Seedling raising method for improving disease resistance of crops by inoculating disease-preventing growth-promoting bacteria

Abstract

The invention relates to the technical field of tissue culture and microorganisms, in particular to a seedling culture method for improving disease resistance of crops by inoculating disease-preventing and growth-promoting bacteria, which comprises the following steps: step 1, culturing tissue culture seedlings to be inoculated with the disease-preventing and growth-promoting bacteria in a tissue culture manner; step 2, inoculating disease-preventing growth-promoting bacteria at the roots of the tissue culture seedlings; step 3, carrying out spot check to determine that the disease-preventing growth-promoting bacteria are successfully negatively colonized in the tissue culture seedling body, if colonization is successful, entering step 5, and if colonization is not successful, entering step 4 and then entering step 5; 4, when the disease-preventing growth-promoting bacteria are inoculated in the step 2, auxiliary bacteria are synchronously inoculated; 5, domesticating and transplanting the tissue culture seedlings successfully colonized with the disease-preventing growth-promoting bacteria; according to the method, disease-preventing growth-promoting bacteria can be colonized into tissue culture seedling bodies in advance, so that pathogenic bacteria entering crop bodies in the later period are reduced, and the purpose of disease-preventing seedling raising is achieved.

Description

Technical Field

[0001] The present invention relates to the technical fields of tissue culture and microbiology, and particularly relates to a seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops. Background Art

[0002] Plant virus diseases have always been a thorny problem in agricultural production, posing a serious threat to the yield and quality of crops. As important cash crops, tomatoes, peppers, ginger, etc. are vulnerable to a variety of viruses.

[0003] Tomato brown rugose fruit virus (ToBRFV) is a new virus that seriously harms solanaceous vegetable crops such as tomatoes and peppers. It has multiple transmission routes and strong economic harmfulness. In recent years, the virus has broken out comprehensively in the main production areas such as Shandong, Jiangsu, and Yunnan in China, seriously affecting the domestic production of tomatoes and peppers and the development of related industries, resulting in extremely heavy losses. The generation and research cycle of this virus is short. So far, there are no targeted resistant varieties and green control technologies in China. Therefore, creating resistant seedlings and establishing a green cultivation and comprehensive prevention and control system is an urgent problem for the planting industries of tomatoes, peppers, etc. in China.

[0004] Ginger uses rhizomes as propagation materials, with a low propagation coefficient, easy transmission of diseases through seed ginger, and long-term asexual reproduction causing degeneration of the variety and easy infection by viruses. In recent years, great progress has been made in the research on virus-free and bacteria-free tissue culture seedlings of ginger at home and abroad. Ginger tissue culture seedlings can solve the problem of viruses and various pathogenic bacteria carried in seed ginger, but it is difficult to solve the problem of quickly adapting to complex soil conditions and pathogenic bacteria infection when transplanting tissue culture seedlings into the natural environment. There is an urgent need to find a method for improving disease resistance that is compatible with tissue culture detoxification technology.

[0005] The limitations of traditional chemical control methods have prompted people to seek more green and effective prevention and control strategies. Therefore, plant tissue culture technology and microbial-induced antiviral technology have emerged. Among them, disease-preventing and growth-promoting bacteria can interact with plants, directly or indirectly help plants obtain nutrients, secrete growth-promoting substances to regulate the growth level of plants, improve the ability of plants to resist pathogenic bacteria, etc., and have become a hot spot in green prevention and control strategies. At present, the application of disease-preventing and growth-promoting bacteria mainly includes seed coating method, rhizosphere irrigation method, foliar spraying method, direct application method to soil (or cultivation substrate), etc., and the combination of tissue culture technology and disease-preventing and growth-promoting bacteria has not been reported yet. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops, that is, to organically combine tissue culture technology with disease-preventing and growth-promoting bacteria, and colonize the disease-preventing and growth-promoting bacteria in the bodies of crops such as tomatoes, peppers, and ginger in advance under sterile conditions, thereby improving the antiviral disease ability of tomato, pepper, and ginger plants and achieving the purpose of disease-resistant seedling raising.

[0007] To solve the above technical problems, the technical solution of the present invention is: Provide a seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops, including the following steps: Step 1, cultivate tissue culture seedlings to be inoculated with disease-preventing and growth-promoting bacteria by tissue culture; Step 2, inoculate the disease-preventing and growth-promoting bacteria at the roots of the tissue culture seedlings; Step 3, randomly inspect to determine whether the disease-preventing and growth-promoting bacteria have successfully colonized in the tissue culture seedlings. If the colonization is successful, enter Step 5; if the colonization is not successful, first enter Step 4 and then enter Step 5; Step 4, add auxiliary bacteria synchronously when inoculating the disease-preventing and growth-promoting bacteria in Step 2; Step 5, domesticate and transplant the tissue culture seedlings in which the disease-preventing and growth-promoting bacteria have successfully colonized.

[0008] As a preferred technical solution, the disease-preventing and growth-promoting bacteria are Serratia marcescens or Bacillus velezensis that are resistant to tomato brown rugose fruit virus disease; among them, The Serratia marcescens is Serratia marcescens WF01, which was deposited at the China General Microbiological Culture Collection Center on April 17, 2024, and its deposit number is: CGMCC No. 30368; The Bacillus velezensis is Bacillus velezensis WF02, which was deposited at the China General Microbiological Culture Collection Center on May 24, 2024, and its deposit number is: CGMCC No. 30756.

[0009] As a preferred technical solution, when the disease-preventing and growth-promoting bacteria are the Bacillus velezensis WF02, the auxiliary bacteria are Bacillus cereus WF04 that is resistant to low temperature. The Bacillus cereus WF04 was deposited at the China General Microbiological Culture Collection Center on May 24, 2024, and its deposit number is: CGMCC No. 30758.

[0010] As a preferred technical solution, the tissue culture seedlings in Step 1 are tomato tissue culture seedlings or pepper tissue culture seedlings, and Step 1 is specifically as follows: Step 1A: Disinfect plump, healthy, and pest-free tomato or pepper seeds under sterile conditions. Then spread the tomato or pepper seeds evenly on the surface of the solid medium for seed germination. When the cotyledons are fully expanded 6 - 8 days after seed germination, proceed to Step 1B. Step 1B: Cut the middle part of the cotyledons as explants with a size of 0.5 cm × 0.5 cm. Inoculate the explants on the surface of the induction medium based on Ms medium supplemented with 1 - 1.5 mg / L 6 - BA, 0.5 - 0.7 mg / L NAA, 30 - 50 g / L sucrose, and 6 - 8 g / L agar. Culture for 7 - 8 weeks to obtain callus and adventitious buds. When the adventitious buds grow to about 2 cm, proceed to Step 1C. Step 1C: Transfer the adventitious buds obtained in Step 1B to a medium based on Ms medium supplemented with 30 - 50 g / L sucrose and 6 - 8 g / L agar to induce rooting. After rooting, continue to culture for 10 days to obtain the tomato tissue culture seedlings or the pepper tissue culture seedlings.

[0011] As a preferred technical solution, the tissue culture seedlings in Step 1 are ginger tissue culture seedlings, and the ginger tissue culture seedlings are virus - free ginger tissue culture seedlings obtained through three stages of callus culture, multiple shoot proliferation culture, and rooting culture from the shoot tips of ginger buds.

