Method for preparing composite antibiological inoculant from Shuanghuanglian medicine residues and application of composite antibiological inoculant

Abstract

The invention relates to a method for preparing a composite antibiological inoculant by using Shuanghuanglian herb residues. The method comprises the following steps: 1) preparing a seed solution; 2) preparing a Shuanghuanglian medicine residue solid fermentation culture medium; and (3) preparing the biocontrol inoculant: uniformly inoculating the seed solution prepared in the step (1) into a Shuanghuanglian decoction dreg solid fermentation culture medium according to the inoculum size of 8-12%, fermenting at 33-37 DEG C for 9-11 days, and drying at room temperature to obtain the composite biocontrol inoculant. The preparation method is simple, low in cost and convenient to use, the Shuanghuanglian medicine residues with bacteriostatic activity serve as a solid culture medium to conduct solid fermentation on antagonistic strains F-1 and HT-9 to prepare the composite biocontrol inoculant, the obvious biocontrol effect is achieved, the obvious prevention and treatment effect on the peanut southern blight is achieved, and the social and economic benefits are obvious.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a method for preparing a composite biocontrol agent by utilizing Shuanghuanglian medicinal residues and application thereof. Background Art

[0002] Peanut (Arachis hypogaea Linn.) is one of the most important oil crops in the world, with high edible value and economic value. In recent years, with the rapid development of agricultural production in my country, the planting area of ​​peanuts has continued to expand, and the phenomenon of continuous cropping of peanuts has become increasingly serious. At the same time, the large-scale use of chemical pesticides has caused low soil organic matter content, reduced soil fertility, soil compaction, etc., resulting in the increasing severity of peanut diseases year by year. In particular, the white rot of peanut caused by Sclerotium rolfsii Sacc. has caused serious damage to my country's peanut industry. At present, the prevention and control methods of white rot of peanuts at home and abroad mainly include crop rotation, breeding resistant varieties and the use of chemical agents. However, in actual production, the prevention and control of white rot mainly relies on chemical pesticides. However, the long-term use of chemical pesticides will cause the white rot pathogen to develop drug resistance, making the prevention and control effect gradually worse. In addition, the large-scale use of chemical pesticides will not only cause pesticide residue problems affecting food safety, but also destroy the ecological balance and biodiversity of the soil. Therefore, it is urgent to find an economical, safe and effective biological control measure.

[0003] Shuanghuanglian dregs refer to the dregs produced during the production of Shuanghuanglian, and the output is relatively large. Some enterprises engaged in the production of traditional Chinese medicine are not environmentally friendly in the treatment of dregs, such as direct incineration, open-air stacking, landfilling, etc., which cause serious pollution to our surrounding living environment. Cellulose, the main component of the cell wall of traditional Chinese medicine plants, seriously hinders the precipitation of effective drug components in the plant cytoplasm. According to relevant research and determination, the extraction rate of effective ingredients in Shuanghuanglian medicinal materials is only 30% to 70%. The dregs not only retain crude protein, crude polysaccharides, starch and other nutrients, but also contain a variety of effective ingredients such as baicalin, chlorogenic acid, forsythiaside, etc. that have not been utilized. Therefore, how to make full use of Shuanghuanglian dregs to reduce resource waste and environmental pollution is a problem that needs to be solved urgently. Summary of the invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a method and application of preparing a composite biocontrol agent using Shuanghuanglian residues, which can effectively solve the problem of medication for peanut white rot disease and the problem of recycling Shuanghuanglian residue resources.

[0005] To achieve the above object, the technical solution provided by the present invention is a method for preparing a composite biocontrol agent using Shuanghuanglian drug residues, comprising the following steps:

[0006] 1) preparing seed solution: inoculating the activated Bacillus subtilis F-1 and Bacillus Velez HT-9 into beef extract peptone liquid culture medium respectively, shaking and culturing at 28-32° C. and 170-190 r / min for 22-26 h, mixing in a ratio of 1:1 to obtain seed solution for standby use;

