Compound microbial agent and application thereof in prevention and control of tomato brown fruit wrinkling virus disease

Abstract

The invention relates to the technical field of microorganisms, in particular to a compound microbial agent and application thereof to prevention and control of tomato brown fruit wrinkling virus diseases, and the compound microbial agent comprises a microbial agent formed by compounding bacillus velezensis WF02 and low-temperature-resistant bacillus WF04, can improve rhizosphere soil of tomato plants and micro-ecological environment in the plants, and has the effects of improving tomato yield and preventing and controlling tomato brown fruit wrinkling virus diseases. The systematic resistance of plants for preventing the tomato brown fruit crinkling virus is activated, the field control effect on the tomato brown fruit crinkling virus can reach 73.06%-79.64%, and the influence of the tomato brown fruit crinkling virus on the tomato yield and quality is effectively reduced; the low-temperature-resistant bacillus WF04 can promote the growth and development of root systems of tomato seedlings in low-temperature and drought environments and assist the bacillus velezensis WF02 to quickly enter tomato plants.

Description

Technical Field

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

[0002] Tomato is one of the most important vegetable crops grown in my country and even in the world. Tomatoes are often infected by various viruses throughout their growth cycle, which have a serious impact on tomato yield and quality. It is difficult to prevent and control tomato viruses after they are infected, which is an important factor restricting tomato production.

[0003] Tomato brown rugose fruit virus (ToBRFV) is a new species of the genus Tobacco mosaic virus, belonging to the family Brucoviridae. The virus infects tomatoes, causing mosaic, dark green protrusions, narrow leaves, and severe necrosis of veins, a reduction in the number of flowers and fruits, yellow or brown spots on the fruits, smaller fruits, and wrinkles. In severe cases, the fruit stalks will necrotize, causing a significant reduction in fruit yield. In 2015, Jordanian scientists discovered that tomato fruits showed severe brown shrunken symptoms, with an incidence rate of nearly 100% of the fruits. They described and identified it as ToBRFV.

[0004] ToBRFV has strong tolerance to the environment, is not easy to inactivate, and is extremely easy to spread. Therefore, it is urgent to conduct systematic research and prevention and control of ToBRFV. However, there is currently a lack of effective targeted agents for chemical control of ToBRFV, and there is even less green control technology for ToBRFV. Therefore, it is of great application value to explore specific microorganisms from healthy tomato plants in resistant habitats and conduct research on green control technology based on specific microorganisms to control Tomato Brown Fruit Virus. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a composite microbial agent capable of improving the microecological environment in tomato plants and activating the resistance of the defense system of tomato plants, and the application of the composite microbial agent 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: In a first aspect, the present invention claims protection for a composite microbial agent, which is compounded by Bacillus Velez WF02 and psychrophilic Bacillus WF04.

[0007] The Velez subtilis WF02 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on May 24, 2024, with a deposit number of CGMCC NO. 30756, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified and named as Velez subtilisBacillus velezensis ; The low-temperature resistant Bacillus sp. WF04 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on May 24, 2024, with the deposit number CGMCC NO. 30758, the deposit address being No. 1, Beichen West Road, Chaoyang District, Beijing, and the taxonomic name being Bacillus sp. Peribacillus frigoritolerans .

[0008] Furthermore, the effective viable count of the compound microbial inoculum is 5.1×10 9 ~1.01×10 10 cfu / mL.

[0009] Furthermore, the ratio between the effective viable count of Bacillus velezensis WF02 and the effective viable count of the low-temperature resistant Bacillus sp. WF04 in the compound microbial inoculum is 50~100:1.

[0010] In a second aspect, the present invention claims to protect the application of the Bacillus velezensis WF02 inoculum, the low-temperature resistant Bacillus sp. WF04 inoculum, and the compound microbial inoculum in tomato cultivation respectively.

[0011] In a third aspect, the present invention claims to protect the application of the Bacillus velezensis WF02 inoculum, the low-temperature resistant Bacillus sp. WF04 inoculum, and the compound microbial inoculum in preventing and controlling tomato brown rugose fruit virus disease respectively.

[0012] In a fourth aspect, the present invention claims to protect the application of the Bacillus velezensis WF02 inoculum and the compound microbial inoculum in inducing systemic defense of tomato plants against tomato brown rugose fruit virus respectively.

[0013] In a fifth aspect, the present invention claims to protect the application of the low-temperature resistant Bacillus sp. WF04 and the compound microbial inoculum in promoting the root growth and development of tomato plants, thereby improving the resistance of tomato plants to tomato brown rugose fruit virus.

[0014] In a sixth aspect, the present invention claims to protect the application of the low-temperature resistant Bacillus sp. WF04 and the compound microbial inoculum in promoting the root growth and development of tomato seedlings in low-temperature and / or arid soil environments respectively.

[0015] In a seventh aspect, the present invention claims to protect a method for preventing and controlling tomato brown rugose fruit virus disease, which is to apply the compound microbial inoculum to the rhizosphere soil of tomato seedlings.

