Big tree inoculation method of Lijiang truffle

Abstract

The invention relates to a big tree inoculation method of Lijiang truffle, and belongs to the technical field of biology. The method comprises the seven steps of environment and inoculated tree requirements, inoculation time selection, preparation before inoculation, strain preparation, big tree inoculation, management after inoculation and truffle harvesting. Compared with a truffle conservation and propagation promotion technology and a mycorrhizal seedling cultivation and planting technology, the period is short, a small amount of truffle sporocarp is found to be formed in winter in the current year of big tree inoculation, the success rate of big tree inoculation reaches up to 80% or above, and the effect is obvious.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a large tree inoculation method of Lijiang white truffles. Background Art

[0002] Truffles, also known as truffles, belong to the Pezizales and Tuberaceae. They are a famous wild edible fungus. Among them, the Italian white truffle and the French black truffle are the most precious. They are known as "diamonds in the kitchen" and "underground gold". They are often called "pig arch fungus" by people in southwest my country, and are often called truffles in business. With unique fragrance, taste and nutritional value, humans have been eating truffles for thousands of years. In developed countries such as Europe and the United States, truffles are known as "black diamonds" and are the best wild fungi. They are called the three treasures together with caviar and foie gras. Truffles have multiple therapeutic effects and are rich in 17 amino acids, 8 vitamins, appropriate amounts of protein, and more than 50 physiologically active ingredients such as androstenone, sterols, sphingolipids, fatty acids, amino acids and trace elements. It also contains 8 kinds of amino acids that the human body cannot synthesize, as well as essential nutrients such as zinc, manganese, iron, calcium, phosphorus, and selenium. It has medicinal value such as enhancing immunity, anti-aging, benefiting the stomach, refreshing the mind, stopping bleeding, and treating hemorrhoids. It has anti-cancer activity, has a certain inhibitory effect on cancer cells, and can stimulate the vitality of brain cells.

[0003] Truffles are fragrant and delicious, and are one of the favorite mushrooms in Europe and the United States. They are mainly eaten fresh, made into food seasonings, or extracted for use in cosmetics. At present, the market price of black truffles in Japan, Europe and the United States is between 50 and 1,000 US dollars per kilogram, and the domestic market price of black truffles is between 500 and 1,000 yuan per kilogram. The price of high-quality Italian white truffles can reach more than 5,000 euros per kilogram, making it one of the most expensive mushrooms.

[0004] Lijiang White Truffle was officially named in 2011. It is a kind of white truffle grown in Yongsheng County, Lijiang City, Yunnan Province. It has a diameter of 0.5-3cm, is subspherical or irregularly spherical, is white in the young stage, and gradually turns grayish white with increasing maturity. When fully mature, it has a strong fragrance. The spores are gray-brown with white to grayish white veins. The size of the ascus is: 60-90×50-80µm, and the spore size is 25-42.5µm. Compared with black truffles, Lijiang White Truffles have a more delicate, dense and soft taste. The commodity value is 5-8 times that of black truffles, and it has great development and utilization prospects. However, at present, except for local farmers in Lijiang collecting wild Lijiang White Truffles for sale, there has been no development and research reports. Wild Lijiang White Truffles are scarce and the yield is extremely low, far from meeting people's needs.

[0005] Truffles are rich in nutrients, high in functional ingredients, strong in fragrance and high in value, so wild resources are often over-exploited, and wild truffle ponds are destroyed in large quantities, resulting in a decrease in production year by year. At present, there are two ways to increase truffle production and improve quality: 1. Wild truffle conservation and propagation technology. The necessary condition for this technology is to protect and improve the ecological conditions of the truffle ponds in the forest, so that the ponds can produce more truffles. However, this technology has little effect on forests with very few ponds, and it cannot be implemented at all for forests without ponds. 2. Truffle mycorrhizal seedling cultivation and planting technology. This technology requires the cultivation of sterile seedlings from tree seeds, and then inoculation of truffle spores to form mycorrhizal seedlings. After the cultivation is completed, the seedlings are planted and managed. After 5 to 8 years, the seedlings grow up, and the ponds are formed near the roots of the trees to naturally grow truffles. The whole process requires a long period of time and consumes a lot of manpower, material resources and time costs.

[0006] Therefore, how to overcome the shortcomings of existing technologies is an urgent problem to be solved in the field of biotechnology. Summary of the invention

[0007] The invention aims to solve the deficiencies in the prior art and to provide a method for inoculating large trees with Lijiang white truffles.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for inoculating a large tree of Lijiang white truffle comprises the following steps: Step 1, Environmental and inoculated tree requirements: Environmental requirements are: sloping or flat forests with an altitude range of 1500-2800m; annual average temperature of 13-20℃, annual rainfall of 600-1800mm, annual sunshine hours of 1600-3000h; distinct dry and rainy seasons, with a daily temperature difference of 8-22℃; soil types of red soil, brown soil or brown soil, and forest soil thickness >40cm; Requirements for inoculated forest trees: adult Yunnan pine forests, or mixed forests dominated by adult Yunnan pine mixed with one or more of Armand pine, Quercus, Quercus, Castanea mollissima, Platycarya styracifolia, Hazelnut, Populus yunnanensis and Cyclobalanopsis yunnanensis, with a forest canopy density of 6.5-7.5; Step 2: Choose the vaccination time: Vaccination is done in March-April or June-July every year; Before inoculation in March and April, water should be sprinkled to ensure that the surface soil is moist at a depth of 3-5 cm. After inoculation, water should be sprinkled every 1 m 2 The amount of water is 3-5kg, water once every three days until the rain comes; In June and July, inoculation should be carried out just after the rain falls to the ground or after the rain has penetrated 40cm of the soil layer; Step 3: Preparation before vaccination: Remove the small shrubs and weeds in the forest, then shovel away the humus on the surface of the forest; then shovel away the 2-4cm thick soil under the humus; then loosen the 10-20cm thick soil underground to expose part of the epidermal root system of Yunnan pine and other large trees; then spread quicklime for disinfection and sterilization; inoculate the next day after disinfection and sterilization; Step 4: Preparation of strains: Sterile water adjusted to a pH between 7.5 and 8.0 using slaked lime was used as a solvent; Select Lijiang white truffle fruiting bodies with a diameter of more than 1 cm, no insect infestation, no mildew and a maturity of greater than or equal to 95%, add 595-605 mL of solvent for every 99-101 g of fruiting bodies, then put them into a blender and crush them to make a spore stock solution; store at 0°C-4°C; Before inoculation, the spore stock solution is diluted with the solvent to obtain a spore solution with a volume concentration of 0.5%-1.0%, and then slaked lime is added to adjust the pH to 7.0-7.5 for standby use; Step 5: Large tree inoculation: On the forest land treated in step 3, the spore solution prepared in step 4 is used to spray the surface of the forest land, and 500-600 kg of spore solution is sprayed per mu of land; after spraying, a layer of stone powder is first sprinkled, 3-5 kg ​​of stone powder is sprinkled per square meter; then a layer of slaked lime is sprinkled, 0.5-1.0 kg per square meter; then the soil shoveled in step 3 is evenly backfilled, and then the humus shoveled in step 3 is evenly spread on it; After inoculation, soil exchangeable calcium Ca was tested 2+ Content and soil pH value, soil exchangeable calcium Ca 2+ The content should reach 30-80cmol / kg, and the soil pH should be 7.5-8.5; Step 6: Post-vaccination management: After vaccination, animals and strangers are prohibited from entering the vaccination area. Check the formation of truffle mycorrhiza 6 months after inoculation; if mycorrhiza has not formed, mark the trunk so that it can be inoculated again next year; After vaccination, ants and earwigs are collectively trapped and killed in the forest in July and August every year; Step 7: Truffle harvesting: The harvesting time is from January to February of the third year after inoculation, and the truffles are harvested when their maturity is greater than or equal to 95%.

