Biocontrol bacterial system JW for preventing and treating buckwheat stem canker and application of biocontrol bacterial system JW

Abstract

The invention relates to a biocontrol strain JW for preventing and treating buckwheat stem canker and application thereof, and belongs to the field of agricultural microorganism and plant disease biological prevention and treatment. The biocontrol bacterial system provided by the invention is formed by combining bacillus amyloliquefaciens and bacillus velezensis, and the biocontrol bacterial system is formed by combining bacillus amyloliquefaciens and bacillus velezensis. When the strain is used for seed dressing treatment of buckwheat seeds, the strain is safe to buckwheat, the prevention effect on the buckwheat stem canker is 60.8%, the yield is increased by 20.7 kg / 667m < 2 >, the buckwheat stem canker can be effectively prevented and treated, the yield is increased, and the strain has good development and application prospects.

Description

Technical Field

[0001] The invention relates to the field of biological control, and in particular to a biological control bacteria system JW for controlling buckwheat stem canker and an application thereof. Background Art

[0002] Buckwheat (Fagopyrum esculentum) belongs to the genus Fagopyrum of the Polygonaceae family. It is a multi-purpose grain economic crop that integrates nutrition, health care, medicine, feed and honey. The main cultivated species are sweet buckwheat and bitter buckwheat. According to statistics in 2021, the cultivated area in my country reached 30,000 hectares and the output reached 300,000 to 500,000 tons. my country is a major producer and exporter of buckwheat. In recent years, the world's demand for buckwheat has been on the rise, which has also put forward higher requirements on the quality and yield of buckwheat.

[0003] With the development of the buckwheat industry, the degree of intensive production and the increase in the multiple cropping index, the occurrence of diseases has become more and more serious. Buckwheat stem canker (Rhizoctonia spp.) is an important factor affecting buckwheat yield and quality, and is the main disease in production. There are few studies on this disease at home and abroad, there are no effective prevention and control measures, and no prevention and control measures are taken in the field. Using microorganisms themselves or secondary metabolites produced by microorganisms to inhibit soil-borne pathogens has the advantages of green safety and continuous prevention and control, and is increasingly favored by people. Biological control is a fundamental measure for the prevention and control of soil-borne diseases.

[0004] The present invention takes soil samples from a field where buckwheat stem canker disease occurs, isolates antagonistic bacteria from the rhizosphere soil, identifies them from the perspectives of morphology, physiology, biochemistry and molecular biology, mixes strains that have no antagonistic effect on each other, constructs a composite bacterial system, and uses pot tests and field tests to determine the control effect of the composite bacterial system, aiming to find efficient and stable biocontrol resources for the biological control of buckwheat stem canker disease. Summary of the invention

[0005] The invention aims to provide a biocontrol fungus system JW which can be used for preventing and treating buckwheat stem canker, comprising Bacillus amyloliquefaciens JGII5 and Bacillus velezensis WXB10.

[0006] Another object of the present invention is to provide a biocontrol bacteria system made using the above-mentioned bacterial composition.

[0007] The third object of the present invention is to provide the use of the above-mentioned bacterial strain in preventing and treating buckwheat stem canker.

[0008] The present invention discloses a strain of Bacillus amyloliquefaciens JGII5, which is isolated and screened from rhizosphere soil of a field where buckwheat stem canker occurs, has a significant inhibitory effect on buckwheat stem canker pathogens, is identified as Bacillus amyloliquefaciens through morphology, physiology, biochemistry and molecular biology, and is deposited in the China Center for Type Culture Collection on May 22, 2023, with a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and a registration number of the collection center of CCTCC M 2023808. The strain is referred to as strain JGII5 in the present invention.

[0009] The present invention discloses a strain of Bacillus velezensis WXB10 (Bacillus velezensis), which is isolated and screened from the rhizosphere soil of a field where buckwheat stem canker disease occurs, has a significant inhibitory effect on buckwheat stem canker pathogens, is identified as Bacillus velezensis through morphology, physiology, biochemistry and molecular biology, and is deposited in the China Center for Type Culture Collection on May 19, 2022, with a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and a deposit number of CCTCC M 2022680. The strain is referred to as strain WXB10 in the present invention.

