Method for driving rhizosphere bacterial enrichment to improve cucumber resistance to fusarium wilt by using trichoderma asperellum fj035

Abstract

The application discloses a method for driving rhizosphere bacteria enrichment by Trichoderma asperellum FJ035 to improve cucumber resistance to fusarium wilt, and belongs to the technical field of biological control of plant diseases. The method is characterized in that Trichoderma asperellum FJ035, Pseudomonas nitroreducens 22 and Bacillus alvei 33 are applied to the rhizosphere of cucumber by root irrigation. The method has stable prevention and treatment effects, is environment-friendly, can replace or reduce the use of chemical pesticides, and has a potting prevention and treatment effect of 77.97% and a field prevention and treatment effect of 68.42%, which is equivalent to the prevention and treatment effects of the commonly used chemical pesticides hymexazol and thiophanate-methyl, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for plant diseases, specifically to a method for improving cucumber resistance to Fusarium wilt by driving the accumulation of rhizosphere bacteria using Trichoderma hygroscopicum FJ035. Background Technology

[0002] Cucumber wilt is caused by *Fusarium oxysporum* f.sp. *cucumerinum* (FOC), with an average annual incidence rate of 10%-30%, reaching as high as 80%-90% in severely affected areas. It is one of the major soil-borne diseases leading to cucumber yield loss. Currently, production mainly relies on chemical pesticides for control, but long-term use can easily lead to pesticide residues, increased pathogen resistance, and soil microecological imbalance.

[0003] Biological control has become a research hotspot due to its advantages such as being environmentally friendly and less prone to developing resistance. However, existing biological control technologies still have the following shortcomings: the efficacy of single strains is unstable and easily affected by environmental conditions; the combination and ratio of multiple strains are complex, with competition or antagonism between strains, resulting in poor colonization and high production costs.

[0004] Trichoderma asperellum, an important biocontrol fungus, can induce systemic resistance in plants and regulate the structure of the rhizosphere microbiota. However, current technologies have not yet elucidated the complete mechanism by which Trichoderma asperellum specifically recruits functional bacteria through root exudates to enhance crop disease resistance, and there is also a lack of standardized application methods that can be industrially applied. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving cucumber resistance to Fusarium wilt by driving the enrichment of rhizosphere bacteria through Trichoderma echinococcus FJ035. This method has high efficacy, strong stability, and is easy to operate, making it suitable for large-scale production and large-scale field application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for improving the resistance of cucumber to Fusarium wilt, wherein the method involves applying *Trichoderma asperellum* FJ035, *Pseudomonas nitroreducens* 22, and *Bacillus nitroreducens* 33 to the rhizosphere of cucumber plants via root irrigation; the *Trichoderma asperellum* FJ035 has the accession number CGMCC NO.40942 and was deposited at the China General Microbiological Culture Collection Center on November 30, 2023, and is classified as *Trichoderma asperellum*; the *Pseudomonas nitroreducens* 22 has the accession number CGMCC No.29051 and was deposited at the China General Microbiological Culture Collection Center on November 20, 2023, and is classified as *Pseudomonas nitroreducens*; the *Bacillus nitroreducens* 33 has the accession number CGMCC... No. 29055 was deposited at the China General Microbiological Culture Collection Center on November 20, 2023, and classified as Paenibacillus alvei.

[0007] Furthermore, the application concentration of *Trichoderma hydathodes* FJ035 is 1.0 × 10⁻⁶. 6 Spores / mL; the application concentration of both *Pseudomonas nitroreductoides* 22 and *Bacillus vesicularis* 33 was 1.0 × 10⁻⁶. 7 CFU / mL.

[0008] Furthermore, it can be applied throughout the entire growth period of cucumbers, once each during the seedling stage (7-10 days after transplanting), the vine-growing stage, the flowering stage, and the early fruit-setting stage.

[0009] Beneficial effects: Compared with the prior art, the present invention has the following beneficial effects: 1. The complete mechanism by which Trichoderma echinococcus FJ035 enhances cucumber wilt resistance by regulating root exudate content and specifically recruiting certain beneficial bacteria has been clarified, providing a theoretical basis for the research and application of biocontrol agents.

[0010] 2. The control effect is stable and significant, with a potted plant control effect of 77.97% and a field control effect of 68.42%, which is comparable to the control effect of chemical pesticides (hymexazol and thiophanate-methyl).

[0011] 3. Environmentally friendly, it can replace or significantly reduce the use of chemical pesticides.

[0012] 4. The method is simple and highly standardized, making it suitable for industrial production and large-scale application. Attached Figure Description

[0013] Figure 1This is a schematic diagram illustrating the mechanism by which Trichoderma echinosporum FJ035 of the present invention drives the enrichment of beneficial bacteria in the rhizosphere and enhances the resistance of cucumber to Fusarium wilt.

