Microbial agent for preventing and treating tomato fusarium wilt caused by fusarium oxysporum and promoting plant growth

Abstract

The invention relates to a microbial agent for preventing and treating tomato fusarium wilt caused by fusarium oxysporum and promoting plant growth. The microbial agent comprises a microbial flora constructed by five strains including bacillus amyloliquefaciens, bacillus velezensis, paenibacillus polymyxa, bacillus megatherium and bacillus subtilis. The synthetic flora provided by the invention can effectively antagonize Fusarium oxysporum, the inhibition rate of the Fusarium oxysporum can reach 64-99%, the inhibition capability of the synthetic flora is obviously superior to that of single strains A, B, C, D and E, and the biomass of tomatoes can be obviously improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a microbial inoculant for preventing and controlling tomato wilt caused by Fusarium oxysporum and promoting plant growth. Background Art

[0002] In the soil-crop system, preventing and controlling biological pollutants is a complex and multi-level task. To effectively control pathogenic microorganisms and ensure the healthy growth of crops, various prevention and control strategies have been developed in the agricultural field. Among them, the biological control technology of introducing beneficial organisms to inhibit pathogenic microorganisms has received extensive attention for its environmental protection, safety, and sustainability characteristics. Biological pollutants mostly refer to biological organisms that can cause adverse effects on humans or the environment, including harmful bacteria, fungi, viruses, parasites, and insects, etc. These pollutants not only affect the yield and quality of crops but may also be transmitted through the food chain, endangering the health of animals and even humans. Among them, pathogenic bacteria, as an important part of biological pollutants, have attracted particular attention. They can cause various plant and animal diseases, thus threatening the sustainable development of the farmland ecosystem. Therefore, in-depth research on the transmission and control mechanisms of farmland biological pollutants, including pathogenic bacteria, is crucial for ensuring agricultural sustainable development and public health.

[0003] It is estimated that the annual global agricultural losses caused by pests and diseases are one-third, and soil-borne pathogens alone can cause 90% of the important economic crop diseases. Plant diseases caused by soil-borne pathogens are the main threat to food security. There are many types of pathogenic bacteria, and common ones include Ralstonia solanacearum, Ralstonia spp., Fusarium oxysporum, Alternaria alternata, Candida albicans, Brevibacillus brevis, and Trichophyton rubrum, etc. After these pathogenic bacteria contaminate the soil, they usually cause plant diseases of various plants such as tomatoes, tomatoes, peppers, potatoes, tobacco, Arabidopsis thaliana, cotton, and eggplants. Wilt disease is one of the "cancer" diseases of plants and is a typical plant disease. Because of its characteristics of fast onset and high incidence rate, it seriously threatens crop production around the world. China is a major tomato-growing country, and tomato wilt caused by Fusarium oxysporum f.sp. Lycopersici is particularly serious and urgently needs to be effectively solved.

[0004] Microbial community construction, namely synthetic microbial community (SynCom), is a new direction in the field of microbiology research in recent years. Instead of studying a single microorganism alone, it constructs an artificial synthetic community containing multiple microorganisms to utilize the synergy and interaction among microorganisms to maintain the stability and functional diversity of the community. The microbiota helps to promote plant growth and health, including enhancing the resistance of plants to various diseases. The application of synthetic beneficial microbiota in farmland systems aims to improve soil quality, promote crop growth, enhance crop stress resistance, and reduce pests and diseases by artificially introducing and regulating specific microbial communities. By effectively utilizing these beneficial microbial communities, the efficient and sustainable development of agricultural production can be promoted. Compared with chemical pesticides and fertilizers, the application of synthetic beneficial microbiota is more environmentally friendly and sustainable, helps to reduce agricultural non-point source pollution, and protects the ecological environment, with very distinct ecological significance.

[0005] There are still relatively few studies on the inhibition of biological pollutants by beneficial microbiota, but some existing research results have shown that constructing beneficial microbiota to inhibit biological pollutants has good effects and broad application prospects. Based on the characteristics and functions of different beneficial bacteria, this study constructs beneficial microbiota to provide a new solution for the prevention and control of the biological pollutant disease - tomato wilt. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a microbial agent for preventing and treating tomato wilt caused by Fusarium oxysporum and promoting plant growth, which can effectively prevent and treat tomato wilt caused by Fusarium oxysporum.