[0010] One embodiment of the present invention is a bacterial composition consisting of Bacillus amyloliquefaciens JGII5 and Bacillus velezensis WXB10.

[0011] Preferably, the optimal ratio of the number of viable bacteria of Bacillus amyloliquefaciens to that of Bacillus velezensis in the composition is 4:3.

[0012] One embodiment of the present invention is a bacterial agent containing any one of the bacterial compositions described in claim 1, wherein the bacterial agent is a liquid preparation and the number of viable bacteria of the bacterial agent is 10 7 -10 8 CFU / mL.

[0013] For the above-mentioned bacterial agent, Bacillus amyloliquefaciens strain JGII5 and Bacillus velezensis strain WXB10 were cultured on LB medium at 28°C for 3 days, the cultured bacteria and the medium were crushed and mixed together, and then diluted with water to a concentration of 107 ~10 8 CFU / mL, compounded in proportion.

[0014] The invention further discloses application of the bacterial composition and the biocontrol bacteria system in preventing and treating buckwheat stem canker disease.

[0015] The method for using the biocontrol bacteria system in preventing and controlling buckwheat stem canker is to dilute the obtained bacteria system with water until the number of viable bacteria is 10 7 ~10 8 CFU / mL. The diluted bacterial system is used for seed dressing, and the seed dressing is to mix the diluted bacterial system with buckwheat seeds and place them for 12 hours before use.

[0016] The present invention has the advantages that:

[0017] The potted plant control effect of the biocontrol fungus system JW on buckwheat stem canker is 46.7%, and the field control effect is 60.1%. The biocontrol fungus system JW provided by the present invention is used for the prevention and treatment of buckwheat stem canker, has good prevention and treatment effect, has no pollution to the environment, and has good development and application prospects.

[0018] Storage Instructions:

[0019] The Bacillus amyloliquefaciens of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on May 22, 2023. The deposit address is China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The registration number of the deposit center is CCTCC M2023808. It is referred to as strain JGII5 in the present invention and has been tested for survival.

[0020] The Bacillus velezensis WXB10 (Bacillus velezensis) of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on May 19, 2022. The deposit address is China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The registration number of the collection center is CCTCC M 2022680. It is referred to as strain WXB10 in the present invention and has been tested for survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Inhibitory effects of two strains on buckwheat stem canker pathogen ( Figure 1 a is the control, 1b is strain JGII5, 1c is strain WXB10)

[0022] Figure 2 Construction of phylogenetic tree of strain JGII5

[0023] Figure 3 Construction of phylogenetic tree of strain WXB10 DETAILED DESCRIPTION

[0024] The following examples are convenient for better illustrating the present invention, but are not intended to limit the scope of the present invention. The test methods in the following examples are conventional methods unless otherwise specified. The materials used in the following examples can be purchased from conventional biochemical reagent companies unless otherwise specified.

[0025] The specific methods for isolating and screening antagonistic bacteria include the following:

[0026] The culture medium and pathogenic bacteria in the following examples:

[0027] LB solid medium: peptone 10 g, yeast extract powder 5 g, NaCl 10 g, agar 15 g, distilled water 1000 mL.

[0028] LB liquid medium: peptone 10 g, yeast extract powder 5 g, NaCl 10 g, distilled water 1000 mL.

[0029] PDA medium: 200 g peeled potatoes, 18 g agar, 20 g glucose, 1000 mL distilled water.

[0030] PDB medium: 200 g peeled potatoes, 20 g glucose, 1000 mL distilled water.

[0031] Pathogens: CBWR3, CBWR4, ZBWR4, TBWR2, and WBWR21. Pathogens CBWR3, CBWR4, and WBWR21 belong to Rhizoctonia solani AG-4, which are dominant strains in Inner Mongolia. Pathogens ZBWR4 and TBWR2 belong to Binucleate Rhizoctonia AG-A, which were isolated and identified by our research group and were found to be highly pathogenic to buckwheat through pathogenicity tests.

