Core microbiome for strongly preventing and treating cucumber fusarium wilt as well as preparation method and application thereof

Abstract

The invention relates to the technical field of agricultural disease control, in particular to a core microbiome for strongly controlling cucumber fusarium wilt as well as a preparation method and application of the core microbiome. According to the invention, three biocontrol bacteria with high antagonistic ability to fusarium oxysporum of cucumber are screened from rhizosphere soil of cucumber, and the three biocontrol bacteria are respectively bacillus velezensis VJH5041, paenibacillus polymyxa SXC20-B62 and bacillus subtilis YJZL20-1 through whole genome sequencing and genetic evolution analysis; however, it is found that the strain is significantly different from the reported strain in the aspects of genomic sequence and function, and it is detected that the three strains are not reported, and microbial strain preservation is completed. Meanwhile, culture conditions and proportions of mixed fermentation of core microorganisms of the three biocontrol strains are optimally designed, so that a better culture effect is achieved. Through condition optimization, the core microbiome has a remarkable biocontrol effect on cucumber fusarium wilt, the biocontrol effect of the core microbiome is obviously higher than that of a single strain, and the core microbiome can remarkably promote growth of cucumber seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural disease prevention and control, and in particular to a core microbial group for strongly preventing and controlling cucumber wilt disease, and a preparation method and application thereof. Background Art

[0002] Cucumber Fusarium wilt is called the "cancer" of plants. It is a highly destructive soil-borne disease in the cucumber production process, which has a great impact on the yield and quality of cucumbers. The pathogen of cucumber wilt is Fusarium oxysporum f.sp.cucumerium (FOC), which belongs to the genus Fusarium (Fusarium) of the Hyphomycetes of the Deuteromycotina subdivision and the Tuberculariaceae family of the Moniliales order. Four physiological subspecies of Fusarium oxysporum f.sp.cucumerium have been identified so far, and the pathogen in my country belongs to physiological subspecies No. 4. At 25-30℃, the pathogen invades the cucumber rhizome from the wound or root hair growth point and parasitizes and reproduces in the vascular bundle, blocking the duct, hindering the plant's absorption of nutrients and water, and can produce toxins to destroy the plant's defense system, causing the plant to die. Since cucumber wilt disease is highly explosive, destructive, and difficult to control, its current control is mainly based on prevention. Specific methods include: breeding of disease-resistant varieties, agricultural control, chemical pesticide control, and biological control.

[0003] Biological control has become a research hotspot for the prevention and control of soil-borne crop diseases because of its environmental protection and sustainability. With the rapid progress of microbiome science, the construction strategy of the core microbiome in biological control has attracted more and more attention. The rhizosphere soil microbiome is the first line of defense against pathogens invading plant roots. Inoculation of beneficial microbial groups (groups) can form a biological barrier around the crop rhizosphere, inhibit harmful microorganisms, and induce crops to produce immune functions, thereby providing consistent and lasting protection for plants. However, current research mainly focuses on the use of a single beneficial strain, ignoring the direct or synergistic effects of the microbiome on the rhizosphere of plants, resulting in a single beneficial bacteria often unable to achieve stable disease suppression in the field due to poor rhizosphere colonization ability.

[0004] Therefore, in order to solve the above problems, the present application provides a core microbial group with strong control over cucumber wilt disease and its preparation method and application. The constructed microbial group can not only avoid resource competition or direct interference that leads to mutual cancellation of effects, but also can cooperate with each other and promote each other to effectively inhibit cucumber wilt disease. Summary of the invention

[0005] The invention provides a core microbial group for effectively preventing and controlling cucumber wilt disease, and a preparation method and application thereof.

[0006] A core microbial group for effectively preventing and controlling cucumber wilt of the present invention comprises the following strains:

[0007] 1) Bacillus velezensis VJH5041, which was deposited in China Center for Type Culture Collection on May 10, 2024, with the deposit number of CCTCC NO: M2024902;

[0008] 2) Paenibacillus polymyxa SXC20-B62, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 23, 2020, with the deposit number CGMCC No. 21540;

[0009] 3) Bacillus subtilis YJZL20-1, the strain was deposited in the General Microbiology Center of China Culture Collection Administration on December 23, 2020, with the deposit number CGMCC No.21531.

[0010] Preferably, the inoculation amount of the above three strains in the culture medium is 4%-12%.

[0011] Preferably, the method is used to prepare a core microbial group for strong prevention and control of cucumber wilt, and the specific steps are:

[0012] S1: Biocontrol strains with strong inhibitory effects on cucumber wilt pathogens were screened from healthy cucumber soil. There were three biocontrol strains, named VJH5041, SXC20-B62 and YJZL20-1. The cucumber wilt pathogen was Fusarium oxysporum, a cucumber-specific strain.

[0013] S2: Verify the disease prevention effects of three biocontrol strains on cucumber wilt;

[0014] S3: Identify the specific species of the three biocontrol strains;

[0015] S4: Cultivate the three biocontrol strains and determine the optimal culture conditions;

[0016] S5: Determine the composition and ratio of the core microbiome.

[0017] Preferably, the disease prevention effects of the three biocontrol strains in step S3 on cucumber wilt are verified respectively, and the specific verification steps are:

[0018] 1) Soil samples were collected from the rhizosphere of healthy cucumber plants using the five-point sampling method;

[0019] 2) The pathogen used for the test was selected as Fusarium oxysporumf.sp.cucumerinum (FOC)

[0020] 3) Select detection kits and biocontrol strain culture media;

[0021] 4) Isolation, purification and preservation of biocontrol strains;

[0022] 5) Screening of biocontrol strains, screening strains with obvious antibacterial effects;

[0023] 6) Detection of the effect of biocontrol strains on FOC mycelial growth;

[0024] 7) Determination of the biocontrol and growth-promoting effects of biocontrol strains on cucumber wilt;

[0025] 8) The activity of cucumber-related defense enzymes induced by biocontrol strains was determined using a kit.

[0026] Preferably, the method for identifying the specific types of the three biocontrol strains in step S4 is:

[0027] 1) To identify the molecular biology of biocontrol strains;

[0028] 2) Determine the whole genome of the biocontrol strain, the specific steps are: extraction, sequencing and assembly of genomic DNA of the biocontrol strain; average nucleotide consistency analysis of the biocontrol strain; genome annotation analysis of the biocontrol strain;

[0029] 3) The specific types of the three biocontrol strains after identification are: Bacillus Velez, Paenibacillus polymyxa and Bacillus subtilis; therefore, the three biocontrol strains are specifically named: Bacillus Velez VJH5041, Paenibacillus polymyxa SXC20-B62 and Bacillus subtilis YJZL20-1.

