Method for shortening fruiting time of termitomyces albuminosus and application thereof

Abstract

The invention belongs to the technical field of termitomyces albuminosus breeding, and particularly relates to a method for shortening fruiting time of termitomyces albuminosus and application of the method. According to the method, termitomyces albuminosus spores are subjected to single spore culture to obtain mycelia of different mating types, the mycelia are added into newly-cultivated termite nests, after termite-termitomyces albuminosus symbiotic ant nests are formed, bacterial slurry matched with the mycelia in the mating types is added, formation of sexual mycelia in the ant nests is artificially promoted, and the survival rate of the termite-termitomyces albuminosus symbiotic ant nests is increased. Further, the number of sexual small white balls is increased, the fruiting rate is increased, and the fruiting time is shortened. According to the method disclosed by the invention, after the collybia albuminosa pulp is inoculated, fruiting is realized within 6 months, so that the fruiting time of the collybia albuminosa is greatly shortened; in addition, the used raw materials are cheap and easy to obtain, and the production cost of termitomyces albuminosus domestication is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of Alstroemeria albuminosa propagation, and particularly relates to a method for shortening the fruiting time of Alstroemeria albuminosa and an application thereof. Background Art

[0002] Termitomyces R. Heim and termites of the Macrotermitinae subfamily have evolved a strict in vitro mutualistic symbiotic relationship. The mycelium of Termitomyces R. Heim grows on a fungus bed structure composed of plant materials that have been digested by the termites' intestines. The termites that cultivate the fungus can provide a rich nutrient matrix and a suitable growth environment for Termitomyces R. Heim, and also eat the spherical structures formed by the mycelium of Termitomyces R. Heim on the fungus bed (called small white balls, including sexual small white balls and asexual small white balls) as an important source of nutrition to help themselves digest the cellulose of plant materials. It is not until the termite mating season every year that a large number of termites leave the nest, causing a sharp decrease in the consumption of small white balls in the fungus bed. At this time, the sexual small white balls of Termitomyces R. Heim continuously absorb nutrients to develop into Termitomyces R. Heim primordia and produce columnar mycelium, and finally form fruiting bodies, and Termitomyces R. Heim mushrooms break out of the ground.

[0003] Most of the Alternaria albuminosa mushrooms only produce mushrooms in the rainy season, and the limited supply cannot meet the current market demand. Previously, there have been many studies on the artificial domestication technology of Alternaria albuminosa mushrooms. The cultivation technology of making termite-Alternaria albuminosa symbiotic seedlings and then artificially cultivating termite nests in the wild to achieve the expansion of Alternaria albuminosa mushrooms has achieved mushroom production, but there are still many problems to be solved, such as: 1) Alternaria albuminosa mushrooms take too long to produce mushrooms, and mushrooms may only be produced about 2 years after the ant nest matures; 2) The long-term maintenance process in the wild increases the production cost; 3) The number of sexual white balls in the termite nest fungus nursery is extremely small, resulting in a low mushroom production rate; 4) Although the termites are fed with fungi during the cultivation process, unlike the domesticated Alternaria albuminosa fungi in the termite nest, the mushroom strains are less stable. Summary of the invention

[0004] The purpose of the present invention is to provide a method for shortening the fruiting time of Alpinia terrestris and an application thereof. The method of the present invention can promote the increase of the number of sexual primordia in an ant nest mushroom nursery, and fruiting can be achieved within 6 months after inoculation, thereby greatly shortening the fruiting time of Alpinia terrestris and improving the fruiting rate; at the same time, the raw materials used are cheap and easily available, thereby reducing the production cost of domestication of Alpinia terrestris.

[0005] The present invention provides a method for shortening the fruiting time of Alternaria albuminata, comprising the following steps:

[0006] Using the spores of Aldrichthyes albuminii to carry out single spore culture to obtain mycelium; adding the obtained mycelium to a newly cultivated termite nest to obtain a termite-Aldrichthyes albuminii symbiotic ant nest;

[0007] The mycelium is fermented to obtain a bacterial slurry; the mycelium in the bacterial slurry contains a mating type that can be combined with the mycelium used to construct the symbiotic ant nest;

[0008] The bacterial slurry is added to the termite-Alstroemeria symbiotic ant nest to achieve the mating type of the mycelium in the bacterial slurry and the mating type of the mycelium in the termite-Alstroemeria symbiotic ant nest.

[0009] Preferably, the time of adding is after the first batch of worker ants appear; the number of adding is ≥5 times; based on the floor area of ​​the termite nest, the amount of mycelium added each time is 20-100g of mycelium per square meter of the termite nest.