[0032] Example 1 Soil sample collection and isolation and screening of antagonistic bacteria to buckwheat stem canker pathogen

[0033] The rhizosphere soil of the buckwheat field in the New District Farm of Inner Mongolia Agricultural University was collected in 2021. 10 g of soil sample was weighed and placed in a triangular flask containing 90 mL of sterile water. The soil sample was shaken at 28 °C and 150 r / min for 15 min and diluted to a concentration of 10 -2 , 10 -3 , 10 -4 , 10 -5The bacterial suspension was prepared by the plate spreading method. 200 μL of bacterial suspension of different concentrations was evenly spread on each LB plate medium to separate the bacteria. Three replicates were set up and cultured in a 28°C incubator for 24 to 48 hours. Different types of single colonies were selected for purification based on the colony morphology and color. The purified strains were counted, numbered and preserved.

[0034] The filter paper method was used to screen strains with good antagonistic effects on buckwheat stem canker. The isolated bacterial strains were inoculated into LB liquid culture medium and cultured in a shaking incubator at 25°C and 180 r / min for 2 days. 8 CFU / mL. Take a 1cm diameter pathogen cake cultured on PDA medium at 25℃ for 5 days and place it in the center of the PDA plate. Place 8 layers of 1cm diameter sterilized filter paper in four symmetrical areas on the periphery. Add 150μL of the bacterial solution to be tested on the filter paper. Use sterile water as a control. Repeat each treatment 3 times. Culture in a constant temperature incubator at 25℃ for 5 days, measure the width of the inhibition zone and calculate the inhibition rate.

[0035] The results are shown in Table 1. The two bacterial strains JGII5 and WXB10 had inhibitory effects on several strains of buckwheat stem canker pathogens, with inhibition rates greater than 50%. The inhibition rates on the dominant species CBWR3 of buckwheat stem canker pathogen in Inner Mongolia were 51.4% and 55.8%.

[0036] Table 1 Inhibitory effects of antagonistic bacterial strains on different species of pathogens

[0037]

[0038] Example 2 Construction of antagonistic bacterial strain

[0039] The strains with better inhibitory effects on the pathogenic bacteria of buckwheat stem canker were screened out, and the strain compatibility test was carried out by the filter paper method. Three antagonistic strain cakes with a diameter of 1 cm were added to LB liquid culture medium and cultured in a shaking incubator at 25°C and 180 r / min for 2 days to prepare a concentration of 1×10 8 CFU / mL of antagonistic strain suspension, evenly spread 150 μL of one of the antagonistic strain suspensions on LB solid culture medium, place 8 layers of sterilized filter paper with a diameter of 1 cm in four symmetrical areas on the periphery, inoculate 150 μL of other antagonistic strain suspensions on the filter paper respectively, set 3 replicates, and culture in a constant temperature incubator at 28°C for 3 days to observe whether there is an antagonistic effect.

[0040] The results showed that there was no antagonism between strains JGII5 and WXB10, and they could be used in combination and named JW. The antagonistic effect was best when the combination ratio of the two strains was 4:3.

[0041] Example 3 Identification of strains JGII5 and WXB10

[0042] Morphological identification

[0043] The strains JGII5 and WXB10 were spread on LB solid medium by dilution method to grow single colonies. The colonies of strain JGII5 were round or oval, milky white, often with bulges in the middle, and sticky. The colonies of strain WXB10 were round or oval, milky white, with obvious white outer circle, bulges on the edge, wrinkles, sticky, and could be pulled into filaments.

[0044] Physiological and biochemical identification

[0045] The experiment was conducted with reference to the Manual of Identification of Common Bacteria Systems, and the physiological and biochemical indicators of the strains, such as carbon source, nitrogen source utilization, malonate utilization, methyl red test, starch hydrolysis, oxidase test and catalase test, were determined. The results are shown in Table 2. Both strains are Gram-positive bacteria, and can utilize malonate; hydrolyze starch; decompose glucose to produce acidic substances to lower the pH, making the methyl red indicator yellow; can utilize maltose, glucose, sucrose, mannitol and raffinose as the only carbon source; and can utilize ammonium nitrate as the only nitrogen source.

