A seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops

Abstract

The present invention relates to the fields of tissue culture and microbial technology, and particularly to a seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops, comprising the following steps: Step 1, culturing tissue culture seedlings to be inoculated with disease-preventing and growth-promoting bacteria by means of tissue culture; Step 2, inoculating disease-preventing and growth-promoting bacteria at the roots of the tissue culture seedlings; Step 3, randomly inspecting to determine whether the disease-preventing and growth-promoting bacteria have successfully colonized in the tissue culture seedlings. If the colonization is successful, proceed to Step 5. If the colonization is unsuccessful, first proceed to Step 4 and then to Step 5; Step 4, synchronously inoculating auxiliary bacteria when inoculating the disease-preventing and growth-promoting bacteria in Step 2; Step 5, domesticating and transplanting the tissue culture seedlings in which the disease-preventing and growth-promoting bacteria have successfully colonized; The present invention can colonize the disease-preventing and growth-promoting bacteria in the tissue culture seedlings in advance, reduce the entry of late pathogens into the crop body, and achieve the purpose of disease-resistant seedling raising.

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 various 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, this 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, with 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 are urgent problems in 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 applications of disease-preventing and growth-promoting bacteria mainly include 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, so as to improve the antiviral disease ability of tomato, pepper and ginger plants and achieve the purpose of disease-resistant seedling raising.

[0007] To solve the above technical problem, the technical solution of the present invention is:

[0008] Provide a seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the disease resistance of crops, including the following steps:

[0009] Step 1, cultivate tissue culture seedlings to be inoculated with disease-preventing and growth-promoting bacteria by means of tissue culture;

[0010] Step 2, inoculate the disease-preventing and growth-promoting bacteria at the roots of the tissue culture seedlings;

[0011] Step 3, randomly check to determine whether the disease-preventing and growth-promoting bacteria are 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;

[0012] Step 4, add auxiliary bacteria synchronously when inoculating the disease-preventing and growth-promoting bacteria in Step 2;

[0013] Step 5, domesticate and transplant the tissue culture seedlings in which the disease-preventing and growth-promoting bacteria are successfully colonized.

[0014] 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; among them,

[0015] 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;

[0016] 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.

[0017] As a preferred technical solution, when the disease-preventing and growth-promoting bacteria are 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.

[0018] As a preferred technical solution, the tissue culture seedlings in step one are tomato tissue culture seedlings or pepper tissue culture seedlings, and step one is specifically as follows:

[0019] Step 1A, under aseptic conditions, disinfect plump, healthy, and pest-free tomato or pepper seeds, and then spread the tomato or pepper seeds on the surface of the seed germination solid medium. When the cotyledons are fully unfolded 6 - 8 days after seed germination, proceed to step 1B;

[0020] Step 1B, cut the middle section of the cotyledon as the explant, with the size of the explant being 0.5 cm × 0.5 cm, and inoculate it onto 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;

[0021] 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.

[0022] As a preferred technical solution, the tissue culture seedlings in step one are ginger tissue culture seedlings, and the ginger tissue culture seedlings are virus-free ginger tissue culture seedlings obtained by successively passing through three stages of callus culture, multiple shoot proliferation culture, and rooting culture from the shoot tip of ginger buds.

[0023] As a preferred technical solution, step two is specifically as follows:

[0024] 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 aseptic conditions, inoculate the bacterial solution onto the roots of the tissue culture seedlings in step one, and continue to culture for 24 h before proceeding with the operation of step three.

[0025] As a preferred technical solution, step three is specifically as follows:

[0026] Step 3A, randomly select the tissue culture seedlings inoculated with the disease-preventing and growth-promoting bacteria in step two, cut the leaves of the tissue culture seedlings under aseptic conditions, mash them, suck 50 μL of the juice and evenly spread it on the surface of NA solid medium, and culture in an inverted position at 28°C for 24 h;

[0027] Step 3B: Observe whether there are single colonies on the NA solid medium coated with leaf sap. 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 disease-preventing and growth-promoting bacteria single colonies. If the colony morphology comparison results are similar, proceed to Step 3C;

[0028] Step 3C: Pick the single colonies to be tested from the NA solid medium coated with leaf sap and inoculate them into NB liquid medium. Cultivate them at 28 °C with shaking at 180 r / min for 12 h to obtain bacterial solutions;

[0029] Step 3D: Take the bacterial solutions and perform 16S rDNA gene sequencing. Compare the sequencing results with the gene sequences 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.

[0030] As a preferred technical solution, Step Four is specifically as follows:

[0031] Inoculate the activated disease-preventing and growth-promoting bacteria and the auxiliary bacteria into NB liquid medium. Cultivate them at 28 °C with shaking at 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 solution and the auxiliary bacteria solution. Under sterile conditions, mix the disease-preventing and growth-promoting bacteria solution and the auxiliary bacteria solution evenly according to the volume ratio of 1:1, inoculate the mixed bacteria solution to the roots of the tissue culture seedlings in Step One, and continue to cultivate for 24 h, then proceed to Step Five.