[0010] Preferably, based on the floor area of ​​the termite-Album polypore symbiotic ant nest, the amount of the bacterial slurry added is 1 to 2 L of bacterial slurry per square meter of the termite-Album polypore symbiotic ant nest.

[0011] Preferably, the medium for monospore culture is COMB medium; the COMB medium includes 50-100 g of termite mound soil, 10-20 g of glucose, 5-10 g of peptone, 15-20 g of agar and 1 L of distilled water.

[0012] Preferably, the fermentation temperature is 25-28° C.; the fermentation time is 28-37 days.

[0013] Preferably, the fermentation medium comprises water, sugar, starch and termite mound soil; and the mass ratio of the fermentation medium to the mycelium is 1000:20-150.

[0014] Preferably, the substrate of the newly cultivated termite nest is soil that has not been exposed to the sun and has not been added with chemical substances.

[0015] Preferably, after adding the bacterial slurry into the termite-Altissima symbiotic ant nest, the method further comprises covering the adding position with organic materials; the organic materials include agricultural waste and / or plant leaves.

[0016] Preferably, the humidity of the termite-Altissima symbiotic ant nest is 35% to 55%.

[0017] The present invention also provides application of the method described in the above scheme in artificial domestication of Alpinia albuminata.

[0018] The invention provides a method for shortening the fruiting time of Aldrinia albuminii, comprising the following steps: using Aldrinia albuminii spores to perform monospore culture to obtain mycelium; adding the obtained mycelium into a newly cultivated termite nest to obtain a termite-Aldrinia albuminii symbiotic ant nest; fermenting the mycelium to obtain a bacterial pulp; the mycelium in the bacterial pulp contains a mating type that can be combined with a mycelium used to construct a symbiotic ant nest; adding the bacterial pulp into the termite-Aldrinia albuminii symbiotic ant nest to achieve mating of the mycelium in the bacterial pulp and the mating type of the mycelium in the termite-Aldrinia albuminii symbiotic ant nest. The present invention utilizes the spores of Alternaria albuminosa to perform monospore culture to obtain mycelium, and the mycelium is added to a newly cultivated termite nest, which can be captured by termite workers and brought back to the nest for cultivation, forming a termite-Alternaria albuminosa symbiotic system; then, a bacterial slurry paired with the mating type of the mycelium is added to artificially promote the formation of sexual mycelium in the nest, thereby promoting the increase in the number of sexual small white balls, improving the fruiting rate, and shortening the fruiting time. Using the method of the present invention, after the termite-Alternaria albuminosa symbiotic nest is inoculated with Alternaria albuminosa slurry, fruiting is achieved within 6 months, greatly shortening the fruiting time of Alternaria albuminosa; in addition, the raw materials used are cheap and easy to obtain, reducing the production cost of Alternaria albuminosa domestication. The present invention can be used for large-scale cultivation of Alternaria albuminosa under indoor controllable conditions to achieve increased production of Alternaria albuminosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 This is a diagram of the process of sexual mycelium formation of Alternaria albuminosa, where a: haploid spores of mating type + and mating type -; b: spore germination to form monokaryotic mycelium; c: monokaryotic mycelium of different mating types fused to form binucleate mycelium; d: binucleate mycelium proliferation;

[0021] Figure 2 The spores of Alternaria japonica observed under a microscope in Example 1, wherein the magnification of the eyepiece is 10×, and the values ​​in the picture represent the magnification of the objective lens;

[0022] Figure 3 The monospore culture colonies of different mating types of the Termitomyces cerevisiae in Example 1;

[0023] Figure 4 This is a schematic diagram of screening mating type + and mating type - spores in Example 1. The inability of mycelium to fuse indicates that the spores are of the same mating type, and the ability of mycelium to fuse indicates that the spore combination is of different mating types;

[0024] Figure 5Schematic diagram of the fusion of the mating type + and mating type - spores selected in Example 1 into sexual hyphae and proliferation, wherein a is a schematic diagram of sexual hyphae fusion, b is a schematic diagram of observation under a microscope, and the arrow indicates the lock-like joint structure of the sexual hyphae after proliferation;

[0025] Figure 6 The termite nest newly cultivated indoors in Example 1;

[0026] Figure 7 It is a schematic diagram of harvesting the fruiting bodies of Alpinia terrestris on the artificially cultivated ant nest in the indoor in Example 1, wherein a is a schematic diagram of Alpinia terrestris in the termite nest, the arrow indicates the fruiting body of the mushroom, and b is a schematic diagram of the size of Alpinia terrestris;