[0014] Figure 2 This refers to the regulatory effect of the core bacterial community on cucumber wilt disease in Example 1 of the present invention, wherein... Figure 2 A: Phenotypes of cucumbers after 21 days of treatment with the ten different treatments in Table 1; Figure 2 B: A bar chart of disease indexes for ten different treatments.

[0015] Figure 3 This is an example of verifying the regulatory effect of the core bacterial community on cucumber wilt disease in Example 1 of the present invention. Figure 3 A: Phenotypes of cucumbers after 21 days of treatment with the four different treatments in Table 2; Figure 3 B: A bar chart of the disease index for four different treatments.

[0016] Figure 4 This describes the effect of different concentrations of core bacterial groups on cucumber wilt disease in Example 1 of the present invention. Figure 4 A: Phenotypes of cucumbers after 21 days of treatment with the six different treatments in Table 3; Figure 4 B: A bar chart of the disease index for six different treatments.

[0017] Figure 5 This refers to the effect of FJ035 on the motility of the core bacterial community in Example 2 of the present invention. Figure 5 A: Colony sizes of Pseudomonas 22 and Paenibacillus 33 grown in SMM medium after adding 500 μL, 1 mL, and 2 mL of FJ035 metabolic solution, respectively, for 48 h; Figure 5 B: Pseudomonas 22 and Paenibacillus 33 in addition Figure 5 Diameter bar graph of each A treatment grown on SMM medium for 48 h.

[0018] Figure 6 This refers to the effect of FJ035 on the motility of the core bacterial community in Example 2 of the present invention. Figure 6 C: The colony size of cucumber rhizosphere exudates treated with 25 mg / mL, 50 mg / mL, and 100 mg / mL of pure cucumber rhizosphere exudates, and cucumber rhizosphere exudates treated with 25 mg / mL, 50 mg / mL, and 100 mg / mL of FJ035, respectively, after 48 h of growth in SMM medium for Pseudomonas 22 and Paenibacillus 33. Figure 6 D- Figure 6 E: Pseudomonas 22 and Paenibacillus 33 in addition Figure 6 The diameter bar graph of each C treatment on SMM medium after 48 h of growth; CRE: pure cucumber rhizosphere exudate; SR: rhizosphere exudate of cucumber rooted with added FJ035 (25, 50, and 100 are the amount of rhizosphere exudate added at 25 mg / mL, 50 mg / mL, and 100 mg / mL, respectively).

[0019] Figure 7 This is the effect of FJ035 on the chemotaxis of the core bacterial community in Example 2 of the present invention, wherein CRE: pure cucumber rhizosphere exudate; SR: cucumber rhizosphere exudate collected from the sterile water side after applying FJ035 to one side and then treating it with sterile water (25, 50, and 100 are the amount of rhizosphere exudate added, which are 25 mg / mL, 50 mg / mL, and 100 mg / mL, respectively).

[0020] Figure 8 This refers to the effect of FJ035 on the colonization of the core bacterial community in cucumber roots, as described in Example 2 of the present invention. Figure 8 A: The biocolonization of cucumber roots by Pseudomonas 22 in the core microbial community; Figure 8 B: Cucumber root biocolonization of Paenibacillus 33 in the core microbial community; Figure 8 Biocolony of C:Pseudomonas 22 alone in cucumber roots; Figure 8 D:Paenibacillus 33 alone in cucumber root biocolonization.

[0021] Figure 9 This refers to the rhizosphere exudate metabolome of cucumber roots treated with FJ035 in Example 2 of this invention, wherein... Figure 9 A: Scatter plot of the metabolome of cucumber rhizosphere exudates; Figure 9 B: Cluster diagram of the metabolome of cucumber rhizosphere exudates; Figure 9 C: The types of substances significantly upregulated by the rhizosphere exudate metabolome of cucumber.

[0022] Figure 10 This is a pot experiment on the control efficacy of the core bacterial group against cucumber wilt disease in Example 3 of the present invention. Figure 10 A: Cucumber phenotype after potted plants were treated with core microbial communities and biological and chemical treatments, followed by FOC growth for 21 days; Figure 10 B: Figure 10 The bar chart of the prevention efficacy of each treatment in A.

[0023] Figure 11 This is a pot experiment in Example 3 of the present invention on the effect of the core bacterial community on cucumber wilt disease at different concentration gradients, wherein... Figure 11 A: Cucumbers grown in pots after being treated with different concentrations of core bacteria and then with FOC for 21 days; Figure 11B: Figure 11 The bar chart of the prevention efficacy of each treatment in A.

