Streptomyces BRS-24 and its application in controlling clubroot and promoting growth of cruciferous crops

Abstract

The present invention discloses a strain of Streptomyces BRS-24 capable of preventing and treating clubroot of Chinese cabbage and cabbage, as well as a fermentation liquid and a bacterial suspension. The Streptomyces BRS-24 capable of preventing and treating clubroot of cruciferous crops and promoting growth of the present invention has a preservation number of CCTCC NO.M 20221182. The fermentation liquid or bacterial suspension made by the strain has an inhibitory effect on clubroot. In addition, the Streptomyces can be well colonized in the root system of plants and promote the growth of plants. Compared with traditional chemical pesticides, the use of the Streptomyces biocontrol agent is environmentally friendly, and the prevention and control is green and efficient, and has good prospects in the future application of biological control and green fertilizers.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically relates to a Streptomyces BRS-24 capable of promoting the growth of cruciferous crops and preventing and controlling clubroot disease and a biocontrol agent thereof. Background Art

[0002] Clubroot is one of the most destructive soil-borne diseases of cruciferous crops, severely limiting crop yield and quality. Although disease-resistant breeding is still an option, the rapid evolution of pathogens has greatly restricted the application of disease-resistant varieties. In many cases, chemical prevention and control of clubroot is also far from ideal, and only the traditional lime method has achieved good results. However, chemical methods have seriously damaged the field ecology and threatened food quality and safety. Therefore, environmentally friendly alternatives or more means are needed to make the resistance response of crops more stable and lasting.

[0003] The development and application of biocontrol microorganisms is an environmentally friendly strategy for plant disease control. However, the colonization of biocontrol strains in host plants and soil and the inefficient inhibition of pathogen growth limit their disease control function. Streptomyces can synthesize a variety of metabolites that have plant growth-promoting and biocontrol functions. In addition, they can colonize various parts and growth stages of plants in the form of mycelium. Therefore, the development of biocontrol streptomyces has broad prospects. However, there are currently few biocontrol streptomyces that can control root knot fungi. Summary of the invention

[0004] In order to solve the above problems, the present invention provides Streptomyces BRS-24 and its application in preventing and controlling clubroot disease and promoting growth of cruciferous crops.

[0005] The invention provides a Streptomyces BRS-24 capable of preventing and controlling clubroot of cruciferous crops and promoting growth, wherein the strain is characterized in that the strain has a preservation number of CCTCC NO.M 20221182.

[0006] The invention also provides a cruciferous crop clubroot control agent containing the streptomyces BRS-24.

[0007] In a specific embodiment of the present invention, the control agent contains the fermentation broth of the Streptomyces BRS-24.

[0008] The control agent may be in a dosage form well known in the art, such as a solid, liquid or capsule preparation.

[0009] The control agent may further contain any one or more of a bacterial agent carrier, a thickener, a dispersant, an emulsifier, a thickener, a preservative, a defoaming agent and an antifreeze agent.

[0010] Wherein, the bacterial agent carrier is any one or more selected from vermiculite, peat, zeolite, diatomaceous earth, fungus bran, corn flour, soybean meal, bran, and rice bran; the dispersant is any one or more selected from sodium lignin sulfonate, calcium lignin sulfonate, sodium naphthalene sulfonate formaldehyde condensate NNO, fatty amide-N-methyl taurine, alkyl sulfosuccinic acid sodium salt, sodium dibutyl tea sulfonate, sodium dodecyl sulfate, polyarylphenol polyoxyethylene ether, and ethylenediaminetetraacetate; the emulsifier is any one or more selected from alkylbenzene sulfonate, alkyl alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, phenethylphenol polyoxyethylene ether, sorbic acid ester, and polyol fatty acid ester; the enhancing agent is any one or more selected from sodium lignin sulfonate, calcium lignin sulfonate, sodium naphthalene sulfonate formaldehyde condensate NNO, fatty amide-N-methyl taurine, alkyl sulfosuccinic acid sodium salt, sodium dibutyl tea sulfonate, sodium dodecyl sulfate, polyarylphenol polyoxyethylene ether, and ethylenediaminetetraacetate; the emulsifier is any one or more selected from alkylbenzene sulfonate, alkyl alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, phenethylphenol polyoxyethylene ether, sorbic acid ester, and polyol fatty acid ester; The thickener is one or more of xanthan gum, gelatin, gum arabic, guar gum, sodium alginate, soluble starch, heteropolysaccharide, sodium carboxymethyl cellulose, sodium carboxyethyl cellulose, polyvinyl alcohol, sodium polyacrylate, polyacrylamide, urea, magnesium aluminum silicate, silicon dioxide, diatomaceous earth, bentonite and attapulgite; the preservative is one or more of sodium benzoate, potassium sorbate and ethyl parahydroxybenzoate; the defoamer is one or more of isooctyl alcohol, isopentanol, stearic acid, lauric acid, lipid ethers and silicone; the antifreeze is one or more of ethylene glycol, propylene glycol, glycerol, diethylene glycol, triethylene glycol, polyethylene glycol and urea.