[0027] Figure 8 It is the result of the molecular biological sequence comparison of ITS of the fruiting body of Alternaria japonica in Example 1;

[0028] Fig. 9 A new termite nest artificially reared in Comparative Example 1 without inoculation of the slurry of mating type +;

[0029] Fig.10 This is a schematic diagram of the transfer of artificially raised new ant nests that were not inoculated with mating type + slurry in Comparative Example 2 to wild conditions, and the fruiting of Alternaria albuminosa after 2 years and 6 months after transplantation, wherein a is a schematic diagram of the termite nest transplanted to the wild, b is a schematic diagram of the foraging of termite workers after transplantation, c is the collected Alternaria albuminosa, and d is the fruiting Alternaria albuminosa. DETAILED DESCRIPTION

[0030] The invention provides a method for shortening the fruiting time of Aldrinia albuminii, comprising the following steps: using Aldrinia albuminii spores to perform monospore culture to obtain mycelium; adding the obtained mycelium into a newly cultivated termite nest to obtain a termite-Aldrinia albuminii symbiotic ant nest; fermenting the mycelium to obtain a bacterial pulp; the mycelium in the bacterial pulp contains a mating type that can be combined with a mycelium used to construct a symbiotic ant nest; adding the bacterial pulp into the termite-Aldrinia albuminii symbiotic ant nest to achieve mating of the mycelium in the bacterial pulp and the mating type of the mycelium in the termite-Aldrinia albuminii symbiotic ant nest.

[0031] In the present invention, the spores of the chicken mushroom can preferably be prepared by themselves. As a preferred embodiment of the present invention, the preparation method of the chicken mushroom spores preferably includes: under sterile conditions, removing the stipes of the same kind of chicken mushroom to obtain the chicken mushroom cap. After obtaining the chicken mushroom cap, the present invention preferably places the chicken mushroom cap with the folds facing down on a sterile weighing paper, leaves it at room temperature for 24 hours, removes the chicken mushroom cap, and obtains a chicken mushroom spore print. Preferably, the present invention places a cup of distilled water near the sterile weighing paper. The present invention places a cup of distilled water near the sterile weighing paper to maintain air humidity and promote spore release. After obtaining the chicken mushroom spore print, the present invention preferably dissolves the chicken mushroom spore print in distilled water to obtain an initial spore liquid, and dilutes the initial spore liquid to obtain a single chicken mushroom spore. The dissolution of the present invention preferably comprises cutting the weighing paper carrying the spore print of Alternaria albuminosa, adding the cut weighing paper into distilled water, and shaking and mixing; the area of ​​the cut weighing paper is preferably 1 cm 2 ; The volume of the distilled water is preferably 10 mL. The dilution of the present invention preferably includes taking 1 mL of the initial spore mixture and adding it to a test tube containing 9 mL of distilled water for dilution, and continuously diluting it 9 times to obtain a diluted spore mixture. After obtaining the diluted spore mixture, the present invention preferably takes 1 mL of the diluted spore mixture and observes it under a microscope to obtain a single Alpinia spore.

[0032] After obtaining a single Album spore, the present invention preferably transfers the single Album spore to COMB medium for dark culture to obtain monospore mycelium. In the present invention, the COMB medium preferably includes 50-100g of termite mound soil, 10-20g of glucose, 5-10g of peptone, 15-20g of agar and 1L of distilled water, and further preferably includes 100g of termite mound soil, 10g of glucose, 5gL of peptone, 20g of agar and 1L of distilled water; the temperature of the dark culture is preferably 25°C to 28°C, more preferably 25°C to 27°C, and more preferably 26°C.

[0033] After obtaining the monospore mycelium, the present invention preferably screens the mycelium that can be joined. In the present invention, the screening method preferably includes: under sterile conditions, placing the samples of the two monospore mycelium obtained by culture on both sides of the same COMB culture medium, culturing in the dark, and the mycelium of different mating types that fuse in the culture medium are capable of joining, recorded as mycelium of mating type + and mycelium of mating type -. The preparation of the sample of the present invention includes taking a mycelium block with a diameter of 5 mm from the monospore mycelium to obtain the sample; the temperature of the light-proof culture is preferably 25°C to 28°C, more preferably 25°C to 27°C, and more preferably 26°C. In the present invention, the inability of two monospore mycelia to fuse indicates that they are of the same mating type, and no lock-like joint structure can be observed under a microscope; the ability of two monospore mycelia to fuse is indicated as a mycelium of mating type + and a mycelium of mating type -, and a lock-like joint structure can be observed under a microscope.

