Quality control method in mycorrhizal edible mushroom seedling production

Abstract

The invention discloses a quality control method in mycorrhizal edible mushroom seedling production, and belongs to the technical field of forest seedling production. The method comprises the steps of strain detection, sterilized planting matrix detection, planting environment sterilization and disinfection treatment, temperature and humidity control, personnel control, nutrition supply and target seedling detection. According to the quality control method in mycorrhizal edible mushroom seedling production, quality control is carried out on multiple aspects in seedling cultivation production at the same time, particularly nutrient supply and target seedling detection means are adopted, the quality of mycorrhizal edible mushroom seedlings is improved, the adaptability of the mycorrhizal edible mushroom seedlings in the actual cultivation process is improved, and the yield of the mycorrhizal edible mushroom seedlings in the actual cultivation process is increased. The method is simple to operate, systematic, easy to popularize and suitable for actual production. The method is suitable for production of mycorrhizal edible mushroom seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of forest tree seedling production and relates to a quality control method, in particular to a quality control method in the production of mycorrhizal edible fungus seedlings. Background Art

[0002] There are approximately 900 species of edible fungi in my country. According to the "List of Mycorrhizal Edible Fungi in China," 532 of them are mycorrhizal, including truffles, matsutake mushrooms, lactarius, boletus, chanterelles, tricholoma, and tricholoma. Mycorrhizal fungi are fungi that form a unique symbiotic relationship: their hyphae tightly surround the root hairs of specific plant roots, forming a cap or Harty's network. They do not invade the inner cells of the roots, but instead spread and grow in the intercellular spaces between them as ectomycorrhizae. Ectomycorrhizae replace the function of plant root hairs, increasing the absorption area of the root hairs and improving the plant's absorption of various nutrients. Furthermore, mycorrhizal fungi secrete auxins, which are absorbed and utilized by the plant, while the plant also provides nutrients to the mycorrhizal fungi, forming a symbiotic relationship between the mycorrhizal fungi and the plant roots.

[0003] Mycorrhizal edible fungi are not only delicious and nutritious, but also have high medicinal value. Their unique symbiotic relationships regulate plant growth and development, making them crucial for forestry seedling cultivation and the greening of wasteland. However, the vast majority of mycorrhizal edible fungi, including matsutake and porcini, currently struggle to grow on artificial culture media. Even when they do grow, there are no comprehensive, industrially suitable management protocols for their subsequent cultivation. This makes artificial cultivation of mycorrhizal edible fungi difficult, resulting in extremely low yields and the need for wild collection, which is expensive.

[0004] The applicant has long been engaged in the research of mycorrhizal edible fungi. Through years of research and production experience, they have discovered that the quality of mycorrhizal edible fungi seedlings directly affects the growth, development, and yield of the fungi. Currently, seedling quality is often unstable during seedling production due to factors such as the environment and strain management, seriously affecting the yield of mycorrhizal edible fungi after planting. Therefore, it is of great significance to develop a quality control method for mycorrhizal edible fungi seedling production systems that can ensure the stable quality of mycorrhizal edible fungi seedlings and improve their adaptability and yield during actual cultivation. Summary of the Invention

[0005] The purpose of the present invention is to provide a quality control method for the production of mycorrhizal edible fungi seedlings, so as to solve the problems of lack of quality control methods and unstable seedling quality in the current seedling production process, which affect the yield of mycorrhizal edible fungi, ensure the high quality and health of mycorrhizal edible fungi seedlings, and promote their rapid growth and high yield.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: A quality control method for the production of mycorrhizal edible fungus seedlings includes: fungus strain detection, sterilized planting substrate detection, planting environment sterilization and disinfection treatment, temperature and humidity control, personnel control, nutrient supply and target seedling detection.

[0007] As a limitation, the bacterial strain detection is specifically as follows: the bacterial strain is identified, and the bacterial strain that is identified as consistent with the target bacteria and germinates within 7 days is qualified.

[0008] As another limitation, the sterilized planting substrate is tested, specifically, if there is no contamination of the planting substrate 5 days after sterilization, it is qualified.