[0030] Preferably, the specific cultivation steps of the three biocontrol strains in step S5 are:

[0031] 1) Select test strains and test culture medium. The test strains are: Bacillus velez VJH5041, Paenibacillus polymyxa SXC20-B62 and Bacillus subtilis YJZL20-1;

[0032] 2) Preparation of seed solution of biocontrol strains;

[0033] 3) Compatibility determination of three biocontrol strains;

[0034] 4) Optimize the ratio of the three biocontrol strains through orthogonal experiments. The process is based on the cell density OD of the core microbiome. 600 The OD value and the inhibition rate of FOC are used as the measurement indicators, among which OD 600 The value is an indicator of bacterial density or bacterial population density, which indirectly reflects the concentration of bacterial cells in liquid culture medium by measuring the light absorption intensity of the sample at a wavelength of 600 nanometers;

[0035] 5) Screening and optimization of the fermentation basal medium for the core microbiome to inhibit cucumber wilt, including: screening of carbon source species and concentration optimization, screening of nitrogen source species and concentration optimization, screening of inorganic salt species and concentration optimization, and orthogonal experiments for strain basal medium optimization;

[0036] 6) Optimization of the culture conditions of the core microbiome, including: response surface experiments on the optimization of the culture conditions of the core microbiome and comparative experiments on the fermentation medium and culture conditions of the core microbiome before and after optimization. The response surface experiment on the optimization of the culture conditions of the core microbiome is designed based on the inoculation amount, culture time, culture temperature, culture speed and initial pH screened by the single factor experiment. Each culture medium combination listed in the program is a treatment for strain fermentation culture. Each treatment is repeated 3 times, and the cell density OD of the fermentation broth is measured. 600 The values ​​were analyzed to determine the optimal conditions for the cultivation of the core microbiome.

[0037] Preferably, the culture conditions of the core microbial group in step S5 are: the carbon source of the culture medium is selected as glucose, the nitrogen source is selected as yeast extract powder, the inorganic salt is selected as dipotassium hydrogen phosphate, the solvent is double distilled water, the culture temperature is 20-35°C, the rotation speed is 150-200rpm, the time is 18h-24h, and the initial pH is 5.0-7.0.

[0038] Preferably, the culture conditions of the core microbial group in step S5 are preferably: the culture medium is 30g glucose, 20g yeast extract powder, 1g potassium dihydrogen phosphate, 1000mL double distilled water, the culture temperature is 30°C, the rotation speed is 171rpm, the time is 21.6h, the initial pH is 7.0, and the inoculation amounts of Bacillus velez VJH5041, Bacillus polymyxa SXC20-B62 and Bacillus subtilis YJZL20-1 are set to: 4%, 4% and 4%, respectively.

[0039] The invention discloses an application of a core microbial group for strongly controlling cucumber wilt disease. The core microbial group for strongly controlling cucumber wilt disease is applied to control cucumber wilt disease, and the core microbial group is also used to promote the growth of cucumber seedlings.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The invention screens out three biocontrol bacteria with high antagonistic ability to cucumber wilt pathogen from cucumber rhizosphere soil, namely VJH5041, SXC20-B62 and YJZL20-1, and identifies them as Bacillus velez, Paenibacillus polymyxa and Bacillus subtilis respectively through whole genome sequencing. The culture conditions and proportions of the three biocontrol strains are optimized and designed to have high-quality culture effects. The core microbial groups constructed by the three biocontrol strains have good biocontrol effects on cucumber wilt and can significantly promote the growth of cucumber seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a flow chart of the core microbiome construction method of the present invention;

[0043] Figure 2 This is a diagram showing the antagonistic effect of some of the screened strains of the present invention on FOC;

[0044] Figure 3 It is a graph showing the effect of strain VJH5041 of the present invention on FOC mycelium;

[0045] Figure 4 This is a graph showing the growth-promoting effect of the strain VJH5041 of the present invention on cucumber;

[0046] Figure 5 is a compatibility diagram of B. velezensis VJH5041, P. polymyxa SXC20-B62 and B. subtilis YJZL20-1 of the present invention;

[0047] Figure 6 Graph showing the effects of different carbon sources on the cell density and antibacterial activity of the core microbial group of the present invention;

[0048] Figure 7 Graph showing the effects of different nitrogen sources on the cell density and antibacterial activity of the core microbial group of the present invention;

[0049] Figure 8 Graph showing the effects of different inorganic salts on the cell density and antibacterial activity of the core microbial group of the present invention;

[0050] Fig. 9 Graph showing the effects of different inoculation amounts on the bacterial density and antibacterial activity of the core microbial group of the present invention;

[0051] Fig.10 is a graph showing the effect of different fermentation times on the bacterial density and antibacterial activity of the core microbial group of the present invention;

[0052] Fig.11 Graph showing the effects of different culture temperatures on the cell density and antibacterial activity of the core microbial group of the present invention;

[0053] Fig.12 This is a graph showing the effects of different culture speeds on the cell density and antibacterial activity of the core microbial group of the present invention;

[0054] Fig.13 : is a graph showing the effect of different initial pH values ​​on the bacterial density and antibacterial activity of the core microbial group of the present invention;

[0055] Fig.14 The different culture factors of the present invention interact with each other to affect OD 600 Response surface and contour plots of the influence of .

[0056] Fig.15 This is a potted plant control effect diagram of the core microbial group of the present invention;

[0057] Fig.16 It is a diagram of the prevention and treatment effect of the core microbial group of the present invention. DETAILED DESCRIPTION

[0058] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0059] First, experiments have confirmed the presence of microorganisms in healthy cucumber soil that inhibit the pathogen of cucumber wilt. The following examples describe the process of screening, effect verification, species identification, cultivation, and determination of the core microbial group of biocontrol strains.

[0060] 1. Embodiment 1

[0061] Disease prevention effect and whole genome analysis of the antagonistic bacterium VJH5041 against cucumber wilt;

[0062] 1. Experimental Materials

[0063] 1.1 Soil samples

[0064] Soil samples were collected from the rhizosphere of healthy cucumber plants in the cucumber planting area of ​​Jingdian Town, Neihuang County, Anyang City, Henan Province. A total of 50 soil samples were collected using the five-point sampling method. The sampling table is as follows:

[0065]

[0066]

[0067] 1.2. Test pathogens

[0068] Fusarium oxysporum f.sp.cucumerinum (FOC) was isolated and preserved in our laboratory.

[0069] 1.3 Reagents and culture media

[0070] Phenylalanine ammonia-lyase (PAL) activity detection kit, lipoxygenase (LOX) activity detection kit, catalase (CAT) activity detection kit, polyphenol oxidase (PPO) activity detection kit, superoxide dismutase (SOD) activity detection kit.

[0071] LB liquid medium: peptone 10 g, yeast extract powder 5 g, NaCl 1 g, add double distilled water to 1 000 mL, adjust the pH to 7.0 with NaOH, and sterilize at 121 °C for 25 min.

[0072] LB solid medium: peptone 10 g, yeast extract powder 5 g, NaCl 1 g, agar powder 10 g, add double distilled water to 1000 mL, adjust the pH to 7.0 with NaOH, and sterilize at 121 °C for 25 min.