[0009] Furthermore, preferably, in step one, the environment requires that the landform is karst landform.

[0010] Furthermore, preferably, in step 1, in the inoculated forest, more than 60% of the trees are 10-35 years old, the tree diameter is 10-40 cm, and the length and width of the tree spacing are both 2-5 meters.

[0011] Furthermore, it is preferred that in step 2, inoculation is carried out in June or July. If there is no rain for 20 consecutive days after inoculation, watering should be carried out by spraying. The amount of watering is 100% per 1m 2 3-5kg, water once every three days.

[0012] Furthermore, preferably, in step three, the humus on the surface of the forest land is shoveled and piled into a pile of 5 meters × 5 meters; and then the 2-4 cm thick soil below the humus is shoveled and piled into a pile of 3 meters × 3 meters.

[0013] Further, preferably, in step 3, every 667m 2 The amount of quicklime spread on the forest land is 200-300kg.

[0014] Further, preferably, in step 4, the fruiting bodies of Lijiang white truffles are obtained from the end of December to February of the following year, the soil and debris on the surface of the truffle fruiting bodies are cleaned, sealed and packaged, and frozen in a -20°C refrigerator for standby use; the fruiting bodies are taken out when preparing the spore stock solution; When preparing the spore stock solution, the speed of the mixer is 8000rpm / min-15000rpm / min, and the crushing time is 15s-30s.

[0015] Furthermore, preferably, in step five, the thickness of the humus is 3.0-5.0 cm.

[0016] Furthermore, preferably, in step 6, the secondary supplementary inoculation method is to carry out the secondary supplementary inoculation in March-April or May-June of the second year after the inoculation; with the large tree without mycorrhizal formation as the center and the area with a radius of 1.0-1.5m as the inoculation area, first shovel away the backfilled humus, then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder, and then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder at 0.85-0.90kg / m 2 Spray the spore solution in the amount of spores; then backfill the shoveled soil and the soil layer sprinkled with slaked lime and stone powder again, and then backfill the shoveled humus; after inoculation, the soil should be refilled every 1m 2 The amount of water used is 3-5kg, watered once every three days until the rain comes.

[0017] Furthermore, preferably, in step seven, during harvesting, the raking depth shall not exceed 10 cm.

[0018] The technology of the invention is to improve the soil directly through human intervention and to carry out large-scale tree inoculation. The technology has high precision and high inoculation success rate. After the Lijiang white truffle is inoculated, truffle fruiting bodies are formed in the same year, and the effect is quick.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a method for inoculating large trees with Lijiang white truffles. The method is to inoculate the entire forest with Lijiang white truffles, and artificially improve the soil by covering it with stone powder and lime, so that the pH value and exchangeable Ca 2+ The content can meet the growth requirements of truffles, so the present invention does not need to ensure that there are a large number of truffle ponds in the inoculated forest land, that is, the method of the present invention can still be implemented in the absence of truffle ponds; the implementation scope of the method of the present invention is greatly increased compared with the conservation and propagation promotion technology of truffles; (2) Compared with truffle conservation and propagation promotion technology and mycorrhizal seedling cultivation and planting technology, the present invention has a shorter cycle. A small amount of truffle fruiting bodies are found in the winter of the year when the big trees are inoculated. The success rate of big tree inoculation is as high as more than 80%, and the effect is obvious. (3) The present invention simultaneously implemented the tree inoculation technology for Indian truffles, Chinese truffles and Lijiang white truffles, and the inoculation success rate of Indian truffles was 21%, that of Chinese truffles was 15%, and that of Lijiang white truffles was greater than 80%. It was found that only Lijiang white truffles performed well and had the highest inoculation success rate, and the tree inoculation technology for Lijiang white truffles was the first technology and has not been reported so far.

[0020] The method of the present invention is compared with the prior art, as shown in Table 1.

[0021] Table 1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the inoculated forest map selected in the application example of the present invention; Figure 2 These are pictures of ascospores of Lijiang white truffle; (a) shows mature spores observed under a 400x microscope; (b) shows partially mature and partially immature spores observed under a 100x microscope; Figure 3 The mycorrhizal morphology of the white truffle of Lijiang was observed visually; Figure 4 The mycorrhizal morphology of Lijiang white truffle observed with a 20x handheld magnifying glass; Figure 5 This is a microscopic observation (400 times) of a large number of hyphae of the mycorrhizal slice of the white truffle of Lijiang; Figure 6 This is a picture of the burn circle formed under the Yunnan pine forest in the experimental site; the upper left part of the picture is the uninoculated area, with lush herbaceous plants on the surface, and the lower right part is the inoculated area, forming a whole burn circle; Figure 7 This is a diagram showing a large number of young truffle fruiting bodies formed in the inoculated forest selected in the application example of the present invention. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with embodiments.

