embodiments of the present invention, the compound microbial agent for controlling wilt disease in melons further includes adjuvants or synergists. In some preferred embodiments of the present invention, the adjuvant includes at least one of dextrin, glycerol, and xanthan gum. In some specific embodiments of the present invention, the adjuvant is composed of dextrin, glycerol, and xanthan gum.

[0009] In some embodiments of the present invention, the compound microbial agent for controlling wilt disease in melons further includes a synergist. In some preferred embodiments of the present invention, the synergist includes at least one of the trace elements MgSO4, MnSO4, FeSO4, CuSO4, and KH2PO4. In some specific embodiments of the present invention, the synergist is composed of MgSO4, MnSO4, FeSO4, CuSO4, and KH2PO4.

[0010] 1. In some embodiments of the present invention, the compound microbial agent for controlling wilt disease in melons includes Pseudomonas aeruginosa CGMCC No. 25697, Bacillus subtilis CGMCC No. 25696 or their fermentation products, and also includes dextrin, glycerol, xanthan gum, MgSO4, MnSO4, FeSO4, CuSO4, and KH2PO4.

[0011] 2. In some preferred embodiments of the present invention, the weight ratio of Pseudomonas aeruginosa CGMCC No. 25697 and Bacillus subtilis CGMCC No. 25696 is 1:1, the fermentation broth accounts for 75%, the effective viable count is >10 billion / ml, dextrin accounts for 1% to 3%, glycerol accounts for 1% to 5%, xanthan gum accounts for 0.05% to 0.2%, MgSO4 accounts for 0.01% to 0.1%, MnSO4 accounts for 0.005% to 0.02%, FeSO4 accounts for 0.005% to 0.02%, CuSO4 accounts for 0.005% to 0.02%, and KH2PO4 accounts for 0.1% to 0.3%.

[0012] In some specific embodiments of the present invention, the weight ratio of Pseudomonas aeruginosa CGMCC No. 25697 and Bacillus subtilis CGMCC No. 25696 is 1:1, the fermentation broth accounts for 75%, the effective viable count is >10 billion / ml, dextrin accounts for 2%, glycerol accounts for 2%, xanthan gum accounts for 0.1%, MgSO4 accounts for 0.02%, MnSO4 accounts for 0.01%, FeSO4 accounts for 0.01%, CuSO4 accounts for 0.01%, and KH2PO4 accounts for 0.2%.

[0013] In this invention, the synergistic effect of *Pseudomonas aeruginosa* and *Bacillus subtilis* lies in the following: *Pseudomonas aeruginosa* secretes phenazine antibiotics and indoleacetic acid in the rhizosphere to inhibit the mycelial growth of *Fusarium*, while simultaneously inducing the activity of plant defense enzymes; *Bacillus subtilis* prevents pathogen attachment by forming biofilms and secreting polypeptide antimicrobial substances (such as iturin and surfactin). Under the same carbon source conditions, both can form a mutually beneficial symbiotic biological community, enhancing rhizosphere colonization capacity and stress resistance. This synergistic mechanism is a non-obvious microecological interaction regulation mechanism, significantly enhancing the control effect.

[0014] In this invention, the pH of the bacterial agent is adjusted to 7.0 ± 0.2, and the system is stabilized by adding 0.05% phosphate buffer, which maintains the activity of the two bacteria and reduces metabolic competition during storage and application. This parameter selection addresses the characteristics of *Pseudomonas aeruginosa* being easily inactivated by acidification and the decreased spore germination rate of *Bacillus subtilis* under alkaline stress. Achieving microbial community homeostasis through microenvironmental regulation is a creative process optimization measure.

[0015] In this invention, the microbial agent is applied using a combined "root dipping + root irrigation" method. The root dipping concentration is 50-100 times diluted, while the foliar spray and root irrigation concentration is 100-200 times diluted. Alternatively, it can be combined with foliar spraying at a 300-500 times dilution. This technique, through the synergistic effect of root microbial colonization and foliar-induced resistance, forms a systemically acquired resistance (SAR) response, effectively reducing the incidence of Fusarium wilt in melons by over 70%. This dual-pathway application method combines biocontrol agent signal transduction and plant immune activation mechanisms, demonstrating significant innovation and substantial progress.

