Large-scale inoculation cultivation method for tuber indicum mycorrhizal seedlings

Abstract

The invention discloses a large-scale inoculation cultivation method for tuber indicum mycorrhizal seedlings, and belongs to the technical field of tuber indicum mycorrhizal seedling infection cultivation, the method comprises the following steps: (1) cultivating pinus armandii or Chinese chestnut seedlings to obtain aseptic seedlings; preparing the fruiting body of the tuber indicum into an inoculant; (2) uniformly mixing peat, vermiculite and perlite according to the equal proportion in percentage by volume to obtain a substrate; filling the 1 / 3-1 / 2 height position of the seedling pot with the matrix; (3) wrapping the roots of the cultivated physochlaina armandii or Chinese chestnut aseptic seedlings in the prepared inoculant by using an inoculation clamp; (4) lightly placing the pinus armandii or Chinese chestnut seedlings wrapped with the inoculant in a seedling pot, filling the periphery with the prepared matrix, and lightly pressing; and (5) placing the inoculated seedlings of the pinus armandii or the Chinese chestnuts in a controllable greenhouse, and starting to spray water at regular time. The problems that the host seedling stage is inconsistent with the sporocarp mature stage, the infection success rate is low, and the mycorrhizal seedling quality is poor are solved, meanwhile, the operation steps are simplified, and the mycorrhizal seedling cultivation time is shortened.

Description

Technical Field

[0001] The invention relates to the technical field of plants and microorganisms, and in particular to a large-scale inoculation and cultivation method of Indian truffle mycorrhizal seedlings. Background Art

[0002] Indian truffle (Tuber indicum Cooke&Massee), commonly known as "truffle" and "pig arch mushroom", is a precious underground edible (medicinal) fungus. It is famous all over the world for its unique flavor and taste. It has important economic, edible, medicinal and ecological value. It is widely distributed in southwest my country and is a relatively successful truffle variety in commercial cultivation.

[0003] Indian truffles are typical ectomycorrhizal edible fungi. Their cultivation requires the cultivation of mycorrhizal seedlings first and then the fruiting bodies through bionic cultivation for 3-5 years. Traditional Indian truffle mycorrhizal seedling cultivation mainly focuses on oak and pine plants, especially Yunnan pine, Armand pine, cork oak, chestnut and walnut. In commercial cultivation, the cultivation of Indian truffle mycorrhizal seedlings faces the problem of inconsistency between the seedling stage and the fruiting body maturity stage. To solve this problem, Indian truffle fruiting bodies are often frozen in production and thawed and inoculated after the sterile seedlings are prepared. However, this practice will greatly reduce the activity of truffle spores, resulting in a low success rate of truffle infection and mycorrhizal quality. In addition, truffle inoculation agents mostly use spore suspensions to dip roots, fruiting bodies or matrix mixtures to inoculate young roots of Armand pine or chestnut. In order to control the cost of fungus, the amount of fungus inoculated into single Armand pine or chestnut seedlings is very limited. As the roots of the seedlings extend rapidly, the newly grown roots can no longer effectively contact the spores, resulting in a low infection rate of Armand pine or chestnut seedlings and a small number of mycorrhizae formed. In order to improve the quality of mycorrhizae, the co-cultivation time of the seedlings is forced to be extended, which greatly increases the cost of cultivating Indian truffle mycorrhizal seedlings and has become an important limiting factor for the rapid and large-scale cultivation of Indian truffle mycorrhizal seedlings.

[0004] Therefore, based on the traditional synthesis of Indian truffle mycorrhizal seedlings, it is very important to find a method for rapid and large-scale cultivation of mycorrhizal seedlings in a short period of time for the commercial promotion of Indian truffle mycorrhizal seedlings. This is also of great significance for the adjustment of tree species structure in southwestern regions such as Guizhou and improving the quality of local forest economy. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a large-scale inoculation and cultivation method of Indian truffle mycorrhizal seedlings to solve the problems of inconsistency between the host seedling stage and the fruiting body maturity stage, low infection success rate and poor quality of mycorrhizal seedlings, while streamlining the operating steps and reducing the mycorrhizal seedling cultivation time.