[0007] 2) Preparing Shuanghuanglian medicinal residue solid fermentation medium: After the Shuanghuanglian medicinal residue is crushed, it is sieved through a 30-mesh sieve to obtain Shuanghuanglian medicinal residue powder, and 45-55g of Shuanghuanglian medicinal residue powder, 0.8-1.2g of KH2PO4, 0.8-1.2g of MgSO4·7H2O, 0.4-0.6g of urea and 0.8-1.2g of corn flour are mixed, the pH value is natural, and sterilized at 120-122°C for 1.8-2.2h to obtain Shuanghuanglian medicinal residue solid fermentation medium; the Shuanghuanglian medicinal residue is prepared by mixing honeysuckle medicinal residue, scutellaria medicinal residue and forsythia medicinal residue in a mass ratio of 1:1:2;

[0008] 3) Preparation of biocontrol agent: The seed solution prepared in step 1) was evenly inoculated into the solid fermentation medium of Shuanghuanglian drug residue at an inoculation rate of 8-12%, and fermented at 33-37°C for 9-11 days. After drying at room temperature, the composite biocontrol agent was obtained, and the effective viable count was 2×10 8 cfu / g or above.

[0009] The Bacillus subtilis F-1 has been disclosed in patent application number 202011429616.2, and is classified and named Bacillus subtilis. It has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with a deposit number of CGMCC No. 20856 and a deposit date of October 12, 2020. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0010] The Bacillus velezensis HT-9 has been disclosed in patent application number 202111482053.8, and is classified and named Bacillus velezensis. It has been deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 22072. The deposit date is March 25, 2021, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0011] The composite biocontrol agent prepared by the method is used in preparing medicine for preventing and controlling peanut white rot.

[0012] The preparation method of the invention is simple, low in cost and easy to use. The shuanghuanglian drug residue with antibacterial activity is used as a solid culture medium to carry out solid fermentation on antagonistic strains F-1 and HT-9 to prepare a composite biocontrol agent, which achieves a significant biocontrol effect, has a significant control effect on peanut white rot, and has significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the compatibility test result of Bacillus subtilis F-1 and Bacillus velez HT-9 of the present invention. DETAILED DESCRIPTION

[0014] The specific implementation of the present invention is described in detail below with reference to the accompanying drawings and examples.

[0015] The present invention is specifically implemented by the following examples.

[0016] Example 1

[0017] A method for preparing a composite biocontrol agent using Shuanghuanglian drug residues comprises the following steps:

[0018] 1) preparing seed solution: inoculating the activated Bacillus subtilis F-1 and Bacillus Velez HT-9 into beef extract peptone liquid culture medium respectively, shaking and culturing at 30° C. and 180 r / min for 24 h, mixing in a ratio of 1:1 to obtain seed solution for later use;

[0019] 2) Treatment of drug residues: After the Shuanghuanglian drug residues were crushed, they were sieved through a 30-mesh sieve to obtain Shuanghuanglian drug residue powder. 50 g of Shuanghuanglian drug residue powder, 1 g of KH2PO4, 1 g of MgSO4·7H2O, 0.5 g of urea and 1 g of corn flour were mixed, the pH was natural, and sterilized at 121°C for 2 h to obtain the Shuanghuanglian drug residue solid fermentation medium;

[0020] 3) Preparation of biocontrol agent: The seed solution prepared in step 1) was evenly inoculated into the solid fermentation medium of Shuanghuanglian drug residue at a 10% inoculation amount, and fermented at 35°C for 10 days. After drying at room temperature, the composite biocontrol agent was obtained, and the effective viable count was 2×10 8 cfu / g or above.

[0021] Example 2

[0022] A method for preparing a composite biocontrol agent using Shuanghuanglian drug residues comprises the following steps:

[0023] 1) Preparing seed solution: inoculate the activated Bacillus subtilis F-1 and Bacillus Velez HT-9 strains into beef extract peptone liquid culture medium respectively, shake and culture at 28° C. and 170 r / min for 26 h, mix them in a ratio of 1:1 to obtain seed solution for later use;

[0024] 2) Preparation of Shuanghuanglian medicinal residue solid fermentation medium: After the Shuanghuanglian medicinal residue is crushed, it is passed through a 30-mesh sieve to obtain Shuanghuanglian medicinal residue powder, 45 g of Shuanghuanglian medicinal residue powder, 0.8 g of KH2PO4, 0.8 g of MgSO4·7H2O, 0.4 g of urea and 0.8 g of corn flour are mixed, the pH is natural, and sterilized at 120°C for 2.2 h to obtain Shuanghuanglian medicinal residue solid fermentation medium;

[0025] 3) Preparation of biocontrol agent: The seed solution prepared in step 1) was evenly inoculated into the solid fermentation medium of Shuanghuanglian drug residue at an inoculum amount of 8%, and fermented at 33°C for 11 days. After drying at room temperature, the composite biocontrol agent was obtained, and the effective viable bacteria count was 2×10 8 cfu / g or above.

