A Streptomyces synbiotic for controlling soil-borne Ralstonia solanacearum and its application

Abstract

The present invention discloses a Streptomyces symbiotic composite for preventing and controlling soil-borne Ralstonia solanacearum, which is composed of at least one selected from Streptomyces strain SBL, Streptomyces strain SHS or Streptomyces strain SNP and at least one selected from L-citrulline, tannic acid, ascorbic acid, glutathione, spermidine, L-methionine, L-phenylalanine or L-alanine. The Streptomyces strain SBL has a deposit number of CGMCC No. 25283. The Streptomyces strain SHS has a deposit number of CGMCC No. 25281. The Streptomyces strain SNP has a deposit number of CGMCC No. 25282. The present invention also discloses a method for preventing and controlling tomato soil-borne bacterial wilt by using a resource substance in combination with Streptomyces, including: applying the Streptomyces symbiotic composite into the soil by the root irrigation method. The Streptomyces symbiotic composite can reduce the incidence of tomato bacterial wilt and prevent and control soil-borne bacterial wilt.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and relates to Streptomyces strains SBL, SHS, SNP, and the application of Streptomyces strains SBL, SHS, or SNP in controlling Ralstonia solanacearum. Specifically, it relates to a Streptomyces synbiotic for controlling soil-borne Ralstonia solanacearum, and the application of the Streptomyces synbiotic in controlling tomato bacterial wilt. Specifically, it relates to the application of L-citrulline or tannic acid in combination with Streptomyces SBL, ascorbic acid (VC), glutathione, or spermidine in combination with Streptomyces SHS, L-methionine, L-phenylalanine, or L-alanine in combination with Streptomyces SNP in controlling soil-borne bacterial wilt. Background Art

[0002] Bacterial wilt is a soil-borne disease caused by Ralstonia solanacearum (hereinafter referred to as Ralstonia solanacearum), which mostly occurs in the eastern and southern regions of China. It can cause irreversible wilting and death of crops such as tomatoes, eggplants, and tobacco, resulting in serious crop yield reduction and restricting the development of agricultural economy. Traditional methods such as applying pesticides or fumigation can kill soil-borne Ralstonia solanacearum in a short time and effectively slow down the occurrence of the disease. However, these methods will also kill indigenous microorganisms in the soil, reducing soil species diversity or abundance.

[0003] Currently, applying Streptomyces is one of the relatively green and sustainable measures for controlling bacterial wilt, which can inhibit the growth of soil-borne Ralstonia solanacearum and reduce the occurrence of the disease to a certain extent. During the process of screening Streptomyces and verifying its antibacterial effect, people often use culture media with relatively rich nutrients, and Streptomyces can proliferate in large numbers or show good antibacterial ability. However, the types and quantities of resources in the soil are relatively limited. After Streptomyces is applied to the soil, it cannot obtain sufficient and appropriate resources, resulting in slowed growth or even inability to survive in the soil environment, and its biocontrol efficiency will inevitably decline.

[0004] Therefore, it is necessary to find resources that can promote the growth of Streptomyces, provide a suitable growth resource environment for Streptomyces in the soil, and enhance its ability to inhibit soil-borne Ralstonia solanacearum. The inventor intends to screen resource substances that can efficiently improve the control of soil-borne bacterial wilt by Streptomyces, and apply Streptomyces in combination with resource substances (synbiotic) to the soil to improve the control effect on soil-borne bacterial wilt. Summary of the Invention

[0005] In view of the problem that when controlling soil-borne diseases by applying Streptomyces, Streptomyces is often difficult to multiply in large numbers in the soil and exert its due control effect due to limited nutrients in the soil, the inventors found through experiments that in an indoor microplate system, L-citrulline can significantly promote the growth of Streptomyces SBL and enhance its inhibitory ability against Ralstonia solanacearum, ascorbic acid (VC) can significantly promote the growth of Streptomyces SHS and enhance its inhibitory ability against Ralstonia solanacearum, and L-methionine can significantly promote the growth of Streptomyces SNP and enhance its inhibitory ability against Ralstonia solanacearum; in greenhouse pot experiments, when L-citrulline is used in combination with Streptomyces SBL, ascorbic acid is used in combination with Streptomyces SHS, and L-methionine is used in combination with Streptomyces SNP, and at the same time, they are applied into the soil by the root irrigation method, the disease index of tomato soil-borne bacterial wilt can be reduced. It shows that the combined application of L-citrulline and Streptomyces SBL or the combined application of ascorbic acid and Streptomyces SHS or the combined application of L-methionine and Streptomyces SNP can all reduce the incidence of tomato bacterial wilt and control tomato soil-borne bacterial wilt.