[0016] The beneficial effects of the present invention are as follows: the Velez Bacillus WF02 of the present invention has a broad spectrum of antibacterial effects, and the metabolites and volatile substances produced have a strong inhibitory effect on soil-borne pathogens such as ginger stem base rot bacteria, asparagus stem rot bacteria, and tomato wilt bacteria; Velez Bacillus WF02 has a strong ability to produce biofilms, can quickly colonize tomato roots, and then transfer to plant leaves and other internal locations, improve the microecological environment in the tomato plant, and reduce the incidence of tomato brown wrinkled fruit virus disease. Low-temperature resistant Bacillus WF04 can promote the growth and development of tomato seedling roots in low temperature and drought environments, and assist Velez Bacillus WF02 to quickly enter the tomato plant body. The composite microbial agent of the present invention is composed of a double bacteria that has a wide range of adaptability to growth environments and is salt-resistant and resistant to severe drought environments. It 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 the field prevention effect on tomato brown wrinkled fruit virus disease can reach 73.06%-79.64%, effectively reducing the impact of tomato brown wrinkled fruit virus on tomato yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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. Figure 1 is a colony morphology diagram of Bacillus Velez WF02 of the present invention; Figure 2 is a phylogenetic tree of Bacillus Velez WF02 of the present invention based on 16S rDNA sequence; Figure 3 This is a colony morphology diagram of the low-temperature resistant Bacillus WF04 of the present invention; Figure 4 is a phylogenetic tree of the low-temperature resistant Bacillus WF04 of the present invention based on 16S rDNA sequence; Figure 5 The diagram is a diagram of the antibacterial effect of the Velez subsp. 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 fuscata (CK), and B on the right is an antibacterial effect diagram; C on the left is Magnaporthe oryzae (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; 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); Figure 7 This is a diagram of the antibacterial effect of the volatile substances of Bacillus Velez WF02 of the present invention (left: control diagram; right: antibacterial diagram of the volatile substances of Bacillus Velez WF02); Figure 8This 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); Fig. 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; Fig.10 It 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

[0018] 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, unless otherwise specified, can be obtained from commercial channels.

[0019] The various culture media involved in the following examples are all standard culture media commonly used in the art, for example: LB liquid medium: peptone 10.0 g, yeast extract 5.0 g, sodium chloride 5.0 g, pH 7.0-7.2, distilled water to 1000 mL; NA solid medium: peptone 10.0g, beef extract 5.0g, sodium chloride 5.0g, agar 20g, pH 7.0-7.2, distilled water to 1000mL; PDA medium: 6.0 g potato powder, 20.0 g glucose, 20.0 g agar, pH 5.5-6.0, and distilled water to 1000 mL; NB liquid culture medium: peptone 10.0g, beef extract powder 3.0g, sodium chloride 5.0g, pH 7.0-7.2, distilled water to 1000mL; Basal culture medium (1 / 2Ms+3% sucrose+0.6% agar): 950mg potassium nitrate, 825mg ammonium nitrate, 85mg potassium dihydrogen phosphate, 185mg magnesium sulfate, 220mg calcium chloride, 0.83mg potassium iodide, 6.2mg boric acid, 22.3mg manganese sulfate, 8.6mg zinc sulfate, 0.25mg sodium molybdate, 0.025mg copper sulfate, 0.025mg cobalt chloride, 37.3mg disodium ethylenediaminetetraacetic acid, 27.8mg ferrous sulfate, 100mg inositol, 2mg glycine, 0.1mg thiamine hydrochloride, 0.5mg pyridoxine hydrochloride, 0.5mg niacin, 30g sucrose, and 7g agar.

[0020] Example 1: Screening and identification of Bacillus Velezii WF02 1. Strain screening The virus was isolated from healthy tomato plants collected from a greenhouse where the Tomato Brown Fruit Virus was found on a large scale in the tomato growing area of ​​Anqiu City, Shandong Province. The sampling and isolation methods are as follows: Select healthy plants, use a sampling shovel to dig out the entire root system, rinse with tap water first, 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℃ for 30 days; put the tomato roots, stems, and leaves in a sterilized mortar, add 5 to 10 mL of 1×PBS buffer, and then grind the juice evenly 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 out for isolation and purification to obtain strain WF02.

[0021] 2. Strain identification The morphological identification of strain WF02 was performed, and the results are as follows: 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.

[0022] Molecular biological identification of strain WF02: The total DNA of strain WF02 was extracted and prepared according to the conventional bacterial DNA extraction method. The 16S rDNA gene was amplified by PCR using the bacterial universal primers 27f and 1492r. The PCR reaction system was: DNA template 1µl, 2×MastarMix 25µl, 27f2µl, 1492r 2µl, and ultrapure water was added to 50µl; the amplified product was detected by 0.8% agarose gel electrophoresis and sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing.

[0023] 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 using the neighbor-joining method (NJ). The 16S rDNA sequence of strain WF02 was compared with that of Bacillus velez. (Bacillus velezensis ) is 99% similar (e.g. Figure 2 As shown); strain WF02 was identified as Bacillus Velezii ( Bacillus velezensis ), named Bacillus velez WF02.

[0024] The Bacillus Velezii WF02 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on May 24, 2024, with a deposit number of CGMCC NO. 30756, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified and named Bacillus velezensis .

[0025] Example 2: Screening and identification of low-temperature resistant Bacillus WF04 1. Strain screening It was isolated from soil collected from the Ali region of Tibet at an average altitude of more than 4,500 meters. The isolation method is as follows: The collected soil was adjusted to an appropriate moisture content and frozen at -80°C for 35 days (freeze and thaw once every 7 days). After repeated freeze-thaw for 5 times, 1 g of soil was placed in 100 mL of sterile water and shaken for 20 minutes to obtain the sample stock solution. The sample stock solution was diluted in a gradient manner, and the dilution was 10 -3 , 10 -4 , 10 -5 100 μL of the dilution was spread on NA solid culture medium and inverted for 2 days at 10°C. A single colony of the fast-growing dominant bacteria was picked out for isolation and purification to obtain strain WF04.

[0026] 2. Strain identification The morphology of strain WF04 was identified, and the results are as follows: The colonies of strain WF04 on NA solid medium are off-white, round, oily, without wrinkles on the surface, and opaque; Gram staining is positive, the cells are rod-shaped, not in chains, and produce spores, such as Figure 3 shown.

