Composite microbial agent and its application in preventing and controlling tomato brown wrinkled fruit virus disease

Abstract

The present invention relates to the field of microbial technology, and in particular to a composite microbial agent and application thereof to preventing and controlling tomato brown wrinkled fruit virus disease. The composite microbial agent comprises a composite microbial agent composed of Bacillus velez WF02 and psychrotolerant Bacillus velez WF04, which can improve the rhizosphere soil of tomato plants and the microecological environment within the plants, activate the systemic resistance of the plants to tomato brown wrinkled fruit virus, and achieve a field prevention effect of 73.06% to 79.64% against tomato brown wrinkled fruit virus disease, effectively reducing the impact of tomato brown wrinkled fruit virus on tomato yield and quality; the psychrotolerant Bacillus velez WF04 can promote the growth and development of the root system of tomato seedlings in low temperature and drought environments, and assist Bacillus velez WF02 in quickly entering the tomato plants.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a composite microbial agent and application thereof in preventing and controlling tomato brown wrinkled fruit virus disease. Background Art

[0002] Tomatoes are one of the most important vegetable crops grown in my country and around the world. Throughout their growth cycle, tomatoes are frequently infected by various viruses, which severely impact both yield and quality. The difficulty in controlling viral infections in tomatoes is a major constraint on tomato production.

[0003] Tomato brown rugose fruit virus (ToBRFV) is a new species of the genus Tobacco mosaic virus, belonging to the family Brucoviridae. This virus infects tomatoes, causing mosaic, dark green bumps, narrowed leaves, and severe venation and necrosis. This virus reduces the number of flowers and fruits, develops yellow or brown patches on the fruit, reduces the size of the fruit, and develops wrinkles. In severe cases, it can cause necrosis of the fruit stem, significantly reducing fruit yield. In 2015, Jordanian scientists discovered severe brown shrivel symptoms on tomato fruit, with an incidence rate of nearly 100%. They described and identified the virus as ToBRFV.

[0004] ToBRFV is highly tolerant to environmental factors, difficult to inactivate, and highly transmissible. Therefore, systematic research and control of ToBRFV are urgent. However, there is currently a lack of effective targeted agents for chemical control of ToBRFV, and even less so, green control technologies for ToBRFV. Therefore, identifying specific microorganisms from healthy tomato plants in resistant habitats and conducting research on green control technologies for Tomato Brown Fruit Virus based on these specific microorganisms has enormous application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a composite microbial agent that can improve the microecological environment in tomato plants and activate the resistance of the tomato plant defense system, and its application in preventing and controlling tomato brown wrinkled fruit virus disease.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] In a first aspect, the present invention claims protection for a composite microbial agent, which is formed by compounding Bacillus Velez WF02 agent and cold-resistant Bacillus WF04 agent.

[0008] The Bacillus velez WF02 was deposited in the General Microbiology Center of the China Culture Collection Administration on May 24, 2024, with the deposit number CGMCC NO. 30756, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified as Bacillus velez Bacillus velezensis ;

[0009] The refractory Bacillus WF04 was deposited in the General Microbiology Center of China Culture Collection Administration on May 24, 2024, with the deposit number CGMCC NO. 30758, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified as refractory Bacillus Peribacillus frigoritolerans .

[0010] Furthermore, the effective viable count of the composite microbial agent is 5.1×10 9 ~1.01×10 10 cfu / mL.

[0011] Furthermore, the ratio of the effective viable bacteria count of the Bacillus Velez subtilis WF02 to the effective viable bacteria count of the low-temperature-resistant Bacillus subtilis WF04 in the composite microbial agent is 50-100:1.

[0012] In the second aspect, the present invention claims protection for the use of the Bacillus Velez WF02 agent, the low-temperature-resistant Bacillus WF04 agent and the composite microbial agent in tomato cultivation.

[0013] In a third aspect, the present invention claims protection for the use of the Bacillus Velez WF02 agent, the low-temperature-resistant Bacillus WF04 agent and the composite microbial agent for the prevention and control of tomato brown wrinkled fruit virus disease.

[0014] In a fourth aspect, the present invention claims protection for the use of the Bacillus Velez WF02 agent and the composite microbial agent respectively for inducing systemic defense of tomato plants against tomato brown wrinkle fruit virus.

[0015] In a fifth aspect, the present invention claims protection for the cold-resistant Bacillus WF04 and the composite microbial agent for use in promoting the growth and development of tomato plant roots, thereby improving the resistance of tomato plants to tomato brown fruit virus.

[0016] In a sixth aspect, the present invention claims protection for the application of the cold-resistant Bacillus WF04 and the composite microbial agent respectively in low temperature and / or drought soil environments to promote the growth and development of tomato seedling roots.

[0017] In a seventh aspect, the present invention claims protection for a method for preventing and controlling tomato brown wrinkled fruit virus disease, wherein the composite microbial agent is applied to the rhizosphere soil of tomato seedlings.

[0018] The present invention has the following beneficial effects: The Bacillus Velez WF02 strain has a broad spectrum of antibacterial activity, and the metabolites and volatile substances it produces have strong inhibitory effects on soil-borne pathogens such as ginger stem rot, asparagus stem rot, and tomato wilt. Bacillus Velez WF02 also has a strong biofilm-forming ability, rapidly colonizing tomato roots and then migrating to internal locations such as leaves, improving the microecological environment within tomato plants and reducing the incidence of tomato brown fruit virus. The low-temperature-tolerant Bacillus Velez WF04 strain can promote root growth and development in tomato seedlings in low-temperature, drought-prone environments, helping Bacillus Velez WF02 quickly enter the tomato plant. The composite microbial agent of the present invention is composed of a pair of bacteria that have a wide range of adaptability to growth environments, are salt-resistant, and are resistant to severe drought environments. It can improve the rhizosphere soil of tomato plants and the microecological environment within the plants, activate the plant's systemic resistance to tomato brown wrinkled fruit virus, and achieve a field prevention effect of 73.06%-79.64% against tomato brown wrinkled fruit virus disease, effectively reducing the impact of tomato brown wrinkled fruit virus on tomato yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings are intended only to illustrate and explain the present invention, and are not intended to limit the scope of the present invention.