[0006] The object of the present invention is to provide the application of Streptomyces strains SBL, SHS, SNP and Streptomyces strains SBL, SHS or SNP in controlling Ralstonia solanacearum, as well as a Streptomyces synbiota for controlling soil-borne Ralstonia solanacearum containing the above Streptomyces, and the application of the Streptomyces synbiota in controlling tomato bacterial wilt.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] Streptomyces strain SBL, classified and named as Streptomyces bellus, was deposited in the General Microbiology Center of the China Microbial Culture Collection Center on July 11, 2022, at the address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is CGMCC No. 25283.

[0009] Streptomyces strain SHS, classified and named as Streptomyces torulosus, was deposited in the General Microbiology Center of the China Microbial Culture Collection Center on July 11, 2022, at the address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is CGMCC No. 25281.

[0010] Streptomyces strain SNP, classified and named as Streptomyces rishiriensis, was deposited in the General Microbiology Center of the China Microbial Culture Collection Center on July 11, 2022, at the address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is CGMCC No. 25282.

[0011] Another object of the present invention is to provide the application of the Streptomyces strain SBL, the Streptomyces strain SHS or the Streptomyces strain SNP in controlling soil-borne bacterial wilt.

[0012] Another object of the present invention is to provide the application of the combination of the Streptomyces strain SBL, the Streptomyces strain SHS or the Streptomyces strain SNP and a resource substance in controlling soil-borne bacterial wilt.

[0013] The resource substance is L-citrulline, tannic acid, ascorbic acid (VC), glutathione, spermidine, L-methionine, L-phenylalanine or L-alanine.

[0014] The application is the application of the combined application of L-citrulline or tannic acid and Streptomyces SBL, ascorbic acid, glutathione or spermidine and Streptomyces SHS, L-methionine, L-phenylalanine or L-alanine and Streptomyces SNP in controlling soil-borne bacterial wilt.

[0015] Preferably, the application is to apply L-citrulline or tannic acid and Streptomyces SBL, ascorbic acid, glutathione or spermidine and Streptomyces SHS, L-methionine, L-phenylalanine or L-alanine and Streptomyces SNP into the soil by the root irrigation method.

[0016] More preferably, the application is to apply L-citrulline and Streptomyces SBL, ascorbic acid and Streptomyces SHS, L-methionine and Streptomyces SNP into the soil by the root irrigation method.

[0017] The soil-borne bacterial wilt is tomato soil-borne bacterial wilt, eggplant soil-borne bacterial wilt, tobacco soil-borne bacterial wilt.

[0018] Another object of the present invention is to provide a Streptomyces synbiotic for controlling soil-borne Ralstonia solanacearum, which is composed of at least one selected from the Streptomyces strain SBL, the Streptomyces strain SHS or the Streptomyces strain SNP and at least one selected from L-citrulline tannic acid, ascorbic acid, glutathione, spermidine, L-methionine, L-phenylalanine or L-alanine.

[0019] Preferably, the Streptomyces synbiotic is applied by the root irrigation method, and 0.05-0.15 mmol of the resource substance and 5 mL-15 mL of a Streptomyces spore suspension with a concentration of 1×10 7 ~1×10 9 CFU / mL are applied to each tomato seedling.

[0020] Preferably, the Streptomyces synbiotic for controlling soil-borne Ralstonia solanacearum is composed of L-citrulline and Streptomyces SBL, or composed of ascorbic acid and Streptomyces SHS, or composed of L-methionine and Streptomyces SNP.

[0021] Another object of the present invention is to provide a method for preventing and controlling soil-borne bacterial wilt of tomatoes by combining resource substances and Streptomyces, comprising: applying the Streptomyces synbiotic into the soil by root irrigation; wherein, 0.05-0.15 mmol of resource substances and 5 mL-15 mL of 1×10 7 ~1×10 9 CFU / mL of Streptomyces spore suspension.

[0022] Preferably, 7 to 10 days after the tomato seedlings with 3 to 5 true leaves are transplanted, L-citrulline and a spore suspension of Streptomyces SBL, ascorbic acid and a spore suspension of Streptomyces SHS, or L-methionine and a spore suspension of Streptomyces SNP are applied to the soil by root irrigation.

[0023] Preferably, 0.1 mmol of resource material and 10 mL of 1×10 8 CFU / mL of Streptomyces spore suspension.

[0024] The resource material is applied in the form of a solution. Generally, the concentration of the resource material solution is 0.1 mol / L. The preparation method of the resource material solution is: the resource material is dissolved in deionized water or warm water, and sucked with a sterile syringe, filtered with a 0.22 μm filter membrane, and the resource material solution is obtained.

[0025] The Streptomyces spore suspension is Streptomyces SBL spore suspension, Streptomyces SBL spore suspension or Streptomyces SHS spore suspension.