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

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

[0008] (1) cultivating Armand pine or chestnut seedlings from September to November each year to obtain sterile seedlings; preparing an inoculant from Indian truffle fruiting bodies harvested from December each year to February of the following year;

[0009] (2) Peat, vermiculite, and perlite are mixed in a volume percentage of 4:3:3, and then tap water that has been allowed to stand for more than 24 hours is added and mixed to obtain a matrix; the matrix is ​​filled to a position of 1 / 3 to 1 / 2 of the height of the seedling pot;

[0010] (3) using an inoculation clamp to wrap the roots of the cultivated sterile seedlings of Armand pine or chestnut in the prepared inoculant;

[0011] (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 matrix, and gently press;

[0012] (5) Place the inoculated Armand pine or chestnut seedlings in a controllable greenhouse at 15-24°C for 48 hours, and then start spraying water regularly, three times a week, for 15 minutes each time;

[0013] (6) After the inoculated seedlings were co-cultivated for 3 months, the roots of the Armand pine or chestnut mycorrhizal seedlings were placed under a stereoscope to observe whether mycorrhizae were produced. After washing, the presence of the target fungus species was identified to obtain mycorrhizal seedlings infected with Indian truffles.

[0014] The invention cultivates Armand pine or chestnut seedlings from September to November each year, and uses Indian truffle fruiting bodies harvested from December each year to February of the following year to prepare inoculating agents, so as to provide seedlings with optimal conditions for infection, and solve the problem of low infection rate caused by inconsistency between the host seedling stage and the fruiting body maturity stage.

[0015] Further, the inoculation clamp in step (3) is a cavity double-ball series inoculation clamp, the inoculation clamp includes a gripping end and a cavity double-ball clamping portion, the gripping end includes a first gripping end and a second gripping end that are rotatably connected, the cavity double-ball clamping portion includes a chuck and a gourd connected to the chuck to form a molding cavity, the molding 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 chuck, and when the first gripping end and the second gripping end are gripped, the molding cavity can be driven to be clamped;

[0016] 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 stem base of the Armand pine or chestnut seedling is placed in the upper chamber of the molding cavity, and the stem is 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 seedling, and at the same time, the Armand pine or chestnut seedling with the inoculated fungus is obtained after inoculation.

[0017] The inoculation clamp used in the present invention selects a special cavity double ball series inoculation clamp, which is gourd-shaped, 10-13 cm in height, and has an upper chamber and a lower chamber, wherein the upper chamber has a diameter of about 4 cm and the lower chamber has a diameter of 5-6 cm, and the stem base of the seedling is placed in the inoculation clamp filled with inoculation agent, and after clamping, the root and stem parts of the Armand pine or chestnut seedlings are covered with inoculation agent all around. The root system of the root seedling formed under the inoculation clamp of this size can contact the inoculation agent as long as possible during the downward and surrounding growth period, and the blades around the molding cavity can cut off the redundant rhizomes of the seedlings, promote the growth of lateral roots, and allow more roots to infect truffles as much as possible. Compared with the traditional kneading inoculation method, it can prevent the main root from growing rapidly downward and extending out of the inoculation agent infection range. In addition, the inoculation clamp of this shape can save the amount of inoculation agent as much as possible, and can save costs compared with the traditional direct planting of seedlings in the seedling pot filled with inoculation agent. Furthermore, the particle size of the peat in step (2) is 1-20 mm, the particle size of the vermiculite is 2-3 mm, and the particle size of the perlite is 3-6 mm.

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

[0019] Fresh and mature Indian truffle fruiting bodies are picked from December to February of the following year. After the surface of the fresh and mature Indian truffle fruiting bodies is disinfected with 1% potassium permanganate for 2-5 minutes, they are cleaned with sterile water, and then placed on a sterilized stainless steel table and cut into 5-10 mm cubes. Distilled water is added for pulping, and then sterile vermiculite is added, mixed, and placed at 4°C for standby use. The storage time when in use shall not exceed 8 hours.

[0020] Furthermore, the mass 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 alternately beaten at a speed of 2000-5000 r / min for 3-5 minutes.

[0022] The beating time at a speed greater than 3500 r / min does not exceed 2 minutes, which can prevent the spore suspension from being heated during the beating process and thus affecting the spore activity.