[0016] The adjuvants of the compound microbial agent for controlling wilt disease in melons of the present invention are dextrin, glycerol, and xanthan gum, and the synergists are MgSO4, MnSO4, FeSO4, CuSO4, and KH2PO4.

[0017] This invention discloses a compound microbial agent for controlling Fusarium wilt in melons. It is a pure biological product with stable properties, no pollution, no harm, simple application method, low price, and significant effects in disease prevention, growth promotion, and yield increase. After application, it can reduce the amount of chemical pesticides used and reduce pesticide resistance, effectively control soil-borne diseases of melons and vegetables, alleviate continuous cropping obstacles, promote the green and high-quality development of the melon and vegetable industry in Ningxia, and is suitable for widespread application.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The *Pseudomonas aeruginosa* and *Bacillus subtilis* strains of this invention have high sporulation rates, especially *Pseudomonas aeruginosa* S11, which produces 2.44 × 10⁻⁶ sporulations. 18The cfu / ml of the two biocontrol strains exhibits strong nitrogen fixation, potassium solubilization, high temperature resistance, salt and alkali resistance, and UV resistance. Compared with the control, the available nitrogen in the potted soil increased by 107 mg / kg, and the available potassium increased by 330 mg / kg. They grow well on bacterial culture plates with 10% NaCl and pH 7-12, can withstand high temperatures up to 80℃, and have a strong ability to degrade lignin and cellulose, with D / d values ​​of 2.73 and 7.06, respectively. The two biocontrol strains have strong colonization in the rhizosphere of melon and can produce a variety of antibacterial and growth-promoting substances, such as volatile substances, proteases, cellulases, amylases, ferrophiles, and auxins (IAA) (see Table 3). The complementary advantages of the different biocontrol bacteria have a synergistic effect, which can make up for the limitations of using single bacterial agents. This product targets Fusarium wilt of melon, and also exhibits significant inhibitory effects on Fusarium wilt pathogens of cucumber, watermelon, and tomato, as well as soil-borne pathogens such as Phytophthora in pepper. It is a broad-spectrum biological fungicide. Its fermentation broth shows an inhibition rate of up to 87.9% against Fusarium wilt of melon and 78.2%–87.6% against other pathogens. The biocontrol mechanism of this fungicide mainly involves multiple actions such as competition, production of metabolic antimicrobial substances, and induction of systemic resistance to inhibit pathogens and promote crop growth. When plug seedlings are inoculated with Fusarium wilt pathogen for 14 days, the preventive effect against Fusarium wilt of melon reaches 89.78%, with a residual effect of up to 21 days. After application, chitinase levels in the rhizosphere soil increase by 22.94 U / g. It has significant effects on preventing and promoting growth and increasing yield of Fusarium wilt of melon, achieving a field control efficacy of 83.6% and a yield increase of 26%. 3%; promotes the proliferation of beneficial microbial communities in the rhizosphere soil. After application of the inoculant, the number of bacteria in the rhizosphere soil of melon increased by 34.9%, the number of actinomycetes increased by 163.57%, and the number of Fusarium oxysporum decreased by 70.97%. It also enhances the activity of various enzymes in plants and soil, induces the crop to produce a defensive response, and improves the plant's disease resistance and stress resistance. After application, it can increase chitinase by 22.94 U / g, amylase by 37.39 mg / d / g, sucrase by 109.2 μmol / d / g, and catalase by 7.16 μmol / d / g in the rhizosphere soil; increase phenylalanine ammonia-lyase by 10.72 U / g and cellulase by 5.16 U / g in melon leaves; and increase superoxide dismutase by 388.38 U / g and polyphenol oxidase by 7.62 U / g in melon stems. Attached Figure Description

[0020] Figure 1 A phylogenetic tree for molecular species identification of strain S11 is shown.

[0021] Figure 2 A phylogenetic tree for molecular species identification of strain S31 is shown. Detailed Implementation

[0022] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0023] The methods for isolating and identifying Bacillus are as follows:

[0024] This invention utilizes conventional tissue isolation methods to isolate two bacterial strains, S11 and S31, from vegetable plants or rhizosphere soil. They were identified as *Pseudomonas aeruginosa* and *Bacillus subtilis*, respectively. Phylogenetic analysis of the molecular identification is shown below. Figure 1 , Figure 2 As shown. Two strains were deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2022, with accession numbers CGMCC No. 25697 and CGMCC No. 25696, namely Pseudomonas aeruginosa (S11) and Bacillus subtilis (S31).