[0034] The present invention preferably performs wing removal treatment on the detached termite reproductive ants to obtain wing-removed termite reproductive ants.

[0035] After obtaining the wingless reproductive termites, the present invention preferably places the wingless reproductive termites in pairs of male and female in the matrix of the termite nest, and cultivates them in an indoor environment to obtain a newly cultivated termite nest. In the present invention, the termite nest is preferably artificially prepared; the matrix of the termite nest is preferably soil that has not been exposed to the sun and has not been added with chemical substances, and the humidity of the termite nest is preferably 35% to 55%. In the present invention, the soil that has not been exposed to the sun and has not been added with chemical substances is used as the matrix of the termite nest, which can be beneficial to the reproduction of termites and the survival of young termites. The termites in the newly cultivated termite nest of the present invention do not form a symbiotic system with the Alpha Colletotrichum, which is beneficial to the formation of a termite-Alpha Colletotrichum symbiotic system with the mycelium of different mating types added later.

[0036] After obtaining the newly cultivated termite nest and mycelium, the present invention adds the mycelium to the newly cultivated termite nest to obtain a termite-Altissima symbiotic ant nest. In the present invention, the time of the addition is preferably after the first batch of worker ants appear; the number of additions is preferably ≥5 times; based on the floor area of ​​the termite nest, the amount of the mycelium added each time is preferably 20 to 100 g of mycelium per square meter of the termite nest, more preferably 40 to 100 g of mycelium per square meter of the termite nest, and more preferably 50 to 100 g of mycelium per square meter of the termite nest. The mycelium added to the newly cultivated termite nest of the present invention is preferably a mycelium block carrying a culture medium. In the present invention, the mycelium added to the termite nest can be a mating type + mycelium, a mating type - mycelium, or a mating type + mycelium and a mating type - mycelium. In the present invention, mycelium is added after the first batch of worker ants, which can be brought back to the nest by the worker ants to promote the establishment of a termite-Altissima symbiotic system; and the culture medium carried on the mycelium can provide nutrients for the termites and promote the growth and reproduction of the termites.

[0037] In the present invention, the humidity of the termite-Altissima symbiotic nest is preferably 35% to 55%, more preferably 35% to 50%, and more preferably 35% to 45%. In the present invention, the appropriate humidity of the termite-Altissima symbiotic nest is conducive to the fruiting of Altissima.

[0038] The present invention ferments monosporic mycelium to obtain a bacterial slurry. In the present invention, the fermentation medium preferably includes water, sugar, starch and termite mound soil; the mass ratio of the sugar, starch and termite mound soil is preferably 60-120:60-120:30-60, and more preferably 100:100:50; the sugar is preferably derived from Saccharum spp. plants and / or purple-leafed beets (Beta vulgaris), the water content of the sugar is preferably 2wt%-4wt%, and the particle size is preferably 0.1-1.2mm; the starch is preferably crushed grains; the grains are preferably rice (Oryza sativa), rye (Secalecereale), wheat (Triticum aestivum) or corn (Zea mays) one or more; the particle size of the starch is preferably 6-8mm, more preferably 7mm; the particle size of the termite mound soil is preferably ≤1mm, and the water content is preferably 35wt%-55wt%; the mass ratio of the fermentation medium to the mycelium is preferably 1000:20-150, more preferably 1000:60-120, more preferably 1000:80-100; the fermentation temperature is preferably 25-28°C, more preferably 25-27°C, more preferably 27°C; the fermentation time is preferably 28-37d. When the mycelium of mating type + is fermented, the mycelium obtained is recorded as the mycelium of mating type +, and the mycelium obtained by fermenting the mycelium of mating type - is recorded as the mycelium of mating type -.

[0039] After obtaining the slurry, the present invention adds the slurry to the termite-convolvulus symbiotic ant nest, and the mycelium in the slurry contains a mating type that can be joined with the mycelium used to construct the symbiotic ant nest. As a preferred embodiment of the present invention, when the mycelium added to the termite-symbiotic ant nest is a mycelium of mating type +, the slurry is a slurry of mating type -, or a slurry of mating type - and a slurry of mating type +; when the mycelium added to the termite-symbiotic ant nest is a mycelium of mating type -, the slurry is a slurry of mating type +, or a slurry of mating type + and a slurry of mating type -; when the mycelium added to the termite-symbiotic ant nest is a mycelium of mating type + and a mycelium of mating type -, the slurry is a slurry of mating type +, or a slurry of mating type -, or a slurry of mating type - and a slurry of mating type +. In the present invention, based on the floor area of ​​the termite-Album symbiotic ant nest, the amount of the slurry added is preferably 1 to 2 L of slurry per square meter of the termite-Album symbiotic ant nest, and more preferably 1.3 to 1.8 L of slurry per square meter of the termite-Album symbiotic ant nest.