[0023] Further, the method for cultivating sterile seedlings of Armand pine or chestnut is as follows:

[0024] 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, controlled-temperature 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.

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

[0026] Furthermore, during the cultivation of the aseptic seedlings of Armand pine or chestnut, the ambient temperature of the controllable greenhouse is not less than 3°C at the lowest temperature, not more than 32°C at the highest temperature, and the humidity is 50-85% RH.

[0027] Beneficial effects:

[0028] The present invention adjusts the cultivation method of Armand pine and chestnut seedlings under controllable artificial conditions, and inoculates after the seedlings and fruiting bodies reach the optimal state, so that mycorrhizal seedlings with high infection rate can be formed, and the mycorrhizal quality is also high. At the same time, the disadvantages of traditional Indian truffle solid mushroom blocks and spore suspension inoculation are effectively broken, the pollution rate is reduced, and the quality of the truffle is improved. At the same time, the cavity double-ball series inoculation clamp is adopted, which provides a new technical key point for the large-scale cultivation of Indian truffle Armand pine and chestnut mycorrhizal seedlings in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Schematic diagram of a cavity double-ball series inoculation clamp; wherein, the clamp 1, the molding cavity 2, the upper chamber 21, the reserved groove 211, the lower chamber 22, the holding end 3, the first holding end 31, and the second holding end 32;

[0030] Figure 2 : Schematic diagram of chestnut inoculation, schematic diagram of Armand pine inoculation;

[0031] Figure 3 : Chestnut mycorrhiza infected by Indian truffle, Armand pine mycorrhiza infected by Indian truffle. DETAILED DESCRIPTION

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

[0033] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0034] like Figure 1As shown, the inoculation clamp is a cavity double-ball series inoculation clamp, the inoculation clamp includes a gripping end 3 and a cavity double-ball clamping portion, the gripping end 3 includes a first gripping end 31 and a second gripping end 32 that are rotatably connected, the cavity double-ball clamping portion includes a chuck 1 and a gourd-shaped cavity 2 connected to the chuck 1, the molding cavity 2 includes an upper chamber 21 and a lower chamber 22 that are integrally formed, the first gripping end 31 and the second gripping end 32 are respectively connected to the chuck 1, and when the first gripping end 31 and the second gripping end 32 are gripped, the molding cavity 2 can be driven to be clamped;

[0035] The edge of the molding cavity 2 is sharpened, and the upper chamber 21 of the molding cavity 2 is provided with a reserved groove 211. After the upper and lower cavities of the molding cavity 2 are filled with the inoculating agent, the root base of the Armand pine 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 the holding end 3 is rotated to clamp the molding cavity 2, the edge of the molding cavity 2 cuts off the excess root system of the seedling, and at the same time, the Armand pine or chestnut seedling with the inoculating agent after inoculation is obtained.

[0036] The inoculation clamp used in the present invention is a special cavity double ball series inoculation clamp, which is gourd-shaped, with a height of 10-13 cm, preferably 13 cm, and has an upper chamber 21 and a lower chamber 22, wherein the diameter of the upper chamber 21 is about 3-4 cm, preferably 4 cm, and the diameter of the lower chamber 22 is 5-6 cm, preferably 5 cm. The rhizome of the seedling is placed in the inoculation clamp filled with the inoculation agent, and after clamping, the rhizome of the Chinese pine or chestnut seedling is covered with the inoculation agent all around. The root system of the root seedling formed under the inoculation clamp of this size can contact the inoculation agent as much as possible and for a long time during the downward and surrounding growth period. At the same time, the blades around the molding cavity 2 can cut off the redundant rhizome of the seedling, promote the growth of the lateral roots, and allow more roots to infect the truffle as much as possible, and prevent the main root from growing rapidly downward and extending out of the inoculation agent infection range. In addition, the inoculation clamp of this shape can save the amount of inoculation agent as much as possible, and can save costs compared with the traditional method of directly planting the seedlings in the seedling pot filled with the inoculation agent.

