A method for harmless prevention and control of rhizoctonia solani of rhodiola crenulata

Abstract

The application provides a Rhodiola kirilowii root rot harmless prevention and control method and relates to the technical field of plant disease prevention and control. The Rhodiola kirilowii root rot harmless prevention and control method is characterized by the following steps: after seed disinfection, the seed is treated by immersion in a solution of a Yunnan sclerotia of Sclerotinia spore powder extract, and a fermentation substrate of Bacillus subtilis and Bacillus amyloliquefaciens is used, and then the roots are irrigated with a solution of a Yunnan sclerotia of Sclerotinia spore powder extract and a Trichoderma harzianum liquid. The method can effectively prevent and control the Rhodiola kirilowii root rot, reduce the use of chemical pesticides, reduce pesticide residues, and improve the safety of planting.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to a method for the harmless control of root rot in Rhodiola rosea. Background Technology

[0002] Rhodiola rosea ( Rhodiola crenulata Rhodiola rosea (also known as large-flowered rhodiola) is a perennial herbaceous plant belonging to the genus Rhodiola in the family Crassulaceae. Its roots and rhizomes possess medicinal properties such as anti-fatigue, anti-hypoxia, and immune-enhancing effects, making it a rare medicinal plant resource in my country. With increasing market demand, the artificial cultivation of Rhodiola rosea has gradually expanded. Greenhouse cultivation, due to its ability to precisely control environmental conditions such as temperature, humidity, and light, has become one of the main methods for large-scale cultivation. However, the enclosed environment, high humidity, and relatively limited ventilation in greenhouses easily induce root rot. Root rot in Rhodiola rosea is mainly caused by soil-borne pathogens such as Fusarium spp., Pythium spp., and Phytophthora spp. These pathogens can spread through seed-borne transmission and substrate contamination. Early symptoms include root discoloration and rotting, followed by the withering and death of the entire plant. In severe cases, the incidence rate can reach 30% to 50%, causing significant economic losses to growers.

[0003] Currently, the control of root rot in Rhodiola rosea relies heavily on chemical fungicides (such as metalaxyl and carbendazim). However, long-term use of these chemicals can lead to drug resistance in pathogens and leaves residues in the plant, affecting its medicinal quality and failing to meet the requirements for harmless cultivation. Furthermore, existing technologies do not adequately address the standardization of seed collection, the targeted nature of seed treatment, and the thoroughness of substrate treatment: some seed collections fail to screen healthy mother plants, resulting in high initial bacterial loads; seed treatment often employs a single disinfection method, leading to incomplete sterilization; and substrate treatment only involves simple sterilization without considering aeration and fertility, hindering root growth and further reducing plant disease resistance. Therefore, developing a harmless control method for root rot in Rhodiola rosea that combines standardized seed collection, efficient seed treatment, and high-quality substrate treatment, suitable for greenhouse conditions, is crucial for solving current cultivation challenges. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for the harmless control of root rot in Rhodiola rosea, which can effectively prevent and control root rot in Rhodiola rosea, reduce the use of chemical pesticides, lower pesticide residues, and improve planting safety.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for the harmless control of root rot in Rhodiola rosea, the method comprising the following steps:

[0007] S1. Collect the seeds of Rhodiola rosea, remove the pericarp and impurities, select healthy seeds, wash and dry the surface moisture, and then disinfect them to obtain disinfected seeds for later use.

[0008] S2. Soak the sterilized seeds in a solution of Yunnan puffball spore powder extract with a concentration of 0.01-0.03 g / mL for 2-4 hours to obtain seeds ready for sowing.

[0009] S3. Peat moss, vermiculite and perlite are mixed to obtain a basic substrate. Then, the basic substrate is inoculated with Bacillus subtilis and Bacillus amyloliquefaciens for composting and fermentation to obtain a fermented antibacterial seedling substrate.

[0010] S4. Fill the seedling tray with fermented antibacterial seedling substrate, then sow the seeds to be sown on the surface of the fermented antibacterial seedling substrate, cover with another layer of fermented antibacterial seedling substrate, then immediately spray water to thoroughly water the soil, and finally cover with a transparent seedling cover for planting management.

[0011] S5. When the seedlings have 2-3 true leaves after seed germination, drench the roots for the first time with a solution of Yunnan sclerotium spore powder extract at a concentration of 0.009-0.02 g / mL. Seven days later, drench the roots again with Trichoderma harzianum solution. After that, drench the roots with Trichoderma harzianum solution every 20-25 days until the seedlings are established.