[0046] Table 2 Physiological and biochemical characteristics of strains

[0047]

[0048]

[0049] Note: "+" indicates positive, "-" indicates negative

[0050] Molecular Biology Identification

[0051] DNA extraction was performed according to the instructions of TIAN GEN Bacterial Genomic DNA Extraction Kit (spin column type) (Beijing, China), which was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The PCR primers for strains JGII5 and WXB10 were 27F and 1492R, 7F and 1540R, rpoBF and rpoBR, trpBF and trpBR, respectively. The primer sequences are shown in Table 3. The 25 μL reaction system contained: 17 μL of ddH2O, 2.5 μL of 10×PCR Buffer, 2 μL of dNTP, 1 μL of upstream primer, 1 μL of downstream primer, and 0.25 μL of TaqDNA polymerase. PCR reaction conditions: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s; annealing temperature for 30 s; 72℃ for 1 min; 34 cycles; 72℃ for 10 min. The PCR products of the four genes were detected by 1% agarose gel electrophoresis and sent to Shanghai Sangon Biotechnology Co., Ltd. for 16S rDNA sequencing. The sequencing results of the four PCR products were spliced, aligned using the Blast program in the NCBI database, and a phylogenetic tree was constructed using MEGA7.0 software.

[0052] The results showed that strain JGII5 and Bacillus amyloliquefaciens were grouped together with a similarity of 100% and the accession number was (CP038028.1); strain WXB10 and Bacillus velezensis were grouped together with a similarity of 99.93% and the accession number was (MG461457.1). Based on the results of morphological, physiological, biochemical and molecular biological identification, strain JGII5 was identified as Bacillus amyloliquefaciens and strain WXB10 was identified as Bacillus velezensis.

[0053] Table 3 Main primers and sequences

[0054]

[0055] Example 4 Potted plant control effect of antagonistic bacteria on buckwheat stem canker

[0056] Test strains and fungicides:

[0057] Test pathogen: CBWR3 (Rhizoctonia solani AG-4).

[0058] Antagonistic bacteria system: Antagonistic bacteria system JW, including Bacillus amyloliquefaciens JGII5 (Bacillus amyloliquefaciens) and Bacillus velezensis WXB10 (Bacillus velezensis), with a composite ratio of 4:3.

[0059] Test fungicide: myclobutanil.

[0060] Buckwheat varieties tested:

[0061] The buckwheat variety was Wuchuan sweet buckwheat, a commonly used local variety in Wuchuan County, Hohhot City, Inner Mongolia Autonomous Region.

[0062] Test method:

[0063] (1) Preparation of pathogen inoculum: 300 g of wheat bran was mixed with 600 mL of distilled water and placed in a 1000 mL Erlenmeyer flask. The mixture was sterilized at 121°C for 40 min. After cooling, each flask was inoculated with a 5 mm diameter pathogen cake cultured on a PDA plate for 5 days. The cake was cultured in the dark at 25°C for 30 days. When a large number of sclerotia were produced on the bran, the cake was dried naturally in the sun, rubbed into powder and mixed for later use.