[0012] As a preferred technical solution, Step 2 is specifically as follows: Inoculate the activated disease - preventing and growth - promoting bacteria into NB liquid medium, and culture with shaking at 28°C and 180 r / min until the optical density value of the culture solution reaches 0.8 - 1.0 to obtain a bacterial solution. Under sterile conditions, inoculate the bacterial solution onto the roots of the tissue culture seedlings in Step 1, and continue to culture for 24 h before proceeding to the operation of Step 3.

[0013] As a preferred technical solution, Step 3 is specifically as follows: Step 3A: Randomly select the tissue culture seedlings inoculated with the disease - preventing and growth - promoting bacteria in Step 2. Under sterile conditions, cut the leaves of the tissue culture seedlings, mash them, suck 50 μL of the juice, and evenly spread it on the surface of NA solid medium. Incubate the medium upside - down at 28°C for 24 h. Step 3B: Observe whether there are single colonies on the NA solid medium coated with leaf juice. If there are single colonies and the number of the same type of single colonies is not less than 50, compare the morphological characteristics of the selected single colonies with those of the original disease - preventing and growth - promoting bacteria single colonies. If the colony morphology comparison results are similar, proceed to Step 3C. Step 3C: Pick the test single colonies on the NA solid medium coated with leaf juice and inoculate them into NB liquid medium. Culture with shaking at 28°C and 180 r / min for 12 h to obtain a bacterial solution. Step 3D: Take the bacterial liquid, perform 16S rDNA gene sequencing, and compare the sequencing results with the gene sequence of the original disease-preventing and growth-promoting bacteria. If the gene sequencing results are consistent, it is determined that the colonization is successful; if the gene sequencing results are inconsistent, it is determined that the colonization is unsuccessful. If the colonization is successful, proceed to step five; if the colonization is unsuccessful, proceed to step four.

[0014] As a preferred technical solution, step four is specifically as follows: Inoculate the activated disease-preventing and growth-promoting bacteria and the auxiliary bacteria into NB liquid medium, and culture them with shaking at 28 °C and 180 r / min until the optical density value of the culture solution is 0.8 - 1.0 to obtain the disease-preventing and growth-promoting bacteria liquid and the auxiliary bacteria liquid. Under aseptic conditions, mix the disease-preventing and growth-promoting bacteria liquid and the auxiliary bacteria liquid evenly according to a volume ratio of 1:1, inoculate the mixed bacteria liquid onto the roots of the tissue culture seedlings in step one, and continue to culture for 24 h, then proceed to step five.

[0015] As a preferred technical solution, step five is specifically as follows: Step 5A: For the tissue culture seedlings with successful colonization of the disease-preventing and growth-promoting bacteria, acclimatize them with the tissue culture bottle cap half-opened in an incubator for 1 - 2 days, then fully open the tissue culture bottle cap and acclimatize for 1 - 2 days, and finally acclimatize in the natural environment for 1 - 2 days; Step 5B: Transplant the acclimatized tissue culture seedlings from the tissue culture bottle into a sterilized cultivation substrate and grow for 28 - 30 days to obtain the disease-resistant tissue culture seedlings.

[0016] The present invention also provides a seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of tomatoes or peppers, which is characterized by including the following steps: Step a: Under aseptic conditions, disinfect plump, healthy, and pest-free tomato or pepper seeds, and then spread the tomato seeds or the pepper seeds on the surface of the solid medium in a tissue culture bottle. After the seeds germinate for 10 days, obtain the seedlings; Step b: Inoculate the activated Serratia marcescens WF01 into NB liquid medium, and culture it with shaking at 28 °C and 180 r / min until the optical density value of the culture solution is 0.8 - 1.0 to obtain the bacterial liquid. Under aseptic conditions, inoculate the bacterial liquid onto the roots of the seedlings in step a and continue to culture for 24 h; Step c: Randomly select the seedlings inoculated with the Serratia marcescens WF01 in step b. Under sterile conditions, cut the leaves of the seedlings, mash them, suck 50 μL of the juice and evenly coat it on the NA solid medium, and incubate it upside down at 28 °C for 24 h; observe whether there are bright red single colonies on the NA solid medium coated with the leaf juice. If the colonization is not successful, there will be no bright red single colonies. If the colonization is successful, there will be bright red single colonies and no less than 50 single colonies of the same type. Domesticate and transplant the seedlings with successful colonization of the Serratia marcescens WF01.

[0017] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: By organically combining the crop tissue culture technology with the disease-preventing and growth-promoting bacteria, the disease-preventing and growth-promoting bacteria are colonized in the bodies of crops such as tomatoes, peppers, and ginger in advance under the condition of no interference from other microorganisms, which can not only promote the growth of tissue culture seedlings and improve the in-vivo directional inoculation efficiency of the disease-preventing and growth-promoting bacteria, but also improve the transplanting survival rate of tissue culture seedlings and the disease resistance of crops after transplantation; The Serratia marcescens WF01 or Bacillus velezensis WF02 can effectively improve the resistance of crop plants such as tomatoes to ToBRFV disease, achieving the purpose of disease-resistant ToBRFV seedling raising; For tomatoes and peppers, without using the tissue culture method, directly cultivating seedlings with seeds and inoculating disease-preventing and growth-promoting bacteria at the roots of the seedlings can also achieve successful colonization; The Serratia marcescens WF01 can secrete dark red pigments, and the used colonization detection method is simple and feasible. Brief Description of the Drawings

[0018] The following drawings are only intended to illustrate and explain the present invention schematically and do not limit the scope of the present invention. Among them: Figure 1 It is a flowchart of a seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops according to the present invention; Figure 2 It is a diagram of tomato tissue culture seedlings inoculated with Serratia marcescens WF01 in Example 1 of the present invention (right figure) and a diagram of control tomato tissue culture seedlings (left figure); Figure 3 It is a diagram of the colony detection culture results in the leaf juice of tomato tissue culture seedlings in Example 1 of the present invention; the left figure is a control diagram without inoculation with Serratia marcescens WF01; the right figure is a detection culture result diagram after inoculation with Serratia marcescens WF01; Figure 4 It is a diagram of the gene sequencing alignment results of Serratia marcescens WF01 in Example 4 of the present invention; Figure 5 It is a diagram of the effect comparison of tomato tissue culture seedlings against ToBRFV infection in Example 4 of the present invention; the left side is inoculated with Serratia marcescens WF01, and the right side is not inoculated with Serratia marcescens WF01; Figure 6 is Figure 5Partial enlarged view in the circle; the left side is the enlarged view of the left circle; the right side is the enlarged view of the right circle; Figure 7 It is the colony detection and culture result diagram of the leaves juice of tomato tissue culture seedlings in Example 5 of the present invention; the left figure is the control diagram without inoculating Bacillus velezensis WF02 and the auxiliary bacterium Bacillus cereus WF04 with low temperature tolerance; the right figure is the colony detection and culture result diagram after inoculating Bacillus velezensis WF02 and the auxiliary bacterium Bacillus cereus WF04 with low temperature tolerance, where the large single colony is Bacillus velezensis WF02 and the small single colony is the auxiliary bacterium Bacillus cereus WF04 with low temperature tolerance; Figure 8 It is the gene sequencing alignment result of Bacillus velezensis WF02 in Example 5 of the present invention; Figure 9 It is the effect comparison diagram of the anti - ToBRFV ability of tomato tissue culture seedlings in Example 5 of the present invention; among them, the left side is the infection diagram of tomato tissue culture seedlings inoculated with Bacillus velezensis WF02, and the right side is the infection diagram of tomato tissue culture seedlings not inoculated with Bacillus velezensis WF02; Figure 10 It is the colony growth diagram of the pathogen of ginger basal rot for 7 days in Example 8 of the present invention; Figure 11 It is the antagonistic effect diagram of Bacillus velezensis WF02 and the pathogen of ginger basal rot in Example 8 of the present invention; Figure 12 It is the colony detection and culture result diagram of the leaves juice of ginger tissue culture seedlings in Example 8 of the present invention; the left figure is the control diagram without inoculating Bacillus velezensis WF02; the right figure is the colony detection and culture result diagram after inoculating Bacillus velezensis WF02; Figure 13 It is the colony activation diagram of Bacillus velezensis WF02 of the present invention; Figure 14 It is the phylogenetic tree of Bacillus velezensis WF02 based on the 16S rDNA sequence of the present invention; Figure 15 It is the colony activation diagram of Bacillus cereus WF04 with low temperature tolerance of the present invention; Figure 16 It is the phylogenetic tree of Bacillus cereus WF04 with low temperature tolerance based on the 16S rDNA sequence of the present invention; Figure 17 It is the colony activation diagram of Serratia marcescens WF01 of the present invention; Figure 18 It is the phylogenetic tree of Serratia marcescens WF01 based on the 16S rDNA sequence of the present invention. Detailed implementation manners