[0027] Molecular biological identification of strain WF04: The total DNA of strain WF04 was extracted and prepared according to the conventional bacterial DNA extraction method. The 16SrDNA gene was amplified by PCR using the bacterial universal primers 27f and 1492r. The PCR reaction system was: DNA template 1µl, 2×MastarMix 25µl, 27f 2µl, 1492r 2µl, and ultrapure water was added to 50µl; the amplified product was detected by 0.8% agarose gel electrophoresis and sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing.

[0028] 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 using the neighbor-joining method (NJ). The 16S rDNA sequence comparison results of strain WF04 (such as Figure 4 ) was identified as a psychrotolerant Bacillus (Peribacillus frigoritolerans ), named as psychrotolerant Bacillus WF04.

[0029] The cold-resistant Bacillus WF04 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on May 24, 2024, with the deposit number CGMCC NO. 30758, the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and the classification name is Peribacillus frigoritolerans .

[0030] Example 3: Determination of the inhibitory effect of Bacillus Velezii WF02 on plant pathogens This example provides the antibacterial effect of Bacillus Velezii WF02 on the pathogen of ginger stem rot: Fusarium oxysporum; the pathogen of asparagus stem rot: Fusarium fujikura; the pathogen of rice blast: Aspergillus grisea; and the pathogen of tomato wilt: tomato-specific Fusarium oxysporum.

[0031] The test method is as follows: Antagonistic pathogenic fungi test: Place a pathogen cake (7 mm) in the center of a 90 mm diameter PDA solid plate, inoculate Bacillus Velez subtilis WF02 25 mm above, below, left and right from the center, and leave no inoculation on the control plate. Culture the plates in a constant temperature incubator at 28°C for 5 to 7 days, and count the diameters of the pathogens in the control group and the treatment group, and calculate the inhibition rate.

[0032] The antibacterial rate calculation formula is as follows: Inhibition rate = (pathogen diameter in the control group - pathogen diameter in the treatment group) / (pathogen diameter in the control group - bacterial cake diameter) × 100%.

[0033] Table 1 Antibacterial effect of Bacillus velezensis WF02 on plant pathogens

[0034] As shown in Table 1, Bacillus Velezii 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 Velezii WF02 of the present invention has a wide antibacterial spectrum and strong antibacterial ability, and has potential disease prevention ability.

[0035] Example 4: Antibacterial Activity Detection of Sterile Metabolites and Volatile Substances of Bacillus Velezii WF02 This example provides the antibacterial activity detection of sterile metabolites and volatile substances of Bacillus Velezii WF02, and the method is as follows: 1. Antibacterial activity of sterile metabolites: Bacillus Velez WF02 was inoculated into LB liquid culture medium, and the fermentation liquid of Bacillus Velez WF02 was obtained after shaking at 28°C and 180r / min for 48h; 1.5mL of the fermentation liquid was taken into a 2mL sterilized centrifuge tube, and centrifuged at 4°C and 12000r / min for 5min; the supernatant was sterilized and filtered through a 0.22μm pinhole filter to obtain a sterile filtrate of Bacillus Velez WF02; a Fusarium oxysporum cake (7mm) was placed in the center of a PDA solid plate with a diameter of 90mm, and holes were punched at 25mm from the center, and then 50μL 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 5-7d, and the diameters of the pathogens in the control group and the treatment group were counted respectively, and the inhibition rate was calculated; The results are as follows Figure 6 As shown, 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.

[0036] 2. Antibacterial activity of volatile substances: The inhibitory effect of volatile substances of Bacillus Velez WF02 on the pathogen of ginger stem base rot (Fusarium oxysporum) was determined by using a two-divided plate method and a 90 mm two-divided culture dish. 10 mL of PDA medium was added to one side of the second divided culture dish for culturing Fusarium oxysporum; 10 mL of NA solid medium was added to the other side for culturing Bacillus Velez WF02. One side was not cultured with Bacillus Velez WF02, and the other side was cultured with Fusarium oxysporum as the control. The culture dish was sealed with Parafilm to cut off the air circulation inside and outside the dish. Five replicates were used for each treatment. After sealing, the dishes were inverted and cultured at 28°C in the dark for 7 days, and then the growth of pathogenic fungi was observed.

[0037] The results are as follows Figure 7 As shown, the growth of Fusarium oxysporum in the two-grid 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.

[0038] Example 5: Growth characteristics and stress resistance of Bacillus velez WF02 and Bacillus thuringiensis WF04 This example provides growth characteristics and stress resistance detection of Bacillus Velezii WF02 and psychrotolerant Bacillus WF04. The test method is as follows: 1. Determination of optimal growth temperature The seed solutions of Bacillus Velezii WF02 and psychrophilic Bacillus WF04 were prepared respectively, inoculated into 250mL Erlenmeyer flasks containing 50mL LB liquid medium at a 5% inoculum rate, and cultured with shaking (speed 180r / min) in a constant temperature shaking incubator at 4℃, 10℃, 20℃, 30℃, 40℃, 50℃, and 60℃ for 2d, respectively. Repeat 3 times, observe and record the growth conditions (whether it is turbid or not).

[0039] 2. Acid and alkali resistance determination 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 5% seed liquid of Bacillus velez WF02 and psychrotolerant Bacillus WF04 were inoculated respectively. The culture was shaken at 30℃ and 180r / min for 2d, and repeated 3 times. The growth conditions (whether it was turbid or not) were observed and recorded.

