Serratia marcescens WF01 for Degrading Pesticides and Its Application

Abstract

The present invention relates to the technical field of agricultural microorganisms, and particularly relates to Serratia marcescens WF01 for degrading pesticides and its application. Serratia marcescens WF01 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on April 17, 2024, with the deposit number of CGMCC NO. 30368. A high-yield prodigiosin strain was obtained by induction with beta-cypermethrin. This strain has strong growth-promoting properties such as IAA production, siderophore production, nitrogen fixation activity, organic phosphorus solubilization, and inorganic phosphorus solubilization. At the same time, it has a wide tolerance to pyrethroid pesticides, especially a high degradation rate for beta-cypermethrin remaining in pepper leaves and soil. This strain can rapidly and effectively colonize the crop roots and enter the plant body, and has broad application prospects in the degradation of pyrethroid pesticides and the prevention and control of tomato brown rugose fruit virus.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and specifically relates to Serratia marcescens WF01 for degrading pesticides and its application. Background Art

[0002] With the banning of traditional pesticides such as organochlorines and organophosphates that are highly toxic and have slow degradation, pyrethroid insecticides are used to control agricultural and forest pests and sanitary pests due to their broad-spectrum insecticidal activity, low residue, high efficiency (fast knockdown speed), and strong activity. They have become one of the most widely used pesticides in China today. With the increase in their usage, the residues of pyrethroid pesticides in the environment are continuously accumulating, causing pollution to the ecological environment and directly affecting the quality of agricultural products and food safety.

[0003] Pyrethroid insecticides are a class of insecticides artificially synthesized by mimicking the chemical structure of natural pyrethrins, mainly composed of cypermethrin, beta-cypermethrin, deltamethrin, bifenthrin, cyfluthrin, lambda-cyhalothrin, fenpropathrin, fenvalerate, etc. As of November 19, 2024, there are a total of 6,709 pyrethroid insecticide products with valid registration in China, involving 42 common varieties, among which lambda-cyhalothrin products account for 13.54% of the total number of pyrethroid insecticides.

[0004] After the use of pyrethroid insecticides, they will remain in the soil, water, and air, causing long-term effects on the environment and ecosystem. These chemical substances may enter other organisms through the food chain, leading to bioaccumulation and ultimately disrupting the ecological balance. In addition, they may also contaminate groundwater and have toxic effects on aquatic organisms. Therefore, it is urgent to eliminate the impact on the ecological environment caused by the residues. For example, cypermethrin has stomach toxicity and contact toxicity. Although it is safe for crops, it is moderately toxic to humans and livestock and highly toxic to aquatic organisms, bees, and silkworms.

[0005] Currently, the methods for solving pesticide residues in the ecological environment mainly include banning, physical and chemical methods, and bioremediation methods, etc. Among them, physical and chemical methods are costly, have large side effects, are prone to secondary pollution, and the elimination is not thorough; bioremediation methods have the advantages of high efficiency, safety, low cost, no secondary pollution, wide application range, etc., mainly including phytoremediation, microbial remediation, etc. Microbial degradation is an important means to eliminate pesticide pollution. It has the advantages of low cost, high efficiency, and no secondary pollution, and is generally considered the most effective and feasible method. However, the current microbial strain resources for degrading pyrethroid insecticides are insufficient, restricting the practical application of this remediation technology.

[0006] Tomato brown rugose fruit virus (ToBRFV), a newly discovered RNA virus, is extremely harmful and highly transmissible. It mainly infects crops such as tomatoes, peppers, and tobacco, and can be transmitted through seeds and fruits. In severe cases of disease, the economic loss can reach up to 100%. Since its discovery in 2014, it has rapidly spread across more than 30 countries in Europe, the Americas, and Asia. In China, ToBRFV was first detected infecting tomatoes in Shandong in 2019. Subsequently, the virus was successively discovered in Shaanxi, Jiangsu, Yunnan, Hebei, Henan, Anhui, Guangdong, Liaoning and other places, causing extremely serious harm. In April 2021, ToBRFV was listed in the Catalogue of Quarantine Pests for Inbound Plants of the People's Republic of China. Due to the short time of research on ToBRFV, current prevention and control technology development involves aspects such as the development of detection methods, the excavation of resistance genes, and the breeding of resistant varieties, while the prevention and control technology using microorganisms to induce resistance to ToBRFV has not been reported.

[0007] Therefore, it is of great significance to breed a microorganism that can not only degrade pyrethroid insecticides but also control tomato brown rugose fruit virus, and it has broad application prospects. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide Serratia marcescens WF01 for degrading pesticides, which can not only degrade pyrethroid insecticides but also control tomato brown rugose fruit virus, and its application.

[0009] To solve the above technical problems, the technical solution of the present invention is:

[0010] Provide Serratia marcescens WF01 for degrading pesticides. The Serratia marcescens WF01 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on April 17, 2024, with the deposit number CGMCC NO.30368, and the deposit address is Beichen West Road, Chaoyang District, Beijing, and the taxonomic name is Serratia marcescens Serratia marcescens .

[0011] Provide an antibacterial agent induced by tomato brown rugose fruit virus, which contains the Serratia marcescens WF01 for degrading pesticides.

[0012] As a preferred technical solution, the antibacterial agent induced by tomato brown rugose fruit virus has the function of degrading pyrethroid insecticides.

[0013] As a preferred technical solution, the active ingredient of the pyrethroid insecticide is beta-cypermethrin or lambda-cyhalothrin or cypermethrin.

[0014] As a preferred technical solution, the beta-cypermethrin is the beta-cypermethrin remaining in the pepper planting soil and / or the pepper plant.

[0015] As a preferred technical solution, the tomato brown rugose fruit virus inducing antibacterial agent is used during the growth period of pepper and is applied by root irrigation.

[0016] A method for preparing the tomato brown rugose fruit virus inducing antibacterial agent is provided.