[0073] PDA medium: 20 g glucose, 200 g potato, 15 g agar powder, add double distilled water to 1 000 mL, adjust the pH to 7.0, and sterilize at 121°C for 25 min.

[0074] PDB medium: 20 g glucose, 200 g potato, add double distilled water to 1 000 mL, adjust the pH to 7.0, and sterilize at 121°C for 25 min.

[0075] Skim milk medium: CaCl2 0.1g, NaCl 0.1g, peptone 10g, skim milk powder 10g, agar powder 18g, add double distilled water to 1 000mL, adjust pH to 7.0.

[0076] Starch agar medium: 10 g soluble starch, 10 g tryptone, 5 g glucose, 5 g NaCl, 5 g beef extract, 18 g agar powder, add double distilled water to 1 000 mL, and adjust the pH to 7.0.

[0077] Carboxymethyl cellulose agar medium: CMC-Na 5g, MgSO4·7H2O 0.1g, K2HPO4 0.25g, agar powder 18g, add double distilled water to 1 000mL, and adjust the pH to 7.0.

[0078] β-1,3-Glucan agar medium: 0.05 g glucose, 0.5 g yeast extract, 1 g peptone, 0.5 g NaCl, 0.01 g Congo red, 18 g agar powder, add double distilled water to 1 000 mL, and adjust the pH to 7.0.

[0079] Chrome Azurol S blue agar medium: 20% sucrose solution 10mL, 10% acid hydrolyzed casein 30mL, 1mmol·L-1CaCl2 1mL, 0.1mol·L-1 phosphate buffered saline (pH=6.8) 5mL, CAS staining solution 50mL, agar powder 18g, add double distilled water to 1000mL, adjust pH to 7.0.

[0080] L-tryptophan NB medium: 3 g beef extract, 10 g peptone, 5 g NaCl, 0.5 g L-tryptophan, add double distilled water to 1000 mL, and adjust the pH to 7.0.

[0081] Salkowski dye: 150 mL of concentrated sulfuric acid, 250 mL of distilled water, 7.5 mL of 0.5 mol·L-1 ferric chloride solution.

[0082] 1.4 Experimental instruments

[0083] Refrigerated centrifuge, clean bench, water purifier, high-pressure steam sterilizer, electric blast drying oven, electronic analytical balance, Monad PCR instrument and UV spectrophotometer.

[0084] 2. Experimental methods

[0085] 2.1. Isolation, purification and preservation of biocontrol strains

[0086] Weigh 10g of each collected soil sample into a 250mL conical flask and add 100mL of sterile distilled water. Oscillate to fully suspend the soil sample. Then heat the suspension in a 62℃ water bath for 25min. After standing for 15min, take 100μL of the suspension and evenly spread it on the LB solid culture medium plate. Invert the plate in a 30℃ constant temperature mold incubator and culture for 2d. Select colonies of different morphologies, streak and purify 3 times until the new colonies grown are all single colonies, and use a microscope for microscopic examination and verification. Store the purified strain in a 4℃ refrigerator for subsequent experiments.

[0087] 2.2. Biocontrol strain screening

[0088] The plate confrontation method was used for initial screening. FOC was used as the indicator bacteria. A 5 mm bacterial cake was punched with a sterile puncher and connected to the center of the PDA plate. A single colony of the purified isolated strain was picked with a sterile toothpick and inoculated 2.5 cm on both sides of the FOC bacterial cake. The PDA plate inoculated with only FOC was used as a blank control. After 7 days of culture in a 28°C mold incubator, the antibacterial zone was measured to screen out the strain with the most obvious antibacterial effect.

[0089] 2.3. Detection of the effect of strain VJH5041 on FOC mycelial growth

[0090] Co-culture of FOC and VJH5041 strain: Use a 5mm diameter puncher to punch pathogenic bacteria cakes on the edge of the FOC plate and insert them into the center of the PDA plate. Inoculate bacteria 25mm away from both sides of the cake, and be on the same straight line as the midpoint of the cake. The control is a PDA plate inoculated with only FOC but no bacteria. Cut the FOC hyphae from the antagonistic part of the antagonistic plate, and the sample volume should not exceed 3mm 3 After gently rinsing the sample with phosphate buffer sterile (PBS), the sample was quickly placed in electron microscopy fixative at room temperature for 2 hours and then transferred to 4°C for storage. The fixed sample was observed by scanning electron microscopy.

[0091] 2.4. Determination of the biocontrol and growth-promoting effects of strain VJH5041 on cucumber wilt

[0092] The VJH5041 strain was cultured in LB medium at 37°C with shaking at 180 rpm for 24 h, and the cells were collected by centrifugation at 4000×g for 5 min. The cells were diluted to 1×10 8 CFU / mL.

[0093] FOC was grown on PDA plates at 30°C for 5 days, and then 10 bacterial cakes were punched at the edge of the pathogen using a 5 mm puncher and transferred to 100 mL PDB. The culture was cultured on a shaker at 28°C at 180 rpm for 3 days, and the FOC culture was filtered through 4 layers of sterile gauze and diluted with sterile water to a concentration of 1 × 10 6 CFU / mL of pathogen spore suspension.

[0094] Select the robust, plump and uniform-sized cucumber seeds of variety Bojie 616, disinfect the surface with 75% ethanol (v / v) for 30 seconds, and wash with sterile water 5 times. Soak the seeds in warm water at 55°C for 10 minutes. Then soak the seeds in distilled water for 4 hours, rinse the soaked cucumber seeds 3 times, moisten the gauze with sterile water, place the cucumber seeds on the gauze and place them in a 28°C constant temperature incubator for germination. After the seeds turn white, sow the germinated cucumber seeds in a seedling tray (40 holes) filled with sterile substrate. After 10 days of sowing, transfer the seedlings with two leaves and one heart to a pot with a diameter of 10 cm, a height of 10 cm and 400 g of sterile substrate.

[0095] The completely randomized design was as follows: Treatment 1: 20 mL of sterile distilled water was used to irrigate the cucumber seedlings, once every two days, for a total of 3 times; Treatment 2: 20 mL of sterile distilled water was used to irrigate the cucumber seedlings for 3 times, and then the FOC spore suspension was used for root irrigation; Treatment 3: 20 mL of 0.1% oxadipate was used to irrigate the roots once, and 20 mL of FOC spore suspension was used to irrigate the roots 24 hours later; Treatment 4: 20 mL of VJH5041 suspension was used to irrigate the roots for 3 times, and then 20 mL of FOC spore suspension was used to irrigate the roots; Treatment 5: 20 mL of VJH5041 suspension was used to irrigate the cucumber seedlings, once every two days, for a total of 3 times. Each treatment had 5 replicates, and each replicate had 5 pots of plants. Under greenhouse conditions, the ambient temperature was between 14-25°C, and the photoperiod was 16 hours of light and 8 hours of darkness.

