Mushroom waste substrate-based tobacco soil-borne disease prevention and control method and application

Abstract

The invention discloses a prevention and control method for tobacco soil-borne diseases based on a mushroom waste substrate and application. Belongs to the technical field of tobacco soil-borne disease prevention and control. According to the method, the tobacco soil-borne diseases are prevented and controlled by combining the mushroom waste substrate with the biological agent, the added mushroom waste substrate can increase the antibacterial activity of biocontrol bacteria, the control effect of the biocontrol bacteria is remarkably improved, the application direction of the mushroom waste substrate is widened, and maximum utilization of mushroom waste substrate resources is achieved on the premise of environment friendliness.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco soil-borne disease prevention and control, and more particularly to a tobacco soil-borne disease prevention and control method based on mushroom waste substrate and application. Background Art

[0002] Tobacco (Nicotiana tabacum) is a plant of the Solanaceae family native to South America. It is an annual or limited perennial herb. Its leaves, after drying and fermentation, are widely used to make tobacco products such as cigarettes, cigars, and pipe tobacco. Tobacco requires a warm and humid climate and fertile soil to grow. It is usually sown in spring and has a strong adaptability to the pH value of the soil, but prefers slightly acidic soil. Tobacco cultivation requires sufficient sunlight and water, as well as regular fertilization and prevention of pests and diseases. In the process of tobacco cultivation, soil-borne diseases such as tobacco bacterial wilt and black leg disease often occur, causing huge economic losses to the tobacco planting industry. Tobacco soil-borne diseases are diseases in which pathogens survive in the soil or on the soil surface and attack the stems of plants in the soil and above the ground. Since the occurrence of soil-borne diseases is based on the relationship between the three factors of host, parasite and soil microorganisms and uses a complex soil environment as a culture medium, their prevention and control is quite difficult.

[0003] The traditional method is to use chemical pesticides to control soil-borne diseases. However, long-term excessive use of chemical pesticides to control soil-borne diseases will not only reduce the population density of microbial communities suitable for tobacco growth in the soil, affect the balance of soil microbial communities, and affect the growth and development of tobacco plants to varying degrees, but also increase the drug resistance of disease strains, resulting in poor control effects.

[0004] Mushroom waste matrix is ​​mainly formed by a series of easily fermented materials such as cottonseed hulls, rice straw, cow dung, etc., which are composted. It is rich in organic matter and nutrients. It is mostly used to improve soil fertility, increase soil enzyme activity and reduce soil bulk density. There are few examples of using mushroom waste matrix in combination with biological agents for the prevention and control of soil-borne diseases.

[0005] Therefore, how to provide a method for combining mushroom waste substrate with biological agents to effectively prevent and control tobacco soil-borne diseases is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0006] In view of this, the present invention provides a method and application of preventing and controlling tobacco soil-borne diseases based on mushroom waste matrix. After the soil and mushroom waste matrix are mixed in a certain proportion, biological agents are used in combination to effectively improve the resistance of tobacco to bacterial wilt disease and the prevention and control effect. The added mushroom waste matrix can increase the antibacterial activity of biocontrol bacteria, significantly improving its prevention and control effect.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] Application of mushroom waste substrate in combination with Bacillus subtilis and Oligotrophic Monospora maltophilia in controlling soil-borne diseases of tobacco.

[0009] Preferably, the Bacillus subtilis is Bacillus subtilis HNA1; the Stenotrophomonas maltophilia is Stenotrophomonas maltophilia 93-17. Another object of the present invention is to provide: a method for preventing and controlling tobacco soil-borne diseases based on mushroom waste substrate, comprising the following steps:

[0010] (1) Preparation of biological agent: Bacillus subtilis HNA1 and Oligotrophic Monospora maltophilia 93-17 were activated and cultured respectively, and the cultured bacterial liquids were mixed in a volume ratio of 2:3-3:2 to prepare a biological agent;

[0011] (2) Preparation of mushroom waste matrix: removing impurities from the mushroom waste matrix, spreading it out to dry naturally, and crushing it to obtain the mushroom waste matrix;

[0012] (3) Prevention and control of soil-borne tobacco diseases: Mix the spent mushroom substrate with the soil and then plant tobacco. At the same time, add biological agents by root irrigation.