[0017] Streak the Serratia marcescens WF01 on a solid medium with beta-cypermethrin as the sole carbon source, and incubate it upside down at 32 °C for 36 - 40 h; pick a bright red single colony and inoculate it into a liquid medium with beta-cypermethrin as the sole carbon source, and shake culture it at 32 °C and 185 r / min for 48 h to obtain the seed liquid of Serratia marcescens WF01; inoculate the seed liquid into a liquid medium without any carbon source at a volume ratio of 10%, shake culture it at 28 °C and 185 r / min for 3 - 5 h, then add yeast extract, and continue to shake culture it at 28 °C and 200 r / min for 48 h to obtain the tomato brown rugose fruit virus inducing antibacterial agent, and the tomato brown rugose fruit virus inducing antibacterial agent is a deep red fermentation broth of Serratia marcescens WF01.

[0018] As a preferred technical solution, the content of prodigiosin in the fermentation broth of Serratia marcescens WF01 reaches 13.5 - 17.2 g / L, and the effective viable count is 0.5×10 9 cfu / mL - 1.0×10 9 cfu / mL.

[0019] As a preferred technical solution, the tomato brown rugose fruit virus inducing antibacterial agent can prevent and control tomato brown rugose fruit virus disease.

[0020] Due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0021] (1) The Serratia marcescens WF01 of the present invention is a high-yield prodigiosin strain obtained by induction with beta-cypermethrin from an initial strain, and the initial strain is isolated from the wheat leaves in an environment where beta-cypermethrin pesticide has been used for a long time. After induction, the prodigiosin yield increases from the initial 9.5 g / L to 17.2 g / L, and the yield increases by 81.1%.

[0022] (2) In the KEGG functional classification of the whole genome of this strain, there are 16 related to xenobiotic metabolism and degradation, and 41 genes regulating xenobiotic biological metabolism and degradation;

[0023] (3) This strain has a strong IAA-producing ability, and the IAA production can reach 56.18 mg / L;

[0024] (4)This strain has a strong ability to produce siderophores;

[0025] (5)This strain has strong abilities such as nitrogen fixation, organic phosphorus solubilization, and inorganic phosphorus solubilization;

[0026] (6)This strain has a wide tolerance to pyrethroid pesticides. In particular, it has a good degradation effect on beta-cypermethrin. Under indoor soil cultivation conditions, the degradation rate of beta-cypermethrin can reach 75.28%. This strain can quickly colonize the crop roots and enter the plant body. The degradation rate of residual beta-cypermethrin in pepper leaves can reach 76.80%, and the degradation rate of residual beta-cypermethrin in pepper rhizosphere soil can reach 78.95%. It has broad application prospects in the degradation of residual pyrethroid pesticides;

[0027] (7)This strain has a significant control effect on tomato brown rugose fruit virus on field pepper crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings are only intended to illustrate and explain the present invention and do not limit the scope of the present invention. Among them:

[0029] Figure 1 is the colony morphology diagram of Serratia marcescens WF01 for degrading pesticides of the present invention on NA solid medium;

[0030] Figure 2 is the phylogenetic tree of Serratia marcescens WF01 for degrading pesticides of the present invention;

[0031] Figure 3 is the effect diagram of organic phosphorus solubilization of Serratia marcescens WF01 for degrading pesticides of the present invention;

[0032] Figure 4 is the effect diagram of siderophore production of Serratia marcescens WF01 for degrading pesticides of the present invention;

[0033] Figure 5 is the diagram of beta-cypermethrin residue at different times of Serratia marcescens WF01 for degrading pesticides of the present invention;

[0034] Figure 6 is the genomic circular diagram of Serratia marcescens WF01 for degrading pesticides of the present invention;

[0035] Figure 7 is the annotation diagram of KEGG functional analysis results of Serratia marcescens WF01 for degrading pesticides of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0036] 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 techniques or conditions in the embodiments, they shall be carried out according to the techniques 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.

[0037] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods.

[0038] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels.

[0039] All kinds of culture media involved in the following embodiments are standard culture media commonly used in the art.

[0040] Example 1: Obtaining Serratia marcescens WF01 with high yield of prodigiosin induced by beta-cypermethrin

[0041] 1. Screening of the initial strain of Serratia marcescens WF01

[0042] The initial strain of Serratia marcescens WF01 in this embodiment 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:

[0043] Select healthy and robust wheat plants, cut off the above-ground wheat plants with scissors, first rinse 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 with sterile water 4 - 5 times, then soak in 75% alcohol for 4 - 5 min, after rinsing with sterile water 4 - 5 times, 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 solution. Perform gradient dilution on the enriched culture solution, and the dilution factors are 10 -5 、10 -6 、10 -7100 μL of the dilution was evenly spread on NA solid medium (5 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, 20 g / L agar, pH 7.0 - 7.2) and cultured in an inverted position at 28 °C for 48 h. Then, the grown red or pink single colonies (dominant colonies) were picked for purification.

[0044] 2. Identification of the initial strain of Serratia marcescens WF01

[0045] The isolated strain was identified morphologically. The results showed that after culturing on NA solid medium at 28 °C for 24 h, it formed dark red colonies (as Figure 1 shown), with a diameter of 2 - 4 mm. The colony surface was smooth, moist, with regular edges, protruding and opaque. The cells were Gram-negative, nearly spherical or short rod-shaped, 0.4 - 0.5 μm wide and 0.4 - 0.8 μm long.

[0046] The isolated strain was identified by molecular biology. The results showed that as Figure 2 shown, universal bacterial primers 27f and 1492r were used for PCR amplification of the 16S rDNA gene. The amplified product was sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing. 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.

[0047] 3. Obtaining high-yield prodigiosin-producing Serratia marcescens WF01 induced by beta-cypermethrin

[0048] The initial strain of Serratia marcescens WF01 was gradually induced with different concentrations of beta-cypermethrin to make the strain's tolerance to beta-cypermethrin reach 1.5 g / L.