[0032] As a preferred technical solution, Step Five is specifically as follows:

[0033] Step 5A: For the tissue culture seedlings with successful colonization of the disease-preventing and growth-promoting bacteria, acclimatize them in the tissue culture bottle with the cap half-open for 1 - 2 d in the incubator, then acclimatize them with the cap fully open for 1 - 2 d, and finally acclimatize them in the natural environment for 1 - 2 d;

[0034] Step 5B: Transplant the acclimatized tissue culture seedlings from the tissue culture bottle into the sterilized cultivation substrate and grow for 28 - 30 d to obtain the disease-resistant tissue culture seedlings.

[0035] 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:

[0036] Step a: Under sterile conditions, disinfect the 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 the tissue culture bottle. After the seeds germinate for 10 d, obtain the seedlings;

[0037] 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 reaches 0.8 - 1.0 to obtain a bacterial solution; under aseptic conditions, inoculate the bacterial solution onto the roots of the seedlings in step a, and continue culturing for 24 h;

[0038] Step c: Randomly select the seedlings inoculated with Serratia marcescens WF01 in step b, cut the leaves of the seedlings under aseptic conditions, mash them, suck 50 μL of the juice and evenly spread it on NA solid medium, and culture it upside down at 28°C for 24 h; observe whether there are bright red single colonies on the NA solid medium coated with 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 the number of the same type of single colonies is not less than 50. Domesticate and transplant the seedlings with successful colonization of Serratia marcescens WF01.

[0039] 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 the transplanted crops; 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 disease 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 on the roots of the seedlings can also achieve successful colonization; Serratia marcescens WF01 can secrete dark red pigments, and the used colonization detection method is simple and feasible. Description of the Drawings

[0040] 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:

[0041] Figure 1 is a flow chart 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;

[0042] Figure 2 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);

[0043] Figure 3 is a diagram of the colony detection culture results of the leaf juice of tomato tissue culture seedlings in Example 1 of the present invention; the left figure is a control diagram without inoculation of Serratia marcescens WF01; the right figure is a detection culture result diagram after inoculation of Serratia marcescens WF01;

[0044] Figure 4 It is the gene sequencing alignment result diagram of Serratia marcescens WF01 in Example 4 of the present invention;

[0045] Figure 5 It is the effect comparison diagram of the resistance of tomato tissue culture seedlings to ToBRFV infection in Example 4 of the present invention; The left side was inoculated with Serratia marcescens WF01, and the right side was not inoculated with Serratia marcescens WF01;

[0046] Figure 6 is Figure 5 The partial enlarged diagram in the circle; The left side is the enlarged diagram of the left circle; The right side is the enlarged diagram of the right circle;

[0047] Figure 7 It is the colony detection culture result diagram of the leaves of tomato tissue culture seedlings in Example 5 of the present invention; The left figure is the control figure without inoculating Bacillus velezensis WF02 and the auxiliary bacterium Bacillus cereus WF04; The right figure is the colony detection culture result diagram after inoculating Bacillus velezensis WF02 and the auxiliary bacterium Bacillus cereus WF04, where the large single colony is Bacillus velezensis WF02 and the small single colony is the auxiliary bacterium Bacillus cereus WF04;

[0048] Figure 8 It is the gene sequencing alignment result of Bacillus velezensis WF02 in Example 5 of the present invention;

[0049] Figure 9 It is the effect comparison diagram of the resistance of tomato tissue culture seedlings to ToBRFV 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;

[0050] 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;

[0051] 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;

[0052] Figure 12 It is the colony detection culture result diagram of the leaves of ginger tissue culture seedlings in Example 8 of the present invention; The left figure is the control figure without inoculating Bacillus velezensis WF02; The right figure is the colony detection culture result diagram after inoculating Bacillus velezensis WF02;

[0053] Figure 13 It is the colony activation diagram of Bacillus velezensis WF02 of the present invention;

[0054] Figure 14 It is the phylogenetic tree of Bacillus velezensis WF02 based on the 16S rDNA sequence of the present invention;

[0055] Figure 15 It is the colony activation diagram of the low-temperature-tolerant Bacillus WF04 of the present invention;

[0056] Figure 16 It is the phylogenetic tree of the low-temperature-tolerant Bacillus WF04 of the present invention based on the 16S rDNA sequence;

[0057] Figure 17 It is the colony activation diagram of Serratia marcescens WF01 of the present invention;

[0058] Figure 18 It is the phylogenetic tree of Serratia marcescens WF01 of the present invention based on the 16S rDNA sequence. Detailed implementation manners

[0059] In order 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. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; for those not specifying specific technologies or conditions in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0060] All the culture media without special instructions involved in the following embodiments are standard culture media commonly used in the art, for example:

[0061] 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.

[0062] 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.