[0019] To make the objectives and technical solutions of the present invention clearer and easier to understand, the present invention will be further described in detail below. Unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods; for those without specific technologies or conditions specified in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials can be obtained from commercial channels.

[0020] All the culture media without specific instructions involved in the following embodiments are standard culture media commonly used in this field. For example: 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, made up to 1000 mL with distilled water.

[0021] NB liquid medium: peptone 10.0 g, beef extract powder 3.0 g, sodium chloride 5.0 g, pH 7.0 - 7.2, made up to 1000 mL with distilled water.

[0022] Example 1: Screening and identification of Bacillus velezensis WF02 1. Strain screening It was isolated from healthy tomato plants collected from a greenhouse in the tomato-growing area of Anqiu City, Shandong Province where tomato brown rugose fruit virus occurred on a large scale. The sampling and isolation methods are as follows: Select healthy plants, dig out the entire root system intact with a sampling shovel, first rinse it thoroughly with tap water to remove surface attachments such as soil, then soak the roots, stems and leaves of the plants in 2% sodium hypochlorite in a laminar flow bench for 5 - 10 min, rinse with sterile water 3 - 5 times, then soak in 75% alcohol for 3 - 5 min, rinse with sterile water 3 - 5 times, then blot the surface moisture of the tomato roots, stems and leaves with sterile filter paper, and place the tomato roots, stems and leaves in a -80 °C ultra-low temperature environment for 30 d; put the tomato roots, stems and leaves into a sterilized mortar respectively, add 5 - 10 mL of 1×PBS buffer solution and grind evenly to obtain the sample stock solution. Gradient dilute the sample stock solution, and take 100 μL of the dilution solutions with dilution factors of 10 -2 、10 -3 、10 -4 and coat them on the NA solid medium, and incubate them upside down at 30 °C for 2 d, then pick the single colonies of the dominant bacteria to isolate and purify to obtain strain WF02.

[0023] 2. Strain identification Morphological identification was carried out on strain WF02, and the results are as follows: The colonies of strain WF02 on the NA solid medium are milky white, convex in the center, round, with wrinkled surfaces and opaque; Gram staining is positive, the cells are rod-shaped, not in chains, and produce spores, as shown in Figure 13 shown.

[0024] Molecular biological identification of strain WF02: Extract and prepare the total DNA of strain WF02 according to the conventional bacterial DNA extraction method. Use the universal bacterial primers 27f and 1492r for PCR amplification of the 16S rDNA gene. The PCR reaction system is as follows: 1 μl of DNA template, 25 μl of 2×MastarMix, 2 μl of 27f, 2 μl of 1492r, and make up to 50 μl with ultrapure water; after the amplification product is detected by 0.8% agarose gel electrophoresis, it is sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing.

[0025] The sequencing results of strain WF02 were compared on the NCBI and EzBio Cloud websites, and Mega 6.0 software was used for comparative analysis. At the same time, a phylogenetic tree was constructed by the neighbor-joining method (Neighbor-Joining, abbreviated as NJ). The 16S rDNA sequence alignment result of strain WF02 was (Bacillus velezensis 99% similar to that of Bacillus velezensis Figure 14 (as shown in Bacillus velezensis ); then strain WF02 was identified as Bacillus velezensis

[0026] The above-mentioned Bacillus velezensis WF02 was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on May 24, 2024, with the deposit number CGMCC NO. 30756 and the deposit address at No. 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name is Bacillus velezensis .

[0027] Example 2: Screening and identification of low-temperature tolerant Bacillus WF04 1. Strain screening It was isolated from the soil collected in the Ali area of Tibet with an average altitude of over 4500 meters. The isolation method is as follows: Adjust the collected soil to an appropriate moisture content, freeze it in an ultra-low temperature environment of -80°C for 35 days (during which freeze-thaw once every 7 days). Take 1 g of the soil after repeated freeze-thawing 5 times and place it in 100 mL of sterile water and shake for 20 min to obtain the sample stock solution. Gradient dilute the sample stock solution, and take 100 μL of the dilution solutions of 10 -3 , 10 -4 , 10 -5 and coat them on the NA solid medium, and incubate them upside down at 10°C for 2 days. Pick the single colonies of the dominant bacteria with rapid growth and isolate and purify to obtain strain WF04.

[0028] 2. Strain identification Identify the morphology of strain WF04, and the results are as follows: 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, do not form chains, and produce spores, as Figure 15 shown.

[0029] Molecular biological identification of strain WF04 was carried out: Total DNA of strain WF04 was extracted and prepared by the conventional bacterial DNA extraction method. PCR amplification of the 16S rDNA gene was carried out using the universal bacterial primers 27f and 1492r. The PCR reaction system was: 1 μl of DNA template, 25 μl of 2×MastarMix, 2 μl of 27f, 2 μl of 1492r, and made up to 50 μl with ultrapure water; the amplified product was detected by 0.8% agarose gel electrophoresis and then sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing.

[0030] The sequencing results of strain WF04 were compared on the NCBI and EzBio Cloud websites, and Mega 6.0 software was used for comparative analysis. At the same time, a phylogenetic tree was constructed by the neighbor-joining method (Neighbor-Joining, abbreviated as NJ). The 16S rDNA sequence comparison results of strain WF04 (as Figure 16 shown) were identified as Bacillus cereus group psychrotolerans (Peribacillus frigoritolerans ), named Bacillus cereus group psychrotolerans WF04.

[0031] The above-mentioned Bacillus cereus group psychrotolerans WF04 was deposited in the China General Microbiological Culture Collection Center 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 the taxonomic name is Peribacillus frigoritolerans .