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

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

[0039] The fresh Armand pine and chestnut seeds harvested that year were placed in a cold storage at 0℃. When sowing, they were soaked and disinfected with 1% potassium permanganate for 5 minutes. After disinfection, the chestnuts were placed in a 50-hole PS material deep seedling tray, with each hole measuring 4.7cm above × 1.8cm below × 9.0cm high. The chestnut seeds were small chestnuts for breeding, and one chestnut and one Armand pine were sown in each hole.

[0040] The breeding matrix is ​​a vermiculite / perlite (1:1, v / v) mixed matrix. The sowing time is September of the same year. The culture is carried out in a controllable greenhouse. The environmental temperature and humidity are controlled by water curtains, exhaust fans, sprays, heating lamps and other measures. The minimum temperature of the controllable greenhouse is not lower than 3°C, the maximum temperature is not higher than 32°C, and the humidity is in the range of 50% to 85% RH.

[0041] After 4 months of cultivation, most of the seedlings basically met the inoculation requirements. To unify the specifications of sterile seedlings, the Armand pine seedlings were required to be 5 cm tall, and the chestnut seedlings were required to have 4 leaves (germinating seeds were retained to continuously provide nutrition and improve the survival rate of seedlings). To avoid contamination by competitive bacteria in the greenhouse, this batch of seedlings was sealed and sterilized with an ozone generator for 48 hours before and after the seedlings were raised.

[0042] (2) Preparation of Indian Truffle Inoculation Agent

[0043] The Indian truffle fruiting body maturity period (December each year to February of the following year) is to avoid the high weather temperature in the initial stage of co-cultivation of seedlings and reduce the temperature control cost. In this embodiment, mature Indian truffle fruiting bodies in mid-December are purchased, with a size of 3-4 cm. 2-3 mm vermiculite is sterilized at 125 ° C for 2 h and then cooled to room temperature for standby use, and 2 L of distilled water is prepared at the same time.

[0044] 5g of fresh and mature Indian truffle fruiting bodies were surface disinfected with 1% potassium permanganate for 2 minutes, then washed with sterile water, placed on a sterilized stainless steel table, cut into 5-10mm cubes, placed in a wall breaking machine, added with part of 2L of distilled water, and alternately pulped at a speed of 2000-5000r / min in the wall breaking machine for 3 minutes, wherein the pulping time at a speed greater than 3500r / min did not exceed 2min. After pulping, 1L of sterile vermiculite was added to a stainless steel basin, and the Indian truffle spore suspension was poured in while stirring, and the remaining distilled water was added. After stirring evenly, it was moved into a 4°C cold storage for standby use. The storage time from pulping to use should not exceed 8 hours.

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

[0046] Peat (1-20mm), vermiculite (2-3mm), and perlite (3-6mm) were mixed in a volume percentage of 4:3:3 (v / v / v), and tap water was added in a volume ratio equal to the total volume of the matrix and allowed to stand for more than 24 hours, and the matrix was obtained. The matrix was filled in about 1 / 3 of the square seedling pot (upper opening 10cm×lower opening 7cm×height 13cm). The roots of Armand pine and chestnut were wrapped in the truffle inoculant using an inoculation clip (a gourd-shaped hollow double-ball series inoculation clip with an upper chamber of 4cm, a lower chamber of 5cm, and a total height of 13cm).

[0047] The inoculated pine and chestnut seedlings were gently placed in a square seedling pot filled with seedling substrate, and the surrounding area was filled with substrate and lightly pressed. After the inoculated pine and chestnut seedlings were placed in a 15-24℃ controllable greenhouse for 48 hours, the timed watering was turned on, and tap water was sprayed regularly 3 times a week for 15 minutes each time.

[0048] After 6 months of co-cultivation of the seedlings, the roots of the mycorrhizal seedlings of Armand pine and chestnut were placed under a stereoscope to observe whether mycorrhizae were produced. After cleaning, ITS molecular identification technology was used. The primers used were ITS universal primers, ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. Sequence analysis was performed after PCR amplification to remove double peaks and special structure sequences. Qualified sequences were compared by BLAST in the NCBI database. If the comparison similarity was more than 99%, it was determined to be Indian truffle, that is, the infection was successful.