[0026] The concentration of the Streptomyces SBL spore suspension is 1×10 7 ~10 9 CFU / mL, preferably 1×10 8 CFU / mL; the concentration of Streptomyces SHS spore suspension is 1×10 7 ~10 9 CFU / mL, preferably 1×10 8 CFU / mL; the concentration of Streptomyces SNP spore suspension is 1×10 7 ~10 9 CFU / mL, preferably 1×10 8 CFU / mL.

[0027] The Streptomyces SBL spore suspension is prepared by the following method: the strain SBL is activated by using M1 solid culture medium; a single SBL colony is picked up and placed in M1 liquid culture medium, and cultured at 30°C and 170rpm for 3d; 100μL of fresh bacterial liquid is spread on ISP3 solid culture medium, and cultured at 30°C for 5d to 7d, and the bacterial bodies are scraped and placed in sterile physiological saline; and the concentration of the bacterial liquid is adjusted by using sterile physiological saline to obtain the Streptomyces SBL spore suspension.

[0028] The Streptomyces SHS spore suspension is prepared by the following method: The strain SHS is activated using M1 solid medium; a single colony of SHS is picked and inoculated into M1 liquid medium, and cultured at 30 °C and 170 rpm for 3 days; 100 μL of fresh bacterial liquid is spread on ISP3 solid medium and cultured at 30 °C for 5 to 7 days, and the bacterial cells are scraped into sterile physiological saline; the concentration of the bacterial liquid is adjusted with sterile physiological saline to obtain the Streptomyces SHS spore suspension.

[0029] The Streptomyces SNP spore suspension is prepared by the following method: The strain SNP is activated using M1 solid medium; a single colony of SNP is picked and inoculated into M1 liquid medium, and cultured at 30 °C and 170 rpm for 3 days; 100 μL of fresh bacterial liquid is spread on ISP3 solid medium and cultured at 30 °C for 5 to 7 days, and the bacterial cells are scraped into sterile physiological saline; the concentration of the bacterial liquid is adjusted with sterile physiological saline to obtain the Streptomyces SNP spore suspension.

[0030] Advantages of the present invention:

[0031] The present invention combines L-citrulline with Streptomyces SBL, ascorbic acid with Streptomyces SHS, or L-methionine with Streptomyces SNP to form a streptomycin synbiotic for controlling soil-borne Ralstonia solanacearum, which can significantly promote the growth of Streptomyces and enhance its inhibitory ability against Ralstonia solanacearum. At the same time, it can reduce the incidence of tomato bacterial wilt and control soil-borne bacterial wilt. Description of the Drawings

[0032] Figure 1 Shows the effects of L-citrulline on the growth of Streptomyces SBL, ascorbic acid VC on the growth of Streptomyces SHS, and L-methionine on the growth of Streptomyces SNP.

[0033] Figure 2 Shows the effects of L-citrulline on the growth inhibition of soil-borne Ralstonia solanacearum by Streptomyces SBL, ascorbic acid VC on the growth inhibition of soil-borne Ralstonia solanacearum by Streptomyces SHS, and L-methionine on the growth inhibition of soil-borne Ralstonia solanacearum by Streptomyces SNP; the dotted line in the figure represents the treatment with only Ralstonia solanacearum added.

[0034] Figure 3 Shows the effects of L-citrulline, ascorbic acid VC, and L-methionine on the disease index of tomato soil-borne bacterial wilt respectively; the dotted line in the figure represents the treatment with only Ralstonia solanacearum added.

[0035] Biological Material Preservation Information

[0036] SHS, classified and named as Streptomyces torulosus, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 11, 2022. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number of the strain is CGMCC No. 25281.

[0037] SNP, taxonomically named Streptomyces rishiriensis, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 11, 2022. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number of the strain is CGMCC No. 25282.

[0038] SBL, taxonomically named Streptomyces bellus, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 11, 2022. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number of the strain is CGMCC No. 25283. Specific Embodiments

[0039] The technical solutions of the present invention will be further described below in conjunction with specific embodiments.

[0040] M1 Liquid Medium: 20.0 g of yeast powder, 1.5 g of KH2PO4, 0.5 g of MgSO4·7H2O, made up to 1 L with distilled water, pH 8.0, sterilized at 115 °C for 30 min.

[0041] M1 Solid Medium: 20.0 g of yeast powder, 1.5 g of KH2PO4, 0.5 g of MgSO4·7H2O, 20 - 30 g of agar strips, made up to 1 L with distilled water, pH 8.0, sterilized at 115 °C for 30 min.

