A Streptomyces synbiotic for preventing and controlling angelica root rot and its application

Abstract

The invention discloses a streptomyces synbiotic for preventing and controlling root rot, which is composed of streptomyces strain S1, streptomyces strain S3, streptomyces strain S15 and at least one resource material selected from methyl jasmonate and brassinolide; the streptomyces strain S1 is classified and named as Streptomyces peucetius, and the strain collection number is CGMCC NO.30458; the streptomyces strain S3 is classified and named as Streptomyces pratensis, and the strain collection number is CGMCC NO.30459; the streptomyces strain S15 is classified and named as Streptomyces avidinii, and the strain collection number is CGMCC NO.30460. The streptomyces synbiotic can significantly reduce the incidence of angelica root rot and effectively prevent and control angelica root rot.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to Streptomyces strain S1, Streptomyces strain S3 and Streptomyces strain S15 which have antagonistic effects on Fusarium oxysporum, to resource substances methyl jasmonate and brassinolide which specifically promote the growth of Streptomyces strain S1, Streptomyces strain S3 and Streptomyces strain S15, to a Streptomyces synbiotic for preventing and controlling angelica root rot, and application of the Streptomyces synbiotic for preventing and controlling angelica root rot. Background Art

[0002] Angelica sinensis is a medicinal plant of the Umbelliferae family. Its root is used as medicine and has the function of nourishing blood and activating blood circulation. Angelica sinensis is rich in nutrients, and its main medicinal ingredients are volatile oils, polysaccharides, amino acids, and volatile substances. As a rhizome medicinal material, Angelica sinensis is very susceptible to root rot. Angelica sinensis root rot harms the roots and stems of Angelica sinensis, whether in the seedling stage or the adult stage, and has a great impact on the quality and yield of Angelica sinensis. During the disease period, the plant gradually withers and the root tissue turns brown, which causes the plant to rot, leaving only a fibrous empty shell, completely losing its medicinal value.

[0003] The pathogenic bacteria that cause plant root rot are mainly Fusarium. Because Fusarium has strong ecological adaptability, wide distribution, and can be both parasitic and saprophytic in nutrition, it often causes wilting, root rot, bark rot, and ear rot in the field, and causes dry rot and wet rot during product transportation and storage. Traditional methods such as applying pesticides or fumigation can kill pathogenic Fusarium in a short time and effectively slow down the occurrence of diseases. However, the above methods will also kill indigenous microorganisms in the soil and reduce soil species diversity or abundance.

[0004] At present, the application of Streptomyces is one of the more green and sustainable measures for preventing and controlling plant diseases. Streptomyces is a higher actinomycete, one of the actinomycetes with a large number of species and numbers, and plays an important role in preventing and controlling plant diseases. The live preparation of Streptomyces (Mycostop) can prevent and control some soil-borne plant diseases caused by Rhizoctonia spp., Pythium spp., Fusariun spp., Phytophthora spp., etc., and reduce the occurrence of diseases to a certain extent. In the process of screening Streptomyces and verifying its antibacterial effect, people often use a culture medium with rich nutrition to ensure that Streptomyces can proliferate in large quantities 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 suitable resources, which leads to its growth slowing down, and even cannot survive in the soil environment, and its biological control efficiency is bound to decrease.

[0005] Therefore, it is necessary to find resources that can promote the growth of Streptomyces, provide a suitable resource environment for the growth of Streptomyces in the soil, and enhance its ability to inhibit Fusarium oxysporum. At present, there are studies on the targeted promotion of beneficial bacteria by rhizosphere prebiotics to reduce the biological barriers of Fusarium rubus, but different crops have different root secretions and different rhizosphere microbial community compositions. As angelica is a relatively special local medicinal material, it is urgent to screen for resources that can effectively promote Streptomyces to prevent and control root rot, and combine Streptomyces with resources (synbiotics) and apply them to the soil to improve the prevention and control effect of angelica root rot. Summary of the invention

[0006] The inventors have aimed at the problem that due to the limited nutrients in the soil, when Streptomyces is applied to prevent and control soil-borne diseases, it is often difficult for Streptomyces to proliferate in large quantities in the soil and exert its due preventive effect. Through experiments, it was found that in the indoor microplate system, brassinolide and methyl jasmonate can significantly promote the inhibitory ability of Streptomyces S1, S3, and S15 when used in combination against Fusarium oxysporum; in the greenhouse potted experiment, a resource material selected from brassinolide and methyl jasmonate was used in combination with Streptomyces S1, S3, and S15, and applied to the soil by root irrigation at the same time, which can reduce the disease index of Angelica root rot caused by Fusarium oxysporum. It shows that the combined application of a probiotic group selected from at least one of brassinolide or methyl jasmonate and Streptomyces S1, S3, and S15 can reduce the incidence of Angelica root rot and prevent and control Angelica root rot.