[0049] Embodiment 2:

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

[0051] The fresh Armand pine and chestnut seeds harvested that year were placed in a cold storage at 0℃. When sowing, they were soaked and disinfected with 1% potassium permanganate for 3 minutes. After disinfection, the chestnuts were placed in a 50-hole PS material deep seedling tray, with each hole measuring 4.7cm above × 1.8cm below × 9.0cm high. The chestnut seeds were small chestnuts for breeding, and one chestnut and one Armand pine were sown in each hole.

[0052] The breeding matrix is ​​a vermiculite / perlite (1:1, v / v) mixed matrix. The sowing time is October of the same year. The culture is carried out in a controllable greenhouse. The environmental temperature and humidity are controlled by water curtains, exhaust fans, sprinklers, heating lamps and other measures. The minimum temperature of the controllable greenhouse is not lower than 3°C, the maximum temperature is not higher than 32°C, and the humidity is in the range of 50% to 85% RH.

[0053] After 4 months of cultivation, most of the seedlings basically meet the inoculation requirements. To unify the specifications of sterile seedlings, Armand pine seedlings are required to be 7cm tall, and chestnut seedlings are required to have 4 leaves (germinating seeds are retained to provide continuous nutrition). To avoid contamination by competitive bacteria in the greenhouse, an ozone generator is used to sterilize the greenhouse for 48 hours after each batch of seedlings is completed.

[0054] (2) Preparation of Indian Truffle Inoculation Agent

[0055] According to the maturity period of Indian truffle fruiting bodies (December to February of the following year), in order to avoid high weather temperature in the initial stage of co-cultivation of seedlings and reduce temperature control costs, this embodiment purchases mature Indian truffle fruiting bodies in mid-January with a size of 3-4 cm. 2-3 mm vermiculite is sterilized at 125 ° C for 2 h and then cooled to room temperature for standby use, and 2 L of distilled water is prepared at the same time.

[0056] 7g of fresh mature Indian truffle fruiting bodies were surface disinfected with 1% potassium permanganate for 5 minutes, then washed with sterile water, placed on a sterilized stainless steel table, cut into 5-10mm cubes, placed in a wall breaking machine, added with part of 2L of distilled water, and alternately pulped at a speed of 2000-5000r / min in the wall breaking machine for 5 minutes, wherein the pulping time at a speed greater than 3500r / min did not exceed 2min. After the pulping was completed, 1L of sterile vermiculite was added to a stainless steel basin, and the Indian truffle spore suspension was poured in while stirring, and the remaining distilled water was added. After stirring evenly, it was moved into a 4°C cold storage for standby use. The storage time from pulping to use should not exceed 8 hours.

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

[0058] Peat (1-20mm), vermiculite (2-3mm), and perlite (3-6mm) were mixed in a volume percentage of 4:3:3 (v / v / v), and tap water that had been standing for more than 24 hours was added in a volume ratio of 1:1.5 (total volume of the matrix: water), and mixed to obtain the matrix. The matrix was filled into about 1 / 3 of the square seedling pot (upper opening 10cm×lower opening 7cm×height 13cm). The roots of Armand pine and chestnut were wrapped in the truffle inoculant using an inoculation clip (a gourd-shaped hollow double-ball series inoculation clip with an upper chamber of 4cm, a lower chamber of 5cm, and a total height of 13cm).

[0059] The inoculated pine and chestnut seedlings were gently placed in a square seedling pot filled with seedling medium, and the surrounding area was filled with mixed medium and lightly pressed. After the inoculated pine and chestnut seedlings were placed in a 15-24℃ controllable greenhouse for 48 hours, the timed watering was turned on, and tap water was sprayed regularly 3 times a week for 15 minutes each time.

[0060] After 6 months of co-culture, the roots of Armand pine and chestnut mycorrhizal seedlings were placed under a stereoscope to observe whether mycorrhizae were produced. After cleaning, ITS molecular identification technology was used. The primers used were ITS universal primers, ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. After PCR amplification, sequence analysis was performed to remove double peaks and special structure sequences. Qualified sequences were compared by BLAST in the NCBI database. If the comparison similarity was more than 99%, it was determined to be Indian truffle. That is, the infection was successful.