[0024] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0025] Example 1 A method for inoculating a large tree of Lijiang white truffle comprises the following steps: Step 1, Environmental and inoculated tree requirements: Environmental requirements are: sloping or flat forests with an altitude range of 1500-2800m; annual average temperature of 13-20℃, annual rainfall of 600-1800mm, annual sunshine hours of 1600-3000h; distinct dry and rainy seasons, with a daily temperature difference of 8-22℃; soil types of red soil, brown soil or brown soil, and forest soil thickness >40cm; Requirements for inoculated forest trees: adult Yunnan pine forests, or mixed forests dominated by adult Yunnan pine mixed with one or more of Armand pine, Quercus, Quercus, Castanea mollissima, Platycarya styracifolia, Hazelnut, Populus yunnanensis and Cyclobalanopsis yunnanensis, with a forest canopy density of 6.5-7.5; Step 2: Choose the vaccination time: Vaccination is done in March-April or June-July every year; Before inoculation in March and April, water should be sprinkled to ensure that the surface soil is moist at a depth of 3.5-4.5 cm. After inoculation, water should be sprinkled every 1 m 2 The watering amount is 4kg, watering once every three days until the rain comes; In June and July, inoculation should be carried out just after the rain falls to the ground or after the rain has penetrated 40cm of the soil layer; Step 3: Preparation before vaccination: Remove the small shrubs and weeds in the forest, then shovel the humus on the surface of the forest; then shovel the 2.5-3.5cm thick soil under the humus; then loosen the 13-18cm thick soil underground; then sprinkle quicklime for disinfection and sterilization; inoculate the next day after disinfection and sterilization; Step 4: Preparation of strains: Sterile water adjusted to pH 7.7 using slaked lime was used as solvent; Select Lijiang white truffle fruiting bodies with a diameter of more than 1 cm, no insect infestation, no mildew and a maturity of greater than or equal to 95%, add 600 mL of solvent to every 100 g of fruiting bodies, then put them into a blender and crush them to make spore stock solution; store at 0°C-4°C; Before inoculation, the spore stock solution is diluted with the solvent to a spore solution with a volume concentration of 0.8%, and then slaked lime is added to adjust the pH to 7.2 for standby use; Step 5: Large tree inoculation: On the forest land treated in step 3, the spore solution prepared in step 4 is used to spray the surface of the forest land, and 550 kg of spore solution is sprayed per mu of land; after spraying, a layer of stone powder is first sprinkled, 4 kg of stone powder is sprinkled per square meter; then a layer of slaked lime is sprinkled, 0.6 kg per square meter; then the soil shoveled in step 3 is evenly backfilled, and then the humus shoveled in step 3 is evenly spread on it; After inoculation, soil exchangeable calcium Ca was tested 2+ Content and soil pH value, soil exchangeable calcium Ca 2+ The content should reach 30-80cmol / kg, and the soil pH should be 7.5-8.5; Step 6: Post-vaccination management: After vaccination, animals and strangers are prohibited from entering the vaccination area. Check the formation of truffle mycorrhiza 6 months after inoculation; if mycorrhiza has not formed, mark the trunk so that it can be inoculated again next year; After vaccination, ants and earwigs are collectively trapped and killed in the forest in July and August every year; Step 7: Truffle harvesting: The harvesting time is from January to February of the third year after inoculation, and the truffles are harvested when their maturity is greater than or equal to 95%.

[0026] Example 2 A method for inoculating a large tree of Lijiang white truffle comprises the following steps: Step 1, Environmental and inoculated tree requirements: Environmental requirements are: sloping or flat forests with an altitude range of 1500-2800m; annual average temperature of 13-20℃, annual rainfall of 600-1800mm, annual sunshine hours of 1600-3000h; distinct dry and rainy seasons, with a daily temperature difference of 8-22℃; soil types of red soil, brown soil or brown soil, and forest soil thickness >40cm; Requirements for inoculated forest trees: adult Yunnan pine forests, or mixed forests dominated by adult Yunnan pine mixed with one or more of Armand pine, Quercus, Quercus, Castanea mollissima, Platycarya styracifolia, Hazelnut, Populus yunnanensis and Cyclobalanopsis yunnanensis, with a forest canopy density of 6.5-7.5; Step 2: Choose the vaccination time: Vaccination is done in March-April or June-July every year; Before inoculation in March and April, water should be sprinkled to ensure that the surface soil is moist at a depth of 3-4 cm. After inoculation, water should be sprinkled every 1m 2 The watering amount is 3kg, watering once every three days until the rain comes; In June and July, inoculation should be carried out just after the rain falls to the ground or after the rain has penetrated 40cm of the soil layer; Step 3: Preparation before vaccination: Remove the small shrubs and weeds in the forest, then shovel the humus on the surface of the forest; then shovel the 2-3cm thick soil under the humus; then loosen the 10-15cm thick soil underground; then sprinkle quicklime for disinfection and sterilization; inoculate the next day after disinfection and sterilization; Step 4: Preparation of strains: Sterile water adjusted to pH 7.5 using slaked lime was used as solvent; Select Lijiang white truffle fruiting bodies with a diameter of more than 1 cm, no insect infestation, no mildew and a maturity of greater than or equal to 95%, add 595 mL of solvent to every 99 g of fruiting bodies, then put them into a blender and crush them to make a spore stock solution; store at 0°C-4°C; Before inoculation, the spore stock solution is diluted with the solvent to obtain a spore solution with a volume concentration of 0.5%, and then slaked lime is added to adjust the pH to 7.0 for standby use; Step 5: Large tree inoculation: On the forest land treated in step 3, the spore solution prepared in step 4 is used to spray the surface of the forest land, and 50 kg of spore solution is sprayed per mu of land; after spraying, a layer of stone powder is first sprinkled, 3 kg of stone powder is sprinkled per square meter; then a layer of slaked lime is sprinkled, 0.5 kg per square meter; then the soil shoveled in step 3 is evenly backfilled, and then the humus shoveled in step 3 is evenly spread on it; After inoculation, soil exchangeable calcium Ca was tested 2+ Content and soil pH value, soil exchangeable calcium Ca 2+ The content should reach 30-80cmol / kg, and the soil pH should be 7.5-8.5; Step 6: Post-vaccination management: After vaccination, animals and strangers are prohibited from entering the vaccination area. Check the formation of truffle mycorrhiza 6 months after inoculation; if mycorrhiza has not formed, mark the trunk so that it can be inoculated again next year; After vaccination, ants and earwigs are collectively trapped and killed in the forest in July and August every year; Step 7: Truffle harvesting: The harvesting time is from January to February of the third year after inoculation, and the truffles are harvested when their maturity is greater than or equal to 95%.

[0027] In step 1, the environment requires that the landform be karst.