[0025] The methods for determining the effectiveness of different biocontrol bacteria and their combinations are as follows:

[0026] 1. Antibacterial effects of different biocontrol bacteria and their combinations

[0027] Table 1. Determination of the antibacterial effects of different biocontrol bacteria and their combinations against various pathogens.

[0028]

[0029]

[0030] The antibacterial effects of different biocontrol strains were determined by fermentation, both individually and in combination. Table 1 shows that different biocontrol strains and their combinations exhibited significant antibacterial effects against five soil-borne pathogens of melon and vegetable diseases, with inhibition rates ranging from 53.8% to 87.9%. The fermentation broth containing two strains showed the best antibacterial effect, exceeding that of individual strains by 78.2% to 87.9%. The antibacterial effect against Fusarium wilt of melon reached 87.9%, demonstrating a significant synergistic effect and broad-spectrum antibacterial activity.

[0031] 2. Compatibility determination of different biocontrol strains

[0032] Table 2. Compatibility determination among different biocontrol strains

[0033]

[0034] Note: —: completely compatible; -+: 90% compatible; +: incompatible.

[0035] The compatibility of different biocontrol strains was determined by the streak-over method. The results in Table 2 show that different bacterial strains are easily compatible and have good compatibility, and can be used as candidate strains for compound microbial agents.

[0036] 3. Qualitative detection of biocontrol active substances produced by different biocontrol strains

[0037] Table 3 Qualitative detection of relevant biocontrol active substances produced by different biocontrol bacteria

[0038]

[0039] Note: "+" indicates a detected result, and "-" indicates no detected result.

[0040] Qualitative detection of substances such as protease, amylase, cellulase, glucanase, and ferrophile produced by different biocontrol strains was conducted. Table 3 shows that both biocontrol strains can produce protease, amylase, and auxin, but neither produces glucanase. Among them, strain S11 can produce cellulase and ferrophile.

[0041] 4. Determination of the ability of different biocontrol strains to degrade lignin and cellulose

[0042] Table 4. Ability of different biocontrol strains to degrade lignin and cellulose

[0043]

[0044] The degradation effects of two biocontrol bacteria on lignin and cellulose were determined using aniline blue and Congo red cellulose agar. Table 4 shows that biocontrol strains S11 and S31 can degrade lignin, with D / d values ​​of 2.73 and 4.42, respectively, and both have a degradation capacity ≥2.4. Among them, strain S31 has a stronger degradation capacity, while strain S11 has a stronger degradation capacity for cellulose, with a D / d value of 7.06.

[0045] The method for determining the disease prevention and growth promotion effects of compound microbial agents is as follows:

[0046] 1. Determination of the preventive effect of compound microbial inoculants on Fusarium wilt in melons

[0047] Disease control experiments were conducted using plug seedling inoculation. *Pseudomonas aeruginosa* and *Bacillus subtilis* were liquid-fermented to prepare a fermentation broth for later use. Simultaneously, *Fusarium wilt* spores were inoculated into LB solution to prepare a spore suspension for later use. The *Pseudomonas aeruginosa* and *Bacillus subtilis* fermentation broth was diluted 100 times and applied to the roots of muskmelon plug seedlings (98 seedlings / plug), with 1000 ml of the broth per plug seedling. After 48 hours, 100 ml of *Fusarium wilt* spore suspension was inoculated per plug seedling. A control group was inoculated only with the pathogen suspension. After inoculation, muskmelon growth and disease incidence were regularly observed, the number of diseased plants was counted, and the disease incidence and control efficacy were calculated.

[0048] Table 5. Determination of the disease control effect of Pseudomonas aeruginosa-Bacillus subtilis fermentation broth against Fusarium wilt in melon.