[0063] Example 1: Screening and identification of Bacillus velezensis WF02

[0064] 1. Strain screening

[0065] It was isolated from healthy tomato plants collected from a greenhouse where tomato brown rugose fruit virus occurred on a large scale in the tomato planting area of Anqiu City, Shandong Province. The sampling and isolation methods are as follows:

[0066] Select healthy plants and use a sampling shovel to dig out the entire root system. Rinse with tap water first to remove surface soil and other attachments. Then soak the roots, stems, and leaves of the plants in 2% sodium hypochlorite in a clean bench for 5 to 10 minutes, rinse with sterile water 3 to 5 times, then soak in 75% alcohol for 3 to 5 minutes, rinse with sterile water 3 to 5 times, then use sterile filter paper to absorb the moisture on the surface of the tomato roots, stems, and leaves, and place the tomato roots, stems, and leaves in an ultra-low temperature environment of -80°C for 30 days; place the tomato roots, stems, and leaves in a sterilized mortar, add 5 to 10 mL of 1×PBS buffer, and evenly grind to obtain the juice to obtain the sample stock solution. The sample stock solution was gradient diluted, and the dilution was 10 -2 , 10 -3 , 10 -4 100 μL of the dilution was spread on NA solid culture medium and cultured upside down at 30°C for 2 days. A single colony of the dominant bacteria was picked and isolated and purified to obtain strain WF02.

[0067] 2. Strain identification

[0068] The morphological identification of strain WF02 was performed, and the results are as follows:

[0069] 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 13 shown.

[0070] Molecular biological identification of strain WF02:

[0071] Total DNA from strain WF02 was extracted using conventional bacterial DNA extraction methods. 16S rDNA gene PCR amplification was performed using universal bacterial primers 27f and 1492r. The PCR reaction system consisted of 1 µl of DNA template, 25 µl of 2× Mastar Mix, 2 µl of 27f, 2 µl of 1492r, and ultrapure water to 50 µl. The amplified product was analyzed by 0.8% agarose gel electrophoresis and then sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing.

[0072] The sequencing results of strain WF02 were compared on NCBI and EzBio Cloud websites, and the comparison analysis was performed using Mega 6.0 software. At the same time, the phylogenetic tree was constructed by the neighbor-joining method (NJ). The 16S rDNA sequence of strain WF02 was aligned with that of Bacillus velezensis. (Bacillus velezensis ) has a similarity of 99% (e.g. Figure 14 As shown); strain WF02 was identified as Bacillus velezinis ( Bacillus velezensis ), named Bacillus velez WF02.

[0073] The Bacillus velezensis WF02 was deposited at the China General Microbiological Culture Collection Center on May 24, 2024, with the deposit number CGMCC NO. 30756, and the deposit address is No. 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name is Bacillus velezensis .

[0074] Example 2: Screening and identification of low-temperature resistant Bacillus sp. WF04

[0075] 1. Strain screening

[0076] It was isolated from the soil collected from the Ali area of Tibet Autonomous Region with an average altitude of over 4500 meters. The isolation method is as follows:

[0077] The collected soil was adjusted to an appropriate moisture content and frozen in an ultra-low temperature environment of -80°C for 35 days (during which it was freeze-thawed once every 7 days). 1 g of the soil after being repeatedly freeze-thawed 5 times was placed in 100 mL of sterile water and shaken for 20 min to obtain the sample stock solution. The sample stock solution was serially diluted, and 100 μL of the diluted solutions with dilution factors of 10 -3 , 10 -4 , 10 -5 were spread on NA solid medium and cultured in an inverted position at 10°C for 2 days. Single colonies of the dominant bacteria with rapid growth were picked and isolated and purified to obtain strain WF04.

[0078] 2. Strain identification

[0079] The morphology of strain WF04 was identified, and the results are as follows:

[0080] The colonies of strain WF04 on NA solid medium were off-white, round, oily, with no wrinkles on the surface and opaque; Gram staining was positive, the cells were rod-shaped, not in chains, and produced spores, as shown in Figure 15 .

[0081] Molecular biological identification of strain WF04:

[0082] The total DNA of strain WF04 was extracted and prepared according to the conventional bacterial DNA extraction method. The 16S rDNA gene was amplified by PCR 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 amplification product was detected by 0.8% agarose gel electrophoresis and then sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing.

[0083] The sequencing results of strain WF04 were compared on the NCBI and EzBio Cloud websites, and Mega 6.0 software was used for alignment analysis. At the same time, a phylogenetic tree was constructed by the Neighbor-Joining method (abbreviated as NJ). The alignment results of the 16S rDNA sequence of strain WF04 (as Figure 16 shown) were identified as Bacillus psychrotolerans (Peribacillus frigoritolerans ), named Bacillus psychrotolerans WF04.

[0084] The above-mentioned Bacillus psychrotolerans WF04 was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on May 24, 2024, with the deposit number CGMCC NO. 30758, and the deposit address is No. 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name is Peribacillus frigoritolerans .

[0085] Example 3. Screening and identification of Serratia marcescens WF01

[0086] 1. Strain screening

[0087] 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, which had been using beta-cypermethrin for a long time. The screening method is as follows:

[0088] Select healthy and robust wheat plants, cut off the above-ground wheat plants with scissors, first rinse them with tap water to remove surface dirt and other attachments, 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 out the juice as 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 an enriched culture solution. Gradient dilute the enriched culture solution, and spread 100 μL of the dilution solutions with dilution factors of 10 -5 、10 -6 、10 -7 uniformly on 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 it upside down at 28 °C for 48 h, then pick out the grown red or pink single colonies (dominant colonies) for purification.

[0089] 2. Strain Identification

[0090] The isolated strain was identified morphologically. The results showed that after culturing on NA solid medium at 28 °C for 24 h, bright red colonies ( Figure 17 ) were formed, with a diameter of 2 - 4 mm. The colony surface was smooth, moist, with regular edges, protruding and opaque. The bacteria were Gram-negative, with a nearly spherical or short rod-shaped morphology, 0.4 - 0.5 μm wide and 0.4 - 0.8 μm long.

