A method for producing high-viability termite-Aconitum symbiotic nests by large-scale inoculation

Abstract

The present invention discloses a method for producing a high-vitality termite symbiotic nest of Albizia albuminii on a large scale, which belongs to the field of termite symbiotic nest cultivation, and mainly includes: intestinal microbial flora culture, synergistic microbial flora culture, high-purity Albizia albuminii liquid strain preparation, preparation of inoculation feed, ant seedling inoculation and post-inoculation management. The present invention prepares inoculation feed suitable for the stable growth of bacterial culture termites, and selects ant seedling cups 1 day before the activity of the first generation of worker ants to 15 days after the activity as ant nests for inoculation, and the ant nest inoculation success rate is as high as 98%, and the average space volume of the formed fungus garden reaches 3.0-10mL, and the larvae of all grades and ages of termites develop healthily, the proportion is sustainable, there is no risk of labor grade interruption, and still has a high growth and development vitality after stocking, which can be used for the large-scale inoculation production of high-vitality termite symbiotic nest of Albizia albuminii.

Description

Technical Field

[0001] The invention belongs to the field of termite Albizia albuminosa cultivation, and specifically provides a method for producing high-vitality termite Albizia albuminosa symbiotic nests by large-scale inoculation. Background Art

[0002] Termites belong to the order Blattodea, suborder Isoptera, and are close relatives of cockroaches. Fungus-growing termites are important soil-improving insects that play a key role in the stability of the regional ecosystem. Adult termites mainly feed on dead plants and animals, providing rich nitrogen nutrients to the environment while also providing rich food for other anteaters (such as birds, predatory ants, bats, etc.); the nitrogen nutrients produced by the decomposition of dead adult termites provide fertilizer for some shallow-rooted plants and seedlings. However, with the changes in the environment and the excessive use of pesticides, the number of fungus-growing termite nests has been rapidly decreasing year by year, affecting the stability of the ecosystem.

[0003] In the long-term evolution process, termites and Altissima albuminosa gradually evolved into a symbiotic ecosystem. Termites spread the spores and mycelium of Altissima albuminosa, cultivate mycelium, and also eat the cultivated mycelium of Altissima albuminosa as food. Therefore, in order to protect and develop the ecological service functions of fungus-growing termites, fungus-growing termites can be cultivated with Altissima albuminosa strains, among which the inoculation of ant seedlings is the key link to achieve efficient cultivation. The key to the successful nest construction of fungus-growing termites is the newly produced nest colony of the first generation of worker ants, which can obtain the fungus strains of Altissima albuminosa and produce a fungus bed.

[0004] In the existing methods of cultivating termite-collar mushroom, no stable synergistic microbial community is introduced into the new ant nest. Similarly, without controlling the culture conditions, the microbial community in the ant nest is disordered. This means that even if the ant nest can be successfully inoculated and a small mushroom garden is produced, it will still be invaded by antagonistic bacteria in the later stage or after stocking, and eventually the mushroom garden will shrink and the nest construction will fail. In particular, the survival rate of ant nests of the main symbiotic ant species of colarugina is low, resulting in a low success rate of natural establishment of mushroom gardens, which limits the scale development of the termite-collar mushroom industry. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art, provide a method for large-scale inoculation and production of high-vitality termite-Albino symbiotic nests, prepare inoculation feed suitable for the stable growth of bacterial-cultured termites, select ant seedling cups from 1 day before to 15 days after the activity of primary worker ants as ant nests for inoculation, the ant nest inoculation success rate is as high as 98%, the average spatial volume of the formed fungus garden reaches 3.0-10 mL, the larvae of all grades and ages of termites develop healthily, the proportion is sustainable, there is no risk of generation interruption of the labor grade, and the termites still have high growth and development vitality after stocking, and can be used for large-scale inoculation and production of high-vitality termite-Albino symbiotic nests.

[0006] In order to solve the above technical problems, the present invention provides a method for producing a highly active termite-Albino symbiotic nest by large-scale inoculation, comprising the following steps:

[0007] (1) Cultivation of intestinal microbial flora: The intestinal segments of the cultured termites were microdissected and inoculated into intestinal microorganism culture matrix under a sterile environment. The pH of the intestinal segments was adjusted to 3.0-7.0 by buffering. A mixed gas of 0.5-10 v% CO2 / N2 was filled for replacement protection. The culture was anaerobically cultured at 21-45 ℃ for 0.5-3 days with stirring at 5-200 rpm until the bacterial count concentration reached 10 5 ~10 8 When the cell count is 100 / mL, store it at 4.0-15.0°C for later use, then amplify it to 0.1-100 L at a ratio of 1:1-1:100 in multiple stages, fill it with a 0.5-10 v% CO2 / N2 mixed gas to replace it into an anaerobic environment, and store it at 4.0-15.0°C for later use;

[0008] The method for obtaining the intestinal microorganism culture matrix is ​​as follows: fresh cow stomach or fresh healthy cow dung fed with clean grass is extracted with sterile water to retain the extract, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained and quickly pasteurized at 60-90°C for 5-20 seconds.

[0009] (2) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the termite-Agaricus collifolius garden were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources. The culture was carried out in a low oxygen environment of 10-20 v% O2 / N2 at a pH of 3.0-7.0 and a temperature of 21-45 °C for 0.1-3.0 days until the bacterial count concentration reached 10 5 ~10 9 When the concentration is 0.1447g / mL, store at 8-15℃ for future use.

[0010] The synergistic aerobic bacteria include Bacillus species A 0-100%, B 0-100%, C 0-100%, D 0-100% and E 0-100%, Clostridium species F 0.1-5%, and Actinomycetes species G 0-1.0%.