[0032] Example 3, Screening and Identification of Serratia marcescens WF01 1. Strain Screening The initial strain of Serratia marcescens WF01 in this example was isolated from the leaves of wheat plants in saline-alkali land in Weifang, Shandong, where beta-cypermethrin has been used for a long time. The screening method is as follows: Select healthy and robust wheat plants, cut off the above-ground wheat plants with scissors, first rinse them with tap water to remove surface attachments such as soil, soak the wheat plant leaves in 3% sodium hypochlorite in a laminar flow hood for 8 - 10 min, rinse them 4 - 5 times with sterile water, then soak them in 75% alcohol for 4 - 5 min, after rinsing 4 - 5 times with sterile water, dry the surface moisture of the leaves with sterile filter paper, put the leaves into a sterilized mortar, add 5 - 10 ml of 1×PBS buffer (sterile) and grind to obtain the sample stock solution. Pipette 100 μL of the sample stock solution into a 250 mL Erlenmeyer flask containing 50 mL of culture medium (beta-cypermethrin 0.1 g / L, dipotassium hydrogen phosphate 5.8 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 1.0 g / L, distilled water 1 L, pH 7.0 - 7.2), shake and culture at 30 °C and 185 r / min for 48 h to obtain the enriched culture medium. Perform gradient dilution on the enriched culture medium, and spread 100 μL of the diluted solutions with dilution factors of 10 -5 、10 -6 、10 -7 uniformly on the NA solid medium (beef extract 5 g / L, peptone 10 g / L, sodium chloride 5 g / L, agar 20 g / L, pH 7.0 - 7.2) and incubate in an inverted position at 28 °C for 48 h, then pick out the grown red or pink single colonies (dominant colonies) for purification.

[0033] 2. Strain identification Perform morphological identification on the isolated strain. The results are as follows: After culturing the strain on the NA solid medium at 28 °C for 24 h, bright red colonies ( Figure 17 ) are formed, with a diameter of 2 - 4 mm. The colony surface is smooth and moist, the edge is neat, protruding and opaque. The bacteria are Gram-negative, with a morphology nearly spherical or short rod-shaped, 0.4 - 0.5 μm wide and 0.4 - 0.8 μm long.

[0034] Perform molecular biological identification on the isolated strain. The results are as follows: As Figure 18 shown, use the universal bacterial primers 27f and 1492r for 16S rDNA gene PCR amplification. The amplified product is sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing, and then the sequencing results are compared and analyzed on the NCBI and EzBio Cloud websites. After identification, this strain is Serratia marcescens, which is the initial strain of Serratia marcescens WF01.

[0035] Serratia marcescens WF01 was deposited in the China General Microbiological Culture Collection Center on April 17, 2024, with the deposit number CGMCC NO. 30368 and the deposit address at Beichen West Road, Chaoyang District, Beijing, and the taxonomic name is Serratia marcescens Serratia marcescens .

[0036] Example 4: Method for inoculating tomato tissue culture seedlings with Serratia marcescens WF01 to improve the ability of tomatoes to resist ToBRFV As Figure 1 shown, it includes the following steps: Step 1, disinfect and process tomato seeds, and germinate the seeds in a tissue culture bottle containing a basic medium to produce tomato tissue culture seedlings; The specific steps of Step 1 are as follows: Step 1A, disinfection and treatment of tomatoes: Under sterile conditions, disinfect plump, healthy, and pest-free tomato seeds. First, treat them with 75% alcohol for 30 - 60 s, wash them 3 times with sterile water, treat them with 3% - 5% NaClO for 8 - 10 min, and wash them 8 - 10 times with sterile water; spread the tomato seeds on the surface of a medium based on Ms supplemented with 30 - 50 g / L of sucrose and 6 - 8 g / L of agar, and wait for the seeds to germinate for 6 - 8 d until the cotyledons are fully unfolded; Step 1B, inducing callus and adventitious buds from tomato cotyledons: When the tomato cotyledons are fully unfolded, cut the middle section (0.5 cm × 0.5 cm) of the cotyledons as explants, and inoculate them on the surface of a medium based on Ms supplemented with 1 - 1.5 mg / L of 6 - BA, 0.5 - 0.7 mg / L of NAA, 30 - 50 g / L of sucrose, and 6 - 8 g / L of agar, and culture for 7 - 8 weeks; Step 1C, inducing the adventitious buds to root; inoculate the adventitious buds about 2 cm long into a medium based on Ms supplemented with 30 - 50 g / L of sucrose and 6 - 8 g / L of agar to induce rooting, and culture them into complete tomato tissue culture seedlings.

[0037] Step 2, inoculate Serratia marcescens WF01 at the roots of tomato tissue culture seedlings; The specific steps of Step 2 are as follows: Step 2A, activate Serratia marcescens WF01 on NA solid medium 2 days in advance, and culture it in an inverted position at 28°C for 24 h; the activated Serratia marcescens WF01 is as Figure 17 shown; Step 2B, pick a single colony of activated Serratia marcescens WF01 and inoculate it into NB liquid medium, and culture it at 28°C and 180 r / min with shaking until the optical density value (OD value) of the culture solution is 0.8 - 1.0 to obtain a bacterial solution; Step 2C, under sterile conditions, inoculate the bacterial solution at the roots of the tomato tissue culture seedlings in Step 1, and continue to culture for 24 h. As Figure 2 shown.

[0038] Step 3, detect whether Serratia marcescens WF01 has successfully colonized in the tomato tissue culture seedlings; The specific steps of Step 3 are as follows: Step 3A: Under sterile conditions, cut the leaves of the tomato tissue culture seedlings inoculated with Serratia marcescens WF01 in Step 2 and place them in a 1.5 mL sterile centrifuge tube; Step 3B: Mash the leaves with a sterile glass rod, aspirate 50 μL of the juice, and evenly spread it on the NA solid medium; Step 3C: Incubate the NA solid medium coated with the leaf juice at 28 °C for 24 h; Step 3D: Observe whether there are single colonies on the surface of the NA solid medium coated with the leaf juice. If there are single colonies and the number of the same type of single colonies is not less than 50, compare the morphological characteristics of the selected single colonies with those of the original Serratia marcescens WF01 single colonies.

[0039] Step 3E: If the results of the colony morphology comparison are similar, pick the single colonies to be tested from the NA solid medium coated with the leaf juice and inoculate them into the NB liquid medium, and culture them with shaking at 180 r / min for 12 h to obtain a bacterial solution; Step 3F: Take the bacterial solution, perform 16S rDNA gene sequencing, compare the sequencing results with the sequence of the original Serratia marcescens using DNAMAN software, and determine whether the colonization is successful based on the morphological and gene sequencing results. As Figure 3 shown, the morphological characteristics of the single colonies in the right figure are all similar to those of the original Serratia marcescens WF01 single colonies, and the color of the single colonies is bright red. As Figure 4 shown is the result of the gene sequencing alignment of Serratia marcescens. The alignment results are consistent, indicating that Serratia marcescens WF01 has successfully colonized in the tomato tissue culture seedlings. Then directly enter Step 5; Step 5: Domestication and transplantation of the tomato tissue culture seedlings with successful colonization of Serratia marcescens WF01.