[0020] Figure 1 This is a colony morphology diagram of Bacillus Velez WF02 of the present invention;

[0021] Figure 2 is a phylogenetic tree of Bacillus velez WF02 of the present invention based on 16S rDNA sequence;

[0022] Figure 3 This is a colony morphology diagram of the cold-resistant Bacillus WF04 of the present invention;

[0023] Figure 4 This is a phylogenetic tree of the cold-resistant Bacillus WF04 of the present invention based on the 16S rDNA sequence;

[0024] Figure 5 The diagram shows the antibacterial effect of the Bacillus Velez WF02 of the present invention on plant pathogens, wherein A on the left is Fusarium oxysporum (CK), and A on the right is an antibacterial effect diagram; B on the left is Fusarium fujikura (CK), and B on the right is an antibacterial effect diagram; C on the left is Rice blast fungus (CK), and C on the right is an antibacterial effect diagram; D on the left is Tomato-specific Fusarium oxysporum (CK), and D on the right is an antibacterial effect diagram;

[0025] Figure 6 : This is a diagram showing the antibacterial effect of the sterile metabolites of Bacillus Velez WF02 of the present invention (left: control diagram; right: antibacterial diagram of the sterile metabolites of Bacillus Velez WF02);

[0026] Figure 7 Graphs showing the antibacterial effects of the volatile substances of Bacillus velez WF02 of the present invention (left: control graph; right: antibacterial graph of the volatile substances of Bacillus velez WF02);

[0027] Figure 8 This is a graph showing the biofilm characteristics of Bacillus Velez WF02 and Bacillus Velez WF04 of the present invention (A is Bacillus Velez WF04; B is Bacillus Velez WF02; C is Bacillus Velez WF04:Bacillus Velez WF02 = 1:1);

[0028] Figure 9 This is a diagram showing the effect of the composite microbial agent of the present invention on improving the cold resistance of tomato plants;

[0029] Figure 10 This is a diagram showing changes in enzyme activity of the composite microbial agent of the present invention in preventing and controlling tomato brown wrinkled fruit virus disease. DETAILED DESCRIPTION

[0030] In order to make the purpose and technical solution of the present invention clearer and easier to understand, the present invention is further described in detail below. The experimental methods described in the following examples are all conventional methods unless otherwise specified. If no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The reagents and materials described are all commercially available unless otherwise specified.

[0031] The various culture media involved in the following examples are all standard culture media commonly used in the art, for example:

[0032] LB liquid medium: peptone 10.0 g, yeast extract 5.0 g, sodium chloride 5.0 g, pH 7.0-7.2, and distilled water to 1000 mL;

[0033] NA solid medium: peptone 10.0 g, beef extract 5.0 g, sodium chloride 5.0 g, agar 20 g, pH 7.0-7.2, and distilled water to 1000 mL;

[0034] PDA medium: 6.0 g potato extract powder, 20.0 g glucose, 20.0 g agar, pH 5.5-6.0, and distilled water to 1000 mL;

[0035] NB liquid medium: peptone 10.0 g, beef extract powder 3.0 g, sodium chloride 5.0 g, pH 7.0-7.2, and distilled water to 1000 mL;

[0036] Basal culture medium (1 / 2Ms + 3% sucrose + 0.6% agar): potassium nitrate 950 mg, ammonium nitrate 825 mg, potassium dihydrogen phosphate 85 mg, magnesium sulfate 185 mg, calcium chloride 220 mg, potassium iodide 0.83 mg, boric acid 6.2 mg, manganese sulfate 22.3 mg, zinc sulfate 8.6 mg, sodium molybdate 0.25 mg, copper sulfate 0.025 mg, cobalt chloride 0.025 mg, disodium edetate 37.3 mg, ferrous sulfate 27.8 mg, inositol 100 mg, glycine 2 mg, thiamine hydrochloride 0.1 mg, pyridoxine hydrochloride 0.5 mg, niacin 0.5 mg, sucrose 30 g, agar 7 g.

[0037] Example 1: Screening and identification of Bacillus velez WF02

[0038] 1. Strain screening

[0039] The virus was isolated from healthy tomato plants collected from a greenhouse in a tomato growing area in Anqiu City, Shandong Province, where a large-scale outbreak of Tomato Brown Fruit Virus occurred. The sampling and isolation methods are as follows:

[0040] Select healthy plants and use a sampling shovel to dig out the entire root system. Rinse with tap water first to remove surface soil and other attachments. Then soak the roots, stems, and leaves of the plants in 2% sodium hypochlorite in a clean bench for 5 to 10 minutes, rinse with sterile water 3 to 5 times, then soak in 75% alcohol for 3 to 5 minutes, rinse with sterile water 3 to 5 times, then use sterile filter paper to absorb the moisture on the surface of the tomato roots, stems, and leaves, and place the tomato roots, stems, and leaves in an ultra-low temperature environment of -80°C for 30 days; place the tomato roots, stems, and leaves in a sterilized mortar, add 5 to 10 mL of 1×PBS buffer, and evenly grind to obtain the juice to obtain the sample stock solution. The sample stock solution was gradient diluted, and the dilution was 10 -2 , 10 -3 , 10 -4 100 μL of the dilution was spread on NA solid culture medium and cultured upside down at 30°C for 2 days. A single colony of the dominant bacteria was picked and isolated and purified to obtain strain WF02.

[0041] 2. Strain identification

[0042] The morphological identification of strain WF02 was performed, and the results are as follows:

[0043] The colonies of strain WF02 on NA solid medium are milky white, with a raised center, round, wrinkled surface, and opaque; Gram staining is positive, the cells are rod-shaped, not in chains, and produce spores, such as Figure 1 shown.

[0044] Molecular biological identification of strain WF02:

[0045] Total DNA from strain WF02 was extracted using conventional bacterial DNA extraction methods. 16S rDNA gene PCR amplification was performed using universal bacterial primers 27f and 1492r. The PCR reaction system consisted of 1 µl of DNA template, 25 µl of 2× Mastar Mix, 2 µl of 27f, 2 µl of 1492r, and ultrapure water to 50 µl. The amplified product was analyzed by 0.8% agarose gel electrophoresis and then sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing.

[0046] The sequencing results of strain WF02 were compared on NCBI and EzBio Cloud websites, and the comparison analysis was performed using Mega 6.0 software. At the same time, the phylogenetic tree was constructed by the neighbor-joining method (NJ). The 16S rDNA sequence of strain WF02 was aligned with that of Bacillus velezensis. (Bacillus velezensis ) has a similarity of 99% (e.g. Figure 2 As shown); strain WF02 was identified as Bacillus velezinis ( Bacillus velezensis ), named Bacillus velez WF02.

[0047] The Bacillus velezensis WF02 was deposited in the General Microbiology Center of China Culture Collection Administration on May 24, 2024, with the deposit number CGMCC NO. 30756, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and the classification name is Bacillus velezensis .