[0009] As a third limitation, the sterilization and disinfection treatment of the planting environment specifically includes: disinfecting the host plant seeds, sterilizing the irrigation water, establishing a seedling space and configuring a fresh air system, disinfecting the seedling space, anti-pollution treatment of the ground of the seedling space, establishing a disinfection system at the entrance and exit of the seedling space, and disinfecting tools.

[0010] As a further limitation, the disinfecting of the host plant seeds specifically comprises: soaking the seeds in 30% concentration hydrogen peroxide or 84 disinfectant for 30-50 minutes, washing with sterile water, and then germinating under sterile conditions; Sterilize the irrigation water, including sterilizing the irrigation water using ozone sterilization, membrane filtration sterilization, ultraviolet sterilization, or radiation sterilization; Disinfect the nursery space 1-2 times a day before inoculating the strain, using 84 disinfectant spray, hypochlorous acid spray, ozone sterilization, or ultraviolet sterilization. Carry out anti-pollution treatment on the ground of the seedling cultivation space, including: covering with mulch or disinfecting the soil to prevent fungal contamination, and flushing the ground with irrigation water 1-2 times a day during the seedling cultivation period; The disinfection system includes an automatic spray disinfection system or an automatic air shower disinfection system; Disinfect the tools, specifically including: wiping the tool surface with diluted 84 disinfectant or hypochlorous acid; or placing the tools in a confined space and disinfecting them with ozone sterilization or ultraviolet sterilization.

[0011] As the fourth limitation, the temperature of the seedling growth environment is controlled at 15-25℃ and the humidity is controlled at 30%-70%.

[0012] As the fifth limitation, the personnel control specifically includes: operators wearing sterile clothing, caps and masks, washing hands and disinfecting before operation, and non-operating personnel are prohibited from entering and exiting.

[0013] As the sixth limitation, the nutritional supplementation specifically refers to supplementing the nutritional elements required by the seedlings and the trace elements required by the bacteria 3-7 days after inoculation of the bacteria.

[0014] After a large number of experimental studies, it was found that if nutritional supplementation is carried out after 7 days, the bacteria and seedlings will lack the elements needed for growth for a long time, and they will easily suffer from malnutrition, poor body development, or even death; if nutritional supplementation is carried out before 3 days, or even immediately after inoculation, the bacteria and seedlings will absorb nutrients separately, produce rejection reactions, and cannot achieve a symbiotic state.

[0015] As a further limitation, the target seedling detection specifically includes: 7-9 months after inoculation, checking the seedlings one by one, making a preliminary judgment based on the color and morphology of the mycorrhiza, and marking the seedlings with consistent color and morphology; Select representative seedlings with consistent color and morphology and test them using a microscope, DNA or PCR kit to determine whether the target bacteria are growing on the seedlings; Based on the labeling and test results, the seedlings of the target bacterial species are retained and the seedlings of the non-target bacterial species are eliminated.

[0016] After a large number of experimental studies, it was found that the growth state of most mycorrhizal edible fungi will be stable 7-9 months after inoculation. If the test is carried out before 7 months, the growth state of the fungus will be unstable, which may easily lead to contamination by foreign bacteria or affect the growth of mycorrhiza; if the test is carried out after 9 months, the mycorrhiza will be aged, with too many colors, making it difficult to distinguish the colors with the naked eye and impossible to mark. Judging each plant will waste a lot of resources.

[0017] Due to the adoption of the above technical solution, the present invention has the following technical advancements compared with the prior art: The present invention provides a quality control method for the production of mycorrhizal edible fungus seedlings. By simultaneously controlling the quality of multiple aspects in seedling cultivation and production, in particular by adopting nutritional supplementation and target seedling detection means, the quality of mycorrhizal edible fungus seedlings is improved, and their adaptability and yield in the actual cultivation process are improved. The operation is simple, systematic, easy to promote, and suitable for actual production. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below by way of specific examples. It should be understood that the described examples are only used to illustrate the present invention and are not intended to limit the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0020] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0021] Example 1 This embodiment discloses a mycorrhizal edible fungus (Truffles spp., Latin name Tuber aestivum Vittad. ) A quality control method for seedling production (host plant is Quercus coneflower), specifically comprising the following steps in sequence: 1) After washing mature, preliminarily confirmed summer truffle fruiting bodies based on appearance, soak them in 70% alcohol for 1 minute, label them individually, and bury them in sterile sand in a sealed container at 4-5°C until ready for use.