[0011] (3) Preparation of high-purity liquid culture of Alternaria albuminosa: After the surface of fresh Alternaria albuminosa fruiting bodies is disinfected, the internal tissues are dissected and obtained, and inoculated on a special solid culture medium under sterile conditions. Purification culture is carried out at 15-25 v% low O2 and 19-31 ℃ for 3-30 days. The Alternaria albuminosa strains after each purification culture are taken and the operation is repeated 1-5 times to remove antagonistic bacteria to obtain high-purity Alternaria albuminosa strains; the obtained high-purity Alternaria albuminosa strains are then inoculated into a special liquid culture medium at a weight ratio of 1:10-1:100 under sterile conditions, and cultured at 10-25 v% low O2 and 0.5-10 v% high CO2 gas atmosphere at 21-35 ℃ and 10-120 rpm for 3-30 days to obtain a liquid culture of Alternaria albuminosa with a mycelium content of 0.001-0.1 g / mL, which is inoculated by horizontal subculture method;

[0012] The Termitomyces symbioticus fungus is a Termitomyces fungus, including individual large rhizoids with swollen spherical ant nests. T. globulus , Thick-handled Myrmecophaga T. robustus , bulbous ant nest umbrella T. bulborhizus , Myrmecophaga spp. T. eurrhizum , medium-sized white-handled ant nest umbrella T. albiceps 、Columnar ant nest umbrella T. cylindrieus 、Golden Ant Nest Umbrella T. macrocarpus 、Striped ant nest umbrella T. striatus 、Black-capped ant nest umbrella T. entolomoides , Shield-tipped Ant Nest T. shielded , Intermorpha Alternaria T. intermedius , Gallus heim T. heimi and Alternaria japonica T. mammiform wait;

[0013] The special solid culture medium comprises 0.001-1 wt% of synergistic microbial flora, 0.001-0.1 wt% of essential synergistic trace element components and the balance of acidic PDA or improved Martin solid culture medium with a pH of 3.0-7.0;

[0014] The special liquid culture medium comprises 0.001-0.1 wt% of essential synergistic trace element components and the balance of acidic PDA or modified Martin solid culture medium with a pH of 3.0-7.0;

[0015] The essential synergistic trace element components include 0.001-0.1% of Fe, Mn, Mg, Zn and other elements.

[0016] (4) Preparing a feed matrix: The feed matrix comprises component A and component B, wherein component A is a raw material of lignin, hemi-lignin and hemicellulose that can be efficiently utilized by Albizia albuminosa, and component B is a raw material of cellulose and hemicellulose that is suitable for carbon and water energy supply to termites;

[0017] Component A is derived from fresh cow dung that has been fed clean grass and pasteurized within 0 to 3 days at 60 to 90 degrees Celsius for 10 to 40 seconds, or dead plant materials that have been fermented by brown rot fungi and sterilized at high temperature and high pressure at 100 to 120 degrees Celsius for 1 to 5 hours.

[0018] Ingredient B comes from fresh cow dung fed with clean grass, which has been sterilized under high temperature and high pressure at 100-120°C for 1-5 hours, or dead plant materials fermented by other fungi.

[0019] (5) Preparing inoculated feed; mixing the intestinal microorganisms with quantitative bacterial count concentration, synergistic microorganisms, purified cultured liquid strains of Alternaria japonica with quantitative mycelium content, and sterilized feed matrix in a specific proportion under sterile conditions to prepare an inoculated feed with a stable bacterial community, and storing it in a sealed low-oxygen environment of 10-20 v% O2 / N2.

[0020] (6) Inoculation of ant seedlings: Select the ant seedling cups that were cultivated 1 day before to 15 days after the activity of the first generation of termites as the ant nest to be inoculated. Under sterile conditions, take 1-20 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated for ant seedling inoculation;

[0021] (7) Management after inoculation: After inoculation, the ant nest must ensure the normal breathing of the termites and prevent the disappearance of the synergistic bacteria, which may lead to contamination by other bacteria. The ant nest can be sealed with an oxygen semipermeable membrane with small holes, and the oxygen content can be quantitatively controlled at 10-25% through the oxygen semipermeable membrane and the small holes. The culture temperature must meet the needs of rapid growth of microorganisms and ensure the safety of termites. The temperature range is 16-32°C. The absolute moisture content of the culture medium is maintained at 8-20% (saturated), and gradually decreases within 0-60 days as the vitality of the ant colony changes. The microenvironment is gradually changed to allow different life forms of microorganisms to be gradually colonized in the seedlings as the microenvironment changes, forming a sound symbiotic microecological community. After cultivation, a fungus bed is formed in the ant nest. After cultivation, a fungus bed is formed in the ant nest.

[0022] As a further description of the present invention, the intestinal microbial flora described in step (1) can also be obtained from fresh cow stomachs or fresh healthy cow dung fed with clean grass, specifically: fresh cow stomachs or fresh healthy cow dung fed with clean grass are taken, extracted with sterile water, and the extract is retained, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained, and sterile water is added to adjust the bacterial count concentration to 10 5 ~10 9 / mL, fill with a mixed gas of (1-5):(1-5):(15-40) O2 / CO2 / N2 to replace it into a low oxygen environment, adjust the pH to 3.0-7.0, and store at 4.0-15.0 ℃.

[0023] As a further description of the present invention, in step (3), when the ant nest fungus species is determined, the termites themselves still retain the Gallinarum species and are vertically inoculated by a synergistic bacteria growth promotion method. The synergistic bacteria species are directly derived from the intestines of healthy cattle fed clean grass or cultured bacterial species.

[0024] As a further description of the present invention, step (3) can also be performed by disinfecting the surface of the freshly opened Alpinia terrestris fruiting body, adding a sterile water-diluted bacterial solution of the synergistic microbial flora at a ratio of 0.001 to 1 wt% of the fresh weight of Alpinia terrestris in a sterile or natural environment to prepare a Alpinia terrestris tissue suspension, and buffering and adjusting the pH to 3.0 to 7.0, which is directly used for inoculation.

[0025] As a further description of the present invention, the raw materials of lignin, hemi-lignin and hemicellulose efficiently utilized by the Alpinia terrestris in step (4) are dead plant materials fermented by brown rot fungi after high temperature and high pressure sterilization at 120°C for 3h, or sawdust fermented by fungi such as Ganoderma lucidum, Auricularia auricula, Lentinula edodes, and White ginseng.

[0026] As a further description of the present invention, the feed matrix of step (4) can also be obtained from chemically added lignin powder, semi-lignin powder, cellulose powder and hemicellulose powder by high temperature and high pressure sterilization at 100-120°C for 1-5 h, wherein the weight ratio of cellulose, hemicellulose, lignin and semi-lignin is 1:1:1:1.

[0027] As a further description of the present invention, the cellulose powder is cotton cellulose powder that termites like to eat.

[0028] As a further description of the present invention, the specific process of step (5) is as follows: under sterile or natural conditions, the intestinal microbial flora and the synergistic microbial flora are mixed, and then mixed with the liquid strain of Alpinia terrestris at a volume ratio of 1:10 to 1:1000 to form a flora mixture, and finally inoculated into the feed matrix at a volume dry weight ratio of 0 to 30% mL / g to obtain an inoculated feed, which is sealed and stored in a low-oxygen environment, wherein the strain count ratio of the intestinal microbial flora to the synergistic microbial flora is 10:1 to 1:100.

