A large-scale inoculation and cultivation method for Indian truffle mycorrhizal seedlings

Abstract

The invention discloses a large-scale inoculation and cultivation method of Indian truffle mycorrhizal seedlings, and belongs to the technical field of infection and cultivation of Indian truffle mycorrhizal seedlings. The method is as follows: (1) cultivating Armand pine or chestnut seedlings to obtain sterile seedlings; preparing Indian truffle fruiting bodies into inoculation agents; (2) mixing peat, vermiculite and perlite in equal proportions by volume percentage to obtain a matrix; filling the matrix at a position of 1 / 3 to 1 / 2 of the height of a nursery pot; (3) using an inoculation clamp to wrap the roots of the cultivated Armand pine or chestnut sterile seedlings in the prepared inoculation agent; (4) gently placing the Armand pine or chestnut seedlings wrapped with the inoculation agent in a nursery pot, filling the surrounding area with the prepared matrix, and lightly pressing; (5) placing the inoculated Armand pine or chestnut seedlings in a controllable greenhouse, and starting to spray water at a fixed time. The method solves the problem of inconsistency between the host seedling stage and the fruiting body maturity stage, the low infection success rate and the poor quality of mycorrhizal seedlings, and at the same time simplifies the operation steps and reduces the mycorrhizal seedling cultivation time.

Description

Technical Field

[0001] This invention relates to the field of plant and microbial technology, and in particular to a method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings. Background Technology

[0002] Indian truffle (Tuber indicum Cooke & Masse), commonly known as "truffle" or "pig truffle," is a valuable underground edible (and medicinal) fungus. It is renowned worldwide for its unique flavor and taste and has significant economic, edible, medicinal, and ecological value. It is widely distributed in southwestern my country and is a commercially successful truffle variety.

[0003] Indian truffles are typical ectomycorrhizal edible fungi. Their cultivation requires first cultivating mycorrhizal seedlings and then using biomimetic cultivation for 3-5 years to obtain fruiting bodies. Traditional Indian truffle mycorrhizal seedling cultivation mainly uses oak and pine species, particularly Yunnan pine, Huashan pine, cork oak, chestnut, and pecan. In commercial cultivation, Indian truffle mycorrhizal seedling cultivation faces the problem of inconsistent seedling and fruiting body maturity periods. To solve this problem, production often involves freezing Indian truffle fruiting bodies until aseptic seedlings are prepared, then thawing and inoculating. However, this practice greatly reduces truffle spore viability, resulting in low truffle infection success rates and low mycorrhizal quality. Furthermore, the inoculation agents for truffles often involve dipping roots in spore suspensions, fruiting body blocks, or substrate mixtures to inoculate young roots of Pinus armandii or Chestnut. To control the cost of the inoculum, the amount of inoculum inoculated per Pinus armandii or Chestnut seedling is very limited. As the seedling roots extend rapidly, the newly grown roots can no longer effectively contact the spores, resulting in a low infection rate and a small number of mycorrhizae formed in Pinus armandii or Chestnut seedlings. To improve the quality of mycorrhizae, it is necessary to extend the co-culture time of the mycorrhizae and seedlings, which greatly increases the cost of cultivating Indian truffle mycorrhizal seedlings and has become an important limiting factor for the rapid large-scale cultivation of Indian truffle mycorrhizal seedlings.

[0004] Therefore, based on the traditional synthesis of Indian truffle mycorrhizal seedlings, finding a rapid and large-scale method for cultivating mycorrhizal seedlings is crucial for the commercial promotion of Indian truffle mycorrhizal seedlings. This is also of great significance for adjusting the tree species structure and improving the quality of the local understory economy in southwestern regions such as Guizhou. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings to solve the problems of inconsistent host seedling stage and fruiting body maturity stage, low infection success rate and poor quality of mycorrhizal seedlings, while simplifying the operation steps and reducing the cultivation time of mycorrhizal seedlings.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions:

[0007] A method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings, wherein the inoculation and cultivation method is as follows:

[0008] (1) Cultivate Huashan pine or chestnut seedlings from September to November each year to obtain sterile seedlings; prepare inoculum from Indian truffle fruiting bodies harvested from December to February of the following year;

[0009] (2) Mix peat, vermiculite and perlite in a volume ratio of 4:3:3, then add tap water that has been left to stand for more than 24 hours, mix well, and obtain the substrate; fill the substrate to 1 / 3 to 1 / 2 of the height of the seedling pot;

[0010] (3) Wrap the roots of the cultivated aseptic seedlings of Pinus armandii or chestnut in the prepared inoculum using an inoculation clip;

[0011] (4) Gently place the Huashan pine or chestnut seedlings wrapped with inoculant into the seedling pot in step (2), fill the surrounding area with the prepared substrate, and press lightly.