[0022] 2) Build a clean room with a fresh air system for seedling cultivation. Requirements include: automatic air showers and restrooms at the entrances and exits; seedbeds 50 cm above the ground and a sprinkler system; a 1 cm thick layer of slaked lime, flattened, followed by a 0.15 mm thick mulch film; drain pipes with elbows and check valves; and an ultraviolet sterilizer for the irrigation water inlet. Twenty-four hours before inoculation, dilute 84 disinfectant at a ratio of 1:200 and spray it in a sprayer for air disinfection. Disinfection should be performed twice daily. Maintain a clean and hygienic environment during use, flushing the floor with irrigation water daily. A tool cabinet with an ozone generator should be installed to regularly sterilize tools used in the nursery. The fresh air system must operate 24 hours a day.

[0023] 3) Only trained operators are allowed to enter and exit the nursery space. When entering the nursery space, operators must wear sterile clothing, caps and masks, and wash and disinfect their hands in the restroom.

[0024] 4) Mix soil, perlite, vermiculite, peat soil, and dolomite sand in a ratio of 1:1:1:2:0.25 to prepare a planting medium. Adjust the pH value of the planting medium to 7.5. Place the medium in a sterilization bag and sterilize it under high temperature and high pressure for 1 hour. After cooling to room temperature, take 0.2-1g of the medium under a sterile environment and inoculate it onto a plate culture medium. If no bacteria germinate after 5 days, the medium is qualified.

[0025] 5) Soak the seeds of Quercus truncatula in 30% 84 disinfectant for 30 minutes, rinse with sterile water, and germinate in a nursery space. 40-50 days after germination, transplant them into a sterilized nursery container (patent number: CN219459866U) containing a qualified planting medium for cultivation.

[0026] 6) 20 days after the germination of Quercus cone seeds, remove the fruiting bodies of the summer truffle and cut them into pieces less than 0.5 cm in a sterile environment. 2 Take a small amount of spores from the epidermis and activate them on a flat culture medium. The strains that germinate within 7 days are qualified, and the unqualified summer truffle fruiting bodies are eliminated according to the code.

[0027] 7) Perform ITS sequencing on the strains that have passed the strain activity test, compare the sequencing results with the sequences of summer truffles in the fungal species database, and retain and cultivate the fruiting bodies that are consistent with the target bacteria, that is, qualified summer truffles ( Tuber aestiv About Vittad. ) strains and eliminate unqualified fruiting bodies.

[0028] 8) After the seedlings are transplanted and cultivated, use a household pulper to crush the qualified truffle fruiting bodies under a sterile environment, dilute them into a spore suspension of 35,000 spores / mL, and inject them into the roots of the seedlings using the injection method at an inoculum volume of 1 mL per plant. 9) Use a spray system to control the growth environment temperature of the seedlings to 15-25℃ and the humidity to 30%-70%.

[0029] 10) Start fertilizing 7 days after inoculation of the bacteria to provide nutritional supplements: dilute the fertilizer with a nitrogen, phosphorus and potassium ratio of 15:9:20 with water to 1000-2000 times, add 15g of chelated trace elements, mix and spray, and fertilize once every 7-10 days.

[0030] 11) After 9 months of inoculation, examine the mycorrhizae growing on each seedling. Visually observe the color and morphology of the mycorrhizae to make a preliminary judgment: from a morphological perspective, group the seedlings with apical swellings and group the seedlings with those without apical swellings. For those with apical swellings, mark them according to the color with the largest number of apical swellings, such as light yellow, yellow-brown, light brown, gray-brown, dark brown, black-brown, or other colors.

[0031] 12) Select one representative seedling with swollen root tips for ITS sequencing. Compare the sequencing results with the summer tuber sequence in the fungal species database to determine whether the target species is growing on the seedling. Retain qualified seedlings and eliminate seedlings with non-target species and seedlings without swollen root tips.

[0032] The quality qualification rate of the root edible fungi seedlings of Tuber serrata and Quercus truncatula cultured by the method reaches more than 95%, and the economic benefits are significant.