[0040] 3. Salt resistance test Prepare LB liquid culture medium with different NaCl contents (1%, 5%, 10%, 15%, 20%), inoculate 5% seed liquid of Bacillus Velez WF02 and Bacillus thuringiensis WF04 respectively, shake and culture for 2 days at 30℃ and 180r / min, set 3 replicates, observe and record the growth (whether it is turbid or not). The judgment method is as follows: non-salt-tolerant strains: NaCl concentration is less than 1.17%; low salt-tolerant strains: NaCl concentration is between 1.17% and 2.93%; medium salt-tolerant strains: NaCl concentration is between 2.93% and 14.63%; highly salt-tolerant strains: NaCl concentration is between 14.63% and 30.4%.

[0041] 4. Determination of drought resistance Prepare LB liquid culture medium with different concentrations of PEG6000 (0g / L, 100g / L, 200g / L, 300g / L, 400g / L, 500g / L, 600g / L), inoculate 5% of Bacillus Velezii WF02 and Bacillus thuringiensis WF04 seed solution respectively, culture at 30℃ and 180r / min for 2d, set 3 replicates, observe and record the growth (whether it is turbid). Measure the OD value at 700nm by spectrophotometry. Judgment method: PEG6000 concentration of 0-60g / L represents mild drought resistance, PEG6000 concentration of 90-150g / L represents moderate drought resistance, and PEG6000 concentration greater than 150g / L represents severe drought resistance.

[0042] Table 2 Growth and stress resistance characteristics of Bacillus velezensis WF02

[0043] Note: + indicates growth, ++ indicates better growth, +++ indicates good growth, - indicates no growth Table 3 Growth and stress resistance characteristics of low-temperature resistant Bacillus sp. WF04

[0044] Note: + indicates growth, ++ indicates better growth, +++ indicates good growth, - indicates no growth It can be seen from Table 2 and Table 3 that the growth temperature range of Bacillus velezensis WF02 is 20 - 50 °C, and the optimum growth temperature is 30 °C; the growth temperature range of low-temperature resistant Bacillus sp. WF04 is 4 - 40 °C, and the optimum growth temperature is 30 °C; the growth pH value range of Bacillus velezensis WF02 is 5.0 - 9.0, and the optimum pH value is 7.0 - 8.0; the growth pH value range of low-temperature resistant Bacillus sp. WF04 is 6.0 - 9.0, and the optimum pH value is 7.0 - 8.0; Bacillus velezensis WF02 can tolerate 10% concentration of NaCl and is a moderately salt-tolerant strain, and can grow under 50% PEG6000, tolerating severe drought conditions; low-temperature resistant Bacillus sp. WF04 can tolerate 5% concentration of NaCl and is a low-salt-tolerant strain, and can grow under 60% PEG6000, tolerating severe drought conditions; the above results indicate that Bacillus velezensis WF02 and low-temperature resistant Bacillus sp. WF04 of the present invention have a wide range of growth environment adaptability, salt tolerance, and tolerance to severe drought environment.

[0045] Example 6: Determination of biofilm production performance of Bacillus velezensis WF02 and low-temperature resistant Bacillus sp. WF04 This example provides a method for determining the biofilm production of Bacillus velezensis WF02 and low-temperature resistant Bacillus sp. WF04. The test method is as follows: Bacillus Velez WF02 and psychrotolerant Bacillus WF04 were inoculated into LB liquid culture medium and cultured at 30°C and 180r / min for 24h. 4mL of single bacterial solution of Bacillus Velez WF02 and psychrotolerant Bacillus WF04 and composite bacterial solution (2mL of each of Bacillus Velez WF02 and psychrotolerant Bacillus WF04 were mixed) were respectively aspirated into 10mL centrifuge tubes and placed in a 30°C incubator for 2d. The bacterial solution was poured out after taking out, washed twice with 1×PBS to remove the unabsorbed bacterial solution, and air-dried naturally; 4mL of 1% crystal violet staining solution was added to the centrifuge tube and stained for 30min; the crystal violet staining solution was aspirated, and the surface floating color was washed with 1×PBS, and air-dried naturally; then 4mL of 95% ethanol was added and allowed to stand for 15min to dissolve the crystal violet, and the biofilm content was analyzed by detecting the absorbance value (As) of the sample at 590nm, and LB (Ac) was used as a control.

[0046] Based on the OD value generated by the bacterial biofilm at 590nm, the ability of bacteria to form biofilm was evaluated: if As≤Ac, there is no biofilm production function; if Ac<As≤(2×Ac), there is a low biofilm production ability; if (2×Ac)<As≤(4×Ac), there is a medium biofilm production ability; if (4×Ac)<As, there is a strong biofilm production ability.

[0047] Table 4 OD values ​​of different treatments at 590 nm

[0048] As shown in Table 4 and Figure 8 As shown, Ac(CK)=0.615, As(WF02)=1.690, As(WF04)=0.930, that is, (2×Ac)<As(WF02), Ac<As(WF04), As(WF02+WF04)>(4×Ac). The results showed that Bacillus Velezii WF02 had a moderate biofilm production ability, and Bacillus pyrrotolerant WF04 had a lower biofilm production ability. After co-culture, Bacillus Velezii WF02 and Bacillus pyrrotolerant WF04 both had a strong biofilm production ability. This property plays an important role in improving the colonization ability of the two strains in crop roots and synergistic defense against diseases.

[0049] Example 7: Preparation of composite microbial agent 1. Activation of bacteria Bacillus Velezii WF02 and psychrotolerant Bacillus WF04 preserved in -80°C glycerol tubes were inoculated on NA solid culture medium, activated and cultured at 28°C for 2-3 days to obtain solid seed cultures.