[0007] The purpose of the present invention is to provide Streptomyces strain S1, Streptomyces strain S3, Streptomyces strain S15 and the use of Streptomyces strain S1, Streptomyces strain S3, Streptomyces strain S15 in preventing and controlling root rot, as well as a Streptomyces synbiotic for preventing and controlling root rot containing the above-mentioned Streptomyces, and the use of the Streptomyces synbiotic in preventing and controlling root rot.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] Streptomyces strain S1, classified and named Streptomyces peucetius, was deposited on April 26, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC NO.30458.

[0010] Streptomyces strain S3, classified and named Streptomyces pratensis, was deposited on April 26, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC NO.30459.

[0011] Streptomyces strain S15, classified and named Streptomyces avidinii, was deposited on April 26, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC NO.30460.

[0012] Streptomyces strain S1, Streptomyces strain S3, and Streptomyces strain S15 all showed antagonistic effects against Fusarium oxysporum.

[0013] Another object of the present invention is to provide the use of at least one of the Streptomyces strain S1, the Streptomyces strain S3 and the Streptomyces strain S15 in preventing and controlling root rot.

[0014] Another object of the present invention is to provide an application of the combined use of the Streptomyces strain S1, the Streptomyces strain S3 and the Streptomyces strain S15 in preventing and controlling root rot.

[0015] Another object of the present invention is to provide an application of at least one Streptomyces selected from the Streptomyces strain S1, Streptomyces strain S3, and Streptomyces strain S15 in combination with resource materials for preventing and controlling root rot.

[0016] The resource material is selected from at least one of methyl jasmonate and brassinolide.

[0017] Preferably, the application is the application of at least one selected from brassinolide and methyl jasmonate in combination with the Streptomyces strain S1, Streptomyces strain S3 and Streptomyces strain S15 for the prevention and control of root rot.

[0018] Preferably, the application is to apply at least one selected from brassinolide and methyl jasmonate and the Streptomyces strain S1, Streptomyces strain S3 and Streptomyces strain S15 into the rhizosphere soil of the plant by root irrigation.

[0019] Another object of the present invention is to provide a Streptomyces synbiotic for preventing and controlling root rot, which is composed of the Streptomyces strain S1, the Streptomyces strain S3, the Streptomyces strain S15 and at least one resource material selected from methyl jasmonate and brassinolide, or is composed of the Streptomyces strain S1, the Streptomyces strain S3 and the Streptomyces strain S15.

[0020] Preferably, the Streptomyces synbiotic for preventing and controlling root rot is composed of the Streptomyces strain S1, the Streptomyces strain S3, the Streptomyces strain S15 and methyl jasmonate, or is composed of the Streptomyces strain S1, the Streptomyces strain S3, the Streptomyces strain S15 and brassinolide.

[0021] Another object of the present invention is to provide a method for preventing and controlling root rot, comprising: applying the Streptomyces synbiotic into the soil by root irrigation; wherein, when the Streptomyces synbiotic consists of the Streptomyces strain S1, the Streptomyces strain S3, the Streptomyces strain S15 and at least one resource material selected from methyl jasmonate and brassinolide, 0.05-0.15 mmoL of the resource material and 100 mL of a concentration of 1×10 8 ~1×10 10 CFU / mL of Streptomyces liquid; when the Streptomyces synbiotics consist of the Streptomyces strain S1, the Streptomyces strain S3 and the Streptomyces strain S15, 100 mL of a concentration of 1×10 8 ~1×10 10 CFU / mL of Streptomyces bacterial solution.

[0022] Preferably, when the Streptomyces synbiotics are composed of the Streptomyces strain S1, the Streptomyces strain S3, the Streptomyces strain S15 and at least one resource substance selected from methyl jasmonate and brassinolide, 0.1 mmol of the resource substance and 100 mL of a concentration of 1×10 9 CFU / mL of Streptomyces liquid; when the Streptomyces synbiotics consist of the Streptomyces strain S1, the Streptomyces strain S3 and the Streptomyces strain S15, 100 mL of a concentration of 1×10 9 CFU / mL of Streptomyces bacterial solution.