[0029] As a further description of the present invention, the specific process of step (5) is: first, the intestinal microbial flora and the synergistic microbial flora are mixed at a bacterial count ratio of 10:1 to 1:100, then mixed with the feed matrix at a volume dry weight ratio of 0 to 30% mL / g, and finally, the liquid strain of Alternaria albuminosa is inoculated alone at a ratio of 0.1 to 1 mL / g of the feed matrix.

[0030] As a further description of the present invention, the inoculated feed in step (5) may not be inoculated with the liquid strain of Aldrinia albuminosa, but the liquid strain of Aldrinia albuminosa may be placed on the ant tunnels where termites are active and inoculated separately, or the liquid strain of Aldrinia albuminosa may not be inoculated to stimulate the longitudinal inoculation of the strains carried by adult termites.

[0031] As a further description of the present invention, when the inoculated feed in step (5) may not be inoculated with the liquid strain of Aldrinia albuminosa, 0.1-2 mL of the liquid strain of Aldrinia albuminosa is still required to be dripped onto the ant tunnel.

[0032] As a further description of the present invention, step (6) can also be carried out in a sterile workshop by using an inoculation feed assembly line with a stable bacterial population to quantitatively add inoculation feed and liquid strains of Alternaria albuminosa into the ant seedling cup.

[0033] As a further description of the present invention, the fungus-cultivating termites in step (6) are of the genus Termitomyces Toothed termites , Macrotermes Macrotermes , Subtermes Microterms Or Hook-termes Ancistrotermes Any one of .

[0034] The termite genus is black-winged termite O. formosanus , Yunnan termites O. yunnanensis Or Coptotermes hainanensis O. hainanensis Macrotermes flavotermes M. barneyi 、Yunnan termite M. from Yunnan Macrotermes truncatus M. annandalei ; The genus of the termite is Menglun termite Microterms from Menglun ; Hook-shaped termite genus is Microcephala A. dimorphus.

[0035] As a further description of the present invention, the oxygen semipermeable membrane in step (7) is a PE, PVC, PVDC, or PMP membrane of non-toxic material with a thickness of 2 to 50 µm, and the ratio of the pore diameter to the ant nest volume is 0.2 to 1.0 mm / 50 mL.

[0036] The basis proposed by the present invention is:

[0037] 1) The life history of the termite-like Alternaria is characterized by the fecal fungus type. The Alternaria fungus garden built by termites not only includes Alternaria as the main species, but also includes some synergistic bacteria. The main species include Bacillus species A, B, C, D and E of the Bacillota (formerly Firmicutes), Clostridium species F, Actinobacteria species G, etc. These species can inhibit the miscellaneous bacteria in the garden, absorb the nutrients produced by the previous anaerobic intestinal bacteria fermentation, fix nitrogen and digest cellulose and lignin structures externally, and grow synergistically with Alternaria;

[0038] 2) The metabolic physiology of Alternaria albuminosa mainly includes the metabolic characteristics of efficient utilization of lignin and semi-lignin. Alternaria albuminosa is most suitable for utilizing lignin and semi-lignin that have been initially decomposed by intestinal microorganisms or other fungi;

[0039] 3) The intestinal microorganisms of anaerobic termites are the primary metabolites of termites, while the intestinal tracts of pure herbivores have similar intestinal microorganisms and metabolites. The secondary metabolites produced after being utilized by the synergistic microorganisms in the fungus garden can be better utilized by Alstroemeria albuminata.

[0040] 4) The main carbon and water energy supply of fungus-cultivating termites is lignocellulose, hemicellulose, and polysaccharides, oligosaccharides and monosaccharides produced by the decomposition of synergistic microorganisms;

[0041] 5) In addition to food materials, the nitrogen source in the fungus garden mainly comes from the nitrogen fixation of symbiotic bacteria;

[0042] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0043] The inoculation success rate of the termite-Albino mushroom symbiotic nest obtained by the method of the present invention can reach 98%. After 1-2 months of cultivation, the average spatial volume of the fungus bed produced in the ant nest reaches 3.0-10 mL; the larvae and adults of all grades and ages of termites develop healthily, the proportion is sustainable, there is no risk of generation interruption of the working grade, and they have high growth and development vitality after stocking, so that the termite-Albino mushroom symbiotic nest can be used for large-scale inoculation production of high-vitality termite-Albino mushroom symbiotic nests. DETAILED DESCRIPTION

[0044] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0045] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the invention belongs.

[0046] Example 1

[0047] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0048] (1) Intestinal microbial flora culture: Black-winged termites O. formosanus The intestinal segments were removed by microdissection, and the intestinal microorganisms in the intestinal segments were inoculated into the intestinal microbial culture matrix under a sterile environment. The pH was adjusted to 5.0 by buffering, and a 2 v% CO2 / N2 mixed gas was filled for replacement protection. The culture was anaerobically cultured at 35 °C for 1 day with stirring at 50 rpm until the bacterial count concentration reached 10 7 When the concentration of the cells was 100 / mL, they were stored at 8°C for future use. After being amplified to 10.0 L at a ratio of 1:10, they were filled with a 5 v% CO2 / N2 mixed gas to replace the anaerobic environment and stored at 8.0°C for future use.

[0049] The method for obtaining the intestinal microorganism culture matrix is ​​as follows: fresh cow stomach or fresh healthy cow dung fed with clean grass is extracted with sterile water to retain the extract, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained and low-temperature pasteurized at 80°C for 15 seconds.

[0050] (2) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the black-winged termites and the true root ant nests were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured in a low-oxygen environment of pH = 5.0, 31 °C, and 15% O2 for 1 day until the bacterial count concentration reached 10 8 / mL, stored at 12℃ for future use;

[0051] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0052] (3) High-purity true root anthill T. eurrhizus Preparation of liquid culture: After the surface of fresh Albizia albuminosa fruiting bodies was disinfected, the internal tissues were dissected and obtained, and inoculated on a special solid culture medium under sterile conditions, and naturally aerobically cultivated at 28°C for 10 days; then the Albizia albuminosa strains purified twice were inoculated into a special liquid culture medium at a weight ratio of 1:50 under sterile conditions, and cultivated at 28°C and 60 rpm for 15 days in a 20 v% low O2 and 2 v% high CO2 gas atmosphere, to obtain a liquid culture of Albizia albuminosa with a mycelium content of 0.01 g / mL;

[0053] The special solid culture medium comprises 0.05 wt% of synergistic microbial flora, 0.01 wt% of FeSO4 and the balance of acidic modified Martin solid culture medium with a pH of 5.0;

[0054] The special liquid culture medium comprises 0.01 wt % of FeSO4 and the remainder of an acidic modified Martin liquid culture medium with a pH of 2.0.