[0049] The induced concentrations of beta-cypermethrin were 0.2 g / L, 0.4 g / L, 0.5 g / L, 0.8 g / L, 1.0 g / L, and 1.5 g / L respectively. The initial strain of Serratia marcescens WF01 was inoculated into a basal salt liquid medium (5.8 g / L dipotassium hydrogen phosphate, 2.0 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 1.0 g / L sodium chloride, 1 L distilled water, pH 7.0 - 7.2) with beta-cypermethrin as the sole carbon source, and cultured with shaking at 28°C and 180 r / min for 3 days. Then, 100 μl was taken and evenly spread on a basal salt solid medium (5.8 g / L dipotassium hydrogen phosphate, 2.0 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 1.0 g / L sodium chloride, 20 g / L agar, 1 L distilled water, pH 7.0 - 7.2) containing the same concentration of beta-cypermethrin. After normal growth, it was transferred to the next lower concentration for induction until a strain that could rapidly form bright red colonies on the solid medium with a beta-cypermethrin concentration of 1.5 g / L was obtained. This strain was named Serratia marcescens WF01. At the same time, the initial strain and the strains obtained after each induction were inoculated into a liquid fermentation medium (5.5 g / L sucrose, 5 g / L sodium citrate, 8 g / L ammonium chloride, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.04 g / L ferric chloride heptahydrate, 0.15 g / L calcium chloride anhydrous, pH 7.0 - 7.2), and the liquid fermentation broths of the initial strain and the induced strains were obtained after culturing at 28°C and 180 r / min for 2 days; referring to the reference "Optimization of Fermentation Conditions for the Production of Prodigiosin by a New Marine Serratia marcescens Strain", the prodigiosin in the liquid fermentation broths of the initial strain and the induced strains was extracted and detected by a normal-phase liquid chromatography-mass spectrometry (HPLC-APCI-MS; Agilent 1200 series).

[0050] The prodigiosin yield of Serratia marcescens WF01 obtained after stepwise induction with beta-cypermethrin increased from 9.5 g / L of the initial strain to 17.2 g / L.

[0051] Serratia marcescens WF01 obtained after induction with beta-cypermethrin was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 17, 2024, with the deposit number CGMCC NO. 30368 and the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0052] Example 2: Determination of the growth-promoting performance of Serratia marcescens WF01

[0053] In this example, the growth-promoting performances of Serratia marcescens WF01, such as the ability to secrete indole-3-acetic acid (IAA), the ability to produce siderophores, and nitrogen fixation activity, were determined.

[0054] The test method is as follows:

[0055] 1. Determination of siderophore production:

[0056] Inoculate Serratia marcescens WF01 onto the CAS detection solid medium (CAS detection medium: 60.5 mg of chrome azurol, 72.9 mg of cetyltrimethylammonium bromide, 2.645 mg of ferric chloride, 295.25 mg of sodium dihydrogen phosphate, 1213.5 mg of disodium hydrogen phosphate dodecahydrate, 125 mg of ammonium chloride, 37.5 mg of potassium dihydrogen phosphate, 62.5 mg of sodium chloride, 9 g of agar, pH 6.8, made up to 1 L with distilled water, pH 6.8), and culture at 30 °C for 6 d; if a yellow-green halo appears, it indicates siderophore production, and the larger the halo, the stronger the siderophore production ability.

[0057] 2. Determination of nitrogen fixation activity:

[0058] Inoculate Serratia marcescens WF01 onto the Ashby nitrogen-free solid medium (0.2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.2 g of sodium chloride, 5.0 g of calcium carbonate, 10.0 g of mannitol, 0.1 g of calcium sulfate, 1 L of distilled water, 15 g of agar, pH 7.0), and culture at 30 °C for 70 - 72 h, then observe the colony growth; if the strain can grow on the Ashby nitrogen-free solid medium, it means the strain has nitrogen fixation activity, and the nitrogen fixation activity size is judged according to the colony growth diameter.

[0059] 3. Determination of the performance of degrading organic phosphorus and inorganic phosphorus:

[0060] Inoculate Serratia marcescens WF01 onto the Mengjinna inorganic phosphorus solid medium ((NH4)2SO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 50 g, Ca3(PO4)2 10.0 g, CaCO3 2.5 g, glucose 2.0 g, MnSO4 0.02 g, FeSO4 0.02 g, agar 20.0 g, pH 7.2, 1000 mL of distilled water) and the lecithin organic phosphorus solid medium (glucose 10.0 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, MgSO4·7H2O 0.3 g, MnSO4 0.02 g, KCl 0.3 g, FeSO4 0.03 g, lecithin 2.0 g, agar 15.0 g, pH 7.2, 1000 mL of distilled water), and culture in an inverted position at 28 °C for 7 - 10 d, then observe whether a transparent phosphorus-degrading halo appears in the strain, measure the diameter D of the phosphorus-degrading halo and the diameter d of the colony, and qualitatively judge the performance of the strain in degrading organic phosphorus and inorganic phosphorus by the ratio (D / d).

[0061] 4. Determination of the ability to secrete indole-3-acetic acid (IAA):

[0062] The ability to produce indole-3-acetic acid (IAA) was determined by the Salkowski method. Specifically, Serratia marcescens WF01 was inoculated into King's B liquid medium containing 500 mg / L tryptophan and cultured with shaking at 28 °C and 180 r / min for 70 - 72 h. Then, 2 mL of the bacterial suspension was centrifuged at 10,000 r / min for 15 min, and the supernatant was taken and added with Salkowski reagent (2 mL of Salkowski reagent was added to every 1 mL of the supernatant). The color was developed in the dark at room temperature for 30 min, and the OD530 value was measured. The blank medium was used as a control, and the OD530 value corresponding to pure IAA was used to make a standard curve to calculate the IAA content (mg / L) in the culture solution.

[0063] Table 1 Growth-promoting performance of Serratia marcescens WF01

[0064]

[0065] Note: + indicates general performance; ++ indicates medium performance; +++ indicates strong performance.

[0066] As Figure 3 、 Figure 4 shown, Serratia marcescens WF01 has strong abilities in nitrogen fixation, organic phosphorus solubilization, inorganic phosphorus solubilization, and siderophore production, and its IAA production can reach 56.18 mg / L. This shows that Serratia marcescens WF01 of the present invention has strong potential for promoting plant growth.