[0007] The present invention solves its technical problems by adopting the following technical solutions:

[0008] A microbial community for preventing and treating tomato wilt caused by Fusarium oxysporum and promoting plant growth, comprising a microbial group constructed by five strains of Bacillus amyloliquefaciens, Bacillus velezensis, Paenibacillus polymyxa, Bacillus megaterium, and Bacillus subtilis.

[0009] Moreover, the strain name of the Bacillus amyloliquefaciens is: GXJ-12, the taxonomic name is: Bacillus amyloliquefaciens, the preservation number is: CGMCC NO.33171, the preservation date is: December 23, 2024, and the preservation unit is: China General Microbiological Culture Collection Center, and the preservation address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0010] The strain name of the Bacillus velezensis is: PYX-12, the classification name is: Bacillus velezensis, the preservation number is: CGMCC NO.33174, the preservation date is: December 23, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing;

[0011] The strain name of the polymyxa bacillus is: ZLZ-12, the classification name is: polymyxa bacillus Paenibacillus polymyxa, the preservation number is: CGMCC NO.33227, the preservation date is: December 30, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing;

[0012] The strain name of the Bacillus megaterium is: YQF-12, the classification name is: Bacillus megaterium, the preservation number is: CGMCC NO.33172, the preservation date is: December 23, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing;

[0013] The strain name of the Bacillus subtilis is: LYH-12, the classification name is: Bacillus subtilis, the preservation number is: CGMCC NO.33173, the preservation date is: December 23, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0014] Moreover, the ratio of the number of bacteria of the five strains is 1:1-10:1-10:1-10:1-10.

[0015] Moreover, the dosage of microbial flora was 1×10 7 CFU / g soil for soil mixing.

[0016] A method for screening a microbial flora for preventing and controlling tomato wilt caused by Fusarium oxysporum and promoting plant growth, characterized in that it comprises the following steps:

[0017] Soil samples were collected from the rhizosphere of healthy tomato plants. The healthy tomato seedlings with soil were dug out, the soil at the roots was gently shaken off, and the samples were placed in sterile paper bags and brought back to the laboratory for later use. The rhizosphere soil was fully mixed with sterile water to make 10 -1Soil suspension. Then gradient dilution was carried out to obtain soil dilutions with different concentrations. The diluted soil suspension was spread on nutrient agar (NA) plates, and three replicate plates were made for each dilution. It was cultured at 30 °C for 24 - 48 hours, the colony morphology was observed, and the colonies with spore characteristics were selected for purification. The initially screened colonies were purified by the quadrant streak method to obtain single colonies. The purified strains were stored in a 4 °C refrigerator; through 16S rRNA sequence phylogenetic analysis and morphological analysis, by sequencing the genes of the strains, the obtained sequences were subjected to BLAST alignment analysis on the NCBI website to identify five key strains: Bacillus amyloliquefaciens, Bacillus velezensis, Paenibacillus polymyxa, Bacillus megaterium, and Bacillus subtilis.

[0018] Use of the above-mentioned microbial flora for preventing and treating tomato wilt caused by Fusarium oxysporum and promoting plant growth in the preparation of a microbial agent for preventing and treating tomato wilt.

[0019] A preparation method of a microbial agent for preventing and treating tomato wilt with the above-mentioned microbial flora, characterized in that it includes the following steps: The specific process is as follows:

[0020] (1) Strain activation

[0021] The strains Bacillus amyloliquefaciens GXJ-12, Bacillus velezensis PYX-12, Paenibacillus polymyxa ZLZ-12, Bacillus megaterium YQF-12, and Bacillus subtilis LYH-12 were streaked on NA plate medium and cultured at 30 °C for 24 - 48 hours. After single colonies grew, single colonies were picked and inoculated into NA liquid medium, and cultured on a shaker at 30 °C and 150 rpm for 12 - 24 hours to obtain activated bacterial liquid;

[0022] (2) Fermentation broth preparation

[0023] The concentration of the bacterial cells in the activated bacterial liquid was determined by a flow cytometer, and initial bacteria were added in equal amounts at a ratio of 1:1 - 10:1 - 10:1 - 10:1 - 10, and the added concentration was 5×10 7 CFU, and the ABCDE bacterial liquid was co-cultured in NA liquid medium, cultured on a shaker at 30 °C and 150 rpm for 24 - 48 hours, and subcultured to the third generation to obtain the ABCDE microbial agent fermentation broth, and the microbial agent was obtained after separating the bacterial cells.