[0050] 2. Preparation of single bacterial agent The solid seed culture of Bacillus Velez subtilis WF02 and the solid seed culture of Bacillus pyrogenus WF04 were inoculated into LB liquid culture medium, respectively, and cultured under shaking conditions of 30°C and 180 rpm for 48 h to obtain liquid primary fermentation seeds; the liquid primary fermentation seeds were inoculated into the liquid culture medium of the seed fermentation tank at an inoculation amount of 5%, and fermented and cultured for 48 h at a temperature of 30°C, a rotation speed of 180 rpm and a ventilation volume of 5% to obtain liquid secondary fermentation seeds; the liquid secondary fermentation seeds were inoculated into the fermentation tank filled with liquid fermentation culture medium at an inoculation amount of 8%, and fermented for 48 h at 30°C, 150 rpm and a ventilation volume of 5% to obtain Bacillus Velez subtilis WF02 fermentation liquid and Bacillus pyrogenus WF04 fermentation liquid, wherein the effective viable count of Bacillus Velez subtilis WF02 fermentation liquid was 1.2×10 10 cfu / mL, the effective viable count in the fermentation broth of low-temperature resistant Bacillus WF04 was 4.0×10 9 cfu / mL.

[0051] The liquid culture medium of Bacillus Velez WF02 seed tank and fermentation tank is: 4.5g / L glucose, 5g / L maltose, 3.5g / L peptone, 1.5g / L ammonium sulfate, 0.3g / L magnesium sulfate, 0.1g / L potassium dihydrogen phosphate, 0.1g / L potassium hydrogen phosphate, 5g / L corn flour, 8g / L soybean meal, 1g / L light calcium carbonate, pH7.2; The liquid culture medium for the cryoresistant 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.

[0052] 3. Preparation of composite microbial agents The fermentation broth of Bacillus Velezii WF02 and the fermentation broth of Bacillus pyrrolate WF04 prepared by the above method were fully mixed at a volume ratio of 50:3 to obtain 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 velez WF02 to that of the low-temperature-resistant Bacillus WF04 was 50:1.

[0053] Example 8: Preparation of composite microbial agent 1. Activation of bacteria Bacillus Velezii WF02 and psychrotolerant Bacillus WF04 preserved in -80°C glycerol tubes were inoculated on NA solid culture medium, activated and cultured at 28°C for 2-3 days to obtain solid seed cultures.

[0054] 2. Preparation of single bacterial agent The solid seed culture of Bacillus Velez subtilis WF02 and the solid seed culture of Bacillus pyrogenus WF04 were inoculated into LB liquid culture medium, respectively, and cultured under shaking conditions of 30°C and 180 rpm for 48 h to obtain liquid primary fermentation seeds; the liquid primary fermentation seeds were inoculated into the liquid culture medium of the seed fermentation tank at an inoculation rate of 5%, and fermented and cultured for 48 h at a temperature of 30°C, a rotation speed of 180 rpm, and a ventilation volume of 5% to obtain liquid secondary fermentation seeds; the liquid secondary fermentation seeds were inoculated into the fermentation tank filled with liquid fermentation culture medium at an inoculation rate of 8%, and fermented for 48 h at 30°C, 150 rpm, and a ventilation volume of 5% to obtain Bacillus Velez subtilis WF02 fermentation liquid and Bacillus pyrogenus WF04 fermentation liquid; the effective viable count in the fermentation liquid of Bacillus Velez subtilis WF02 was 1.2×10 10 cfu / mL, the effective viable count in the fermentation broth of low-temperature resistant Bacillus WF04 was 4.0×10 9 cfu / mL.

[0055] The liquid culture medium of Bacillus Velez WF02 seed tank and fermentation tank is: 4.5g / L glucose, 5g / L maltose, 3.5g / L peptone, 1.5g / L ammonium sulfate, 0.3g / L magnesium sulfate, 0.1g / L potassium dihydrogen phosphate, 0.1g / L potassium hydrogen phosphate, 5g / L corn flour, 8g / L soybean meal, 1g / L light calcium carbonate, pH7.2; The liquid culture medium for the cryoresistant 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.

[0056] 3. Preparation of composite microbial agents The fermentation broth of Bacillus Velezii WF02 and the fermentation broth of Bacillus pyrrolate 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, the ratio of the effective viable count of Bacillus velez WF02 to that of Bacillus thuringiensis WF04 was 80:1.

[0057] Example 9: Preparation of composite microbial agent 1. Activation of bacteria Bacillus Velezii WF02 and psychrotolerant Bacillus WF04 preserved in -80°C glycerol tubes were inoculated on NA solid culture medium, activated and cultured at 28°C for 2-3 days to obtain solid seed cultures.

[0058] 2. Preparation of single bacterial agent The solid seed culture of Bacillus Velez subtilis WF02 and the solid seed culture of Bacillus pyrogenus WF04 were inoculated into LB liquid culture medium, respectively, and cultured under shaking conditions of 30°C and 180 rpm for 48 h to obtain liquid primary fermentation seeds; the liquid primary fermentation seeds were inoculated into the liquid culture medium of the seed fermentation tank at a 5% inoculation amount, and fermented and cultured for 48 h at a temperature of 30°C, a rotation speed of 180 rpm, and a ventilation volume of 5% to obtain liquid secondary fermentation seeds; the liquid secondary fermentation seeds were inoculated into the fermentation tank filled with liquid fermentation culture medium at a 8% inoculation amount, and fermented for 48 h at 30°C, 150 rpm, and a ventilation volume of 5% to obtain Bacillus Velez subtilis WF02 fermentation liquid and Bacillus pyrogenus WF04 fermentation liquid; the effective viable bacteria count in the fermentation liquid of Bacillus Velez subtilis WF02 was 1.2×10 10 cfu / mL, the effective viable count in the fermentation broth of low-temperature resistant Bacillus WF04 was 4.0×10 9 cfu / mL.