[0096] At 20d and 40d after inoculation of the strain, plant height, stem diameter, leaf area, chlorophyll content, aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight were measured, and the seedling index was calculated. The leaf chlorophyll content was measured using a SPAD-502Plus chlorophyll content meter. Root length and branch height were measured with a ruler. The fresh weight of roots and stems was recorded using an analytical balance (ME203E, Mettler Toledo, Shanghai, China). Stem diameter was measured at 2cm from the crown using a vernier caliper (MNT-200, Shanghai Mettler Metal Instrument Co., Ltd., Shanghai, China). Seedling index = (stem diameter / plant height + root dry weight / aboveground dry weight) × whole plant dry weight. The severity of the disease and the disease index of the plant at 40d were recorded. The disease level of cucumber wilt is divided into 5 levels: level 0, no symptoms on leaves; level 1, the leaves wilt less than 1 / 4 of the cucumber seedlings; level 2, 1 / 4-1 / 2 of the cucumber seedlings wilt leaves; level 3, more than 1 / 2 of the cucumber seedlings wilt leaves; level 4, the whole cucumber plant wilts and dies. Incidence = [N1+N2+N3+N4] / T×100%, where N is the number of cucumber seedlings scored for each disease, and T is the total number of cucumber seedlings. The health index (HI) of cucumbers is evaluated: healthy plants are "2", plants infected with wilt are "1", and plants severely infected with wilt are "0". The disease index is calculated using DI = [[(0×N0)+(1×N1)+(2×N2)+(3×N3)+(4×N4)] / T×4]×100, where N is the number of cucumber seedlings scored for each disease, and T is the total number of cucumber seedlings. Morbidity = [N1 + N2 + N3 + N4] / T × 100%. Relative protective efficacy = (control DI - treatment DI) / control DI × 100%.

[0097] 2.5. Determination of the activity of cucumber-related defense enzymes induced by strain VJH5041

[0098] 20 days after the antagonistic bacteria were inoculated, plants with the same growth were selected. Cucumber leaves of each treatment were collected, 1 g each, and the samples were quickly frozen with liquid nitrogen, packed in tin foil, and stored in a refrigerator at -80°C. According to the requirements of the kit instructions, the crude enzyme solution of leaf tissue was extracted, and the enzyme activity of PAL, LOX, CAT, PPO, and SOD was determined using the enzyme activity kit provided by Beijing Solebow Technology Co., Ltd. The enzyme activity is expressed as per gram of fresh weight of leaf tissue (U·g -1 ·FW -1 ).

[0099] 2.6 Molecular biological identification of strain VJH5041

[0100] A single colony of the VJH5041 strain was inoculated in LB medium and shaken at 37°C and 150 rpm for 18 h. The genomic DNA was extracted using the Mini BEST Bacterial Genomic DNA Extraction Kit (V 3.0) according to the manufacturer's instructions. The extracted strain genomic DNA was used as a template, and the universal primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and U1492R (5'-GGTTACCTTGTTACGACTT-3') were used for 16S rDNA sequence amplification. The amplified product was purified by Zhengzhou Qingke Biotechnology Co., Ltd. and sequenced. The measured sequence was compared with the known gene sequences in the database by BLAST, homology analysis was performed, homologous sequences were downloaded, and finally the phylogenetic tree was constructed using the neighbor-joining method of MEGA software.

[0101] 3. Results and analysis

[0102] 3.1. Screening of biocontrol strains

[0103] A total of 322 strains were isolated by plate dilution method, including 218 bacteria and 104 fungi, and they were numbered respectively. The subsequent plate confrontation method screened out 41 bacteria with strong antagonism to FOC. Figure 2 The antagonistic effect of some strains on FOC, among which the strains with the most obvious antagonistic effect on cucumber wilt pathogen are VJH5041, SXC20-B62 and YJZL20-1. Their inhibition rates are calculated as 87.96%, 88.36% and 86.52% respectively.

[0104] 3.2 Effect of strain VJH5041 on FOC mycelial growth

[0105] The mycelial morphology of the antagonized FOC was observed using a scanning electron microscope, and it was found that the mycelial morphology of the VJH5041 strain antagonized showed a wrinkled morphology, while the mycelial morphology of the control treatment was smooth, and no abnormal changes were observed, such as Figure 3 shown.

[0106] 3.3. Control effect of strain VJH5041 in potted plants

[0107] 40 days after inoculation of FOC, the incidence rate, disease index and relative control efficacy of different treatments showed that the VJH5041 strain significantly reduced the wilt symptoms caused by FOC, with the incidence rate and disease index reduced by 77.12% and 92.26% respectively. The control effect of the VJH5041 strain on the wilt pathogen was higher than that of the chemical fungicide, oxazolidinone, as shown in the following table:

[0108]

[0109] Note: Values ​​are mean ± standard error (SE), n = 3. Different letters in the column indicate significant differences between the means (p < 0.05).

[0110] 3.4. Growth-promoting effect of strain VJH5041 on cucumber

[0111] At 20 and 40 days, the chlorophyll content, plant height, stem diameter, root length, leaf area, aboveground fresh weight and dry weight, and underground fresh weight and dry weight of cucumber plants inoculated with VJH5041 strain increased significantly. At 20 days, all indicators of cucumber plants inoculated with VJH5041 strain were significantly higher than those of the control treatment. At 20 days, the increase in leaf area and aboveground fresh weight was the largest. The area of ​​the first true leaf treated with VJH5041 increased by 99.16% compared with the control, and the area of ​​the second true leaf increased by 385.1% compared with the control; the aboveground fresh weight increased by 70.78% compared with the control, as shown in Table 1 below. At 40 days, the most obvious difference between the two treatments was that the average area of ​​the fourth true leaf of the cucumber seedlings treated with VJH5041 could reach 65.5mm 2 , but the fourth true leaf area of ​​the control treatment was less than 5mm 2 , as shown in Table 2. At 20 days, the seedling index of VJH5041 treatment (0.065) was significantly higher than that of the control (0.052); at 40 days, the seedling index of VJH5041 treatment (0.115) was significantly higher than that of the control (0.073). Figure 4 shown.

[0112] Table 1

[0113]

[0114]

[0115] Note: Values ​​are mean ± standard error (SE), n = 3. Different letters in the column indicate significant differences between the means (p < 0.05).

[0116] Table 2

[0117]

[0118]

[0119] Note: Values ​​are mean ± standard error (SE), n = 3. Different letters in the column indicate significant differences between means (p < 0.05).

[0120] 3.5 Effect of strain VJH5041 on the activity of cucumber defense enzymes

[0121] As shown in the table below, at 20 days, the activities of LOX, PAL, CAT, PPO and SOD in cucumber seedlings treated with VJH5041 strain were significantly higher than those in the control treatment, increasing by 48.75%, 48.68%, 24.28%, 227.83% and 50.49%, respectively. Compared with the control treatment, inoculation with FOC also significantly increased the activities of these five defense-related enzymes, with increases of 236.03%, 81.64%, 43.84%, 223.98% and 51.67%, respectively. It was observed that the activities of all enzymes in seedlings inoculated with VJH5041 strain and then inoculated with FOC were significantly higher than those inoculated with FOC alone. However, compared with inoculation with FOC alone, the activities of PAL, CAT and SOD increased significantly after inoculation with FOC and benomyl, but LOX and PPO decreased significantly.