[0024] A microbial inoculum for preventing and treating tomato wilt disease, comprising the five strains described in claim 1.

[0025] An application of the above microbial flora in preventing and treating Fusarium oxysporum, the pathogenic bacterium in tomato wilt disease.

[0026] An application of the above microbial flora in promoting the germination of tomato seeds.

[0027] An application of the above microbial flora in the growth of tomato seedlings.

[0028] The advantages and positive effects of the present invention are:

[0029] 1. The synthetic flora ABCDE provided by the present invention can effectively antagonize Fusarium oxysporum, with an inhibition rate of up to 64-99%, and the inhibition ability is significantly better than that of individual strains A, B, C, D, and E, and can significantly increase the biomass of tomatoes.

[0030] 2. Through pot experiments, the present invention explores the inhibitory effect of the synthetic flora on tomato wilt disease. The results show that after treatment with the ABCDE microbial inoculum, the disease index is significantly lower than that of the control group with only the addition of pathogenic bacteria, and the relative control effect on tomato wilt disease is as high as 80% or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a "V-shaped" plate confrontation test diagram of the present invention.

[0032] Figure 2 It is a semi-swimming test diagram of the present invention.

[0033] Figure 3 It is a boundary confrontation test diagram of the present invention.

[0034] Figure 4 It is a four-corner confrontation test diagram of the present invention.

[0035] Figure 5 It is an antibacterial diagram between different bacterial combinations of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention will be further described in detail below through specific examples. The following examples are only descriptive and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0037] The present invention uses microbial culturomics to isolate five strains from the rhizosphere soil of tomatoes and screen them from the Bacillaceae family: A - Bacillus amyloliquefaciens, B - Bacillus velezensis, C - Paenibacillus polymyxa, D - Bacillus megaterium, and E - Bacillus subtilis for the construction of synthetic microbiota, abbreviated as ABCDE. At the same time, the pathogenic fungus Fusarium oxysporum f.sp Lycopersici of tomato wilt is isolated, abbreviated as F. The above six strains are identified by molecular methods and named Bacillus amyloliquefaciens GXJ - 12, Bacillus velezensis PYX - 12, Paenibacillus polymyxa ZLZ - 12, Bacillus megaterium YQF - 12, Bacillus subtilis LYH - 12, and Fusarium oxysporum FZZ - 12 (preservation number CGMCC NO. 41736, preservation date: December 30, 2024, preservation unit: China General Microbiological Culture Collection Center, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing).

[0038] The preservation information of the above five strains is as follows:

[0039] The strain name of the Bacillus amyloliquefaciens is: GXJ - 12, classified as: Bacillus amyloliquefaciens, preservation number: CGMCC NO. 33171, preservation date: December 23, 2024, preservation unit: China General Microbiological Culture Collection Center, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0040] The strain name of the Bacillus velezensis is: PYX - 12, classified as: Bacillus velezensis, preservation number: CGMCC NO. 33174, preservation date: December 23, 2024, preservation unit: China General Microbiological Culture Collection Center, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0041] The strain name of the Paenibacillus polymyxa is: ZLZ-12, and its taxonomic name is: Paenibacillus polymyxa. The preservation number is: CGMCC NO.33227, the preservation date is: December 30, 2024, and the preservation unit is: China General Microbiological Culture Collection Center. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0042] The strain name of the Bacillus megaterium is: YQF-12, and its taxonomic name is: Bacillus megaterium. The preservation number is: CGMCC NO.33172, the preservation date is: December 23, 2024, and the preservation unit is: China General Microbiological Culture Collection Center. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0043] The strain name of the Bacillus subtilis is: LYH-12, and its taxonomic name is: Bacillus subtilis. The preservation number is: CGMCC NO.33173, the preservation date is: December 23, 2024, and the preservation unit is: China General Microbiological Culture Collection Center. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] Isolation and purification of the pathogen Diseased tomato plants for testing were collected from the tomato planting area in Tianjin Wuqing. The pathogen of tomato wilt was isolated by the conventional tissue isolation method. The tomato roots and stems with wilt disease were rinsed with running water. After surface disinfection, small pieces of 5 mm were cut from the tissue at the junction of diseased and healthy parts with a sterile scalpel, and the tissue was soaked in 5% sodium hypochlorite solution for 5 min for surface disinfection, rinsed with sterile water several times, inoculated on PDA medium, and three pieces of tissue were inoculated in each petri dish. Then it was placed in an incubator at 25°C for 72 h. After purification of the strain, it was placed in an incubator at 25°C for cultivation, and the morphology was observed. The strain was stored in a refrigerator at 4°C for standby.