[0040] The specific steps of Step 5 are as follows: Step 5A: Select the tomato tissue culture seedlings with successful colonization of Serratia marcescens WF01, domesticate them in the incubator with the tissue culture bottle cap half-open for 1 - 2 days, then with the cap fully open for 1 - 2 days, and finally in the natural environment for 1 - 2 days; Step 5B: Remove the domesticated tomato tissue culture seedlings from the tissue culture bottle, transplant them into the cultivation substrate, and grow for 30 days to obtain the tomato tissue culture seedlings colonized with Serratia marcescens WF01.

[0041] Perform the detection of resistance to ToBRFV on the tomato tissue culture seedlings in Step 5B. The detection method is as follows: (1) Cut 1 g of the leaves of the tomato plants identified as carrying ToBRFV by RT-PCR, grind them, and add 9 mL of 1xPBS to obtain a crude extract containing ToBRFV; (2)Filter the crude extract to remove the leaf residues in the crude extract, and obtain a tomato extract containing ToBRFV; (3)Gently rub the tomato leaves of the experimental group (tomato tissue culture seedlings colonized successfully by Serratia marcescens WF01) and the control group (tomato tissue culture seedlings not colonized by Serratia marcescens WF01) with sandpaper, dip a cotton swab in the tomato extract containing ToBRFV, and smear it on the rubbed parts of the leaves of the experimental group and the control group respectively; (4)After 8 d, it was observed that the leaves of the tomato tissue culture seedlings in the control group showed obvious leaf drying and yellowing; see Figure 5 the right picture in Figure 6 and Figure 5 the left picture in Figure 6 as shown in the left picture; while the leaves of the tomato tissue culture seedlings in the experimental group did not show obvious symptoms; see

[0042] Thus, it can be seen that Serratia marcescens WF01 colonizes the tomato in advance, enhances the ability of tomato tissue culture seedlings to resist ToBRFV infection, and further achieves the purpose of anti-ToBRFV seedling raising.

[0043] Example 5: Method for inoculating tomato tissue culture seedlings with Bacillus velezensis WF02 to improve the ability of tomatoes to resist ToBRFV As Figure 1 shown, it includes the following steps: Step 1, disinfect and process tomato seeds, and germinate the seeds in a tissue culture bottle containing a basic medium to produce tomato tissue culture seedlings; The specific steps of Step 1 are as follows: Step 1A, disinfection and treatment of tomatoes: Under sterile conditions, disinfect plump, healthy, and pest-free tomato seeds. First, treat them with 75% alcohol for 30 - 60 s, wash them 3 times with sterile water, treat them with 3% - 5% NaClO for 8 - 10 min, and wash them 8 - 10 times with sterile water; spread the tomato seeds on the surface of a medium based on Ms supplemented with 30 - 50 g / L sucrose and 6 - 8 g / L agar, and wait for the seeds to germinate for 6 - 8 d until the cotyledons are fully expanded; Step 1B, induce the cotyledons of tomatoes to form callus and adventitious buds: When the cotyledons of tomatoes are fully expanded, cut the middle section (0.5 cm × 0.5 cm) of the cotyledons as explants, and inoculate them on the surface of a medium based on Ms supplemented with 1 - 1.5 mg / L 6 - BA, 0.5 - 0.7 mg / L NAA, 30 - 50 g / L sucrose, and 6 - 8 g / L agar, and culture for 7 - 8 weeks; Step 1C, induce the adventitious buds to root; Inoculate the adventitious buds about 2 cm long into a medium based on Ms supplemented with 30 - 50 g / L sucrose and 6 - 8 g / L agar to induce rooting, and culture them into complete tomato tissue culture seedlings.

[0044] Step 2: Inoculate Bacillus velezensis WF02 at the roots of tomato tissue culture seedlings; The specific steps of Step 2 are as follows: Step 2A: Activate Bacillus velezensis WF02 on NA solid medium 2 days in advance, and incubate it in an inverted position at 28 °C for 24 h; Step 2B: Pick a single colony of activated Bacillus velezensis WF02 and inoculate it into NB liquid medium, and incubate it with shaking at 28 °C and 180 r / min until the optical density value (OD value) of the culture solution is 0.8 - 1.0 to obtain a bacterial solution; Step 2C: Under sterile conditions, inoculate the bacterial solution at the roots of the tissue culture seedlings in Step 1, and continue to culture for 24 h.

[0045] Step 3: Randomly check whether Bacillus velezensis WF02 has successfully colonized in tomato tissue culture seedlings; The specific steps of Step 3 are as follows: Step 3A: Under sterile conditions, cut the leaves of the tomato tissue culture seedlings inoculated with Bacillus velezensis WF02 in Step 2, and place them in a 1.5 mL sterile centrifuge tube; Step 3B: Mash the leaves with a sterile glass rod, suck 50 μL of the juice, and evenly spread it on the NA solid medium; Step 3C: Incubate the NA solid medium coated with leaf juice in an inverted position at 28 °C for 24 h; Step 3D: Observe whether there are single colonies on the surface of the NA solid medium coated with leaf juice. After detection, it is found that no colonies similar to the original Bacillus velezensis WF02 grow on the NA solid medium, and it is determined that Bacillus velezensis WF02 has not entered the tomato tissue culture seedlings, so enter Step 4.

[0046] Step 4: Inoculate Bacillus velezensis WF02 and the auxiliary bacterium Bacillus cereus WF04 that is resistant to low temperature at the roots of tomato tissue culture seedlings; The specific steps of Step 4 are as follows: Step 4A: Activate Bacillus velezensis WF02 (accession number: CGMCC No. 30756) and the auxiliary bacterium Bacillus cereus WF04 that is resistant to low temperature (accession number: CGMCC No. 30758) on NA solid medium 2 days in advance, and incubate them in an inverted position at 28 °C for 24 h; As Figure 13 Shown is the activation diagram of Bacillus velezensis WF02; Figure 15 Shown is the activation diagram of the auxiliary bacterium Bacillus cereus WF04 that is resistant to low temperature.

[0047] Step 4B: Pick single colonies of the activated Bacillus velezensis WF02 and the cold-tolerant Bacillus sp. WF04 respectively and inoculate them into NB liquid medium. Incubate with shaking at 28 °C and 180 r / min until the optical density (OD value) of the culture broth reaches 0.8 - 1.0 to obtain the bacterial solution. Step 4C: Mix the NB liquid medium inoculated with Bacillus velezensis WF02 and the cold-tolerant Bacillus sp. WF04 according to a volume ratio of 1:1. Under sterile conditions, inoculate the mixed bacterial solution onto the roots of the tissue-cultured seedlings in Step 1 and continue culturing for 24 h.

[0048] Then proceed to Step 3 to detect whether Bacillus velezensis WF02 has successfully colonized in the tomato tissue-cultured seedlings. The specific steps of Step 3 are as follows: Step 3A: Under sterile conditions, cut the leaves of the tomato tissue-cultured seedlings inoculated with Bacillus velezensis WF02 and the auxiliary cold-tolerant Bacillus sp. WF04 in Step 2 and place them in a 1.5 mL sterile centrifuge tube. Step 3B: Mash the leaves with a sterile glass rod, aspirate 50 μL of the juice, and evenly spread it on the NA solid medium. Step 3C: Invert and culture the NA solid medium coated with the leaf juice at 28 °C for 24 h. Step 3D: Observe whether there are single colonies on the surface of the NA solid medium coated with the leaf juice. If there are single colonies and the number of the same type of single colonies is not less than 50, compare the morphology of the selected single colonies with that of the original single colonies of Bacillus velezensis WF02.