[0012] Preferably, the method for screening healthy seeds in step S1 is to use wind separation and 5% NaCl brine flotation to remove chaff, residue and potentially bacteria-carrying seeds, and collect the plump seeds that sink to the bottom.

[0013] Preferably, the disinfection method in step S1 is to place the seeds in a 0.03% potassium permanganate aqueous solution, stir continuously and soak for 18 minutes, then remove the seeds and wash away the residual potassium permanganate aqueous solution with clean water to complete the disinfection.

[0014] Preferably, the preparation method of the Yunnan puffball spore powder extract includes the following steps:

[0015] S2-1. Add dried Yunnan hard-skinned puffball spore powder to 1 mol / L NaOH solution at a material-to-liquid mass ratio of 1:10. After stirring evenly, place the mixture on a shaker and extract at 150 rpm and 50℃ for 24 h to obtain the extract for later use.

[0016] S2-2. Centrifuge the extract at 6000 rpm for 10 min using a high-speed centrifuge to remove fungal tissue and collect the supernatant for later use.

[0017] S2-3. Adjust the pH of the supernatant to 2.0 with 1 mol / L HCl to obtain a black precipitate. Then centrifuge at 6000 rpm for 10 min using a high-speed centrifuge, retain the precipitate and discard the supernatant. Resuspend the precipitate with 6 mol / L HCl and hydrolyze it in a 90℃ water bath for 2 h. After 2 h, centrifuge at 6000 rpm for 10 min using a high-speed centrifuge, retain the precipitate and discard the supernatant to obtain the crude extract for later use.

[0018] S2-4. The crude extract was washed repeatedly with pure water 5 times, then washed 3 times each with chloroform, ethyl acetate and ethanol, and finally dried at 35℃ to obtain Yunnan hard-skinned puffball spore powder extract.

[0019] Preferably, the specific volume ratio of the basic matrix components in step S3 is peat moss:vermiculite:perlite = 4:2:1.

[0020] Preferably, the specific preparation method of the fermentation antibacterial seedling substrate in step S3 includes the following steps:

[0021] S3-1. Pile the base substrate into a stack 1 meter high, adjust the moisture content to 55% ~ 60%, and set aside.

[0022] S3-2. Inoculate 2 kg of compound microbial agent per cubic meter of substrate, with the viable counts of Bacillus subtilis and Bacillus amyloliquefaciens in the agent both ≥ 5 × 10⁻⁶. 8 Take CFU / g, cover with plastic sheeting, and compost for 15-20 days, turning the pile 2-3 times during the process to obtain a fermented antibacterial seedling substrate.

[0023] Preferably, the planting management method in step S4 is to maintain soil moisture at 60%-70% and temperature at 15℃-20℃ during the seed germination stage; maintain soil moisture at 40%-50% and temperature at 18℃-22℃ during the seedling growth stage; and ensure at least 6 hours of sufficient diffused light every day, and use shade nets during strong sunlight in summer; the shading rate of the shade nets is 30%-40%.

[0024] Preferably, in step S4, the sowing amount is 2-3 seeds per square centimeter; the thickness of the covering fermented antibacterial seedling substrate is 0.4-0.5 cm.

[0025] Preferably, in step S5, the Trichoderma harzianum solution has an effective viable count of 5 × 10⁻⁶ cells / day. 8 A 250-fold dilution of Trichoderma harzianum powder at CFU / g.

[0026] Preferably, in steps S2 and S5, when preparing the Yunnan hard-skinned puffball spore powder extract solution, it should first be dissolved in a small amount of dimethyl sulfoxide (DMSO) and then added to the target volume of water and stirred evenly.

[0027] This invention provides a method for the harmless control of root rot in Rhodiola rosea, which has the following advantages compared with the prior art:

[0028] This invention uses extracts from the spores of *Lycoperdon perlatum* from Yunnan to soak and drench the roots of *Rhodiola rosea*, inducing disease resistance in the plant. It also combines this with biocontrol fungi such as *Trichoderma harzianum* to create an environment conducive to the growth and development of *Rhodiola rosea* while inhibiting root rot. This effectively reduces the incidence of root rot in *Rhodiola rosea*, with no chemical pesticide residues, ensuring healthy seedlings and safe and high-quality medicinal materials. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the Yunnan hard-skinned puffball spore powder extract of the present invention;

[0030] Figure 2 This invention relates to the extraction of Yunnan puffball spore powder from puffballs against the soil-borne pathogen Fusarium solani (…). Fusarium solani ) Schematic diagram of the inhibition of mycelial growth;

[0031] Figure 3 The images show the growth status and root development of Embodiment 2 and Comparative Example 1 at the time of transplanting. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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.