[0122]

[0123] Note: Values ​​are mean ± standard error (SE), n = 3. Different letters in the column indicate significant differences between means (p < 0.05).

[0124] 3.6. Species identification of strain VJH5041

[0125] The 16S rDNA sequencing results of strain VJH5041 were compared for homology using the NCBI database, and a phylogenetic tree was constructed. The results showed that strain VJH5041 had the highest homology with Bacillus velezensis FZB42 and G431. At the same time, the whole genome comparison results of VJH5041 and other strains showed that the average nucleotide identity (ANI) between VJH5041 strain and B. velezensis FZB42, B. velezensis G341 and B. velezensis BvL03 strains were 98.12%, 98.05% and 97.55%, respectively, all higher than the 95% threshold for bacterial species. In contrast, the ANI values ​​between VJH5041 strain and the other 10 Bacillus strains were much lower (66.53%-72.03%). Therefore, strain VJH5041 was identified as Bacillus velezensis.

[0126] 2. Embodiment 2

[0127] The technical ideas for disease prevention effects and whole genome analysis of cucumber wilt antagonistic bacteria SXC20-B62 and cucumber wilt antagonistic bacteria YJZL20-1 are the same as those in Example 1. Through experiments, it was found that cucumber wilt antagonistic bacteria SXC20-B62 and cucumber wilt antagonistic bacteria YJZL20-1 are Bacillus polymyxa SXC20-B62 and Bacillus subtilis YJZL20-1, respectively. Both Bacillus polymyxa SXC20-B62 and Bacillus subtilis YJZL20-1 can significantly reduce the incidence of cucumber wilt and increase the activity of cucumber defense enzymes.

[0128] 3. Embodiment 3

[0129] Core microbiome construction and culture condition optimization

[0130] 1. Experimental Materials

[0131] 1.1. Test strains

[0132] The strains Bacillus velezensis VJH5041, Paenibacillus polymyxa SXC20-B62 and Bacillus subtilis YJZL20-1, hereinafter referred to as B. velezensis VJH5041, P. polymyxa SXC20-B62 and B. subtilis YJZL20-1, were isolated and screened by the above experiment.

[0133] 1.2 Test culture medium

[0134] LB solid culture medium, LB liquid culture medium, and PDA culture medium are the same as those in Example 1.

[0135] 2. Experimental methods

[0136] 2.1. Preparation of strain seed solution

[0137] The B. velezensis VJH5041, P. polymyxa SXC20-B62 and B. subtilis YJZL20-1 strains stored at -20°C were activated by plate streaking. A single colony of the cultured strain was inoculated into LB liquid medium using a sterile inoculation needle and placed in a shaker at 30°C and 180 rpm for shaking culture. The seed liquid OD 600 Adjust the value to 1.0.

[0138] 2.2. Compatibility determination of antagonistic strains

[0139] Single colonies of B. velezensis VJH5041, P. polymyxa SXC20-B62 and B. subtilis YJZL20-1 were picked in LB liquid medium and cultured in a shaker at 30°C and 150rpm for 24h. The plate streak method was used to inoculate one biocontrol bacterium horizontally on the LB solid medium plate, and then the second antagonistic bacterium was inoculated vertically, and finally the third antagonistic bacterium was inoculated at the intersection of the first two streaks. After the plate was inverted and cultured in a 30°C incubator for 24h, it was observed whether an antibacterial band appeared between the antagonistic strain combinations. If no antibacterial band appeared, it indicated that the strains had good compatibility and could be used to construct the core microbiome for preventing cucumber wilt.

[0140] 2.3. Orthogonal experiment to optimize the ratio of three strains

[0141] The density of the core microbiome OD 600 The value and the inhibition rate of FOC were used as the measurement indicators, and the orthogonal experiment was used to optimize the ratio of B. velezensis VJH5041, P. polymyxa SXC20-B62 and B. subtilis YJZL20-1 strains in the core microbiome. B. velezensis VJH5041, P. polymyxa SXC20-B62 and B. subtilis YJZL20-1 strains were selected as factors, and 4%, 8% and 12% of each strain were selected as the inoculation level, respectively, and a three-factor and three-level L 9 (3 4 ) Orthogonal experiment, as shown in the following table. The seed liquid of the three strains was mixed in LB liquid medium according to the proportions in the orthogonal experiment table, placed in a shaker at 30°C and 180 rpm for 24 hours, and a portion was taken out and the OD was measured using a UV spectrophotometer 600 The other part was used for plate antagonism experiment with FOC strain.

[0142]

[0143]

[0144] 2.4 Screening and optimization of fermentation basal medium for inhibiting cucumber wilt by core microbiome

[0145] change

[0146] Single-factor experiments on carbon source, nitrogen source and inorganic salt were carried out on the constructed core microbial group in turn to determine the composition of the fermentation medium of the core microbial group that inhibits cucumber wilt.

[0147] 2.4.1. Screening of carbon source types and optimization of their concentration

[0148] Glucose, maltose, fructose, sucrose and soluble starch were used as carbon sources in the culture medium, and the experimental treatments were as follows:

[0149] (1) 1 g peptone, 1 g glucose, 0.1 g sodium chloride, 100 mL double distilled water.

[0150] (2) 1 g peptone, 1 g maltose, 0.1 g sodium chloride, 100 mL double distilled water.

[0151] (3) 1 g peptone, 1 g fructose, 0.1 g sodium chloride, 100 mL double distilled water.

[0152] (4) 1 g peptone, 1 g sucrose, 0.1 g sodium chloride, 100 mL double distilled water.

[0153] (5) 1 g peptone, 1 g soluble starch, 0.1 g sodium chloride, 100 mL double distilled water.

[0154] LB medium was used as the control, each carbon source was used as one treatment, and each treatment was repeated 3 times. 600 Value and suppression rate of FOC.

[0155] 2.4.2. Screening of nitrogen sources and optimization of their concentration

[0156] Beef extract, peptone, yeast extract powder, tryptone, soy peptone, and ammonium sulfate were used as nitrogen sources in the culture medium, respectively. The experimental treatments were as follows:

[0157] (1) 1 g beef extract, 1 g glucose, 0.1 g sodium chloride, 100 mL double distilled water.

[0158] (2) 1 g peptone, 1 g glucose, 0.1 g sodium chloride, 100 mL double distilled water.

[0159] (3) 1 g yeast extract powder, 1 g glucose, 0.1 g sodium chloride, 100 mL double distilled water.