[0023] The resource material is applied in the form of a solution. Generally, the concentration of the resource material solution is 0.5-1.5 mmol / L. The preparation method of the resource material solution is: the resource material is dissolved in deionized water or hot water, and sucked with a sterile syringe, filtered with a 0.22 μm filter membrane, and the resource material solution is obtained.

[0024] The Streptomyces bacterial liquid is obtained by cultivating Streptomyces strain S1, Streptomyces strain S3 and Streptomyces strain S15 separately to obtain Streptomyces S1 bacterial liquid, Streptomyces S3 bacterial liquid and Streptomyces S15 bacterial liquid, and then mixing the Streptomyces S1 bacterial liquid, Streptomyces S3 bacterial liquid and Streptomyces S15 bacterial liquid in a volume ratio of 1:1:1.

[0025] The concentration of the Streptomyces S1 bacterial solution is 1×10 8 ~10 10 CFU / mL, preferably 1×10 9 CFU / mL.

[0026] The concentration of the Streptomyces S3 bacterial solution is 1×10 8 ~10 10 CFU / mL, preferably 1×10 9 CFU / mL.

[0027] The concentration of the Streptomyces S15 bacterial solution is 1×10 8 ~10 10 CFU / mL, preferably 1×10 9 CFU / mL.

[0028] The streptomyces bacterial solution is prepared by the following method:

[0029] The Streptomyces strain S1 was activated using ISP2 solid culture medium, and a single colony of Streptomyces S1 was picked and placed in ISP2 liquid culture medium, and cultured at 26-30° C. and 170-180 rpm for 18-40 hours to obtain a Streptomyces S1 bacterial liquid;

[0030] The Streptomyces strain S3 is activated by using ISP2 solid culture medium; a single colony of Streptomyces S3 is picked up and placed in ISP2 liquid culture medium, and cultured at 26-30° C. and 170-180 rpm for 18-40 hours to obtain a Streptomyces S3 bacterial liquid;

[0031] The Streptomyces strain S15 is activated by using ISP2 solid culture medium; a single colony of Streptomyces S15 is picked up and placed in ISP2 liquid culture medium, and cultured at 26-30° C. and 170-180 rpm for 18-40 hours to obtain a Streptomyces S15 bacterial liquid;

[0032] A bacterial solution is obtained by mixing a bacterial solution of Streptomyces S1, a bacterial solution of Streptomyces S3, and a bacterial solution of Streptomyces S15 in a volume ratio of 1:1:1.

[0033] The root rot diseases are angelica root rot, angelica root rot, salvia root rot and astragalus root rot.

[0034] The root rot is mainly caused by the pathogen Fusarium oxysporum.

[0035] When the root rot is angelica root rot, 21 days after the angelica rhizomes with uniform germination degree and healthy and full body are sown in the seedling pot, brassinolide or methyl jasmonate and Streptomyces bacterial solution are applied to the rhizosphere soil of the plant by root irrigation method.

[0036] Beneficial effects of the present invention:

[0037] The present invention combines brassinolide or methyl jasmonate with Streptomyces S1, S3, and S15 to form a Streptomyces synbiotic for preventing and controlling angelica root rot, which can significantly improve the inhibitory ability of Streptomyces on Fusarium oxysporum, reduce the number of Fusarium oxysporum in the rhizosphere soil of angelica planting, reduce the incidence of angelica root rot, and effectively prevent and control angelica root rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 These are the results of the antagonism study of Streptomyces strains S1, S3, and S15 against Fusarium oxysporum on PDA plates.

[0039] Figure 2 Phylogenetic tree of Streptomyces strains S1, S3, and S15.

[0040] Figure 3 This graph shows the promoting effect of resource substances on the growth of Streptomyces strains S1, S3, and S15.

[0041] Figure 4 The effect of brassinolide and methyl jasmonate on the growth of Fusarium oxysporum; gray represents the control group and white represents the experimental group.

[0042] Figure 5 The figure shows the effect of brassinolide and methyl jasmonate on the inhibition of Fusarium oxysporum growth by Streptomyces S1, S3, and S15; the dotted line in the figure indicates the treatment with only Fusarium oxysporum added.

[0043] Figure 6 This is the effect of brassinolide and methyl jasmonate on the disease index of Angelica root rot; the dotted line in the figure represents the treatment with only Fusarium spp.