[0061] Embodiment 3:

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

[0063] The fresh Armand pine and chestnut seeds harvested that year were placed in a cold storage at 0℃. When sowing, they were soaked and disinfected with 1% potassium permanganate for 5 minutes. After disinfection, the chestnuts were placed in a 50-hole PS material deep seedling tray, with each hole measuring 4.7cm above × 1.8cm below × 9.0cm high. The chestnut seeds were small chestnuts for breeding, and one chestnut / Arand pine was sown in each hole.

[0064] The breeding matrix is ​​a vermiculite / perlite (1:1, v / v) mixed matrix. The sowing time is November of the same year. The culture is carried out in a controllable greenhouse. The environmental temperature and humidity are controlled by water curtains, exhaust fans, sprays, heating lamps and other measures. The minimum temperature of the controllable greenhouse is not lower than 3°C, the maximum temperature does not exceed 32°C, and the humidity is in the range of 50% to 85% RH.

[0065] After 4 months of cultivation, most of the seedlings basically meet the inoculation requirements. To unify the specifications of sterile seedlings, Armand pine seedlings are required to be 10 cm tall, and chestnut seedlings are required to have 6 leaves (germinating seeds are retained to provide continuous nutrition). To avoid contamination by competitive bacteria in the greenhouse, an ozone generator is used to sterilize the greenhouse for 48 hours after each batch of seedlings is completed.

[0066] (2) Preparation of Indian Truffle Inoculation Agent

[0067] According to the maturity period of Indian truffle fruiting bodies (December to February of the following year), in order to avoid high weather temperature in the initial stage of co-cultivation of seedlings and reduce temperature control costs, this embodiment purchases mature Indian truffle fruiting bodies in mid-February, with a size of 3-4 cm. 2-3 mm vermiculite is sterilized at 125 ° C for 2 h and then cooled to room temperature for standby use, and 2 L of distilled water is prepared at the same time.

[0068] 10g of fresh mature Indian truffle fruiting bodies were surface disinfected with 1% potassium permanganate for 5 minutes, then washed with sterile water, placed on a sterilized stainless steel table, cut into 5-10mm cubes, placed in a wall breaking machine, added with part of 2L distilled water, and alternately pulped at a speed of 2000-5000r / min in the wall breaking machine for 4 minutes, wherein the pulping time at a speed greater than 3500r / min did not exceed 2min. After pulping, 1L of sterile vermiculite was added to a stainless steel basin, and the Indian truffle spore suspension was poured in while stirring, and the remaining distilled water was added. After stirring evenly, it was moved into a 4°C cold storage for standby use. The storage time from pulping to use should not exceed 8 hours.

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

[0070] Peat (1-20mm), vermiculite (2-3mm), and perlite (3-6mm) were mixed in a volume percentage of 4:3:3 (v / v / v), and tap water that had been standing for more than 24 hours was added in a volume ratio of 1:1 (total volume of the matrix: water), and mixed to obtain the matrix. The matrix was filled into about 1 / 3 of the square seedling pot (upper opening 10cm×lower opening 7cm×height 13cm). The roots of Armand pine and chestnut were wrapped in the truffle inoculant using an inoculation clip (a gourd-shaped hollow double-ball series inoculation clip with an upper chamber of 4cm, a lower chamber of 5cm, and a total height of 13cm).

[0071] Place the inoculated Armand pine and chestnut seedlings gently in a square seedling pot filled with a seedling mixed matrix, and fill and lightly press the surrounding area with the mixed matrix. After the inoculated Armand pine and chestnut seedlings are placed in a 15-24℃ controllable greenhouse for 48 hours, start the timed watering, and spray tap water regularly 3 times a week for 15 minutes each time.

[0072] After 6 months of co-cultivation of the seedlings, the roots of the mycorrhizal seedlings of Armand pine and chestnut were placed under a stereoscope to observe whether mycorrhizae were produced. After cleaning, ITS molecular identification technology was used. The primers used were ITS universal primers, ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. Sequence analysis was performed after PCR amplification to remove double peaks and special structure sequences. Qualified sequences were compared by BLAST in the NCBI database. If the comparison similarity was more than 99%, it was determined to be Indian truffle, that is, the infection was successful.

[0073] Comparative Example 1:

[0074] The cultivation method of this comparative example is similar to that of Example 1, except that the Armand pine and chestnut seeds harvested that year were not promptly refrigerated at 0°C, but directly stored in a cool and shaded place and then used for seedling cultivation.

