Compound microbial agent for preventing and treating clubroot of cruciferae crops

Abstract

The invention discloses a compound microorganism bacterium agent for preventing and treating clubroot of cruciferae crops. The compound microorganism bacterium agent comprises a streptomyces roseosporus bacterium agent with the serial number of B98035, a trichoderma citrinoviride bacterium agent with the serial number of BMZ102895, and a bacillus sororensis bacterium agent with the serial number of BMZ065932, and the compound microorganism bacterium agent is prepared from a compound microorganism bacterium agent with the serial number of BMZ065932, a compound microorganism bacterium agent with the serial number of BMZ065932, a compound microorganism bacterium agent with the serial number of BMZ065932 and a compound microorganism bacterium agent with the serial number of BMZ065932 and a compound microorganism bacterium agent with the serial number of BMZ065932. The volume ratio of the streptomyces roseosporus microbial inoculum to the trichoderma citrinoviride microbial inoculum to the bacillus sorola is (1: 2)-(4: 2), the number of effective viable bacteria in the streptomyces roseosporus microbial inoculum is not less than 1 * 10 < 8 > CFU / ml, the number of effective viable bacteria in the trichoderma citrinoviride microbial inoculum is not less than 1 * 10 < 8 > CFU / ml, and the number of effective viable bacteria in the bacillus sorola microbial inoculum is not less than 1 * 10 < 9 > CFU / ml. The complex microbial inoculant disclosed by the invention can be used for effectively preventing and treating the clubroot of the cruciferae crops, which is caused by plasmodiophora brassicae, and can be used for preventing and treating the clubroot of the cruciferae crops, which is caused by the plasmodiophora brassicae.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a composite microbial agent for preventing and treating clubroot disease of cruciferous crops. Background Art

[0002] Clubroot disease of cruciferous crops is caused by Brassica rapa ( Plasmodiophorabrassicae Woron.) is a global disease caused by infection, which mainly harms the roots of cruciferous plants such as cabbage, kale, and rapeseed, forming tumors and causing the plants to wilt and die, seriously affecting the yield of cruciferous crops.

[0003] At present, chemical agents such as thiophanate-methyl wettable powder or benomyl wettable powder can be used to prevent and control clubroot. However, chemical control can easily bring about pesticide residues and environmental pollution, endangering the environment and food safety, and the harm to human body cannot be ignored. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the present invention proposes a composite microbial agent for preventing and treating clubroot disease of cruciferous crops.

[0005] To achieve the above object, the present invention provides a composite microbial agent for preventing and treating clubroot disease in cruciferous crops, the composite microbial agent comprising: Streptomyces roseosporus numbered B98035 ( Streptomyces roseosporus ) fungal agent, Trichoderma viride numbered BMZ102895 ( Trichoderma citrinoviride ) and Sonora Desert Bacillus spp. BMZ065932 ( Bacillus sonorensis ) agent; the volume ratio of the Streptomyces roseosporus agent, the Trichoderma citriodora agent and the Sonora desert Bacillus agent is 1:2-4:2, wherein the effective viable cell count of the Streptomyces roseosporus agent is not less than 1×10 8 CFU / ml, the effective viable bacteria count in the Trichoderma viride agent is not less than 1×10 8 CFU / ml, the effective viable bacteria count in Sonora Desert Bacillus inoculum is not less than 1×10 9 CFU / ml.

[0006] Furthermore, the preparation method of the Streptomyces roseosporus agent comprises the following steps: A1, inoculate the activated Streptomyces roseosporus into a liquid seed culture medium and culture it aerobically at 30°C for 72 hours to obtain liquid seeds; the liquid seed culture medium contains 4.0 g of yeast-soaked manure, 10.0 g of malt extract powder, 4.0 g of glucose, and 1 L of water, with a pH of 7.2±0.2; A2: Inoculate the liquid seed into the liquid fermentation medium at a volume ratio of 1:50. The culture conditions are: 30°C, 150-180 rpm, and aerobic culture for 72 hours to obtain the liquid strain. The liquid fermentation medium is the same as the liquid seed medium. A3: Filter the liquid culture and dilute it to make the effective viable bacteria count about 1×10 8 CFU / ml, and the Streptomyces roseosporus agent is obtained.

