A compound microbial agent for controlling clubroot of cruciferous crops

Abstract

The application discloses a kind of for preventing and treating cruciferous crops root rot complex microbial inoculant, comprising: the rose chain-forming Streptomyces agent numbered B98035, the Trichoderma citrinoviride agent numbered BMZ102895 and the Bacillus sonorensis agent numbered BMZ065932;The volume ratio of the rose chain-forming Streptomyces agent, Trichoderma citrinoviride agent and Bacillus sonorensis agent is 1:2-4:2, wherein the effective viable count in the rose chain-forming Streptomyces agent, Trichoderma citrinoviride agent is not less than 1×10 8 CFU / ml, and the effective viable count in Bacillus sonorensis agent is not less than 1×10 9 CFU / ml.The complex microbial inoculant of the application can effectively prevent and treat cruciferous crops root rot caused by Plamodiophora brassicae.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a compound microbial agent for the prevention and control of clubroot disease in cruciferous crops. Background Technology

[0002] Clubroot disease in cruciferous crops is caused by *Platycodon brassicae* (…). Plasmodiophorabrassicae Woron. is a global disease that mainly affects the roots of cruciferous plants such as cabbage, kale, and rapeseed, forming tumors that cause the plants to wilt and die, severely impacting the yield of cruciferous crops.

[0003] Currently, chemical agents such as methyl thiophanate wettable powder or carbendazim wettable powder can be used to control clubroot disease. However, chemical control can easily lead to pesticide residues and environmental pollution, endangering the environment and food safety, and the harm to human health cannot be ignored. Summary of the Invention

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

[0005] To achieve the above objectives, the first aspect of the present invention provides a compound microbial agent for controlling clubroot disease in cruciferous crops, the compound microbial agent comprising: Streptomyces roseosus (numbered B98035) Streptomyces roseosporus ) inoculant, Trichoderma viride with the serial number BMZ102895 ( Trichoderma citrinoviride ) bacterial agent and Sonora desert Bacillus (number BMZ065932) Bacillus sonorensis The volume ratio of the *Streptomyces roseum* inoculant, *Trichoderma citrinum* inoculant, and *Bacillus desertica* inoculant is 1:2-4:2, wherein the *Streptomyces roseum* inoculant contains an effective viable count of not less than 1×10⁻⁶. 8 CFU / ml, the effective viable count of Trichoderma viride in the fungicide is not less than 1×10⁻⁶. 8 The effective viable count of Sonora desert Bacillus inoculum is not less than 1×10⁻⁶ CFU / ml. 9 CFU / ml.

[0006] Furthermore, the preparation method of the *Streptomyces roseosus* inoculant includes the following steps:

[0007] A1. After activating Streptomyces roseosus, it was inoculated into liquid seed culture medium and cultured aerobicly at 30°C for 72 hours to obtain liquid seeds. The liquid seed culture medium contained 4.0 g yeast extract, 10.0 g malt extract, 4.0 g glucose, 1 L water, and pH 7.2 ± 0.2.

[0008] A2. Inoculate the liquid seed culture medium with a volume ratio of 1:50; culture conditions are: 30℃, 150r / min-180r / min, aerobic culture for 72h to obtain the liquid inoculum; the liquid fermentation medium is the same as the liquid seed culture medium.

[0009] A3, the liquid bacterial culture was filtered and diluted to achieve an effective viable count of approximately 1×10⁻⁶. 8 CFU / ml yields the Streptomyces roseosus inoculum.

[0010] Furthermore, the preparation method of the *Trichoderma viride* agent includes the following steps:

[0011] B1. After activating Trichoderma viride, it was inoculated onto the surface of a PDA plate and incubated at 28°C for 48 hours.

[0012] B2. The Trichoderma citrinum obtained in step B1 was inoculated into liquid seed culture medium and cultured in a shake flask at 28℃ and 120r / min for 72h to obtain liquid seed; wherein the liquid seed culture medium was PDB medium, containing 200g potato, 15g glucose and 1L water.

[0013] B3. The liquid seed obtained in step B2 is inoculated into the liquid fermentation medium at a volume ratio of 0.05-0.1:1. The fermentation culture conditions are 28℃ and 120-150r / min for 72h to obtain the liquid strain. The liquid fermentation medium is PDB medium, which contains 200g of potato, 15g of glucose and 1L of water.