[0091] The isolated strain was identified by molecular biology. The results were as follows: As Figure 18 shown, universal bacterial primers 27f and 1492r were used for PCR amplification of the 16S rDNA gene. The amplification product was sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing, and then the sequencing results were compared and analyzed on the NCBI and EzBio Cloud websites. The strain was identified as Serratia marcescens, which was the initial strain of Serratia marcescens WF01.

[0092] 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 was Serratia marcescens Serratia marcescens .

[0093] Example 4: Method for inoculating tomato tissue culture seedlings with Serratia marcescens WF01 to improve the resistance of tomatoes to ToBRFV

[0094] As Figure 1 shown, it includes the following steps:

[0095] Step 1: Disinfect and process tomato seeds, and germinate the seeds in a tissue culture bottle containing a basal medium to produce tomato tissue culture seedlings;

[0096] The specific steps of Step 1 are as follows:

[0097] 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 unfolded;

[0098] Step 1B, inducing callus and adventitious buds from tomato cotyledons: When the tomato cotyledons are fully expanded, cut the middle section (0.5 cm × 0.5 cm) of the cotyledons as explants and inoculate them onto the surface of a medium with Ms as the basal 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, and culture for 7 - 8 weeks;

[0099] Step 1C, inducing rooting of the adventitious buds; inoculate the adventitious buds about 2 cm long into a medium with Ms as the basal medium supplemented with 30 - 50 g / L sucrose and 6 - 8 g / L agar to induce rooting, and culture into complete tomato tissue culture seedlings.

[0100] Step two, inoculating Serratia marcescens WF01 at the roots of tomato tissue culture seedlings;

[0101] The specific steps of step two are as follows:

[0102] Step 2A, activating Serratia marcescens WF01 on NA solid medium 2 days in advance, and culturing it upside down at 28°C for 24 h; the activated Serratia marcescens WF01 is as Figure 17 shown;

[0103] Step 2B, picking a single colony of the activated Serratia marcescens WF01 and inoculating it into NB liquid medium, and culturing 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;

[0104] Step 2C, under sterile conditions, inoculate the bacterial solution onto the roots of the tomato tissue culture seedlings in step one, and continue to culture for 24 h. As Figure 2 shown.

[0105] Step three, detecting whether Serratia marcescens WF01 has successfully colonized in tomato tissue culture seedlings;

[0106] The specific steps of step three are as follows:

[0107] Step 3A, under sterile conditions, cut the leaves of the tomato tissue culture seedlings inoculated with Serratia marcescens WF01 in step two and place them in a 1.5 mL sterile centrifuge tube;

[0108] Step 3B, crush the leaves with a sterile glass rod, suck 50 μL of the juice, and evenly spread it on the NA solid medium;

[0109] Step 3C, culture the NA solid medium coated with leaf juice at 28°C for 24 h;

[0110] Step 3D: Observe whether there are single colonies on the surface of the NA solid medium coated with leaf sap. 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 Serratia marcescens WF01 single colonies.

[0111] 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 leaf sap and inoculate them into NB liquid medium, and culture them with shaking at 180 r / min for 12 h to obtain a bacterial solution.

[0112] Step 3F: Take the bacterial solution for 16S rDNA gene sequencing, and compare the sequencing results with the sequence of the original Serratia marcescens using DNAMAN software, and judge whether the colonization is successful based on the morphological and gene sequencing results. As Figure 3 shown, the single colony morphologies in the right figure are all single colonies similar to the single colony morphology of the original Serratia marcescens WF01, 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. Directly enter Step Five;

[0113] Step Five: Domesticate and transplant the tomato tissue culture seedlings in which Serratia marcescens WF01 has successfully colonized.

[0114] The specific steps of Step Five are as follows:

[0115] Step 5A: Select the tomato tissue culture seedlings in which Serratia marcescens WF01 has successfully colonized, domesticate them with the tissue culture bottle caps half-open in the incubator for 1 - 2 d, then fully open the tissue culture bottle caps and domesticate them for 1 - 2 d, and finally domesticate them in the natural environment for 1 - 2 d;

[0116] Step 5B: Remove the domesticated tomato tissue culture seedlings from the tissue culture bottles, transplant them into the cultivation substrate, and grow for 30 d to obtain tomato tissue culture seedlings colonized with Serratia marcescens WF01.

[0117] Detect the tomato tissue culture seedlings in Step 5B for resistance to ToBRFV, and the detection method is as follows:

[0118] (1) Cut 1 g of the leaves of the tomato plants identified with ToBRFV by RT-PCR, grind them, and add 9 mL of 1xPBS to obtain a crude extract containing ToBRFV;

[0119] (2) Filter the crude extract to remove the leaf residues in the crude extract to obtain a tomato extract containing ToBRFV.

[0120] (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 areas of the leaves of the experimental group and the control group respectively;

[0121] (4) After 8 d, it was observed that the leaves of the tomato tissue culture seedlings in the control group showed obvious leaf withering and yellowing; see Figure 5 the right picture in Figure 6 and the right picture shown; while the leaves of the tomato tissue culture seedlings in the experimental group did not show obvious symptoms; see Figure 5 the left picture in Figure 6 and the left picture shown.