[0048] Example 2: Screening and identification of low-temperature resistant Bacillus WF04

[0049] 1. Strain screening

[0050] It was isolated from soil collected from the Ngari region of Tibet at an average altitude of more than 4,500 meters. The isolation method is as follows:

[0051] The collected soil was adjusted to a moderate moisture content and frozen at -80℃ for 35 days (freeze and thaw once every 7 days). After repeated freeze and thaw for 5 times, 1g of soil was placed in 100mL sterile water and shaken for 20 minutes to obtain the sample stock solution. The sample stock solution was diluted stepwise, and the dilution was 10 -3 , 10 -4 , 10 -5 100 μL of the dilution was spread on NA solid culture medium and cultured upside down at 10°C for 2 days. A single colony of the rapidly growing dominant bacteria was picked and isolated and purified to obtain strain WF04.

[0052] 2. Strain identification

[0053] The morphology of strain WF04 was identified, and the results are as follows:

[0054] The colonies of strain WF04 on NA solid medium are off-white, round, oily, with no wrinkles on the surface and are opaque; Gram staining is positive, the cells are rod-shaped, not in chains, and produce spores, such as Figure 3 shown.

[0055] Molecular biological identification of strain WF04:

[0056] Total DNA from strain WF04 was extracted using conventional bacterial DNA extraction methods. 16S rDNA gene PCR amplification was performed using universal bacterial primers 27f and 1492r. The PCR reaction system consisted of 1 µl of DNA template, 25 µl of 2× MastarMix, 2 µl of 27f, 2 µl of 1492r, and ultrapure water to 50 µl. The amplified product was analyzed by 0.8% agarose gel electrophoresis and then sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing.

[0057] The sequencing results of strain WF04 were compared on NCBI and EzBio Cloud websites, and the comparison analysis was performed using Mega 6.0 software. At the same time, the phylogenetic tree was constructed by the neighbor-joining method (NJ). The 16S rDNA sequence comparison results of strain WF04 (such as Figure 4 ) were identified as psychrotolerant Bacillus (Peribacillus frigoritolerans ), named as psychrotolerant Bacillus WF04.

[0058] The cold-resistant Bacillus WF04 was deposited in the General Microbiology Center of China Culture Collection Administration on May 24, 2024, with the deposit number CGMCC NO. 30758, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified as Peribacillus frigoritolerans .

[0059] Example 3: Determination of the inhibitory effect of Bacillus velezensis WF02 on plant pathogens

[0060] This example provides the antibacterial effects of Bacillus Velez WF02 on the pathogen of ginger stem rot: Fusarium oxysporum; the pathogen of asparagus stem rot: Fusarium fujikura; the pathogen of rice blast: Aspergillus oryzae; and the pathogen of tomato wilt: Tomato-specific Fusarium oxysporum.

[0061] The test method is as follows:

[0062] Antagonistic pathogenic fungi test: A pathogen cake (7 mm) was placed in the center of a 90 mm diameter PDA solid plate, and Bacillus Velez WF02 was inoculated 25 mm above, below, left, and right from the center. The control plate was not inoculated and cultured in a constant temperature incubator at 28°C for 5 to 7 days. The diameters of the pathogens in the control group and the treatment group were counted, and the inhibition rate was calculated.

[0063] The formula for calculating the inhibition rate is as follows:

[0064] Inhibition rate = (pathogen diameter in the control group - pathogen diameter in the treatment group) / (pathogen diameter in the control group - bacterial cake diameter) × 100%.

[0065] Table 1 Antibacterial effect of Bacillus velezensis WF02 on plant pathogens

[0066]

[0067] As shown in Table 1, Bacillus velezensis WF02 has a strong antibacterial ability against plant pathogenic fungi, with an inhibition rate of 82.5% to 91.3% (e.g. Figure 5 As shown), it shows that the Bacillus Velez WF02 of the present invention has a broad antibacterial spectrum and strong antibacterial ability, and has potential disease prevention ability.

[0068] Example 4: Antibacterial Activity Detection of Sterile Metabolites and Volatile Substances of Bacillus Velezii WF02

[0069] This example provides a method for detecting the antibacterial activity of sterile metabolites and volatile substances of Bacillus velezensis WF02, as follows:

[0070] 1. Antibacterial activity of sterile metabolites:

[0071] Bacillus Velez WF02 was inoculated into LB liquid culture medium and shaken at 28°C and 180 rpm for 48 hours to obtain Bacillus Velez WF02 fermentation liquid; 1.5 mL of fermentation liquid was aspirated into a 2 mL sterile centrifuge tube and centrifuged at 4°C and 12,000 rpm for 5 minutes; the supernatant was sterilized and filtered through a 0.22 μm pinhole filter to obtain a sterile filtrate of Bacillus Velez WF02; a 7 mm Fusarium oxysporum cake was placed in the center of a 90 mm diameter PDA solid plate, and holes were punched 25 mm from the center on the top, bottom, left, and right sides, and then 50 μL of the sterile filtrate of Bacillus Velez WF02 was inoculated. The control plate was not inoculated and cultured in a constant temperature incubator at 28°C for 5 to 7 days. The diameters of the pathogens in the control group and the treatment group were counted, and the inhibition rate was calculated;

[0072] The results are as follows Figure 6As shown in the figure, the growth of Fusarium oxysporum inoculated with the sterile filtrate of Bacillus Velez WF02 was significantly inhibited, with an inhibition rate of 65.4%, indicating that Bacillus Velez WF02 can produce antibacterial metabolites to inhibit the growth of pathogens.

[0073] 2. Antibacterial activity of volatile substances:

[0074] The inhibitory effect of volatile compounds from Bacillus Velez WF02 against Fusarium oxysporum, the pathogen of ginger stem rot, was determined using a two-divided plate method using 90 mm two-divided Petri dishes. 10 mL of PDA medium was added to one side of the second Petri dish for the cultivation of Fusarium oxysporum; 10 mL of NA solid medium was added to the other side for the cultivation of Bacillus Velez WF02. A control was used in which Bacillus Velez WF02 was not cultured on one side and Fusarium oxysporum was cultured on the other side. The Petri dishes were sealed with Parafilm to prevent air circulation. Five replicates were used for each treatment. After sealing, the plates were incubated upside down at 28°C in the dark for 7 days, and the growth of the pathogen was observed.

[0075] The results are as follows Figure 7 As shown in the figure, the growth of Fusarium oxysporum in the two-divided culture dish inoculated with Bacillus Velez WF02 was inhibited, with an inhibition rate of 25%, indicating that Bacillus Velez WF02 can produce volatile substances to inhibit the growth of pathogens.