[0044] Biodeposit Information:

[0045] Streptomyces strain S1, classified and named Streptomyces peucetius, was deposited on April 26, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC NO.30458.

[0046] Streptomyces strain S3, classified and named Streptomyces pratensis, was deposited on April 26, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC NO.30459.

[0047] Streptomyces strain S15, classified and named Streptomyces avidinii, was deposited on April 26, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC NO.30460. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0049] ISP2 liquid culture medium: 10.0 g malt extract powder, 4.0 g yeast powder, 4.0 g glucose, distilled water to 1 L, pH 7.5, sterilize at 115°C for 30 min.

[0050] ISP2 solid medium: Add 20 g / L agar to ISP2 liquid medium, pH 7.5, and sterilize at 115℃ for 30 min.

[0051] PDB medium: 200.0 g peeled potatoes, 20.0 g glucose, distilled water to 1 L, pH 7.5, sterilized at 115°C for 30 min.

[0052] PDA medium: 6 g / L potato flour, 20 g / L glucose, 20 g / L agar, dilute to 1 L with deionized water, and sterilize at 115°C for 30 min.

[0053] Carbon source-free culture medium: 3.0 g / L potassium dihydrogen phosphate, 3.0 g / L sodium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L calcium chloride, dilute to 1 L with deionized water, and sterilize at 115°C for 30 min.

[0054] Resource materials: Brassinolide (catalog number B794084) and methyl jasmonate (catalog number M813622) were purchased from MacLean Reagent Company.

[0055] The soil used in the Angelica potted experiment was peat soil, purchased from company.

[0056] Root rot pathogen: Fusarium oxysporun 220, isolated from the rhizosphere soil of angelica root rot in the laboratory. The screening process is as follows: weigh 1g of diseased soil sample, put it into a 50mL Erlenmeyer flask, add 9mL of sterile water, mix, and seal; place the Erlenmeyer flask in a 30℃ constant temperature shaker, shake at a speed of 180r / min for 30min, let it stand on a clean bench for 30min, draw 1mL of supernatant into a 1.5mL sterilized centrifuge tube, take 100μL of supernatant, and dilute to 10% by volume ratio of supernatant to water 1:9. -2 , 10-3 Isogradient. Use a pipette to draw 100 μL of supernatant with different dilution gradients and spread it on PDA solid culture medium, and invert the coated plate in a constant temperature incubator at 28°C for 3 days; pick mycelium of different morphologies and inoculate it on a new PDA solid culture medium, and culture it in a 28°C incubator for 3 days for purification. Based on morphological microscopic identification, a strain was selected for genomic DNA extraction, amplified with ITS1-ITS2 primers, and compared through NCBI. It was identified as Fusarium oxysporum (ITS sequence similarity>97). Based on the verification, it was found that the Fusarium oxysporum 220 strain is the model invasive pathogen of the present invention, which can cause root rot outbreaks of various rhizome medicinal materials such as Angelica sinensis, Angelica dahurica, Salvia miltiorrhiza and Astragalus membranaceus. It is a root rot model pathogenic fungus that adapts to multiple hosts and can grow on PDA culture medium.

[0057] Experimental Example 1

[0058] Isolation, Screening and Identification of Streptomyces

[0059] Isolation of Streptomyces

[0060] The rhizosphere soil samples of Angelica sinensis for isolation of microorganisms were collected from Zhuoluo Village, Dingxi County, Gansu Province (104.20°E, 34.52°N).

[0061] Isolate Streptomyces by dilution plate spreading method: 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 Angelica rhizosphere soil sample and put it into the above conical flask, shake at 28°C and 180 r / min for 30 min, and use a pipette to take 10 -1 1 mL of the gradient soil dilution was transferred into a test tube containing 9 mL of sterile water, mixed, and then gradiently diluted to obtain soil suspensions of different dilutions. 100 μL of the dilution gradient of 10 -7 The soil suspension was spread on ISP2 plates and cultured in a 30°C incubator for 2 days. Single colonies of Streptomyces were picked out for purification on new ISP2 plates and stored on slant surfaces.