[0012] (5) After placing the inoculated Pinus armandii or chestnut seedlings in a controlled greenhouse at 15-24℃ for 48 hours, turn on the timed water sprayer and spray tap water 3 times a week for 15 minutes each time.

[0013] (6) After the seedlings were cultured for 3 months after inoculation, the roots of the mycorrhizal seedlings of Pinus armandii or Prunus chestnut were placed under a stereomicroscope to observe whether mycorrhizae were produced. After cleaning, the presence of the target fungus was identified, and mycorrhizal seedlings infected with Tuberculia praecox were obtained.

[0014] This invention cultivates Huashan pine or chestnut seedlings from September to November each year, and uses Indian truffle fruiting bodies harvested from December to February of the following year to make inoculum. This provides seedlings with optimal conditions for infection and solves the problem of low infection rate caused by the inconsistency between the host seedling stage and the fruiting body maturity stage.

[0015] Furthermore, the inoculation clamp in step (3) is a hollow double-ball series inoculation clamp. The inoculation clamp includes a gripping end and a hollow double-ball clamping part. The gripping end includes a first gripping end and a second gripping end that are rotatably connected. The hollow double-ball clamping part includes a clamp and a gourd-shaped cavity connected to the clamp. The forming cavity includes an integrally formed upper chamber and a lower chamber. The first gripping end and the second gripping end are respectively connected to the clamp. When the first gripping end and the second gripping end are gripped tightly, the forming cavity can be clamped.

[0016] The molding cavity has a sharpened edge, and the upper chamber of the molding cavity has a reserved groove. After the inoculant is filled into the upper and lower cavities of the molding cavity, the stem base of the Pinus armandii or chestnut seedling is placed in the upper chamber of the molding cavity, and the stem is placed in the reserved groove. After the gripping end is rotated to clamp the molding cavity, the edge of the molding cavity cuts off the excess roots of the seedling, and at the same time, Pinus armandii or chestnut seedlings with inoculant are obtained.

[0017] This invention utilizes a special hollow double-sphere tandem inoculation clip, gourd-shaped with a height of 10-13cm. It has an upper chamber (approximately 4cm in diameter) and a lower chamber (5-6cm in diameter). The base of the seedling stem is placed into the clip filled with inoculum, and after clamping, the rootstock of the *Pinus armandii* or *Chestnut* seedling is surrounded by the inoculum. Under this size clip, the root system of the seedling can be in contact with the inoculum for as long as possible during its downward and lateral growth. Simultaneously, the sharp edges around the forming cavity cut off excess rootstock, promoting lateral root growth and maximizing root infection of the truffle. Compared to traditional pinching inoculation methods, this prevents the taproot from rapidly growing downwards and extending beyond the inoculum's infection range. Furthermore, this clip design minimizes the amount of inoculum used, saving costs compared to directly planting seedlings in inoculum-filled seedling pots. Furthermore, in step (2), the peat has a particle size of 1-20 mm, the vermiculite has a particle size of 2-3 mm, and the perlite has a particle size of 3-6 mm.

[0018] Furthermore, the preparation method of the inoculum in step (3) is as follows:

[0019] Fresh, mature Indian truffle fruiting bodies are harvested from December to February of the following year. After surface disinfection with 1% potassium permanganate for 2-5 minutes, the fruiting bodies are cleaned with sterile water, placed on a sterilized stainless steel table and cut into 5-10mm cubes. Distilled water is added to make a pulp, then sterile vermiculite is added, mixed well, and stored at 4℃ for later use. The storage time should not exceed 8 hours before use.

[0020] Furthermore, the mass-to-volume ratio of the Indian truffle fruiting body to distilled water and sterile vermiculite is (5-10) g: 2 L: 1 L.

[0021] Furthermore, the fresh and mature Indian truffle fruiting bodies are refrigerated at 4°C for no more than 120 hours before use, and the Indian truffle fruiting bodies are pulped alternately at a speed of 2000-5000 r / min for 3-5 minutes.

[0022] When pulping, the pulping time should not exceed 2 minutes at a speed greater than 3500 r / min to prevent the spore suspension from being heated during pulping, which would affect spore viability.

[0023] Furthermore, the method for cultivating aseptic seedlings of Pinus armandii or chestnut is as follows:

[0024] After disinfecting the surface of fresh Huashan pine or chestnut seeds harvested that year, sow them in seedling trays filled with a mixture of vermiculite and perlite. Sowing time is from September to November of that year. Place them in a sterilized, controlled greenhouse to cultivate until the Huashan pine seedlings are 5-10cm tall and the chestnut seedlings have 4-6 leaves. When inoculating the chestnut seedlings, be sure to retain the germinated seeds.

[0025] Furthermore, the chestnut seeds harvested that year need to be refrigerated at 0-1℃.