[0007] Furthermore, the preparation method of the Trichoderma citriodora agent comprises the following steps: B1, after activation, Trichoderma virens was inoculated on the surface of a PDA plate and cultured at 28°C for 48 h; B2, inoculating the Trichoderma virens obtained in step B1 into a liquid seed culture medium, and culturing the culture in a shake flask at 28°C and 120 rpm for 72 hours to obtain liquid seeds; wherein the liquid seed culture medium is PDB medium containing 200 g of potatoes, 15 g of glucose, and 1 L of water; B3, inoculating the liquid seeds obtained in step B2 into a liquid fermentation medium at a volume ratio of 0.05-0.1:1, and fermenting under conditions of 28° C. and 120-150 rpm for 72 h to obtain a liquid strain; wherein the liquid fermentation medium is PDB medium containing 200 g of potatoes, 15 g of glucose, and 1 L of water; B4: Filter the liquid culture and dilute it to make the effective viable bacteria count about 1×10 8 CFU / ml, and the citrus green Trichoderma agent is obtained.

[0008] Furthermore, the preparation method of the Sonora Desert Bacillus inoculum comprises the following steps: C1, inoculating the activated Sonora Desert Bacillus into liquid LB medium, and culturing at 28-37°C and 100-300 rpm for 16-18 hours to obtain liquid seeds; C2, inoculating the liquid seeds into a fermenter filled with liquid LB medium at an inoculum rate of 2-5%, and culturing at 28-37°C and 100-300 rpm for 16-24 hours to obtain a Sonora Desert Bacillus culture liquid; C3, the Sonora Desert Bacillus liquid was filtered and diluted to make the effective viable bacterial count about 1×10 9 CFU / ml, that is, the Sonora Desert Bacillus agent.

[0009] The second aspect of the present invention provides an application of the composite bacterial agent of the first aspect in preventing and treating clubroot disease of cruciferous crops, wherein the pathogen of clubroot disease is Brassica rapa ( Plasmodiophorabrassicae Woron.).

[0010] Through the above technical solution, the present invention can achieve at least the following beneficial effects: The composite bacterial agent of the present invention can effectively prevent and control Brassica rapa ( Plasmodiophorabrassicae Woron.) caused by clubroot disease of cruciferous crops. DETAILED DESCRIPTION

[0011] Unless otherwise stated, all materials and reagents used in the present invention are commercially available.

[0012] In the present invention, Streptomyces roseosporus numbered B98035 ( Streptomyces roseosporus ), Trichoderma virens numbered BMZ102895 ( Trichoderma citrinoviride ) and Sonora Desert Bacillus BMZ065932 were purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0013] In the present invention: The preparation method of the Streptomyces roseosporus agent comprises the following steps: A1, inoculate the activated Streptomyces roseosporus into a liquid seed culture medium and culture it aerobically at 30°C for 72 hours to obtain liquid seeds; the liquid seed culture medium contains 4.0 g of yeast-soaked manure, 10.0 g of malt extract powder, 4.0 g of glucose, and 1 L of water, with a pH of 7.2±0.2; A2: Inoculate the liquid seed into the liquid fermentation medium at a volume ratio of 1:50. The culture conditions are: 30°C, 150-180 rpm, and aerobic culture for 72 hours to obtain the liquid strain. The liquid fermentation medium is the same as the liquid seed medium. A3: Filter the liquid culture and dilute it to make the effective viable bacteria count about 1×10 8 CFU / ml, and the Streptomyces roseosporus agent is obtained.

[0014] The preparation method of Trichoderma virens agent comprises the following steps: B1, after activation, Trichoderma virens was inoculated on the surface of a PDA plate and cultured at 28°C for 48 h; B2, inoculating the Trichoderma virens obtained in step B1 into a liquid seed culture medium, and culturing the culture in a shake flask at 28°C and 120 rpm for 72 hours to obtain liquid seeds; wherein the liquid seed culture medium is PDB medium containing 200 g of potatoes, 15 g of glucose, and 1 L of water; B3, inoculating the liquid seeds obtained in step B2 into a liquid fermentation medium at a volume ratio of 0.05-0.1:1, and fermenting under conditions of 28° C. and 120-150 rpm for 72 h to obtain a liquid strain; wherein the liquid fermentation medium is PDB medium containing 200 g of potatoes, 15 g of glucose, and 1 L of water; B4: Filter the liquid culture and dilute it to make the effective viable bacteria count about 1×10 8 CFU / ml, and the citrus green Trichoderma agent is obtained.