[0040] The present invention adds the bacterial slurry described in the above technical solution to the termite-Albino Mushroom symbiotic ant nest to achieve the mating type of the mycelium in the bacterial slurry and the mating type of the mycelium in the termite-Albino Mushroom symbiotic ant nest to obtain the sexual mycelium of Albino Mushroom. The sexual mycelium of Albino Mushroom is formed by the fusion of monokaryotic mycelia of different mating types through cells to form a lock-like joint structure, and then form sexual binucleate secondary mycelia, see Figure 1 . Further, the sexual mycelium of Alpinia terrestris is interwoven and transformed into sexual small white balls, which gradually develop into primordia and form fruiting bodies. The present invention adds bacterial pulp to the termite-Alpinia terrestris symbiotic ant nest, so that the mating type of the mycelium in the bacterial pulp is paired with the mating type of the mycelium in the termite-Alpinia terrestris symbiotic ant nest, thereby artificially promoting the formation of sexual mycelium in the ant nest, thereby promoting the increase in the number of sexual small white balls, improving the mushroom fruiting rate, and shortening the mushroom fruiting time. Using the method provided by the present invention, after the termite-Alpinia terrestris symbiotic ant nest is inoculated with Alpinia terrestris bacterial pulp, mushrooms are fruited within 6 months, which greatly shortens the fruiting time of Alpinia terrestris; and the cultivation process is completed indoors, which causes less damage to the environment; in addition, the raw materials used are cheap and easy to obtain, which reduces the production cost of Alpinia terrestris domestication. The present invention can be used for large-scale cultivation of Alpinia terrestris under indoor controllable conditions to achieve increased production of Alpinia terrestris.

[0041] After the fungus slurry is added to the termite-Altissima symbiotic ant nest, the present invention preferably further comprises covering the adding position with organic materials; the organic materials preferably comprise agricultural waste and / or plant leaves. The thickness of the organic material of the present invention is preferably >10 cm; the agricultural waste of the present invention preferably comprises one or more of rice, wheat and corn straw. The present invention covers the fungus slurry adding position with organic materials, which can maintain the temperature and humidity of the ant nest and can serve as a food source for termites. The present invention uses cheap and easily available raw materials, which reduces the production cost of domestication of Altissima.

[0042] The present invention also provides an application of the method described in the above scheme in the artificial domestication of Alpinia terrestris. The method of the present invention artificially constructs a termite-Alpinia terrestris symbiotic system by adding mating type paired mycelium and bacterial pulp to the newly cultivated termite nest, effectively increases the number of sexual small white balls, can be used for large-scale artificial cultivation of Alpinia terrestris under indoor controllable conditions, and increases the yield of Alpinia terrestris and shortens the fruiting time.

[0043] In order to further illustrate the present invention, a method for shortening the fruiting time of Alpinia terrestris provided by the present invention and its application are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] (1) Collecting spores of Alternaria japonica

[0046] Take the fruiting body of fresh same species of Alternaria albuminosa, spray a paper towel with 75% ethanol under aseptic conditions, gently wipe the surface of the fruiting body for disinfection, remove the Alternaria albuminosa stipe, and leave only the Alternaria albuminosa cap part. Place the Alternaria albuminosa cap part with the folds facing down, gently put it on a sterile weighing paper (sulfuric acid paper), and place a cup of distilled water near the weighing paper to maintain air humidity and promote spore release. Remove the cap after placing it at room temperature for 24 hours, and collect the Alternaria albuminosa spore print.

[0047] (2) Single spore culture of Alternaria albuminosa

[0048] Under sterile conditions, cut the weighing paper with spore print into 1 cm 2 Size, respectively, into a test tube containing 10mL of distilled water, shake and mix, to obtain the initial spore mixture. Take 1mL of the initial spore mixture, dissolve it in 9mL of distilled water, shake and mix, to complete the dilution operation; repeat the above dilution operation, dilute it 9 times in a row, to obtain the diluted spore mixture. Take 1mL of the diluted spore mixture, drop it on a microscope slide for observation, see Figure 2 After observing the single spores, extract the single spores and inoculate them into different COMB medium respectively, and culture them at 26℃ in the dark. Figure 3 The components of the COMB medium are 100 g of termite mound soil, 10 g of glucose, 5 g of peptone, 20 g of agar and 1 L of distilled water.