[0049] Step 3E: If the results of the colony morphology comparison are similar, pick the single colonies of the Bacillus velezensis to be tested from the NA solid medium coated with the leaf juice and inoculate them into NB liquid medium. Incubate with shaking at 28 °C and 180 r / min for 12 h to obtain the bacterial solution. Step 3F: Take the bacterial solution for 16S rDNA gene sequencing. Compare the sequencing results with the sequence of the original Bacillus velezensis using DNAMAN software, and judge whether the colonization is successful based on the morphological and gene sequencing results.

[0050] As Figure 7 shown, large single colonies with a morphology similar to that of the original single colonies of Bacillus velezensis WF02 were found in the right figure, and the number of the same type of single colonies was not less than 50. As Figure 8 shown is the result of the gene sequencing alignment of Bacillus velezensis. The two gene sequencing results are consistent, indicating that Bacillus velezensis WF02 has successfully colonized in the tomato tissue-cultured seedlings, and directly proceed to Step 5.

[0051] Step 5: Domestication and transplantation of tomato tissue culture seedlings colonized successfully by Bacillus velezensis WF02.

[0052] The specific steps of Step 5 are as follows: Step 5A: Select tomato tissue culture seedlings colonized successfully by Bacillus velezensis WF02, domesticate them with the tissue culture bottle caps half-opened in an incubator for 1 - 2 days, then domesticate them with the tissue culture bottle caps fully opened in the incubator for 1 - 2 days, and finally domesticate them in the external natural environment for 1 - 2 days; Step 5B: Remove the domesticated tomato tissue culture seedlings from the tissue culture bottles, transplant them into nutrient soil, and grow for 30 days to obtain tomato tissue culture seedlings colonized successfully by Bacillus velezensis WF02.

[0053] Detect the tomato tissue culture seedlings in Step 5B, and the detection method is as follows: (1) Cut 1 g of leaves of tomato plants identified as carrying ToBRFV by RT-PCR, grind them, and add 9 mL of 1xPBS to obtain a crude extract containing ToBRFV; (2) Filter the crude extract to remove the leaf residues in the crude extract to obtain a tomato extract containing ToBRFV; (3) Gently rub the tomato leaves of the experimental group (tomato tissue culture seedlings colonized successfully by Bacillus velezensis) and the control group (tomato tissue culture seedlings not colonized by Bacillus velezensis) with sandcloth, dip a cotton swab in the tomato extract containing ToBRFV, and smear it on the rubbed parts of the leaves of the experimental group and the control group respectively; (4) After 8 days, it is observed that the leaves of the tomato tissue culture seedlings in the control group show obvious leaf withering and yellowing, as shown in the right figure in Figure 9 ; while the leaves of the tomato tissue culture seedlings in the experimental group do not show obvious symptoms, as shown in the left figure in Figure 9 .

[0054] Thus, Bacillus velezensis WF02 colonizes the tomato body in advance under the action of the auxiliary bacterium Bacillus cereus group WF04 that is resistant to low temperature, enhances the ability of tomato tissue culture seedlings to resist ToBRFV infection, and further achieves the purpose of anti-ToBRFV seedling raising.

[0055] Example 6: Growth-promoting effect of inoculating Serratia marcescens WF01 and Bacillus velezensis WF02 on tomato seedlings Select tomatoes of the variety Provence as the experimental materials.

[0056] (1) Under sterile conditions, disinfect plump, healthy, and pest-free tomato seeds: first treat them with 75% alcohol for 30 - 60 s, wash them 3 times with sterile water, treat them with 3% - 5% NaClO for 10 min, and wash them 10 times with sterile water; (2)Sow the disinfected seeds into flower pots filled with sterilized seedling substrate, with 3 tomato seeds in each pot, 5 replicates for each treatment, and raise seedlings in a light incubator room; (3)When the seeds germinate and grow to the stage of two leaves and one heart, pour 10 mL / plant of the fermentation broth of Serratia marcescens WF01 and Bacillus velezensis WF02 at the roots of the tomato plants, and conduct secondary irrigation after 7 days. The control group is irrigated with the same volume of sterile water; (4)Observe the growth of tomato plants under different treatments throughout the growth period. After 20 days, count the plant height, stem diameter, leaf area, and fresh weight indexes of tomatoes under different treatments. The statistical results are shown in Table 1: Table 1 Effects of Serratia marcescens WF01 and Bacillus velezensis WF02 on the growth of tomato plants

[0057] As can be seen from Table 1, after the fermentation broth of Serratia marcescens WF01 and Bacillus velezensis WF02 is poured at the roots of tomato seedlings, the plant height and stem diameter are greatly increased, and the leaf area and fresh weight are also significantly increased. It can be seen that inoculating Serratia marcescens WF01 and Bacillus velezensis WF02 has a growth-promoting effect on tomato seedlings.

[0058] Example 7: Inhibitory effect of inoculating Serratia marcescens WF01 and Bacillus velezensis WF02 on tomato brown rugose fruit virus Treatment 1: Control (virus negative), without any treatment; Treatment 2: Control (virus positive), inoculated with virus; Treatment 3: Inoculated with Serratia marcescens WF01 bacterial solution with OD600 = 0.85 + virus; Treatment 4: Inoculated with a mixed bacterial solution of Bacillus velezensis WF02 with OD600 = 0.82 and Bacillus cereus WF04 with OD600 = 0.86 (volume ratio 1:1) + virus; Adopt the whole-leaf inoculation method

[0059] (1)Under sterile conditions, disinfect plump, healthy, and pest-free tomato seeds: first treat with 75% alcohol for 30 - 60 s, wash 3 times with sterile water, treat with 3% - 5% NaClO for 10 min, and wash 10 times with sterile water; (2)Sow the sterile seeds into flower pots filled with sterilized seedling substrate, with 3 tomato seeds in each pot, 5 replicates for each treatment, and raise seedlings in a light incubator room.

[0060] (3)When the tomatoes grow to the 5 true leaf stage, rub and inoculate the virus solution (virus inoculation concentration is 1.2×10 8(copies / μL). For each treatment, 10 plants were inoculated, and 4 leaves of each plant were inoculated with the virus, with 20 μL of the virus inoculated on each leaf. After 5 days of inoculation, the number of necrotic spots was counted, and the inhibition rate was calculated as follows: Inhibition rate = (number of necrotic spots in the control - number of necrotic spots in the treatment) / number of necrotic spots in the control × 100%.

[0061] (4)Three days after inoculation, the third functional leaf from the upper part of the leaf was picked, RNA was extracted, and the expression level of the virus was detected by fluorescence quantitative PCR.

[0062] The inhibitory effects of Serratia marcescens WF01 and Bacillus velezensis WF02 on tomato plants ToBRFV are shown in Table 2.