[0011] The present invention also provides application of the Streptomyces BRS-24 or the control agent in controlling clubroot disease of cruciferous crops.

[0012] In a specific embodiment of the present invention, the application comprises the following steps: soaking the cruciferous crops with a Streptomyces BRS-24 bacterial solution for 10-30 minutes before sowing, wherein the number of viable bacteria of the Streptomyces BRS-24 bacterial solution is 1×10 5 -1×10 8 cfu / ml; and / or watering with Streptomyces BRS-24 solution after planting, 5-20ml / tree, concentration 1×10 5 -1×10 8 cfu / ml.

[0013] The present invention also provides application of the Streptomyces BRS-24 or the control agent in controlling clubroot disease of cruciferous crops.

[0014] In a specific embodiment of the present invention, the application comprises the following steps: soaking the cruciferous crops with a Streptomyces BRS-24 bacterial solution for 10-30 minutes before sowing, wherein the number of viable bacteria of the Streptomyces BRS-24 bacterial solution is 1×10 5 -1×10 8 cfu / ml; and / or watering with Streptomyces BRS-24 solution after planting, 5-20ml / tree, concentration 1×10 5 -1×10 8cfu / ml.

[0015] The Streptomyces BRS-24 of the present invention has a significant effect on the prevention and treatment of clubroot of cabbage and Chinese cabbage, while the disease in the control group is serious and the growth of plants is restricted. The study of the embodiments shows that the biocontrol agent of the present invention can significantly inhibit the occurrence of clubroot and can effectively promote plant growth. In the potted plant experiment inoculated with pathogens, compared with the control, it decreased by 53.3% and 53.2%, respectively. In addition, in the potted plant experiment without inoculation of pathogens, the application of the BRS-24 biocontrol agent significantly increased the biomass of the plants. Finally, compared with chemical prevention and control methods, the prevention and control of cabbage and cabbage clubroot by Streptomyces BRS-24 is greener and more efficient, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the colony morphology of Streptomyces BRS-24 of the present invention on ISP4 solid culture medium.

[0017] Figure 2 The present invention is a phylogenetic tree and related sequences constructed based on the 16S rRNA sequence of the BRS-24 strain.

[0018] Figure 3 The figure is a diagram showing the effect of Streptomyces BRS-24 of the present invention on inhibiting clubroot disease of cabbage and Chinese cabbage.

[0019] Figure 4 It is the incidence index and disease index of clubroot disease of cabbage and Chinese cabbage after inoculation of Streptomyces BRS-24 of the present invention.

[0020] Figure 5 The plant morphology diagram of the Streptomyces BRS-24 of the present invention promoting the growth of cabbage and Chinese cabbage.

[0021] Figure 6 It is a graph showing relevant indicators of the Streptomyces BRS-24 of the present invention promoting the growth of cabbage and Chinese cabbage. DETAILED DESCRIPTION

[0022] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0023] Example 1 Strain Identification

[0024] (1) Isolation of strains

[0025] Buckwheat roots were collected in a sterilized 50 ml centrifuge tube and 50 ml PBS was added on a clean bench. The centrifuge tube was placed on a shaker at 200 rpm for 30 min and centrifuged at 4000 rpm for 10 min. Roots and other plant residues were removed on a clean bench and pure water (mass ratio 1:100) was added according to the amount of soil after centrifugation. After gradient dilution of the soil suspension (10 1 , 102 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 ), 100 μl was spread on ISP4 medium. After 7 days of constant temperature culture at 28°C, colonies with Streptomyces characteristics on the medium were picked and purified.

[0026] (2) Morphological characteristics

[0027] After the BRS-24 strain was cultured on ISP4 solid medium (soluble starch 10.0 g, potassium dihydrogen phosphate 1.0 g, magnesium sulfate 1.0 g, sodium chloride 1.0 g, ammonium sulfate 2.0 g, calcium sulfate 2.0 g, ferrous sulfate 0.001 g, manganese chloride 0.001 g, zinc sulfate 0.001 g, agar 20.0 g, pH 7.2±0.2) for 3-5 days, the colony texture was dense and round, the surface was dry and wrinkled, not easy to pick up and not easy to break after picking up, no spores were produced on ISP4 medium, no pigment was produced, and the colony color was light yellow. Figure 1 shown.

