Preparation method of stropharia rugoso-annulata culture medium and application of stropharia rugoso-annulata culture medium in flue-cured tobacco production

Abstract

The invention discloses a preparation method of a stropharia rugoso-annulata culture medium and application of the stropharia rugoso-annulata culture medium in flue-cured tobacco production, and belongs to the technical field of tobacco growth and disease control. The invention discloses a preparation method of a stropharia rugoso-annulata culture medium. The preparation method comprises the following steps: crushing corn straws, rice husks, rice straws and wheat bran, uniformly mixing, and fermenting; after fermentation is conducted for 25-30 days, 1-3 parts of lime is mixed, stropharia rugoso-annulata hyphae are inoculated, after stropharia rugoso-annulata is mature, a medium after stropharia rugoso-annulata cultivation is collected, and a medium after stropharia rugoso-annulata cultivation is obtained; after stropharia rugosoannulata is cultivated, a substrate is subjected to greenhouse closing and drying, and the dried substrate is crushed into clusters. According to the stropharia rugoso-annulata culture medium prepared by the method, the disease index of tobacco bacterial wilt is reduced, the soil environment is adjusted to promote the growth of tobacco seedlings, secondary pollution is avoided, pathogenic bacteria do not generate drug resistance, the treatment capacity is large, and the operation is simple.

Description

Technical Field

[0001] This invention relates to the field of tobacco growth and disease control technology, and more specifically to a method for preparing a cultivation substrate for *Agaricus bisporus* and its application in flue-cured tobacco production, using the *Agaricus bisporus* cultivation substrate to inhibit the occurrence of bacterial wilt and promote tobacco growth. Background Technology

[0002] Tobacco is an important economic crop and a major source of national tax revenue. According to data from the National Bureau of Statistics, in 2023, my country's tobacco planting area was 1.04 million hectares. 2 The total industrial and commercial tax revenue reached 1.52 trillion yuan, which is of great significance to agricultural development and my country's economic development. However, with the gradual increase in planting area and planting years, the phenomenon of continuous cropping of tobacco plants has become increasingly obvious, leading to continuous cropping obstacles. Continuous cropping of tobacco plants leads to a continuous decline in soil fertility. At the same time, due to the long-term planting of tobacco plants, tobacco diseases such as bacterial wilt accumulate in the soil, resulting in disease and quality decline in subsequent tobacco plants.

[0003] Bacterial wilt is caused by Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia solanacearum Bacterial wilt, caused by soil-borne pathogens, is a common soil-borne disease in tobacco cultivation. It is widely distributed in my country, occurring in tobacco-growing areas of the middle and upper reaches of the Yangtze River, Southwest China, Southeast China, the Yellow River and Huai River regions, and Northern China. Bacterial wilt is a systemic vascular disease that infects the entire tobacco plant, damaging the roots, stems, and leaves. It causes the plant to die by blocking the vascular bundles. The pathogen spreads through the soil and can survive for extended periods in the soil and plant debris.

[0004] Currently, the main approach to controlling severe bacterial wilt caused by continuous tobacco cropping is through chemical pesticides. Traditional chemical pesticides are primarily synthetic agents such as thiamethoxam and tebuconazole. While these pesticides can temporarily suppress the disease index in the short term, pathogens develop resistance over time, requiring either switching to a different pesticide or increasing the dosage. Furthermore, the continuous accumulation of chemical pesticides negatively impacts the soil environment, including soil and water pollution, killing beneficial soil bacteria, and disrupting the soil ecosystem.

[0005] Therefore, providing a method for preparing a cultivation substrate for *Agaricus bisporus* and its application in flue-cured tobacco production is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing a substrate for the cultivation of giant puffball mushrooms and its application in flue-cured tobacco production, thereby achieving biological control of tobacco bacterial wilt in a green and environmentally friendly manner.

[0007] Current chemical control methods for tobacco bacterial wilt have problems such as the development of pathogen resistance and secondary pollution caused by treatment. This invention mainly uses a mushroom cultivation substrate to control tobacco bacterial wilt, reduces the disease index, adjusts the soil environment to promote the growth of tobacco seedlings, and prevents the development of pathogen resistance and secondary pollution.