[0042] ISP3 Solid Medium: 20.0 g of oatmeal, 0.2 g of MgSO4, 0.2 g of KNO3, 0.5 g of K2HPO4, 20.0 g of agar powder, 1000 mL of deionized water, pH 7.5, sterilized at 115 °C for 30 min.

[0043] ISP3 Liquid Medium: 20.0 g of oatmeal, 0.2 g of MgSO4, 0.2 g of KNO3, 0.5 g of K2HPO4, 1000 mL of deionized water, pH 7.5, sterilized at 115 °C for 30 min.

[0044] NA Liquid Medium: 3 g / L of beef extract, 0.5 g / L of yeast powder, 5 g / L of peptone, 10 g / L of glucose, made up to 1 L with deionized water, sterilized at 115 °C for 30 min.

[0045] NA Solid Medium: 15 g / L of agar was added to the NA solid medium and sterilized at 115 °C for 30 min.

[0046] Experimental Example 1

[0047] Isolation, Screening and Identification of Streptomyces

[0048] Isolation of Streptomyces griseus from soil

[0049] The dilution plate coating method was used to isolate Streptomyces. Isolation of Streptomyces: Measure 90 mL of distilled water and add it to a 250 mL conical flask (put 10 - 12 glass beads in the flask), sterilize at 121 °C for 30 min. Take 10 g of tomato rhizosphere soil and put it into the above conical flask, sterilize by shaking at 28 °C and 180 r / min for 30 min. Use a pipette to aspirate 1 mL of the soil dilution solution with a -1 10-fold gradient and transfer it into a test tube containing 9 mL of sterile water, mix well, and sequentially dilute it by gradient to obtain soil suspensions with different dilution degrees. Aspirate 100 μL of the soil suspension and spread it on an ISP3 plate, culture at 28 °C for 5 days, then pick out the Streptomyces colonies for purification and preservation on a slant.

[0050] Screening and identification of antagonistic Streptomyces

[0051] The plate confrontation method was used to determine the antibacterial activity of the isolated Streptomyces, and the strain with the best effect was screened out. After culturing the isolated Streptomyces for 5 days, use a puncher (Φ = 6 mm) to punch out bacterial cakes, transfer them to the center of a plate containing NA medium, and culture at 28 °C for 2 days. Shake-flask culture Ralstonia solanacearum in NA liquid medium, measure the OD 600 value after 24 h, prepare a bacterial suspension with sterile water (OD 600 = 1.0), use a sterile atomizer to evenly spray the bacterial solution on the plate, and measure the diameter of the inhibition zone after culturing at 30 °C for 24 h. Strains with good antagonistic effects were screened out: strain SHS from the tomato rhizosphere soil of Wuquanshan, Lanzhou City (120.46°E, 49.10°N), strain SNP from the tomato rhizosphere soil of Wuquanshan, Lanzhou City (120.46°E, 49.10°N), strain SBL from the tomato rhizosphere soil of Qiyan Mountain, Erguna City (120.76°E, 52.19°N), and 16S rRNA identification was carried out.

[0052] Strain SHS, classified and named as Streptomyces torulosus, was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 11, 2022. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The strain deposit number is CGMCC No. 25281.

[0053] Strain SNP, classified and named as Streptomyces rishiriensis, was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 11, 2022. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The strain deposit number is CGMCC No. 25282.

[0054] The strain SBL, classified and named as Streptomyces bellus, was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 11, 2022. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number of the strain is CGMCC No. 25283.

[0055] Example 2

[0056] Investigate the indoor effect of resource substances on the growth of Streptomyces

[0057] The strain SBL was activated with M1 solid medium; a single colony of SBL was picked and inoculated into M1 liquid medium, and cultured at 30 °C and 170 rpm for 3 days; 100 μL of fresh bacterial liquid was spread on ISP3 solid medium and cultured at 30 °C for 5 days. The bacterial cells were scraped into sterile physiological saline; the concentration of the bacterial liquid was adjusted to 1×10 8 CFU / mL with sterile physiological saline to obtain a spore suspension of Streptomyces SBL.

[0058] The strain SHS was activated with M1 solid medium; a single colony of SHS was picked and inoculated into M1 liquid medium, and cultured at 30 °C and 170 rpm for 3 days; 100 μL of fresh bacterial liquid was spread on ISP3 solid medium and cultured at 30 °C for 5 days. The bacterial cells were scraped into sterile physiological saline; the concentration of the bacterial liquid was adjusted to 1×10 8 CFU / mL with sterile physiological saline to obtain a spore suspension of Streptomyces SHS.

[0059] The strain SNP was activated with M1 solid medium; a single colony of SNP was picked and inoculated into M1 liquid medium, and cultured at 30 °C and 170 rpm for 3 days; 100 μL of fresh bacterial liquid was spread on ISP3 solid medium and cultured at 30 °C for 5 days. The bacterial cells were scraped into sterile physiological saline; the concentration of the bacterial liquid was adjusted to 1×10 8 CFU / mL with sterile physiological saline to obtain a spore suspension of Streptomyces SNP.