[0033] Example 2 This embodiment discloses a method for quality control in the production of mycorrhizal edible mushroom (Lactarius pine) seedlings (host plant is Yunnan pine), which specifically includes the following steps performed in sequence: 1) Purchase pure strains of Lactarius pine from multiple channels, inoculate them onto plate culture media and expand the culture at a temperature of 20-22°C. Keep strains that germinate within 7 days and eliminate those that do not germinate.

[0034] 2) Perform ITS sequencing on strains that germinate within 7 days and compare the sequencing results with the sequences of Lactarius pine in the fungal species database. Retain and cultivate strains that match the target bacteria and eliminate unqualified strains.

[0035] 3) Build a clean room with a fresh air system as a nursery area. Requirements include: entrances and exits equipped with automatic spray disinfection systems and restrooms; seedbeds 50 cm above the ground and a spray system; the ground should be covered with a 1.5 cm thick layer of quicklime, then watered and flattened, and then covered with a 0.15 mm thick mulch film; sewer pipes should have elbows and check valves; and the irrigation water inlet should have a membrane filtration sterilization purification system. Air disinfection should be performed using an ozone generator combined with ultraviolet light 24 hours before inoculation, with disinfection occurring once every evening. Before disinfection, the nursery area should be sprayed with a spray system to increase humidity. The nursery area should be kept clean and hygienic during use, and the floor should be flushed with irrigation water twice daily. A tool cabinet equipped with ultraviolet light should be installed to regularly sterilize tools used in the nursery area. The fresh air system should operate 18 hours daily.

[0036] 4) Only trained operators are allowed to enter and exit the nursery space. When entering the nursery space, operators must wear sterile clothing, caps and masks, and wash and disinfect their hands in the restroom.

[0037] 5) Mix soil, perlite, vermiculite, peat soil, and lawn grass fermentation product in a ratio of 1:1:1:1:1 to prepare a planting medium. Adjust the pH value of the medium to 6.5. Sterilize the medium in a sterilization bag at 121°C twice. After cooling to room temperature, take 0.1-0.5g of the medium in a sterile environment and inoculate it onto a plate culture medium. If no bacteria germinate after 5 days, the medium is qualified.

[0038] 6) Qualified Lactarius pine strains are cultured in a shaking liquid culture under sterile conditions for 20 days. The strain concentration is ≥1g / L of dry mycelium weight.

[0039] 7) Disinfect the Yunnan pine seeds by soaking them in 30% hydrogen peroxide for 30 minutes. Rinse with sterile water and plant three seeds per hole in a seedling tray filled with a qualified planting medium for cultivation.

[0040] 8) 30 days after the Yunnan pine seeds germinate, remove the cultured Lactarius pine liquid spawn, break up the pellets with a grinder, and dilute with sterile water at a ratio of 1:500 to prepare an inoculum. Inject 6 mL per plant into the roots of the seedlings. 9) Use a spray system to control the growth environment temperature of the seedlings to 15-25℃ and the humidity to 40%-60%.

[0041] 10) Place 2-3 slow-release fertilizers on each acupoint 5 days after inoculation, and add 10 grams of chelated trace elements per ton of water for spraying. Spray the chelated trace elements once every 10 days.

[0042] 11) After 7 months of inoculation, check the mycorrhizae of each seedling. Visually observe the color and morphology of the mycorrhizae to make a preliminary judgment. That is, from a morphological point of view, the ones with swollen root tips are grouped together, and the ones with no swollen root tips are grouped together. For those with swollen root tips, mark them according to the color with the largest number of swollen root tips, such as milky white, light yellow, light brown, yellowish brown, tan, reddish brown, or other colors.

[0043] 12) Select one representative seedling each with enlarged root tips, clean it thoroughly, and separate most of the soil attached to the roots. Use a brush and tweezers to remove the finest soil particles. Observe the morphology under a binocular microscope and compare it with the known mycorrhizal morphology of the target fungus. Acceptable seedlings should be retained, while unqualified seedlings and those with non-enlarged root tips should be eliminated.

[0044] 13) For multiple plants that germinate, keep the largest seedling and discard the remaining seedlings.

[0045] The qualified rate of the seedlings of the edible mycorrhizal mushrooms of Lactarius pine and P. yunnanensis cultivated by the method reaches more than 96%, and the economic benefits are significant.