[0026] Furthermore, during the cultivation of sterile Huashan pine or chestnut seedlings, the minimum temperature of the controllable greenhouse is not lower than 3℃, the maximum temperature is not higher than 32℃, and the humidity is 50-85%RH.

[0027] Beneficial effects:

[0028] This invention adjusts the cultivation method of Pinus armandii and chestnut seedlings under controlled artificial conditions. After both the seedlings and fruiting bodies reach their optimal state, inoculation is carried out, which can form mycorrhizal seedlings with high infection rate and high mycorrhizal quality. At the same time, it effectively overcomes the drawbacks of traditional inoculation with solid mycelium blocks and spore suspensions of Truffle India, reduces the contamination rate, and improves the quality of Truffle. In addition, the use of hollow double-sphere tandem inoculation clips provides a new technical point for the large-scale cultivation of mycorrhizal seedlings of Pinus armandii and chestnut in the future. Attached Figure Description

[0029] Figure 1 : Schematic diagram of cavity double ball series inoculation clip; wherein, clip 1, forming cavity 2, upper chamber 21, reserved groove 211, lower chamber 22, gripping end 3, first gripping end 31, second gripping end 32;

[0030] Figure 2 Schematic diagrams of chestnut inoculation and Chinese pine inoculation;

[0031] Figure 3 Chestnut mycorrhizae infected with Indian truffles, and Pinus armandii mycorrhizae infected with Indian truffles. Detailed Implementation

[0032] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings:

[0033] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0034] like Figure 1As shown, the inoculation clip is a hollow double-ball series inoculation clip. The inoculation clip includes a gripping end 3 and a hollow double-ball clamping part. The gripping end 3 includes a first gripping end 31 and a second gripping end 32 that are rotatably connected. The hollow double-ball clamping part includes a clamp head 1 and a gourd-shaped cavity 2 connected to the clamp head 1. The forming cavity 2 includes an integrally formed upper chamber 21 and a lower chamber 22. The first gripping end 31 and the second gripping end 32 are respectively connected to the clamp head 1. When the first gripping end 31 and the second gripping end 32 are gripped tightly, the forming cavity 2 can be clamped.

[0035] The edges of the molding cavity 2 are sharpened, and the upper chamber 21 of the molding cavity 2 is provided with a reserved groove 211. After the inoculation agent is filled into the upper and lower cavities of the molding cavity 2, the root base of the Pinus armandii or chestnut seedling is placed in the upper chamber 21 of the molding cavity 2, and the stem is placed in the reserved groove 211. After rotating the gripping end 3 to clamp the molding cavity 2, the edge of the molding cavity 2 cuts off the excess roots of the seedling, and at the same time, Pinus armandii or chestnut seedlings with inoculation agent are obtained.

[0036] The inoculation clip used in this invention is a special hollow double-sphere series inoculation clip, which is gourd-shaped with a height of 10-13cm, preferably 13cm. It has an upper chamber 21 and a lower chamber 22, where the upper chamber 21 has a diameter of approximately 3-4cm, preferably 4cm, and the lower chamber 22 has a diameter of 5-6cm, preferably 5cm. The seedling rhizomes are placed into the inoculation clip filled with inoculum, and after clamping, the rhizomes of the Pinus armandii or Chestnut seedlings are covered with inoculum. Under this size inoculation clip, the root system of the seedling can be in contact with the inoculum as much as possible and for as long as possible during its downward and outward growth. Simultaneously, the edges of the forming chamber 2 cut off excess rhizomes, promoting lateral root growth and allowing more roots to be infected with the truffle, preventing the taproot from growing rapidly downwards and extending beyond the inoculum's infection range. Furthermore, this design of the inoculation clip can minimize the amount of inoculum used, saving costs compared to the traditional method of directly planting seedlings in seedling pots filled with inoculum.

[0037] Example 1: Inoculation and cultivation of mycorrhizal seedlings inoculated with Indian truffles

[0038] (1) Cultivation of sterile seedlings of Pinus armandii and chestnut

[0039] Freshly harvested Huashan pine and chestnut seeds were refrigerated at 0℃ until sowing. They were then disinfected by soaking in a 1% potassium permanganate solution for 5 minutes. After disinfection, the chestnut seeds were placed in 50-well deep PS seedling trays, each measuring 4.7cm x 1.8cm x 9.0cm. The chestnut seeds used were small chestnuts for breeding; one chestnut and one Huashan pine seed were sown per hole.

[0040] The breeding substrate is a mixture of vermiculite and perlite (1:1, v / v). Sowing time is September of the same year. The plants are cultivated in a controlled greenhouse. The ambient temperature and humidity are controlled by measures such as water curtains, exhaust fans, spraying, and heating lamps. The minimum ambient temperature in the controlled greenhouse is not lower than 3℃ and the maximum temperature is not higher than 32℃. The humidity is in the range of 50% to 85% RH.