[0028] (3) Molecular identification

[0029] Based on the 16S rDNA sequence of the BRS-24 strain, the 22 strains closest to it at the species level were selected, and the phylogenetic tree was constructed using the NJ (Neighbor-Joining) method using the MEGA6.0 software. Figure 2 As shown. There are two strains (strain genome ID: GCF_000331005.1; GCF_003032475.1) whose 16S rDNA is similar to BRS-24 is higher than 98.65%. Therefore, it is necessary to calculate the average nucleotide identity (ANI) of the whole genome based on their genome sequences to obtain accurate species identification results. The ANI analysis results based on the GTDA database showed that the ANI values ​​of BRS-24 and GCF_000331005.1 and GCF_003032475.1 were 91.6848% and 83.7049%, respectively, which were all less than the threshold of 95%. Therefore, BRS-24 was determined to be a new species of Streptomyces sp. (see Table 1-2).

[0030] BRS-24 (Streptomyces sp.) was deposited in the China Center for Type Culture Collection on July 26, 2022. The address of the depository is Wuhan University, Wuhan, China, CCTCC, and the deposit number is: CCTCC NO.M 20221182.

[0031] Table 1 Identification results of BRS-24 strain based on genome ANI value

[0032]

[0033] Table 2 Similarity between different strains and BRS-24 16s rDNA gene

[0034] Strain genome ID Similarity (%) RS_GCF_000331005.1 98.95 RS_GCF_003032475.1 98.817 RS_GCF_001280005.1 98.642 RS_GCF_003664315.1 98.621 RS_GCF_004151105.1 98.62 RS_GCF_001746365.1 98.555 RS_GCF_001418565.1 98.555 RS_GCF_002846625.1 98.493 RS_GCF_003112535.1 98.49 RS_GCF_000718095.1 98.489 RS_GCF_900129855.1 98.424 RS_GCF_002803085.1 98.424 RS_GCF_001514235.1 98.359 RS_GCF_001428885.1 98.357 RS_GCF_003627815.1 98.295 RS_GCF_001279695.1 98.295 RS_GCF_004217285.1 98.292 RS_GCF_001550375.1 98.231 RS_GCF_000091305.1 98.231 RS_GCF_000772895.1 98.23 RS_GCF_001514305.1 98.228 RS_GCF_001514205.1 98.228

[0035] Example 2 Experiment on prevention and control of clubroot disease in cabbage and Chinese cabbage

[0036] Preparation of Streptomyces BRS-24 fermentation broth and bacterial suspension: Streptomyces was activated on TSA solid medium, cultured at 28°C for 3 days, single colonies were picked and cultured in YEME liquid medium at 28°C and 200 rpm for 3 days, and then transferred to MS liquid medium and cultured at 28°C and 200 rpm for 3-5 days to prepare Streptomyces BRS-24 fermentation broth. After centrifugation of Streptomyces BRS-24 fermentation broth at 4000 rpm / s, it was resuspended in distilled water and repeated 3 times to prepare BRS-24 bacterial suspension.

[0037] Two groups of experiments were set up for each of the two crops, cabbage and Chinese cabbage, with three replicates in each group and 30 plants in each replicate. Control group: seeds were soaked in distilled water for 30 minutes before sowing and seedlings. After 15 days, the plants were transplanted to 10×10 cm plastic pots inoculated with root-knot bacteria, and then irrigated with 10 ml of clean water every 7 days. Treatment group: seeds were soaked in Streptomyces BRS-24 suspension for 30 minutes before sowing and seedlings. After 15 days, the plants were transplanted to 10×10 cm plastic pots inoculated with root-knot bacteria, and then irrigated with 10 ml of bacterial suspension every 7 days. The incidence of the disease was counted after 35 days. Figure 3 and 4 It can be seen that the control group of cabbage or Chinese cabbage was greatly affected by root knot fungi, with obvious root disease, and the disease index was as high as 81.7% and 86.4% respectively, and the growth of the aboveground part was greatly restricted; while the disease of plants treated with Streptomyces BRS-24 suspension was significantly improved, the rhizosphere was well developed, and the aboveground growth was normal. Compared with the control, the disease index decreased by 53.3% and 53.2% respectively. The results show that the treatment with BRS-24 suspension can significantly improve the incidence of root knot disease and has a strong biocontrol effect.

[0038]

[0039] Example 3 Experiment on promoting the growth of cabbage and Chinese cabbage

[0040] The preparation of the fermentation broth and bacterial suspension of Streptomyces BRS-24 is the same as in Example 2.