[0059] The liquid culture medium of Bacillus Velez WF02 seed tank and fermentation tank is: 4.5g / L glucose, 5g / L maltose, 3.5g / L peptone, 1.5g / L ammonium sulfate, 0.3g / L magnesium sulfate, 0.1g / L potassium dihydrogen phosphate, 0.1g / L potassium hydrogen phosphate, 5g / L corn flour, 8g / L soybean meal, 1g / L light calcium carbonate, pH7.2; The liquid culture medium for the cryoresistant 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.

[0060] 3. Preparation of composite microbial agents The fermentation broth of Bacillus Velezii WF02 and the fermentation broth of Bacillus pyrrolate WF04 prepared by the above method were fully mixed at a volume ratio of 100:3 to obtain 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 velez WF02 to that of the low-temperature-resistant Bacillus WF04 was 100:1.

[0061] Comparative Example 1: The above method is basically the same as that of Example 7 except that Bacillus Velez CGMCC1.923 purchased from China General Microorganism Collection Center is used to replace Bacillus Velez WF02 in Example 7. The bacterial count in the fermentation broth of Bacillus Velez CGMCC1.923 is 1.2×10 10 cfu / mL, the bacterial count in the fermentation broth of low-temperature resistant Bacillus WF04 was 4.0×10 9 cfu / mL, and the effective viable count of the prepared composite microbial inoculant was 5.1×10 9 cfu / mL.

[0062] Experimental Example 1: Colonization performance of Bacillus Velezii WF02 and Psychrotolerant Bacillus WF04 in tomato plants This example provides a test for the colonization performance of the Bacillus Velezii WF02 fermentation liquid, the psychrophilic Bacillus WF04 fermentation liquid and the composite microbial agent prepared in Example 7 in tomato plants, with a total of 4 treatments; Control group: blank control; Treatment 1: fermentation broth of Bacillus velez WF02 prepared in Example 7; Treatment 2: fermentation broth of the low-temperature resistant Bacillus WF04 prepared in Example 7; Treatment 3: The composite microbial agent prepared in Example 7.

[0063] The test method is as follows: Select plump, healthy and pest-free tomato seeds and soak them in cold water for 24 hours; disinfect the tomato seeds under sterile conditions, first treat them with 75% alcohol for 30-60 seconds, then wash them with sterile water for 3 times, then treat them with 3-5% NaClO for 8-10 minutes, and finally wash them with sterile water for 8-10 times; spread the disinfected tomato seeds in a tissue culture bottle filled with 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.

[0064] The bacterial solutions of treatment 1, treatment 2 and treatment 3 were diluted with sterile water to OD600 = 0.8 respectively; centrifuged at 10000rpm 4℃ for 10min, the supernatant was removed, the bacteria were collected, and 1mL of sterile water was used to fully mix to obtain a bacterial suspension; under sterile conditions, the bacterial solution was inoculated into the roots of tomato seedlings respectively, and after 12h, 24h, and 36h of inoculation, the roots, stems and leaves of tomato seedlings of each treatment were cut, weighed and placed in a 1.5mL sterile centrifuge tube, and the surface was 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 Velez buds in different parts of the tomato plants were counted respectively. The colony counts of Bacillus subtilis WF02 and psychrophilic Bacillus subtilis WF04 were measured according to the method for determining the number of effective live 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. Those with the same morphology were preliminarily determined to be 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 was performed using DNA man.

[0065] Table 5 Colonization performance of different treated strains in tomato

[0066] 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. The strain and Bacillus Velez WF02 were subjected to 16S rDNA sequencing. After sequence comparison, the 16S rDNA sequence of the strain isolated from tomato leaves was consistent with the 16S rDNA sequence of Bacillus Velez WF02. Therefore, the isolated strain was Bacillus Velez WF02. This result indicated that Bacillus Velez WF02 could colonize in tomato plants within 36 hours; while no bacteria were detected in the roots, stems and leaves of tomatoes inoculated with low-temperature-resistant Bacillus WF04 (treatment 2), indicating that the strain could not colonize in tomato plants within at least 36 hours.

[0067] As shown in Table 5, the tomato seedlings inoculated with Bacillus Velez subtilis WF02 (treatment 1) can be detected in the roots 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 the tomatoes inoculated with the low-temperature-resistant Bacillus WF04 (treatment 2), indicating that the strain cannot colonize in the tomato plants within at least 36 hours; Bacillus Velez subtilis WF02 can be detected in the roots and stems of the tomato seedlings inoculated with the composite microbial agent (treatment 3) 12 hours after inoculation, and the number of the bacteria after 12 hours, 24 hours and 36 hours of inoculation is significantly increased compared with that of the single inoculation of Bacillus Velez subtilis WF02 (treatment 1), indicating that the low-temperature-resistant Bacillus WF04 can promote the colonization of Bacillus Velez subtilis WF02 in the tomato plants. This result is basically consistent with the strong biofilm production ability of the two bacteria after co-culture in Example 6.

[0068] Experimental Example 2: Treatment with different microbial agents improves the low temperature tolerance of tomato plants This example provides the application of the fermentation liquid of Bacillus Velezii WF02, the fermentation liquid of Bacillus pyrrotolerant WF04 and the composite microbial agent prepared in Example 7 in improving the low temperature tolerance of tomato plants. A total of 5 treatments are provided, and the treatment groups and test methods are as follows: Control group: sterile water control; Treatment 1: microbial agent prepared in control example 1; Treatment 2: fermentation broth of the low-temperature resistant Bacillus WF04 prepared in Example 7; Treatment 3: fermentation broth of Bacillus velez WF02 prepared in Example 7; Treatment 4: The composite microbial agent prepared in Example 7.