[0045] Identification of the pathogen After the above isolation and purification, 1 kind of pathogenic fungus was focused on and subjected to PCR molecular identification. DNA was extracted according to the fungal genome kit method, and PCR amplification and sequencing were carried out using the fungal universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’ SEQ ID NO.2) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’ SEQ ID NO.3). The sequencing results were subjected to Blast alignment on the NCBI website and homology comparison in the GenBank database, and it was identified as Fusarium oxysporum.

[0046] The strain name of the Fusarium oxysporum is: FZZ-12, classified and named as: Fusarium oxysporum, the preservation number is CGMCC NO.41736, the preservation date is: December 30, 2024, the preservation unit is: China General Microbiological Culture Collection Center, and the preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0047] The synthetic bacterial community ABCDE provided by the present invention has strong inhibitory activity against Fusarium oxysporum FZZ-12, the main root rot pathogen of tomatoes, and its inhibitory ability is significantly better than that of single strains A, B, C, D, and E.

[0048] Example 1

[0049] This example is to explore the relationship and motility among beneficial bacteria through the "V-shaped" plate confrontation test and semi-swimming test.

[0050] 1. To explore the interaction relationship among bacteria A, B, C, D, and E, the steps are as follows:

[0051] Prepare a solid agar NA medium according to the growth requirements of bacteria: peptone 10.0 g, beef powder 3.0 g, sodium chloride 5.0 g, agar 15.0 g, distilled water 1000 ml, pH: 7.3 ± 0.1. Sterilize at high temperature, pour the sterilized agar medium into a square petri dish, fill it to about 1 / 3 to 1 / 2 of the height, and let it solidify naturally to form a solid state. Under sterile conditions, use a sterile inoculation needle to dip a small amount of bacterial solutions of A, B, C, D, and E respectively, and inoculate A and B, A and C, A and D, A and E, B and C, B and D, B and E, C and D, C and E, D and E, A, B, C, D, and E on the diagonal of the petri dish, a total of eleven treatments. The inoculation points are gradually approaching to form a "V" shape, and each treatment is repeated three times. Place the inoculated square petri dish in a constant temperature incubator and incubate it in the dark at 28°C for 48 hours, then take out the square petri dish and observe the growth of the colonies.

[0052] It can be seen from Figure 1 that there is no mutual inhibition of growth among strains A, B, C, D, and E.

[0053] 2. Bacterial motility test, the steps are as follows:

[0054] Prepare semi-solid agar NA medium according to the growth requirements of bacteria: peptone 10.0 g, beef powder 3.0 g, sodium chloride 5.0 g, distilled water 1000 ml, pH: 7.3 ± 0.1, and use an agar concentration of 0.3% to 0.6% to ensure that the medium has appropriate semi-solid properties. Pour the sterilized agar medium into a six-well plate, filling it to about 1 / 3 to 1 / 2 of the height, and let it solidify naturally to form a semi-solid state. Under sterile conditions, use an inoculation needle to dip a small amount of bacterial solutions of A, B, C, D, E, and ABCDE respectively, and puncture-inoculate the bacteria into the center of the semi-solid agar medium, with the puncture depth being about 1 / 3 to 1 / 2 of the height of the medium. Repeat each treatment six times. Place the inoculated six-well plate in an incubator, set the temperature at 28 °C, and incubate for 48 hours. After the incubation is completed, take out the six-well plate and observe the growth of the colonies.