[0014] B4, the liquid bacterial culture was filtered and diluted to achieve an effective viable count of approximately 1×10⁻⁶. 8 CFU / ml yields the Trichoderma viride fungicide.

[0015] Furthermore, the preparation method of the Sonora desert Bacillus inoculum includes the following steps:

[0016] C1. Activated Sonora desert Bacillus was inoculated into liquid LB medium and cultured at 28-37℃ with shaking at 100-300r / min for 16-18h to obtain liquid seed.

[0017] C2, inoculate the liquid seed into a fermenter containing liquid LB medium at an inoculation rate of 2-5%, and culture at 28-37℃ and 100-300r / min for 16-24h to obtain Sonora desert Bacillus bacterial culture;

[0018] C3, after filtering and diluting the Sonora desert Bacillus bacterial suspension, the effective viable count was approximately 1×10⁻⁶. 9 CFU / ml yields Sonora desert Bacillus inoculum.

[0019] The second aspect of this invention provides the application of the compound microbial agent of the first aspect in the control of clubroot disease in cruciferous crops, wherein the pathogen of clubroot disease is *Platycodon brassicae* (…). Plasmodiophorabrassicae Woron.).

[0020] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0021] The compound microbial agent of this invention can effectively control clubroot fungus (Bacteroides brassicae). Plasmodiophorabrassicae Clubroot disease in cruciferous crops caused by Woron. Detailed Implementation

[0022] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0023] In this invention, *Streptomyces roseosus* with the serial number B98035 ( Streptomyces roseosporus Trichoderma viride with serial number BMZ102895 Trichoderma citrinoviride Both the Sonora desert bacillus (with serial number BMZ065932) and the Sonora desert bacillus were purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0024] In this invention:

[0025] The preparation method of Streptomyces roseosus inoculum includes the following steps:

[0026] A1. After activating Streptomyces roseosus, it was inoculated into liquid seed culture medium and cultured aerobicly at 30°C for 72 hours to obtain liquid seeds. The liquid seed culture medium contained 4.0 g yeast extract, 10.0 g malt extract, 4.0 g glucose, 1 L water, and pH 7.2 ± 0.2.

[0027] A2. Inoculate the liquid seed culture medium with a volume ratio of 1:50; culture conditions are: 30℃, 150r / min-180r / min, aerobic culture for 72h to obtain the liquid inoculum; the liquid fermentation medium is the same as the liquid seed culture medium.

[0028] A3, the liquid bacterial culture was filtered and diluted to achieve an effective viable count of approximately 1×10⁻⁶. 8 CFU / ml yields the Streptomyces roseosus inoculum.

[0029] The preparation method of Trichoderma viride agent includes the following steps:

[0030] B1. After activating Trichoderma viride, it was inoculated onto the surface of a PDA plate and incubated at 28°C for 48 hours.

[0031] B2. The Trichoderma citrinum obtained in step B1 was inoculated into liquid seed culture medium and cultured in a shake flask at 28℃ and 120r / min for 72h to obtain liquid seed; wherein the liquid seed culture medium was PDB medium, containing 200g potato, 15g glucose and 1L water.

[0032] B3. The liquid seed obtained in step B2 is inoculated into the liquid fermentation medium at a volume ratio of 0.05-0.1:1. The fermentation culture conditions are 28℃ and 120-150r / min for 72h to obtain the liquid strain. The liquid fermentation medium is PDB medium, which contains 200g of potato, 15g of glucose and 1L of water.

[0033] B4, the liquid bacterial culture was filtered and diluted to achieve an effective viable count of approximately 1×10⁻⁶. 8 CFU / ml yields the Trichoderma viride fungicide.

[0034] The preparation method of Sonora desert Bacillus inoculum includes the following steps:

[0035] C1. Activated Sonora desert Bacillus was inoculated into liquid LB medium and cultured at 28-37℃ with shaking at 100-300r / min for 16-18h to obtain liquid seed.