[0060] Preparation of the mother liquor of resource substances: Each resource substance was accurately weighed with a ten-thousandth balance, dissolved with deionized water or hot water, and aspirated with a sterile syringe, then filtered through a 0.22 μm filter membrane into a 5 mL sterile centrifuge tube to obtain a sterile mother liquor of resource substances with a concentration of 1 mM, which was stored in a -4 °C refrigerator for later use.

[0061] Experimental setup: Experimental group: Add resource substances and Streptomyces SBL or Streptomyces SHS or Streptomyces SNP; Control group 1: Only add Streptomyces SBL and replace the resource substances with an equal volume of water; Control group 2: Only add Streptomyces SHS and replace the resource substances with an equal volume of water; Control group 3: Only add Streptomyces SNP and replace the resource substances with an equal volume of water.

[0062] Add 1335 μL of M1 liquid medium into a 24-well plate (Costar), and add 15 μL of Streptomyces spore suspension (final concentration is 1×10 6 CFU / mL). Add 150 μL of the resource stock solution (final concentration is 0.1 mM) to the experimental group, and add an equal volume of sterile water to the control group. Each treatment has 4 replicates. After adding the samples, place the 24-well plate in a constant temperature (30 °C) shaker and shake continuously at 170 r / min for 5 days. Transfer it to a 2 mL sterile centrifuge tube and dry it in an oven. Use a ten-thousandth balance to calculate the biomass of Streptomyces.

[0063] The promoting effect of resource substances on the growth of Streptomyces SBL can be seen in Figure 1 and Table 1. The promoting effect of resource substances on the growth of Streptomyces SHS can be seen in Figure 1 and Table 2. The promoting effect of resource substances on the growth of Streptomyces SNP can be seen in Figure 1 and Table 3. The results show that: compared with the control group, L-citrulline can significantly promote the increase in the biomass of Streptomyces SBL (P<0.05), ascorbic acid (VC) can significantly promote the increase in the biomass of Streptomyces SHS (P<0.05), and L-methionine can significantly promote the increase in the biomass of Streptomyces SNP (P<0.05).

[0064] Table 1. Effects of resource substances on the biomass of Streptomyces SBL

[0065]

[0066] Note: Different letters in the same column in the table indicate a significant level of 0.05. The same applies hereinafter.

[0067] Table 2. Effects of resource substances on the biomass of Streptomyces SHS

[0068]

[0069] Table 3. Effects of resource substances on the biomass of Streptomyces SNP

[0070]

[0071] Example 3

[0072] Investigate the indoor effect of resource substances on Streptomyces inhibiting soil-borne Ralstonia solanacearum

[0073] Material: The Ralstonia solanacearum strain QL-Rs1115 isolated from Qilin Town, Nanjing (Wei Z, Yang XM, Yin SX, et al. Efficacy of Bacillus-fortified organic fertiliser in controlling bacterial wilt of tomato in the field[J]. Applied Soil Ecology, 2011, 48, 152-159.). The red fluorescent protein gene mCherry was transferred into the Ralstonia solanacearum strain QL-Rs1115 through the plasmid pYC12 to obtain the red fluorescent protein-labeled strain RS-RFP. This strain is the model invasive pathogen of the present invention and can grow on NA medium plates containing 30 μg / mL gentamicin (Yang T, Wei Z, Friman V-P, et al. Resource availability modulates biodiversity-invasion relationships by altering competitive interactions[J]. Environmental Microbiology, 2017, 19(8): 2984-2991).

[0074] For the preparation of the Streptomyces SBL spore suspension, Streptomyces SHS spore suspension, and Streptomyces SNP spore suspension, see Example 2.

[0075] The Ralstonia solanacearum RS-RFP was resuscitated on NA solid medium containing 30 μg / mL gentamicin. The plate was placed in an incubator at 30 °C for 2 d until single colonies appeared; single colonies on the plate were picked and transferred to NA liquid medium, and cultured overnight at 30 °C and 170 rpm; fresh bacterial liquid was taken, centrifuged at room temperature at 4500 rpm for 5 min, the cells were collected, washed 3 times with sterile normal saline (0.85% NaCl) to remove the medium, and the concentration of the Ralstonia solanacearum bacterial liquid was adjusted to OD 600 = 0.5 to obtain the Ralstonia solanacearum RS-RFP suspension for use.