[0041] After four months of cultivation, most seedlings basically met the inoculation requirements. To ensure uniform aseptic seedling specifications, the Chinese pine seedlings were required to be 5cm tall, and the chestnut seedlings were required to have 4 leaves (to retain germinating seeds to continuously provide nutrition and improve seedling survival rate). To avoid competitive bacterial contamination in the greenhouse, the greenhouse was sterilized for 48 hours with an ozone generator before and after the seedling cultivation.

[0042] (2) Preparation of inoculum for Indian Truffle

[0043] The fruiting bodies of Indian truffle mature from December to February of the following year. To avoid high temperatures during the initial stage of co-culturing the mycelium and reduce temperature control costs, this embodiment purchases mature Indian truffle fruiting bodies in mid-December, with a size of 3-4 cm. 2-3 mm vermiculite is sterilized at 125°C for 2 hours and then cooled to room temperature for later use. 2 L of distilled water is also prepared.

[0044] After surface sterilization of 5g of fresh, mature Indian truffle fruiting bodies with 1% potassium permanganate for 2 minutes, rinse them with sterile water, place them on a sterilized stainless steel table, cut them into 5-10mm cubes, put them in a high-speed blender, add part of 2L of distilled water, and blend at 2000-5000 rpm for 3 minutes, with the blending time above 3500 rpm not exceeding 2 minutes. After blending, add 1L of sterile vermiculite to a stainless steel basin, pour in the Indian truffle spore suspension while stirring, add the remaining distilled water, stir well, and then transfer to a 4℃ cold storage for later use. The storage time from blending to use should not exceed 8 hours.

[0045] (3) Inoculation and cultivation of Indian truffle mycorrhizal seedlings

[0046] Mix peat moss (1-20mm), vermiculite (2-3mm), and perlite (3-6mm) in a volume ratio of 4:3:3 (v / v / v). Add an equal volume of tap water (let it stand for at least 24 hours) and mix thoroughly to obtain the substrate. Fill a square seedling pot (10cm top × 7cm bottom × 13cm height) to approximately one-third full. Using an inoculation clip (a gourd-shaped, hollow, double-ball, tandem inoculation clip with an upper chamber of 4cm, a lower chamber of 5cm, and a total height of 13cm), wrap the roots of *Pinus armandii* and *Pinus koraiensis* in the truffle inoculum.

[0047] Gently place the Huashan pine and chestnut seedlings coated with inoculant into square seedling trays filled with seedling substrate, and fill the surrounding area with substrate and press lightly. After inoculation, place the Huashan pine and chestnut seedlings in a controlled greenhouse at 15-24℃ for 48 hours, then turn on the timed watering and spray with tap water 3 times a week for 15 minutes each time.

[0048] After six months of co-culturing with mycorrhizal fungi, the roots of *Pinus armandii* and *Pinus thunbergii* seedlings were observed under a stereomicroscope to check for mycorrhizal formation. After washing, the mycorrhizal fungi were identified using ITS molecular identification technology. The primers used were universal ITS primers: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. After PCR amplification, sequence analysis was performed, and double-peaked and special structural sequences were removed. Qualified sequences were compared with BLAST in the NCBI database. If the similarity was above 99%, it was determined to be *Truffle indicum*, indicating successful infection.

[0049] Example 2:

[0050] (1) Cultivation of sterile seedlings of Pinus armandii and chestnut

[0051] Freshly harvested Huashan pine and chestnut seeds were refrigerated at 0℃ until sowing. They were then disinfected by soaking in a 1% potassium permanganate solution for 3 minutes. After disinfection, the chestnut seeds were placed in 50-well deep PS seedling trays, each measuring 4.7cm x 1.8cm x 9.0cm. The chestnut seeds used were small chestnuts for breeding; one chestnut and one Huashan pine seed were sown per hole.

[0052] The breeding substrate is a mixture of vermiculite and perlite (1:1, v / v). Sowing time is October of the same year. The plants are cultivated in a controlled greenhouse. The ambient temperature and humidity are controlled by water curtains, exhaust fans, sprayers, and heating lamps. The minimum ambient temperature in the controlled greenhouse is no lower than 3℃ and the maximum temperature is no higher than 32℃. The humidity is in the range of 50% to 85% RH.

[0053] After four months of cultivation, most seedlings basically met the inoculation requirements. To ensure uniform aseptic seedling specifications, the Chinese pine seedlings were required to be 7cm tall, and the chestnut seedlings were required to have 4 leaves (to retain germinating seeds for continuous nutrition). To avoid competitive microbial contamination in the greenhouse, the greenhouse was sterilized for 48 hours using an ozone generator after each batch of seedlings was completed.