[0024] Figure 12 This is a field plot efficacy test of the core bacterial group against cucumber wilt disease in Example 3 of the present invention. Figure 12 A: Phenotypic and root longitudinal section of cucumber plants at different stages of growth, compared with those treated with biological agents and chemical pesticides. Figure 12 B: Figure 12 The bar chart of the prevention efficacy of each treatment in A. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the embodiments. The following embodiments are preferred embodiments of the present invention and are not intended to limit the present invention.

[0026] Example 1: Construction of the core microbial community

[0027] (1) Test materials Tested strains: *Fusarium oxysporum* f.sp. cucumerinum (FOC), designated SCCFO1; *T. asperellum*, designated FJ035; *Sinomonas atrocyanea* B1-1; *Streptomyces griseorubiginosus* B1-3; *Ensifer adhaerens* B1-9; *Pseudomonas nitroreducens* B1-22 (abbreviated as *Pseudomonas 22*); *Paenibacillus alvei* B1-33 (abbreviated as *Paenibacillus 33*); and *Paenibacillus alvei* B1-35 (abbreviated as *Paenibacillus 35*). These strains were isolated, identified, and preserved. The above-mentioned test strains were effective strains isolated from the rhizosphere of cucumber after applying *T. asperellum* FJ035 to the rhizosphere through root irrigation.

[0028] Cucumber: The variety is Chuanlv 21, a hybrid F1 generation, from Chengdu Haote Horticulture Co., Ltd.

[0029] (2) Test culture medium and experimental solution PDA medium: 200g potato, 18g glucose, 18g agar, and deionized water to a final volume of 1L.

[0030] PD medium: 200g potato, 18g glucose, deionized water to a final volume of 1L.

[0031] TSB medium: 17g tryptone, 3g soybean peptone, 5g sodium chloride, 2.5g glucose, 2.5g dipotassium hydrogen phosphate, and deionized water to a final volume of 1L. Adjust the pH to neutral.

[0032] SMM semi-solid medium: glucose 0.1g, peptone 0.1g, EDTA-2Na 38mg, neutral phosphate buffer 10mL, agar 5.0g, deionized water to a final volume of 1L.

[0033] MS culture medium: sucrose and agar-free, 3.0 g MS powder dissolved in deionized water.

[0034] Chemotaxis buffer: 10 mM neutral potassium phosphate buffer, 0.1 mM EDTA-2Na.

[0035] The above culture media and experimental solutions were sterilized at 121℃ for 20 minutes before use.

[0036] (3) Preparations for the pot experiment Cucumber cultivation method: Disinfect cucumber seeds with 75% ethanol for 1 minute, rinse repeatedly with sterile water 3 times, then place the cucumber seeds on sterile filter paper. Add 3 mL of distilled water to a petri dish and place the filter paper with seeds inside. Seal the petri dish before germination to prevent contamination. Cultivate in a light incubator at 28℃ for 10 / 14 hours of day / night cycle. When the cucumber plants reach a height of 15 cm, fix them in a transplanting basket and then transfer them to a 24-well hydroponic incubator. The hydroponic incubator contains distilled water and 1 / 4 concentration MS culture medium. The total liquid in the incubator should completely cover the transplanting basket to ensure that the cucumber roots can absorb water normally.

[0037] The bacterial strains used were inoculated into TSB medium and cultured at 37℃ and 180 rpm for 72 h with shaking. The bacterial concentration was then adjusted to an OD600 of 1.0. After Trichoderma echinosporum strain FJ035 was inoculated into PDA medium and grown for 7 days, mycelia were scraped to prepare spore suspensions. The number of spores was observed under a microscope, and the concentration of the spore suspension was calculated.

[0038] The disease index of cucumber wilt is as follows: Grade 0: No symptoms found in the plant or leaves; Grade 1: The leaves of the plant below 1 / 4 show wilting symptoms, while the base of the stem is asymptomatic and the plant grows normally. Grade 2: 1 / 4 to 1 / 2 of the plant leaves show wilting symptoms, the lower 1 / 2 of the stem shows browning, and the plant is stunted; Level 3: More than half of the plant's leaves show wilting symptoms, more than half of the stem base shows browning, and the plant is significantly stunted; Level 4: The entire plant wilts and dies.

[0039] Disease index = ∑ (disease level × number of affected plants) / highest disease level × total number of plants × 100.

[0040] (4) Simplified experiments on six types of bacteria The tested bacterial strains were inoculated into TSB liquid medium and cultured at 37℃ and 180 rpm with shaking for 36 h. *Trichoderma echinococcus* strain FJ035 was inoculated onto PDA solid plates and cultured at 28℃ for 7 days. Mycelia and spores were scraped to prepare a spore suspension. A pot experiment was conducted using hydroponics. Seven days after cucumber transplanting and seedling establishment, the prepared bacterial suspension and spore suspension were added to the hydroponic system to achieve a final concentration of 1.0 × 10⁻⁶. 6 CFU / mL (bacteria) or 1.0 × 10⁻⁶ 6 1 spore / mL (fungus). The experiment consisted of 10 treatment groups (Table 1), each inoculated with different combinations of FJ035 and 6 test bacteria strains. The treatment inoculated solely with *Fusarium oxysporum* cucumber-specific type (FOC) served as a control. Each treatment had 8 seedlings, replicated 3 times. Disease incidence was assessed 21 days after inoculation, disease index was recorded, and control efficacy was calculated.