[0008] This invention addresses the negative impacts of continuous tobacco cropping by applying a *Stropharia masticata* cultivation substrate. *Stropharia masticata* is a fast-growing and economically viable edible fungus, and its cultivation substrate is rich in organic matter and other nutrients, providing more nourishment for the tobacco plants. Simultaneously, it contains beneficial bacteria that inhibit the growth of pathogens. This *Stropharia masticata* cultivation substrate can resolve the issue of continuous tobacco cropping without damaging soil microorganisms or the soil ecosystem due to the accumulation of chemical substances.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a cultivation substrate for *Agaricus bisporus* includes the following steps: (1) By weight, crush and mix 40-60 parts of corn stalks, 30-40 parts of rice husks, 10-20 parts of rice straw and 3-6 parts of wheat bran, add water to adjust the compost material to a moisture content of 60%-70%, and pile it up to a height of 30-40cm. Then cover it with plastic film and leave ventilation holes for fermentation. (2) After fermentation for 25-30 days, mix in 1-3 parts lime and inoculate with the mycelium of *Stropharia macrocarpa*. When *Stropharia macrocarpa* matures, collect its cultivation substrate to obtain the cultivation substrate of *Stropharia macrocarpa*. (3) After the substrate for the cultivation of giant king mushrooms is dried in an idle vegetable greenhouse with the maximum temperature not exceeding 50°C, the dried substrate is broken into clumps to obtain the cultivation substrate for giant king mushrooms.

[0010] Furthermore, the method described above prepares a culture substrate for *Agaricus bisporus*.

[0011] Furthermore, the application of the aforementioned *Agaricus bisporus* cultivation substrate in flue-cured tobacco production.

[0012] Furthermore, the application of the aforementioned *Agaricus bisporus* cultivation substrate in promoting the growth of flue-cured tobacco.

[0013] Furthermore, the application of the aforementioned *Agaricus bisporus* cultivation substrate in suppressing bacterial wilt disease.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for preparing a cultivation substrate for giant puffball mushrooms and its application in flue-cured tobacco production, which reduces the disease index of tobacco bacterial wilt, adjusts the soil environment to promote the growth of tobacco seedlings, has no secondary pollution, does not cause pathogens to develop drug resistance, has a large processing capacity, and is simple to operate. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: Effects of different formulations of *Agaricus bisporus* cultivation substrate on inhibiting bacterial wilt and promoting tobacco plant growth in a pot experiment. The four formulas are: S1: 60 parts corn stalks, 40 parts rice husks, 20 parts rice straw, and 6 parts wheat bran are crushed and mixed evenly. Water is added to adjust the compost material to a moisture content of 70%, and the pile height is 30-40cm. Then, a plastic film is covered with ventilation holes for fermentation. After 30 days of fermentation, 3 parts lime are mixed in, and then *Agaricus bisporus* mycelium is inoculated. Once the *Agaricus bisporus* matures, its post-cultivation substrate is collected to obtain the *Agaricus bisporus* post-cultivation substrate. The *Agaricus bisporus* post-cultivation substrate is then dried in an unused vegetable greenhouse under a fumigation system, with the maximum temperature not exceeding 50℃. The dried substrate is then broken into clumps to obtain the *Agaricus bisporus* cultivation substrate.

[0017] S2: 50 parts corn stalks, 40 parts rice husks, 15 parts rice straw, and 4 parts wheat bran are crushed and mixed evenly. Water is added to adjust the compost material to a moisture content of 65%, and the pile height is 30-40cm. Then, a plastic film is covered with ventilation holes for fermentation. After 27 days of fermentation, 2 parts lime are mixed in, and then *Stropharia macrocarpa* mycelium is inoculated. Once the *Stropharia macrocarpa* matures, its post-cultivation substrate is collected to obtain the *Stropharia macrocarpa* post-cultivation substrate. The post-cultivation substrate is then dried in an unused vegetable greenhouse under a fumigation system, with the maximum temperature not exceeding 50℃. The dried substrate is then broken into clumps to obtain the *Stropharia macrocarpa* cultivation substrate.

[0018] S3: 55 parts corn stalks, 35 parts rice husks, 10 parts rice straw, and 3 parts wheat bran are crushed and mixed evenly. Water is added to adjust the compost material to a moisture content of 65%, and the pile height is 30-40cm. Then, a plastic film is covered with ventilation holes for fermentation. After 25 days of fermentation, 1 part lime is mixed in, and then *Stropharia macrocarpa* mycelium is inoculated. Once the *Stropharia macrocarpa* matures, its post-cultivation substrate is collected to obtain the *Stropharia macrocarpa* post-cultivation substrate. The *Stropharia macrocarpa* post-cultivation substrate is then dried in an unused vegetable greenhouse under a fumigation system, with the maximum temperature not exceeding 50℃. The dried substrate is then broken into clumps to obtain the *Stropharia macrocarpa* cultivation substrate.