[0055] (4) Preparation of feed matrix: The feed matrix comes from cow dung that has been pasteurized at 80°C for 25 seconds within 1 hour after being fed with clean pure grass.

[0056] (5) Preparing inoculated feed; under sterile or natural conditions, the intestinal microbial flora and the synergistic microbial flora are mixed at a ratio of 1:10 in terms of bacterial species count, and then mixed with the liquid bacterial species of Alternaria albuminosa at a volume ratio of 1:10 to form a bacterial flora mixture, and finally inoculated into the feed matrix at a volume dry weight ratio of 10% mL / g to obtain the inoculated feed.

[0057] (6) Inoculation of ant seedlings: Select the ant seedling cups where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated.

[0058] (7) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0059] In this example, a total of 3000 termite seedlings were inoculated, and 2986 seedlings were successfully inoculated, with an average space volume of 4.5±2.3 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generation interruption of the labor grade, and had strong growth and development vitality after stocking.

[0060] Example 2

[0061] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0062] (1) Intestinal microbial flora culture: Directly use the intestinal flora from fresh cow stomachs fed with clean grass and fresh healthy cow dung extract, add sterile water to adjust the bacterial count concentration to 10 7 / mL, filled with a mixed gas of O2 / CO2 / N2 in a volume ratio of 9:1:40 to replace it into a low oxygen environment, and stored at a pH of 5.0 and a temperature of 10.0℃ for future use.

[0063] (2) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the black-winged termites and the true root ant nests were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured in a low oxygen environment of pH = 5.0, 31 °C, and 15 v% O2 for one day to reach a bacterial count of 10 8 / mL, stored at 12℃ for future use;

[0064] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0065] (3) High-purity true root anthill T. eurrhizus Preparation of liquid culture: After the surface of fresh Albizia albuminosa fruiting bodies was disinfected, the internal tissues were dissected and obtained, and inoculated on a special solid culture medium under sterile conditions, and naturally aerobically cultivated at 28°C for 10 days; then the Albizia albuminosa strains purified twice were inoculated into a special liquid culture medium at a weight ratio of 1:50 under sterile conditions, and cultivated at 28°C and 60 rpm for 15 days in a 20 v% low O2 and 2 v% high CO2 gas atmosphere, to obtain a liquid culture of Albizia albuminosa with a mycelium content of 0.01 g / mL;

[0066] The special solid culture medium comprises 0.05 wt% of synergistic microbial flora, 0.01 wt% of FeSO4 and the balance of acidic modified Martin solid culture medium with a pH of 5.0;

[0067] The special liquid culture medium comprises 0.01 wt % of FeSO4 and the remainder of an acidic modified Martin liquid culture medium with a pH of 2.0.

[0068] (4) Preparation of feed matrix: The feed matrix comes from cow dung that has been pasteurized at 80°C for 25 seconds within 1 hour after being fed with clean pure grass.

[0069] (5) Preparing inoculated feed; under sterile or natural conditions, the intestinal microbial flora and the synergistic microbial flora are mixed at a ratio of 1:10 in terms of bacterial species count, and then mixed with the liquid bacterial species of Alternaria albuminosa at a volume ratio of 1:10 to form a bacterial flora mixture, and finally inoculated into the feed matrix at a volume dry weight ratio of 10% mL / g to obtain the inoculated feed.

[0070] (6) Inoculation of ant seedlings: Select the ant seedling cups where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated.

[0071] (7) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0072] (8) In this example, a total of 3,000 termite seedlings were inoculated, and 2,659 seedlings were successfully inoculated. The average space volume of the fungus bed was 4.2±3.8 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generational interruption of the labor grade, and had strong growth and development vitality after release.

[0073] Example 3

[0074] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0075] (1) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the black-winged termites and the true root ant nests were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured in a low oxygen environment of pH = 5.0, 31 °C, and 15 v% O2 for one day to reach a bacterial count of 10 8 / mL, stored at 12℃ for future use;

[0076] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0077] (2) Preparation of feed matrix: The stomach contents and fresh feces of intestinal microorganisms of large herbivores similar to termites in a ratio of 1:1 are used as the feed matrix. The stomach contents and fresh feces are pasteurized at 60-90°C for 10-40 s.

[0078] (3) Preparing inoculated feed; adding 20% ​​mL / g of the dry weight of the feed matrix to the synergistic microbial flora liquid and 10 wt% of the dry weight of the feed matrix to the feed matrix to mix and prepare the inoculated feed; the cellulose powder is industrial cellulose powder without chemical additives obtained by high temperature and high pressure sterilization at 100-120°C for 1-5 h.

[0079] (4) Inoculation of ant seedlings: Select the ant seedling cups where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated.

[0080] (5) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0081] In this example, a total of 3000 termite seedlings were inoculated, and 1883 seedlings were successfully inoculated, with an average space volume of 2.9±1.5 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generation interruption of the labor grade, and had strong growth and development vitality after stocking.

[0082] Example 4

[0083] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0084] (1) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the black-winged termites and the true root ant nests were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured in a low oxygen environment of pH = 5.0, 31 °C, and 15 v% O2 for one day to reach a bacterial count of 10 8 / mL, stored at 12℃ for future use;

[0085] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0086] (2) High-purity true root anthill T. eurrhizus Preparation of liquid culture: After the surface of fresh Albizia albuminosa fruiting bodies was disinfected, the internal tissues were dissected and obtained, and inoculated on a special solid culture medium under sterile conditions, and naturally aerobically cultivated at 28°C for 10 days; then the Albizia albuminosa strains purified twice were inoculated into a special liquid culture medium at a weight ratio of 1:50 under sterile conditions, and cultivated at 28°C and 60 rpm for 15 days in a 20 v% low O2 and 2 v% high CO2 gas atmosphere, to obtain a liquid culture of Albizia albuminosa with a mycelium content of 0.01 g / mL;

[0087] The special solid culture medium comprises 0.05 wt% of synergistic microbial flora, 0.01 wt% of FeSO4 and the balance of acidic modified Martin solid culture medium with a pH of 5.0;

[0088] The special liquid culture medium comprises 0.01 wt % of FeSO4 and the remainder of an acidic modified Martin liquid culture medium with a pH of 2.0.