[0041] Table 1. Deficiency / reduction experiments of six bacteria in hydroponic cucumbers: 10 treatment groups

[0042] Data from a pot experiment analyzing the function of core microbiota revealed ( Figure 2 Among the ten treatment groups, the disease index of SP1 was 37.5 with an efficacy of 40.98%; the disease index of SP2 was 43.75 with an efficacy of 31.15%; the disease index of SP3 was 36.46 with an efficacy of 42.62%; the disease index of SP4 was 22.92 with an efficacy of 63.93%; the disease index of SP5 was 22.92 with an efficacy of 63.93%; the disease index of SP6 was 19.79 with an efficacy of 68.85%; the disease index of SP7 was 23.96 with an efficacy of 62.29%; the disease index of SP8 was 37.5 with an efficacy of 40.98%; and the disease index of SP9 was 41.67 with an efficacy of 39.39%. The disease index of the control group (CK) was 63.54. Therefore, SP4, SP5, SP6 and SP7, which had high control efficacy, were screened and found to have all added biocontrol bacteria Pseudomonas22, Paenibacillus 33 and Paenibacillus 35. Therefore, it is speculated that the simple community treatment composed of FJ035 and Pseudomonas 22, Paenibacillus 33 and Paenibacillus 35 can reduce the incidence of Fusarium wilt and has stronger disease prevention ability.

[0043] (5) Validation experiment of core microbial community To verify the disease prevention effect of the simplified core bacterial group, a hydroponic method was used for verification experiments, with a total of 4 treatment groups (Table 2). The specific treatments are as follows: SP10 group (inoculated with Trichoderma echinococcus FJ035 and FOC), SP11 group (inoculated with 3 core bacteria and FOC simultaneously), SP12 group (inoculated with 3 core bacteria first, and then inoculated with FOC after 48 h); and the treatment inoculated with FOC only served as a positive control (CK).

[0044] Table 2. Four treatments of hydroponic cucumbers with core microbial communities

[0045] turn out( Figure 3 The disease index of SP10 was 36.46, with a control efficacy of 42.62%; the disease index of SP11 was 31.25, with a control efficacy of 50.82%; the disease index of SP12 was 20.83, with a control efficacy of 67.21%; while the disease index of the control group (CK) was 68.75. The simplified experimental verification showed that treatment SP12 had the highest control efficacy. Therefore, the hydroponic experiment verifying the function of the simplified core bacterial community showed that the simple community treatment consisting of FJ035 and Pseudomonas 22, Paenibacillus 33, and Paenibacillus 35, followed by FOC after 48 hours, significantly reduced the disease index of cucumber wilt. Since Paenibacillus 33 and Paenibacillus 35 are the same species of bacteria, to simplify the bacterial community, the following treatment groups were constructed: SPa (treatment group with FJ035, FOC, Pseudomonas 22 and Paenibacillus 35), SPb (treatment group with FJ035, FOC, Pseudomonas 22 and Paenibacillus 33), SPc (treatment group with FJ035, FOC, Pseudomonas 22, Paenibacillus 33 and Paenibacillus 35), SPd (treatment group with FJ035, FOC, Pseudomonas 22 and double Paenibacillus 35), Spe (treatment group with FJ035, FOC, Pseudomonas 22 and double Paenibacillus 33), and a control group with only FOC added (CK) (Table 3).

[0046] Table 3 Experimental treatments for constructing the core microbial community

[0047] turn out( Figure 4The disease index of SPa was 29.17, with a control efficacy of 58.82%; the disease index of SPb was 20.83, with a control efficacy of 70.59%; the disease index of SPc was 25, with a control efficacy of 64.7%; the disease index of SPd was 31.25, with a control efficacy of 55.88%; the disease index of Spe was 27.08, with a control efficacy of 61.77%; while the disease index of the control group CK was 70.83. Simplified experiments with different concentrations of core bacterial groups revealed that the control efficacy of SPb was the highest, demonstrating that when Paenibacillus 33 and Pseudomonas 22, at OD600=1.0 and the same addition amount, the core bacterial group composed of Paenibacillus 33, Pseudomonas 22, and FJ035 had the best control effect on cucumber wilt.