[0055] From Figure 2 the confrontation test of the demarcation line, it can be seen that the ABCDE synthetic bacterial community has the best motility.

[0056] Example 2

[0057] This example is an experiment on the ABCDE synthetic bacterial community against pathogenic bacteria, and the steps are as follows:

[0058] Prepare solid agar PDA medium according to the growth requirements of bacteria: potato powder 6.0 g, glucose 20.0 g, agar 20.0 g, distilled water 1000 ml; pH: 6.0 ± 0.2; temperature: 28 °C. Heat and dissolve, pour the sterilized agar medium into a round petri dish, filling it to about 1 / 3 to 1 / 2 of the height, and let it solidify naturally to form a solid state. Under sterile conditions, use a sterile inoculation loop to dip a small amount of bacterial solutions of A, B, C, D, E, and ABCDE respectively and draw a straight line in the middle of the petri dish. Use a punch with a diameter of 4 mm to make a fungal cake from the tested Fusarium oxysporum that has grown well on the PDA medium, and then transfer the fungal cake to the left side of the PDA medium plate. There are a total of 6 treatments, and only inoculating the fungus is used as the control group CK. Repeat each group 3 times. Place the inoculated petri dish in an incubator, set the conditions at 28 °C and incubate in the dark for 5 - 7 days. After the incubation is completed, take out the round petri dish and observe the growth of the bacteria.

[0059] From Figure 3 it can be seen that the ABCDE synthetic bacterial community shows a significant effect of inhibiting Fusarium oxysporum.

[0060] Example 3

[0061] This example is an experiment on the ABCDE synthetic bacterial community against pathogenic bacteria under a simulated soil environment, and the steps are as follows:

[0062] Prepare solid agar PDA medium according to the growth requirements of bacteria: potato powder 6.0 g, glucose 20.0 g, agar 20.0 g, distilled water 1000 ml); pH: 6.0 ± 0.2; temperature: 28 °C. Heat and dissolve, pour the sterilized agar medium into a round petri dish, fill it to about 1 / 3 to 1 / 2 of the height, and let it solidify naturally to form a solid state.

[0063] Prepare solid agar NA medium according to the growth requirements of bacteria: peptone 10.0 g, beef powder 3.0 g, sodium chloride 5.0 g, distilled water 1000 ml, pH: 7.3 ± 0.1.

[0064] To simulate the whole in the soil environment, a soil microbial suspension was extracted and denoted as S. Set 7 treatments including A+S, B+S, C+S, D+S, E+S, A+B+C+D+E+S, S, and 1 control CK, with each group repeated 3 times. Add an equal amount of each bacterial liquid to the liquid medium for subculture (cultured to the 3rd generation). The inhibitory activity of A+S, B+S, C+S, D+S, E+S, A+B+C+D+E+S, S against the mycelial growth of the test Fusarium oxysporum FZZ was determined by the four-corner plate confrontation culture method. Under sterile conditions, use a sterilized inoculation needle to dip a small amount of A+S, B+S, C+S, D+S, E+S, A+B+C+D+E+S, S bacterial liquid respectively and connect the bacterial liquid at the four endpoints of the petri dish. After culturing for 24 h, on the ultra-clean workbench, make a bacterial cake of the test Fusarium oxysporum grown on PDA medium with a puncher with a diameter of 4 mm, and then transfer the bacterial cake to the center of the PDA medium plate. Use the plate with only the pathogen bacterial cake as the control CK, and culture it at 28 °C in the dark and at a constant temperature. Observe the antibacterial effect after 5 - 7 days.

[0065] From Figure 4 the four-corner confrontation test, it can be seen that the synthetic bacterial community of ABCDE shows a significant effect of inhibiting Fusarium oxysporum.