[0069] The main tomato varieties that are intolerant to low temperature and weak light were selected as the experimental materials. Tomato seeds of the same size and growth were selected, and the seeds were soaked in 75% alcohol for 15 seconds, rinsed with sterile water 1-2 times, and then soaked in 3% sodium hypochlorite for 15 minutes, then the sodium hypochlorite was removed, and rinsed with sterile water 3-5 times. Then the seeds were sown in pots filled with seedling substrates, with one tomato seed per pot, and three replicates for each treatment. The seedlings were cultured in a light culture room, and light and dark culture and dual culture temperatures were alternated (the daytime light time was 16 hours and the culture temperature was 26°C, the rest of the time was dark and the culture temperature was 18°C, the light intensity was 3000lx~10000lx, and the soil moisture was 80%). Normal management was carried out according to planting needs without applying other fertilizers. When the seeds germinate and grow to two leaves and one heart, water them with 50 mL / plant of the diluted bacterial agent (OD600=0.8), and water them again after 7 days. The control group is watered with the same volume of sterile water. 7 days after the second watering, the light culture room is adjusted to a low-temperature simulation environment (16 hours of daylight per day and a culture temperature of 12°C, and the rest of the time is dark and the culture temperature is 8°C), and other conditions remain unchanged. The growth of tomato plants with different treatments was observed throughout the growth period, and the plant height, stem thickness, root weight, leaf area and other indicators of tomatoes with different treatments were investigated after 20 days.

[0070] Table 6 Effects of different treatments of bacterial agents on the growth of tomato plants under low temperature conditions

[0071] From Table 6, Fig. 9 It can be seen that compared with the control group, the application of treatment 2 (the low-temperature-resistant Bacillus WF04 fermentation liquid prepared in Example 7), treatment 3 (the fermentation liquid of 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 resistance of tomato plants, among which treatment 4 (the composite microbial agent prepared in Example 7) has the best effect.

[0072] Experimental Example 3: Inhibitory Effects of Different Treatments on Tomato Brown Fruit Virus This example provides the effects of the fermentation liquid of Bacillus Velezii WF02, the fermentation liquid of Bacillus pyrrotolerant WF04 and the composite microbial agent prepared in Example 7 on the prevention and control of tomato brown wrinkled fruit virus in a tomato pot test. The test has 7 treatment groups. The treatment groups and test methods are as follows: Treatment 1: control (virus negative), no treatment; Treatment 2: control (virus positive), inoculated with virus; Treatment 3: inoculation of virus + fermentation broth of Bacillus velez WF02 prepared in Example 7; Treatment 4: inoculation of virus + fermentation broth of the low-temperature resistant Bacillus WF04 prepared in Example 7; Treatment 5: inoculation of virus + composite microbial agent prepared in Example 7; Treatment 6: inoculation of virus + fermentation broth of Bacillus velez CGMCC1.923 prepared in control example 1; Treatment 7: Inoculation of virus + composite microbial agent prepared in control example 1.

[0073] The whole leaf inoculation method was used. Tomato seeds of each treatment were sown in pots filled with seedling medium, 3 seeds per pot, and 5 pots per treatment. For each treatment, the tomato was inoculated with virus solution (virus inoculation concentration was 1.2×10 8 The number of virus copies / μL was 10 for each treatment, 4 leaves for each plant were inoculated with the virus, and 20μL of virus was inoculated for each leaf. The number of dead spots was counted 5 days after inoculation, and the inhibition rate was calculated. The third functional leaf on the upper part of the leaf was picked 7 days after inoculation, RNA was extracted, and the expression of the virus was detected by fluorescent quantitative PCR. At the same time, the tomato leaves with different treatments were disinfected on the surface, and the total number of culturable bacteria in the leaves was counted. The colony counting method was based on the determination method of effective live bacteria in agricultural microbial agents in GB20287-2006. The whole experiment was repeated 3 times. Inhibition rate = (number of control dead spots - number of treatment dead spots) / number of control dead spots × 100%.

[0074] Tomato plants of each treatment were taken separately, and the leaves at the same position on the top of the tomato were sampled at the 1st, 2nd, 3rd and 4th day after virus inoculation, and then quickly frozen in liquid nitrogen and stored at -80°C. According to the reference "Changes in the activity of defense enzymes in peppers infected with tomato spotted wilt virus", the ELISA method was used to determine the changes in the activities of superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) in the leaves of tomatoes of each treatment. The absorbance (OD value) of each treatment was measured in sequence at a wavelength of 450nm using an ELISA reader, and the activities of SOD, POD, CAT, PAL and PPO were calculated. The relationship between the resistance of tomatoes of different treatments to tomato brown wrinkled fruit virus (ToBRFV) and the activity of defense enzymes was clarified.

[0075] Table 7 Inhibitory effects of different treatments on tomato brown wrinkled fruit virus

[0076] 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 by treatment 3 (Bacillus Velez WF02 fermentation broth prepared in Example 7), treatment 4 (low-temperature resistant Bacillus WF04 fermentation broth prepared in Example 7), and treatment 5 (composite microbial agent prepared in Example 7) had a strong inhibitory effect on tomato brown wrinkled fruit virus, among which the composite microbial agent (treatment 5) of the present invention had the strongest inhibition rate on tomato brown wrinkled fruit virus, which could reach 87.62%.

[0077] The results of the detection of the number of culturable bacteria, virus content and the activities of four defense enzymes in the leaves showed (as shown in Table 7, Fig.10 As shown in the figure, the composite microbial agent (treatment 5) of the present invention can greatly increase the number of cultivable bacteria in tomato leaves and activate the activity of defense enzymes in the leaves; the composite microbial agent (treatment 5) of the present invention for treating tomato seeds can improve the bacterial community structure in the leaves, activate the activity of the plant system defense enzymes, and improve the resistance of tomato plants to viruses, thereby achieving the effect of inhibiting virus proliferation and reducing virus content.