[0048] Regulation of the core flora by Trichoderma echinosporum FJ035

[0049] (1) Preparation and collection of root exudates First, a cucumber root division system was constructed. Seven-day-old cucumber seedlings were selected and transplanted into a sterilized root division device, with the roots evenly distributed in both side compartments. The device was wrapped with aluminum foil to protect it from light. An appropriate amount of 1 / 4 MS nutrient solution (enough to cover the roots) was added to both sides, and the solution was changed every 48 hours until the roots grew evenly on both sides. The experiment included a treatment group and a control group. Treatment group: FJ035 spore suspension (final concentration 1.0 × 10⁻⁶) was inoculated into the left root compartment. 6 In the control group, an equal volume of sterile water was added to both root chambers (spores / mL). After inoculation, residual liquid on the right side was aspirated, and the roots were rinsed with sterile water. Then, an appropriate amount of sterile water was added to begin collecting root exudates. Exudates from the uninoculated side (right side) of the treatment group and the control group were collected daily, with fresh sterile water added, for 4 consecutive days. The collected liquid was filtered through a 0.22 μm microporous membrane for sterilization and then freeze-dried for concentration. Standardization was performed based on root dry weight; the concentrated exudate corresponding to each gram of root dry weight was reconstituted in 1 mL of sterile water and stored at -20℃ for later use. Each treatment was repeated 3 times, with 5 plants per replicate.

[0050] (2) Effect of FJ035 on the motility of core bacterial strains Three bacterial discs were made from the 7-day activated FJ035 strain using a 6 mm punch and inoculated into 100 mL of PD medium. The culture was incubated at 28°C and 180 rpm for 7 days with shaking. The culture was centrifuged at 4°C and 10,000 rpm for 10 min to remove bacterial cells. The supernatant was filtered through a 0.22 μm filter to obtain sterile FJ035 metabolic solution, which was stored at 4°C for later use. The 3-day activated Pseudomonas 22 and Paenibacillus 33 strains were inoculated into 100 mL of TSB medium and incubated at 37°C and 180 rpm for 36 h with shaking. The bacterial concentration was adjusted to OD600 = 1.0 to prepare the test bacterial suspensions. Sterilized SMM semi-solid medium was cooled to approximately 45°C, and different treatment solutions were added according to the experimental design. After mixing, the solutions were poured into sterile petri dishes and allowed to solidify before use.

[0051] The specific treatments were as follows: Cucumber rhizosphere exudates were treated with FJ035 sterile metabolic solution at concentrations of 5%, 10%, and 20% (5 mL, 10 mL, and 20 mL of metabolic solution were added per 100 mL of solution), 25 mg / mL, 50 mg / mL, and 100 mg / mL, respectively. Cucumber rhizosphere exudates treated with FJ035 at concentrations of 25 mg / mL, 50 mg / mL, and 100 mg / mL served as treatment groups. The control group consisted of the same volume of PD medium and sterile water. 5 μL of bacterial suspension (OD600 = 1.0) of each core bacterial strain was vertically added to a petri dish. After the bacterial suspension air-dried on the medium, the dishes were sealed and incubated upright at 30°C for 48 h. Two colony diameters were measured from each petri dish, and the average value was taken. Each treatment was repeated three times.

[0052] turn out( Figures 5-6 Both bacteria were grown on SMM medium with each treatment for 48 h. The best motility was observed with 500 μL of FJ035 metabolite. Pseudomonas 22 showed the highest motility with 50 mg / mL and 100 mg / mL of cucumber rhizosphere exudate containing FJ035, resulting in colony diameters of 3.50 cm and 3.75 cm after 48 h on SMM medium, respectively. Paenibacillus 33 also showed the highest motility with 50 mg / mL and 100 mg / mL of cucumber rhizosphere exudate, resulting in colony diameters of 0.86 cm and 0.87 cm after 48 h on SMM medium, respectively. Therefore, the addition of FJ035 to cucumber rhizosphere exudate can improve the motility of the core bacteria Pseudomonas 22 and Paenibacillus 33.