[0062] Screening and identification of Streptomyces antagonistic to Fusarium oxysporum

[0063] The plate confrontation method was used to determine the antibacterial activity of the isolated Streptomyces and screen out the strain with the best effect. The specific method is:

[0064] Fusarium oxysporum 220 was inoculated into PDA medium and cultured at 28°C for 5 days. The bacterial cake was punched with a hole puncher (Φ=6 mm) and moved to the center of a plate containing PDA medium and cultured at 28°C for 2 days. Streptomyces was inoculated into a shake flask containing ISP2 liquid medium and cultured at 30°C and 170 rpm for 24 hours. The OD of the Streptomyces liquid was measured.600 Value (OD 600 =1.0), 50 μL of Streptomyces liquid was dripped onto the four positions of the upper, lower, left, and right at a certain distance from the Fusarium oxysporum cake, and cultured at 30°C for 24 hours, and the diameter of the inhibition zone was measured. Three strains with good antagonistic effects were selected: strains S1, S3, and S15 ( Figure 1 ), the strains were identified by 16S rRNA, and the phylogenetic tree of the three strains was constructed based on the identification results ( Figure 2 ).

[0065] Strain S1, classified and named Streptomyces peucetius, has a yellowish-brown colony, white and slightly white aerial hyphae, sometimes with a slightly gray hue, and slow spore production and growth rates. It was deposited on April 26, 2024 at the General Microbiological Center of the China Microbiological Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with a strain collection number of CGMCC NO.30458.

[0066] Strain S3, classified and named as Streptomyces pratensis, has a yellowish colony, slightly yellow aerial hyphae, fast spore production and growth rate. It was deposited on April 26, 2024 at the General Microbiological Center of China Microbiological Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC NO.30459.

[0067] Strain S15, classified and named as Streptomyces avidinii, has a white colony, light white aerial hyphae, fast spore production and growth rate. It was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on April 26, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is CGMCC NO.30460.

[0068] Example 2

[0069] Investigating the indoor effects of resource materials on the growth of Streptomyces

[0070] The strain S1 was activated using ISP2 solid medium. A single colony of S1 was selected and cultured in ISP2 liquid medium at 30°C and 170 rpm for 48 h. The biomass OD600 of the bacterial solution was adjusted to 0.5 (1×10 8 CFU / mL) to obtain the Streptomyces S1 bacterial liquid.

[0071] The strain S3 was activated using ISP2 solid medium. A single colony of S3 was selected and cultured in ISP2 liquid medium at 30°C and 170 rpm for 48 h. The biomass OD600 of the bacterial solution was adjusted to 0.5 (1×108 CFU / mL) to obtain the Streptomyces S3 bacterial liquid.

[0072] The strain S15 was activated using ISP2 solid medium. A single colony of S15 was selected and cultured in ISP2 liquid medium at 30°C and 170 rpm for 48 h. The biomass OD600 of the bacterial solution was adjusted to 0.5 (1×10 8 CFU / mL) to obtain the Streptomyces S15 bacterial liquid.

[0073] Preparation of resource material mother liquor: accurately weigh each resource material (brassinolide or methyl jasmonate) with a 1 / 10,000 balance, dissolve it after heating with deionized water, draw it up with a sterile syringe, filter it under a 0.22μm filter membrane into a 5mL sterile centrifuge tube, and obtain a sterile resource material mother liquor with a concentration of 10mM. Store it in a -4℃ refrigerator for later use.

[0074] Experimental setup and treatment: Experimental group: resource materials and one selected from Streptomyces S1, S3, and S15 were added respectively, specifically: methyl jasmonate + Streptomyces S1, methyl jasmonate + Streptomyces S3, methyl jasmonate + Streptomyces S15, brassinolide + Streptomyces S1, brassinolide + Streptomyces S3, brassinolide + Streptomyces S15; Control group: only Streptomyces S1, S3 or S15 was added, and the resource material mother liquor was replaced with an equal volume of sterile water.

[0075] Experimental group: 194 μL carbon-free medium was added to a 96-well cell culture plate (Costar) by gun, and then 2 μL of sterile resource substance mother solution was added to the culture medium, followed by 4 μL of Streptomyces S1, Streptomyces S3 or Streptomyces S15 with an OD600 of 0.5. The control group replaced the sterile resource substance mother solution with 2 μL of sterile water. Each treatment was repeated 6 times. After the addition, the cell culture plate was continuously cultured in a constant temperature (30°C) shaker at 170 r / min for 120 h, and the growth amount (in log) was determined by the dilution coating method. 10 CFU).