[0160] (4) 1 g tryptone, 1 g glucose, 0.1 g sodium chloride, 100 mL double distilled water.

[0161] (5) 1 g soy peptone, 1 g glucose, 0.1 g sodium chloride, 100 mL double distilled water.

[0162] (6) 1gl ammonium sulfate, 1g glucose, 0.1g sodium chloride, 100mL double distilled water. LB medium was used as the control, each nitrogen source was used as one treatment, and each treatment was repeated 3 times. Determine the cell density OD of the fermentation broth with different nitrogen sources 600 Value and suppression rate of FOC.

[0163] 2.4.3. Screening of inorganic salts and optimization of their concentration

[0164] Sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, calcium carbonate, magnesium sulfate and zinc sulfate were used as inorganic salts in the culture medium, respectively. The experimental treatment was as follows:

[0165] (1) 1 g yeast extract powder, 1 g glucose, 0.1 g sodium chloride, 100 mL double distilled water.

[0166] (2) 1 g yeast extract powder, 1 g glucose, 0.1 g disodium hydrogen phosphate, 100 mL double distilled water.

[0167] (3) 1 g yeast extract powder, 1 g glucose, 0.1 g potassium dihydrogen phosphate, 100 mL double distilled water.

[0168] (4) 1 g yeast extract powder, 1 g glucose, 0.1 g calcium carbonate, 100 mL double distilled water.

[0169] (5) 1 g yeast extract powder, 1 g glucose, 0.1 g magnesium sulfate, 100 mL double distilled water.

[0170] (6) 1g yeast extract powder, 1g glucose, 0.1g zinc sulfate, 100mL double distilled water. LB medium was used as the control, each inorganic salt was used as one treatment, and each treatment was repeated 3 times. Determine the cell density OD of the fermentation broth with different inorganic salts 600 Value and suppression rate of FOC.

[0171] 2.4.4 Orthogonal experiment for optimization of basic culture medium for strains

[0172] The selected carbon sources were divided into five different concentrations of 0.3%, 0.5%, 1.0%, 2.0%, and 3.0%, nitrogen sources were divided into five different concentrations of 0.3%, 0.5%, 1.0%, 2.0%, and 3.0%, and inorganic salts were divided into five different concentrations of 0.03%, 0.05%, 0.10%, 0.20%, and 0.30%, and a three-factor, five-level L 25 (5 6 ) Orthogonal experiment to determine the best ratio. The experimental design is as follows:

[0173]

[0174]

[0175] 2.5. Optimization of culture conditions for core microbiome

[0176] The core microbial group was cultured using the optimal culture medium screened out in the above experiment, and single-factor condition experiments such as inoculation volume, fermentation time, culture temperature, culture speed and initial pH were carried out in sequence. The inoculation volume was 50, 100, 15, 200, and 250 μL respectively; the fermentation time was 12, 15, 18, 21, and 24 h respectively; the culture temperature was 20, 25, 30, 35, and 38 ° C respectively; the culture speed was 120, 150, 180, and 200 rpm respectively; and the initial pH was 5.0, 6.0, 7.0, 8.0, and 9.0 respectively. Each treatment was repeated 3 times, and the OD of the fermentation liquid was measured. 600 The optimal culture conditions of the core microbiome were determined by the inhibition rate of FOC. On this basis, the culture conditions of the core microbiome were further optimized using the response surface methodology, and the optimal culture conditions of the core microbiome for disease prevention and growth promotion were finally determined.

[0177] 2.5.1. Response surface experiment for optimization of core microbiome culture conditions

[0178] According to the inoculation amount, culture time, culture temperature, culture speed and initial pH screened out by the single factor experiment, the response surface program was designed. Each culture medium combination listed in the program was used as a treatment for strain fermentation. Each treatment was repeated 3 times, and the cell density OD of the fermentation liquid was measured. 600 The values ​​were analyzed to determine the optimal conditions for the cultivation of the core microbiome.

[0179] 2.5.2. Comparative test of the fermentation medium and culture conditions of the core microbiome before and after optimization

[0180] The core microbiome was fermented and cultured using the optimized culture medium formula and culture conditions, and the inhibition rate of the fermentation liquid against cucumber wilt was determined. The fermentation medium and fermentation conditions before optimization were used as a control to verify the effect of the optimized culture conditions on the cell density of the core microbiome.

[0181] The bacterial solution of the core microorganism group was applied to cucumber potted plants to verify its control effect on cucumber wilt. The specific experimental operation was the same as 2.4 of Example 2, and the control effect was tested after 15 days of cultivation.

[0182] 3. Results and analysis

[0183] 3.1 Compatibility results analysis

[0184] Compatibility tests were performed on B. velezensis VJH5041, P. polymyxa SXC20-B62 and B. subtilis YJZL20-1. The results are as follows: Figure 5 As shown, no inhibition zone appeared when the three bacteria were co-cultured, indicating that the three bacteria had good compatibility.

[0185] 3.2 Analysis of orthogonal experimental results

[0186] As shown in the table below, when the inoculation amounts of B. velezensis VJH5041, P. polymyxa SXC20-B62, and B. subtilis YJZL20-1 strains were 4%, 4%, and 4%, respectively, after culturing at 30°C and 180rpm for 24 hours, the core microbial group had the highest cell density and the strongest inhibitory ability against cucumber wilt pathogen.

[0187]

[0188] 3.3. Optimization results of the basal medium for core microbiome fermentation

[0189] 3.3.1 Effects of carbon sources on the density and antibacterial activity of the core microbiome

[0190] The carbon source in the culture medium has a vital influence on the growth of microorganisms, because it is not only the main source of energy production for microorganisms, but also an important component of cell synthesis. Figure 6 It can be seen that when glucose is used as the carbon source, the OD of the core microbiome 600 The value was the highest. At the same time, the results of the antagonistic experiment between the core microbiome and FOC also showed that glucose was the best carbon source for the culture medium in this study.

[0191] 3.3.2 Effects of nitrogen sources on the density and antibacterial activity of the core microbiome

[0192] The nitrogen source in the culture medium is also crucial to the growth of microorganisms, because nitrogen is one of the indispensable elements in the process of cell biosynthesis, mainly used to synthesize proteins, nucleic acids, certain components of cell walls and other nitrogen-containing compounds. The type and availability of nitrogen sources have a significant impact on the growth rate, biomass production, metabolic pathways and product synthesis of microorganisms. Figure 7 It can be seen that when yeast extract powder is used as the nitrogen source of the culture medium, the bacterial growth of the core microbiome is the largest and the inhibition rate of FOC is the highest. Therefore, yeast extract powder is used as the nitrogen source of the fermentation medium of the core microbiome.

[0193] 3.3.3 Effects of inorganic salts on the cell density and antibacterial activity of the core microbiome

[0194] Inorganic salts in the culture medium can participate in regulating the ion balance and pH value inside and outside the cells, and affect enzyme activity and metabolic pathways. Figure 8 It can be seen that dipotassium hydrogen phosphate is the best inorganic salt for fermentation basal medium.