[0049] (3) Screening of mycelia of mating type + and mycelia of mating type -

[0050] After the spores of the single spore culture in step (2) grow white mycelium of Alternaria albuminosa, under sterile conditions, mycelium blocks with a diameter of 5 mm are taken from the culture medium as samples and placed in a new COMB medium for combined culture. The combined culture is to place a mycelium block from a different single spore culture on each side of the COMB medium. The culture results are shown in Figure 4 The COMB medium containing mycelium blocks of different monospore cultures was cultured at 26°C in the dark, and the mycelium on the medium was observed to be fused.

[0051] If the mycelium cannot fuse, it means that the spores are of the same mating type (same mating type + or same mating type -), and the lock-like joint structure cannot be observed under the microscope; if the mycelium can fuse, it means that the spores are of different mating types (mating type + and mating type -), and the lock-like joint structure can be observed under the microscope, see Figure 5 , Figure 5 The middle arrow is a lock-shaped joint structure. In this way, mycelia of different mating types are screened and recorded as mycelia of mating type + and mycelia of mating type -.

[0052] (4) Artificially raising new termite nests indoors

[0053] The flying reproductive termites were captured and their wings were removed. A black plastic basin (24 cm in diameter and 10 cm in height) was prepared and filled with 2 kg of substrate, which was soil that had not been exposed to the sun and had no chemicals added, and had a water content of 35%. Male and female adult termites were paired and placed in the basin and cultured in an indoor environment. Figure 6 shown.

[0054] When the first batch of worker ants appear, place dry cow dung and dry wheat bran (about 20g in total) in the basin as food for the termites.

[0055] Cut the mycelium of mating type - in step (3) together with the COMB medium into 1 cm 2 Add 5 mycelium blocks of mating type - to the termite nest to provide nutrition for the termites. Add 5 mycelium blocks every 3 days for 5 times in a row. The worker ants bring the mycelium of mating type - back to the nest to eat and form a fungus bed.

[0056] (5) Preparation of mating type + bacterial pulp

[0057] Preparation of culture medium: 1L of distilled water, 100g of sugar (derived from sugar cane), 100g of starch (derived from rice) and 50g of termite mound soil are added into a conical flask to obtain a fermentation culture medium; the sugar is obtained by drying sugar cane at 40°C and grinding it into particles with a particle size of 1mm, and the water content is maintained at 2wt%; the starch is obtained by removing the husk of rice and grinding it into particles with a particle size of 7mm, and distilled water is added to the particles to keep the water content at 65wt%; the termite mound soil is obtained by crushing termite mound soil into powder with a particle size of ≤1mm, and distilled water is added to keep the water content at 35wt%.

[0058] The mating type + mycelium obtained by screening in step (3) is cut into mycelial blocks with a diameter of 5 mm together with COMB medium, each mycelial block is 1g, and 100 mycelial blocks of mating type + spores are added to the fermentation medium for mixing. The above-mentioned culture medium with mycelial blocks is placed on a shaker to ferment in the dark for 7 days, the shaker speed is 110rpm, the temperature is 26°C, and solid white Albion mycelium is formed on the surface of the liquid surface; after fermentation for 7 days, 9L of distilled water is added to the culture medium, and then fermented in the dark for 21 days, the shaker speed is 110rpm, and the fermentation temperature is 26°C. Solid white Albion mycelium is formed on the surface of the slurry, and the liquid surface is completely covered to obtain a mating type + slurry.

[0059] (6) Inoculation of mating type + bacterial slurry

[0060] After the newly cultivated termite nest in step (4) was artificially cultivated for 4 months under an indoor environment, 10 mL of the slurry of the mating type + of the Alpinia terrestris was evenly applied on the surface of the termite nest using a nozzle with a 2 mm diameter hole, and then corn stalks were used as an organic material covering layer to evenly cover the inoculated soil substrate. The organic material covering thickness was >10 cm, which was used to maintain the temperature and humidity of the ant nest and serve as a food source for the termite colony.

[0061] Carry out routine maintenance and observation on the inoculated ant nest, maintain the soil moisture at 35%, and achieve fruiting 6 months after inoculation. Figure 7 shown.

[0062] (7) Identification of Alternaria albuminata

[0063] Collect the Albizia oleifera from step (6), and gently wipe the surface of the fruiting body with a paper towel wetted with 75% ethanol for disinfection. After disinfection, take 100 mg of the fruiting body tissue of Albizia oleifera and place it in a grinding mortar, add a small amount of liquid nitrogen to the grinding mortar, and fully grind it with a grinding rod.