[0013] Preferably, the viable counts of the Bacillus subtilis HNA1 and the Oligotrophic Monosporus maltophilia 93-17 bacterial cultures after the expansion in step (1) are both 10 9 -10 10 cell / ml; the final concentration of the biological agent is 8.0×10 8 -1.2×10 9 cell / ml.

[0014] Preferably, the expansion culture process of step (1) is as follows: the activated strain is inoculated into a sterile LB culture medium, placed in a constant temperature shaking incubator for shaking culture for 24 hours to obtain a primary seed solution; then the primary seed solution is inoculated into an industrial culture medium, and further cultured for 48 hours under the condition of pH = 8 to obtain a secondary seed solution; then the secondary seed solution is inoculated into the industrial culture medium by pressure inoculation, and fermented in the range of pH 6.5-7.5 for 72 hours to obtain an expansion culture solution.

[0015] Preferably, the LB medium is prepared as follows: 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl are added to 950 ml of deionized water.

[0016] Preferably, the preparation process of the industrial culture medium is as follows: a carbon source and a nitrogen source in a ratio of 3:2 are added to the LB culture medium to prepare an industrial culture medium; the carbon source is glucose; the nitrogen source is trypsin, yeast powder and ammonium sulfate; wherein the mass ratio of trypsin, yeast powder and ammonium sulfate is 2:3:1.

[0017] Preferably, in step (3), the mushroom waste substrate and soil are mixed in a mass ratio of 1:5; and the amount of the biological agent used is 16-25 ml of the biological agent per 1 kg of mushroom waste substrate.

[0018] Another object of the present invention is to provide: application of the above method in preventing and controlling soil-borne diseases of tobacco.

[0019] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The method of the present invention mixes diseased soil and mushroom waste substrate in a certain proportion and then uses a biological agent to effectively improve the resistance and control effect of tobacco to bacterial wilt. The added mushroom waste substrate can increase the antibacterial activity of biocontrol bacteria, significantly improve its control effect, broaden the application direction of mushroom waste substrate, and maximize the utilization of mushroom waste substrate resources under the premise of environmental friendliness.

[0021] (2) At the same time, the mushroom waste substrate will decompose a large amount of organic matter during the mushroom cultivation process, which helps to improve the soil structure, increase the organic matter content in the soil, provide essential nutrients for tobacco, promote its healthy growth, and effectively improve the soil microbial diversity. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] The culture medium used in the embodiments of the present invention is as follows:

[0024] LB medium: prepared by adding 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl to 950 ml of deionized water.

[0025] Industrial culture medium: Add 45 g glucose, 10 g tryptone, 15 g yeast powder and 5 g ammonium sulfate per liter of culture medium to LB medium.

[0026] Bacillus subtilis HNA1 was purchased from China National Center for Cultural Heritage Collection (CCTCC) with the number HB20081306.

[0027] Oligotrophic monospora maltophilia 93-17 was purchased from China General Microbiological Culture Collection Center (CGMCC) with the number CGMCC 1.1844.

[0028] The activation and expansion process of Bacillus subtilis HNA1 and Oligotrophic Monosporus maltophilia 93-17 is as follows:

[0029] (1) Bacillus subtilis HNA1 and Oligotrophic Monospora maltophilia 93-17 were activated on solid culture medium to form original strains. Then, a sterile inoculation loop was used to remove the colonies from the solid culture medium and transferred to 2L of sterile LB culture medium. The LB culture medium was placed in a constant temperature shaking incubator and cultured at 25°C and a shaking speed of 180 r / min for 24 hours to obtain a primary seed solution;

[0030] (2) Add 30 L of industrial culture medium into a 50 L fermenter and sterilize it. Then, inoculate the primary seed liquid into the industrial culture medium and further culture it for 48 h at a pH of 8, 35° C., and an oscillation speed of 180 r / min to obtain a secondary seed liquid.