[0033] The base matrix used in the following examples is a mixture of peat moss, vermiculite, and perlite in a volume ratio of 4:2:1. Example 1:

[0034] 1. Preparation of extract from Yunnan hard-skinned puffball spore powder:

[0035] Weigh 10 g of dried Yunnan puffball spore powder and add 1 mol / L NaOH solution at a material-to-liquid ratio of 1:10. After stirring evenly, extract the product on a shaker (150 rpm, 50℃, 24 h). Centrifuge at 6000 rpm for 10 min to remove fungal tissue. Collect the supernatant and adjust the pH to 2.0 with 1 mol / L HCl to obtain a black precipitate. Centrifuge again at 6000 rpm for 10 min, retain the precipitate and discard the supernatant. Resuspend the precipitate in 6 mol / L HCl and hydrolyze it in a 90℃ water bath for 2 h. After 2 h, centrifuge at 6000 rpm for 10 min, retain the precipitate and discard the supernatant. The precipitate at this point is the crude extract. Wash the final precipitate repeatedly with pure water 5 times to remove acid, then wash 3 times each with chloroform, ethyl acetate, and ethanol to remove lipids and other residues. Finally, dry and purify at 35℃ to obtain the black product (e.g., ...). Figure 1 As shown in the figure, after weighing and labeling, it was stored in a refrigerator (-20℃) as an extract of Yunnan hard-skinned puffball spore powder.

[0036] 2. Antibacterial test of Yunnan puffball spore powder extract:

[0037] (1) Test materials

[0038] The pathogenic fungus tested was *Fusarium solani* (saturates). Fusarium solani It is a fungus belonging to the genus *Fusarium* in the family Rhodiola rosea. It was isolated from diseased *Rhodiola crenulata* plants, identified, and preserved in the laboratory.

[0039] (2) Experimental methods

[0040] Prepare 1 L of PDA medium, autoclave, pour into plates, and dry before use. Use dimethyl sulfoxide (DMSO) as the solvent for the Yunnan puffball spore powder extract, preparing a 0.01 g / mL solution. Use a pipette to add 100 μL of the prepared Yunnan puffball spore powder extract solution to the medium, gently shaking the petri dish to distribute the extract evenly (treatment); for the control, add 100 μL of DMSO solution to the medium. Inoculate the activated *Fusarium solani* spores into the treated medium for an antibacterial test, with 5 replicates. Incubate at 26℃ for 24, 48, and 72 h, observing the pathogen growth and measuring the hyphal diameter using the cross-hatching method to calculate the inhibition rate.

[0041] Inhibition rate (%) = (Coronation diameter of control strain - Coronation diameter of treated strain) / Average diameter of control colonies × 100%.

[0042] (3) Experimental results

[0043] Experimental results showed that a 0.01 g / mL solution of *Lycoperdon yunnanense* spore powder extract had a significant inhibitory effect on *Fusarium solani*, with an inhibition rate of 64.13% after 72 h of treatment (as shown in Table 1). The results for the control and treatment groups were as follows: Figure 2 As shown.

[0044] Table 1

[0045] Example 2:

[0046] Harmless control of root rot in Rhodiola rosea:

[0047] (1) In the late autumn when the capsules are ripe, choose a sunny day and collect mature but unopened brown capsules from healthy mother plants that are free from pests and diseases. Spread the capsules in a cool and ventilated place indoors to dry naturally. After drying, manually rub them to extract the seeds and remove the pericarp and impurities by sieving. Then, use wind selection and 5% NaCl salt water flotation to remove chaff, residue and potentially infected seeds. Collect the plump seeds that sink to the bottom, rinse them with clean water, and place them in a cool place to dry the surface moisture for later use.

[0048] (2) Pack the selected seeds in a breathable mesh bag and place them in a 0.03% potassium permanganate aqueous solution. Stir and soak for 18 minutes. After soaking, remove the seeds, wash off the residual potassium permanganate aqueous solution with clean water, drain the water, and set aside.