[0064] DNA was extracted using the Biospin Fungal Genomic DNA Extraction Kit (BioFlux, Cat#BSC14S1). The extracted DNA was amplified for the ITS gene, which included the ITS1 region, the entire 5.8S region, and the ITS2 region. The universal fungal primers ITS5 and ITS4 were used for amplification.

[0065] ITS5: 5′-GGAAGTAAAAGTCGTAACAAGG-3′, SEQ ID NO.1;

[0066] ITS4: 5′-TCCTCCGCTTATTGATATGC-3′, SEQ ID NO. 2.

[0067] PCR reaction system (25 μL): 2×Tolo Fast Taq Premix (TOLOBIO #21401) 12.5 μL, ddH2O 9.5 μL, forward and reverse primers (final primer concentration 0.4 μM) 1 μL each, DNA 1 μL;

[0068] PCR reaction program: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 40 s, annealing at 54°C for 45 s, extension at 72°C for 1 min, for a total of 34 cycles; final extension at 72°C for 10 min, and the PCR product was stored at 4°C.

[0069] The PCR amplification product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The primers for sequencing were the same as the primers for PCR reaction. The forward ITS sequence obtained was compared in the NCBI gene library. The ITS molecular biology sequence comparison (BLAST) was as follows: Figure 8 As shown. Molecular biology confirmed that the mushrooms were Termitomyces.

[0070] Example 2

[0071] (1) Collection of Alternaria albuminosa spores: The operation method is the same as step (1) of Example 1.

[0072] (2) Monospore culture of Alternaria albuminosa: The operation method is the same as step (2) of Example 1.

[0073] (3) Screening of spores of different mating types and their mycelium: The operation method is the same as step (3) of Example 1.

[0074] (4) Artificially rearing new termite nests indoors: The operation method is the same as step (4) of Example 1, except that mycelium of mating type + is added to the termite nests.

[0075] (5) Preparing mating type-A slurry: The operation method is the same as step (5) of Example 1, except that mycelium of mating type-B is added to the fermentation medium to obtain mating type-B slurry.

[0076] (6) Inoculation of mating type - bacterial slurry: The operation method is the same as step (6) of Example 1.

[0077] Carry out routine maintenance and observation on the inoculated ant nests, maintain the soil moisture at 35%, and achieve mushroom production 6 months after inoculation.

[0078] Example 3

[0079] (1) Collection of Alternaria albuminosa spores: The operation method is the same as step (1) of Example 1.

[0080] (2) Monospore culture of Alternaria albuminosa: The operation method is the same as step (2) of Example 1.

[0081] (3) Screening of spores of different mating types and their mycelium: The operation method is the same as step (3) of Example 1.

[0082] (4) Artificially rearing new termite nests indoors: The operation method is the same as step (4) of Example 1, except that mycelium of mating type + and mycelium of mating type - are added to the termite nests.

[0083] (5) Preparing mating type + slurry and mating type - slurry: The operation method is the same as step (5) of Example 1, except that mycelium of mating type + and mycelium of mating type - are added to the fermentation medium respectively to obtain mating type + slurry and mating type - slurry.

[0084] (6) Inoculating the bacterial slurry of mating type + and the bacterial slurry of mating type -: The operation method is the same as step (6) of Example 1.

[0085] Carry out routine maintenance and observation on the inoculated ant nests, maintain the soil moisture at 35%, and achieve mushroom production 6 months after inoculation.

[0086] Comparative Example 1

[0087] (1) Collection of Alternaria albuminosa spores: The operation method is the same as step (1) of Example 1.

[0088] (2) Monospore culture of Alternaria albuminosa: The operation method is the same as step (2) of Example 1.

[0089] (3) Screening of spores of different mating types and their mycelium: The operation method is the same as step (3) of Example 1.

[0090] (4) Artificially rearing new termite nests indoors: The operation method is the same as step (4) of Example 1.

[0091] The newly cultivated termite nests were observed under conventional indoor conditions and the soil moisture was maintained at 35%. However, the fruiting was not achieved. Fig. 9 shown.

[0092] In Comparative Example 1, only mycelium blocks of mating type - were added, and no mycelium of mating type + was added. There was only one mating type in the termite nest, and sexual mycelium could not be formed, so mushroom production could not be achieved.

[0093] Comparative Example 2

[0094] (1) Collection of Alternaria albuminosa spores: The operation method is the same as step (1) of Example 1.

[0095] (2) Monospore culture of Alternaria albuminosa: The operation method is the same as step (2) of Example 1.