[0122] 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.

[0123] Example 5: Method for inoculating tomato tissue culture seedlings with Bacillus velezensis WF02 to improve the ability of tomatoes to resist ToBRFV

[0124] As Figure 1 shown, it includes the following steps:

[0125] Step 1, disinfect and process tomato seeds, and germinate the seeds in a tissue culture bottle filled with a basic medium to produce tomato tissue culture seedlings;

[0126] The specific steps of Step 1 are as follows:

[0127] 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 unfolded;

[0128] Step 1B, inducing the cotyledons of tomatoes to form callus and adventitious buds: Wait until the cotyledons of tomatoes 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 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;

[0129] Step 1C, inducing rooting of the adventitious buds; inoculating 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 culturing into complete tomato tissue culture seedlings.

[0130] Step two, inoculating Bacillus velezensis WF02 at the roots of the tomato tissue culture seedlings;

[0131] The specific steps of step two are as follows:

[0132] Step 2A, activating Bacillus velezensis WF02 on NA solid medium 2 days in advance and culturing it in an inverted position at 28 °C for 24 h;

[0133] Step 2B, picking a single colony of the activated Bacillus velezensis WF02 and inoculating it into NB liquid medium, culturing 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;

[0134] Step 2C, under sterile conditions, inoculating the bacterial solution at the roots of the tissue culture seedlings in step one and continuing to culture for 24 h.

[0135] Step three, randomly checking whether Bacillus velezensis WF02 has successfully colonized in the tomato tissue culture seedlings;

[0136] The specific steps of step three are as follows:

[0137] Step 3A, under sterile conditions, cutting the leaves of the tomato tissue culture seedlings inoculated with Bacillus velezensis WF02 in step two and placing them in a 1.5 mL sterile centrifuge tube;

[0138] Step 3B, mashing the leaves with a sterile glass rod, sucking 50 μL of the juice, and evenly coating it on the NA solid medium;

[0139] Step 3C, inverting and culturing the NA solid medium coated with the leaf juice at 28 °C for 24 h;

[0140] Step 3D, observing whether there are single colonies on the surface of the NA solid medium coated with the 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 step four is entered.

[0141] Step four, inoculating Bacillus velezensis WF02 and the auxiliary bacterium Bacillus cereus WF04 that is resistant to low temperature at the roots of the tomato tissue culture seedlings; the specific steps of step four are as follows:

[0142] Step 4A: Activate Bacillus velezensis WF02 (accession number: CGMCC No. 30756) and the auxiliary bacterium Bacillus cereus WF04 (accession number: CGMCC No. 30758) on NA solid medium 2 days in advance, and culture them in an inverted position at 28 °C for 24 h; as Figure 13 shown in the activation diagram of Bacillus velezensis WF02; Figure 15 shown in the activation diagram of the auxiliary bacterium Bacillus cereus WF04.

[0143] Step 4B: Pick single colonies of the activated Bacillus velezensis WF02 and Bacillus cereus WF04 and inoculate them into NB liquid medium respectively, and culture them 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;

[0144] Step 4C: Mix the NB liquid medium inoculated with Bacillus velezensis WF02 and Bacillus cereus 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 to culture for 24 h.

[0145] Then enter Step 3 to detect whether Bacillus velezensis WF02 has successfully colonized in the tomato tissue-cultured seedlings;

[0146] The specific steps of Step 3 are as follows:

[0147] Step 3A: Under sterile conditions, cut the leaves of the tomato tissue-cultured seedlings inoculated with Bacillus velezensis WF02 and the auxiliary bacterium Bacillus cereus WF04 in Step 2, and place them in a 1.5 mL sterile centrifuge tube;

[0148] Step 3B: Mash the leaves with a sterile glass rod, suck 50 μL of the juice, and evenly coat it on the NA solid medium;

[0149] Step 3C: Invert and culture the NA solid medium coated with leaf juice at 28 °C for 24 h;

[0150] Step 3D: Observe whether there are single colonies on the surface of 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 morphology of the selected single colonies with that of the original single colonies of Bacillus velezensis WF02.

[0151] Step 3E: If the results of the colony morphology comparison are similar, pick the single colonies of the Bacillus velezensis to be tested in the NA solid medium coated with leaf juice and inoculate them into NB liquid medium, and culture them with shaking at 28 °C and 180 r / min for 12 h to obtain a bacterial solution;

[0152] Step 3F: Take the bacterial liquid and perform 16S rDNA gene sequencing. Compare the sequencing results with the sequence of the original Bacillus velezensis using DNAMAN software, and determine whether the colonization is successful based on the morphological and gene sequencing results.

[0153] As Figure 7 shown, large single colonies similar to the single colonies of the original Bacillus velezensis WF02 were found in the right figure, and there were no less than 50 single colonies of the same type. As Figure 8 shown is the gene sequencing alignment result of Bacillus velezensis. The two gene sequencing results are consistent, indicating that Bacillus velezensis WF02 has successfully colonized in tomato tissue culture seedlings, and directly proceed to Step Five.