[0063] Table 2 Inhibitory effects of Serratia marcescens WF01 and Bacillus velezensis WF02 on tomato plants ToBRFV

[0064] As can be seen from Table 2, the virus inhibition rates calculated from the number of necrotic spots on tomato leaves indicate that Serratia marcescens WF01 and Bacillus velezensis WF02 have strong inhibitory effects on the infection of ToBRFV; compared with the positive control (treatment 2), the virus content in the leaves of tomato plants treated with Serratia marcescens WF01 and Bacillus velezensis WF02 decreased to a certain extent, indicating that Serratia marcescens WF01 and Bacillus velezensis WF02 have significant antagonistic inhibitory effects on ToBRFV.

[0065] Example 8: Inoculating tissue-cultured ginger seedlings with Bacillus velezensis WF02 to improve the ability of ginger to resist ToBRFV; It includes the following steps: Step 1: Cultivate the required tissue-cultured ginger seedlings from the shoot tips of ginger, specifically as follows: Step 1A, Select plump, large, and pest-free ginger, wash the surface soil with tap water, put it in a mesh bag and expose it to the sun for 3 - 4 days, and then carry out sprouting. When the ginger buds grow to 2 - 3 cm, treat them at a high temperature of 55 °C for 5 minutes and place them in a laminar flow hood; Step 1B, disinfect the ginger buds selected in step 1A: soak in 70% alcohol for 15-30 seconds, rinse with sterile water 3 times, disinfect with 0.1% mercuric chloride solution for 8 minutes, and rinse with sterile water 5-6 times. Use sterile filter paper to absorb the moisture on the surface of the stem tip, peel off the stem tip to leave only 1-2 leaf primordia, inoculate in the stem tip callus formation medium, and culture the stem tip callus tissue; wherein, the stem tip callus formation medium is based on Ms, supplemented with 1-1.5 mg / L 6-BA, 0.1-0.2 mg / L NAA, 30 g / L sucrose and 5 g / L agar, and the pH value is adjusted to 5.8-6.0 with NaOH or HCl, and cultured for 4-5 weeks (culture conditions: light intensity 4000lx, light time 16h / d, culture temperature 25°C, relative humidity maintained at 60%-80%); Step 1C, inoculating the shoot apex callus cultured in step 1B into a shoot apex cluster bud proliferation medium to culture cluster buds; wherein the shoot apex cluster bud proliferation medium is based on Ms as a basic medium, supplemented with 2-3 mg / L 6-BA, 0.2-0.3 mg / L NAA, 30 g / L sucrose and 5 g / L agar, and culturing for 8-9 weeks; Step 1D, dividing the clustered buds cultured in step 1C into individual plants, transferring them to a rooting medium, and culturing complete ginger tissue culture seedlings, wherein the rooting medium is based on Ms medium supplemented with 0.1 mg / L NAA, 30 g / L sucrose and 5 g / L agar, and culturing at 28° C. for 15 days; Step 1E, extracting RNA from ginger tissue culture seedling leaves, detecting RNA extraction results by PCR, directly performing reverse transcription, and detecting the detoxification of ginger leaves by RT-PCR to ensure that the ginger tissue culture seedlings are successfully detoxified; Check whether the ginger tissue culture seedlings are successfully detoxified: RNA was extracted from the leaves of ginger tissue culture seedlings, and reverse transcription was performed to obtain cDNA. RT-PCR was used to detect whether the ginger tissue culture seedlings contained tobacco mosaic virus and cucumber mosaic virus. The ginger tissue culture seedlings obtained by ginger stem tip tissue culture were all successfully virus-free. The successfully virus-free tissue culture seedlings were the ginger tissue culture seedlings to be inoculated with disease prevention and growth-promoting bacteria.

[0066] Step 2: Inoculate the roots of ginger tissue culture seedlings with Bacillus Velez WF02 as follows: Step 2A: Activate Bacillus Velezii WF02 on NA solid medium 2 days in advance and culture at 28°C for 24 h. Figure 13 As shown; Step 2B, picking a single colony of the activated Bacillus Velezii WF02 and inoculating it into NB liquid culture medium, shaking and culturing at 28° C. and 180 r / min until the optical density (OD value) of the culture solution is 0.8-1.0, thereby obtaining a bacterial solution; Step 2C: Under aseptic conditions, inoculate the bacterial solution onto the roots of the ginger tissue culture seedlings in Step 1 and continue culturing for 24 h.

[0067] Step 3: Bacillus velezensis WF02 colonizes in the ginger tissue culture seedlings, specifically as follows: Step 3A: Under aseptic conditions, cut the leaves of the ginger tissue culture seedlings inoculated with Bacillus velezensis WF02 in Step 2 and place them in a 1.5 mL sterile centrifuge tube. Step 3B: Mash the leaves with a sterile glass rod, aspirate 50 μL of the juice, and evenly spread it on the NA solid medium. Step 3C: Invert and culture the NA solid medium coated with the leaf juice at 28 °C for 24 h. Step 3D: Observe whether there are single colonies on the surface of the NA solid medium coated with the leaf juice. If there are single colonies and the number of the same type of single colonies is not less than 50, compare the morphology of the selected single colonies with that of the original Bacillus velezensis WF02 single colonies. If the morphology is consistent and there are no colonies in the control, it is determined that Bacillus velezensis WF02 has successfully colonized in the ginger tissue culture seedlings.

[0068] Step 5: Domestication and transplantation of the ginger tissue culture seedlings in which Bacillus velezensis WF02 has successfully colonized are carried out. Specifically as follows: Step 5A: Select the ginger tissue culture seedlings in which Bacillus velezensis WF02 has successfully colonized, half-open the tissue culture bottle cap in the incubator for 1 - 2 d for domestication, then fully open the tissue culture bottle cap in the incubator for 1 - 2 d for domestication, and finally domesticate in the external natural environment for 1 - 2 d. Step 5B: Remove the domesticated ginger tissue culture seedlings from the tissue culture bottle, transplant them into the cultivation substrate, and grow for 28 d to obtain the ginger tissue culture seedlings in which Bacillus velezensis WF02 has successfully colonized.

[0069] In order to test the disease resistance effect of Bacillus velezensis WF02, the following test is carried out with the stem rot pathogen: Figure 10 It is the colony map of the ginger basal rot pathogen growing for 7 d; Figure 11 It is the antagonistic effect diagram of Bacillus velezensis WF02 and the ginger stem rot pathogen; From Figure 10 、 11 It can be seen that Bacillus velezensis WF02 has a strong antagonistic effect on the ginger stem rot pathogen. Figure 12 It is the detection result diagram of the Bacillus velezensis WF02 colonies in the leaf juice of the ginger tissue culture seedlings. The left figure is the control diagram without inoculation of Bacillus velezensis WF02, and the right figure is the colony detection result diagram after inoculation of Bacillus velezensis WF02; From Figure 12It can be seen that Bacillus velezensis WF02 colonizes in the virus-free seedlings during the tissue culture stage of ginger, without the interference of other bacteria; this method for detecting the colonization of Bacillus velezensis WF02 is simple and does not require prior labeling or antibiotic domestication of Bacillus velezensis WF02.