[0028] In step 1, more than 60% of the trees in the inoculated forest are 10-20 years old, with a tree diameter of 10-30 cm and a tree spacing of 2-4 meters in length and width.

[0029] In step 2, inoculation is done in June and July. If there is no rain for 20 consecutive days after inoculation, watering should be done by spraying. The amount of watering is 100 grams per 1m 2 3kg, water once every three days.

[0030] In step three, shovel the humus on the surface of the forest land and pile it into a pile of 5 meters x 5 meters; then shovel the 2-3 cm thick soil under the humus and pile it into a pile of 3 meters x 3 meters.

[0031] In step 3, every 667m 2 The amount of quicklime spread on the forest land is 200kg.

[0032] In step 4, the fruiting bodies of Lijiang white truffles are obtained from the end of December to February of the following year, the soil and debris on the surface of the truffle fruiting bodies are cleaned, sealed and packaged, and frozen in a -20°C refrigerator for use; the fruiting bodies are taken out when preparing the spore stock solution; When preparing the spore stock solution, the speed of the mixer is 8000 rpm / min and the crushing time is 15 s.

[0033] In step 5, the thickness of the humus is 3.0-4.0 cm.

[0034] In step 6, the secondary inoculation method is to carry out the secondary inoculation in March-April or May-June of the second year after the inoculation; with the large tree without mycorrhizal formation as the center and the area with a radius of 1.0-1.2m as the inoculation area, first shovel away the backfilled humus, then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder, and then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder at 0.85kg / m 2 Spray the spore solution in the amount of spores; then backfill the shoveled soil and the soil layer sprinkled with slaked lime and stone powder again, and then backfill the shoveled humus; after inoculation, the soil should be refilled every 1m 2 The watering amount is 3kg, water once every three days until the rain comes.

[0035] In step seven, when harvesting, the depth of raking should not exceed 10cm.

[0036] Example 3 A method for inoculating a large tree of Lijiang white truffle comprises the following steps: Step 1, Environmental and inoculated tree requirements: Environmental requirements are: sloping or flat forests with an altitude range of 1500-2800m; annual average temperature of 13-20℃, annual rainfall of 600-1800mm, annual sunshine hours of 1600-3000h; distinct dry and rainy seasons, with a daily temperature difference of 8-22℃; soil types of red soil, brown soil or brown soil, and forest soil thickness >40cm; Requirements for inoculated forest trees: adult Yunnan pine forests, or mixed forests dominated by adult Yunnan pine mixed with one or more of Armand pine, Quercus, Quercus, Castanea mollissima, Platycarya styracifolia, Hazelnut, Populus yunnanensis and Cyclobalanopsis yunnanensis, with a forest canopy density of 6.5-7.5; Step 2: Choose the vaccination time: Vaccination is done in March-April or June-July every year; Before inoculation in March and April, water should be sprinkled to ensure that the surface soil is moist at a depth of 4-5 cm. After inoculation, water should be sprinkled every 1m 2 The watering amount is 5kg, watering once every three days until the rain comes; In June and July, inoculation should be carried out just after the rain falls to the ground or after the rain has penetrated 40cm of the soil layer; Step 3: Preparation before vaccination: Remove the small shrubs and weeds in the forest, then shovel the humus on the surface of the forest; then shovel the 3-4cm thick soil under the humus; then loosen the 15-20cm thick soil underground; then sprinkle quicklime for disinfection and sterilization; inoculate the next day after disinfection and sterilization; Step 4: Preparation of strains: Sterile water adjusted to pH 8.0 using slaked lime was used as the solvent; Select Lijiang white truffle fruiting bodies with a diameter of more than 1 cm, no insect infestation, no mildew and a maturity of greater than or equal to 95%, add 605 mL of solvent to every 101 g of fruiting bodies, then put them into a blender and crush them to make a spore stock solution; store at 0°C-4°C; Before inoculation, the spore stock solution is diluted with the solvent to a spore solution with a volume concentration of 1.0%, and then slaked lime is added to adjust the pH to 7.5 for standby use; Step 5: Large tree inoculation: On the forest land treated in step 3, the spore solution prepared in step 4 is used to spray the surface of the forest land, and 600 kg of spore solution is sprayed per mu of land; after spraying, a layer of stone powder is first sprinkled, 5 kg of stone powder is sprinkled per square meter; then a layer of slaked lime is sprinkled, 1.0 kg per square meter; then the soil shoveled in step 3 is evenly backfilled, and then the humus shoveled in step 3 is evenly spread on it; After inoculation, soil exchangeable calcium Ca was tested 2+ Content and soil pH value, soil exchangeable calcium Ca 2+ The content should reach 30-80cmol / kg, and the soil pH should be 7.5-8.5; Step 6: Post-vaccination management: After vaccination, animals and strangers are prohibited from entering the vaccination area. Check the formation of truffle mycorrhiza 6 months after inoculation; if mycorrhiza has not formed, mark the trunk so that it can be inoculated again next year; After vaccination, ants and earwigs are collectively trapped and killed in the forest in July and August every year; Step 7: Truffle harvesting: The harvesting time is from January to February of the third year after inoculation, and the truffles are harvested when their maturity is greater than or equal to 95%.

[0037] In step 1, the environment requires that the landform be karst.

[0038] In step 1, more than 60% of the trees in the inoculated forest are 15-35 years old, with a tree diameter of 20-40 cm and a tree spacing of 3-5 meters in length and width.

[0039] In step 2, inoculation is done in June and July. If there is no rain for 20 consecutive days after inoculation, watering should be done by spraying. The amount of watering is 100 grams per 1m 2 5kg, water once every three days.

[0040] In step three, shovel the humus on the surface of the forest land and pile it into a pile of 5 meters x 5 meters; then shovel the 3-4 cm thick soil under the humus and pile it into a pile of 3 meters x 3 meters.

[0041] In step 3, every 667m 2 The amount of quicklime spread on the forest land is 300kg.

[0042] In step 4, the fruiting bodies of Lijiang white truffles are obtained from the end of December to February of the following year, the soil and debris on the surface of the truffle fruiting bodies are cleaned, sealed and packaged, and frozen in a -20°C refrigerator for use; the fruiting bodies are taken out when preparing the spore stock solution; When preparing the spore stock solution, the speed of the mixer is 15000 rpm / min and the crushing time is 30 s.

[0043] In step 5, the thickness of the humus is 5.0 cm.