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

[0076] Comparative Example 3:

[0077] The cultivation method of this comparative example is similar to that of Example 1, except that the co-cultivation environment of the Armand pine and chestnut seedlings after inoculation with the bacterial agent changes with the greenhouse temperature. It was actually recorded that the ambient temperature reached above 30° C. for 2 days within 1 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 Indian truffle inoculation agent is prepared by using 5 g of fresh mature Indian truffle fruiting bodies and 2 L of distilled water to prepare a spore suspension, without adding sterile vermiculite, and the Armand pine and chestnut seedlings are immersed in the spore suspension for 30 min during inoculation.

[0080] Comparative Example 5:

[0081] The cultivation method of this comparative example is similar to that of Example 1, except that when preparing the inoculant of Indian truffle, the surface of the fresh and mature fruiting bodies is not disinfected, and the added vermiculite is not sterilized at high temperature.

[0082] Comparative Example 6:

[0083] The cultivation method of this comparative example is similar to that of Example 1, except that no inoculation clamp is used when wrapping the truffle inoculant with the roots of Armand pine and chestnut, and the inoculant is kneaded with the palm of the hand to form a sphere of 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 inoculated Armand pine and chestnut seedlings are immediately irrigated with rooting water without being left for 48 hours.

[0086] experiment:

[0087] Mycorrhizae were synthesized according to the methods of Examples 1-3 and Comparative Examples 1-7, with 100 plants in each group. After 6 months of co-cultivation, the infection of Indian truffles was sampled and checked using morphological and molecular identification techniques. The survival rate of chestnut and Armand pine seedlings after 6 months of cultivation, the mycorrhizal synthesis rate (infection means successful synthesis) and the quality index of each mycorrhizal root were statistically analyzed to calculate the average quality index. The mycorrhizal infection rate and mycorrhizal quality index of Indian truffles were statistically evaluated with the help of a stereoscope (0-4 as the percentage of mycorrhizae in the total roots, with an index reference of 1<25%, 25≤2<50%, 50≤3<75%, 4≥75%), and the specific results are shown in Table 1 below.

[0088] Table 1

[0089]

[0090]

[0091] From the analysis of Table 1, we can see that:

[0092] (1) Compared with Example 1, the inactivation of chestnut seeds in Comparative Example 1 is serious. Comparative Example 1 found that fresh chestnut seeds should be kept in water and refrigerated in time after harvesting, otherwise the seeds are easily inactivated due to high environmental temperature and physiological heat generation, resulting in a cliff-like 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 is 98%, the mortality rate of 2% is still at a relatively high level for actual large-scale production, and it is still an adverse effect on mass breeding of truffles.

[0093] (2) Compared with Example 1, the inside of the fruiting bodies picked in Comparative Example 2 was dirty white and the spores were not mature enough, which confirmed that the Indian truffles as a species should be collected after the fruiting bodies 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 Armand pine and chestnut.

[0094] (3) Compared with Example 1, Comparative Example 3 shows that after the inoculation of Indian truffles, the ambient temperature should be controlled below 24°C as much as possible. When the ambient temperature is too high, the newly inoculated Armand pine and chestnut seedlings will die and the activity of Indian truffle spores will decrease, thereby affecting the mycorrhizal synthesis rate and mycorrhizal quality.

[0095] (4) Compared with Example 1, Comparative Example 4 found that after inoculation with Indian truffles using spore suspension, the synthesis rate of mycorrhizal seedlings of Armand pine and chestnut was higher, but the young roots extended faster, and the new roots not wrapped in the spore suspension were difficult to form mycorrhizae, resulting in a lower mycorrhizal quality index of the whole plant.

[0096] (5) Compared with Example 1, Comparative Example 5 shows that when preparing the Indian truffle spore inoculation agent, the surface of the fruiting body is not disinfected, and the added vermiculite is not disinfected. The disinfection of the inoculation agent has little effect on the synthesis rate of the mycorrhizal seedlings of Armand pine and chestnut, and the mycorrhizal quality index is even slightly higher. Then, the spores of this group of mycorrhizal seedlings were molecularly identified. 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 cause potential contamination by miscellaneous bacteria.