[0076] Example 5: Growth characteristics and stress resistance of Bacillus velez WF02 and psychrotolerant Bacillus velez WF04

[0077] This example provides growth characteristics and stress resistance testing of Bacillus velezinis WF02 and Bacillus thuringiensis WF04. The test method is as follows:

[0078] 1. Determination of optimal growth temperature

[0079] The seed liquid of Bacillus velezensis WF02 and the low-temperature-resistant Bacillus velezensis WF04 were prepared respectively, and inoculated into a 250-mL Erlenmeyer flask containing 50 mL of LB liquid medium at a 5% inoculum size. The culture was shaken (rotation speed 180 r / min) in a constant temperature shaking incubator at 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, and 60°C for 2 days, respectively. The growth conditions (whether it was turbid) were observed and recorded.

[0080] 2. Acid and alkali resistance determination

[0081] LB liquid culture medium with different pH values ​​(3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) was prepared and inoculated with 5% seed liquid of Bacillus velezensis WF02 and 5% seed liquid of Bacillus thuringiensis WF04, respectively. The culture was shaken at 30°C and 180 rpm for 2 days, with three replicates. The growth status (whether it was turbid) was observed and recorded.

[0082] 3. Salt tolerance test

[0083] Prepare LB liquid media with different NaCl contents (1%, 5%, 10%, 15%, and 20%) and inoculate 5% seed liquid of Bacillus velezensis WF02 and Bacillus thioredophylla WF04, respectively. Cultivate at 30°C, 180 rpm, and shake for 2 days, with three replicates. Observe and record growth (including turbidity). For non-halotolerant strains, determine the NaCl concentration: less than 1.17%; for low-halotolerant strains, determine the NaCl concentration: 1.17% to 2.93%; for moderately tolerant strains, determine the NaCl concentration: 2.93% to 14.63%; and for highly tolerant strains, determine the NaCl concentration: 14.63% to 30.4%.

[0084] 4. Determination of drought resistance

[0085] LB liquid medium containing different concentrations of PEG6000 (0 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, 500 g / L, and 600 g / L) was inoculated with 5% seed liquid of Bacillus velezensis WF02 and Bacillus thiotolerant WF04, respectively. Cultures were held at 30°C and 180 rpm for 2 days, with three replicates. Growth (including turbidity) was observed and recorded. OD values ​​at 700 nm were measured spectrophotometrically. A PEG6000 concentration of 0-60 g / L indicates mild drought tolerance, 90-150 g / L indicates moderate drought tolerance, and greater than 150 g / L indicates severe drought tolerance.

[0086] Table 2 Growth and stress resistance of Bacillus velez WF02

[0087]

[0088] Note: + indicates growth possible, ++ indicates good growth, +++ indicates good growth, - indicates no growth

[0089] Table 3 Growth and stress resistance characteristics of low-temperature tolerant Bacillus WF04

[0090]

[0091] Note: + indicates growth possible, ++ indicates good growth, +++ indicates good growth, - indicates no growth

[0092] As shown in Tables 2 and 3, the growth temperature range of Bacillus velez WF02 is 20-50°C, with an optimum growth temperature of 30°C, and the growth temperature range of Bacillus velez WF04 is 4-40°C, with an optimum growth temperature of 30°C; the growth pH range of Bacillus velez WF02 is 5.0-9.0, with an optimum pH of 7.0-8.0, and the growth pH range of Bacillus velez WF04 is 6.0-9.0, with an optimum pH of 7.0-8.0; Bacillus Velez WF02 can tolerate 10% NaCl concentration, is a moderately salt-tolerant strain, can grow under 50% PEG6000, and tolerates severe drought conditions; the low-temperature-tolerant Bacillus WF04 can tolerate 5% NaCl concentration, is a low-salt-tolerant strain, can grow under 60% PEG6000, and tolerates severe drought conditions; the above results show that the Bacillus Velez WF02 and the low-temperature-tolerant Bacillus WF04 of the present invention have a wide range of adaptability to growth environments, are salt-tolerant, and can tolerate severe drought environments.

[0093] Example 6: Determination of biofilm production performance of Bacillus velez WF02 and psychrotolerant Bacillus velez WF04

[0094] This example provides a method for determining biofilm production by Bacillus velezensis WF02 and psychrotolerant Bacillus velezensis WF04. The test method is as follows:

[0095] Bacillus Velez WF02 and psychrotolerant Bacillus WF04 were inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 24 h. 4 mL of single bacterial suspension of Bacillus Velez WF02 and psychrotolerant Bacillus WF04 and a composite bacterial suspension (2 mL each of Bacillus Velez WF02 and psychrotolerant Bacillus WF04 and mixed) were respectively pipetted into 10 mL centrifuge tubes and placed in a 30°C incubator for 2 d. After removal, the bacterial suspension was poured out, washed twice with 1× PBS to remove unabsorbed bacterial suspension, and air-dried. 4 mL of 1% crystal violet stain was added to the centrifuge tube and stained for 30 min. The crystal violet stain was aspirated, and the surface floating color was washed with 1× PBS, and air-dried. Then, 4 mL of 95% ethanol was added and allowed to stand for 15 min to dissolve the crystal violet. The biofilm content was analyzed by measuring the absorbance value (As) at 590 nm of the sample, and LB (Ac) was used as a control.

[0096] The ability of bacteria to form biofilms was assessed based on the OD value generated by bacterial biofilms at 590 nm: if As≤Ac, there was no biofilm production function; if Ac<As≤(2×Ac), there was a low biofilm production ability; if (2×Ac)<As≤(4×Ac), there was a moderate biofilm production ability; and if (4×Ac)<As, there was a strong biofilm production ability.

[0097] Table 4 OD values ​​of different treatments at 590 nm

[0098]

[0099] As shown in Table 4 and Figure 8 As shown, Ac(CK) = 0.615, As(WF02) = 1.690, and As(WF04) = 0.930, i.e., (2×Ac) < As(WF02), Ac < As(WF04), and As(WF02+WF04) > (4×Ac). The results showed that Bacillus Velez WF02 had moderate biofilm production ability, while psychrotolerant Bacillus WF04 had lower biofilm production. However, co-culture of Bacillus Velez WF02 and psychrotolerant Bacillus WF04 showed strong biofilm production, suggesting that this property is crucial for enhancing the colonization of both strains on crop roots and their synergistic defense against disease.

[0100] Example 7: Preparation of composite microbial agent

[0101] 1. Activation of bacteria

[0102] Bacillus velezensis WF02 and psychrotolerant Bacillus velezensis WF04 stored in -80°C glycerol tubes were inoculated onto NA solid culture medium, respectively, and activated and cultured at 28°C for 2-3 days to obtain solid seed cultures.