[0195] 3.3.4. Orthogonal experimental results of carbon source, nitrogen source and inorganic salt in fermentation basal medium

[0196] The OD of the core microbiome after 24 h of culture was evaluated by different combinations of orthogonal experiments. 600 And the inhibition rate of FOC, as shown in the following table, when the concentrations of glucose, yeast extract powder and potassium dihydrogen phosphate were 3%, 2% and 0.1% respectively, after culturing at 30℃, 180rpm for 24h, the core microbial group had the highest cell density and the strongest inhibition ability against cucumber wilt pathogen.

[0197]

[0198]

[0199] 3.4. Results of optimization of core microbiome culture conditions

[0200] 3.4.1 Effect of inoculum size on the bacterial density and antibacterial activity of the core microbiome

[0201] The inoculum size affects the colony growth of the core microbiome, so controlling the inoculum size is crucial for the cultivation of the core microbiome. Fig. 9 It can be seen that the bacterial density of the core microbiome increases first and then decreases with the increase of the inoculation volume. When the inoculation volume is 100μL and 200μL, the colony OD 600 The value and inhibition rate of FOC were the highest, which were significantly different from those of 50 μL and 250 μL. Therefore, the experiment determined that the best effect was achieved when the inoculation volume of the core microbiome was 100-200 μL.

[0202] 3.4.2 Effects of fermentation time on the bacterial density and antibacterial activity of the core microbiome

[0203] Fermentation time affects the growth rate, metabolic activity, cell composition, and type and amount of the final product of the microorganism. Fig.10 It can be seen that the density of the core microbiome first increased and then decreased with the extension of fermentation time. 600 The value reached a peak of 2.591 at the 18th hour, which was significantly higher than other culture times. The inhibition rate of FOC also showed the same trend. When the culture time was 24 hours, the inhibition rate of the core microbiome on FOC was the highest, reaching 96.79%. Therefore, the optimal time for the core microbiome culture is 18-24 hours.

[0204] 3.4.3 Effect of culture temperature on the bacterial density and antibacterial activity of the core microbiome

[0205] Temperature not only affects the expression of bacterial cell proteins, but also affects the activity of enzymes in the body. Fig.11 It can be seen that when the culture temperature is 30℃, the OD of the core microbiome600 The values ​​and the inhibition rate of FOC reached the maximum value, and were significantly higher than those of other temperature treatments. Therefore, the culture temperature was set at 30℃ as the optimal culture temperature for the core microbiome of this study.

[0206] 3.4.4 Effect of culture speed on the cell density and antibacterial activity of the core microbiome

[0207] like Fig.12 As shown, the OD of the core microbiome 600 The value increases first and then decreases with the increase of speed. When the speed is 200rpm, OD 600 The value reached a maximum value of 2.622; at the same time, the inhibitory effect of the core microbiome on FOC also showed the same trend, but the inhibition rate reached a peak when the rotation speed was 150rpm. Therefore, it was preliminarily determined that the rotation speed range for the cultivation of the core microbiome in this study was 150-200rpm.

[0208] 3.4.5 Effect of initial pH on the bacterial density and antibacterial activity of the core microbiome pH has a crucial impact on the growth of microorganisms. It can directly affect the enzyme activity and stability of biomolecules in microorganisms. Fig.13 It can be seen that pH has a significant effect on the bacterial density of the core microbiome and the inhibition rate of FOC. When the pH value is 5.0-7.0, the bacterial density of the core microbiome and the inhibition rate of FOC both show an upward trend. When the pH is higher than 7.0, the bacterial density of the core microbiome and the inhibition rate of FOC both show a downward trend. Therefore, pH 7.0 is the optimal pH for the cultivation of the core microbiome in this study.

[0209] 3.5 Response surface experiment results

[0210] The single-factor experiment determined that the optimal temperature for the core microbiome mixed culture was 30°C, the optimal initial pH was 7.0, and the optimal inoculation volume, rotation speed, and time were 100-200 μL, 150-200 rpm, and 18-24 h, respectively. To further optimize the culture conditions, the optimal inoculation volume (A), rotation speed (B), and time (C) of the core microbiome culture were determined. The cell density (OD 600 ) is the response value, and the Design-Expert (V 8.0.6) software is used to optimize the response surface experiment with 3 factors and 3 levels. The experimental design is shown in the following table:

[0211]

[0212] The data in the above table are analyzed, and the experimental results are shown in the table below. The core microbiome cell density OD 600 The quadratic multinomial regression equation with inoculation amount (A), rotation speed (B) and time (C):

[0213] OD 600 =-114.94287+0.040150A+0.52191B+6.53470C+2.31400E-

[0214] 004AB-3.63333E-004AC-5.94000E-003BC-2.13010E-004A 2 -1.28004E-003B 2 -0.12598C 2

[0215]

[0216]

[0217] From the variance analysis in the table below, we can see that the model is extremely significant (P=0.0023<0.0500), the P of the lack of fit term is 0.107, which is greater than 0.05, and the difference is not significant, indicating that the model has practical application significance for the cultivation of the core microbiome. 2 is 0.9343, indicating that the model can explain 93.43% of the variation. 2 It is 0.8498, which shows that the model has a good fitting degree, a small experimental error, and can reflect the changes in the response value.

[0218] As shown in the table below, the first-order terms A, B, and C of the model have significant effects (P<0.05), and the second-order term B 2 , C 2 The effect was extremely significant (P<0.01): ANOVA of the regression model of the core microbiome

[0219]

[0220]

[0221] The data in the above table are fitted with quadratic multivariate regression, and the response surface and contour lines of the quadratic regression equation are shown in Fig.14 The contour lines of inoculation volume and time, inoculation volume and rotation speed, and time and rotation speed in the figure are all elliptical, indicating that the interaction between the two factors is significant. Fig.14 It can be seen that there are extreme points in the inoculation volume, time and rotation speed. Finally, the optimal culture conditions were obtained: inoculation volume of 200μL, rotation speed of 171rpm, time of 21.6h, temperature of 30℃, and initial pH of 7.0. Under this optimized condition, the cell density (OD 600 ) takes the maximum value of 3.16.

[0222] Three parallel experiments were conducted using the optimal culture conditions of the core microbiome obtained by response surface analysis, and the experimental results were consistent with the predictions. Fig.15 and Fig.16 The core microbial group has a better control effect on cucumber wilt than the three strains alone. Therefore, the response surface analysis method is reliable, and the experimental verification results are as follows:

[0223]

[0224] 3.6 Summary

[0225] First, the ratio of the three strains of the core microbiome for disease prevention and growth promotion in this study was determined by orthogonal experiments. The inoculation amounts of B. velezensis VJH5041, P. polymyxa SXC20-B62 and B. subtilis YJZL20-1 were 4%, 4% and 4% respectively. The fermentation basal culture of the core microbiome was optimized by single factor experiments and orthogonal experiments. The optimized fermentation basal culture medium was 30g glucose, 20g yeast extract powder, 1g potassium dihydrogen phosphate, and 1000mL double distilled water. The culture conditions of the core microbiome were optimized by single factor experiments and response surface experiments. The final culture conditions were as follows: temperature of 30℃, inoculation amount of 200μL, rotation speed of 171rpm, time of 21.6h, and initial pH of 7.0. The results of verification showed that the above screening results were reliable. And the pot experiment verified that the core microbiome had a better biocontrol effect than a single strain.