[0076] Experimental treatment settings: Experimental group: Streptomyces SBL or SHS or SNP + Resource substance group: The fermentation broth of Streptomyces SBL or SHS or SNP cultured with the addition of resource substances and Ralstonia solanacearum; Control group: Only adding SBL or SHS or SNP group: The fermentation broth of Streptomyces SBL or SHS or SNP cultured without the addition of resource substances and Ralstonia solanacearum, and an equal volume of water was used to replace the resource substances; Blank control group: No Streptomyces fermentation broth was added, only Ralstonia solanacearum and M1 liquid medium were added, and an equal volume of water was used to replace the fermentation broth.

[0077] Add 1335 μL of M1 liquid medium into a 24-well plate (Costar), and add 15 μL of streptomycete spore suspension (final concentration is 1×10 6 CFU / mL). Add 150 μL of the stock solution of the resource substance (final concentration is 0.1 mM) to the experimental group, and add an equal volume of sterile water to the control group. Each treatment has 4 replicates. After adding the samples, place the 24-well plate in a constant temperature (30 °C) shaker and shake continuously at 170 r / min for 5 days. Filter with a sterile 0.22 μm aqueous filter membrane to collect the sterile streptomycete fermentation broth of the experimental group and the sterile streptomycete fermentation broth of the control group.

[0078] Add 20 μL of the sterile streptomycete fermentation broth of the experimental group into a 96-well cell culture plate, and at the same time add 2 μL of the suspension of Ralstonia solanacearum RS-RFP (OD 600 = 0.5), and add 178 μL of M1 liquid medium to make the initial OD of Ralstonia solanacearum in the 96-well cell culture plate 600 value be 0.05. The control group adds 20 μL of the sterile streptomycete fermentation broth of the control group and 2 μL of the suspension of Ralstonia solanacearum RS-RFP (OD 600 = 0.5), and add 178 μL of M1 liquid medium to make the initial OD of Ralstonia solanacearum in the 96-well cell culture plate 600 value be 0.05; Blank control group: without adding fermentation broth, add 2 μL of the suspension of Ralstonia solanacearum RS-RFP, 20 μL of sterile water and 178 μL of M1 liquid medium. Each treatment is set with four replicates. Place the 96-well cell culture plate in a constant temperature shaker at 30 °C and continuously culture at a rotation speed of 170 rpm for 48 h. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the optical density OD 600 value to judge the effect of the streptomycete fermentation broth on Ralstonia solanacearum RS-RFP.

[0079] The results are shown in Figure 2 and Tables 4 - 6. Compared with the control group, L-citrulline can significantly enhance the ability of Streptomyces SBL to inhibit Ralstonia solanacearum (P < 0.05), ascorbic acid can significantly enhance the ability of Streptomyces SHS to inhibit Ralstonia solanacearum (P < 0.05), and L-methionine can significantly enhance the ability of Streptomyces SNP to inhibit Ralstonia solanacearum (P < 0.05). Compared with other resource substances, L-citrulline has the best effect on improving the antibacterial ability of Streptomyces SBL (the lowest biomass of Ralstonia solanacearum, OD 600 = 0.28), ascorbic acid (VC) has the best effect on improving the antibacterial ability of Streptomyces SHS (the lowest biomass of Ralstonia solanacearum, OD 600 = 0.53), and L-methionine has the best effect on improving the antibacterial ability of Streptomyces SNP (the lowest biomass of Ralstonia solanacearum, OD 600 = 0.39).

[0080] Table 4. Effects of Resource Substances on the Ability of Streptomyces SBL to Inhibit the Growth of Ralstonia solanacearum

[0081]

[0082] Table 5. Effects of Resource Substances on the Ability of Streptomyces SHS to Inhibit Ralstonia solanacearum

[0083]

[0084] Table 6. Effects of Resource Substances on the Ability of Streptomyces SNP to Inhibit Ralstonia solanacearum

[0085]

[0086]

[0087] Example 4

[0088] Effects of Resource Substances on the Reduction of Tomato Disease Index by Streptomyces

[0089] According to the ability of resource substances to promote the growth of Streptomyces and assist Streptomyces in inhibiting Ralstonia solanacearum, the relatively excellent resource substances were screened out (Streptomyces SBL, resource substances: L-citrulline, L-pyroglutamic acid, tannic acid; Streptomyces SHS, resource substances: ascorbic acid, glutathione (reduced form), spermidine; Streptomyces SNP, resource substances: L-phenylalanine, L-alanine, L-methionine), and further investigated the effects of the above resource substances on the reduction of tomato disease index by Streptomyces.

[0090] The preparation of Streptomyces SBL spore suspension, Streptomyces SHS spore suspension, and Streptomyces SNP spore suspension is shown in Example 2.