[0044] In step 6, the secondary inoculation method is to carry out the secondary inoculation in March-April or May-June of the second year after the inoculation; with the large tree without mycorrhizal formation as the center and the area with a radius of 1.2-1.5m as the inoculation area, first shovel away the backfilled humus, then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder, and then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder at 0.90kg / m 2 Spray the spore solution in the amount of spores; then backfill the shoveled soil and the soil layer sprinkled with slaked lime and stone powder again, and then backfill the shoveled humus; after inoculation, the soil should be refilled every 1m 2 The amount of water is 5kg, water once every three days until the rain comes.

[0045] In step seven, when harvesting, the depth of raking should not exceed 10cm.

[0046] Example 4 A method for inoculating a large tree of Lijiang white truffle comprises the following steps: Step 1, Environmental and inoculated tree requirements: Environmental requirements are: sloping or flat forests with an altitude range of 1500-2800m; annual average temperature of 13-20℃, annual rainfall of 600-1800mm, annual sunshine hours of 1600-3000h; distinct dry and rainy seasons, with a daily temperature difference of 8-22℃; soil types of red soil, brown soil or brown soil, and forest soil thickness >40cm; Requirements for inoculated forest trees: adult Yunnan pine forests, or mixed forests dominated by adult Yunnan pine mixed with one or more of Armand pine, Quercus, Quercus, Castanea mollissima, Platycarya styracifolia, Hazelnut, Populus yunnanensis and Cyclobalanopsis yunnanensis, with a forest canopy density of 6.5-7.5; Step 2: Choose the vaccination time: Vaccination is done in March-April or June-July every year; Before inoculation in March and April, water should be sprinkled to ensure that the surface soil is moist at a depth of 3-5 cm. After inoculation, water should be sprinkled every 1 m 2 The watering amount is 4kg, watering once every three days until the rain comes; In June and July, inoculation should be carried out just after the rain falls to the ground or after the rain has penetrated 40cm of the soil layer; Step 3: Preparation before vaccination: Remove the short shrubs and weeds in the forest, then shovel the humus on the surface of the forest; then shovel the 2-4cm thick soil under the humus; then loosen the 10-20cm thick soil underground; then sprinkle quicklime for disinfection and sterilization; inoculate the next day after disinfection and sterilization; Step 4: Preparation of strains: Sterile water adjusted to pH 7.8 using slaked lime was used as solvent; Select Lijiang white truffle fruiting bodies with a diameter of more than 1 cm, no insect infestation, no mildew and a maturity of greater than or equal to 95%, add 600 mL of solvent to every 100 g of fruiting bodies, then put them into a blender and crush them to make spore stock solution; store at 0°C-4°C; Before inoculation, the spore stock solution is diluted with the solvent to a spore solution with a volume concentration of 0.8%, and then slaked lime is added to adjust the pH to 7.2 for standby use; Step 5: Large tree inoculation: On the forest land treated in step 3, the spore solution prepared in step 4 is used to spray the surface of the forest land, and 550 kg of spore solution is sprayed per mu of land; after spraying, a layer of stone powder is first sprinkled, 4 kg of stone powder is sprinkled per square meter; then a layer of slaked lime is sprinkled, 0.8 kg per square meter; then the soil shoveled in step 3 is evenly backfilled, and then the humus shoveled in step 3 is evenly spread on it; After inoculation, soil exchangeable calcium Ca was tested 2+Content and soil pH value, soil exchangeable calcium Ca 2+ The content should reach 30-80cmol / kg, and the soil pH should be 7.5-8.5; Step 6: Post-vaccination management: After vaccination, animals and strangers are prohibited from entering the vaccination area. Check the formation of truffle mycorrhiza 6 months after inoculation; if mycorrhiza has not formed, mark the trunk so that it can be inoculated again next year; After vaccination, ants and earwigs are collectively trapped and killed in the forest in July and August every year; Step 7: Truffle harvesting: The harvesting time is from January to February of the third year after inoculation, and the truffles are harvested when their maturity is greater than or equal to 95%.

[0047] In step 1, the environment requires that the landform be karst.

[0048] In step 1, more than 60% of the trees in the inoculated forest are 10-35 years old, with a tree diameter of 10-40 cm and a tree spacing of 2-5 meters in length and width.

[0049] In step 2, inoculation is done in June and July. If there is no rain for 20 consecutive days after inoculation, watering should be done by spraying. The amount of watering is 100 grams per 1m 2 4kg, water once every three days.

[0050] In step three, shovel the humus on the surface of the forest land and pile it into a pile of 5 meters x 5 meters; then shovel the 2-4 cm thick soil under the humus and pile it into a pile of 3 meters x 3 meters.

[0051] In step 3, every 667m 2 The amount of quicklime spread on the forest land is 250kg.

[0052] In step 4, the fruiting bodies of Lijiang white truffles are obtained from the end of December to February of the following year, the soil and debris on the surface of the truffle fruiting bodies are cleaned, sealed and packaged, and frozen in a -20°C refrigerator for use; the fruiting bodies are taken out when preparing the spore stock solution; When preparing the spore stock solution, the speed of the mixer is 10000 rpm / min and the crushing time is 20 s.

[0053] In step 5, the thickness of the humus is 3.0-5.0 cm.

[0054] In step 6, the secondary inoculation method is to carry out the secondary inoculation in March-April or May-June of the second year after the inoculation; with the large tree without mycorrhizal formation as the center and the area with a radius of 1.1-1.4m as the inoculation area, first shovel away the backfilled humus, then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder, and then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder at 0.88kg / m 2Spray the spore solution in the amount of spores; then backfill the shoveled soil and the soil layer sprinkled with slaked lime and stone powder again, and then backfill the shoveled humus; after inoculation, the soil should be refilled every 1m 2 The watering amount is 4kg, water once every three days until the rain comes.

[0055] In step seven, when harvesting, the depth of raking should not exceed 10cm.

[0056] Application Examples 1. Environmental and inoculated tree requirements Environmental requirements: Slope or flat forests with an altitude range of 1500-2800m. Annual average temperature 13-20℃, annual rainfall 600-1800mm, annual sunshine hours 1600-3000h. Distinct dry and rainy seasons, with a daily temperature difference of 8-22℃. Karst landform areas are the best, with red soil, brown soil or brown soil mainly composed of sandstone, basalt, limestone and parent rock. The thickness of the forest soil layer is >40cm, with loose structure and good permeability.