[0103] 2. Preparation of single bacterial agent

[0104] The solid seed culture of Bacillus velezii WF02 and the solid seed culture of Bacillus refractory WF04 were inoculated into LB liquid culture medium, respectively, and cultured at a temperature of 30°C and a rotation speed of 180 rpm for 48 h to obtain liquid first-level fermentation seeds; the liquid first-level fermentation seeds were inoculated into the liquid culture medium of the seed fermentation tank at a 5% inoculation amount, and fermented at a temperature of 30°C, a rotation speed of 180 rpm and a ventilation volume of 5% for 48 h to obtain liquid second-level fermentation seeds; the liquid second-level fermentation seeds were inoculated into the fermentation tank filled with liquid fermentation medium at a 8% inoculation amount, and fermented at 30°C, 150 rpm and a ventilation volume of 5% for 48 h to obtain Bacillus velezii WF02 fermentation liquid and Bacillus refractory WF04 fermentation liquid, wherein the effective viable count of Bacillus velezii WF02 fermentation liquid was 1.2×10 10 cfu / mL, and the effective viable count in the fermentation broth of psychrotrophic Bacillus WF04 was 4.0×10 9 cfu / mL.

[0105] The liquid culture medium for the Bacillus velezensis WF02 seed tank and fermentation tank was as follows: 4.5 g / L glucose, 5 g / L maltose, 3.5 g / L peptone, 1.5 g / L ammonium sulfate, 0.3 g / L magnesium sulfate, 0.1 g / L potassium dihydrogen phosphate, 0.1 g / L potassium hydrogen phosphate, 5 g / L corn flour, 8 g / L soybean meal, 1 g / L light calcium carbonate, pH 7.2;

[0106] The liquid culture medium for the cold-resistant Bacillus WF04 seed tank and fermentation tank is: 5g / L sucrose, 6g / L soluble starch, 3g / L peptone, 1.5g / L ammonium sulfate, 0.5g / L magnesium sulfate, 0.2g / L potassium dihydrogen phosphate, 0.2g / L potassium hydrogen phosphate, 10g / L soybean meal powder, 5g / L light calcium carbonate, pH 7.0.

[0107] 3. Preparation of composite microbial agents

[0108] The fermentation broth of Bacillus velezensis WF02 and the fermentation broth of Bacillus thioredophylla WF04 prepared by the above method were fully mixed at a volume ratio of 50:3 to prepare a composite microbial agent, the effective viable count of which was 5.1×10 9 cfu / mL, and the ratio of the effective viable count of Bacillus velezensis WF02 to that of Bacillus thuringiensis WF04 was 50:1.

[0109] Example 8: Preparation of composite microbial agent

[0110] 1. Activation of bacteria

[0111] Bacillus velezensis WF02 and psychrotolerant Bacillus velezensis WF04 stored in -80°C glycerol tubes were inoculated onto NA solid culture medium, respectively, and activated and cultured at 28°C for 2-3 days to obtain solid seed cultures.

[0112] 2. Preparation of single bacterial agent

[0113] The solid seed culture of Bacillus velezii WF02 and the solid seed culture of Bacillus refractory WF04 were inoculated into LB liquid medium, respectively, and cultured at a temperature of 30°C and a rotation speed of 180 rpm for 48 h to obtain liquid first-level fermentation seeds; the liquid first-level fermentation seeds were inoculated into the liquid culture medium of the seed fermentation tank at a 5% inoculation amount, and fermented at a temperature of 30°C, a rotation speed of 180 rpm and a ventilation volume of 5% for 48 h to obtain liquid second-level fermentation seeds; the liquid second-level fermentation seeds were inoculated into the fermentation tank filled with liquid fermentation medium at a 8% inoculation amount, and fermented at 30°C, 150 rpm and a ventilation volume of 5% for 48 h to obtain Bacillus velezii WF02 fermentation liquid and Bacillus refractory WF04 fermentation liquid; the effective viable bacteria count in the Bacillus velezii WF02 fermentation liquid was 1.2×10 10 cfu / mL, and the effective viable count in the fermentation broth of psychrotrophic Bacillus WF04 was 4.0×10 9 cfu / mL.

[0114] The liquid culture medium for the Bacillus velezensis WF02 seed tank and fermentation tank was as follows: 4.5 g / L glucose, 5 g / L maltose, 3.5 g / L peptone, 1.5 g / L ammonium sulfate, 0.3 g / L magnesium sulfate, 0.1 g / L potassium dihydrogen phosphate, 0.1 g / L potassium hydrogen phosphate, 5 g / L corn flour, 8 g / L soybean meal, 1 g / L light calcium carbonate, pH 7.2;

[0115] The liquid culture medium for the cold-resistant Bacillus WF04 seed tank and fermentation tank is: 5g / L sucrose, 6g / L soluble starch, 3g / L peptone, 1.5g / L ammonium sulfate, 0.5g / L magnesium sulfate, 0.2g / L potassium dihydrogen phosphate, 0.2g / L potassium hydrogen phosphate, 10g / L soybean meal powder, 5g / L light calcium carbonate, pH 7.0.

[0116] 3. Preparation of composite microbial agents

[0117] The fermentation broth of Bacillus velezensis WF02 and the fermentation broth of Bacillus thuringiensis WF04 prepared by the above method were fully mixed at a volume ratio of 80:3 to obtain a composite microbial agent, the effective viable count of which was 8.1×10 9 cfu / mL, and the ratio of the effective viable count of Bacillus velezensis WF02 to that of Bacillus thuringiensis WF04 was 80:1.

[0118] Example 9: Preparation of composite microbial agent

[0119] 1. Activation of bacteria

[0120] Bacillus velezensis WF02 and psychrotolerant Bacillus velezensis WF04 stored in -80°C glycerol tubes were inoculated onto NA solid culture medium, respectively, and activated and cultured at 28°C for 2-3 days to obtain solid seed cultures.

[0121] 2. Preparation of single bacterial agent

[0122] The solid seed culture of Bacillus velezii WF02 and the solid seed culture of Bacillus refractory WF04 were inoculated into LB liquid medium, respectively, and cultured at a temperature of 30°C and a rotation speed of 180 rpm for 48 h to obtain liquid first-level fermentation seeds; the liquid first-level fermentation seeds were inoculated into the liquid culture medium of the seed fermentation tank at a 5% inoculation amount, and fermented at a temperature of 30°C, a rotation speed of 180 rpm and a ventilation volume of 5% for 48 h to obtain liquid second-level fermentation seeds; the liquid second-level fermentation seeds were inoculated into the fermentation tank filled with liquid fermentation medium at a 8% inoculation amount, and fermented at 30°C, 150 rpm and a ventilation volume of 5% for 48 h to obtain Bacillus velezii WF02 fermentation liquid and Bacillus refractory WF04 fermentation liquid; the effective viable bacteria count in the Bacillus velezii WF02 fermentation liquid was 1.2×10 10 cfu / mL, and the effective viable count in the fermentation broth of psychrotrophic Bacillus WF04 was 4.0×10 9 cfu / mL.