[0076] The promoting effect of resource substances on the growth of Streptomyces S1, S3, and S15 is shown in Figure 3 The results showed that compared with the control group, brassinolide and methyl jasmonate had a significant effect on the growth of Streptomyces S1, S3, and S15 (P<0.05), indicating that these two resource substances meet the screening criteria of prebiotics to promote the growth of beneficial bacteria.

[0077] Example 3

[0078] Investigating the indoor effects of resource materials on the growth of pathogenic fungus Fusarium oxysporum

[0079] Pathogen: Fusarium oxysporun 220.

[0080] Preparation of resource substance mother liquor: accurately weigh each resource substance (methyl jasmonate or brassinolide) with a 1 / 10,000 balance, dissolve it in deionized water after heating, draw it up with a sterile syringe, filter it under a 0.22μm filter membrane into a 5mL sterile centrifuge tube, obtain a sterile resource substance mother liquor with a concentration of 5mM, store it in a -4℃ refrigerator for later use.

[0081] Preparation of Streptomyces bacterial solution: Referring to the method of Example 2, Streptomyces S1, Streptomyces S3 and Streptomyces S15 were cultivated separately to prepare Streptomyces S1 bacterial solution (1×10 9 CFU / mL), Streptomyces S3 culture (1×10 9 CFU / mL), Streptomyces S15 culture liquid (1×10 9 CFU / mL); Streptomyces S1, Streptomyces S3, and Streptomyces S15 were mixed at a volume ratio of 1:1:1 to obtain a total Streptomyces concentration of 1×10 9 CFU / mL of bacterial solution.

[0082] Preparation of Fusarium oxysporum culture liquid: Fusarium oxysporum 220 was revived on PDA medium, and the plate was placed in an incubator and cultured at 30°C for 2 days until a single colony appeared. The plate was punched with a hole puncher (Φ=6 mm) to make a bacterial cake for standby use; a single colony on the plate was picked and transferred to PDB medium, and cultured at 30°C and 180rpm for 48 hours to obtain a fresh Fusarium oxysporum culture liquid for standby use.

[0083] Experimental setting treatment: Experimental group: adding resource substances and Fusarium oxysporum; control group: adding Fusarium oxysporum and replacing resource substances with an equal volume of sterile anhydrous ethanol.

[0084] Add 880 μL PDB liquid medium to a 24-well plate (Costar), and add 50 μL Fusarium oxysporum bacterial solution. Experimental group: Add 20 μL resource substance mother solution (concentration of 5 mM), and the control group adds an equal volume of sterile water to replace the resource substance mother solution, and each treatment is repeated 9 times. After adding the sample, place the 24-well plate in a constant temperature (30°C) shaker, shake continuously at 170 r / min for 3 days, weigh the corresponding number of centrifuge tubes of appropriate size, carefully transfer the bacterial solution to the centrifuge tube, place it in a 60°C oven until completely dried, weigh it, and obtain the total weight of the bacteria.

[0085] Results Figure 4, gray represents the control group without adding resource substances, and white represents the experimental group. Compared with the control group, brassinolide and methyl jasmonate had no significant effect on the growth of Fusarium oxysporum, indicating that these two resource substances meet the screening criteria that prebiotics cannot promote the growth of pathogens.

[0086] Example 4

[0087] Effects of resource materials on the reduction of disease index of Angelica sinensis root rot by Streptomyces

[0088] Based on the ability of resource substances to promote the growth of Streptomyces and assist Streptomyces in inhibiting Fusarium oxysporum, better resource substances (Streptomyces S1, S3, S15, resource substances: brassinolide and methyl jasmonate) were screened out. A pot experiment was conducted to further investigate the effects of the above resource substances on Streptomyces reducing the disease index of Angelica root rot.

[0089] Preparation of resource substance solution: Use a 1 / 10,000 balance to accurately weigh each resource substance (methyl jasmonate or brassinolide), heat it with deionized water to dissolve it, draw it up with a sterile syringe, filter it through a 0.22 μm filter membrane into a 5 mL sterile centrifuge tube, and obtain a sterile resource substance solution with a concentration of 1 mmol / L. Store it in a -4°C refrigerator for later use.

[0090] The preparation of Streptomyces bacterial solution is shown in Experimental Example 3. The total concentration of Streptomyces in the Streptomyces bacterial solution is 1×10 9 CFU / mL.