[0057] Requirements for inoculating forest trees: To standardize the mature Yunnan pine ( Pinus yunnanensis ) forests, or mainly Yunnan pine, mixed with Armand pine ( Pinus armandii )、Quercus Quercus aliena )、Quercus australis( Quercus franchetii ),Chestnut( Castanea mollissima )、Plaxatidis Platycarya strobilacea ),hazelnut( Corylus heterophylla )、Yunnan Populus( Populus yunnanensis )、Cyclobalanopsis yunnanensis( Quercus glaucoides ) Mixed forests of one or more of the above tree species. In the inoculated forest, more than 60% of the trees are 10-35 years old, with a tree diameter of 10-40 cm, and the length and width of the tree spacing are 2-5 meters. Preferably, the forest canopy density is 6.5-7.5 (see Figure 1 ).

[0058] 2. Vaccination time selection There are two best time periods for vaccination in Yunnan.

[0059] Period 1: Inoculation takes place in March and April each year. At this time, the grass and trees recover, branches sprout and fibrous roots grow, and the mycorrhizal formation rate of the inoculated is relatively high. However, in Yunnan, March and April are severe drought periods. To ensure that the inoculated spores form mycorrhizae with the fibrous roots of large trees, water must be sprinkled before inoculation to ensure that the surface soil is moist at a depth of 3-5 cm. After inoculation, water must be sprinkled every 1 m 2 The amount of water used is 3-5kg, watered once every three days until the rain comes.

[0060] Period 2: Vaccination is carried out from June to July every year. In Yunnan, vaccination is carried out just after the rain falls to the ground or after the rain penetrates 40cm of the soil layer. When the rain comes, everything is full of vitality, and the vaccination success rate is also higher at this time.

[0061] 3. Preparation before vaccination Use tools to remove small shrubs and weeds in the forest, shovel the humus on the surface of the forest, and pile it up every 5 meters by 5 meters; then shovel the 2-4cm thick soil under the humus and pile it up every 3 meters by 3 meters; then use loosening tools to fully loosen the 10-20cm thick soil underground at this time, exposing the epidermal roots of Yunnan pine and other large trees, and sprinkle quicklime for disinfection. 2 The amount of quicklime applied is 200-300 kg, which kills a large number of bacteria and insect eggs, and increases the exchangeable calcium Ca in the soil. 2+ The content of the soil should be inoculated in time the next day after the soil treatment. During all preparatory operations, do not damage or break the large trees in the forest.

[0062] 4. Preparation of strains 1. Selection of strains: Fruiting bodies of Lijiang white truffles with a diameter of more than 1 cm, no insect damage, no mold and rot, and a maturity of greater than or equal to 95%. The maturity test method is to observe under a microscope and test the number of ascospores of mature truffles / the number of ascospores of the tested truffles × 100%. Figure 2 .

[0063] 2. Fungus processing: Collect or purchase Lijiang white truffle fruiting bodies from the end of December to February of the following year, clean the dirt and debris on the surface of the truffle fruiting bodies, seal them in polyethylene plastic bags, and freeze them in a -20℃ refrigerator for later use.

[0064] 3. Preparation of spore solution: Take out the fruiting body when using, use slaked lime to adjust the pH value of sterile water to between 7.5-8.0 as solvent, add 600mL of solvent for every 100g of fruiting body, then put it into a blender, and crush it at 8000rpm / min-15000rpm / min for 15s-30s to make spore solution. Put it into a sterilized bottle, seal it and store it at 0℃-4℃. Before inoculation, use the spore solution to add the above-prepared solvent to dilute it to a spore solution with a volume concentration of 0.5%-1.0% (0.5-1mL of spore solution per 100mL of spore solution), add slaked lime to adjust the pH to 7.0-7.5 for standby use.

[0065] 5. Large tree inoculation On the treated inoculated forest land, use a spray pot to hold the spore solution and then spray the bacteria on all the cleared forest land surfaces, directly spray on the exposed roots or soil on the surface of the big trees, spray until the soil is slightly wet, and spray 500-600kg of spore solution per mu of land. After spraying, first sprinkle a layer of stone powder (the powder is crushed from stones, and the present invention does not limit the mesh size of the powder, as long as it is in powder form), 3-5kg per square meter, and then sprinkle a layer of slaked lime, 0.5-1.0kg per square meter, evenly backfill the previously shoveled soil, and then evenly spread the shoveled humus on top, with a thickness of 3.0-5.0cm; After inoculation, the soil exchangeable calcium Ca should be tested 2+ Content and soil pH value, soil exchangeable calcium Ca 2+ The content should reach 30-80cmol / kg, and the soil pH should be 7.5-8.5, and the highest can reach 9.5. If the inspection does not meet the standard, if the exchangeable calcium Ca 2+ If the content does not meet the standard, stone powder should be sprinkled on the soil surface. If the pH value does not meet the standard, slaked lime should be sprinkled on the soil surface until it meets the requirement.

[0066] 6. Post-vaccination management 1. Build simple fencing facilities to enclose the vaccination area, prohibit livestock and other personnel from entering the vaccination area, and prohibit grazing and grabbing humus soil.

[0067] 2. If the inoculation time is from March to April and it does not rain after inoculation, water should be sprayed 3 days after inoculation. The amount of water should be 1m 2 3-5kg, spray water once every three days until rain comes. For forest land without watering conditions, try to choose June-July for inoculation. If there is no rain for 20 consecutive days after inoculation, spray water to ensure successful inoculation. The amount of water is 100 grams per 1m 2 3-5kg, water once every three days.

[0068] 3. Check the formation of truffle mycorrhiza 6 months after inoculation. Dig up the topsoil 10-30cm away from the trunk of the tree to find the fibrous roots of the tree. First, visually observe the roots of the tree. You can see a bunch of small grape-like protrusions on the fibrous roots (see Figure 3 When mycorrhizae are present, use a handheld magnifying glass with a magnification of 20 times or more to observe the mycorrhizal morphology (see Figure 4 ), and then take the mycorrhizal slices for microscopic examination. The microstructure should show a large number of truffle hyphae and have the typical characteristics of truffle mycorrhiza, such as mosaic or square structures (see Figure 5 If mycorrhizae have not formed, the trunks need to be marked and re-inoculated next year; 4. The secondary inoculation method is carried out in March-April or May-June of the second year after inoculation. The inoculation area is 1.0-1.5m in radius with the tree without mycorrhizal formation as the center. First, shovel away the surface humus, then shovel away the surface soil and the soil layer sprinkled with slaked lime and stone powder, and then shovel at 0.85-0.90kg / m 2 Spray the spore solution in the amount of spore solution. Then fill the soil layer with slaked lime and stone powder again, and then fill the humus layer with shoveled soil. 2 The amount of water used is 3-5kg, watered once every three days until the rain comes.