[0067] Example 3: Preparation of Sample 1 of the antibacterial agent against Tomato brown rugose fruit virus

[0068] Streak Serratia marcescens WF01 onto a solid medium with beta-cypermethrin as the sole carbon source (beta-cypermethrin 1.5 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, agar 18 g / L, distilled water 1 L, pH 7.0 - 7.2), and incubate it in an inverted position at 32 °C for 36 h; pick a bright red single colony and inoculate it into a liquid fermentation medium with beta-cypermethrin as the sole carbon source (K2HPO4 5.8 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, NaCl 1.0 g / L, distilled water 1 L, pH 7.0 - 7.2), and shake-culture it at 32 °C and 185 r / min for 48 h to obtain the Serratia marcescens WF01 seed solution; inoculate the seed solution into a liquid fermentation medium without any carbon source (NH4Cl 8 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 1 g / L, FeCl3·7H2O 0.04 g / L, anhydrous CaCl2 0.15 g / L, pH 7.0 - 7.2) at a volume ratio of 10%, shake-culture it at 28 °C and 185 r / min for 3 h, then add yeast extract (final concentration 3 g / L) to the fermentation broth, and continue to shake-culture it at 28 °C and 200 r / min for 48 h to obtain the first sample of the antibacterial agent against tomato brown rugose fruit virus, and the first sample of the antibacterial agent against tomato brown rugose fruit virus is the fermentation broth of Serratia marcescens WF01 in dark red color.

[0069] Using the method for determining the effective viable count of agricultural microbial inoculants in GB20287 - 2006, the effective viable count of Serratia marcescens in the first sample of the antibacterial agent against tomato brown rugose fruit virus was measured to be 0.5×10 9 cfu / mL; referring to the reference "Optimization of Fermentation Conditions for the Production of Prodigiosin by a New Marine Serratia marcescens Strain", extract the prodigiosin in the first sample of the antibacterial agent against tomato brown rugose fruit virus, and detect it by a normal-phase liquid chromatography-mass spectrometry (HPLC-APCI-MS; Agilent 1200 series). The yield of prodigiosin in the first sample of the antibacterial agent against tomato brown rugose fruit virus was 13.5 g / L.

[0070] Example 4: Preparation of the second sample of the antibacterial agent against tomato brown rugose fruit virus

[0071] Streptomyces marcescens WF01 was streaked onto a solid medium with beta-cypermethrin as the sole carbon source (beta-cypermethrin 1.5 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, agar 18 g / L, distilled water 1 L, pH 7.0 - 7.2), and cultured in an inverted position at 32 °C for 38 h; bright red single colonies were picked and inoculated into a liquid fermentation medium with beta-cypermethrin as the sole carbon source (K2HPO4 5.8 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, NaCl 1.0 g / L, distilled water 1 L, pH 7.0 - 7.2), and cultured with shaking at 32 °C and 185 r / min for 48 h to obtain a Streptomyces marcescens WF01 seed solution; the seed solution was inoculated into a liquid fermentation medium without any carbon source at a volume ratio of 10% (NH4Cl 8 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 1 g / L, FeCl3·7H2O 0.04 g / L, anhydrous CaCl2 0.15 g / L, pH 7.0 - 7.2), cultured with shaking at 28 °C and 185 r / min for 5 h, then yeast extract (final concentration 3 g / L) was added to the fermentation broth, and the culture was continued with shaking at 28 °C and 200 r / min for 48 h to obtain a sample two of the antibacterial agent against tomato brown rugose fruit virus, and the sample two of the antibacterial agent against tomato brown rugose fruit virus was a deep red fermentation broth of Streptomyces marcescens WF01.

[0072] Using the method for determining the effective viable count of agricultural microbial inoculants in GB20287-2006, the effective viable count of Streptomyces marcescens in the sample two of the antibacterial agent against tomato brown rugose fruit virus was 0.8×10 9 cfu / mL; referring to the reference "Optimization of Fermentation Conditions for the Production of Prodigiosin by a New Marine Streptomyces marcescens", the prodigiosin in the sample two of the antibacterial agent against tomato brown rugose fruit virus was extracted and detected by a normal-phase liquid chromatography-mass spectrometry (HPLC-APCI-MS; Agilent 1200 series). The yield of prodigiosin in the sample two of the antibacterial agent against tomato brown rugose fruit virus was 16.0 g / L.

[0073] Example 5: Preparation of Sample Three of the Antibacterial Agent against Tomato Brown Rugose Fruit Virus

[0074] Streptomyces marcescens WF01 was streaked onto a solid medium with beta-cypermethrin as the sole carbon source (beta-cypermethrin 1.5 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, agar 18 g / L, distilled water 1 L, pH 7.0 - 7.2), and cultured in an inverted position at 32 °C for 40 h; bright red single colonies were picked and inoculated into a liquid fermentation medium with beta-cypermethrin as the sole carbon source (K2HPO4 5.8 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, NaCl 1.0 g / L, distilled water 1 L, pH 7.0 - 7.2), and cultured with shaking at 32 °C and 185 r / min for 48 h to obtain the Streptomyces marcescens WF01 seed solution; the seed solution was inoculated into a liquid fermentation medium without any carbon source at a volume ratio of 10% (NH4Cl 8 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 1 g / L, FeCl3·7H2O 0.04 g / L, anhydrous CaCl2 0.15 g / L, pH 7.0 - 7.2), cultured with shaking at 28 °C and 185 r / min for 4 h, then yeast extract (final concentration 3 g / L) was added to the fermentation broth, and continued to be cultured with shaking at 28 °C and 200 r / min for 48 h to obtain the antibacterial agent sample III against tomato brown rugose fruit virus. The antibacterial agent sample III against tomato brown rugose fruit virus is a deep red fermentation broth of Streptomyces marcescens WF01.

[0075] Using the method for determining the effective viable count of agricultural microbial inoculants in GB20287-2006, the effective viable count of Streptomyces marcescens in the antibacterial agent sample III against tomato brown rugose fruit virus is 1.0×10 9 cfu / mL; Referring to the reference "Optimization of Fermentation Conditions for the Production of Prodigiosin by a New Marine Streptomyces marcescens", the prodigiosin in the antibacterial agent sample III against tomato brown rugose fruit virus was extracted and detected by a normal-phase liquid chromatography-mass spectrometry (HPLC-APCI-MS; Agilent 1200 series). The yield of prodigiosin in the antibacterial agent sample III against tomato brown rugose fruit virus was 17.2 g / L.