[0089] (3) Preparation of feed matrix: The stomach contents and fresh feces of intestinal microorganisms of large herbivores similar to termites in a ratio of 1:1 are used as the feed matrix. The stomach contents and fresh feces are pasteurized at 60-90°C for 10-40 s.

[0090] (4) Preparing inoculated feed; adding 20% ​​mL / g of the dry weight of the feed matrix to the synergistic microbial flora liquid and 10 wt% of the dry weight of the feed matrix to the feed matrix to mix and prepare the inoculated feed; the cellulose powder is industrial cellulose powder without chemical additives obtained by high temperature and high pressure sterilization at 100-120°C for 1-5 h.

[0091] (5) Inoculation of ant seedlings: Select the ant seedling cup where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated, and add 1 mL of liquid fungus of Alternaria albuminosa next to the inoculation feed.

[0092] (6) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0093] In this example, a total of 3000 termite seedlings were inoculated, and 2199 seedlings were successfully inoculated, with an average space volume of 3.2±2.7 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generation interruption of the labor grade, and had strong growth and development vitality after stocking.

[0094] Example 5

[0095] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0096] (1) Intestinal microbial flora culture: The intestinal segments of Yunnan termites were microdissected and inoculated into intestinal microorganism culture matrix under a sterile environment. The pH of the intestinal segments was adjusted to 5.0, and a 2 v% CO2 / N2 mixed gas was filled for replacement protection. The culture was carried out anaerobically at 35 °C with stirring at 50 rpm for 1 day until the bacterial count concentration reached 10. 7 When the concentration of the cells was 100 / mL, they were stored at 8°C for future use. After being amplified to 10.0 L at a ratio of 1:10, they were filled with a 5 v% CO2 / N2 mixed gas to replace the anaerobic environment and stored at 8.0°C for future use.

[0097] The method for obtaining the intestinal microorganism culture matrix is ​​as follows: fresh cow stomach or fresh healthy cow dung fed with clean grass is extracted with sterile water to retain the extract, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained and low-temperature pasteurized at 80°C for 15 seconds.

[0098] (2) Synergistic microbial flora culture: Synergistic aerobic bacterial strains isolated from Yunnan termites and Heim ant nests were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured for 1 day in a hypoxic environment of pH = 5.0, 31 °C, and 15% O2 until the flora count concentration reached 10 8 / mL, stored at 12℃ for future use;

[0099] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0100] (3) Preparation of high-purity liquid culture of Mycelium hymenii: After the surface of fresh Altissima fruiting body was disinfected, the internal tissue was dissected and obtained, and inoculated on a special solid culture medium under sterile conditions, and naturally aerobically cultured at 28°C for 10 days; then, the Altissima strain purified twice was inoculated into a special liquid culture medium at a weight ratio of 1:50 under sterile conditions, and cultured at 28°C and 60 rpm for 15 days in an atmosphere of 20 v% low O2 and 2 v% high CO2, to obtain a liquid culture of Altissima with a mycelium content of 0.01 g / mL;

[0101] The special solid culture medium comprises 0.05 wt% of synergistic microbial flora, 0.01 wt% of FeSO4 and the balance of acidic modified Martin solid culture medium with a pH of 5.0;

[0102] The special liquid culture medium comprises 0.05 wt% of cooperative microbial flora, 0.01 wt% of FeSO4 and the remainder of acidic modified Martin liquid culture medium with a pH of 5.0.

[0103] (4) Preparation of feed matrix: The feed matrix comes from cow dung that has been pasteurized at 80°C for 25 seconds within 1 hour after being fed with clean pure grass.

[0104] (5) Preparing inoculated feed; under sterile or natural conditions, the intestinal microbial flora and the synergistic microbial flora are mixed at a ratio of 1:10 in terms of bacterial species count, and then mixed with the liquid bacterial species of Alternaria albuminosa at a volume ratio of 1:10 to form a bacterial flora mixture, and finally inoculated into the feed matrix at a volume dry weight ratio of 10% mL / g to obtain the inoculated feed.

[0105] (6) Inoculation of ant seedlings: Select the ant seedling cups where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated.

[0106] (7) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0107] In this example, a total of 3000 termite seedlings were inoculated, and 2913 seedlings were successfully inoculated, with an average space volume of 4.5±2.3 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generation interruption of the labor grade, and had strong growth and development vitality after stocking.

[0108] Example 6

[0109] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0110] (1) Intestinal microbial flora culture: The intestinal segments of Yunnan termites were microdissected and inoculated into intestinal microorganism culture matrix under a sterile environment. The pH of the intestinal segments was adjusted to 5.0, and a 2 v% CO2 / N2 mixed gas was filled for replacement protection. The culture was carried out anaerobically at 35 °C with stirring at 50 rpm for 1 day until the bacterial count concentration reached 10. 7 When the concentration of the cells was 100 / mL, they were stored at 8°C for future use. After being amplified to 10.0 L at a ratio of 1:10, they were filled with a 5 v% CO2 / N2 mixed gas to replace the anaerobic environment and stored at 8.0°C for future use.

[0111] The method for obtaining the intestinal microorganism culture matrix is ​​as follows: fresh cow stomach or fresh healthy cow dung fed with clean grass is extracted with sterile water to retain the extract, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained and low-temperature pasteurized at 80°C for 15 seconds.

[0112] (2) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the mushroom beds of Yunnan termites and golden ant nests were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources. The culture was carried out in a low oxygen environment of pH = 5.0, 31 °C, and 15% O2 for 1 day until the bacterial count concentration reached 10 8 / mL, stored at 12℃ for future use;

[0113] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0114] (3) Preparation of high-purity liquid culture of P. truncatum: After the surface of fresh fruiting bodies of Albizia albuminosa were disinfected, the internal tissues were dissected and obtained, and inoculated on a special solid culture medium under sterile conditions, and naturally aerobically cultured at 28°C for 10 days; then, the Albizia albuminosa strain purified twice was inoculated into a special liquid culture medium at a weight ratio of 1:50 under sterile conditions, and cultured at 28°C and 60 rpm for 15 days in an atmosphere of 20 v% low O2 and 2 v% high CO2, to obtain a liquid culture of Albizia albuminosa with a mycelium content of 0.01 g / mL;

[0115] The special solid culture medium comprises 0.05 wt% of synergistic microbial flora, 0.01 wt% of FeSO4 and the balance of acidic modified Martin solid culture medium with a pH of 5.0;

[0116] The special liquid culture medium comprises 0.05 wt% of cooperative microbial flora, 0.01 wt% of FeSO4 and the remainder of acidic modified Martin liquid culture medium with a pH of 5.0.