[0069] 5. After inoculation, collective trapping and killing of ants and earwigs in the forest land should be carried out from July to August every year; for example, dipterex can be mixed into the fried bran (use after simmering for 12 hours) to make a poison bait to kill earwigs, and dipterex can be dropped into honey or sugar water to make a poison bait to kill ants, so as to prevent pests from eating the young fruiting bodies of truffles and causing losses.

[0070] 7. Truffle Harvest Harvest time: Harvest in January or February of the third year after inoculation, when the truffles are 95% or more mature and have a strong aroma. A trained truffle dog can be used to assist in harvesting. When using a toothed rake for harvesting, the depth of the rake should not exceed 10 cm to avoid damaging a large number of mycorrhizae.

[0071] On March 12, 2023, the inoculation of Lijiang white truffle trees was carried out at the truffle conservation base at an altitude of 2,300m in Yongsheng County, Lijiang City, Yunnan Province. The inoculation standard is 7 acres of Yunnan pine forest, with 55-65 Yunnan pine trees per acre of forest. On September 15, the mycorrhizal formation was checked, and it was found that 50%-52% of the trees had formed truffle mycorrhiza. On September 16, 2023, 15 large trees with formed mycorrhiza in the forest were investigated, and young truffle fruiting bodies were found in the soil at the roots of 5 large trees. Molecular testing was performed on the fruiting bodies. In January 2024, mature truffle fruiting bodies were found under 10%-15% of the large trees. They were not harvested and kept in the original place.

[0072] On March 16, 2024, the remaining large trees that had not formed mycorrhizae were inoculated for the second time. On August 17, 2024, it was found that the inoculated forest had formed a large area of ​​burnt circles (see Figure 6 ), the burn circle is the area of ​​the forest where the fruiting bodies of truffles grow and develop, which is formed by the intersection of truffle hyphae, mycorrhiza, soil, organic matter, minerals, etc. The mushroom pond can inhibit the growth of surface herbs and even kill them, eventually forming an area similar to a burnt area, called a "burn circle". Figure 6 In the middle, the upper left part of the picture is the uninoculated area with lush herbaceous plants on the surface, and the lower right part is the inoculated area, forming a whole burn circle.

[0073] The mycorrhizal formation of all the trees was re-examined and it was found that 90%-95% of the trees had formed mycorrhizae. A survey of 15 large trees with mycorrhizal formation in the forest was conducted and young truffle fruiting bodies were found in the soil at the roots of 13 large trees (see Figure 7 ), and molecular detection of the fruiting bodies.

[0074] Detection method: Cut an appropriate amount of young truffle fruiting bodies, grind them with liquid nitrogen (or grinder), and use a plant genomic DNA extraction kit (Shanghai Shenggong Biotechnology Co., Ltd.) to extract total DNA according to the steps in the kit manual. Use RNA polymerase chain reaction (PCR) technology to select ITS gene fragments for amplification: Fungal universal primers ITS4: 5'-tcctccgcttattgatatgc-3' (SEQ ID NO. 1), ITS5: 5'-ggaagtaaaagtcgtaacaagg-3' (SEQ ID NO. 2), PCR amplification of ITS fragments; 25 μL PCR reaction system includes: 2×PCRmix 12.5 μL, 10 μmol·L -1 1 μL each of forward and reverse primers, 1 μL DNA template, ddHO 2 O 9.5 μL.

[0075] The PCR reaction conditions were as follows: 94°C pre-denaturation for 5 min, followed by 94°C denaturation for 30 s, 57°C annealing for 30 s, and 72°C extension for 1 min, 35 cycles; finally, 72°C extension for 7 min. The amplified product was stored at 4°C.

[0076] Use an electronic balance to accurately weigh 1g of agarose and dissolve it in 100mL of 0.5×TBE buffer (Tris-borate-EDTA). Heat it in a microwave oven for 2.0 min on medium heat to make it clear and transparent. When the temperature reaches 65°C, add 1μL of nucleic acid dye and shake it thoroughly. Pour it into a silica gel plate, cool it for later use, put it into an electrophoresis pool and use a pipette to spot the sample. The spotting volume is 5μL. The voltage during electrophoresis is 160V, the current is 90A, and the time is 15min. After electrophoresis in 1.0×TBE buffer, observe, take pictures and record the results under a UV gel imager, detect the concentration of the extracted DNA, and send the positive products to Shanghai Bioengineering for sequencing.

[0077] After sequencing the amplified fragments, the sequences were compared by Blast in GeneBank, and it was determined that the truffle fruiting body detected was Lijiang white truffle.

[0078] The detected ITS sequence is: (SEQ ID NO.3) The present invention implements the tree inoculation technology for Indian truffles, Chinese truffles and Lijiang white truffles at the same time (using the method of the present invention, the only difference is the type of truffles), and the inoculation success rate of Indian truffles is 21%, that of Chinese truffles is 15%, and that of Lijiang white truffles is greater than 80%. It is found that only Lijiang white truffles perform excellently and have the highest inoculation success rate, and the tree inoculation technology of Lijiang white truffles is an original technology and has not been reported so far.

[0079] Comparative test 1 This experiment is a comparative experiment of inoculation time and watering requirements; the only difference is the inoculation time, watering frequency and watering amount, and the rest is the same as the method of the present invention.

[0080] Each test set up 20 Yunnan pine trees for inoculation. The test site was Yongsheng County, Lijiang City, Yunnan Province. The experimental results are shown in Table 2. As can be seen from Table 2, the inoculation effect is better in spring, and watering should be done in small amounts and frequently. Large amounts of watering are not conducive to the formation of mycorrhizae. However, it is best to choose rainy season for inoculation in forests without watering conditions, and rainy season inoculation forests do not have strict requirements for watering.

[0081] Table 2

[0082] Comparative test 2 This experiment is a comparative experiment of soil treatment. The only difference is the amount of quicklime, stone powder and slaked lime used, and the rest is the same as the method of the present invention.

[0083] Each test set up 20 Yunnan pine trees for inoculation. The test site was Yongsheng County, Lijiang City, Yunnan Province. The inoculation time was March 15, 2023, and the watering measure was watering once every 3 days, 1m 2 The watering amount is 3-5kg. The results are shown in Table 3. The test results show that the experimental group E has the best effect.

[0084] Table 3

[0085] Comparative test 3 This test is a comparative test of the amount of inoculated spore solution. The only difference is the amount of inoculated spore solution, and the rest is the same as the method of the present invention.