[0036] C2, inoculate the liquid seed into a fermenter containing liquid LB medium at an inoculation rate of 2-5%, and culture at 28-37℃ and 100-300r / min for 16-24h to obtain Sonora desert Bacillus bacterial culture;

[0037] C3, after filtering and diluting the Sonora desert Bacillus bacterial suspension, the effective viable count was approximately 1×10⁻⁶. 9 CFU / ml yields Sonora desert Bacillus inoculum.

[0038] Example 1

[0039] A compound microbial agent for controlling clubroot disease in cruciferous crops, the compound microbial agent comprising: Streptomyces roseosus (numbered B98035) Streptomyces roseosporus ) inoculant, Trichoderma viride with the serial number BMZ102895 ( Trichoderma citrinoviride ) bacterial agent and Sonora desert Bacillus (number BMZ065932) Bacillus sonorensis The inoculant is prepared in the following manner: the mass ratio of the *Streptomyces roseosus* inoculant, *Trichoderma citrinum* inoculant, and *Bacillus desertica* inoculant is 1:2:2.

[0040] Example 2

[0041] A compound microbial agent for controlling clubroot disease in cruciferous crops, the compound microbial agent comprising: Streptomyces roseosus (numbered B98035) Streptomyces roseosporus ) inoculant, Trichoderma viride with the serial number BMZ102895 ( Trichoderma citrinoviride ) bacterial agent and Sonora desert Bacillus (number BMZ065932) Bacillus sonorensis The inoculant is prepared in the following manner: the mass ratio of the *Streptomyces roseosus* inoculant, *Trichoderma citrinum* inoculant, and *Bacillus desertica* inoculant is 1:3:2.

[0042] Example 3

[0043] A compound microbial agent for controlling clubroot disease in cruciferous crops, the compound microbial agent comprising: Streptomyces roseosus (numbered B98035) Streptomyces roseosporus ) inoculant, Trichoderma viride with the serial number BMZ102895 ( Trichoderma citrinoviride ) bacterial agent and Sonora desert Bacillus (number BMZ065932) Bacillus sonorensis The inoculant is prepared in the following manner: the mass ratio of the *Streptomyces roseosus* inoculant, *Trichoderma citrinum* inoculant, and *Bacillus desertica* inoculant is 1:4:2.

[0044] Comparative Example 1

[0045] The difference between Comparative Example 1 and Example 1 is that the compound microbial agent does not contain Streptomyces roseosus agent.

[0046] Comparative Example 2

[0047] The difference between Comparative Example 2 and Example 1 is that the compound microbial agent does not contain Trichoderma viride agent.

[0048] Comparative Example 3

[0049] The difference between Comparative Example 3 and Example 1 is that the compound microbial agent does not contain Sonora desert Bacillus agent.

[0050] Experimental Example

[0051] Select healthy, uniformly sized Chinese cabbage seedlings for transplanting, using a concentration of 1×10⁻⁶. 8 CFU / mL of *Plasmodiophora brassicae* ( Plasmodiophorabrassicae Woron. bacterial suspension was used to drench the roots of Chinese cabbage, with 200 mL per plant. Seven days after drenching, the Chinese cabbage plants were treated with a compound microbial agent according to Examples 1 to 3 and Comparative Examples 1 to 3, 200 mL per plant. Each treatment was replicated 50 times. The disease index was investigated 14 days after the application of the compound microbial agent. The results are shown in Table 1 below.

[0052] Analysis of the condition:

[0053] Grade 0: No root swelling at the base;

[0054] Grade 1: Small root swellings on lateral roots.

[0055] Grade 3: The taproot is swollen, and its diameter is less than twice that of the stem base;

[0056] Grade 5: The main root is swollen, and its rhizome is 2-3 times the size of the stem base;

[0057] Level 7: The main root is swollen, and its rhizome is 3-4 times the size of the base of the stem;

[0058] Level 9: The main root is swollen, and its rootstock is more than 4 times the size of the base of the stem, or the swollen root turns black.

[0059] Disease index = [Σ(number of diseased plants at each level × relative level value) / highest level × total number of plants surveyed] × 100%;

[0060] Table 1. Disease Indices for Each Treatment

[0061]

[0062] As can be seen from Table 1 above, the compound microbial agent of this application can effectively control *Cladosporium brassicae* (Cladosporium spp.). Plasmodiophorabrassicae Clubroot disease in cruciferous crops caused by Woron.