[0066] Example 4

[0067] This example is a tomato pot experiment to explore the control effect of the ABCDE microbial inoculant on tomato wilt. The steps are as follows:

[0068] First, prepare the ABCDE microbial agent: Streak inoculate the strains of Bacillus amyloliquefaciens GXJ-12, Bacillus velezensis PYX-12, Paenibacillus polymyxa ZLZ-12, Bacillus megaterium YQF-12, and Bacillus subtilis LYH-12 on the NA plate medium and culture at 30°C for 24 - 48 hours. After single colonies grow, pick the single colonies and inoculate them into the NA liquid medium, and culture on a shaker at 30°C and 150 rpm for 12 - 24 hours to obtain the activated bacterial solution. Subsequently, determine the cell concentration of the activated bacterial solution by flow cytometry, add the initial bacteria in equal amounts at a ratio of 1:1:1:1:1 of the cell number, with an addition concentration of 5×10⁷ CFU, and co-culture the ABCDE bacterial solution in the NA liquid medium, culture on a shaker at 30°C and 150 rpm for 24 - 48 hours, and subculture to the third generation to obtain the fermentation broth of the ABCDE microbial agent.

[0069] Prepare the spore suspension of Fusarium oxysporum FZZ-12 and count it using a hemocytometer. The ABCDE synthetic bacterial community solution is counted by flow cytometry. Use the field soil of the long-term cultivated tomato experiment, and use plastic flowerpots (diameter 37.2 cm, height 34.5 cm, bottom diameter 26.8 cm), and fill each pot with 25 kg of soil. A total of two treatments are set: ① Control group, only add the pathogenic bacteria; ② Experimental group, add the ABCDE synthetic bacterial community + pathogenic bacteria; each treatment has 3 replicates. Mix the prepared ABCDE bacterial solution into the soil at 1×10 7 CFU / g soil, transplant tomato seedlings after 7 days, and irrigate the roots of the tomatoes with the spore suspension of Fusarium oxysporum FZZ-12 at 1×10 6 CFU / g soil. Investigate the disease incidence at 40 days, and calculate the disease index and relative control effect. The disease index is scored from 0 - 4 points: 0 points, no disease symptoms; 1, slight wilting on true leaves or cotyledons, but normal growth; 2, obvious necrotic patches appear on true leaves or cotyledons, root disease, and growth retardation; 3, root necrosis and partial plant withering or growth stiffness; 4, the whole plant withers, the plant dies or completely wilts. Relative control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0070] Table 1 Control effect of ABCDE microbial agent on tomato fusarium wilt

[0071] Treatment Disease index Relative control efficacy (%) ABCDE synthetic flora + pathogenic bacteria 0.33±0.27* 64.1-96.9 Only pathogenic bacteria added 1.67±0.27 -

[0072] *Significantly different from the control (p < 0.01).

[0073] Referring to Table 1, it can be seen that after treatment with the ABCDE microbial agent, the disease index was significantly lower than that of the control with only the pathogenic bacteria added, and the relative control effect on tomato wilt could reach 64.1% - 96.9%.

[0074] Table 2 Control and growth promotion effects of the ABCDE microbial agent on tomato wilt

[0075] Treatment Plant height (cm) Plant dry weight (g) ABCDE synthetic flora + pathogenic bacteria 30.5±2.1* 2.29±0.42* Only pathogenic bacteria added 25.8±1.4 1.24±0.13

[0076] *Significantly different from the control (p < 0.01).

[0077] Referring to Table 2, it can be seen that after treatment with the ABCDE microbial agent, there was an obvious growth promotion effect on tomatoes.

[0078] Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art can understand that: within the spirit and scope of the present invention and the appended claims, various substitutions, changes, and modifications are possible. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A microbial flora for preventing and controlling tomato wilt caused by Fusarium oxysporum and promoting plant growth, characterized in that: The microbial flora includes five strains: Bacillus amyloliquefaciens, Bacillus velez, Paenibacillus polymyxa, Bacillus megaterium and Bacillus subtilis.