[0091] Inoculate Ralstonia solanacearum QL-Rs1115 into NA liquid medium and shake culture at 30 °C and 170 rpm for 48 h; take the fresh bacterial liquid, centrifuge at room temperature and 4500 rpm for 5 min, collect the bacterial cells, wash the bacterial cells 3 times with sterile normal saline (0.85% NaCl) to remove the medium, and finally adjust the bacterial liquid concentration to about 1×10 7 CFU / mL to obtain the Ralstonia solanacearum QL-Rs1115 suspension for use.

[0092] Tomato variety: Red Dwarf Tomato

[0093] After surface sterilization, tomato seeds were placed on a sterile petri dish lined with filter paper moistened with sterile deionized water and germinated at 30 °C for 2 days. Seeds with uniform germination were planted into a 50-hole seedling tray filled with substrate; after the tomato plants grew 3 true leaves, seedlings with consistent growth vigor were transplanted into a 6-hole seedling tray containing 100 g of substrate. Seven days after seedling transplantation, Streptomyces and resource substances were evenly poured into the soil around the tomato roots using the root irrigation method. Each tomato seedling was inoculated with 1 mL of 0.1 mol / L resource substance solution (so that the amount of resource substance applied to each tomato seedling was 0.1 mmol), and at the same time, 10 mL of a Streptomyces spore suspension with a concentration of 10 8 CFU / mL was inoculated (so that each tomato seedling was inoculated with 10 9 CFU of Streptomyces); The following treatments were set up in the experiment: 1) SBL + resource substance group: resource substance, Streptomyces SBL spore suspension and Ralstonia solanacearum were added; 2) SBL only group: only Streptomyces SBL spore suspension and Ralstonia solanacearum were added, and the resource substance was replaced with an equal volume of water; 3) SHS + resource substance group: resource substance, Streptomyces SHS spore suspension and Ralstonia solanacearum; 4) SHS only group: only Streptomyces SHS spore suspension and Ralstonia solanacearum were added, and the resource substance was replaced with an equal volume of water; 5) SNP + resource substance group: resource substance, Streptomyces SNP spore suspension and Ralstonia solanacearum; 6) SNP only group: only Streptomyces SNP spore suspension and Ralstonia solanacearum were added, and the resource substance was replaced with an equal volume of water. 7) Control group: only an equal amount of Ralstonia solanacearum was added, and the resource substance and Streptomyces were replaced with an equal volume of water. Seven days later, each tomato seedling was also inoculated with 10 mL of a Ralstonia solanacearum suspension with a concentration of 1×10 7 CFU / mL (so that each tomato seedling was inoculated with 10 8 CFU of Ralstonia solanacearum). Once the tomato plants showed disease symptoms after inoculation with Ralstonia solanacearum, the disease incidence was recorded. Based on the results of Examples 2 and 3, the 3 resource substances with the best relative effects were selected for each strain for the pot experiment.

[0094] The plant disease severity levels are divided into 5 levels (Ciampi - Panno L, Fernandez C, Bustamante P, et al. Biological control of bacterial wilt of potatoes caused by Pseudomonas solanacearum[J]. American Potato Journal, 1989, 66(5):315 - 332.): 0 = no disease symptoms, 1 = disease symptoms appear in 1% - 25% of the plant parts, 2 = disease symptoms appear in 26% - 50% of the plant parts, 3 = disease symptoms appear in 51% - 75% of the plant parts, 4 = disease symptoms appear in 76% - 100% of the plant parts or the plant has died. The disease index is used to characterize the disease occurrence, and the calculation formula is (Kempe J. Biological control of bacterial wilt of potatoes: attempts to induce resistance by treating tubers with bacteria.[J]. Plant Disease, 1983, 67(5):499 - 503):

[0095] Plant disease index = [∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × highest level value)]

[0096] The results are as Figure 3 shown in Table 7 - Table 9, and it is found that: the combined use of L - citrulline or tannic acid and Streptomyces SBL, the combined use of ascorbic acid, glutathione (reduced form) or spermidine and Streptomyces SHS, and the combined use of L - methionine, L - phenylalanine or L - alanine and Streptomyces SNP all have significantly better control effects on tomato soil - borne bacterial wilt than the use of a single Streptomyces; especially compared with other resource substances, the addition of resource substances L - citrulline, ascorbic acid, and ascorbic acid can significantly reduce the disease index of tomato soil - borne bacterial wilt (P < 0.05) and reduce the severity of bacterial wilt.