[0049] (3) Soak the disinfected seeds in an aqueous solution of Yunnan hard-skinned puffball spore powder extract with a concentration of 0.01 g / mL for 2 hours.

[0050] (4) Stack the base substrate into a pile 1 meter high and adjust the moisture content to 60%; inoculate 2 kg of Bacillus subtilis (effective viable count ≥ 5 × 10⁻⁶) per cubic meter of substrate. 8 CFU / g) and Bacillus amyloliquefaciens (effective viable count ≥5×10⁻⁶) 8 The microbial agent (CFU / g) was used; the compost was covered with plastic sheeting and fermented for 18 days, during which the compost was turned 3 times. During the composting process, the internal temperature of the substrate reached a maximum of 62℃ (generally, the internal temperature of Bacillus subtilis compost can reach above 45℃ about 2-4 days after inoculation, entering the high-temperature period of fermentation, and then the temperature continues to rise, reaching about 55-65℃. Turning the compost ensures that the compost substrate is in full contact with oxygen, achieving high-temperature aerobic fermentation, and the high-temperature fermentation can generally last for 13-16 days), resulting in a fermented and sterilized substrate.

[0051] (5) Sowing: Fill the seedling tray with fermented sterilized substrate, lightly compact it, and then evenly scatter the seeds treated in step (3) on the surface of the substrate, about 2 seeds per square centimeter. Then cover the seeds with a layer of fermented sterilized substrate about 0.5 cm thick, and then immediately spray water to thoroughly irrigate the soil. Finally, cover with a transparent seedling cover to prevent water loss.

[0052] (6) After sowing, during the seed germination stage, maintain soil moisture at 65% and indoor temperature at 20℃; during the seedling growth period, maintain soil moisture at 50% and indoor temperature at 22℃; after sowing, ensure at least 6 hours of sufficient diffused light every day, and use a shade net with a shading rate of 35% during the strong sunlight in summer.

[0053] (7) When the seedlings have 2-3 true leaves, drench the roots for the first time with an aqueous solution of Yunnan sclerotium spore powder extract at a concentration of 0.009 g / mL. Seven days later, apply Trichoderma harzianum powder (with an effective viable count of 5×10⁻⁶). 8 Drench the roots again with a 250-fold dilution of Trichoderma harzianum powder. Then, drench the roots once every 20-25 days with a 250-fold dilution of Trichoderma harzianum powder until seedlings have established.

[0054] (8) Two weeks before leaving the nursery, open the seedling cover, gradually increase ventilation, and reduce temperature and humidity to harden the seedlings and improve their resistance to stress. Example 3:

[0055] Harmless control of root rot in Rhodiola rosea:

[0056] The prevention and control method in this embodiment is basically the same as that in embodiment 2, except that steps (3) and (7) are different:

[0057] In this embodiment, step (3) is as follows: the disinfected seeds are soaked in an aqueous solution of Yunnan hard-skinned puffball spore powder extract with a concentration of 0.02 g / mL for 3 hours;

[0058] Step (7) is as follows: When the seedlings have grown 2-3 true leaves, drench the roots for the first time with an aqueous solution of Yunnan sclerotium spore powder extract at a concentration of 0.01 g / mL. Seven days later, apply Trichoderma harzianum powder (with an effective viable count of 5×10⁻⁶). 8 Drench the roots again with a 250-fold dilution of Trichoderma harzianum powder. Then, drench the roots once every 20-25 days with a 250-fold dilution of Trichoderma harzianum powder until seedlings have established. Example 4:

[0059] Harmless control of root rot in Rhodiola rosea:

[0060] The prevention and control method in this embodiment is basically the same as that in embodiment 2, except that steps (3) and (7) are different:

[0061] In this embodiment, step (3) is as follows: the disinfected seeds are soaked in an aqueous solution of Yunnan puffball spore powder extract with a concentration of 0.03 g / mL for 4 hours;

[0062] Step (7) is as follows: When the seedlings have grown 2-3 true leaves, drench the roots for the first time with an aqueous solution of Yunnan sclerotium spore powder extract at a concentration of 0.02 g / mL. Seven days later, apply Trichoderma harzianum powder (with an effective viable count of 5×10⁻⁶). 8 Drench the roots again with a 250-fold dilution of Trichoderma harzianum powder. Then, drench the roots once every 20-25 days with a 250-fold dilution of Trichoderma harzianum powder until seedlings have established.