[0019] S4: 40 parts corn stalks, 30 parts rice husks, 20 parts rice straw, and 4 parts wheat bran are crushed and mixed evenly. Water is added to adjust the compost material to a moisture content of 60%, and the pile height is 30-40cm. Then, a plastic film is covered with ventilation holes for fermentation. After 25 days of fermentation, 1 part lime is mixed in, and then *Stropharia macrocarpa* mycelium is inoculated. Once the *Stropharia macrocarpa* matures, its post-cultivation substrate is collected to obtain the *Stropharia macrocarpa* post-cultivation substrate. The post-cultivation substrate is then dried in an unused vegetable greenhouse under a fumigation system, with the maximum temperature not exceeding 50℃. The dried substrate is then broken into clumps to obtain the *Stropharia macrocarpa* cultivation substrate.

[0020] Prepare a suspension of Ralstonia solanacearum pathogen (Ralstonia solanacearum), with a bacterial concentration of 1.0 × 10⁻⁶. 8 cfu / mL.

[0021] Next, a pot experiment was conducted, with five experimental groups: control group CK (tobacco plants + bacterial wilt pathogen), S1 (tobacco plants + bacterial wilt pathogen + S1-prepared *Agaricus bisporus* cultivation substrate), S2 (tobacco plants + bacterial wilt pathogen + S2-prepared *Agaricus bisporus* cultivation substrate), S3 (tobacco plants + bacterial wilt pathogen + S3-prepared *Agaricus bisporus* cultivation substrate), and S4 (tobacco plants + bacterial wilt pathogen + S4-prepared *Agaricus bisporus* cultivation substrate). Each experimental group had 10 replicates. 5 kg of soil was added to each pot, along with *Agaricus bisporus* cultivation substrate, which was added at 15% of the soil volume. The bacterial suspension was added at a rate of 2 ml of bacterial wilt pathogen suspension per kilogram of soil mixture (soil + *Agaricus bisporus* cultivation substrate).

[0022] Tobacco seedlings (Yunyan 87) with similar growth status were selected and transplanted into pots containing different substrates for the cultivation of giant king mushrooms. One seedling was placed in each pot. After the growth status was stabilized and the activity was confirmed, the roots of the tobacco seedlings were irrigated with a suspension of bacterial wilt pathogen according to the above grouping. The temperature in the pots was maintained at 20-25℃ and the humidity at 90%. The bacterial wilt disease index (disease investigation refers to GB / T 23222-2008) and agronomic traits were statistically analyzed at 7 days and 14 days. The results are shown in Tables 1-3.

[0023] Table 1. Disease index of tobacco plant bacterial wilt

[0024] Table 2. Average values ​​of agronomic traits of tobacco plants (7 days)

[0025] Table 3. Average agronomic traits of tobacco plants (14 days)

[0026] The results in Tables 1-3 show that the S4 formulation of the *Agaricus bisporus* substrate has the strongest inhibitory effect on bacterial wilt of tobacco plants, with a disease index of 16.67, while the control group has an index of 27.33. At the same time, the tobacco plants cultivated under the S4 formulation have the best development among the four *Agaricus bisporus* substrate formulations.

[0027] Example 2: Effects of different amounts of *Agaricus bisporus* cultivation substrate on inhibiting bacterial wilt and promoting tobacco plant growth in a pot experiment. In this embodiment, the above-mentioned S4 substrate formula for *Agaricus bisporus* cultivation was selected for pot experiments. Five experimental groups were set up: control group CK (tobacco plants + bacterial wilt pathogen), A1 (tobacco plants + bacterial wilt pathogen + *Agaricus bisporus* cultivation substrate prepared with 10% v / v S4), A2 (tobacco plants + bacterial wilt pathogen + *Agaricus bisporus* cultivation substrate prepared with 15% v / v S4), A3 (tobacco plants + bacterial wilt pathogen + *Agaricus bisporus* cultivation substrate prepared with 20% v / v S4), and A4 (tobacco plants + bacterial wilt pathogen + *Agaricus bisporus* cultivation substrate prepared with 25% v / v S4). Each experimental group had 10 replicates. Tobacco seedlings with similar growth stages were selected and transplanted into pots containing different substrates for *Agaricus bisporus* cultivation. After the seedlings stabilized and their activity was confirmed, a suspension of bacterial wilt pathogen was applied to the roots of the tobacco plants according to the above-mentioned groupings. The temperature in the pots was maintained at 20-25℃, and the humidity at 90%, with other parameters the same as in Example 1. Bacterial wilt disease index and agronomic traits were then statistically analyzed at 7 and 14 days. The results are shown in Tables 4-6.