[0076] Example VI: Degradation Effect of the Antibacterial Agent against Tomato Brown Rugose Fruit Virus on Beta-cypermethrin

[0077] The high-performance liquid chromatography method was used to determine the degradation effect of the antibacterial agent against tomato brown rugose fruit virus on beta-cypermethrin. The method is as follows:

[0078] Accurately weigh 0.05 g of beta-cypermethrin standard product, dissolve it with acetonitrile, and make up the volume to 10 mL in a volumetric flask. The final concentration is 5 g / L. Use a high performance liquid chromatograph (HPLC) to determine and prepare a standard curve. Add beta-cypermethrin emulsifiable concentrate to the inorganic salt medium (so that the final concentration of beta-cypermethrin is 0.8 g / L), inoculate 1 mL of the prepared tomato brown rugose fruit virus-induced antibacterial agent sample, and shake and culture at 28 °C and 180 r / min. Samples are taken and the beta-cypermethrin content is determined at 0, 12 h, and 24 h respectively, with the sample without inoculating the tomato brown rugose fruit virus-induced antibacterial agent sample as the control. Add an equal amount of petroleum ether to 50 mL of the fermentation broth, shake and extract for 1.5 h, transfer it to a separating funnel, take the lower organic phase, transfer the upper aqueous phase to a triangular flask, and extract 3 times. Combine the organic phases, add anhydrous sodium sulfate to remove water, then transfer it to a 250 mL flask and rotary evaporate to dryness under negative pressure at 56 °C. Add petroleum ether to dissolve it in 3 portions and make up the volume to 20 mL. HPLC detection: The chromatographic column is Elite C18 column with a particle size of 5 μm. The mobile phase is water + acetonitrile (80:20), the flow rate is 1 mL / min, the detection wavelength is 235 nm, the injection volume is 10 μL, and the column temperature is 35 °C.

[0079] The results are as Figure 5 shown. After 12 h of inoculating sample 1, the residual rate of beta-cypermethrin in the medium is 54.62%, which has a significant difference compared with the control without inoculating sample 1 (P < 0.05). After 24 h of inoculating sample 1, the residual rate of beta-cypermethrin in the medium is 10.51%, and the residual rate of the control without inoculating sample 1 is 81.75%, showing an extremely significant difference (P < 0.01). Serratia marcescens WF01 can use beta-cypermethrin as a carbon source for degradation and utilization in the inorganic salt medium.

[0080] Example 7: Determination of the tolerance of Serratia marcescens WF01 to other pyrethroid pesticides

[0081] The test method of this example is as follows:

[0082] Determine the tolerance of Serratia marcescens WF01 to 5 pyrethroid pesticides (permethrin, deltamethrin, lambda-cyhalothrin, fenvalerate, bifenthrin) other than beta-cypermethrin: Add different concentrations of permethrin (25% powder), deltamethrin (2.5% powder), lambda-cyhalothrin (10% powder), fenvalerate (20% emulsifiable concentrate), and bifenthrin (5% emulsifiable concentrate) to 50 mL of inorganic salt medium (without carbon source) respectively, and prepare final concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, 350 mg / L, 400 mg / L, 500 mg / L. Then inoculate 1 mL of the prepared tomato brown rugose fruit virus-induced antibacterial agent sample 1, and culture at 28 °C and 180 r / min for 48 h. Observe the tolerance of Serratia marcescens WF01 to 5 pesticides such as permethrin, deltamethrin, lambda-cyhalothrin, fenvalerate, and bifenthrin. Judgment method: Add different concentrations of permethrin, deltamethrin, lambda-cyhalothrin, fenvalerate, and bifenthrin to the inorganic salt medium for culture, and judge whether Serratia marcescens WF01 can tolerate the pyrethroid pesticides at this concentration by observing whether the inoculated Serratia marcescens WF01 grows in it, that is, whether the culture solution becomes turbid.

[0083] Table 2 Tolerance of Serratia marcescens WF01 to other pyrethroid pesticides

[0084]

[0085] Add pyrethroid pesticides with different concentrations to the MSM inorganic salt medium, and judge whether Serratia marcescens WF01 can tolerate the pyrethroid pesticides at this concentration by observing whether the inoculated Serratia marcescens WF01 grows in it, that is, whether the solution becomes turbid. The test results show that Serratia marcescens WF01 can use the 5 tested pyrethroid pesticides for growth and reproduction, and the highest tolerance concentration is in the range of 400 - 500 mg / L, indicating that it has the potential to widely degrade other pyrethroid pesticides.

[0086] Example VIII: Gene cluster analysis of Serratia marcescens WF01

[0087] This example provides the whole-genome sequencing results of Serratia marcescens WF01 and the analysis results of secondary metabolite gene clusters. The specific methods and results are as follows:

[0088] The whole-genome sequencing of this example was completed by Shanghai Bioengineering Co., Ltd. The second-generation Illumina and third-generation PacBio combined sequencing technology was used, and the sequencing results were subjected to functional analysis against NR, Swiss-Prot, Pfam, EggNOG, GO, and KEGG databases.

[0089] As Figure 6 shown, analysis of the sequencing results revealed that the whole genome of Serratia marcescens WF01 is a circular DNA molecule, assembled into 1 scaffold in total, with a full length of 5,089,176 bp, a GC content of 59.67%, 4,879 predicted coding genes, with a length of 4,767,972 bp and an average length of 937 bp; among them, the genes related to transcription are the most, and the genes related to amino acid transport and metabolism reach 416, and the genes related to carbohydrate transport and metabolism are 333; 468 genes are for general function prediction, related to the biosynthesis, transport, and catabolism of secondary metabolites, etc.

[0090] The abundances of each functional level in KEGG are as Figure 7 shown. The whole genome of Serratia marcescens WF01 has a total of 2,424 genes, which are respectively annotated into five major categories in the KEGG database: cellular processes, metabolism, genetic information processing, organismal systems, and environmental information processing; in the secondary functional classification, a total of 174 metabolic pathways are enriched to 29 KEGG functional classifications, and 16 of them are related to xenobiotic metabolism and degradation (see Table 3), including benzoate metabolism, aminobenzoate metabolism, dioxin metabolism, ethylbenzene metabolism, and the metabolism of the three-class carcinogen atrazine; among them, there are a total of 41 genes regulating xenobiotic metabolism and degradation in strain WF01. These results indicate that WF01 has strong potential for xenobiotic metabolism and degradation and has great application potential in degrading organic pollutants and other aspects.