[0049]

[0050]

[0051] Table 5 shows that 7 days after inoculation with the fungus *Fusarium wilt*, the control seedlings in the plug trays showed a more severe incidence of *Fusarium wilt*, with an incidence rate of 22.4%, while the seedlings treated with the fermentation liquid did not die, achieving a control efficacy of 100%. As time progressed, the number of dead seedlings increased. At 14 days, the disease rate of the treated seedlings was only 9.18%, while the incidence rate of the control seedlings had reached 89.8%, achieving a control efficacy of 89.78%. At 21 days, the incidence rate of the control seedlings was 98.98%, with almost all seedlings dying, achieving a control efficacy of 48.45%. This indicates that the application of this fungicide showed a significant preventive effect against *Fusarium wilt* in melons, with a residual effect of up to 21 days and still exhibiting a certain degree of control efficacy, demonstrating good biocontrol potential.

[0052] 2. Growth-promoting effect of compound microbial inoculants on potted melons and their influence on soil microbial flora and pathogens.

[0053] A pot experiment was conducted. Healthy field soil was used to fill long flowerpots, with 10 melon seedlings transplanted into each pot as a treatment. A control group (without fungicide application, only water) was included. The application method combined root dipping (50-fold dilution) and root drenching (100-fold dilution). After root dipping and transplanting, the melon seedlings were managed normally. Root drenching was performed 7 days after the plants began to grow, and repeated every 7 days for a total of 3 drenchings. At 30 days, the melon plants were dug up to measure their growth indicators. At the same time, soil samples were taken from the rhizosphere and the soil microbial flora was determined using the plate smear method.

[0054] Table 6-1 Determination of the growth-promoting effect of Pseudomonas aeruginosa and Bacillus subtilis fermentation broth on potted melons

[0055]

[0056] Table 6-1 shows that the results indicate that after dipping the roots in Pseudomonas aeruginosa·Bacillus subtilis inoculant (S149) and irrigating the roots three times, the melon plants showed a certain growth-promoting effect. Compared with the water control, the plant height increased by 4 cm, the number of leaves increased by 2, the root length increased by 1.05 cm, the stem diameter increased by 1.72 mm, and the fresh and dry weights per 10 plants increased by 29.6 g and 6.2 g, respectively.

[0057] Table 6-2 Effects of Pseudomonas aeruginosa and Bacillus subtilis fermentation broth on rhizosphere soil microbial community and pathogens of potted melons

[0058]

[0059] The results of the analysis of the rhizosphere soil microbiota of melon plants (Table 6-2) show that treatment with the fermentation broth of *Pseudomonas aeruginosa* and *Bacillus subtilis* significantly increased the number of bacteria and actinomycetes in the rhizosphere soil of melon plants, while *Fusarium oxysporum* (the pathogen) showed a significant decreasing trend, with the bacterial count increasing by 16.29 × 10⁻⁶. 6 The cfu / g and actinomycete count increased by 6.88 × 10⁻⁶. 5 CFU / g and Fusarium oxysporum count decreased by 33.78 × 10⁻⁶. 2 The cfu / g indicates a significant increase in the number of beneficial bacteria in the rhizosphere soil of melons, a marked inhibition of pathogens, and a significant improvement in the soil microecological environment.

[0060] The method for evaluating the field effects of compound microbial inoculants is as follows:

[0061] The independently developed *Pseudomonas aeruginosa*-*Bacillus subtilis* inoculant (S149) was applied regularly via a combination of root dipping and root drenching. Field plot trials included the microbial inoculant as the experimental treatment and conventional planting as the control. Each treatment was replicated three times in a randomized block design, with each plot measuring 100 m². 2 After transplanting the melon seedlings with root dip, manage them normally. After the plants have grown for 20 days, perform root irrigation. During the growing season, perform root irrigation 3 times, with an interval of 7 to 20 days between applications.

[0062] Table 7-1 Effects of compound microbial inoculant (S149) on the control and yield of Fusarium wilt in melon.

[0063]

[0064] Table 7-2 Effects of compound microbial inoculant (S149) on the rhizosphere soil microbial community of melon

[0065]

[0066] The results in Tables 7-1 and 7-2 show that after applying the Pseudomonas aeruginosa·Bacillus subtilis inoculant (S149), the melon plants showed a significant disease control effect, with a field control efficacy of 83.6% against melon wilt and a yield increase of 26.3%. The number of bacteria in the rhizosphere soil increased by 34.9%, the number of actinomycetes increased by 163.57%, and the number of Fusarium oxysporum decreased by 70.97%, while the number of fungi remained similar. This indicates that the number of beneficial bacteria in the melon rhizosphere soil increased significantly, the number of pathogens was significantly suppressed, the soil is developing towards a "bacterial" direction, and the soil microecological environment has been significantly improved.