[0154] Step Five: Domesticate and transplant the tomato tissue culture seedlings in which Bacillus velezensis WF02 has successfully colonized.

[0155] The specific steps of Step Five are as follows:

[0156] Step 5A: Select the tomato tissue culture seedlings in which Bacillus velezensis WF02 has successfully colonized, half-open the tissue culture bottle caps in the incubator for 1 - 2 days for domestication, then fully open the tissue culture bottle caps in the incubator for 1 - 2 days for domestication, and finally domesticate in the external natural environment for 1 - 2 days;

[0157] 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 in which Bacillus velezensis WF02 has successfully colonized.

[0158] Detect the tomato tissue culture seedlings in Step 5B, and the detection method is as follows:

[0159] (1) Cut 1 g of tomato plant leaves identified with ToBRFV by RT-PCR, grind them, and add 9 mL of 1xPBS to obtain a crude extract containing ToBRFV;

[0160] (2) Filter the crude extract to remove the leaf residues in the crude extract to obtain a tomato extract containing ToBRFV;

[0161] (3) Gently rub the tomato leaves of the experimental group (tomato tissue culture seedlings in which Bacillus velezensis has successfully colonized) and the control group (tomato tissue culture seedlings without Bacillus velezensis colonization) with sandcloth, and 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;

[0162] (4) After 8 days, it was observed that the leaves of the tomato tissue culture seedlings in the control group showed obvious leaf drying and yellowing, as Figure 9 shown in the right figure; while the leaves of the tomato tissue culture seedlings in the experimental group did not show obvious symptoms, as Figure 9 shown in the left figure.

[0163] It can be seen that Bacillus velezensis WF02 colonizes the tomato plant in advance under the action of the auxiliary bacterium, psychrotolerant Bacillus WF04, enhancing the ability of tomato tissue culture seedlings to resist ToBRFV infection, and thus achieving the purpose of anti-ToBRFV seedling raising.

[0164] Example 6: Growth promotion effect of inoculating Serratia marcescens WF01 and Bacillus velezensis WF02 on tomato seedlings

[0165] Tomatoes of the variety Provence were selected as the experimental materials.

[0166] (1) Disinfect the plump, healthy and pest-free tomato seeds under sterile conditions: 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;

[0167] (2) Sow the disinfected seeds into flower pots filled with sterilized seedling-raising substrate, with 3 tomato seeds in each pot, 5 replicates for each treatment, and raise seedlings in a light incubator;

[0168] (3) When the seeds germinate and grow to two leaves and one heart, pour 10 mL / plant of the fermentation broth of Serratia marcescens WF01 and Bacillus velezensis WF02 on the roots of tomato plants, and perform secondary irrigation after 7 days. The control group is irrigated with the same volume of sterile water;

[0169] (4) Observe the growth of tomato plants under different treatments throughout the growth period, and 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:

[0170] Table 1 Effects of Serratia marcescens WF01 and Bacillus velezensis WF02 on the growth of tomato plants

[0171]

[0172] As can be seen from Table 1, after the tomato seedlings are irrigated with the fermentation broth of Serratia marcescens WF01 and Bacillus velezensis WF02 at the roots, both 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 promotion effect on tomato seedlings.

[0173] Example 7: Inhibitory effect of inoculating Serratia marcescens WF01 and Bacillus velezensis WF02 on tomato brown rugose fruit virus

[0174] Treatment 1: Control (virus negative), no treatment;

[0175] Treatment 2: Control (virus positive), inoculated with virus;

[0176] Treatment 3: Inoculate the bacterial solution of Serratia marcescens WF01 with OD600 = 0.85 + virus;

[0177] Treatment 4: Inoculate the mixed bacterial solution (volume ratio 1:1) of Bacillus velezensis WF02 with OD600 = 0.82 and Bacillus cereus WF04 with OD600 = 0.86 + virus;

[0178] The whole - leaf disease - inoculation method was adopted

[0179] (1) Under sterile conditions, disinfect plump, healthy, and pest - free tomato seeds: first, treat with 75% alcohol for 30 - 60 s, wash with sterile water 3 times, treat with 3% - 5% NaClO for 10 min, and wash with sterile water 10 times;

[0180] (2) Sow the sterile seeds into flower pots filled with sterilized seedling - raising substrate, with 3 tomato seeds in each pot, 5 replicates for each treatment, and raise seedlings in a light - incubating room.

[0181] (3) When the tomatoes grow to the 5 - true - leaf stage, friction - inoculate the virus solution (virus inoculation concentration is 1.2×10 8 copies / μL), inoculate 10 plants for each treatment, inoculate 4 leaves of each plant with the virus, inoculate 20 μL of the virus on each leaf. After 5 days of inoculation, count the number of necrotic spots and calculate the inhibition rate. Inhibition rate = (number of necrotic spots in the control - number of necrotic spots in the treatment) / number of necrotic spots in the control × 100%.

[0182] (4) After 7 days of inoculation, pick the 3rd functional leaf above the leaf, extract RNA, and detect the virus expression level by fluorescence quantitative PCR.

[0183] The inhibitory effects of Serratia marcescens WF01 and Bacillus velezensis WF02 on tomato plants against ToBRFV are shown in Table 2.