[0091] Table 3 Pathways related to xenobiotic metabolism and degradation in Serratia marcescens WF01

[0092]

[0093] The results of AntiSMASH analysis showed (Table 4): there are 158 NRPS (non-ribosomal peptide synthetase) genes, indicating stronger stress resistance in harsh environments; there are 32 ribosomally synthesized peptide analog genes, 19 β-lactone genes, 26 prodigiosin genes, and 18 thiopeptide antibiotic genes, which may play a key role in the interaction between plants and microorganisms; there are 19 redox cofactor genes, which play a key role in maintaining intracellular redox homeostasis and have important effects on cell metabolism, growth performance, and biosynthesis ability.

[0094] Table 4 Identification results of secondary metabolite gene clusters of Serratia marcescens WF01

[0095]

[0096] Experimental Example 1: Colonization performance of different Serratia marcescens strains in pepper plants

[0097] This example provides a comparative experiment on the colonization performance of Serratia marcescens WF01 and other existing Serratia marcescens strains (i.e., control strain 1 and control strain 2) in pepper plants. The experimental methods and each treatment are as follows:

[0098] Control group: sterile water;

[0099] Treatment 1: Sample 1 of the tomato brown rugose fruit virus-induced antibacterial agent prepared in Example 3 (0.5×10 9 cfu / mL);

[0100] Treatment 2: Liquid bacterium agent (0.5×10 9 cfu / mL) prepared from control strain 1 (Serratia marcescens CGMCC1.4256, purchased from China General Microbiological Culture Collection Center) using the liquid fermentation medium in Example 3;

[0101] Treatment 3: Liquid bacterium agent (0.5×10 9 cfu / mL) prepared from control strain 2 (Serratia marcescens CICC 10370, purchased from China Center for Industrial Culture Collection) using the liquid fermentation medium in Example 3;

[0102] Select plump and healthy pepper seeds (variety: thread pepper), soak them in cold water for 24 h; under sterile conditions, disinfect the pepper seeds. First, treat them with 75% alcohol for 30 - 60 s, then wash them 3 times with sterile water, then treat them with 3% - 5% NaClO for 8 - 10 min, and finally wash them 8 - 10 times with sterile water; lay the disinfected pepper seeds flat in a tissue culture bottle containing a basal medium (950 mg of potassium nitrate, 825 mg of ammonium nitrate, 85 mg of potassium dihydrogen phosphate, 185 mg of magnesium sulfate, 220 mg of calcium chloride, 0.83 mg of potassium iodide, 6.2 mg of boric acid, 22.30 mg of manganese sulfate, 8.6 mg of zinc sulfate, 0.25 mg of sodium molybdate, 0.025 mg of copper sulfate, 0.025 mg of cobalt chloride, 37.3 mg of disodium ethylenediaminetetraacetate, 27.8 mg of ferrous sulfate, 100 mg of inositol, 2 mg of glycine, 0.1 mg of thiamine hydrochloride, 0.5 mg of pyridoxine hydrochloride, 0.5 mg of nicotinic acid, 30 g of sucrose, 7 g of agar, made up to 1 L with distilled water), and wait for the seeds to germinate into pepper seedlings in 6 d.

[0103] Treatments 1 (effective viable count: 0.5×10 9 cfu / mL), 2 (effective viable count: 0.5×10 9 cfu / mL), and 3 (effective viable count: 0.5×10 9 cfu / mL) were diluted with sterile water to OD600 = 0.8; centrifuged at 10000 rpm for 10 min at 4°C, the supernatant was removed, the cells were collected, and resuspended in 1 mL of sterile water to obtain cell suspensions; under sterile conditions, the cell suspensions were inoculated onto the roots of pepper seedlings respectively. At 12 h, 24 h, and 36 h after inoculation, the roots, stems, and leaves of pepper seedlings treated with each inoculant and non-inoculated (control group) were cut, weighed, placed in 1.5 mL sterile centrifuge tubes, surface disinfected in the same way as pepper seeds; mashed with a sterile glass rod, diluted to different multiples and evenly spread on NA solid medium, and the total number of Serratia marcescens in different parts of pepper was counted. The colony counting method referred to the method for determining the effective viable count in agricultural microbial inoculants in GB20287-2006, and the counting results of Serratia marcescens were counted after culturing at 28°C for 24 h.

[0104] Table 5 Colonization performance of different Serratia marcescens strains in pepper plants

[0105]

[0106] The results are shown in Table 5. Serratia marcescens was not detected in the roots, stems, and leaves of the control group peppers; Serratia marcescens was detected in the roots, stems, and leaves of Treatment 1 (inoculated with Serratia marcescens WF01). Serratia marcescens could be detected in the roots and stems of peppers 12 h after inoculation, and in the leaves 24 h after inoculation, indicating that this strain could enter the roots in a short time and then colonize in the stems and leaves; within 36 h after inoculation, Serratia marcescens was only detected in the roots of Treatment 2 (inoculated with control strain 1), and not detected in the stems and leaves; within 36 h after inoculation, Serratia marcescens was not detected in the roots, stems, and leaves of Treatment 3 (inoculated with control strain 2). The results showed that Serratia marcescens WF01 could quickly colonize in the roots of peppers after being inoculated into the pepper roots, and then quickly transfer and colonize in the stems and leaves of peppers.