[0046] Example 3 This embodiment discloses a method for quality control in the production of mycorrhizal edible fungus (Boletus edulis) seedlings (host plant is Quercus acutissima), which specifically includes the following steps performed in sequence: 1) During the investigation, multiple fruiting bodies that are morphologically consistent with Boletus roseus were collected and identified by a fungal taxonomy expert. Fruiting bodies that were confirmed to be the target fungus were retained. Pure strains were isolated using tissue separation methods and inoculated onto plate culture media. Cultures were maintained at 23-25°C. Those that germinated within 7 days and showed strong vitality were retained, and those that did not germinate or had weak vitality were eliminated.

[0047] 2) Perform whole-genome sequencing on the bacterial species retained after identification.

[0048] 3) Build a clean room with a fresh air system for seedling cultivation. Requirements include: automatic air showers and restrooms at the entrances and exits; seedbeds 50 cm above the ground and a sprinkler system; two layers of 0.15 mm thick mulch film after applying 80 times the amount of Weibamu fungicide diluted in water; drain pipes with elbows and check valves; and an ozone generator at the irrigation water inlet. Disinfect the air in the seedling cultivation space with a 200 ppm hypochlorous acid solution 24 hours before seedling inoculation, once or twice daily. Maintain cleanliness within the seedling cultivation space during use, flushing the floor with irrigation water once daily. A tool cabinet with an ozone generator is required to regularly sterilize tools used in the seedling cultivation space. Wipe tool surfaces with diluted hypochlorous acid before and after use. The fresh air system must operate 24 hours a day.

[0049] 4) Only trained operators are allowed to enter and exit the nursery space. When entering the nursery space, operators must wear sterile clothing, caps and masks, and wash and disinfect their hands in the restroom.

[0050] 5) Mix red soil, perlite, vermiculite, peat soil and coconut coir in a ratio of 1:1:1:1:1 to prepare a planting matrix. Adjust the pH value of the matrix to 6.5. Place the matrix in a sterilization bag and place it in a sterilization bag at 121°C and 1.5 kg / cm 2 Sterilize under pressure for 2 hours, cool to room temperature, take 0.2g of matrix under sterile environment, inoculate it on the plate culture medium for testing, and it is qualified if no foreign bacteria germinate after 5 days.

[0051] 6) Qualified rose yellow boletus strains are cultured in a shaking liquid culture under sterile conditions for 30 days. The strain concentration is qualified when the dry weight of mycelium is ≥2g / L.

[0052] 7) Soak the seeds of Quercus acutissima in 30% hydrogen peroxide for 50 minutes, rinse with sterile water, and germinate in a clean room.

[0053] 8) Plant the germinated Quercus acutissima seeds one seed per acupuncture point in a sterilized seedling container (patent number: CN219459866U) containing a qualified planting medium for cultivation.

[0054] 9) 60 days after the oak seeds germinate, remove the cultured boletus edulis liquid spawn, break up the pellets, and dilute with sterile water at a ratio of 1:600 to prepare an inoculum. Inject 10 mL per plant into the roots of the seedlings. 10) Use a spray system to control the growth environment temperature of the seedlings to 18-24℃ and the humidity to 30%-60%.

[0055] 11) Place 2-3 slow-release fertilizers on each acupoint 7 days after inoculation, and add 10 grams of chelated trace elements per ton of water for spraying. Spray the chelated trace elements once every 10 days.

[0056] 12) After 9 months of inoculation, check the mycorrhizae growing on each seedling. Use the following steps: S1. Making auxiliary labels: By spot-checking 100 seedlings, the color of mycorrhizae was determined by visual observation, and the standard color of different mycorrhizal colors was determined. Bamboo sticks were dyed in white, light yellow, and light brown to make auxiliary labels. The auxiliary bamboo sticks with mycorrhizal colors of other colors were natural and sterilized for later use. S2. Inspect each plant's morphology and label it. Seedlings with apical swellings will have auxiliary labels inserted into their containers. Seedlings without apical swellings will not have auxiliary labels. Compare the color of the mycorrhizae on the seedlings with apical swellings to the color of the auxiliary labels. Seedlings with the most apical swellings will have auxiliary bamboo sticks of the same or similar color inserted. S3. Select one seedling each with swollen root tips and representative mycorrhizal colors, collect the swollen root tips, amplify the DNA using a PCR kit, and perform bidirectional sequencing on the amplified products. Compare the sequencing results with the gene sequencing results of the target bacterial species, retain qualified seedlings based on the auxiliary label color, and eliminate unqualified seedlings and seedlings without labels.