[0117] (4) Preparation of feed matrix: The feed matrix comes from cow dung that has been pasteurized at 80°C for 25 seconds within 1 hour after being fed with clean pure grass.

[0118] (5) Preparing inoculated feed; under sterile or natural conditions, the intestinal microbial flora and the synergistic microbial flora are mixed at a ratio of 1:10 in terms of bacterial species count, and then mixed with the liquid bacterial species of Alternaria albuminosa at a volume ratio of 1:10 to form a bacterial flora mixture, and finally inoculated into the feed matrix at a volume dry weight ratio of 10% mL / g to obtain the inoculated feed.

[0119] (6) Inoculation of ant seedlings: Select the ant seedling cups where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated.

[0120] (7) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0121] In this example, a total of 3000 termite seedlings were inoculated, and 2654 seedlings were successfully inoculated, with an average space volume of 3.5±2.1 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generation interruption of the labor grade, and had strong growth and development vitality after stocking.

[0122] Example 7

[0123] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0124] (1) Intestinal microbial flora culture: The intestinal segment of the termite was microdissected and inoculated into the intestinal microorganism culture matrix under a sterile environment. The pH of the intestinal segment was adjusted to 5.0, and a 2 v% CO2 / N2 mixed gas was filled for replacement protection. The culture was carried out anaerobically at 35 °C with stirring at 50 rpm for 1 day until the bacterial count concentration reached 10. 7 When the concentration of the cells was 100 / mL, they were stored at 8°C for future use. After being amplified to 10.0 L at a ratio of 1:10, they were filled with a 5 v% CO2 / N2 mixed gas to replace the anaerobic environment and stored at 8.0°C for future use.

[0125] The method for obtaining the intestinal microorganism culture matrix is ​​as follows: fresh cow stomach or fresh healthy cow dung fed with clean grass is extracted with sterile water to retain the extract, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained and low-temperature pasteurized at 80°C for 15 seconds.

[0126] (2) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the mushroom beds of the nests of Coptis sphaerocephalus and Coptis sphaerocephalus were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured for 1 day in a hypoxic environment of pH = 5.0, 31 °C, and 15% O2 until the flora count concentration reached 10 8 / mL, stored at 12℃ for future use;

[0127] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0128] (3) Preparation of high-purity liquid culture of black-capped ant nest fungus: After the surface of fresh Altissima albuminosa fruiting bodies was disinfected, the internal tissues were dissected and obtained, and inoculated on a special solid culture medium under sterile conditions, and naturally aerobically cultivated at 28°C for 10 days; then, the Altissima albuminosa strain purified twice was inoculated into a special liquid culture medium at a weight ratio of 1:50 under sterile conditions, and cultivated at 28°C and 60 rpm for 15 days in an atmosphere of 20 v% low O2 and 2 v% high CO2, to obtain a liquid culture of Altissima albuminosa with a mycelium content of 0.01 g / mL;

[0129] The special solid culture medium comprises 0.05 wt% of synergistic microbial flora, 0.01 wt% of FeSO4 and the balance of acidic modified Martin solid culture medium with a pH of 5.0;

[0130] The special liquid culture medium comprises 0.05 wt% of cooperative microbial flora, 0.01 wt% of FeSO4 and the remainder of acidic modified Martin liquid culture medium with a pH of 5.0.

[0131] (4) Preparation of feed matrix: The feed matrix comes from cow dung that has been pasteurized at 80°C for 25 seconds within 1 hour after being fed with clean pure grass.

[0132] (5) Preparing inoculated feed; under sterile or natural conditions, the intestinal microbial flora and the synergistic microbial flora are mixed at a ratio of 1:10 in terms of bacterial species count, and then mixed with the liquid bacterial species of Alternaria albuminosa at a volume ratio of 1:10 to form a bacterial flora mixture, and finally inoculated into the feed matrix at a volume dry weight ratio of 10% mL / g to obtain the inoculated feed.

[0133] (6) Inoculation of ant seedlings: Select the ant seedling cups where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated.

[0134] (7) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0135] In this example, a total of 300 termite seedlings were inoculated, and 264 seedlings were successfully inoculated, with an average space volume of 2.5±1.1 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generation interruption of the labor grade, and had strong growth and development vitality after stocking.

[0136] Example 8

[0137] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0138] (1) Intestinal microbial flora culture: The intestinal segment of the yellow-winged termite was microdissected and inoculated into the intestinal microorganism culture matrix under a sterile environment. The pH of the intestinal segment was adjusted to 5.0, and a 2 v% CO2 / N2 mixed gas was filled for replacement protection. The culture was carried out anaerobically at 35 °C with stirring at 50 rpm for 1 day until the bacterial count concentration reached 10. 7 When the concentration of the cells was 100 / mL, they were stored at 8°C for future use. After being amplified to 10.0 L at a ratio of 1:10, they were filled with a 5 v% CO2 / N2 mixed gas to replace the anaerobic environment and stored at 8.0°C for future use.

[0139] The method for obtaining the intestinal microorganism culture matrix is ​​as follows: fresh cow stomach or fresh healthy cow dung fed with clean grass is extracted with sterile water to retain the extract, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained and low-temperature pasteurized at 80°C for 15 seconds.

[0140] (2) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the agaric beds of yellow-winged termites and bulb ant nests were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured for 1 day in a hypoxic environment of pH = 5.0, 31 °C, and 15% O2 until the flora count concentration reached 10 8 / mL, stored at 12℃ for future use;

[0141] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0142] (3) Preparation of high-purity liquid culture of anthill mushroom: After the surface of fresh Altissima albuminosa fruiting bodies was disinfected, the internal tissues were dissected and obtained, inoculated on a special solid culture medium under sterile conditions, and cultured naturally aerobically at 28°C for 10 days; then, the Altissima albuminosa strain purified twice was inoculated into a special liquid culture medium at a weight ratio of 1:50 under sterile conditions, and cultured at 28°C and 60 rpm for 15 days in an atmosphere of 20 v% low O2 and 2 v% high CO2, to obtain a liquid culture of Altissima albuminosa with a mycelium content of 0.01 g / mL;

[0143] The special solid culture medium comprises 0.05 wt% of synergistic microbial flora, 0.01 wt% of FeSO4 and the balance of acidic modified Martin solid culture medium with a pH of 5.0;

[0144] The special liquid culture medium comprises 0.05 wt% of cooperative microbial flora, 0.01 wt% of FeSO4 and the remainder of acidic modified Martin liquid culture medium with a pH of 5.0.