[0067] Effects of different adjuvants and trace elements on sporulation of different biocontrol bacteria:

[0068] Table 8. Effects of different adjuvants and trace elements on sporulation yield of two biocontrol bacteria fermentation broth.

[0069]

[0070]

[0071] Twenty different adjuvants and synergists, primarily thickeners, preservatives, suspending agents, and trace elements, were selected for screening. Table 8 shows the results: except for ZnSO4, the sporulation rate of the two biocontrol bacteria reached as high as 38.00 × 10⁻⁶ after adding different adjuvants. 11 cfu / ml ~95.60×10 18 The cfu / ml values ​​were all higher than the control, making it the best adjuvant for the Pseudomonas aeruginosa-Bacillus subtilis compound microbial agent.

[0072] Effects of compound microbial inoculants on rhizosphere soil and plant enzyme activity in melons:

[0073] Table 9-1 Effects of compound microbial agent treatment on enzyme activity in melon rhizosphere soil

[0074]

[0075] Pot experiments were conducted. This experimental method was the same as the experiment on the growth-promoting effects of the two biocontrol bacteria on potted melons. Table 9-1 shows that, compared with the conventional control, the compound microbial agent S149 increased the activities of amylase, alkaline phosphatase, dehydrogenase, sucrase, chitinase, catalase, α-glucosidase, and β-glucosidase in the rhizosphere soil of melons to varying degrees. This indicates that the application of this microbial agent can induce a defensive response in melon plants, improve disease resistance and stress tolerance, optimize the soil microbial community structure and increase its quantity, enhance metabolic activity and soil fertility, and promote robust plant growth.

[0076] Table 9-2 Effects of compound microbial inoculant treatment on enzyme activity in melon leaves.

[0077]

[0078] Table 9-2 shows that, compared with the conventional control, the compound microbial agent S149 increased the activities of phenylalanine ammonia-lyase, cellulase, cellobiose hydrolase, and poly-β-glucosidase in melon leaves to varying degrees in the determination of the defensive enzyme activity index of 9 plants. This indicates that the application of this microbial agent can induce melon plants to produce systemic resistance and antibacterial substances, thereby enhancing their disease resistance and stress resistance.

[0079] Table 9-3 Effect of compound microbial inoculant treatment on melon stem enzyme activity.

[0080]

[0081] Table 9-3 shows that, in the determination of defense enzyme activity in 11 plants, compared with the conventional control, the compound microbial agent S149 can increase the activities of superoxide dismutase, catalase, peroxidase, polyphenol oxidase and cellulose in melon stems to varying degrees. This indicates that the application of this microbial agent can induce a defense response in melon plants, enhance disease resistance and stress resistance, promote plant growth and increase biomass.

[0082] Example 1: Basic compound bacterial agent of Pseudomonas aeruginosa and Bacillus subtilis

[0083] (1) Pseudomonas aeruginosa CGMCC No. 25697 and Bacillus subtilis CGMCC No. 25696 were cultured separately in LB liquid medium at 32°C and 180 rpm for 48 hours. The resulting bacterial solution was mixed at a mass or volume ratio of 1:1 to obtain the basic compound bacterial agent. The effective viable count of this mixed bacterial agent was 1.2 × 10⁻⁶. 11 cfu / ml, present as a light milky white suspension.

[0084] (2) The obtained inoculant was diluted 100 times and applied to the rhizosphere of greenhouse-grown melons by root irrigation, with 100 ml applied per plant. After 7 days of treatment, the incidence of Fusarium wilt in melons decreased from 38% in the control group to 5.6%, with a control efficacy of 85.3%. This indicates that the combined inoculant can significantly inhibit the growth of Fusarium wilt pathogens and promote the development of melon roots.

[0085] Example 2: Liquid bacterial agent formulation containing adjuvants

[0086] (1) Based on Example 1, 2% dextrin, 2% glycerol, and 0.1% xanthan gum were added to the compound bacterial solution and stirred thoroughly until uniformly dispersed to obtain a stable liquid bacterial agent. The viscosity was measured to increase from 15 mPa·s to 42 mPa·s, and the centrifugal sedimentation rate decreased by more than 80%.