[0028] Table 4. Disease index of bacterial wilt in tobacco plants under different amounts of *Agaricus bisporus* substrate.

[0029] Table 5. Average agronomic traits of tobacco plants under different amounts of *Pleurotus ostreatus* substrate treatment (7 days)

[0030] Table 6. Average agronomic traits of tobacco plants under different amounts of *Pleurotus ostreatus* substrate treatment (14 days)

[0031] Table 4-6 shows that the S4 formulation of the mushroom cultivation substrate showed the strongest inhibition of bacterial wilt disease in tobacco plants at a volume ratio of 20%, with a disease index of 15.11, while the control group had an index of 27.33. Meanwhile, the tobacco plants cultivated in the A3 formulation of the mushroom cultivation substrate had the best development among the four mushroom volume ratios.

[0032] Example 3: A field plot experiment on a cultivation substrate for *Agaricus bisporus* that controls tobacco bacterial wilt and promotes tobacco plant growth. This embodiment selects the S4 giant king mushroom substrate from Example 1 and the A3 content from Example 2 for field plot experiments. Two experimental groups are set up: control group CK (tobacco plants + bacterial wilt diseased soil) and BK (tobacco plants + bacterial wilt diseased soil + mushroom substrate prepared with 20% v / v S4). Each experimental group has one experimental field with about 100 tobacco plants.

[0033] Tobacco seedlings that had reached the three-leaf stage were selected and transplanted into two experimental fields, both of which had been continuously cropped for many years and contained tobacco plant debris infected with bacterial wilt. One experimental field was left untreated as a control (CK), while the other experimental field had 1 kg of S4 substrate added to each tobacco plant. After 45 and 60 days, the disease index and agronomic traits were statistically analyzed. Soil samples from both experimental fields were also collected for soil physicochemical property testing. The results are shown in Tables 7-10.

[0034] Table 7. Disease index of bacterial wilt in tobacco plants in field plot experiment

[0035] Table 8. Average agronomic traits of tobacco plants in field plot experiments (45 days)

[0036] Table 9. Average agronomic traits of tobacco plants in field plot experiments (60 days)

[0037] Table 10. Soil physicochemical properties of tobacco plants in field plots (60 days)

[0038] Table 7-10 shows that the S4 formula of mushroom cultivation substrate significantly reduced the bacterial wilt disease index in a field experiment with a 20% volume ratio, with an index of 28.91, compared to 40.17 in the control group. At the same time, it significantly promoted the growth of tobacco plants and added new nutrients to the soil, increasing the available potassium, available phosphorus, organic matter and nitrogen in the soil, and also improving the soil pH, which was beneficial to the growth of tobacco plants.

[0039] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a cultivation substrate for *Agaricus bisporus*, characterized in that, Includes the following steps: (1) By weight, crush and mix 40-60 parts of corn stalks, 30-40 parts of rice husks, 10-20 parts of rice straw and 3-6 parts of wheat bran, add water to adjust the compost material to a moisture content of 60%-70%, and pile it up to a height of 30-40cm. Then cover it with plastic film and leave ventilation holes for fermentation. (2) After fermentation for 25-30 days, mix in 1-3 parts lime and inoculate with the mycelium of *Stropharia macrocarpa*. When *Stropharia macrocarpa* matures, collect its cultivation substrate to obtain the cultivation substrate of *Stropharia macrocarpa*. (3) After the substrate for the cultivation of giant king mushrooms is dried in an idle vegetable greenhouse with the maximum temperature not exceeding 50°C, the dried substrate is broken into clumps to obtain the cultivation substrate for giant king mushrooms.

2. The mushroom cultivation substrate prepared by the method of claim 1.

3. The application of the mushroom cultivation substrate according to claim 2 in flue-cured tobacco production.

4. The application of the mushroom cultivation substrate according to claim 2 in promoting the growth of flue-cured tobacco.

5. The application of the mushroom cultivation substrate according to claim 2 in inhibiting bacterial wilt disease.

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