[0145] (4) Preparation of feed matrix: The feed matrix comes from sugarcane bagasse that has been pasteurized at 120 ℃ for 25 s.

[0146] (5) Preparing inoculated feed; under sterile or natural conditions, the intestinal microbial flora and the synergistic microbial flora are mixed at a ratio of 1:10 in terms of bacterial species count, and then mixed with the liquid bacterial species of Alternaria albuminosa at a volume ratio of 1:10 to form a bacterial flora mixture, and finally inoculated into the feed matrix at a volume dry weight ratio of 10% mL / g to obtain the inoculated feed.

[0147] (6) Inoculation of ant seedlings: Select the ant seedling cups where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated.

[0148] (7) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0149] In this example, a total of 300 termite seedlings were inoculated, and 284 seedlings were successfully inoculated, with an average space volume of 4.3±2.2 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generation interruption of the labor grade, and had strong growth and development vitality after stocking.

[0150] Example 9

[0151] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0152] (1) Intestinal microbial flora culture: The intestinal segment of the yellow-winged termite was microdissected and inoculated into the intestinal microorganism culture matrix under a sterile environment. The pH of the intestinal segment was adjusted to 5.0, and a 2 v% CO2 / N2 mixed gas was filled for replacement protection. The culture was carried out anaerobically at 35 °C with stirring at 50 rpm for 1 day until the bacterial count concentration reached 10. 7 When the concentration of the cells was 100 / mL, they were stored at 8°C for future use. After being amplified to 10.0 L at a ratio of 1:10, they were filled with a 5 v% CO2 / N2 mixed gas to replace the anaerobic environment and stored at 8.0°C for future use.

[0153] The method for obtaining the intestinal microorganism culture matrix is ​​as follows: fresh cow stomach or fresh healthy cow dung fed with clean grass is extracted with sterile water to retain the extract, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained and low-temperature pasteurized at 80°C for 15 seconds.

[0154] (2) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the agaric beds of yellow-winged termites and bulb ant nests were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured for 1 day in a hypoxic environment of pH = 5.0, 31 °C, and 15% O2 until the flora count concentration reached 10 8 / mL, stored at 12℃ for future use;

[0155] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0156] (3) Preparation of high-purity liquid culture of anthill mushroom: After the surface of fresh Altissima albuminosa fruiting bodies was disinfected, the internal tissues were dissected and obtained, inoculated on a special solid culture medium under sterile conditions, and cultured naturally aerobically at 28°C for 10 days; then, the Altissima albuminosa strain purified twice was inoculated into a special liquid culture medium at a weight ratio of 1:50 under sterile conditions, and cultured at 28°C and 60 rpm for 15 days in an atmosphere of 20 v% low O2 and 2 v% high CO2, to obtain a liquid culture of Altissima albuminosa with a mycelium content of 0.01 g / mL;

[0157] The special solid culture medium comprises 0.05 wt% of synergistic microbial flora, 0.01 wt% of FeSO4 and the balance of acidic modified Martin solid culture medium with a pH of 5.0;

[0158] The special liquid culture medium comprises 0.05 wt% of cooperative microbial flora, 0.01 wt% of FeSO4 and the remainder of acidic modified Martin liquid culture medium with a pH of 5.0.

[0159] (4) Preparation of feed matrix: The feed matrix comes from sugarcane bagasse that has been pasteurized at 120 ℃ for 25 s.

[0160] (5) Prepare inoculated feed; under sterile or natural conditions, mix the intestinal microbial flora and the synergistic microbial flora at a ratio of 1:10 based on the bacterial species count, and then inoculate them into the feed matrix at a volume-to-dry weight ratio of 10% mL / g to obtain the inoculated feed. The liquid strain of Alternaria albuminosa is not inoculated, and the strains carried by the secondary adults are vertically subcultured.

[0161] (6) Inoculation of ant seedlings: Select the ant seedling cups where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated.

[0162] (7) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0163] In this example, a total of 300 termite seedlings were inoculated, and 214 seedlings were successfully inoculated, with an average space volume of 2.8±2.2 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generation interruption of the labor grade, and had strong growth and development vitality after stocking.

[0164] Example 10

[0165] This embodiment provides a method for producing a high-vitality termite-Albino symbiotic nest by inoculation on a large scale, comprising the following steps:

[0166] (1) Intestinal microbial flora culture: Black-winged termites O. formosanus The intestinal segments were removed by microdissection, and the intestinal microorganisms in the intestinal segments were inoculated into the intestinal microbial culture matrix under a sterile environment. The pH was adjusted to 5.0 by buffering, and a 2 v% CO2 / N2 mixed gas was filled for replacement protection. The culture was anaerobically cultured at 35 °C for 1 day with stirring at 50 rpm until the bacterial count concentration reached 10 7 When the concentration of the cells was 100 / mL, they were stored at 8°C for future use. After being amplified to 10.0 L at a ratio of 1:10, they were filled with a 5 v% CO2 / N2 mixed gas to replace the anaerobic environment and stored at 8.0°C for future use.

[0167] The method for obtaining the intestinal microorganism culture matrix is ​​as follows: fresh cow stomach or fresh healthy cow dung fed with clean grass is extracted with sterile water to retain the extract, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained and low-temperature pasteurized at 80°C for 15 seconds.

[0168] (2) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the mushroom beds of black-winged termites and shield-pointed ants were inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured for 1 day in a hypoxic environment of pH = 5.0, 31 °C, and 15% O2 until the flora count concentration reached 10 8 / mL, stored at 12℃ for future use;

[0169] The synergistic aerobic bacterial species include Bacillus species A 19%, B 19%, C 19%, D 19% and E 19%, Clostridium species F 4%, and Actinomycetes species G 1%.