[0054] (2) Preparation of inoculum for Indian Truffle

[0055] Based on the ripening period of Indian truffle fruiting bodies (December to February of the following year), and to avoid high temperatures during the initial stage of co-culturing of seedlings and reduce temperature control costs, this embodiment purchases Indian truffle fruiting bodies that mature in mid-January, with a size of 3-4 cm. 2-3 mm vermiculite is sterilized at 125°C for 2 hours and then cooled to room temperature for later use. 2 L of distilled water is also prepared.

[0056] After surface sterilization of 7g of fresh, mature Indian truffle fruiting bodies with 1% potassium permanganate for 5 minutes, rinse them with sterile water, place them on a sterilized stainless steel table, cut them into 5-10mm cubes, and put them in a high-speed blender. Add 2L of distilled water and blend at 2000-5000 rpm for 5 minutes, with the blending time at speeds above 3500 rpm not exceeding 2 minutes. After blending, add 1L of sterile vermiculite to a stainless steel bowl, and pour in the Indian truffle spore suspension while stirring. Add the remaining distilled water, stir well, and then transfer to a 4℃ cold storage for later use. The storage time from blending to use should not exceed 8 hours.

[0057] (3) Inoculation and cultivation of Indian truffle mycorrhizal seedlings

[0058] Mix peat moss (1-20mm), vermiculite (2-3mm), and perlite (3-6mm) in a volume ratio of 4:3:3 (v / v / v). Add tap water that has been left to stand for at least 24 hours at a volume ratio of 1:1.5 (total substrate volume: water), and mix well to obtain the substrate. Fill approximately 1 / 3 of a square seedling pot (10cm top × 7cm bottom × 13cm height) with the substrate. Using an inoculation clip (a gourd-shaped, hollow, double-ball, tandem inoculation clip with an upper chamber of 4cm, a lower chamber of 5cm, and a total height of 13cm), wrap the roots of the Chinese pine and chestnut trees in the truffle inoculation agent.

[0059] Gently place the Huashan pine and chestnut seedlings coated with inoculant into square seedling trays filled with seedling substrate, and fill the surrounding area with mixed substrate and press lightly. After inoculation, place the Huashan pine and chestnut seedlings in a controlled greenhouse at 15-24℃ for 48 hours, then turn on the timed watering and spray with tap water 3 times a week for 15 minutes each time.

[0060] After 6 months of co-culturing with mycorrhizal fungi, the roots of *Pinus armandii* and *Chestnut sinensis* seedlings were observed under a stereomicroscope to check for mycorrhizal formation. After washing, ITS molecular identification technology was used, employing universal ITS primers: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. Following PCR amplification, sequence analysis was performed, removing double peaks and special structural sequences. Qualified sequences were then compared against the NCBI database using BLAST. A similarity of 99% or higher was considered indicative of *Truffle indicum*, signifying successful infection.

[0061] Example 3:

[0062] (1) Cultivation of sterile seedlings of Pinus armandii and chestnut

[0063] Freshly harvested Huashan pine and chestnut seeds were refrigerated at 0℃ until sowing. They were then disinfected by soaking in a 1% potassium permanganate solution for 5 minutes. After disinfection, the chestnut seeds were placed in 50-well deep PS seedling trays, each measuring 4.7cm x 1.8cm x 9.0cm. The chestnut seeds used were small chestnuts for breeding; one chestnut / Huashan pine seed was sown per hole.

[0064] The breeding substrate is a mixture of vermiculite and perlite (1:1, v / v). Sowing time is November of the same year. The plants are cultivated in a controlled greenhouse. The ambient temperature and humidity are controlled by water curtains, exhaust fans, sprayers, and heating lamps. The minimum ambient temperature in the controlled greenhouse is not lower than 3℃ and the maximum temperature does not exceed 32℃. The humidity is in the range of 50% to 85% RH.

[0065] After four months of cultivation, most seedlings basically met the inoculation requirements. To ensure uniform aseptic seedling specifications, the Chinese pine seedlings were required to be 10cm tall, and the chestnut seedlings were required to have 6 leaves (to retain germinating seeds for continuous nutrition). To avoid competitive microbial contamination in the greenhouse, the greenhouse was sterilized for 48 hours using an ozone generator after each batch of seedlings was completed.

[0066] (2) Preparation of inoculum for Indian Truffle

[0067] Based on the ripening period of Indian truffle fruiting bodies (December to February of the following year), and to avoid high temperatures during the initial stage of co-culturing of mycelial seedlings and reduce temperature control costs, this embodiment purchases Indian truffle fruiting bodies that mature in mid-February, with a size of 3-4 cm. 2-3 mm vermiculite is sterilized at 125°C for 2 hours and then cooled to room temperature for later use. 2 L of distilled water is also prepared.