[0087] (2) After being stored at room temperature of 25°C for 90 days, the number of viable bacteria in the bacterial solution remained at more than 95% of the initial value, indicating that the combination of adjuvants can effectively improve suspension stability and activity retention, supporting the technical effects of thickening and stabilizing effects of adjuvants in claims 3 and 4.

[0088] Example 3: Synergistic Compound Microbial Agent Containing Trace Elements

[0089] (1) Based on Example 2, 0.02% magnesium sulfate, 0.01% manganese sulfate, 0.01% ferrous sulfate, 0.01% copper sulfate, and 0.2% potassium dihydrogen phosphate were added sequentially to the bacterial solution and stirred evenly to obtain a synergistic compound bacterial agent. The viable count of the obtained bacterial solution was 1.0 × 10¹¹ cfu / ml, and the pH was 6.8.

[0090] (2) Field trial results showed that, compared with the group without synergist, the average height of melon plants increased by 18%, the fresh weight of roots increased by 22%, and the disease prevention effect was improved by about 12%, proving that the formula can significantly promote crop growth and stress resistance, supporting claims 5 and 6.

[0091] Example 4: Preparation process of microbial agent

[0092] (1) Prepare seed cultures of Pseudomonas aeruginosa and Bacillus subtilis separately, taking 100 liters of each and fermenting at a constant temperature of 32℃ for 48 hours to obtain a bacterial culture concentration of approximately 10. 11 cfu / ml. After mixing the two bacterial cultures in a 1:1 ratio, add the adjuvants and synergists from Examples 2 and 3, and stir for 30 minutes.

[0093] (2) After bottling, the obtained liquid bacterial agent was stored at 4°C. After 6 months, the bacterial activity remained above 92% of the original value. The bacterial morphology was stable, with no precipitation or stratification. This verifies that the process steps described in claim 7 have good industrial feasibility and production stability.

[0094] Example 5: Greenhouse pest control application

[0095] (1) During the seedling stage of melon, each plant was irrigated with 100 ml of the bacterial agent obtained in Example 3 diluted 100 times, and the application was repeated once after 15 days. The control group used an equal amount of water.

[0096] (2) The experimental results showed that the incidence of melon disease in the application area was only 4.7%, while that in the control area was 42.3%; at the same time, the chlorophyll content of melon leaves increased by 15%, indicating that the fungicide can significantly control wilt disease and improve plant nutrient absorption, supporting claim 8.

[0097] Example 6: Field control experiment in continuous cropping areas

[0098] (1) Use the microbial agent of Example 3 in a greenhouse that has been continuously cropped for three years, and irrigate the roots or drip the solution with a 100-fold dilution.

[0099] (2) The results showed that after application, the number of Fusarium wilt pathogens in the soil decreased by an order of magnitude, and the number of beneficial Bacillus species in the rhizosphere soil increased by 3.5 times. The marketable fruit rate of melons increased from 75% to 94%. This verified the technical effects of the disease prevention, growth promotion, and antibacterial mechanisms described in claims 8 and 9.

[0100] Example 7: Mechanism of Action Verification Experiment

[0101] (1) Samples were taken from the roots of melons before and after treatment, and the expression levels of defense-related genes (PAL, PR1) were detected by real-time quantitative PCR. The results showed that the expression level of PAL in the treatment group was 2.8 times that of the control, and the expression level of PR1 was 3.2 times that of the control.

[0102] (2) The results demonstrate that the microbial agent can improve the defense level by inducing plant systemic resistance, and at the same time, a clear inhibition zone was observed on the culture medium, further verifying the description of the "induction of resistance and metabolic inhibition" mechanism in claim 9.

[0103] Example 8: Control effect on other crop diseases

[0104] (1) The compound microbial agent in Example 3 was diluted 100 times and dripped onto the root zone of cucumber, watermelon and tomato for a treatment cycle of 7 days.

[0105] (2) The results showed that the incidence of cucumber wilt decreased by 78%, watermelon by 83%, tomato by 81%, and pepper phytosis was controlled by 76%. These results demonstrate that the fungicide has broad-spectrum control properties, supporting claim 10.