[0063] Comparative Example 1:

[0064] Harmless control of root rot in Rhodiola rosea:

[0065] The control method in this comparative example is basically the same as that in Example 2, except that steps (3) and (7) are different:

[0066] In this embodiment, step (3) is: soaking the disinfected seeds in clean water for 3 hours;

[0067] Step (7) is as follows: When the seedlings have grown 2-3 true leaves, irrigate the roots with clean water for the first time, and 7 days later apply Trichoderma harzianum powder (effective viable count is 5×10⁻⁶). 8 Drench the roots again with a 250-fold dilution of Trichoderma harzianum powder. Then, drench the roots once every 20-25 days with a 250-fold dilution of Trichoderma harzianum powder until seedlings have established.

[0068] Comparative Example 2:

[0069] Harmless control of root rot in Rhodiola rosea:

[0070] The control method in this comparative example is basically the same as that in Example 2, except for step (7):

[0071] In this embodiment, step (7) is as follows: when the seedlings have grown 2-3 true leaves, irrigate the roots with clean water for the first time, and 7 days later apply Trichoderma harzianum powder (effective viable count is 5×10⁻⁶). 8 Drench the roots again with a 250-fold dilution of Trichoderma harzianum powder. Then, drench the roots once every 20-25 days with a 250-fold dilution of Trichoderma harzianum powder until seedlings have established.

[0072] Comparative Example 3:

[0073] Harmless control of root rot in Rhodiola rosea:

[0074] The control method in this comparative example is basically the same as that in Example 2, except for step (3):

[0075] In this embodiment, step (3) is: soaking the disinfected seeds in clean water for 3 hours.

[0076] Comparative Example 4:

[0077] Harmless control of root rot in Rhodiola rosea:

[0078] The control method in this comparative example is basically the same as that in Example 2, except for step (7):

[0079] In this embodiment, step (7) is as follows: when the seedling has grown 2-3 true leaves, apply Trichoderma harzianum powder (effective viable count is 5×10⁻⁶). 8 For the first root irrigation, use a 250-fold dilution of CFU / g solution. Seven days later, irrigate the roots again with an aqueous solution of Yunnan scleroderma spore powder extract at a concentration of 0.009 g / mL. After that, irrigate the roots once every 20-25 days with a 250-fold dilution of Trichoderma harzianum powder until seedlings are established.

[0080] Detection:

[0081] Rhodiola rosea was cultivated according to the methods of Examples 2-4 and Comparative Examples 1-4 above, with other conditions being the same and suitable for the growth of Rhodiola rosea. The number of diseased plants was counted and the incidence rate was calculated when the plants were ready for transplanting. The specific results are shown in Table 2 below:

[0082] Table 2

[0083]

[0084] As shown in Table 2, the method for harmless control of root rot in Rhodiola rosea in the embodiments of the present invention can significantly reduce the incidence of root rot, which is significantly better than the method in the comparative examples. A comparison of Examples 2-4 and Comparative Examples 1-4 shows that the simultaneous use of Yunnan puffball spore powder extract solution for seed soaking and root drenching results in better disease prevention; and the effect is even better when Yunnan puffball spore powder extract solution is used first followed by Trichoderma harzianum wettable powder during root drenching.

[0085] Furthermore, the growth and root development of the Rhodiola rosea seedlings in Example 2 and Comparative Example 1 at the time of transplanting were as follows: Figure 3 As shown, the upper left shows the seedling growth of Example 2 before it leaves the nursery, the lower left shows the root development of the seedlings of Example 2 before it leaves the nursery, the upper right shows the seedling growth of Comparative Example 1 before it leaves the nursery, and the lower right shows the root development of the seedlings of Comparative Example 1 before it leaves the nursery. It can be seen that the seedling growth and root development in Example 2 are significantly better than those in Comparative Example 1.