[0026] Example 3

[0027] A method for preparing a composite biocontrol agent using Shuanghuanglian drug residues comprises the following steps:

[0028] 1) Preparing seed solution: inoculate the activated Bacillus subtilis F-1 and Bacillus Velez HT-9 strains into beef extract peptone liquid culture medium respectively, shake and culture at 32° C. and 190 r / min for 22 h, mix them in a ratio of 1:1 to obtain seed solution for later use;

[0029] 2) Preparation of Shuanghuanglian medicinal residue solid fermentation medium: After the Shuanghuanglian medicinal residue is crushed, it is sieved through a 30-mesh sieve to obtain Shuanghuanglian medicinal residue powder, 55 g of Shuanghuanglian medicinal residue powder, 1.2 g of KH2PO4, 1.2 g of MgSO4·7H2O, 0.6 g of urea and 1.2 g of corn flour are mixed, the pH is natural, and sterilized at 122° C. for 1.8 h to obtain Shuanghuanglian medicinal residue solid fermentation medium;

[0030] 3) Preparation of biocontrol agent: The seed solution prepared in step 1) was evenly inoculated into the solid fermentation medium of Shuanghuanglian drug residue at a 12% inoculation rate, and fermented at 37°C for 9 days. After drying at room temperature, the composite biocontrol agent was obtained, and the effective viable count was 2×10 8 cfu / g or above.

[0031] The Bacillus subtilis F-1 and Bacillus Velezii HT-9 of the present invention both have strong antibacterial activity against Sclerotium rolfsii. At the same time, Shuanghuanglian drug residues also have obvious antagonistic effects on peanut Sclerotium rolfsii. When the water extract of Shuanghuanglian drug residues and PDA culture medium are mixed and poured into a plate, Sclerotium rolfsii can hardly grow on the plate, indicating that the drug residues contain active substances that can inhibit the growth of Sclerotium rolfsii. The present invention uses Shuanghuanglian drug residues with antibacterial activity as a solid culture medium to perform solid fermentation on antagonistic strains F-1 and HT-9 to prepare a composite biocontrol agent, which has obvious control effects on peanut Sclerotium rolfsii. Taking Example 1 as an example, the relevant information is as follows:

[0032] 1. Source of Microbial Strains

[0033] The Bacillus subtilis F-1 of the present invention is an endophytic bacterium isolated from a healthy peanut plant in a peanut production area. The strain was deposited in the China General Microbiological Culture Collection Center on October 12, 2020, with a deposit number of CGMCC No. 20856; Bacillus Velezii HT-9 was isolated from the rhizosphere soil of peanuts. The strain was deposited in the China General Microbiological Culture Collection Center on March 25, 2021, with a deposit number of CGMCC No. 22072. Both strains have high antagonistic activity against peanut white rot fungus, and the specific situation has been disclosed in patents with application numbers 202011429616.2 and 202111482053.8, respectively.

[0034] 2. Compatibility test between strains

[0035] On the beef extract peptone plate, inoculate Bacillus subtilis F-1 by streaking, and at the same time, inoculate Bacillus Velez HT-9 by streaking vertically. Incubate the plate at 30°C, and observe the compatibility reaction between the two strains after 24 hours. If no antibacterial bands are produced between the two strains, they are compatible, otherwise they are incompatible.

[0036] The test results are as follows Figure 1 As shown, it was shown that Bacillus subtilis F-1 and Bacillus Velez HT-9 had good compatibility, the strains did not inhibit each other, and could be mixed and used.

[0037] 3. Experiment on the control effect of composite biocontrol agents on peanut white rot

[0038] The experiment was conducted in a potted manner. The composite biocontrol agent prepared above was added to the natural soil containing peanut white rot fungus at a ratio of 5% and 10% respectively and mixed. An equal amount of natural soil was used instead of the composite biocontrol agent as a control. Peanut seeds of uniform size and fullness were selected and soaked in sterile water and placed in a 30°C incubator for 24 hours for germination. 200g of soil with different treatments was placed in a 12cm×15cm plastic pot, and the germinated peanut seeds were sown in the soil. Ten peanut plants were treated for each treatment, and three replicates were set. 45 days after sowing, the incidence rate and disease index of peanuts in each treatment were counted, and the disease index method was used to determine the control effect. The disease grading standards for peanut white rot are shown in Table 1.