[0097] (6) Compared with Example 1, Comparative Example 6 shows that the inoculation clamp and palm kneading have little effect on the synthesis rate of mycorrhizal seedlings of Armand pine and chestnut, but have a significant effect on the quality of mycorrhiza. This is because the roots of the seedlings in the ordinary hand-kneading method will quickly grow downward and around, quickly extend out of the matrix range with the inoculated microbial agent, and thus cause the mycorrhizal quality index to decrease. At the same time, the inoculation clamp also shears the excess roots, promotes the growth of lateral roots, and promotes more roots to contact more microbial agents. The results of the small test found that when raising seedlings on a large scale, the use of inoculation clamps can make the production of Indian truffle mycorrhizal seedlings more standardized and more efficient, and can also avoid uneven seeding between seedlings, pollution, and damage to young roots.

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

[0099] In summary, after 6 months of inoculation with Indian truffles, the Armand pine and chestnut seedlings inoculated with the method and inoculation clip disclosed in the present invention can form good mycorrhizal seedlings, and the mycorrhizal quality is also high, which effectively breaks the disadvantages of traditional Indian truffle solid block and spore suspension inoculation, and innovates in the details of bacterial agent configuration, seedling cultivation and seedling inoculation technology, providing new technical points for the large-scale cultivation of Indian truffle Armand 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 the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all 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 an inoculant from Indian truffle fruiting bodies harvested from December each year to February of the following year; (2) Peat, vermiculite, and perlite are mixed in a volume percentage of 4:3:3, and then tap water that has been allowed to stand for more than 24 hours is added and mixed to obtain a matrix; the matrix is ​​filled to a position of 1 / 3 to 1 / 2 of the height of the seedling pot; (3) using an inoculation clamp to 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 matrix, and gently press; (5) Place the inoculated Armand pine or chestnut seedlings in a controllable greenhouse at 15-24°C for 48 hours, and then start spraying water regularly, three times a week, for 15 minutes each time; (6) After the inoculated seedlings were co-cultivated for 3 months, the roots of the Armand pine or chestnut mycorrhizal seedlings were placed under a stereoscope to observe whether mycorrhizae were produced. After washing, the presence of the target fungus species was identified to obtain mycorrhizal seedlings infected with Indian truffles.

2. A method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings according to claim 1, characterized in that: 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.

3. A method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings according to claim 2, 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.

4. A method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings according to claim 3, characterized in that: The preparation method of the inoculated bacterial agent in step (1) is as follows: Fresh and mature Indian truffle fruiting bodies are picked from December to February of the following year. After the surface of the fresh and mature Indian truffle fruiting bodies is disinfected with 1% potassium permanganate for 2-5 minutes, they are cleaned with sterile water, pulped with distilled water, and sterile vermiculite is added. The mixture is mixed well and placed at 4°C for use. The storage time shall not exceed 8 hours when used.

5. A large-scale inoculation and cultivation method for Indian truffle mycorrhizal seedlings according to claim 4, characterized in that, 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 alternately beaten at a speed of 2000-5000 r / min for 3-5 minutes.

6. A large-scale inoculation and cultivation method for Indian truffle mycorrhizal seedlings according to claim 5, characterized in that: The mass volume ratio of the Indian truffle fruiting body to distilled water and sterile vermiculite is (5-10) g:2 L:1 L.

7. A large-scale inoculation and cultivation method for 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. Cultivate them in a sterilized, controlled-temperature greenhouse until the Armand pine seedlings are 5-10 cm tall, and take them out for inoculation after the chestnut seedlings have 4-6 leaves. When taking chestnut seedlings, keep the germinated seeds.

8. A method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings according to claim 7, characterized in that: The chestnut seeds harvested that year need to be refrigerated at 0-1°C.

9. A method for large-scale inoculation and cultivation of Indian truffle mycorrhizal seedlings according to claim 8, characterized in that: During the cultivation of the aseptic seedlings of Armand pine or chestnut, the lowest temperature of the controllable greenhouse is not lower than 3° C., the highest temperature is not higher than 32° C., and the humidity is 50-85% RH.

Images