[0063] 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 intended to limit it. 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 to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A compound microbial inoculant for controlling clubroot disease in cruciferous crops, characterized in that: The compound microbial agent contains: *Streptomyces roseosus* with the serial number B98035 (…). Streptomyces roseosporus ) inoculant, Trichoderma viride with the serial number BMZ102895 ( Trichoderma citrinoviride ) bacterial agent and Sonora desert Bacillus (number BMZ065932) Bacillus sonorensis The volume ratio of *Streptomyces roseosus* inoculant, *Trichoderma citrinum* inoculant, and *Bacillus desertica* inoculant is 1:2-4:2, wherein the *Streptomyces roseosus* inoculant contains an effective viable count of not less than 1×10⁻⁶. 8 CFU / ml, the effective viable count of Trichoderma viride in the fungicide is not less than 1×10⁻⁶. 8 The effective viable count of Sonora desert Bacillus inoculum is not less than 1×10⁻⁶ CFU / ml. 9 CFU / ml; among which, Streptomyces roseosus, Trichoderma citrinum and Bacillus sanoraensis were purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

2. The compound microbial agent for controlling clubroot disease in cruciferous crops according to claim 1, characterized in that: The preparation method of the Streptomyces roseum inoculum includes the following steps: A1. After activating Streptomyces roseosus, it was inoculated into liquid seed culture medium and cultured aerobicly at 30°C for 72 hours to obtain liquid seeds. The liquid seed culture medium contained 4.0 g yeast extract, 10.0 g malt extract, 4.0 g glucose, 1 L water, and pH 7.2 ± 0.

2. A2. Inoculate the liquid seed culture medium with a volume ratio of 1:50; culture conditions are: 30℃, 150r / min-180r / min, aerobic culture for 72h to obtain the liquid inoculum; the liquid fermentation medium is the same as the liquid seed culture medium. A3, after filtering and diluting the liquid bacterial culture, the effective viable count is made up to 1×10⁻⁶. 8 CFU / ml yields the Streptomyces roseosus inoculum.

3. The compound microbial agent for controlling clubroot disease in cruciferous crops according to claim 1, characterized in that: The preparation method of the Trichoderma viride agent includes the following steps: B1. After activating Trichoderma viride, it was inoculated onto the surface of a PDA plate and incubated at 28°C for 48 hours. B2. The Trichoderma citrinum obtained in step B1 was inoculated into liquid seed culture medium and cultured in a shake flask at 28℃ and 120r / min for 72h to obtain liquid seed; wherein the liquid seed culture medium was PDB medium, containing 200g potato, 15g glucose and 1L water. B3. The liquid seed obtained in step B2 is inoculated into the liquid fermentation medium at a volume ratio of 0.05-0.1:

1. The fermentation culture conditions are 28℃ and 120-150r / min for 72h to obtain the liquid strain. The liquid fermentation medium is PDB medium, which contains 200g of potato, 15g of glucose and 1L of water. B4, after filtering and diluting the liquid bacterial culture, the effective viable count is made up to 1×10⁻⁶. 8 CFU / ml yields the Trichoderma viride fungicide.

4. A compound microbial agent for controlling clubroot disease in cruciferous crops according to claim 1, characterized in that: The preparation method of the Sonora desert Bacillus inoculum includes the following steps: C1. Activated Sonora desert Bacillus was inoculated into liquid LB medium and cultured at 28-37℃ with shaking at 100-300r / min for 16-18h to obtain liquid seed. C2, inoculate the liquid seed into a fermenter containing liquid LB medium at an inoculation rate of 2-5%, and culture at 28-37℃ and 100-300r / min for 16-24h to obtain Sonora desert Bacillus bacterial culture; C3, after filtering and diluting the Sonora desert Bacillus bacterial suspension, the effective viable count was reduced to 1×10⁻⁶. 9 CFU / ml yields Sonora desert Bacillus inoculum.

5. The application of the compound microbial agent according to any one of claims 1-4 in the control of clubroot disease in cruciferous crops, wherein the pathogen of clubroot disease is *Platycodon brassicae* (…). Plasmodiophora brassicae Woron.).

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