[0184] Table 2 Inhibitory effects of Serratia marcescens WF01 and Bacillus velezensis WF02 on tomato plants against ToBRFV

[0185]

[0186] As can be seen from Table 2, the virus inhibition rate calculated from the number of necrotic spots on tomato leaves indicates that Serratia marcescens WF01 and Bacillus velezensis WF02 have a strong inhibitory effect 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 has decreased to a certain extent, indicating that Serratia marcescens WF01 and Bacillus velezensis WF02 have a significant antagonistic inhibitory effect on ToBRFV.

[0187] Example 8: Inoculate tissue culture seedlings of ginger with Bacillus velezensis WF02 to improve the ability of ginger to resist ToBRFV;

[0188] It includes the following steps:

[0189] Step 1: Cultivate the tissue culture seedlings of ginger required by using the shoot tip of ginger, specifically as follows:

[0190] Step 1A, Select plump, large, and pest-free ginger, wash the soil on the surface with tap water, put it in a mesh bag and expose it to the sun for 3 - 4 days, and then carry out germination promotion. 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 super clean bench;

[0191] Step 1B, Disinfect the ginger buds selected in Step 1A: soak them in 70% alcohol for 15 - 30 s, rinse them 3 times with sterile water, disinfect them with 0.1% mercuric chloride solution for 8 minutes, and rinse them 5 - 6 times with sterile water. Blot the water on the surface of the shoot tip with sterile filter paper, peel off the shoot tip leaving only 1 - 2 leaf primordia, and inoculate it into the shoot tip callus formation medium to cultivate shoot tip callus; among them, the shoot tip callus formation medium is based on Ms medium, 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, adjust the pH value to 5.8 - 6.0 with NaOH or HCl, and culture for 4 - 5 weeks (culture conditions: light intensity 4000 lx, light time 16 h / d, culture temperature 25 °C, relative humidity maintained at 60% - 80%);

[0192] Step 1C, Inoculate the shoot tip callus cultured in Step 1B into the shoot tip cluster bud proliferation medium to cultivate cluster buds; among them, the shoot tip cluster bud proliferation medium is based on Ms 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 culture for 8 - 9 weeks;

[0193] Step 1D, Divide the cluster buds cultured in Step 1C into single plants and transfer them to the rooting medium to cultivate complete tissue culture seedlings of ginger. Among them, 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 culture at 28 °C for 15 days;

[0194] Step 1E, Extract the RNA of the leaves of the tissue culture seedlings of ginger, detect the RNA extraction result by PCR, directly carry out reverse transcription, and detect the virus - free situation of the ginger leaves by RT - PCR to ensure the successful virus - free of the tissue culture seedlings of ginger;

[0195] Detect whether the tissue culture seedlings of ginger are successfully virus - free:

[0196] Extract the RNA of the leaves of ginger tissue culture seedlings, perform reverse transcription to obtain cDNA, and use RT-PCR to detect whether the ginger tissue culture seedlings contain tobacco mosaic virus and cucumber mosaic virus. The ginger tissue culture seedlings obtained through the tissue culture of ginger shoot tips are all successfully detoxified, and the detoxified tissue culture seedlings are the ginger tissue culture seedlings to be inoculated with disease-preventing and growth-promoting bacteria.

[0197] Step 2: Inoculate Bacillus velezensis WF02 at the roots of ginger tissue culture seedlings as follows:

[0198] Step 2A: Activate Bacillus velezensis WF02 on NA solid medium 2 days in advance and culture it at 28 °C for 24 h, as Figure 13 shown;

[0199] Step 2B: Pick a single colony of the activated Bacillus velezensis WF02 and inoculate it into NB liquid medium, and culture 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;

[0200] Step 2C: Under sterile conditions, inoculate the bacterial solution at the roots of the ginger tissue culture seedlings in Step 1 and continue to culture for 24 h.

[0201] Step 3: Colonization of Bacillus velezensis WF02 in ginger tissue culture seedlings is as follows:

[0202] Step 3A: Under sterile 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;

[0203] Step 3B: Mash the leaves with a sterile glass rod, suck 50 μL of the juice, and evenly coat it on the NA solid medium;

[0204] Step 3C: Invert and culture the NA solid medium coated with leaf juice at 28 °C for 24 h;

[0205] Step 3D: Observe whether there are single colonies on the surface of 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 morphology of the selected single colonies with the morphology of the original single colonies of Bacillus velezensis WF02. 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.

[0206] Step 5: Domesticate and transplant the ginger tissue culture seedlings in which Bacillus velezensis WF02 has successfully colonized. The specific steps are as follows:

[0207] Step 5A: Select the tissue-cultured ginger seedlings colonized successfully by Bacillus velezensis WF02, acclimatize them in an incubator with the tissue-culture bottle caps half-opened for 1 - 2 days, then acclimatize them with the caps fully opened for 1 - 2 days, and finally acclimatize them in the external natural environment for 1 - 2 days.

[0208] Step 5B: Remove the acclimatized tissue-cultured ginger seedlings from the tissue-culture bottles, transplant them into the cultivation substrate, and grow for 28 days to obtain the tissue-cultured ginger seedlings colonized successfully by Bacillus velezensis WF02.