[0070] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops, characterized in that: The steps include: Step 1: Cultivating the tissue culture seedlings to be inoculated with disease-preventing and growth-promoting bacteria by tissue culture; Step 2, inoculating the disease-preventing and growth-promoting bacteria at the roots of the tissue culture seedlings; Step 3: random inspection to determine whether the disease prevention and growth promotion bacteria have successfully colonized in the tissue culture seedlings. If colonization is successful, proceed to step 5; if colonization is unsuccessful, proceed to step 4 first and then step 5; Step 4, adding auxiliary bacteria simultaneously with inoculating the disease-preventing and growth-promoting bacteria in step 2; Step five, domesticating and transplanting the tissue culture seedlings that have been successfully colonized with the disease-preventing and growth-promoting bacteria.

2. A seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops as claimed in claim 1, characterized in that: The disease-preventing and growth-promoting bacteria are Serratia marcescens or Bacillus velezine that are resistant to tomato brown wrinkled fruit virus disease; wherein, The Serratia marcescens is Serratia marcescens WF01, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 17, 2024, and its deposit number is: CGMCC No.30368; The Bacillus Velez is Bacillus Velez WF02, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 24, 2024, and its deposit number is: CGMCC No.30756.

3. A method for raising seedlings by inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops as claimed in claim 2, characterized in that: When the disease-preventing and growth-promoting bacteria is the Bacillus Velez subtilis WF02, the auxiliary bacteria is the psychrophilic Bacillus WF04. The psychrophilic Bacillus WF04 was deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration on May 24, 2024, and its deposit number is: CGMCC No.30758.

4. A seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops as claimed in claim 1, characterized in that: The tissue culture seedlings in step 1 are tomato tissue culture seedlings or pepper tissue culture seedlings, and the specific details of step 1 are as follows: Step 1A, sterilize plump, healthy, and pest-free tomato or pepper seeds under sterile conditions, then spread the tomato or pepper seeds on the surface of a solid seed germination medium, and proceed to step 1B 6 to 8 days after the seeds germinate and the cotyledons are fully expanded; Step 1B, cutting the middle section of the cotyledon as an explant, the explant size is 0.5 cm×0.5 cm, inoculating it on the surface of an induction medium with Ms as the basic medium, supplemented with 1-1.5 mg / L 6-BA, 0.5-0.7 mg / L NAA, 30-50 g / L sucrose and 6-8 g / L agar, culturing for 7-8 weeks to obtain callus tissue and adventitious buds, and performing step 1C when the adventitious buds grow to about 2 cm; Step 1C, inoculating the adventitious buds obtained in step 1B into a culture medium with Ms as the basic culture medium and supplemented with 30-50 g / L sucrose and 6-8 g / L agar to induce rooting, and continuing to culture for 10 days after rooting to obtain the tomato tissue culture seedlings or the pepper tissue culture seedlings.

5. A method for raising seedlings by inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops as claimed in claim 1, characterized in that: The tissue culture seedlings in step one are ginger tissue culture seedlings, which are ginger tissue culture seedlings that are successfully detoxified by successively undergoing three stages of callus culture, bud proliferation culture, and rooting culture of ginger bud stem tips.

6. A seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops as claimed in claim 1, characterized in that: The step 2 is specifically as follows: The activated disease-preventing and growth-promoting bacteria are inoculated into NB liquid culture medium, and cultured at 28°C and 180 r / min with shaking until the optical density of the culture solution is 0.8-1.0 to obtain a bacterial solution; under sterile conditions, the bacterial solution is inoculated into the roots of the tissue culture seedlings in step one, and the culture is continued for 24 hours before performing step three.

7. A seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops as claimed in claim 1, characterized in that: The step three is as follows: Step 3A, randomly extract the tissue culture seedlings inoculated with the disease prevention and growth promotion bacteria in step 2, cut the leaves of the tissue culture seedlings under sterile conditions, mash them, absorb 50uL of juice and evenly spread it on the NA solid culture medium, and invert and culture at 28°C for 24h; Step 3B, observe whether there are single colonies on the NA solid culture medium coated with leaf juice. If there are single colonies and there are no less than 50 single colonies of the same type, compare the selected single colony morphology with the single colony morphology of the original disease prevention and growth promotion bacteria. If the colony morphology comparison results are similar, proceed to step 3C; Step 3C, pick a single bacterial colony to be tested from the NA solid medium coated with leaf juice and inoculate it into the NB liquid medium, and culture it at 28°C and 180 rpm for 12 h to obtain a bacterial solution; Step 3D, taking the bacterial liquid, performing 16S rDNA gene sequencing, and comparing the sequencing results with the gene sequence of the original disease-preventing and growth-promoting bacteria. If the gene sequencing results are consistent, it is determined that the colonization is successful, and if the gene sequencing results are inconsistent, it is determined that the colonization is unsuccessful; if the colonization is successful, proceed to step five, and if the colonization is unsuccessful, proceed to step four.

8. The method for raising seedlings by inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops as claimed in claim 1, characterized in that: The step 4 is specifically as follows: The activated disease-preventing and growth-promoting bacteria and the auxiliary bacteria are inoculated into NB liquid culture medium, and cultured at 28°C and 180r / min with shaking until the optical density of the culture solution is 0.8-1.0 to obtain a disease-preventing and growth-promoting bacteria solution and an auxiliary bacteria solution; under sterile conditions, the disease-preventing and growth-promoting bacteria solution and the auxiliary bacteria solution are evenly mixed in a volume ratio of 1:1, and the mixed solution is inoculated into the roots of the tissue culture seedlings in step one, and the culture is continued for 24 hours before performing step five.

9. A seedling raising method for improving crop disease resistance by inoculating disease-preventing and growth-promoting bacteria as claimed in claim 1, characterized in that: The step five is as follows: Step 5A, the tissue culture seedlings successfully colonized with the disease prevention and growth promotion bacteria are acclimated in an incubator with the tissue culture bottle cap half-opened for 1 to 2 days, then acclimated with the tissue culture bottle cap fully opened for 1 to 2 days, and finally acclimated in a natural environment for 1 to 2 days; Step 5B, transplanting the acclimated tissue culture seedlings from the tissue culture bottle into a cultivation medium, growing them for 28 to 30 days, and obtaining the disease-resistant tissue culture seedlings.

10. A seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of tomatoes or peppers, characterized in that: The steps include: Step a: sterilizing plump, healthy, and pest-free tomato or pepper seeds under sterile conditions, and then spreading the tomato seeds or pepper seeds on the surface of the solid culture medium in the tissue culture bottle, and obtaining seedlings after the seeds germinate for 10 days; Step b: inoculating the activated Serratia marcescens WF01 into NB liquid culture medium, culturing at 28° C. and 180 r / min with shaking until the optical density of the culture solution is 0.8-1.0, to obtain a bacterial solution; inoculating the bacterial solution into the roots of the seedlings in step a under sterile conditions, and continuing to culture for 24 hours; Step c: randomly extract the seedlings inoculated with the Serratia marcescens WF01 in step b, cut the leaves of the seedlings under sterile conditions, mash them, absorb 50uL of juice and evenly spread them on the NA solid culture medium, and invert and culture for 24 hours at 28°C; observe whether there are bright red single colonies on the NA solid culture medium coated with leaf juice, if the colonization is unsuccessful, there are no bright red single colonies, if the colonization is successful, there are bright red single colonies and the number of single colonies of the same type is not less than 50, and the seedlings successfully colonized with the Serratia marcescens WF01 are acclimated and transplanted.

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