[0053] (3) Chemotaxis assay of FJ035 on core bacterial strains Using 0.9% NaCl (pH 7.2) as a control, the capillary method was employed to conduct the chemotactic response assay of the core bacterial community. Each strain of the core bacterial community was cultured in TSB medium to the logarithmic developmental phase (OD600 = 1.0). The collected bacterial cells were washed twice with chemotactic buffer and resuspended in the same buffer. 10 mL of the prepared cell suspension was added to a sterile 50 mL centrifuge tube. Glass capillary tubes (0.9 mm inner diameter) containing cucumber rhizosphere exudates at different concentrations (e.g., 25 mg / mL, 50 mg / mL, 100 mg / mL), cucumber rhizosphere exudates treated with FJ035, and the test standard solutions listed in Table 4 were vertically placed into centrifuge tubes containing different bacterial treatment solutions. The control group contained sterile water. The tubes were incubated at room temperature for 60 min. The outer wall of the capillary tubes was rinsed with sterile water to remove any adhering bacterial solution. After breaking the capillary tubes, the contents were transferred to enzyme-free EP tubes, diluted with 40 μL of sterile water, and the solution was aspirated. 2 μL of the diluted suspension was then used to detect the relative bacterial expression levels in the treatment and control groups using qPCR. The qPCR experiment used the non-specific SYBR Green I dye method, and the relative fold change in CT value was measured using... Calculations were performed, and bar charts were plotted using the calculated values ​​in GraphPad Prism 9.5.1. qPCR primers were designed based on the gene sequences of Pseudomonas22 (Pse) and Paenibacillus 33 (Pae) ​​(Table 5).

[0054] Table 4 Test Standards

[0055] Table 5 Primer sequences of core bacteria

[0056] The genome of the core bacteria was extracted using the Tiangen Bacterial DNA Extraction Kit (DP302), as shown in Table 6.

[0057] Table 6. DNA extraction system of core bacteria

[0058] Each sample under different treatments was replicated three times.

[0059] turn out( Figure 7The core bacteria Pseudomonas 22 and Paenibacillus 33 showed significantly upregulated chemotaxis in cucumber rhizosphere exudates at concentrations of 50 mg / mL and 100 mg / mL after FJ035 application, with biomass of 19.52 and 18.26 mg / mL and 10.66 and 14.25 mg / mL, respectively. This was 5.76, 5.93, and 4.28 and 6.12 times higher than the chemotaxis of cucumber rhizosphere exudates without FJ035 at the same concentration. This demonstrates that FJ035 application can alter rhizosphere exudates in cucumber roots, and that these substances, at appropriate concentrations, can recruit the beneficial biocontrol bacteria Pseudomonas 22 and Paenibacillus 33.

[0060] (4) Effects of FJ035 on the colonization of core bacterial strains in cucumber roots According to the cucumber root division system in Example (1), the treatment group was inoculated with FJ035 spore suspension (final concentration of 1.0 × 10⁻⁶). 6 In the left chamber, sterile water was added only to the right chamber, while the control group was incubated with sterile water in both chambers. After 48 hours of incubation, the treated and control groups were inoculated with core bacterial culture and single core bacteria (final concentration of each strain was 1.0 × 10⁻⁶) in the left chamber. 6 (CFU / mL), each treatment was repeated three times, with 20 cucumber plants per treatment. After 48 h, the roots in the right chamber were cut and briefly rinsed with sterile water. The biomass of each strain of the core flora in the treatment and control groups colonizing the cucumber roots was detected by qPCR.

[0061] qPCR primers and systems were designed based on the SCCFO1 (FOC) gene sequence (Tables 7-9). Table 7 Primer sequences for FOC

[0062] Table 8 qPCR system

[0063] Table 9 Reverse Transcription Amplification

[0064] The above steps involved thorough mixing at low temperature, and each treatment was repeated three times. The qPCR amplification program consisted of a 5-minute pre-denaturation at 95°C, followed by 40 cycles: 95°C for 10 seconds, 60°C for 5 minutes, 72°C for 20 seconds, a melting phase of 95°C for 15 seconds, 65°C for 1 minute, a 0.1°C / s ramp-up at 95°C, and a final 15 seconds at 95°C. The non-specific SYBR Green I dye method was used for qPCR, and the relative fold change in CT values ​​was measured using... Calculate and plot the bar chart using the calculated values ​​with the GraphPad Prism 9.5.1 program.

[0065] turn out( Figure 8 After 48 hours, it was found that both treatments with FJ035 increased the biocolonization of cucumber rhizosphere. The biocolonization of the core bacterial community with FJ035 (i.e., Pseudomonas 22 and Paenibacillus 33) was 3560.71 and 567.64, respectively, which were 195.2 and 6.5 times higher than the biocolonization without FJ035. The biocolonization of Pseudomonas 22 with FJ035 was 5529.97, which was 7.3 times higher than the biocolonization without FJ035. The biocolonization of Paenibacillus 33 with FJ035 was 200.85, which was 1.7 times higher than the biocolonization without FJ035. This indicates that adding FJ035 to cucumber roots can promote the colonization of core bacteria, and the colonization of core bacteria is greater than that of core bacteria alone. In other words, core bacteria can recruit more core bacteria and enable them to colonize cucumber roots.