[0039] Incidence rate = (number of diseased plants / total number of plants) × 100%;

[0040] Disease index = ∑ (number of diseased plants at each level × representative value of the disease level) × 100 / (total number of plants surveyed × highest level representative value);

[0041] Control effect = [(control disease index - treatment disease index) / control disease index] × 100%.

[0042] Table 1 Peanut white rot disease classification standards

[0043] Disease level Representative value Grading Standards 1 0 Peanut is healthy and symptom-free 2 1 The area of ​​peanut leaves showing symptoms such as yellowing and wilting shall not exceed 25% of the total area 3 2 The area of ​​peanut leaves showing symptoms such as yellowing and wilting accounts for 25% to 50% of the total area 4 3 The area of ​​peanut leaves showing symptoms such as yellowing and wilting accounts for 50% to 75% of the total area 5 4 The peanut leaves show symptoms such as yellowing and wilting, and the area exceeds 75% of the total area, and the plants die.

[0044] The test results are shown in Table 2. The peanut plants that were not inoculated with the composite biocontrol agent were seriously ill, with a peanut susceptibility rate of 90% and a disease index of 75.83. After 45 days, the control effect of the 5% composite biocontrol agent on peanut white rot was 50.04%, and the control effect of the 10% composite biocontrol agent was 79.24%. It can be seen that the inoculation of the composite biocontrol agent can significantly reduce the susceptibility rate and disease index of the peanut plants.

[0045] Table 2 Control effect of composite biocontrol agents on peanut white rot

[0046]

[0047]

[0048] While conducting experiments on Example 1, the present invention also conducted the same experiments on other examples. Each experiment was repeated 3 times and the average value was taken. The same or similar results were obtained, which will not be repeated one by one.

[0049] In summary, the preparation method of the present invention is simple, low in cost, and easy to use. Compared with the prior art, the composite biocontrol fungicide provided by the present invention has a significant control effect on peanut white rot. Secondly, the fungicide is safe for humans and animals and has low pollution. At the same time, it solves the technical problem of recycling Shuanghuanglian drug residue resources, saves resources, reduces pollution to the environment, has a good application prospect, and has significant social and economic benefits.

Claims

1. A method for preparing a composite biocontrol agent using Shuanghuanglian drug residues, characterized in that: The following steps are involved: 1) Preparation of seed solution: inoculate the activated Bacillus subtilis F-1 and Bacillus Velez HT-9 into beef extract peptone liquid culture medium respectively, shake and culture at 28-32°C and 170-190 r / min for 22-26 h, mix in a ratio of 1:1 to obtain seed solution for later use; 2) Preparing Shuanghuanglian medicinal residue solid fermentation medium: After the Shuanghuanglian medicinal residue is crushed, it is sieved through a 30-mesh sieve to obtain Shuanghuanglian medicinal residue powder, and 45-55 g of Shuanghuanglian medicinal residue powder, 0.8-1.2 g of KH2PO4, 0.8-1.2 g of MgSO4·7H2O, 0.4-0.6 g of urea and 0.8-1.2 g of corn flour are mixed, the pH value is natural, and sterilized at 120-122°C for 1.8-2.2 h to obtain Shuanghuanglian medicinal residue solid fermentation medium; the Shuanghuanglian medicinal residue is prepared by mixing honeysuckle medicinal residue, scutellaria medicinal residue and forsythia medicinal residue in a mass ratio of 1:1:2; 3) Preparation of biocontrol agent: The seed solution prepared in step 1) was evenly inoculated into the solid fermentation medium of Shuanghuanglian drug residue at an inoculation rate of 8-12%, and fermented at 33-37°C for 9-11 days. After drying at room temperature, the composite biocontrol agent was obtained, and the effective viable bacteria count was 2×10 8 cfu / g or above.