[0086] Each test set up 20 Yunnan pines for inoculation. The test site was Yongsheng County, Lijiang City, Yunnan Province. The results are shown in Table 4. The test results show that the best effect is achieved when 500-600 kg of spore solution is inoculated per mu of land. Increasing the amount of spore solution not only increases the cost, but also does not achieve better results.

[0087] Table 4

[0088] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for inoculating large trees of Lijiang white truffles, characterized in that: The steps include: Step 1, Environmental and inoculated tree requirements: Environmental requirements are: sloping or flat forests with an altitude range of 1500-2800m; annual average temperature of 13-20℃, annual rainfall of 600-1800mm, annual sunshine hours of 1600-3000h; distinct dry and rainy seasons, with a daily temperature difference of 8-22℃; soil types of red soil, brown soil or brown soil, and forest soil thickness >40cm; Requirements for inoculated forest trees: adult Yunnan pine forests, or mixed forests dominated by adult Yunnan pine mixed with one or more of Armand pine, Quercus, Quercus, Castanea mollissima, Platycarya styracifolia, Hazelnut, Populus yunnanensis and Cyclobalanopsis yunnanensis, with a forest canopy density of 6.5-7.5; Step 2: Choose the vaccination time: Vaccination is done in March-April or June-July every year; Before inoculation in March and April, water should be sprinkled to ensure that the surface soil is moist at a depth of 3-5 cm. After inoculation, water should be sprinkled every 1 m 2 The amount of water is 3-5kg, water once every three days until the rain comes; In June and July, inoculation should be carried out just after the rain falls to the ground or after the rain has penetrated 40cm of the soil layer; Step 3: Preparation before vaccination: Remove the short shrubs and weeds in the forest, then shovel the humus on the surface of the forest; then shovel the 2-4cm thick soil under the humus; then loosen the 10-20cm thick soil underground; then sprinkle quicklime for disinfection and sterilization; inoculate the next day after disinfection and sterilization; Step 4: Preparation of strains: Sterile water adjusted to a pH between 7.5 and 8.0 using slaked lime as a solvent; Select Lijiang white truffle fruiting bodies with a diameter of more than 1 cm, no insect infestation, no mildew and a maturity of greater than or equal to 95%, add 595-605 mL of solvent for every 99-101 g of fruiting bodies, then put them into a blender and crush them to make a spore stock solution; store at 0°C-4°C; Before inoculation, the spore stock solution is diluted with the solvent to obtain a spore solution with a volume concentration of 0.5%-1.0%, and then slaked lime is added to adjust the pH to 7.0-7.5 for standby use; Step 5: Large tree inoculation: On the forest land treated in step 3, the spore solution prepared in step 4 is used to spray the surface of the forest land, and 500-600 kg of spore solution is sprayed per mu of land; after spraying, a layer of stone powder is first sprinkled, 3-5 kg ​​of stone powder is sprinkled per square meter; then a layer of slaked lime is sprinkled, 0.5-1.0 kg per square meter; then the soil shoveled in step 3 is evenly backfilled, and then the humus shoveled in step 3 is evenly spread on it; After inoculation, soil exchangeable calcium Ca was tested 2+ Content and soil pH value, soil exchangeable calcium Ca 2+ The content should reach 30-80cmol / kg, and the soil pH should be 7.5-8.5; Step 6: Post-vaccination management: After vaccination, animals and strangers are prohibited from entering the vaccination area. Check the formation of truffle mycorrhiza 6 months after inoculation; if mycorrhiza has not formed, mark the trunk so that it can be inoculated again next year; After vaccination, ants and earwigs are collectively trapped and killed in the forest in July and August every year; Step 7: Truffle harvesting: The harvesting time is from January to February of the third year after inoculation, and the truffles are harvested when their maturity is greater than or equal to 95%.

2. The method for inoculating large trees of Lijiang white truffle according to claim 1, characterized in that: In step 1, the environment requires that the landform be karst.

3. The method for inoculating large trees of Lijiang white truffle according to claim 1, characterized in that, In step 1, more than 60% of the trees in the inoculated forest are 10-35 years old, with a tree diameter of 10-40 cm and a tree spacing of 2-5 meters in length and width.

4. The method for inoculating large trees of Lijiang white truffle according to claim 1, characterized in that: In step 2, inoculation is done in June and July. If there is no rain for 20 consecutive days after inoculation, watering should be done by spraying. The amount of watering is 100 grams per 1m 2 3-5kg, water once every three days.

5. The method for inoculating large trees of Lijiang white truffle according to claim 1, characterized in that, In step three, shovel the humus on the surface of the forest land and pile it into a pile of 5 meters x 5 meters; then shovel the 2-4 cm thick soil under the humus and pile it into a pile of 3 meters x 3 meters.

6. The method for inoculating large trees of Lijiang white truffle according to claim 1, characterized in that: In step 3, every 667m 2 The amount of quicklime spread on the forest land is 200-300kg.

7. The method for inoculating large trees of Lijiang white truffle according to claim 1, characterized in that: In step 4, the fruiting bodies of Lijiang white truffles are obtained from the end of December to February of the following year, the soil and debris on the surface of the truffle fruiting bodies are cleaned, sealed and packaged, and frozen in a -20°C refrigerator for use; the fruiting bodies are taken out when preparing the spore stock solution; When preparing the spore stock solution, the speed of the mixer is 8000rpm / min-15000rpm / min, and the crushing time is 15s-30s.

8. The method for inoculating large trees of Lijiang white truffle according to claim 1, characterized in that: In step 5, the thickness of the humus is 3.0-5.0 cm.

9. The method for inoculating large trees of Lijiang white truffle according to claim 1, characterized in that: In step 6, the secondary inoculation method is to carry out the secondary inoculation in March-April or May-June of the second year after the inoculation; with the large tree without mycorrhizal formation as the center and the area with a radius of 1.0-1.5m as the inoculation area, first shovel away the backfilled humus, then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder, and then shovel away the backfilled soil and the soil layer sprinkled with slaked lime and stone powder at 0.85-0.90kg / m 2 Spray the spore solution in the amount of spores; then backfill the shoveled soil and the soil layer sprinkled with slaked lime and stone powder again, and then backfill the shoveled humus; after inoculation, the soil should be refilled every 1m 2 The amount of water used is 3-5kg, watered once every three days until the rain comes.

10. The method for inoculating large trees of Lijiang white truffle according to claim 1, characterized in that: In step seven, when harvesting, the depth of raking should not exceed 10cm.

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