[0068] After surface sterilization of 10g of fresh, mature Indian truffle fruiting bodies with 1% potassium permanganate for 5 minutes, rinse them with sterile water, place them on a sterilized stainless steel table, cut them into 5-10mm cubes, put them in a high-speed blender, add part of 2L of distilled water, and blend at 2000-5000 rpm for 4 minutes, with the blending time at speeds above 3500 rpm not exceeding 2 minutes. After blending, add 1L of sterile vermiculite to a stainless steel basin, pour in the Indian truffle spore suspension while stirring, add the remaining distilled water, stir well, and then transfer to a 4℃ cold storage for later use. The storage time from blending to use should not exceed 8 hours.

[0069] (3) Inoculation and cultivation of Indian truffle mycorrhizal seedlings

[0070] Mix peat moss (1-20mm), vermiculite (2-3mm), and perlite (3-6mm) in a volume ratio of 4:3:3 (v / v / v). Add tap water that has been left to stand for at least 24 hours at a 1:1 volume ratio (total substrate volume: water), and mix well to obtain the substrate. Fill the substrate into square seedling pots (10cm top × 7cm bottom × 13cm height) to about 1 / 3 full. Use an inoculation clip (a gourd-shaped, hollow, double-ball, tandem inoculation clip with an upper chamber of 4cm, a lower chamber of 5cm, and a total height of 13cm) to wrap the roots of Chinese pine and chestnut trees in the truffle inoculation agent.

[0071] Gently place the Huashan pine and chestnut seedlings coated with inoculant into square seedling trays filled with seedling substrate, and fill the surrounding area with the substrate and press it down lightly. After inoculation, place the Huashan pine and chestnut seedlings in a controlled greenhouse at 15-24℃ for 48 hours, then turn on the timed watering and spray them with tap water 3 times a week for 15 minutes each time.

[0072] After six months of co-culturing with mycorrhizal fungi, the roots of *Pinus armandii* and *Pinus thunbergii* seedlings were observed under a stereomicroscope to check for mycorrhizal formation. After washing, the mycorrhizal fungi were identified using ITS molecular identification technology. The primers used were universal ITS primers: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. After PCR amplification, sequence analysis was performed, and double-peaked and special structural sequences were removed. Qualified sequences were compared with BLAST in the NCBI database. If the similarity was above 99%, it was determined to be *Truffle indicum*, indicating successful infection.

[0073] Comparative Example 1:

[0074] The cultivation method in this comparative example is similar to that in Example 1, except that the Huashan pine and chestnut seeds harvested that year were not immediately placed in a 0℃ freezer, but were directly stored in a cool, shaded place before being used for seedling cultivation. Comparative Example 2:

[0075] The cultivation method of this comparative example is similar to that of Example 1, except that the purchased Indian truffle fruiting bodies were collected in August.

[0076] Comparative Example 3:

[0077] The cultivation method of this comparative example is similar to that of Example 1. The difference is that the co-cultivation environment of the Huashan pine and chestnut seedlings after inoculation with the inoculant varies with the greenhouse temperature. In fact, it was recorded that the ambient temperature reached above 30°C for two days within one week after inoculation.

[0078] Comparative Example 4:

[0079] The cultivation method of this comparative example is similar to that of Example 1, except that the inoculum preparation of Indian truffle uses 5g of fresh mature Indian truffle fruiting bodies and 2L of distilled water to prepare a spore suspension, without adding sterile vermiculite. During inoculation, the seedlings of Pinus armandii and chestnut are soaked in the spore suspension for 30 minutes.

[0080] Comparative Example 5:

[0081] The cultivation method of this comparative example is similar to that of Example 1, except that the fresh and mature fruiting bodies were not surface-sterilized and the added vermiculite was not subjected to high-temperature sterilization when preparing the Indian Truffle inoculum.

[0082] Comparative Example 6:

[0083] The cultivation method of this comparative example is similar to that of Example 1, except that when wrapping the roots of the Chinese pine and chestnut with truffle inoculation agent, no inoculation clip was used. Instead, the inoculation agent was simply pinched together with the roots of the seedlings by hand, forming a ball about 5 cm in size.

[0084] Comparative Example 7:

[0085] The cultivation method of this comparative example is similar to that of Example 1, except that the Huashan pine and chestnut seedlings were not left for 48 hours after inoculation before being thoroughly watered to help them establish roots.

[0086] experiment:

[0087] Mycorrhizae were synthesized according to the methods of Examples 1-3 and Comparative Examples 1-7, with 100 seedlings in each group. After 6 months of cultivation, the infection status of *Truffleus indicus* was checked by sampling using morphological and molecular identification techniques. The survival rate of chestnut and *Pinus armandii* seedlings, mycorrhizae synthesis rate (successful synthesis was defined as infection), and the quality index of each mycorrhizae were statistically analyzed after 6 months of cultivation, and the average quality index was calculated. The infection rate and quality index of *Truffleus indicus* were statistically evaluated using stereomicroscopy (0-4 were evaluated as the percentage of mycorrhizae to total roots, with index references of 1 < 25%, 25 ≤ 2 < 50%, 50 ≤ 3 < 75%, and 4 ≥ 75%). The specific results are shown in Table 1 below.