[0091] Preparation of Fusarium oxysporum broth: Fusarium oxysporum 220 was inoculated into a large triangular flask filled with PDB medium and cultured at 30°C and 170 rpm for 48 h to obtain a fermentation broth. After the fermentation broth was centrifuged at 4000 r / min for enrichment, the spores in the supernatant were counted using a hemocytometer under a microscope, and the spores of Fusarium oxysporum were diluted with sterile water to a concentration of 10 4 / mL, set aside.

[0092] Angelica variety: Angelica sinensis from Minxian County, Gansu Province.

[0093] The experiment set up three treatment groups and one control group: 1) Methyl jasmonate+S group: methyl jasmonate, Streptomyces solution and Fusarium oxysporum solution were added; 2) Brassinolide+S group: brassinolide, Streptomyces solution and Fusarium oxysporum solution were added; 3) SynCom group: only Streptomyces solution and Fusarium oxysporum solution were added, and the resource material was replaced by an equal volume of sterile anhydrous ethanol; 4) Control group (Control): only an equal amount of Fusarium oxysporum was added, and the resource material mother solution and Streptomyces solution were replaced by an equal volume of sterile water.

[0094] Fresh rhizomes of Angelica sinensis were placed in a room temperature and ventilated place. Healthy and plump rhizomes of Angelica sinensis with uniform thickness and size were selected and transplanted into seedling pots filled with 1 kg of peat soil (peat soil accounted for about 2 / 3 of the height of the seedling pot). One rhizome of Angelica sinensis was sown in each seedling pot, and the sowing distance from the soil surface was about 3 cm. Seedlings were raised in a greenhouse. After 21 days of seedling raising, the Streptomyces bacterial solution and resource substance solution were evenly poured into the rhizosphere soil of Angelica sinensis by the root irrigation method in the Methyl jasmonate+S group, Brassinolide+S group, and SynCom group. Each Angelica sinensis seedling was applied with 100 mL of 1 mmol / L resource substance solution (so that the amount of resource substance applied to each Angelica sinensis seedling was 0.1 mmoL), and 100 mL of 1×10 9 CFU / mL Streptomyces liquid (each Angelica sinensis seedling is inoculated with 10 11 CFU); the control group used an equal volume of sterile water to replace the resource substance solution and Streptomyces solution. After 7 days, each Angelica seedling in the three treatment groups and the control group was inoculated with 100 mL of Fusarium oxysporum spore solution by root irrigation. After inoculation with Fusarium oxysporum, once Angelica showed symptoms of disease, the disease situation began to be recorded and the plant incidence rate was counted; on the 10th day after the Angelica seedlings began to show symptoms, the rhizosphere soil was collected, and the number of Fusarium oxysporum in the Angelica rhizosphere soil samples was detected by qPCR.

[0095] The plant disease level is divided into 5 levels [1] : 0 = no symptoms, 1 = symptoms appear on 1-2 true leaves, 2 = symptoms appear on 3 leaves to 1 / 3 of the leaves, 3 = symptoms appear on 1 / 3-1 / 2 of the leaves, 4 = symptoms appear on 1 / 2-3 / 4 of the leaves, 5 = death of the entire plant.

[0096] The disease index is used to characterize the disease situation, and the calculation formula is: [2] :

[0097] Plant disease index = [∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × highest level value)]

[0098] The results are as follows Figure 5 , Figure 6 As shown in Table 1, it can be seen that compared with the control group, the combined use of Streptomyces S1, S3, and S15 significantly improved the control effect of Angelica root rot; the combined use of any one of the resource materials selected from brassinolide and methyl jasmonate and Streptomyces S1, S3, and S15 on Angelica root rot was significantly better than the control effect of using a single Streptomyces synthetic community, and can significantly reduce the disease index of Angelica root rot (P<0.05), reduce the number of Fusarium in the rhizosphere of Angelica, and reduce the incidence of Fusarium disease.

[0099] Table 1. Effects of resource materials on the reduction of Angelica sinensis disease index by Streptomyces S1, S3, and S15

[0100]

[0101] References:

[0102] 1. Ciampi-Panno L, Fernandez C, Bustamante P, et al. Biological control of bacterial wilt of potatoes caused by Pseudomonas solanacearum[J]. AmericanPotato Journal, 1989, 66(5): 315-332.

[0103] 2. Kempe J. Biological control of bacterial wilt of potatoes: attempts to induce resistance by treating tubes with bacteria. [J]. Plant Disease, 1983, 67(5): 499-503.