[0086] The above 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 with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for harmless prevention and control of Rhizoctonia solani of Rhodiola crenulata, characterized in that, The method includes the following steps: S1. Collect the seeds of Rhodiola rosea, remove the pericarp and impurities, select healthy seeds, wash and dry the surface moisture, and then disinfect them to obtain disinfected seeds for later use. S2. Soak the sterilized seeds in a solution of Yunnan puffball spore powder extract with a concentration of 0.01-0.03 g / mL for 2-4 hours to obtain seeds ready for sowing. The preparation method of the Yunnan hard-skinned puffball spore powder extract includes the following steps: S2-1. Add dried Yunnan hard-skinned puffball spore powder to 1 mol / L NaOH solution at a material-to-liquid mass ratio of 1:

10. After stirring evenly, place the mixture on a shaker and extract at 150 rpm and 50℃ for 24 h to obtain the extract for later use. S2-2. Centrifuge the extract at 6000 rpm for 10 min using a high-speed centrifuge to remove fungal tissue and collect the supernatant for later use. S2-3. Adjust the pH of the supernatant to 2.0 with 1 mol / L HCl to obtain a black precipitate. Then centrifuge at 6000 rpm for 10 min using a high-speed centrifuge, retain the precipitate and discard the supernatant. Resuspend the precipitate with 6 mol / L HCl and hydrolyze it in a 90℃ water bath for 2 h. After 2 h, centrifuge at 6000 rpm for 10 min using a high-speed centrifuge, retain the precipitate and discard the supernatant to obtain the crude extract for later use. S2-4. The crude extract was washed repeatedly with pure water 5 times, then washed 3 times each with chloroform, ethyl acetate and ethanol, and finally dried at 35℃ to obtain Yunnan hard-skinned puffball spore powder extract. S3. Peat moss, vermiculite and perlite are mixed to obtain a basic substrate. Then, the basic substrate is inoculated with Bacillus subtilis and Bacillus amyloliquefaciens for composting and fermentation to obtain a fermented antibacterial seedling substrate. S4. Fill the seedling tray with fermented antibacterial seedling substrate, then sow the seeds to be sown on the surface of the fermented antibacterial seedling substrate, cover with another layer of fermented antibacterial seedling substrate, then immediately spray water to thoroughly water the soil, and finally cover with a transparent seedling cover for planting management. S5. When the seedlings have 2-3 true leaves after seed germination, drench the roots for the first time with a solution of Yunnan sclerotium spore powder extract at a concentration of 0.009-0.02 g / mL. Seven days later, drench the roots again with Trichoderma harzianum solution. After that, drench the roots with Trichoderma harzianum solution every 20-25 days until the seedlings are established.

2. The method of claim 1, wherein: The method for screening healthy seeds in step S1 is to use wind separation and 5% NaCl brine flotation to remove chaff, residue and potentially bacteria-carrying seeds, and collect the plump seeds that sink to the bottom.

3. The method of claim 1, wherein: The disinfection method in step S1 is to place the seeds in a 0.03% potassium permanganate aqueous solution, stir continuously and soak for 18 minutes. After the soaking, remove the seeds and wash away the residual potassium permanganate aqueous solution with clean water to complete the disinfection.

4. The method of claim 1, wherein: The specific volume ratio of the basic matrix components in step S3 is peat moss: vermiculite: perlite = 4:2:

1.

5. The method of claim 1, wherein, The specific preparation method of the fermented antibacterial seedling substrate in step S3 includes the following steps: S3-1. Pile the base substrate into a stack 1 meter high, adjust the moisture content to 55% ~ 60%, and set aside. S3-2. Inoculate 2 kg of compound microbial agent per cubic meter of substrate, with the viable counts of Bacillus subtilis and Bacillus amyloliquefaciens in the agent both ≥ 5 × 10⁻⁶. 8 Take CFU / g, cover with plastic sheeting, and compost for 15-20 days, turning the pile 2-3 times during the process to obtain a fermented antibacterial seedling substrate.

6. The method of claim 1, wherein: The planting management method in step S4 is to maintain soil moisture at 60%-70% and temperature at 15℃-20℃ during the seed germination stage; maintain soil moisture at 40%-50% and temperature at 18℃-22℃ during the seedling growth period; and ensure at least 6 hours of sufficient diffused light every day. In summer, when there is strong sunlight, a shade net is used, and the shading rate of the shade net is 30%-40%.

7. The method of claim 1, wherein: In steps S2 and S5, when preparing the Yunnan hard-skinned puffball spore powder extract solution, it should first be dissolved in a small amount of dimethyl sulfoxide, and then added to the target volume of water and stirred evenly.

8. The method of claim 1, wherein: In step S4, the sowing amount is 2-3 seeds per square centimeter; the thickness of the covering fermented antibacterial seedling substrate is 0.4-0.5 cm.

9. The method of claim 1, wherein: In step S5, the Trichoderma harzianum solution has an effective viable count of 5 × 10⁻⁶. 8 A 250-fold dilution of Trichoderma harzianum powder at CFU / g.

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