[0078] Test Example 4: Field efficacy of different treatment agents against tomato brown wrinkled fruit virus This example provides the effects of the fermentation liquid of Bacillus Velezii WF02, the fermentation liquid of Bacillus pyrrotolerant WF04 and the composite microbial agent prepared in Example 7 on the prevention and control of tomato brown wrinkled fruit virus in greenhouse experiments. The experiment has 8 treatment groups. The treatment groups and test methods are as follows: Treatment 1: blank control, no treatment; Treatment 2: fermentation broth of Bacillus velez WF02 prepared in Example 7; Treatment 3: fermentation broth of the low-temperature resistant Bacillus WF04 prepared in Example 7; Treatment 4: the composite microbial agent prepared in Example 7; Treatment 5: the composite microbial agent prepared in Example 8; Treatment 6: the composite microbial agent prepared in Example 9; Treatment 7: fermentation broth of Bacillus velez CGMCC1.923 prepared in Control Example 1; Treatment 8: The composite microbial agent prepared in Control Example 1.

[0079] The experiment was carried out in Juneng Village, Weifang City, Shandong Province, and the experimental crop was tomato. The experimental greenhouse was a tomato greenhouse with brown wrinkled fruit virus disease and was not disinfected. The area was 72m 2, randomized block arrangement, repeated 4 times. The experimental plots had uniform fertility, consistent planting and management levels, and severe disease occurrence and damage. Tomato seeds of each treatment were raised as seedlings, and transplanted to the experimental greenhouse when they grew to 2 true leaves. Each treatment was irrigated with bacterial solution dilution (1.0×10 7 cfu / mL) 50mL / tree. Protective rows were set up between each treatment and around the test area. When the negative control group had obvious symptoms, the incidence rate was investigated. 50 plants were continuously investigated along the ridges in each plot, and the classification was recorded by plant, the disease index was calculated, and the prevention effect was calculated. The symptom classification standard of tomato brown wrinkled fruit virus disease investigated the diseased plants, calculated the disease index of each treatment and the relative prevention and control effect.

[0080] Tomato brown fruit virus disease symptom grading standard: Level 0: No symptoms; Level 1: The veins of the heart leaves are clear, and 1-2 true leaves are mosaic; Level 3: The leaves in the middle and upper parts are mosaic; Level 5: Most leaves are mosaic, and a few leaves are deformed; Level 7: Most leaves are severely mosaic, deformed, and wrinkled; Level 9: Almost all leaves are severely mosaic, deformed, and wrinkled, and the plant is obviously dwarfed. The calculation formula for the disease index and relative prevention effect is as follows: Disease index = ∑ (number of diseased plants at each level × value of the disease level) / (total number of plants surveyed × highest level value) × 100 Relative protective efficacy = (disease index of control - disease index of treatment) / disease index of control × 100%.

[0081] Table 8 Field efficacy of different treatments against tomato brown fruit virus disease

[0082] Tomatoes treated with different microbial agents all have a certain preventive effect on tomato brown wrinkled fruit virus occurring in the field. Treatment 2 (Bacillus Velez WF02 fermentation broth prepared in Example 7), treatment 3 (low-temperature resistant Bacillus WF04 fermentation broth 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) have 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) have a better preventive effect on tomato brown wrinkled fruit virus, and the relative preventive effect can reach 73.06% to 79.64%.

[0083] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached 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 Velez subtilis WF02 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on May 24, 2024, with a deposit number of CGMCC NO. 30756, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified and named as Velez subtilis Bacillus velezensis ; The refractory Bacillus WF04 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on May 24, 2024, with a deposit number of CGMCC NO. 30758, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified and named as refractory Bacillus Peribacillus frigoritolerans .

2. The composite microbial agent according to claim 1, characterized in that: The effective viable bacteria count of the composite microbial agent is 5.1×10 9 ~1.01×10 10 cfu / mL.

3. The composite microbial agent according to claim 1, characterized in that: The ratio between the effective viable bacteria count of the Velez subtilis WF02 and the effective viable bacteria count of the low-temperature resistant Bacillus WF04 in the composite microbial agent is 50-100:

1.

4. The composite microbial agent according to any one of claims 1 to 3, characterized in that: The Bacillus Velez WF02 bacterial agent, the low-temperature-resistant Bacillus WF04 bacterial agent and the composite microbial bacterial agent are respectively used in tomato planting.

5. The composite microbial agent according to any one of claims 1 to 3, characterized in that: The Bacillus Velez WF02 bacterial agent, the low-temperature-resistant Bacillus WF04 bacterial agent and the composite microbial bacterial agent are respectively used to prevent and control tomato brown wrinkled fruit virus disease.

6. The composite microbial agent according to any one of claims 1 to 3, characterized in that: The Bacillus Velez WF02 bacterial agent and the composite microbial bacterial agent are respectively used to induce tomato plant systemic defense against tomato brown wrinkled fruit virus.

7. The composite microbial agent according to any one of claims 1 to 3, characterized in that: The low-temperature resistant Bacillus WF04 is used to improve the resistance of tomato plants to tomato brown wrinkled fruit virus by promoting the rapid colonization of Bacillus Velez WF02 in tomato plants.

8. The composite microbial agent according to any one of claims 1 to 3, characterized in that: 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.

9. A method for preventing and controlling tomato brown wrinkled fruit virus disease, characterized in that: The composite microbial agent according to any one of claims 1 to 3 is applied to the rhizosphere soil of tomato seedlings.

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