[0096] (3) Screening of spores of different mating types and their mycelium: The operation method is the same as step (3) of Example 1.

[0097] (4) Artificially rearing new termite nests indoors: The operation method is the same as step (4) of Example 1.

[0098] (5) Artificially cultivating termite nests in the wild: After the new ant nests in the indoor environment in step (4) are artificially cultivated for 4 months, the termite nests are transferred to the wild at a depth of about 50 cm from the soil surface for cultivation, and dry corn stalks are added at a distance from the surface of the ant nest as food for the termites.

[0099] Carry out routine maintenance and observation of termite nests and maintain soil moisture at 35%. After 2 years and 6 months after transplantation, the first batch of Alternaria albuminosa fruiting bodies will be produced. Fig.10 shown.

[0100] In comparative example 2, only mycelial blocks of mating type - were added to the termite nest, and no mating type + slurry was artificially added to form a termite-Alternaria symbiotic system and then transferred to the field for cultivation. During the field cultivation process, termite workers can capture Alternaria spores, form sexual white balls with the original Alternaria spores of termites, and develop into fruiting bodies. The overall cultivation time is long, and the fruiting time is increased by 2 years compared with Example 1.

[0101] In summary, adding the mycelium of Alpinia arborescens to the newly cultivated termite nest can form a termite-Alpinia arborescens symbiotic system; adding different mating type slurry can ensure that the mycelium and slurry in the termite nest are the same type of Alpinia arborescens, realize artificial promotion of the formation of sexual mycelium in the ant nest, and then promote the increase of the number of sexual small white balls, improve the fruiting rate, and shorten the fruiting time. Utilizing the method of the present invention, after inoculating different mating type slurry of Alpinia arborescens, fruiting is achieved within 6 months, which greatly shortens the fruiting time of Alpinia arborescens; and the cultivation process is completed indoors, which causes less damage to the environment; in addition, the raw materials used are cheap and easy to obtain, which reduces the production cost of Alpinia arborescens domestication and reduces the destructiveness of termites to nature. The present invention can be used for the cultivation of large-scale Alpinia arborescens under indoor controllable conditions to achieve increased production of Alpinia arborescens.

[0102] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for shortening the fruiting time of Alpinia terrestris, characterized in that: The following steps are involved: Using the spores of Aldrichthyes albuminii to carry out single spore culture to obtain mycelium; adding the obtained mycelium to a newly cultivated termite nest to obtain a termite-Aldrichthyes albuminii symbiotic ant nest; The mycelium is fermented to obtain a bacterial slurry; the mycelium in the bacterial slurry contains a mating type that can be combined with the mycelium used to construct the symbiotic ant nest; The bacterial slurry is added to the termite-Alstroemeria symbiotic ant nest to achieve the mating type of the mycelium in the bacterial slurry and the mating type of the mycelium in the termite-Alstroemeria symbiotic ant nest.

2. The method according to claim 1, characterized in that The time of adding is after the first batch of worker ants appear; the number of adding is ≥5 times; based on the floor area of ​​the termite nest, the amount of mycelium added each time is 20-100g of mycelium per square meter of the termite nest.

3. The method according to claim 1, characterized in that Based on the floor area of ​​the termite-Alstroemeria symbiotic ant nest, the amount of the bacterial slurry added is 1 to 2 L of bacterial slurry per square meter of the termite-Alstroemeria symbiotic ant nest.

4. The method according to claim 1, characterized in that: The culture medium for single spore culture is COMB culture medium; the COMB culture medium comprises 50-100g of termite mound soil, 10-20g of glucose, 5-10g / L of peptone, 15-20g of agar and 1L of distilled water.

5. The method according to claim 1, characterized in that The fermentation temperature is 25-28° C.; the fermentation time is 28-37 days.

6. The method according to claim 1 or 5, characterized in that: The fermentation medium comprises water, sugar, starch and termite mound soil; the mass ratio of the fermentation medium to the mycelium is 1000:20-150.

7. The method according to claim 1, characterized in that The substrate of the newly cultivated termite nest is soil that has not been exposed to the sun and has no added chemicals.

8. The method according to claim 1, characterized in that: After the fungus slurry is added into the termite-Altissima symbiotic ant nest, the method further comprises covering the adding position with organic materials; the organic materials include agricultural waste and / or plant leaves.

9. The method according to claim 1, characterized in that: The humidity of the termite-Altissima symbiotic ant nest is 35% to 55%.

10. Use of the method according to any one of claims 1 to 9 in the artificial domestication of Alpinia terrestris.

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