[0041] Two groups of experiments were set up for each of the two crops, cabbage and Chinese cabbage, with three replicates in each group and 30 plants in each replicate. Control group: Soak the seeds in distilled water for 30 minutes before sowing and raising seedlings. After 15 days, transplant them to 10×10 cm plastic pots and irrigate them with 10 ml of clean water every 7 days. Treatment group: Soak the seeds in Streptomyces BRS-24 suspension for 30 minutes before sowing and raising seedlings. After 15 days, transplant them to 10×10 cm plastic pots and irrigate them with 10 ml of bacterial suspension every 7 days. The growth was counted after 35 days. Figure 5 and Figure 6 It can be seen that the growth advantages of cabbage and Chinese cabbage plants treated with Streptomyces BRS-24 suspension were significant, and the fresh weight increased by 13.38% and 65.71% respectively compared with the control. The experimental results show that BRS-24 can effectively promote plant growth and increase the fresh weight of plants.

[0042] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A plant that can prevent and treat Cruciferae ( Brassicaceae ) Clubroot and growth-promoting Streptomyces ( Streptomyces sp.) BRS-24, characterized in that Its accession number is: CCTCC NO. M 20221182.

2. A cruciferous crop clubroot control agent containing the Streptomyces BRS-24 according to claim 1.

3. The control agent according to claim 2, characterized in that A fermentation broth containing the Streptomyces BRS-24.

4. The control agent according to claim 2 or 3, characterized in that The control agent is a solid or liquid preparation.

5. The control agent according to claim 2 or 3, characterized in that The control agent is a capsule preparation.

6. The control agent according to claim 4, characterized in that It also contains any one or more of a bacterial agent carrier, a thickener, a dispersant, an emulsifier, a preservative, a defoaming agent and an antifreeze agent.

7. The control agent according to claim 6, characterized in that The bacterial agent carrier is any one or more of vermiculite, peat, zeolite, diatomaceous earth, fungus bran, corn flour, soybean meal, bran, and rice bran; the dispersant is any one or more of sodium lignin sulfonate, calcium lignin sulfonate, sodium naphthalene sulfonate formaldehyde condensate NNO, fatty amide-N-methyl taurine, alkyl sulfosuccinic acid sodium salt, sodium dibutyl tea sulfonate, sodium dodecyl sulfate, polyarylphenol polyoxyethylene ether, and ethylenediaminetetraacetate; the emulsifier is any one or more of alkylbenzene sulfonate, alkyl alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, phenylethylphenol polyoxyethylene ether, sorbic acid ester, and polyol fatty acid ester; the thickener The degreasing agent is any one or more of xanthan gum, gelatin, gum arabic, guar gum, sodium alginate, soluble starch, heteropolysaccharide, sodium carboxymethyl cellulose, sodium carboxyethyl cellulose, polyvinyl alcohol, sodium polyacrylate, polyacrylamide, urea, magnesium aluminum silicate, silicon dioxide, diatomaceous earth, bentonite and attapulgite; the preservative is any one or more of sodium benzoate, potassium sorbate and ethyl parahydroxybenzoate; the defoaming agent is any one or more of isooctyl alcohol, isopentanol, stearic acid, lauric acid, lipid ethers and silicone; the antifreeze agent is any one or more of ethylene glycol, propylene glycol, glycerol, diethylene glycol, triethylene glycol, polyethylene glycol and urea.

8. Use of the Streptomyces BRS-24 according to claim 1 or the control agent according to any one of claims 2 to 7 in controlling clubroot of cruciferous crops.

9. The use according to claim 8, characterized in that The method comprises the following steps: soaking the cruciferous crops with the Streptomyces BRS-24 bacterial solution of claim 1 for 10-30 minutes before sowing, wherein the number of viable bacteria of the Streptomyces BRS-24 bacterial solution is 1×10 5 -1×10 8 cfu / ml; and / or watering with Streptomyces BRS-24 solution after planting, 5-20ml / tree, concentration 1×10 5 -1×10 8 cfu / ml.

10. Use of the Streptomyces BRS-24 according to claim 1 or the control agent according to any one of claims 2 to 7 in promoting the growth of healthy cruciferous crops.

11. The use according to claim 10, characterized in that The method comprises the following steps: soaking the cruciferous crops with the Streptomyces BRS-24 bacterial solution of claim 1 for 10-30 minutes before sowing, wherein the number of viable bacteria of the Streptomyces BRS-24 bacterial solution is 1×10 5 -1×10 8 cfu / ml; and / or watering with Streptomyces BRS-24 solution after planting, 5-20ml / tree, concentration 1×10 5 -1×10 8 cfu / ml.

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