[0088] Table 1

[0089]

[0090]

[0091] Analysis of Table 1 shows that:

[0092] (1) Compared with Example 1, the chestnut seeds in Comparative Example 1 showed severe inactivation. Comparative Example 1 found that fresh chestnut seeds should be kept moist and refrigerated promptly after harvesting; otherwise, they are easily inactivated due to high environmental temperatures and their own physiological heat generation, resulting in a sharp drop in the germination rate of chestnut seeds to only 13%, which has no practical significance for subsequent mycorrhizal synthesis. Although the survival rate of Pinus armandii seedlings was 98%, a mortality rate of 2% is still relatively high for actual large-scale production and is still an adverse effect on the large-scale propagation of truffles.

[0093] (2) Compared with Example 1, the fruiting bodies of Comparative Example 2 were dirty white inside and the spores were not mature enough. This confirms that the Indian truffle, as a fungal strain, should be collected after the fruiting bodies are mature to ensure that the internal spores are fully mature. Immature Indian truffles will significantly reduce the mycorrhizal synthesis rate and mycorrhizal quality index of Pinus armandii and chestnut.

[0094] (3) Compared with Example 1, Comparative Example 3 shows that after inoculation with Indian Truffle, the ambient temperature should be controlled below 24°C as much as possible. When the ambient temperature is too high, it will cause the newly inoculated Pinus armandii and chestnut seedlings to die and the Indian Truffle spores to lose vitality, which will affect the mycorrhizal synthesis rate and mycorrhizal quality.

[0095] (4) Compared with Example 1, Comparative Example 4 found that after inoculation with Indian truffle using spore suspension, the mycorrhizal synthesis rate of Pinus armandii and Pterocarya stenoptera was higher, but the young roots extended faster. The new roots not covered by spore suspension had difficulty forming mycorrhizae, resulting in a lower overall mycorrhizal quality index.

[0096] (5) Compared with Example 1, Comparative Example 5 shows that when preparing the inoculum for Indian truffle spores, the surface of the fruiting body was not disinfected, and the added vermiculite was also not disinfected. Whether the inoculum was disinfected or not had little effect on the synthesis rate of mycorrhizal seedlings of Pinus armandii and Prunus chestnut. The mycorrhizal quality index was even slightly higher. Molecular identification was then performed on the spores of the mycorrhizal seedlings. The identification results showed that 30-40% of the mycorrhizae were not the target species. Therefore, it is speculated that the complex microecological environment formed by the microorganisms on the surface of the fruiting body, the microorganisms in the vermiculite and the Indian truffle spores may have caused potential contamination by miscellaneous bacteria.

[0097] (6) Compared with Example 1, Comparative Example 6 shows that the combination of the inoculation clip and the palm has little effect on the mycorrhizal synthesis rate of Pinus armandii and Pleurotus ostreatus seedlings, but it has a significant impact on the quality of mycorrhizae. This is because the roots of seedlings grow rapidly downwards and outwards when squeezed by hand, quickly extending beyond the substrate containing the inoculum, thus causing a decrease in the mycorrhizal quality index. At the same time, the inoculation clip also cuts off excess roots, promoting the growth of lateral roots and allowing more roots to come into contact with more inoculum. The small-scale test results show that when raising seedlings on a large scale, using the inoculation clip can make the production of Indian truffle mycorrhizal seedlings more standardized and efficient, and can also avoid inconsistent inoculation between seedlings, contamination, and damage to young roots.

[0098] (7) Compared with Example 1, Comparative Example 7 showed that after the newly inoculated Pinus armandii and chestnut seedlings were left to stand for 48 hours, the Indian truffle spores could attach to the young roots better. Although watering the roots earlier could improve the survival rate of the seedlings, excessive water would also take away some of the spores around the roots of the seedlings, resulting in a certain decrease in mycorrhizal synthesis rate and mycorrhizal quality.

[0099] In summary, the method and inoculation clips disclosed in this invention, when used to inoculate Huashan pine and chestnut seedlings with Indian truffles for 6 months, all resulted in the formation of excellent mycorrhizal seedlings with high mycorrhizal quality. This effectively overcomes the drawbacks of traditional Indian truffle solid mycelium blocks and spore suspension inoculation, and innovates in the details of mycelium preparation, seedling cultivation, and seedling inoculation techniques. It provides new technical points for the large-scale cultivation of Indian truffle Huashan pine / chestnut mycorrhizal seedlings in the future.