[0107] Experimental Example 2: Degradation of beta-cypermethrin by different treatments under indoor soil culture conditions

[0108] This example provides a comparative experiment on the degradation of beta-cypermethrin by Serratia marcescens WF01 and other existing Serratia marcescens strains (control strain 1, control strain 2) under indoor soil culture conditions. The experimental method and each treatment are as follows:

[0109] Control group: Sterile water;

[0110] Treatment 1: Sample 1 of the tomato brown rugose fruit virus-induced antibacterial agent prepared in Example 3 (0.5×10 9 cfu / mL);

[0111] Treatment 2: Liquid bacterium agent prepared with the liquid fermentation medium of Example 3 using control strain 1 (Serratia marcescens CGMCC 1.4256, purchased from China General Microbiological Culture Collection Center) (0.5×10 9 cfu / mL);

[0112] Treatment 3: Liquid bacterium agent prepared with the liquid fermentation medium of Example 3 using control strain 2 (Serratia marcescens CICC 10370, purchased from China Center of Industrial Culture Collection) (0.5×10 9 cfu / mL);

[0113] Take the soil from the field vegetable plot (unbaked and non-sterilized) for indoor simulation tests, with each pot weighing about 5 kg. First, add beta-cypermethrin artificially to the soil to make the beta-cypermethrin content reach 500 mg / kg; after standing for 48 h, take 50 mL of the prepared bacterium agents of different treatments, dilute them 100 times with water respectively, and spray them evenly onto the soil in the form of a spray, stirring evenly while spraying; use the spraying of an equal amount of sterile water as a control, and set 3 replicates for each treatment. Detect the original deposition amount of beta-cypermethrin in the soil 2 h after the application of the bacterium agent, keep the indoor temperature at 20 - 34°C, turn the soil gently regularly, spray and supplement water to keep the soil humidity at 40% - 60%, take the soil of each treatment by the "five-point sampling method" after 30 d, analyze and detect the residual amount of beta-cypermethrin in the soil by the method of Example 5, and calculate the degradation rate (%).

[0114] Degradation rate = (1 - wT / w0)×100%, where wT is the residual amount after 30 d of treatment, and w0 is the original deposition amount.

[0115] Table 6 Degradation rates of beta-cypermethrin under different treatments under indoor soil culture conditions

[0116]

[0117] As shown in Table 6, 30 d after inoculating with Sample 1 of the tomato brown rugose fruit virus-induced antibacterial agent, the residual amount of beta-cypermethrin in the soil of Treatment 1 decreased from 500 mg / L to 124.381 mg / L, and the degradation rate reached 75.28%. The degradation rates of beta-cypermethrin in the soils of Treatment 2 (inoculating with control strain 1) and Treatment 3 (inoculating with control strain 2) were 25.97% and 32.14% respectively, indicating that Serratia marcescens WF01 has a strong degradation effect on beta-cypermethrin.

[0118] Experimental Example 3: Degradation of beta-cypermethrin under field trial conditions with different treatments

[0119] This example provides a degradation test of beta-cypermethrin under field trial conditions with different treatments. The test method and each treatment are set as follows:

[0120] Control group: Sterile water;

[0121] Treatment 1: Sample 1 of the tomato brown rugose fruit virus-induced antibacterial agent prepared in Example 3 (0.5×10 9 cfu / mL);

[0122] Treatment 2: Liquid bacterium agent prepared with the liquid fermentation medium of Example 3 using control strain 1 (Serratia marcescens CGMCC 1.4256, purchased from China General Microbiological Culture Collection Center) (0.5×10 9 cfu / mL);

[0123] Treatment 3: Liquid bacterium agent prepared with the liquid fermentation medium of Example 3 using control strain 2 (Serratia marcescens CICC 10370, purchased from China National Center for Industrial Culture Collection) (0.5×10 9 cfu / mL).

[0124] The test was carried out in a pepper planting greenhouse in Shouguang City, Shandong Province. The pepper variety was linear pepper. Beta-cypermethrin was sprayed during the peak growth period of peppers to kill aphids. The recommended spraying amount was 30 mL / mu (the liquid medicine concentration was 450 mg / L). The experiment was divided into 5 plots, each plot was 20m 2 . After spraying the medicine for 2 hours, pepper leaves and rhizosphere soil were collected by plot to determine the initial deposition amount of beta-cyhalothrin; for each treatment, after spraying the medicine for 1 day, a 50-fold liquid bacterium agent (dosage 5 L / mu) was used for root irrigation treatment. The control group was irrigated with sterile water. After 7 days of bacterium agent root irrigation, pepper leaves and rhizosphere soil were taken respectively to determine the residual amount of beta-cypermethrin. The pepper leaves were reduced by the quartering method, and 1 / 4 of the leaves were taken and put into a pulverizer to be broken into samples, which were put into a self-sealing bag and stored in a -20°C refrigerator. Rhizosphere soil samples: They were reduced by the quartering method, weeds and stones were removed, and then sieved. Samples were made, put into a self-sealing bag, and stored in a -20°C refrigerator.

[0125] The determination method of beta-cypermethrin in leaves referred to GB / T 5009.110-2003 "Determination of Residues of Cypermethrin, Fenvalerate and Deltamethrin in Vegetable Foods". The method of Example 2 was used to determine the content of beta-cypermethrin in the soil. Degradation rate = (1−wT / w0)×100%, where wT is the residual amount after 7 days of treatment, and w0 is the initial deposition amount.

[0126] Table 7 Degradation of beta-cypermethrin under different treatments in field trials

[0127]

[0128] The experimental results show that: 7 days after spraying the pesticide, the degradation rates of beta-cypermethrin in the pepper leaves and soil of the control group were 22.09% and 14.58% respectively. The degradation rates of beta-cypermethrin in the pepper leaves and soil of the first sample of the tomato brown rugose fruit virus-induced antibacterial agent prepared in Example III by root irrigation (Treatment 1) were 76.80% and 78.95% respectively. While the degradation rates of beta-cypermethrin in the pepper leaves and soil of the control bacterial agent 1 (Treatment 2) and the control bacterial agent 2 (Treatment 3) were 22.40% and 6.58%, 14.76% and 11.86% respectively. It shows that root irrigation of peppers with Serratia marcescens WF01 of the present invention can achieve a significant degradation of the residual beta-cypermethrin in the pepper planting soil and peppers.

[0129] Test Example IV:

[0130] This example provides the field control effect of different treatments on tomato brown rugose fruit virus. The test method and each treatment are set as follows:

[0131] Control group: blank control;

[0132] Treatment 1: The first sample of the tomato brown rugose fruit virus-induced antibacterial agent prepared in Example III (0.5×10 9 cfu / mL);

[0133] Treatment 2: The second sample of the tomato brown rugose fruit virus-induced antibacterial agent prepared in Example IV (0.8×10 9 cfu / mL);

[0134] Treatment 3: The third sample of the tomato brown rugose fruit virus-induced antibacterial agent prepared in Example V (1.0×10 9 cfu / mL);

[0135] Treatment 4: The liquid bacterial agent (0.5×10 9 cfu / mL) prepared from the control strain 1 (Serratia marcescens CGMCC1.4256, purchased from China General Microbiological Culture Collection Center) using the liquid fermentation medium of Example III;

[0136] Treatment 5: The liquid bacterial agent (0.5×10 9 cfu / mL) prepared from the control strain 2 (Serratia marcescens CICC 10370, purchased from China Center for Industrial Culture Collection) using the liquid fermentation medium of Example III.