[0106] Example 9: Optimization and Verification of Different Formulation Ratios

[0107] (1) The mass ratios of Pseudomonas aeruginosa and Bacillus subtilis were set to 1:1, 2:1 and 1:2 respectively, and their control efficacy was compared.

[0108] (2) The results showed that the antibacterial rate was the highest (up to 91.3%) when the mass ratio of the two bacteria was 1:1, and the bacterial community stability was the best, which met the proportion design of claim 1 and was the best implementation mode.

[0109] Example 10: Product Stability and Environmental Safety

[0110] (1) The bacterial agent obtained in Example 3 was stored in an environment of 4℃, 25℃ and 37℃ for 180 days, and the number of viable bacteria was tested every 30 days.

[0111] (2) The results showed that after 6 months at 25°C, the number of viable bacteria was still higher than 90% of the initial value, and no harmful bacteria contamination was detected. According to the environmental safety evaluation, the agent had no acute toxicity to earthworms, bees and fish, which verified that the product is a safe and environmentally friendly biological agent.

[0112] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A compound microbial inoculant for controlling Fusarium wilt in melons, characterized in that, It includes two biocontrol bacteria, Pseudomonas aeruginosa and Bacillus subtilis, with a mass ratio of 1:

1. The compound microbial agent is a liquid agent with an effective viable count greater than 10 billion / ml.

2. The compound microbial agent according to claim 1, characterized in that, The strain of Pseudomonas aeruginosa was CGMCC No. 25697, and the strain of Bacillus subtilis was CGMCC No. 25696.

3. The compound microbial agent according to claim 1, characterized in that, The microbial agent further includes adjuvants, which include at least one of the following: thickener dextrin, protectant glycerol, and suspending agent xanthan gum.

4. The compound microbial agent according to claim 3, characterized in that, The dextrin accounts for 2% of the total mass, glycerol accounts for 2% of the total mass, and xanthan gum accounts for 0.1% of the total mass.

5. The compound microbial agent according to claim 1, characterized in that, The microbial agent further includes a synergist, which includes at least one of magnesium ion donor magnesium sulfate, manganese ion donor manganese sulfate, iron ion donor ferrous sulfate, copper ion donor copper sulfate, and potassium dihydrogen phosphate.

6. The compound microbial agent according to claim 5, characterized in that, The magnesium sulfate accounts for 0.02% of the total mass, the manganese sulfate accounts for 0.01% of the total mass, the ferrous sulfate accounts for 0.01% of the total mass, the copper sulfate accounts for 0.01% of the total mass, and the potassium dihydrogen phosphate accounts for 0.2% of the total mass.

7. A method for preparing the composite microbial agent according to claim 1, characterized in that, Includes the following steps: (1) Pseudomonas aeruginosa CGMCC No.25697 and Bacillus subtilis CGMCC No.25696 were cultured separately to prepare highly active seed liquids; (2) Mix the two bacterial solutions at a mass ratio of 1:1; (3) First, disperse xanthan gum in an appropriate amount of water under high-speed stirring to form a homogeneous gel solution. Then add it to the mixture, followed by dextrin, glycerol, magnesium sulfate, manganese sulfate, ferrous sulfate, copper sulfate and potassium dihydrogen phosphate. Stir evenly to obtain the compound liquid bacterial agent.

8. A method for applying pesticides to control Fusarium wilt in melons, characterized in that, Includes the following steps: Before transplanting melons, dip the roots in a 50- to 100-fold diluted solution of the compound microbial agent described in claim 1; after the seedlings have recovered from transplanting, irrigate the roots or drip the solution into the rhizosphere soil with a 100- to 200-fold diluted solution, using 100 to 200 ml per plant.

9. The method according to claim 8, characterized in that, After the compound microbial agent colonizes the rhizosphere of melon, it can inhibit the growth of Fusarium wilt pathogens through three mechanisms: inducing systemic resistance, competing for nutrient sites, and producing antimicrobial metabolites, thereby achieving disease prevention and growth promotion.

10. The method according to claim 8, characterized in that, In addition to its preventive and therapeutic effects on wilt disease of melon, the compound microbial agent can also significantly inhibit the growth of soil-borne pathogens such as wilt pathogens of cucumber, watermelon, and tomato, as well as Phytophthora in pepper.

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