2. The microbial flora for preventing and controlling tomato wilt caused by Fusarium oxysporum and promoting plant growth according to claim 1, characterized in that: The strain name of the Bacillus amyloliquefaciens is: GXJ-12, the classification name is: Bacillus amyloliquefaciens, the preservation number is: CGMCC NO.33171, the preservation date is: December 23, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; The strain name of the Bacillus velezensis is: PYX-12, the classification name is: Bacillus velezensis, the preservation number is: CGMCC NO.33174, the preservation date is: December 23, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; The strain name of the polymyxa bacillus is: ZLZ-12, the classification name is: polymyxa bacillus Paenibacillus polymyxa, the preservation number is: CGMCC NO.33227, the preservation date is: December 30, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; The strain name of the Bacillus megaterium is: YQF-12, the classification name is: Bacillus megaterium, the preservation number is: CGMCC NO.33172, the preservation date is: December 23, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; The strain name of the Bacillus subtilis is: LYH-12, the classification name is: Bacillus subtilis, the preservation number is: CGMCC NO.33173, the preservation date is: December 23, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

3. The microbial flora for preventing and controlling tomato wilt caused by Fusarium oxysporum and promoting plant growth according to claim 1, characterized in that: The cell number ratio of the five strains is 1:1-10:1-10:1-10:1-10, and the dosage of the microbial group is 1×10 7 CFU / g soil for soil mixing.

4. A method for screening a microbial flora for preventing and controlling tomato wilt caused by Fusarium oxysporum and promoting plant growth as claimed in claim 1, characterized in that: The steps include: Soil samples were collected from the rhizosphere of healthy tomato plants. The healthy tomato seedlings with soil were dug out, the soil at the roots was gently shaken off, and the samples were placed in sterile paper bags and brought back to the laboratory for later use. The rhizosphere soil was fully mixed with sterile water to make 10 -1 Soil suspension. Then perform gradient dilution to obtain soil dilutions of different concentrations. Spread the diluted soil suspension on beef extract peptone (NA) plates, and make 3 replicate plates for each dilution. Culture at 30°C for 24-48 hours, observe the colony morphology, and select colonies with spore characteristics for purification. Use the partitioning and streaking method to purify the initially screened colonies to obtain single colonies. The purified strains are stored in a 4°C refrigerator; Through 16S rRNA sequence phylogenetic analysis and morphological analysis, five key bacterial species were identified by strain gene sequencing and BLAST comparison analysis of the obtained sequences on the NCBI website: Bacillus amyloliquefaciens, Bacillus velezensis, Paenibacillus polymyxa, Bacillus megaterium and Bacillus subtilis.

5. Use of the microbial flora for preventing and controlling tomato wilt caused by Fusarium oxysporum and capable of promoting plant growth as claimed in claim 1 in preparing a microbial agent for preventing and controlling tomato wilt.

6. A method for preparing a microbial agent for preventing and treating tomato wilt containing the microbial flora of claim 2, characterized in that: The specific process includes the following steps: (1) Bacterial Activation Bacillus amyloliquefaciens GXJ-12, Bacillus velezensis PYX-12, Paenibacillus polymyxa ZLZ-12, Bacillus megaterium YQF-12, and Bacillus subtilis LYH-12 strains were streaked onto NA plate medium, cultured at 30° C. for 24-48 hours, and after a single colony grew out, a single colony was picked and inoculated into NA liquid medium, and cultured at 30° C. and 150 rpm in a shaking incubator for 12-24 hours to obtain an activated bacterial solution; (2) Preparation of fermentation broth The activated bacterial solution was used to determine the bacterial concentration by flow cytometry, and the initial bacteria were added in an equal amount at a ratio of 1:1-10:1-10:1-10:1-10, with an added concentration of 5×10 7 CFU, ABCDE bacterial liquid was co-cultured in NA liquid culture medium, cultured in a shaking incubator at 30°C and 150rpm for 24-48 hours, and subcultured to the third generation to obtain ABCDE microbial agent fermentation liquid, and the microbial agent was obtained after separating the bacteria.

7. A microbial agent for preventing and treating tomato wilt, characterized in that: Including the five strains described in claim 1.

8. Use of the microbial flora according to claim 1 in preventing and treating the pathogenic fungus Fusarium oxysporum of tomato wilt.

9. Use of the microbial flora according to claim 1 in promoting tomato seed germination.

10. Use of the microbial flora according to claim 1 in the growth of tomato seedlings.

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