[0137] The experiment was carried out in Weifang City, Shandong Province. The experimental crop was pepper, and the variety was thread pepper. The experimental greenhouse was a pepper planting greenhouse where tomato brown rugose fruit virus disease occurred and had not been disinfected. The plot area was 72 m 2 , arranged in a randomized block design with 4 replications. The fertility of the experimental field was uniform, the planting and management levels were the same, and the incidence and damage degree of the disease were serious. The pepper seeds were raised in seedlings, and when they grew to 2 true leaves, they were transplanted into the experimental greenhouse. At the transplanting stage and the early flowering stage, the root irrigation of each treatment with the 50-fold liquid of the bacterial agent (dosage 5 L / mu) was carried out, and protective rows were set between each treatment and around the experimental area. When obvious symptoms appeared in the control group, the incidence rate was investigated. In each plot, 50 plants were continuously investigated along the ridges in a fixed-plant manner, and the grading records were made in units of plants. The disease index was statistically calculated, and the control effect was calculated. The disease symptom grading standard of tomato brown rugose fruit virus disease was used to investigate the disease occurrence of diseased plants, and the disease index and relative control effect of each treatment were calculated.

[0138] Disease symptom grading standard of tomato brown rugose fruit virus disease:

[0139] Grade 0: No symptoms; Grade 1: The veins of the heart leaves are clear veins, and 1-2 true leaves show mosaic; Grade 3: Mosaic on the upper and middle leaves; Grade 5: Mosaic on most leaves, and a few leaves are deformed; Grade 7: Most leaves are severely mosaic, deformed, and wrinkled; Grade 9: Almost all leaves are severely mosaic, deformed, and wrinkled, and the plant is significantly dwarfed. The calculation formulas for the disease index and relative control effect are as follows:

[0140] Disease index = ∑(number of diseased plants at each level × the value of that disease level) / (total number of investigated plants × the highest level value) × 100

[0141] Relative control effect = (disease index of the control - disease index of the treatment) / disease index of the control × 100%.

[0142] Table 8 Field control effect of different treatments on tomato brown rugose fruit virus disease

[0143]

[0144] As can be seen from Table 8, Treatment 1 (tomato brown rugose fruit virus-induced antibacterial agent sample 1), Treatment 2 (tomato brown rugose fruit virus-induced antibacterial agent sample 2), and Treatment 3 (tomato brown rugose fruit virus-induced antibacterial agent sample 3) had good control effects on tomato brown rugose fruit virus occurring on field pepper crops, and the relative control effects could reach 71.26% - 76.92%, which were significantly better than Treatment 4 (control strain 1) and Treatment 5 (control strain 2); it was shown that the Serratia marcescens WF01 of the present invention could effectively control tomato brown rugose fruit virus disease by root irrigation during the growth period of pepper.

[0145] Only some exemplary embodiments of the present invention have been described by way of illustration. Undoubtedly, for those of ordinary skill in the art, the described embodiments can be modified in various different ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. Serratia marcescens WF01 for degrading pesticides, characterized in that: The Serratia marcescens WF01 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on April 17, 2024, with the deposit number CGMCC NO. 30368, and the deposit address is Beichen West Road, Chaoyang District, Beijing, and the taxonomic name is Serratia marcescens Serratia marcescens 。 2. Tomato brown rugose fruit virus-induced antibacterial agent, characterized in that: The tomato brown rugose fruit virus-induced antibacterial agent contains Serratia marcescens WF01 for degrading pesticides as described in claim 1.

3. The tomato brown rugose fruit virus-induced antibacterial agent according to claim 2, characterized in that: The tomato brown rugose fruit virus-induced antibacterial agent has the effect of degrading pyrethroid insecticides.

4. The tomato brown rugose fruit virus-induced antibacterial agent according to claim 3, characterized in that: The active ingredient of the pyrethroid insecticide is beta-cypermethrin or lambda-cyhalothrin or cypermethrin.

5. The tomato brown rugose fruit virus-induced antibacterial agent according to claim 4, characterized in that: The beta-cypermethrin is the beta-cypermethrin remaining in the pepper planting soil and / or in the pepper plant.

6. The tomato brown rugose fruit virus-induced antibacterial agent according to claim 2, wherein: The tomato brown rugose fruit virus-induced antibacterial agent is used during the growth period of peppers and is applied by root irrigation.

7. A method for preparing the tomato brown rugose fruit virus-induced antibacterial agent as described in claim 2, characterized in that: Streak the Serratia marcescens WF01 onto a solid medium with beta-cypermethrin as the sole carbon source and incubate it inverted at 32°C for 36 - 40 h; pick a bright red single colony and inoculate it into a liquid medium with beta-cypermethrin as the sole carbon source, and shake culture it at 32°C and 185 r / min for 48 h to obtain the seed liquid of Serratia marcescens WF01; inoculate the seed liquid into a liquid medium without any carbon source at a volume ratio of 10%, shake culture it at 28°C and 185 r / min for 3 - 5 h, then add yeast extract, and continue to shake culture it at 28°C and 200 r / min for 48 h to obtain the tomato brown rugose fruit virus-induced antibacterial agent, and the tomato brown rugose fruit virus-induced antibacterial agent is a deep red fermentation broth of Serratia marcescens WF01.

8. The method according to claim 7, characterized in that: The content of prodigiosin in the antibacterial agent against tomato brown rugose fruit virus reaches 13.5 - 17.2 g / L, and the effective viable bacteria count is 0.5×10 9 cfu / mL - 1.0×10 9 cfu / mL.

9. The method according to claim 8, wherein: The tomato brown rugose fruit virus-induced antibacterial agent can prevent and control tomato brown rugose fruit virus disease.

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