[0123] The liquid culture medium for the Bacillus velezensis WF02 seed tank and fermentation tank was as follows: 4.5 g / L glucose, 5 g / L maltose, 3.5 g / L peptone, 1.5 g / L ammonium sulfate, 0.3 g / L magnesium sulfate, 0.1 g / L potassium dihydrogen phosphate, 0.1 g / L potassium hydrogen phosphate, 5 g / L corn flour, 8 g / L soybean meal, 1 g / L light calcium carbonate, pH 7.2;

[0124] The liquid culture medium for the cold-resistant Bacillus WF04 seed tank and fermentation tank is: 5g / L sucrose, 6g / L soluble starch, 3g / L peptone, 1.5g / L ammonium sulfate, 0.5g / L magnesium sulfate, 0.2g / L potassium dihydrogen phosphate, 0.2g / L potassium hydrogen phosphate, 10g / L soybean meal powder, 5g / L light calcium carbonate, pH 7.0.

[0125] 3. Preparation of composite microbial agents

[0126] The fermentation broth of Bacillus velezensis WF02 and the fermentation broth of Bacillus thuringiensis WF04 prepared by the above method were fully mixed at a volume ratio of 100:3 to prepare a composite microbial agent, the effective viable count of which was 1.01×10 10 cfu / mL, and the ratio of the effective viable count of Bacillus velezensis WF02 to the effective viable count of Bacillus thuringiensis WF04 was 100:1.

[0127] Comparative Example 1:

[0128] The results were basically the same as those in Example 7 except that Bacillus velez CGMCC1.923 purchased from the China General Microorganism Collection Center was used to replace Bacillus velez WF02 in Example 7. The bacterial count in the fermentation broth of Bacillus velez CGMCC1.923 was 1.2×10 10 cfu / mL, the bacterial count in the fermentation broth of psychrotrophic Bacillus WF04 was 4.0×10 9 cfu / mL, and the effective viable bacteria count of the prepared composite microbial inoculant was 5.1×10 9 cfu / mL.

[0129] Experimental Example 1: Colonization Performance of Bacillus Velezii WF02 and Psychrotolerant Bacillus WF04 in Tomato Plants

[0130] This example provides a colonization performance test of the Bacillus Velezii WF02 fermentation broth, the psychrophilic Bacillus WF04 fermentation broth, and the composite microbial agent prepared in Example 7 in tomato plants, with a total of 4 treatments.

[0131] Control group: blank control;

[0132] Treatment 1: fermentation broth of Bacillus velezensis WF02 prepared in Example 7;

[0133] Treatment 2: fermentation broth of the refractory Bacillus sp. WF04 prepared in Example 7;

[0134] Treatment 3: The composite microbial agent prepared in Example 7.

[0135] The test method is as follows:

[0136] Select plump, healthy, and pest-free tomato seeds and soak them in cold water for 24 hours. Disinfect the tomato seeds under sterile conditions by first treating them with 75% alcohol for 30-60 seconds, then washing them three times with sterile water, then treating them with 3-5% NaClO for 8-10 minutes, and finally washing them 8-10 times with sterile water. Spread the disinfected tomato seeds in a tissue culture bottle containing basal culture medium (1 / 2Ms + 3% sucrose + 0.6% agar), and wait for the seeds to germinate for 6 days before growing into tomato seedlings.

[0137] The bacterial solutions of treatments 1, 2, and 3 were diluted with sterile water to OD600 = 0.8; centrifuged at 10000rpm at 4℃ for 10min, the supernatant was removed, the bacteria were collected, and thoroughly mixed with 1mL of sterile water to obtain a bacterial suspension; under sterile conditions, the bacterial solution was inoculated into the roots of tomato seedlings, and 12h, 24h, and 36h after inoculation, the roots, stems, and leaves of the tomato seedlings of each treatment were cut, weighed, and placed in a 1.5mL sterile centrifuge tube, and surface disinfected in the same way as tomato seeds; crushed with a sterile glass rod, diluted into different multiples, and evenly spread on NA solid culture medium, and the number of Velez buds in different parts of the tomato plants was counted. The colony counts of Bacillus subtilis WF02 and psychrophilic Bacillus subtilis WF04 were calculated according to the method for determining the number of effective viable bacteria in agricultural microbial agents (GB20287-2006). The bacteria were counted for each treatment after culturing at 28°C for 24 hours. At the same time, the single colonies grown on the NA solid plates coated with each treatment were compared with the initial colony morphology of the effective bacteria. The strains with the same morphology were preliminarily identified as the strains. The initial strains and the strains coated on the leaves were sent to Sangon (Shanghai) Co., Ltd. for 16S rDNA sequencing and sequence comparison by DNA man.

[0138] Table 5 Colonization performance of different treated strains in tomato

[0139]

[0140] As shown in Table 5, no bacteria were detected in the roots, stems, and leaves of tomato seedlings in the control group; bacteria were detected in the roots, stems, and leaves of tomatoes inoculated with Bacillus Velez WF02 (treatment 1) and the composite microbial agent (treatment 3), and their colony morphology was basically consistent with that of Bacillus Velez WF02. 16S rDNA sequencing was performed on this strain and Bacillus Velez WF02. Sequence comparison showed that the 16S rDNA sequence of the strain isolated from tomato leaves was consistent with that of Bacillus Velez WF02. Therefore, the isolated strain was Bacillus Velez WF02, which indicated that Bacillus Velez WF02 could colonize tomato plants within 36 hours; while no bacteria were detected in the roots, stems, and leaves of tomatoes inoculated with the low-temperature-resistant Bacillus Velez WF04 (treatment 2), indicating that this strain could not colonize tomato plants within at least 36 hours.

[0141] As shown in Table 5, Bacillus Velez WF02 could be detected in the roots of tomato seedlings inoculated with Bacillus Velez WF02 (Treatment 1) 12 hours after inoculation, and in the stems and leaves after 24 hours, indicating that the strain can quickly colonize the root system and then colonize the stems and leaves; no bacteria were detected in the roots, stems, and leaves of tomatoes inoculated with psychrophilic Bacillus WF04 (Treatment 2), indicating that the strain could not colonize the tomato plants within at least 36 hours; Bacillus Velez WF02 could be detected in the roots and stems of tomato seedlings inoculated with the composite microbial agent (Treatment 3) 12 hours after inoculation, and the number of the bacteria was significantly increased after 12 hours, 24 hours, and 36 hours of inoculation compared with that inoculated with Bacillus Velez WF02 alone (Treatment 1), indicating that psychrophilic Bacillus WF04 can promote the colonization of Bacillus Velez WF02 in tomato plants. This result is basically consistent with the strong biofilm production ability of the two bacteria after co-culture in Example 6.