[0064] (2) Experimental methods: There were four treatments in the experiment, namely, blank control, pathogen-only control, pathogen-and-antagonistic bacteria, and pathogen-and-pyraclostrobin. Soil was collected from the New District Farm of Inner Mongolia Agricultural University and mixed with vermiculite at a ratio of 2:1. The soil was sterilized in a dry heat sterilizer at 180°C for 2 h. After cooling, it was placed in a flower pot with an upper diameter of 25 cm and a height of 17 cm. The pot was filled to 2 / 3 of the pot. Then, 2 g of pathogen inoculum was inoculated and mixed with the soil. Buckwheat seeds were mixed with the antagonistic bacteria JW. Two antagonistic bacteria JGII5 and WXB10 were cultured in a shaking incubator at 25°C and 180 r / min for 2 days. Water was added to dilute the soil to a bacterial concentration of 1×10 8 CFU / mL, construct the composite bacterial strain JW according to the ratio of 4:3, take 38mL of composite bacterial strain JW and 30g of seeds in a plastic bag, mix well, and place for 12h. Sow buckwheat seeds in pots, 50 seeds in each pot, and cover them with a layer of 1-3cm thick soil. Set up 4 replicates. The pots were placed in the greenhouse of the new district of Inner Mongolia Agricultural University. During the growth period of buckwheat, 500mL of water was poured every 36h, and the emergence rate was measured after germination. At 45d after sowing, the plant height and stem thickness were measured. According to the disease grading standard of buckwheat stem canker, the incidence of underground stems was investigated, the disease level was recorded, and the disease index and prevention and control effect were calculated.

[0065] The results are shown in Table 4. The antagonistic bacteria strain JW is safe for buckwheat and can significantly reduce the disease index, with a control effect of 40.9%.

[0066] Table 4 The potted control effect of antagonistic bacteria on buckwheat stem canker

[0067]

[0068] Example 5 Field test on the efficacy of antagonistic bacteria against buckwheat stem canker

[0069] The tested strains, fungicides, antagonistic bacteria strains, and buckwheat varieties were the same as those in Example 4.

[0070] Test method:

[0071] The field efficacy test was conducted on June 30, 2022, and the test site was set at the teaching base of Inner Mongolia Agricultural University, Tumote Left Banner, Hohhot City, Inner Mongolia. The test set up 4 treatments, the same as Example 4. The pathogen was inoculated in furrows, and the inoculation amount was 1.0 g / m 2 , the preparation method of the pathogen inoculum and the method of dressing buckwheat seeds with the antagonistic bacteria system are the same as in Example 4. The pathogen was inoculated first during sowing, and then buckwheat was sown, with 5 repetitions. The emergence rate was determined after all the buckwheat seedlings emerged. The first survey was conducted on July 20, 2022, and the plant height and stem thickness were measured to observe whether all the seedlings did not emerge due to the high concentration of pathogens; 60 days after planting, the incidence of underground stems was investigated according to the disease grading standard of buckwheat stem canker, the disease grade was recorded, and the disease index and control effect were calculated.

[0072] The results are shown in Table 5. The germination rate, stem diameter, plant height and 1000-grain weight of buckwheat treated with antagonistic bacteria JW were not significantly different from those of the blank control, indicating that the composite bacteria system is safe for buckwheat. The control effect of antagonistic bacteria JW on buckwheat stem canker was 60.1%, and the yield increased by 20.7 kg / 667 m 2 .

[0073] Table 5 Buckwheat stem canker field control test results

[0074]

Claims

1. A biocontrol bacterial strain JW for preventing and treating buckwheat stem canker, comprising a Bacillus amyloliquefaciens JGII5 and a Bacillus velezensis WXB10. Bacillus amyloliquefaciens JGII5 (Bacillus amyloliquefaciens) is deposited in the China Center for Type Culture Collection, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit date is May 22, 2023, and the deposit number is CCTCC M 2023808. Bacillus velezensis WXB10 (Bacillus velezensis) is deposited in the China Center for Type Culture Collection, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit date is May 19, 2022, and the deposit number is CCTCC M 2022680.

2. The biocontrol fungus strain JW for preventing and controlling buckwheat stem canker according to claim 1, characterized in that The optimal composite bacterial strain composed of a mixture of Bacillus amyloliquefaciens JGII5 and Bacillus velezensis WXB10 is a ratio of 4:3 between the live bacteria counts of strains JGII5 and WXB10.

3. The bacterial composition according to any one of claims 1 to 3, characterized in that It utilizes the application of biological control methods on buckwheat stem canker.

4. A liquid preparation containing the bacterial composition according to any one of claims 1 to 3, wherein the viable bacterial count of the liquid preparation is 10 7 ~10 8 CFU / mL.

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