[0100] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A large-scale inoculation and cultivation method for Indian truffle mycorrhizal seedlings, characterized in that: Described inoculation cultivation method is as follows: (1) Cultivating Armand pine or chestnut seedlings from September to November each year to obtain sterile seedlings; preparing inoculants from Indian truffle fruiting bodies harvested from December each year to February of the following year; (2) Mix peat, vermiculite and perlite in a volume percentage of 4:3:3, then add tap water that has been left to stand for more than 24 hours and mix well to obtain a matrix; fill the matrix to 1 / 3 to 1 / 2 of the height of the seedling pot; (3) Using an inoculation clamp, wrap the roots of the cultivated sterile seedlings of Armand pine or chestnut in the prepared inoculant; (4) Gently place the Armand pine or chestnut seedlings wrapped with the inoculated microbial agent in the seedling pot in step (2), fill the surrounding area with the prepared substrate, and gently press; (5) Place the inoculated Armand pine or chestnut seedlings in a greenhouse set at 15-24°C for 48 hours, then start the scheduled watering. Spray tap water regularly three times a week for 15 minutes each time. (6) After the inoculated seedlings have been co-cultivated for 3 months, the roots of the Armand pine or chestnut mycorrhizal seedlings are placed under a stereoscope to observe whether mycorrhizae are produced. After washing, the presence of the target fungus species is identified to obtain mycorrhizal seedlings infected with Indian truffles; The preparation method of the inoculated microbial agent in step (1) is as follows: fresh and mature Indian truffle fruiting bodies are picked from December to February of the following year, the fresh and mature Indian truffle fruiting bodies are surface disinfected with 1% potassium permanganate for 2-5 minutes, and then cleaned with sterile water, and distilled water is added for pulping, and sterile vermiculite is added, mixed, and placed at 4°C for standby use, and the storage time when used shall not exceed 8 hours; The mass volume ratio of the Indian truffle fruiting body to distilled water and sterile vermiculite is (5-10) g: 2 L: 1 L; The inoculation clamp in step (3) is a cavity double-ball series inoculation clamp, the inoculation clamp comprises a gripping end and a cavity double-ball gripping portion, the gripping end comprises a first gripping end and a second gripping end that are rotatably connected, the cavity double-ball gripping portion comprises a chuck and a gourd-shaped cavity connected to the chuck, the molding cavity comprises an upper chamber and a lower chamber that are integrally formed, the first gripping end and the second gripping end are respectively connected to the chuck, and when the first gripping end and the second gripping end are gripped, the molding cavity can be driven to be clamped; The edge of the molding cavity is sharpened, and the upper chamber of the molding cavity is provided with a reserved groove. After the upper and lower cavities of the molding cavity are filled with the inoculated fungus, the roots of the Armand pine or chestnut seedlings are placed in the upper chamber of the molding cavity, and the stems are placed in the reserved groove. After the holding end is rotated to clamp the molding cavity, the edge of the molding cavity cuts off the excess roots of the seedlings, and at the same time, the Armand pine or chestnut seedlings with the inoculated fungus are obtained after inoculation.

2. A method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings according to claim 1, characterized in that: In step (2), the particle size of peat is 1-20 mm, the particle size of vermiculite is 2-3 mm, and the particle size of perlite is 3-6 mm.

3. A method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings according to claim 2, characterized in that: The fresh and mature Indian truffle fruiting bodies are refrigerated at 4° C. for no more than 120 hours before use. The Indian truffle fruiting bodies are pulped by alternately pulping at a speed of 2000-5000 r / min for 3-5 minutes. The pulping time at a speed greater than 3500 r / min does not exceed 2 minutes.

4. A method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings according to claim 3, characterized in that: The method for cultivating sterile seedlings of Pinus sylvestris or Castanea mollissima in step (1) is as follows: After surface disinfection of fresh Armand pine or chestnut seeds harvested that year, sow them in plug trays filled with a vermiculite / perlite mixed matrix from September to November of that year. Place them in a sterilized greenhouse and cultivate them until the Armand pine seedlings are 5-10 cm tall. When the chestnut seedlings have 4-6 leaves, take them out for inoculation. When taking chestnut seedlings, keep the germinated seeds.

5. A large-scale inoculation and cultivation method for Indian truffle mycorrhizal seedlings according to claim 4, characterized in that, The chestnut seeds harvested that year need to be refrigerated at 0-1°C.

6. A large-scale inoculation and cultivation method for Indian truffle mycorrhizal seedlings according to claim 5, characterized in that: During the cultivation of the aseptic seedlings of Armand pine or chestnut, the minimum ambient temperature of the greenhouse is not less than 3° C., the maximum temperature is not higher than 32° C., and the humidity is 50-85% RH.

Images