[0031] (3) Add 400 L of industrial culture medium to a 500 L fermenter and sterilize it. Then, inoculate 30 L of secondary seed solution (5%-10% of the total volume) into the industrial culture medium by pressure inoculation. Ferment for 72 h at a temperature of 30°C and an oscillation speed of 180 r / min while controlling the pH value at 6.5-7.5. When the number of microorganisms in the culture medium reaches 10 9 ~10 10 When the concentration of bacterial liquid was 10 cell / ml, a storage tank was used to collect the high concentration bacterial liquid, and the collected bacterial liquid of Oligomonas maltophilia and Bacillus subtilis were mixed in a volume ratio of 1:1 to prepare a biological inoculant with a final concentration of 10 9 cell / ml.

[0032] Example 1

[0033] Verification of the biological control effect of discarded mushroom substrate

[0034] (1) After the waste mushroom substrate is harvested, the used culture medium is collected immediately to reduce the loss of nutrients and remove non-organic impurities in the waste substrate, such as plastic fragments, stones, soil, etc., to improve the purity of the substrate; the waste substrate is spread out in a clean, ventilated place to dry naturally, and after drying, the dried waste substrate is crushed using a crusher to increase its surface area, which is conducive to microbial activity and the decomposition of organic matter. Thereafter, the waste substrate is stored in a dry, ventilated place, away from moisture and direct sunlight to maintain its quality and effect.

[0035] (2) After the above method was used to treat the waste mushroom substrate (including: black-skinned chicken fir, big stropharia and straw mushroom, etc.), a plate antagonism experiment of bacterial wilt was carried out to verify the biocontrol effect of the waste mushroom substrate. The experiment was divided into three treatment groups, namely black-skinned chicken fir treatment group, big stropharia treatment group and straw mushroom treatment group, and three parallels were set for each treatment group. A mushroom waste substrate suspension was prepared with mushroom waste substrate and sterile water at a solid-liquid ratio of 1:5. The bacterial wilt pathogen Ralstonia solanacearum (purchased from Biobw platform, platform number Bio-115171) was spread on the solid plate, and then a hole was punched. After punching, 0.5 ml of the above three different combinations were added to the hole. After 7 days of culture, the diameter of the inhibition zone was measured. The results of the inhibition zone diameter are shown in Table 1.

[0036] Table 1 Size of inhibition zone in different treatment groups

[0037]

[0038] Analysis of results: As shown in Table 1, the three mushroom waste substrate treatment groups all produced inhibition zones, indicating that the above three mushroom waste substrates all have biological control effects on tobacco bacterial wilt; in addition, compared with the use of the other two mushroom waste substrate suspensions, the inhibition zone was the largest when the Stropharia capillaris suspension was used, indicating that the Stropharia capillaris suspension had the best inhibitory effect on the pathogen of bacterial wilt.

[0039] Example 2

[0040] The control effect of the method of the present invention on tobacco bacterial wilt

[0041] In order to verify the control effect of the method of the present invention on tobacco bacterial wilt, a pot experiment was carried out, and the tobacco experiment was divided into 8 treatment groups, namely A, B, C, D, E, F, G and H treatment groups (as shown in Table 2). Each treatment group was 5m apart. Each treatment group planted 18 tobacco trees, with a tobacco planting spacing of 0.2m and a row spacing of 0.4m. The test area was treated as follows:

[0042] Table 2 Treatment methods for different groups

[0043] Group deal with A 5kg soil with bacterial wilt B 5kg soil with bacterial wilt disease + 1kg spent substrate of Stropharia rugosodium C 5kg soil with bacterial wilt disease + 10mL Bacillus subtilis D 5kg soil with bacterial wilt disease + 10mL of Oligotrophomonas maltophilia E 5kg soil with bacterial wilt disease + 20mL biological agent F 5kg soil with bacterial wilt disease + 1kg spent substrate of Stropharia rugosotis + 10mL Bacillus subtilis G 5kg soil with bacterial wilt disease + 1kg spent substrate of Stropharia rugosodium + 10mL Oligomonas maltophilia H 5kg soil with bacterial wilt disease + 1kg spent substrate of Stropharia rugosodium + 20mL biological agent