Claims

1. Streptomyces strain S15, classified as Streptomyces avidinophilus ( Streptomyces avidinii ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 26, 2024, with the culture collection number CGMCCNO.30460.

2. The Streptomyces strain S15 according to claim 1, or the Streptomyces strain S15 according to claim 1 in combination with resource substances for preventing and controlling root rot, wherein the resource substances are selected from at least one of methyl jasmonate and brassinolide.

3. The combined use of Streptomyces strain S1, Streptomyces strain S3 and the Streptomyces strain S15 according to claim 1, or the combined application of at least one resource substance selected from brassinolide and methyl jasmonate with Streptomyces strain S1, Streptomyces strain S3 and the Streptomyces strain S15 according to claim 1 in the prevention and control of root rot; Streptomyces strain S1, classified as Streptomyces posse Streptomyces peucetius ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 26, 2024, with the culture collection number of CGMCC NO.30458; Streptomyces strain S3, named Streptomyces praecox ( Streptomyces pratensis ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 26, 2024, with the culture collection number CGMCC NO.30459.

4. The use according to claim 2 or 3, characterized in that: The root rot diseases are angelica root rot, angelica root rot, salvia root rot and astragalus root rot.

5. A Streptomyces synbiotic for preventing and controlling root rot, characterized in that: It is composed of Streptomyces strain S1, Streptomyces strain S3, Streptomyces strain S15 as claimed in claim 1, and at least one resource material selected from methyl jasmonate and brassinolide, or it is composed of Streptomyces strain S1, Streptomyces strain S3, and Streptomyces strain S15 as claimed in claim 1; Streptomyces strain S1, classified as Streptomyces posse Streptomyces peucetius ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 26, 2024, with the culture collection number of CGMCC NO.30458; Streptomyces strain S3, named Streptomyces praecox ( Streptomyces pratensis ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 26, 2024, with the culture collection number CGMCC NO.30459.

6. A method for preventing and controlling root rot, characterized in that: include: Applying the Streptomyces synbiotic according to claim 5 into the soil by root irrigation; When the Streptomyces synbiotics consist of Streptomyces strain S1, Streptomyces strain S3, Streptomyces strain S15 and at least one resource substance selected from methyl jasmonate and brassinolide, 0.05-0.15 mmol of the resource substance and 100 mL of 1×10 8 ~1×10 10 CFU / mL of Streptomyces liquid; when the Streptomyces synbiotics consist of Streptomyces strain S1, Streptomyces strain S3 and Streptomyces strain S15, 100 mL of a concentration of 1×10 8 ~1×10 10 CFU / mL of Streptomyces bacterial solution.

7. The method for preventing and controlling root rot according to claim 6, characterized in that: When the Streptomyces synbiotics consist of Streptomyces strain S1, Streptomyces strain S3, Streptomyces strain S15 and at least one resource substance selected from methyl jasmonate and brassinolide, 0.1 mmol of the resource substance and 100 mL of a 1×10 9 CFU / mL of Streptomyces liquid; when the Streptomyces synbiotics consist of Streptomyces strain S1, Streptomyces strain S3 and Streptomyces strain S15, 100 mL of a concentration of 1×10 9 CFU / mL of Streptomyces bacterial solution.

8. The method for preventing and controlling root rot according to claim 6, characterized in that: The Streptomyces bacterial solution is obtained by culturing Streptomyces strain S1, Streptomyces strain S3 and Streptomyces strain S15 to obtain Streptomyces S1 bacterial solution, Streptomyces S3 bacterial solution and Streptomyces S15 bacterial solution respectively, and then mixing the Streptomyces S1 bacterial solution, Streptomyces S3 bacterial solution and Streptomyces S15 bacterial solution in a volume ratio of 1:1:1 to obtain a bacterial solution; wherein the concentration of the Streptomyces S1 bacterial solution is 1×10 8 ~10 10 CFU / mL; the concentration of the Streptomyces S3 bacterial solution is 1×10 8 ~10 10 CFU / mL; the concentration of the Streptomyces S15 bacterial solution is 1×10 8 ~10 10 CFU / mL.

9. The method for preventing and controlling root rot according to claim 8, characterized in that: The concentration of the Streptomyces S1 bacterial solution is 1×10 9 CFU / mL; the concentration of the Streptomyces S3 bacterial solution is 1×10 9 CFU / mL; the concentration of the Streptomyces S15 bacterial solution is 1×10 9 CFU / mL.

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