[0057] The qualified rate of the seedlings of Lactarius pine and Pseudomonas yunnanensis mycorrhizal edible fungi cultivated by this method reached more than 94%, with significant economic benefits.

Claims

1. A quality control method for the production of mycorrhizal edible mushroom seedlings, characterized in that: The method includes: strain detection, sterilized planting matrix detection, planting environment sterilization and disinfection treatment, temperature and humidity control, personnel control, nutrition supply and target seedling detection.

2. The quality control method in the production of mycorrhizal edible mushroom seedlings according to claim 1, characterized in that: The bacterial strain detection specifically includes: identifying the bacterial strain, and the bacterial strain that is consistent with the target bacteria and germinates within 7 days is qualified.

3. The quality control method in the production of mycorrhizal edible mushroom seedlings according to claim 1, characterized in that: The sterilized planting substrate is tested, specifically, if there is no contamination of the planting substrate 5 days after sterilization, it is qualified.

4. The method for quality control in the production of mycorrhizal edible mushroom seedlings according to claim 1, wherein: The sterilization and disinfection treatment of the planting environment specifically includes: disinfecting the host plant seeds, sterilizing the irrigation water, establishing a seedling space and configuring a fresh air system, disinfecting the seedling space, anti-pollution treatment of the ground of the seedling space, establishing a disinfection system at the entrance and exit of the seedling space, and disinfecting tools.

5. The quality control method in the production of mycorrhizal edible mushroom seedlings according to claim 4, characterized in that: The disinfection of the host plant seeds specifically includes: soaking the seeds in 30% concentration hydrogen peroxide or 84 disinfectant for 30-50 minutes, washing with sterile water and then germinating under sterile conditions; Sterilize the irrigation water, including sterilizing the irrigation water using ozone sterilization, membrane filtration sterilization, ultraviolet sterilization, or radiation sterilization; Disinfect the nursery space 1-2 times a day before inoculating the strain, using 84 disinfectant spray, hypochlorous acid spray, ozone sterilization, or ultraviolet sterilization. Carry out anti-pollution treatment on the ground of the seedling cultivation space, including: covering with mulch or disinfecting the soil to prevent fungal contamination, and flushing the ground with irrigation water 1-2 times a day during the seedling cultivation period; The disinfection system includes an automatic spray disinfection system or an automatic air shower disinfection system; Disinfect the tools, specifically including: wiping the tool surface with diluted 84 disinfectant or hypochlorous acid; or placing the tools in a confined space and disinfecting them with ozone sterilization or ultraviolet sterilization.

6. The method for quality control in the production of mycorrhizal edible mushroom seedlings according to claim 1, characterized in that: Control the seedling growth environment temperature at 15-25℃ and the humidity at 30%-70%.

7. The method for quality control in the production of mycorrhizal edible mushroom seedlings according to claim 1, characterized in that: The personnel control specifically includes: operators wearing sterile clothing, caps and masks, washing hands and disinfecting before operation, and non-operating personnel are prohibited from entering or exiting.

8. A quality control method for the production of mycorrhizal edible mushroom seedlings according to any one of claims 1 to 7, characterized in that: The nutritional supplementation specifically includes replenishing the nutritional elements required by the seedlings and the trace elements required by the bacteria 3-7 days after inoculation of the bacteria.

9. The method for quality control in the production of mycorrhizal edible mushroom seedlings according to claim 8, characterized in that: The target seedling detection specifically includes: 7-9 months after inoculation, checking the seedlings one by one, making a preliminary judgment based on the color and morphology of the mycorrhiza, and marking the seedlings with the same color and morphology; Select representative seedlings with consistent color and morphology and test them using a microscope, DNA or PCR kit to determine whether the target bacteria are growing on the seedlings; Based on the labeling and test results, the seedlings of the target bacterial species are retained and the seedlings of the non-target bacterial species are eliminated.