[0142] Experimental Example 2: Treatment with different microbial agents improves the low temperature tolerance of tomato plants

[0143] This example provides the use of the fermentation broth of Bacillus Velezii WF02, the fermentation broth of Bacillus thioredophylla WF04, and the composite microbial agent prepared in Example 7 in improving the low-temperature tolerance of tomato plants. A total of five treatments were provided, and the treatment groups and test methods are as follows:

[0144] Control group: sterile water control;

[0145] Treatment 1: microbial agent prepared in Control Example 1;

[0146] Treatment 2: fermentation broth of the refractory Bacillus sp. WF04 prepared in Example 7;

[0147] Treatment 3: fermentation broth of Bacillus velez WF02 prepared in Example 7;

[0148] Treatment 4: The composite microbial agent prepared in Example 7.

[0149] Tomato varieties that are primarily cultivated and intolerant of low temperatures and low light levels were selected as experimental materials. Tomato seeds of uniform size and growth were selected and soaked in 75% alcohol for 15 seconds, rinsed once or twice with sterile water, then soaked in 3% sodium hypochlorite for 15 minutes, the sodium hypochlorite removed, and rinsed three to five times with sterile water. The seeds were then sown in pots filled with seedling medium, one tomato seed per pot, with three replicates per treatment. Seedlings were cultured in a light-controlled incubator, alternating between light and dark (16 hours of daylight per day at 26°C, followed by darkness and 18°C ​​for the remainder of the day, light intensity between 3000 and 10000 lx, and soil moisture of 80%). No additional fertilizer was applied, and normal management was maintained according to planting requirements. When the seeds germinated and grew to two leaves and one bud, they were watered with 50 mL of the diluted inoculum solution (OD600 = 0.8) per plant. Seven days later, a second watering was performed. The control group was watered with an equal volume of sterile water. Seven days after the second watering, the light culture chamber was adjusted to a low-temperature simulated environment (16 hours of daylight per day and a culture temperature of 12°C, the rest of the day in darkness and a culture temperature of 8°C). Other conditions remained unchanged. The growth of the tomato plants under different treatments was observed throughout the growth period. After 20 days, indicators such as plant height, stem diameter, root weight, and leaf area were measured.

[0150] Table 6 Effects of different treatments on the growth of tomato plants under low temperature conditions

[0151]

[0152] From Table 6, Figure 9 It can be seen that compared with the control group, the application of treatment 2 (the fermentation liquid of the low-temperature-resistant Bacillus WF04 prepared in Example 7), treatment 3 (the fermentation liquid of the Bacillus Velez WF02 prepared in Example 7) and treatment 4 (the composite microbial agent prepared in Example 7) of the present invention can significantly increase the stem diameter, plant height, leaf area and root fresh weight of tomato plants under low temperature conditions (p < 0.05), indicating that the three treatments can improve the low-temperature tolerance of tomato plants, among which treatment 4 (the composite microbial agent prepared in Example 7) has the best effect.

[0153] Experimental Example 3: Inhibitory Effects of Different Treatment Inoculants on Tomato Brown Fruit Virus

[0154] This example provides information on the effects of the fermentation broth of Bacillus Velezii WF02 and the fermentation broth of Bacillus thioresistant WF04, prepared in Example 7, and a composite microbial agent on the prevention and control of tomato brown fruit virus in a tomato potted plant experiment. The experiment included seven treatment groups. The treatment groups and experimental methods are as follows:

[0155] Treatment 1: control (virus negative), no treatment;

[0156] Treatment 2: control (virus positive), inoculated with virus;

[0157] Treatment 3: inoculation of virus + fermentation broth of Bacillus velez WF02 prepared in Example 7;

[0158] Treatment 4: inoculation of virus + fermentation broth of psychrotrophic Bacillus sp. WF04 prepared in Example 7;

[0159] Treatment 5: inoculation of virus + composite microbial agent prepared in Example 7;

[0160] Treatment 6: inoculation with virus + fermentation broth of Bacillus velezensis CGMCC1.923 prepared in Control Example 1;

[0161] Treatment 7: Inoculation of virus + composite microbial agent prepared in control example 1.

[0162] The whole leaf inoculation method was used. Tomato seeds of each treatment were sown in pots filled with seedling medium, with 3 seeds per pot and 5 pots per treatment. Each treatment was inoculated with virus solution (virus inoculation concentration was 1.2×10 8 The virus was detected at 100 copies / μL per treatment. Each treatment inoculated 10 plants with 4 leaves inoculated with the virus, and each leaf was inoculated with 20 μL of virus. Five days after inoculation, the number of necrotic spots was counted, and the inhibition rate was calculated. Seven days after inoculation, the third functional leaf from the upper part of the leaf was removed for RNA extraction and viral expression was detected using fluorescent quantitative PCR. Simultaneously, tomato leaves treated with different treatments were surface disinfected, and the total number of culturable bacteria on the leaves was counted. The colony count method followed the method for determining the number of effective viable bacteria in agricultural microbial agents (GB 20287-2006). The experiment was repeated three times. Inhibition rate = (number of necrotic spots in the control - number of necrotic spots in the treatment) / number of necrotic spots in the control × 100%.

[0163] Tomato plants from each treatment were sampled from the same location on the top of the tomato leaves on the 1st, 2nd, 3rd, and 4th day after virus inoculation. These samples were quickly frozen in liquid nitrogen and stored at -80°C. The activities of superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia lyase (PAL), and polyphenol oxidase (PPO) in the tomato leaves of each treatment were determined using ELISA, according to the reference "Changes in Defense Enzyme Activities in Pepper Infected with Tomato Spotted Wilt Virus." The absorbance (OD) values ​​of each treatment were measured sequentially at a wavelength of 450 nm using a microplate reader, and the activities of SOD, POD, CAT, PAL, and PPO were calculated. This study aimed to determine the relationship between the resistance of tomatoes to Tomato Brown Rough Fruit Virus (ToBRFV) in different treatments and the activities of these defense enzymes.

[0164] Table 7 Inhibitory effects of different treatments on tomato brown wrinkled fruit virus

[0165]

[0166] As can be seen from Table 7, compared with the positive control (treatment 2), the virus content of tomatoes treated with different microbial agents decreased to a certain extent. Among them, the microbial agents prepared in treatment 3 (fermentation broth of Bacillus Velez WF02 prepared in Example 7), treatment 4 (fermentation broth of psychrophilic Bacillus WF04 prepared in Example 7), and treatment 5 (composite microbial agent prepared in Example 7) had a strong inhibitory effect on tomato brown wrinkle fruit virus. Among them, the composite microbial agent of the present invention (treatment 5) had the strongest inhibition rate on tomato brown wrinkle fruit virus, reaching 87.62%.