[0015] The preparation method of the Sonora Desert Bacillus inoculum comprises the following steps: C1, inoculating the activated Sonora Desert Bacillus into liquid LB medium, and culturing at 28-37°C and 100-300 rpm for 16-18 hours to obtain liquid seeds; C2, inoculating the liquid seeds into a fermenter filled with liquid LB medium at an inoculum rate of 2-5%, and culturing at 28-37°C and 100-300 rpm for 16-24 hours to obtain a Sonora Desert Bacillus culture liquid; C3, the Sonora Desert Bacillus liquid was filtered and diluted to make the effective viable bacterial count about 1×10 9 CFU / ml, that is, the Sonora Desert Bacillus agent.

[0016] Example 1 A composite microbial agent for preventing and treating clubroot disease in cruciferous crops, comprising: Streptomyces roseosporus numbered B98035 ( Streptomyces roseosporus ) fungal agent, Trichoderma viride numbered BMZ102895 ( Trichoderma citrinoviride ) and Sonora Desert Bacillus spp. BMZ065932 ( Bacillus sonorensis ) agent; the mass ratio of the rose spore Streptomyces agent, the citrulline Trichoderma agent and the Sonora desert Bacillus agent is 1:2:2.

[0017] Example 2 A composite microbial agent for preventing and treating clubroot disease in cruciferous crops, comprising: Streptomyces roseosporus numbered B98035 ( Streptomyces roseosporus ) fungal agent, Trichoderma viride numbered BMZ102895 ( Trichoderma citrinoviride ) and Sonora Desert Bacillus spp. BMZ065932 ( Bacillus sonorensis ) agent; the mass ratio of the rose spore Streptomyces agent, the orange viridian Trichoderma agent and the Sonora desert Bacillus agent is 1:3:2.

[0018] Example 3 A composite microbial agent for preventing and treating clubroot disease in cruciferous crops, comprising: Streptomyces roseosporus numbered B98035 ( Streptomyces roseosporus ) fungal agent, Trichoderma viride numbered BMZ102895 ( Trichoderma citrinoviride ) and Sonora Desert Bacillus spp. BMZ065932 ( Bacillus sonorensis ) agent; the mass ratio of the rose spore Streptomyces agent, the citrulline Trichoderma agent and the Sonora desert Bacillus agent is 1:4:2.

[0019] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the composite microbial agent does not contain the Streptomyces roseosporus agent.

[0020] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the composite microbial agent does not contain Trichoderma citriviride agent.

[0021] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the composite microbial agent does not contain the Sonora Desert Bacillus agent.

[0022] Experimental example Select the cabbage seedlings with good growth and basically the same size for transplanting, and use a concentration of 1×10 8 CFU / mL of Brassica rapa ( Plasmodiophorabrassicae Cabbage plants were root-irrigated with a suspension of Woron. 200 mL per plant. Seven days after root irrigation, the composite microbial inoculants described in Examples 1 to 3 and Comparative Examples 1 to 3 were applied to the irrigated cabbage plants at a rate of 200 mL per plant. Each treatment was replicated 50 times, and disease index was assessed 14 days after application. The results are shown in Table 1 below.

[0023] Condition analysis: Grade 0: no root swelling; Grade 1: There are small swellings in the lateral roots.

[0024] Level 3: The main root is swollen, and its diameter is less than 2 times that of the stem base; Level 5: The taproot is swollen, and its rhizome is 2-3 times the size of the stem base; Level 7: The taproot is swollen, and its rhizome is 3-4 times the size of the stem base; Level 9: The main root is swollen, and its rhizome is more than 4 times the size of the stem base, or the swollen root turns black.