2. The method for preparing a composite biocontrol agent using Shuanghuanglian drug residues according to claim 1, characterized in that: The following steps are involved: 1) Preparation of seed solution: inoculate the activated Bacillus subtilis F-1 and Bacillus Velez HT-9 into beef extract peptone liquid culture medium respectively, shake and culture at 30°C and 180 r / min for 24 h, mix in a 1:1 ratio to obtain seed solution for later use; 2) Treatment of drug residues: After the Shuanghuanglian drug residues were crushed, they were passed through a 30-mesh sieve to obtain Shuanghuanglian drug residue powder. 50 g of Shuanghuanglian drug residue powder, 1 g of KH2PO4, 1 g of MgSO4·7H2O, 0.5 g of urea and 1 g of corn flour were mixed, the pH was natural, and sterilized at 121°C for 2 h to obtain the Shuanghuanglian drug residue solid fermentation medium; 3) Preparation of biocontrol agent: The seed liquid prepared in step 1) was evenly inoculated into the solid fermentation medium of Shuanghuanglian drug residue at a 10% inoculation rate, and fermented at 35°C for 10 days. After drying at room temperature, the composite biocontrol agent was obtained, and the effective viable bacteria count was 2×10 8 cfu / g or above.

3. The method for preparing a composite biocontrol agent using Shuanghuanglian drug residues according to claim 1, characterized in that: The following steps are involved: 1) Preparation of seed solution: Inoculate the activated Bacillus subtilis F-1 and Bacillus Velez HT-9 into beef extract peptone liquid culture medium respectively, shake and culture at 28°C and 170 r / min for 26 h, mix in a 1:1 ratio to obtain seed solution for later use; 2) Preparation of Shuanghuanglian residue solid fermentation medium: Shuanghuanglian residue was crushed and passed through a 30-mesh sieve to obtain Shuanghuanglian residue powder. 45 g Shuanghuanglian residue powder, 0.8 g KH2PO4, 0.8 g MgSO4·7H2O, 0.4 g urea and 0.8 g corn flour were mixed, the pH was natural, and sterilized at 120℃ for 2.2 h to obtain Shuanghuanglian residue solid fermentation medium; 3) Preparation of biocontrol agent: The seed solution prepared in step 1) was evenly inoculated into the solid fermentation medium of Shuanghuanglian residue at an inoculum volume of 8%, and fermented at 33°C for 11 days. After drying at room temperature, the composite biocontrol agent was obtained, and the effective viable bacterial count was 2×10 8 cfu / g or above.

4. The method for preparing a composite biocontrol agent using Shuanghuanglian drug residues according to claim 1, characterized in that: The following steps are involved: 1) Preparation of seed solution: Inoculate the activated Bacillus subtilis F-1 and Bacillus Velez HT-9 into beef extract peptone liquid culture medium respectively, shake and culture at 32°C and 190 r / min for 22 h, mix in a 1:1 ratio to obtain seed solution for later use; 2) Preparation of Shuanghuanglian residue solid fermentation medium: Shuanghuanglian residue was crushed and passed through a 30-mesh sieve to obtain Shuanghuanglian residue powder. 55 g Shuanghuanglian residue powder, 1.2 g KH2PO4, 1.2 g MgSO4·7H2O, 0.6 g urea and 1.2 g corn flour were mixed, the pH was natural, and sterilized at 122℃ for 1.8 h to obtain Shuanghuanglian residue solid fermentation medium; 3) Preparation of biocontrol agent: The seed solution prepared in step 1) was evenly inoculated into the solid fermentation medium of Shuanghuanglian residue at a 12% inoculation rate, and fermented at 37°C for 9 days. After drying at room temperature, the composite biocontrol agent was obtained, and the effective viable count was 2×10 8 cfu / g or above.

5. The method for preparing a composite biocontrol agent using Shuanghuanglian residues according to claim 1, characterized in that: The Bacillus subtilis F-1 is classified and named Bacillus subtilis, and has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with a deposit number of CGMCC No. 20856, a deposit date of October 12, 2020, and a deposit address of No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

6. The method for preparing a composite biocontrol agent using Shuanghuanglian drug residues according to claim 1, characterized in that: The Bacillus velezensis HT-9 is classified and named Bacillus velezensis, and has been deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 22072. The deposit date is March 25, 2021. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

7. Use of the composite biocontrol agent prepared by the method according to any one of claims 1 to 6 in the preparation of drugs for preventing and controlling peanut white rot.

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