[0066] (5) Effects of FJ035 on cucumber root exudates The rhizosphere exudate samples collected in Example (1) were subjected to combined determination of non-target metabolites and target hormones by UPLC-QTOF-MS. Chromatographic separation was performed on a Waters ACQUITY UPLC HHS T3 column (2.1 mm × 100 mm, 1.8 μm). Mobile phase A was an aqueous solution containing 5 mmol / L ammonium acetate and 5 mmol / L acetic acid, and mobile phase B was acetonitrile. The sample pan temperature was 4 °C, and the injection volume was 2 μL. The screening threshold for differentially expressed genes was |Fold Change| ≥ 2 and FDR < 0.01.

[0067] turn out( Figure 9 The main upregulated substances in cucumber rhizosphere exudates were terpenoids (22.22%), shikimic acid and phenylpropanoids (18.39%), fatty acids (15.92%), and alkaloids (10.96%). In K-means analysis, after treatment with the core microbial community (FHE), gene expression in clusters 3, 4, 5, 7, and 9 was upregulated compared to CK and FOC, particularly from FHE to FOC. Changes in the early stage (CK-FHE-FOC) were mainly concentrated in terpenoid skeleton biosynthesis, fatty acid metabolism, phenylpropanoid biosynthesis, and lipid metabolism, with the main substances being terpenoids, fatty acids and their derivatives, phenylpropanoids and simple phenolic acids, and alkaloids.

[0068] Example 3: Evaluation of the control efficacy of the core microbial community against cucumber wilt disease

[0069] (1) The effect of the core microbial community on the prevention and control of cucumber wilt disease To verify the efficacy of core microbial communities as a biological control method against cucumber wilt, a comparative efficacy experiment was conducted between commercially available single Trichoderma inoculant (Trichoderma wettable powder) and chemical agents (3% hymexazol) and core microbial communities. After cucumber seeds were disinfected and germinated to show white sprouts, they were sown in seedling pots containing 100 g of sterilized soil. Under sterile soil potting conditions, the following treatments were performed (Table 10): PB1 (treatment group with core microbial communities and FOC), PB2 (treatment group with core microbial communities added 24 h later with FOC), PB3 (treatment group with 3% hymexazol and FOC), PB4 (treatment group with Trichoderma inoculant and FOC), PB5 (treatment group with 3% hymexazol added 24 h later with FOC), PB6 (treatment group with Trichoderma inoculant added 24 h later with FOC), and the treatment with only FOC (CK) served as the control. Each treatment and control was repeated three times, with each replicate including 5 plants. The fungal spore suspension (FJ035 / FOC) resulted in a spore concentration of 1.0 × 10⁻⁶ in the soil after inoculation. 6 The concentration of bacteria in the soil was 1.0 × 10⁶ spores / g, after inoculation with each strain of the core bacterial mixture. 6 CFU / g, Trichoderma agent diluted with sterile water to a spore concentration of 1.0 × 10⁻⁶ CFU / g in the soil after inoculation. 6 1 spore / g, 3% hymexazol diluted with sterile water and added to sterile soil, strictly following the instructions for use. Observe the disease incidence in potted cucumbers 21 days after inoculation of each treatment, calculate the disease index, and determine the control efficacy based on the disease index.

[0070] Table 10 Core Microbial Community and Specific Chemical and Biological Treatments

[0071] The results of the prevention and control results showed that ( Figure 10 The disease index of PB1 was 26.25, with a control efficacy of 61.11%; the disease index of PB2 was 20, with a control efficacy of 70.37%; the disease index of PB3 was 17.5, with a control efficacy of 74.07%; the disease index of PB4 was 53.75, with a control efficacy of 20.37%; the disease index of PB5 was 15, with a control efficacy of 77.78%; and the disease index of PB6 was 51.25, with a control efficacy of 24.07%. The control group (CK) had a disease index of 67.5. The experiment showed that applying FOC 24 hours after applying the core bacterial flora resulted in a 3.7% higher control efficacy than both treatments, a 3.71% lower efficacy than commonly used chemical agents, and a 50% higher efficacy than biological agents, proving that the core bacterial flora can effectively control cucumber wilt.