[0025] Disease index = [Σ (number of diseased plants at each level × relative level value) / highest level × total number of plants surveyed] × 100%; Table 1 Disease index of each treatment As can be seen from Table 1 above, the composite bacterial agent of the present application can effectively control Brassica rapa ( Plasmodiophorabrassicae Woron.) caused by clubroot disease of cruciferous crops.

[0026] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A composite microbial agent for preventing and treating clubroot of cruciferous crops, characterized in that: The composite microbial agent comprises: Streptomyces roseosporus numbered B98035 ( Streptomyces roseosporus ) fungal agent, Trichoderma viride numbered BMZ102895 ( Trichoderma citrinoviride ) and Sonora Desert Bacillus spp. BMZ065932 ( Bacillus sonorensis ) agent; the volume ratio of the Streptomyces roseosporus agent, the Trichoderma citriodora agent and the Sonora desert Bacillus agent is 1:2-4:2, wherein the effective viable cell count of the Streptomyces roseosporus agent is not less than 1×10 8 CFU / ml, the effective viable bacteria count in the Trichoderma viride agent is not less than 1×10 8 CFU / ml, the effective viable bacteria count in Sonora Desert Bacillus inoculum is not less than 1×10 9 CFUml.

2. The composite microbial agent for preventing and treating clubroot in cruciferous crops according to claim 1, characterized in that: The preparation method of the Streptomyces roseosporus agent comprises the following steps: A1, inoculate the activated Streptomyces roseosporus into a liquid seed culture medium and culture it aerobically at 30°C for 72 hours to obtain liquid seeds; the liquid seed culture medium contains 4.0 g of yeast-soaked manure, 10.0 g of malt extract powder, 4.0 g of glucose, and 1 L of water, with a pH of 7.2±0.2; A2: Inoculate the liquid seed into the liquid fermentation medium at a volume ratio of 1:

50. The culture conditions are: 30°C, 150-180 rpm, and aerobic culture for 72 hours to obtain the liquid strain. The liquid fermentation medium is the same as the liquid seed medium. A3: Filter the liquid culture and dilute it to make the effective viable count 1×10 8 CFU / ml, and the Streptomyces roseosporus agent was obtained.

3. The composite microbial agent for preventing and treating clubroot of cruciferous crops according to claim 1, characterized in that: The preparation method of the Trichoderma citriodora agent comprises the following steps: B1, after activation, Trichoderma citriviride was inoculated on the surface of a PDA plate and cultured at 28°C for 48 h; B2, inoculating the Trichoderma virens obtained in step B1 into a liquid seed culture medium, and culturing the culture in a shake flask at 28°C and 120 rpm for 72 hours to obtain liquid seeds; wherein the liquid seed culture medium is PDB medium containing 200 g of potatoes, 15 g of glucose, and 1 L of water; B3, inoculating the liquid seeds obtained in step B2 into a liquid fermentation medium at a volume ratio of 0.05-0.1:1, and fermenting under conditions of 28° C. and 120-150 rpm for 72 h to obtain a liquid strain; wherein the liquid fermentation medium is PDB medium containing 200 g of potatoes, 15 g of glucose, and 1 L of water; B4: Filter the liquid culture and dilute it to make the effective viable count 1×10 8 CFU / ml, and the citrus green Trichoderma agent is obtained.

4. The composite microbial agent for preventing and treating clubroot in cruciferous crops according to claim 1, characterized in that: The preparation method of the Sonora Desert Bacillus inoculum comprises the following steps: C1, inoculating the activated Sonora Desert Bacillus into liquid LB medium, and culturing at 28-37°C and 100-300 rpm for 16-18 hours to obtain liquid seeds; C2, inoculating the liquid seeds into a fermenter filled with liquid LB medium at an inoculum rate of 2-5%, and culturing at 28-37°C and 100-300 rpm for 16-24 hours to obtain a Sonora Desert Bacillus culture liquid; C3, the Sonora Desert Bacillus liquid was filtered and diluted to make the effective viable cell count 1×10 9 CFU / ml, that is, the Sonora Desert Bacillus agent.

5. The use of the composite bacterial agent according to any one of claims 1 to 4 in preventing and treating clubroot of cruciferous crops, wherein the pathogen of clubroot is Clubroot brassicae ( Plasmodiophorabrassicae Woron.).

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