[0226] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A core microbial group for effectively preventing and controlling cucumber wilt, characterized in that: The strains included are: 1) Bacillus velezensis VJH5041, which was deposited in China Center for Type Culture Collection on May 10, 2024, with the deposit number of CCTCC NO: M 2024902; 2) Paenibacillus polymyxa SXC20-B62, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 23, 2020, with the deposit number CGMCC No.21540; 3) Bacillus subtilis YJZL20-1, the strain was deposited in the General Microbiology Center of China Culture Collection Administration on December 23, 2020, with the deposit number CGMCC No.21531.

2. The core microbial group for effectively preventing and controlling cucumber wilt according to claim 1, characterized in that: The inoculation amounts of the above three strains in the culture medium are all 4%-12%.

3. A method for preparing a core microbial group that can effectively prevent and treat cucumber wilt, characterized in that: The method is used to prepare the core microbial group for strong prevention and control of cucumber wilt as described in any one of claims 1-2, and the specific steps are: S1: Biocontrol strains with strong inhibitory effects on cucumber wilt pathogens were screened from healthy cucumber soil. Three biocontrol strains were named VJH5041, SXC20-B62 and YJZL20-1. The cucumber wilt pathogen was selected from Fusarium oxysporum, a cucumber-specific strain. S2: Verify the disease prevention effects of three biocontrol strains on cucumber wilt; S3: Identify the specific species of the three biocontrol strains; S4: Cultivate the three biocontrol strains and determine the optimal culture conditions; S5: Determine the composition and ratio of the core microbiome.

4. The method for preparing the core microbial group for strong prevention and treatment of cucumber wilt according to claim 3, characterized in that: The disease prevention effects of the three biocontrol strains in step S2 on cucumber wilt were verified respectively, and the specific verification steps were as follows: 1) Collect soil samples from the rhizosphere of healthy cucumber plants using the five-point sampling method; 2) The pathogen used for the test was selected as Fusarium oxysporumf.sp.cucumerinum (FOC) 3) Select detection kits and biocontrol strain culture media; 4) Isolation, purification and preservation of biocontrol strains; 5) Screening of biocontrol strains, screening strains with obvious antibacterial effects; 6) Detection of the effect of biocontrol strains on FOC mycelial growth; 7) Determination of the biocontrol and growth-promoting effects of biocontrol strains on cucumber wilt; 8) The activity of cucumber-related defense enzymes induced by biocontrol strains was determined using a kit.

5. The method for preparing the core microbial group for strong prevention and treatment of cucumber wilt according to claim 3, characterized in that: The method for identifying the specific types of the three biocontrol strains in step S3 is: 1) To identify the molecular biology of biocontrol strains; 2) Determine the whole genome of the biocontrol strain, the specific steps are: extraction, sequencing and assembly of genomic DNA of the biocontrol strain; average nucleotide consistency analysis of the biocontrol strain; genome annotation analysis of the biocontrol strain; 3) The specific types of the three biocontrol strains after identification are: Bacillus Velez, Paenibacillus polymyxa and Bacillus subtilis; therefore, the three biocontrol strains are specifically named: Bacillus Velez VJH5041, Paenibacillus polymyxa SXC20-B62 and Bacillus subtilis YJZL20-1.

6. The method for preparing the core microbial group for effectively preventing and controlling cucumber wilt disease according to claim 3, characterized in that: The specific cultivation steps of the three biocontrol strains in step S4 are as follows: 1) Select test strains and test culture medium. The test strains are: Bacillus velez VJH5041, Paenibacillus polymyxa SXC20-B62 and Bacillus subtilis YJZL20-1; 2) Preparation of seed solution of biocontrol strains; 3) Compatibility determination of three biocontrol strains; 4) Optimize the ratio of the three biocontrol strains through orthogonal experiments. The process is based on the cell density OD of the core microbiome. 600 The OD value and the inhibition rate of FOC are used as the measurement indicators, among which OD 600 The value is an indicator of bacterial density or bacterial population density, which indirectly reflects the concentration of bacterial cells in liquid culture medium by measuring the light absorption intensity of the sample at a wavelength of 600 nanometers; 5) Screening and optimization of the fermentation basal medium for the core microbiome to inhibit cucumber wilt, including: screening of carbon source species and concentration optimization, screening of nitrogen source species and concentration optimization, screening of inorganic salt species and concentration optimization, and orthogonal experiments for strain basal medium optimization; 6) Optimization of the culture conditions of the core microbiome, including: response surface experiments on the optimization of the culture conditions of the core microbiome and comparative experiments on the fermentation medium and culture conditions of the core microbiome before and after optimization. The response surface experiment on the optimization of the culture conditions of the core microbiome is designed based on the inoculation amount, culture time, culture temperature, culture speed and initial pH screened by the single factor experiment. Each culture medium combination listed in the program is a treatment for strain fermentation culture. Each treatment is repeated 3 times, and the cell density OD of the fermentation broth is measured. 600 The values ​​were analyzed to determine the optimal conditions for the cultivation of the core microbiome.

7. The method for preparing the core microbial group for strong prevention and treatment of cucumber wilt according to claim 6, characterized in that: The culture conditions of the core microbial group in step S4 are as follows: the carbon source of the culture medium is glucose, the nitrogen source is yeast extract powder, the inorganic salt is dipotassium hydrogen phosphate, the solvent is double distilled water, the culture temperature is 20-35°C, the rotation speed is 150-200rpm, the time is 18h-24h, and the initial pH is 5.0-7.

0.

8. The method for preparing the core microbial group for effectively preventing and controlling cucumber wilt disease according to claim 7, characterized in that: The culture conditions of the core microbial group in step S4 are preferably: the culture medium is 30g glucose, 20g yeast extract powder, 1g potassium hydrogen phosphate, 1000mL double distilled water, the culture temperature is 30°C, the rotation speed is 171rpm, the time is 21.6h, the initial pH is 7.0, and the inoculation amounts of Bacillus velez VJH5041, Bacillus polymyxa SXC20-B62 and Bacillus subtilis YJZL20-1 are set to 4%, 4% and 4%, respectively.

9. An application of a core microbial group for effectively preventing and controlling cucumber wilt, characterized in that: The core microbial group with strong control over cucumber wilt as described in any one of claims 1-2 is applied to control cucumber wilt, and the core microbial group is also used to promote the growth of cucumber seedlings.

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