[0209] 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 days; 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 colonies of Bacillus velezensis WF02 in the leaf juice of the tissue-cultured ginger seedlings. The left picture is the control picture without inoculating Bacillus velezensis WF02, and the right picture is the detection result diagram of the colonies after inoculating Bacillus velezensis WF02; from Figure 12 it can be known that Bacillus velezensis WF02 colonizes in the detoxified 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 acclimatization of Bacillus velezensis WF02.

[0210] 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. What is described in the above embodiments and the specification only illustrates 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 ability of crops to resist Tomato brown rugose fruit virus disease, which is characterized in that, It includes 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 sample to determine whether the disease-preventing and growth-promoting bacteria have successfully colonized in the tissue culture seedlings. If the colonization is successful, proceed to Step 5; if the colonization is not successful, first proceed to Step 4 and then to Step 5; Step 4: When inoculating the disease-preventing and growth-promoting bacteria in Step 2, add auxiliary bacteria synchronously; Step 5: Domesticate and transplant the tissue culture seedlings in which the disease-preventing and growth-promoting bacteria have successfully colonized; The disease-preventing and growth-promoting bacteria are Serratia marcescens or Bacillus velezensis that are resistant to tomato brown rugose fruit virus; among them, The Serratia marcescens is Serratia marcescens WF01, which was deposited in 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 in the China General Microbiological Culture Collection Center on May 24, 2024, and its deposit number is: CGMCC No. 30756; 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 in the China General Microbiological Culture Collection Center on May 24, 2024, and its deposit number is: CGMCC No. 30758; The tissue culture seedlings in Step 1 are tomato tissue culture seedlings or pepper tissue culture seedlings.

2. The seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the ability of crops to resist tomato brown rugose fruit virus disease according to claim 1, characterized in that, The tissue culture seedlings in Step 1 are tomato tissue culture seedlings or pepper tissue culture seedlings. The specific steps of Step 1 are as follows: Step 1A: Under sterile conditions, disinfect plump, healthy, and pest-free tomato or pepper seeds, and then spread the tomato or pepper seeds on the surface of the seed germination solid medium. When the cotyledons are fully unfolded 6 - 8 days after seed germination, proceed to Step 1B; Step 1B: Cut the middle section of the cotyledon as the explant, with the size of the explant being 0.5 cm × 0.5 cm. Inoculate it on the surface of the 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. 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 with Ms as the basic medium and 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.

3. The seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the ability of crops to resist tomato brown rugose fruit virus disease according to claim 1, characterized in that, The specific steps of Step 2 are as follows: Inoculate the activated disease-preventing and growth-promoting bacteria 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 a bacterial solution; 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 before performing the operation in Step 3.

4. The seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the ability of crops to resist tomato brown rugose fruit virus disease according to claim 1, characterized in that, The specific steps of Step 3 are 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 NA solid medium, and culture it 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 morphology of the selected single colonies with that of the original disease-preventing and growth-promoting bacteria single colonies. If the results of the colony morphology comparison are similar, proceed to Step 3C; Step 3C: Pick the single colonies to be tested on the NA solid medium coated with leaf juice and inoculate them into the NB liquid medium, and culture them with shaking at 28 °C and 180 r / min for 12 h to obtain a bacterial solution; Step 3D: Take the bacterial solution and perform 16S rDNA gene sequencing, and compare the sequencing results with the gene sequences 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.

5. A seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the ability of crops to resist tomato brown rugose fruit virus disease as described in claim 1, characterized in that, The specific content of Step Four is as follows:

6. The seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the ability of crops to resist tomato brown rugose fruit virus disease according to claim 1, characterized in that, Inoculate the activated disease-preventing and growth-promoting bacteria and the auxiliary bacteria into the 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 a disease-preventing and growth-promoting bacteria solution and an auxiliary bacteria solution; under sterile conditions, mix the disease-preventing and growth-promoting bacteria solution and the auxiliary bacteria solution evenly according to the volume ratio of 1:1, inoculate the mixed bacteria solution onto the roots of the tissue culture seedlings in Step One, and continue to culture for 24 h and then proceed to Step Five. The specific content of Step Five is 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-open in the incubator for 1 - 2 d, then acclimatize them with the tissue culture bottle cap fully open for 1 - 2 d, and finally acclimatize them in the natural environment for 1 - 2 d; 7. A seedling raising method for inoculating disease-preventing and growth-promoting bacteria to improve the resistance of tomatoes or peppers to tomato brown rugose fruit virus disease, characterized in that, Step 5B: Transplant the acclimatized tissue culture seedlings from the tissue culture bottle into the cultivation substrate and grow for 28 - 30 d to obtain the disease-resistant tissue culture seedlings. It includes the following steps: Step a: Under sterile conditions, disinfect plump, healthy, and pest-free tomato seeds or pepper seeds, and then spread the tomato seeds or pepper seeds on the surface of the solid medium in the tissue culture bottle. After the seeds germinate for 10 d, obtain the seedlings; Step b: Inoculate the activated Serratia marcescens WF01 into the 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 a bacterial solution; under sterile conditions, inoculate the bacterial solution 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 spread 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 unsuccessful, 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; The Serratia marcescens WF01 was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on April 17, 2024, and its deposit number is: CGMCC No. 30368.

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