[0044] The investigation recorded the occurrence of tobacco bacterial wilt in each group from the time the tobacco was transplanted to potted plants to one month later. The investigation method was carried out according to the national standard GB / T23222-2008 for tobacco pest and disease investigation. The disease index was calculated, and the control effect was calculated based on the disease index. The results are shown in Table 3.

[0045] Table 3 Incidence of tobacco bacterial wilt in different treatment groups

[0046]

[0047] Analysis of results: Compared with the control, the control method based on the waste substrate of Stropharia rugosa combined with biological agents significantly improved the control effect of tobacco bacterial wilt, among which the combined use of mixed microbial agents had the best control effect on bacterial wilt. The mushroom waste substrate improved the antibacterial activity of biocontrol bacteria and soil quality, which was beneficial to the control function of the agent.

[0048] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0049] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of mushroom waste substrate in combination with Bacillus subtilis and Oligotrophic Monosporus maltophilia in the prevention and control of tobacco soil-borne diseases.

2. The use according to claim 1, characterized in that: The Bacillus subtilis is Bacillus subtilis HNA1; the Stenotrophomonas maltophilia is Stenotrophomonas maltophilia 93-17.

3. A method for preventing and controlling tobacco soil-borne diseases based on mushroom waste substrate, characterized in that: The steps include: (1) Preparation of biological agent: Bacillus subtilis HNA1 and Oligotrophic Monosporus maltophilia 93-17 were activated and cultured respectively, and the cultured bacterial liquids were mixed in a volume ratio of 2:3 to 3:2 to prepare a biological agent; (2) Preparation of mushroom waste matrix: removing impurities from the mushroom waste matrix, spreading it out to dry naturally, and crushing it to obtain the mushroom waste matrix; (3) Prevention and control of soil-borne tobacco diseases: Mix the spent mushroom substrate with the soil and then plant tobacco. At the same time, add biological agents by root irrigation.

4. The prevention and control method according to claim 3, characterized in that: The viable counts of the Bacillus subtilis HNA1 and the Oligotrophic Monosporus maltophilia 93-17 bacterial cultures after the expansion in step (1) were both 10 9 -10 10 cell / ml; the final concentration of the biological agent is 8.0×10 8 ~1.2×10 9 cell / ml.

5. The prevention and control method according to claim 3, characterized in that: The expansion process of step (1) is as follows: inoculating the activated strain into a sterile LB culture medium, placing it in a constant temperature shaking incubator for shaking culture for 24 hours to obtain a primary seed solution; then inoculating the primary seed solution into an industrial culture medium, further culturing it at pH = 8 for 48 hours to obtain a secondary seed solution; Then the secondary seed liquid is inoculated into the industrial culture medium by pressure inoculation method, and fermented for 72 hours in the range of pH 6.5-7.5 to obtain the expanded culture liquid.

6. The control method according to claim 5, characterized in that: The LB medium was prepared as follows: 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl were added to 950 ml of deionized water.

7. The prevention and control method according to claim 5, characterized in that: The preparation process of the industrial culture medium is as follows: a carbon source and a nitrogen source in a mass ratio of 3:2 are added to the LB culture medium to prepare the industrial culture medium; the carbon source is glucose; the nitrogen source is trypsin, yeast powder and ammonium sulfate; wherein the mass ratio of trypsin, yeast powder and ammonium sulfate is 2:3:

1.

8. The prevention and control method according to claim 3, characterized in that: In step (3), the mushroom waste substrate and soil are mixed in a mass ratio of 1:5; the amount of the biological agent used is 16 to 25 ml of the biological agent per 1 kg of mushroom waste substrate.

9. Use of the method according to any one of claims 3 to 8 in preventing and controlling soil-borne diseases of tobacco.

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