[0097] Table 7. Effects of resource substances on reducing the disease index of tomatoes by Streptomyces SBL

[0098]

[0099] Table 8. Effects of resource substances on reducing the disease index of tomatoes by Streptomyces SHS

[0100]

[0101] Table 9. Effects of resource substances on reducing the disease index of tomatoes by Streptomyces SNP

[0102]

Claims

1. Application of combined application of Streptomyces strain SBL, Streptomyces strain SHS or Streptomyces strain SNP and resource substances in controlling soil-borne bacterial wilt, wherein, The combined application is the combined application of resource substances L-citrulline or tannic acid with Streptomyces SBL, the combined application of resource substances ascorbic acid, glutathione or spermidine with Streptomyces SHS, or the combined application of resource substances L-methionine with Streptomyces SNP; the soil-borne bacterial wilt is tomato soil-borne bacterial wilt; Streptomyces strain SBL, taxonomically named Streptomyces pulcher ( Streptomyces bellus Streptomyces bellus ), was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 11, 2022, with the deposit number of CGMCC No. 25283; Streptomyces strain SHS, classified as Streptomyces circumflexus ( Streptomyces torulosus ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 11, 2022, with the culture collection number of CGMCC No.25281; Streptomyces strain SNP, taxonomically named Streptomyces erimoensis ( Streptomyces rishiriensis ), was deposited at the General Microbiological Center of the China National Culture Collection Center on July 11, 2022, with the accession number CGMCC No. 25282.

2. The application according to claim 1, wherein: The application is to adopt the root irrigation method.

3. A Streptomyces synbiotic for preventing and controlling soil-borne Ralstonia solanacearum, characterized in that: It is composed of Streptomyces strain SBL and at least one resource substance selected from L-citrulline or tannic acid, Streptomyces strain SHS and at least one resource substance selected from ascorbic acid, glutathione or spermidine, or Streptomyces strain SNP and the resource substance L-methionine; The soil-borne bacterial wilt is tomato soil-borne bacterial wilt; Streptomyces strain SBL, taxonomically named Streptomyces pulcher ( Streptomyces bellus Streptomyces bellus ), was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 11, 2022, with the deposit number of CGMCC No. 25283; Streptomyces strain SHS, classified as Streptomyces circumflexus ( Streptomyces torulosus ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 11, 2022, with the culture collection number of CGMCC No.25281; Streptomyces strain SNP, taxonomically named Streptomyces erimoensis ( Streptomyces rishiriensis ), was deposited at the China General Microbiological Culture Collection Center on July 11, 2022, with the accession number CGMCC No. 25282.

4. A method for jointly controlling tomato soil-borne bacterial wilt by using a resource substance and Streptomyces, characterized in that: Apply the Streptomyces symbiotic compound described in claim 3 to the soil by the root irrigation method; apply 0.05 - 0.15 mmol of the resource substance and 5 mL - 15 mL of a Streptomyces spore suspension with a concentration of 1×10 7 - 1×10 9 CFU / mL to each tomato seedling.

5. The method for jointly preventing and controlling tomato soil-borne bacterial wilt by using a resource substance and Streptomyces according to claim 4, characterized in that: Apply 0.1 mmol of resource substances and 10 mL of a Streptomyces spore suspension with a concentration of 1×10 8 CFU / mL to each tomato seedling.

6. The method for preventing and controlling soil-borne bacterial wilt of tomatoes by combining resource materials and Streptomyces according to claim 4, characterized in that: The Streptomyces SBL spore suspension is prepared by the following method: the strain SBL is activated by using M1 solid culture medium; a single SBL colony is picked up and placed in M1 liquid culture medium, and cultured at 30°C and 170rpm for 3 days; 100 μL of fresh bacterial liquid is spread on ISP3 solid culture medium, and cultured at 30°C for 5-7 days, and the bacterial bodies are scraped and placed in sterile saline; the concentration of the bacterial liquid is adjusted with sterile saline to obtain the Streptomyces SBL spore suspension; The Streptomyces SHS spore suspension is prepared by the following method: the strain SHS is activated by using M1 solid culture medium; a single SHS colony is picked up and placed in M1 liquid culture medium, and cultured at 30°C and 170rpm for 3 days; 100 μL of fresh bacterial liquid is spread on ISP3 solid culture medium, and cultured at 30°C for 5-7 days, and the bacterial bodies are scraped and placed in sterile physiological saline; the concentration of the bacterial liquid is adjusted with sterile physiological saline to obtain the Streptomyces SHS spore suspension; The Streptomyces SNP spore suspension was prepared by the following method: the strain SNP was activated using M1 solid culture medium; a single SNP colony was picked up and placed in M1 liquid culture medium, and cultured at 30°C and 170rpm for 3 days; 100 μL of fresh bacterial liquid was spread on ISP3 solid culture medium, and cultured at 30°C for 5 to 7 days, and the bacterial bodies were scraped into sterile saline; the concentration of the bacterial liquid was adjusted with sterile saline to obtain the Streptomyces SNP spore suspension.

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