[0072] (2) The control efficacy of the core microbial community against the concentration gradient of cucumber wilt disease To verify the optimal concentration of the core bacterial flora for controlling cucumber wilt and thus determine the optimal addition amount, a pot experiment with different concentration gradients of the bacterial agent was designed. After cucumber seeds were disinfected and germinated to show white sprouts, they were sown in seedling pots containing 100 g of sterilized soil. Under sterile soil potting conditions, the following treatments were performed (Table 11): PE1 (with 1.0 × 10⁻⁶ FJ035 bacterial solution added) 5 1 spore / g and core bacterial culture 1.0 × 10⁶ 5 Treatment group (CFU / g), PE2 (with 1.0 × 10⁻⁶ FJ035 bacterial solution added) 6 1 spore / g and core bacterial culture 1.0 × 10⁶ 6 Treatment group (CFU / g), PE3 (with 1.0 × 10⁻⁶ FJ035 bacterial solution added) 7 1 spore / g and core bacterial culture 1.0 × 10⁶ 7 Treatment group (CFU / g), PE4 (with 1.0 × 10⁻⁶ FJ035 bacterial solution added) 6 1 spore / g and core bacterial culture 1.0 × 10⁶ 7 Treatment group (CFU / g), PE5 (with 1.0 × 10⁻⁶ FJ035 bacterial solution added) 5 1 spore / g and core bacterial culture 1.0 × 10⁶ 6 Treatment group (CFU / g), PE6 (with 1.0 × 10⁻⁶ FJ035 bacterial solution added) 7 1 spore / g and core bacterial culture 1.0 × 10⁶ 8 The treatment groups (CFU / g) and the control group (CK) were all supplemented with FOC at the same concentration of 1.0 × 10⁻⁶ as the control group. 6 1 spore / g. Treatment and control were repeated three times, with each replicate including 5 plants. Disease incidence was observed and the disease index was calculated 21 days after inoculation, and the control efficacy was calculated based on the disease index.

[0073] Table 11 Specific treatments for different concentrations of core microbiota

[0074] turn out( Figure 11 The disease index of PE1 was 31.25 with a control efficacy of 57.63%; the disease index of PE2 was 18.75 with a control efficacy of 74.58%; the disease index of PE3 was 26.25 with a control efficacy of 64.41%; the disease index of PE4 was 16.25 with a control efficacy of 77.97%; the disease index of PE5 was 21.25 with a control efficacy of 71.19%; and the disease index of PE6 was 27.5 with a control efficacy of 62.71%. The disease index of the control group (CK) was 73.75. Experimental data indicate that the optimal concentration of PE4 (FJ035) was 1.0 × 10⁻⁶. 6The concentration of Pseudomonas 22 and Paenibacillus 33 added was 1.0 × 10⁻⁶ spores / mL. 7 When CFU / mL is applied together with the core bacterial group, the protective efficacy is 77.97%, and the effect of antagonizing pathogen infection is the best.

[0075] (3) Field plot efficacy test of core bacterial group against cucumber wilt disease The field plot experiment selected plots of land that had been continuously cropped with cucumbers for many years. The control group received normal water, fertilizer, and pesticide management, without the application of chemical fungicides. The chemical treatment group received root drenching with a mixture of hymexazol and thiophanate-methyl. The green control treatment group received root drenching with a mixture of Trichoderma, Pseudomonas, and Bacillus subtilis, without the use of any chemical pesticides throughout the entire process. Application methods: Applications were given every 15-20 days after transplanting, during the seedling stage, vine-growing stage, flowering stage, and early fruit-setting stage (adjustments could be made slightly depending on the season). Chemical pesticides were used according to recommended dosages. Green control involved root drenching with a 1000-fold diluted solution (the fungicide was Trichoderma spores). 6 10 live spores / g, bacteria 10 7 (CFU / g, prepared separately into wettable powders and then compounded for use).

[0076] In the control group, chemical treatment group, and green control treatment group, 10 plants were randomly sampled at each of the three points to collect data on the incidence of Fusarium wilt. Figure 12 Disease index: The disease index of the control group was 63.33, the disease index of the chemical fungicide treatment group was 18.33, and the control efficacy against cucumber wilt was 71.05%; the disease index of the green control treatment group was 20.00, and the control efficacy against cucumber wilt was 68.42%.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for improving the resistance of cucumber to Fusarium wilt, characterized in that, The method involves applying *Trichoderma echinococcus* FJ035, *Pseudomonas nitroreductoides* 22, and *Bacillus vesicularis* 33 to the rhizosphere of cucumber plants via root irrigation; the concentration of *Trichoderma echinococcus* FJ035 is 1.0 × 10⁻⁶. 6 The application concentration of both *Pseudomonas nitroreductoides* 22 and *Bacillus vesicularis* 33 was 1.0 × 10⁻⁶ spores / mL. 7 CFU / mL; the *Trichoderma asperellum* FJ035, with accession number CGMCC NO.40942, was deposited at the China General Microbiological Culture Collection Center on November 30, 2023, and classified as *Trichoderma asperellum*; the *Pseudomonas nitroreducens* 22, with accession number CGMCC No.29051, was deposited at the China General Microbiological Culture Collection Center on November 20, 2023, and classified as *Pseudomonas nitroreducens*; the *Paenibacillus alvei* 33, with accession number CGMCC No.29055, was deposited at the China General Microbiological Culture Collection Center on November 20, 2023, and classified as *Paenibacillus alvei*.

2. The method according to claim 1, characterized in that, Apply once during the entire growth period of cucumber, at the seedling stage, vine-growing stage, flowering stage, and early fruit-setting stage.

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