[0167] The results of the detection of the number of culturable bacteria, virus content and the activities of four defense enzymes in the leaves showed (Table 7, Figure 10 As shown in the figure, the composite microbial agent (treatment 5) of the present invention can greatly increase the number of culturable bacteria in tomato leaves and activate the activity of defense enzymes in the leaves; treating tomato seeds with the composite microbial agent (treatment 5) of the present invention can improve the bacterial community structure in the leaves, activate the activity of the plant system's defense enzymes, and improve the resistance of tomato plants to viruses, thereby achieving the effect of inhibiting virus proliferation and reducing virus content.

[0168] Test Example 4: Field efficacy of different treatment agents against tomato brown wrinkle fruit virus

[0169] This example provides a greenhouse experiment to test the effectiveness of the fermentation broth of Bacillus velez WF02 and Bacillus thioredophylla WF04 prepared in Example 7, along with a composite microbial agent, in preventing and controlling tomato brown fruit virus. Eight treatment groups were included in the experiment. The treatment groups and test methods are as follows:

[0170] Treatment 1: blank control, no treatment;

[0171] Treatment 2: fermentation broth of Bacillus velezensis WF02 prepared in Example 7;

[0172] Treatment 3: fermentation broth of the refractory Bacillus sp. WF04 prepared in Example 7;

[0173] Treatment 4: the composite microbial agent prepared in Example 7;

[0174] Treatment 5: the composite microbial agent prepared in Example 8;

[0175] Treatment 6: the composite microbial agent prepared in Example 9;

[0176] Treatment 7: fermentation broth of Bacillus velez CGMCC1.923 prepared in Control Example 1;

[0177] Treatment 8: The composite microbial agent prepared in Control Example 1.

[0178] The experiment was conducted in Juneng Village, Weifang City, Shandong Province, with tomatoes as the test crop. The test greenhouse was a tomato greenhouse with an area of ​​72m2 that had been infected with the tomato brown wrinkle virus and had not been disinfected. 2 The experimental plots were arranged in random blocks and replicated four times. The experimental plots had uniform fertility, consistent planting and management levels, and the disease incidence and damage were severe. Tomato seeds from each treatment were raised as seedlings and transplanted to the experimental greenhouse when they grew to two true leaves. Each treatment was watered with a diluted bacterial solution (1.0×10 7 cfu / mL) 50mL / plant. Protective rows were set up between each treatment and around the experimental plot. The incidence of the negative control group was investigated when obvious symptoms were present. Fifty plants were serially surveyed along the ridges of each plot. Each plant was graded and recorded, and the disease index was calculated. The control efficacy was also calculated. The disease incidence of diseased plants was investigated according to the Tomato Brown Fruit Virus Symptom Grading Standard. The disease index and relative control efficacy of each treatment were calculated.

[0179] Tomato brown wrinkle virus disease symptom grading standard:

[0180] Level 0: No symptoms; Level 1: The veins of the heart leaves are clear, and 1-2 true leaves show mosaic; Level 3: Mosaic leaves on the middle and upper leaves; Level 5: Most leaves are mosaic, and a few leaves are deformed; Level 7: Most leaves are severely mosaic, deformed, or wrinkled; Level 9: Almost all leaves are severely mosaic, deformed, or wrinkled, and the plant is obviously dwarfed. The calculation formula for disease index and relative control effect is as follows:

[0181] Disease index = ∑ (number of diseased plants at each level × disease level) / (total number of plants surveyed × highest level) × 100

[0182] Relative protective effect = (disease index of control - disease index of treatment) / disease index of control × 100%.

[0183] Table 8 Field efficacy of different treatments against tomato brown wrinkled fruit virus

[0184]

[0185] Tomatoes treated with different microbial agents all had a certain preventive effect on tomato brown wrinkled fruit virus occurring in the field. Treatment 2 (fermentation broth of Bacillus Velez subtilis WF02 prepared in Example 7), Treatment 3 (fermentation broth of psychrophilic Bacillus WF04 prepared in Example 7), Treatment 4 (composite microbial agent prepared in Example 7), Treatment 5 (composite microbial agent prepared in Example 8), and Treatment 6 (composite microbial agent prepared in Example 9) had a relative preventive effect of 40.65% to 78.90% on tomato brown wrinkled fruit virus; among them, Treatment 4 (composite microbial agent prepared in Example 7), Treatment 5 (composite microbial agent prepared in Example 8), and Treatment 6 (composite microbial agent prepared in Example 9) had better preventive effect on tomato brown wrinkled fruit virus, with a relative preventive effect of 73.06% to 79.64%.

[0186] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A composite microbial agent, characterized in that: The composite microbial agent is prepared by compounding the Bacillus Velezii WF02 agent and the cold-resistant Bacillus WF04 agent; The Bacillus velez WF02 was deposited in the General Microbiology Center of the China Culture Collection Administration on May 24, 2024, with the deposit number CGMCC NO. 30756, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified as Bacillus velez Bacillus velezensis ; The refractory Bacillus WF04 was deposited in the General Microbiology Center of China Culture Collection Administration on May 24, 2024, with the deposit number CGMCC NO. 30758, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified as refractory Bacillus Peribacillus frigoritolerans ; The effective viable bacteria count of the composite microbial agent is 5.1×10 9 ~1.01×10 10 cfu / mL; The ratio between the effective viable bacteria count of the Bacillus Velez WF02 and the effective viable bacteria count of the psychrophilic Bacillus WF04 in the composite microbial agent is 50-100:1; The composite microbial agent is used for preventing and controlling tomato brown wrinkled fruit virus disease.

2. The composite microbial agent according to claim 1, wherein: The composite microbial agent is used for inducing systemic defense of tomato plants against tomato brown wrinkled fruit virus.

3. The composite microbial agent according to claim 1, wherein: The low-temperature-resistant Bacillus WF04 is used to improve the resistance of tomato plants to tomato brown fruit virus by promoting the rapid colonization of Bacillus Velez WF02 in tomato plants.

4. The composite microbial agent according to claim 1, wherein: The composite microbial agent is applied to a low temperature and / or drought soil environment to promote the growth and development of the root system of tomato seedlings.

5. A method for preventing and controlling tomato brown wrinkled fruit virus disease, characterized in that: Apply the composite microbial agent according to claim 1 to the rhizosphere soil of tomato seedlings.

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