[0170] (3) High-purity shield-tipped ant nest umbrella T. eurrhizus Preparation of liquid culture: After the surface of fresh Albizia albuminosa fruiting bodies was disinfected, the internal tissues were dissected and obtained, and inoculated on a special solid culture medium under sterile conditions, and naturally aerobically cultivated at 28°C for 10 days; then the Albizia albuminosa strains purified twice were inoculated into a special liquid culture medium at a weight ratio of 1:50 under sterile conditions, and cultivated at 28°C and 60 rpm for 15 days in a 20 v% low O2 and 2 v% high CO2 gas atmosphere, to obtain a liquid culture of Albizia albuminosa with a mycelium content of 0.01 g / mL;

[0171] The special solid culture medium comprises 0.05 wt% of synergistic microbial flora, 0.01 wt% of FeSO4 and the balance of acidic modified Martin solid culture medium with a pH of 5.0;

[0172] The special liquid culture medium comprises 0.05 wt% of cooperative microbial flora, 0.01 wt% of FeSO4 and the remainder of acidic modified Martin liquid culture medium with a pH of 5.0.

[0173] (4) Preparation of feed matrix: The feed matrix comes from cow dung that has been pasteurized at 80°C for 25 seconds within 1 hour after being fed with clean pure grass.

[0174] (5) Preparing inoculated feed; under sterile or natural conditions, the intestinal microbial flora and the synergistic microbial flora are mixed at a ratio of 1:10 in terms of bacterial species count, and then mixed with the liquid bacterial species of Alternaria albuminosa at a volume ratio of 1:10 to form a bacterial flora mixture, and finally inoculated into the feed matrix at a volume dry weight ratio of 10% mL / g to obtain the inoculated feed.

[0175] (6) Inoculation of ant seedlings: Select the ant seedling cups where black-winged termites have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 5 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated.

[0176] (7) Management after inoculation: The ant nest was encapsulated with a 10 µm thick PE film, and the temperature was controlled to fluctuate between 24 and 29 °C. The humidity was gradually reduced from the initial 18% to 12% within 30 days. After cultivation, a fungus bed was formed in the ant nest.

[0177] In this example, a total of 3000 termite seedlings were inoculated, and 2992 seedlings were successfully inoculated, with an average space volume of 4.6±2.1 mL. The larvae and larvae of all grades and ages of termites developed healthily, with a sustainable ratio, without the risk of generation interruption of the labor grade, and had strong growth and development vitality after stocking.

[0178] Comparative Example 1

[0179] The ant seedling cups of black-winged termites that paired with adults of the symbiotic nest of true root ant umbrellas during seed collection were taken as the ant nests to be inoculated. Under sterile conditions, 5 g of sterilized corn stalk debris were placed on the ant tunnels of the ant nests to be inoculated. The ant nests were sealed with a 10 µm thick PE film, and the temperature was controlled to fluctuate within the range of 24-29 ℃. The humidity was gradually reduced from the initial 18% to 12% within 30 days.

[0180] A total of 500 termite seedlings were inoculated, and 13 seedlings were successfully inoculated. The average space volume of the fungus garden was 1.9±1.3 mL. The larvae and larvae of all grades and ages of termites developed slowly, and the proportion was unsustainable. There was a risk of generational interruption of the labor grade, and they did not have the vitality to grow and develop after being released.

[0181] Comparative Example 2

[0182] The ant seedling cups of Macroptera sphenops ternata that paired with adults of the symbiotic nest of true root ant umbrellas during seed collection were taken as the ant nests to be inoculated. Under sterile conditions, 5 g of sterilized corn stalk debris were placed on the ant tunnels of the ant nests to be inoculated. The ant nests were sealed with 10 µm thick PE film, and the temperature was controlled to fluctuate within the range of 24-29 ℃. The humidity was gradually reduced from the initial 18% to 12% within 30 days.

[0183] A total of 100 termite seedlings were inoculated, and 73 seedlings were successfully inoculated, with an average space volume of 2.2±2.0 mL. The larvae and juveniles of all castes and ages of termites developed moderately, with an unsustainable ratio, and there was a risk of generational interruption of the labor caste, and the growth and development vitality was low.

[0184] Comparative Example 3

[0185] The ant seedling cups of Microcephalus termites that paired with adults of the symbiotic nest of the shield-pointed ant nest were taken as the ant nest to be inoculated. Under sterile conditions, 5 g of sterilized corn stalk debris were placed on the ant tunnel of the ant nest to be inoculated. The ant nest was sealed with a 10 µm thick PE film, and the temperature was controlled to fluctuate within the range of 24-29 ℃. The humidity was gradually reduced from the initial 18% to 12% within 30 days.

[0186] A total of 100 termite seedlings were inoculated, and 68 seedlings were successfully inoculated, with an average space volume of 1.2±0.9 mL in the fungus garden. The larvae and juveniles of all castes and ages of termites developed moderately, with an unsustainable ratio, and there was a risk of generational interruption in the labor caste, and the growth and development vitality was low.

[0187] It can be seen from the above Examples 1-10 and Comparative Examples 1-3 that the inoculation success rate of the termite-Albino symbiotic nest obtained by the method of the present invention is high, the average spatial volume of the mushroom garden is large, and the larvae and larvae of each grade and age develop slowly, the proportion is unsustainable, there is a risk of labor grade interruption, and there is no growth and development vitality after stocking, so it can be used for large-scale inoculation production of high-vitality termite-Albino symbiotic nests.

[0188] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A method for producing high-viability termite-Albino symbiotic nests by large-scale inoculation, characterized in that: The following steps are involved: (1) Intestinal microbial flora culture: The intestinal segments of the cultured termites were microdissected and inoculated into the intestinal microorganism culture matrix under a sterile environment. The pH was adjusted by buffering. Under the protection of CO2 / N2 mixed gas, anaerobically cultured until the bacterial count concentration reached 10 5 ~10 8 pc / mL, store at low temperature; (2) Synergistic microbial flora culture: The synergistic aerobic bacterial strains isolated from the termite-Agaricus collifolius garden are inoculated into a buffered culture medium or a special culture medium supplemented with carbon and nitrogen sources, and cultured under low oxygen until the bacterial count concentration reaches 10 5 ~10 9 / mL, stored at low temperature; (3) Preparation of high-purity liquid culture of Alternaria albuminosa: After the surface of fresh Alternaria albuminosa fruiting bodies is disinfected, the internal tissues are dissected and obtained, and inoculated on a special solid culture medium under sterile conditions. Low-oxygen purification culture is carried out for 3 to 30 days. The Alternaria albuminosa strains after each purification culture are taken and the operation is repeated 1 to 5 times to remove antagonistic bacteria to obtain high-purity Alternaria albuminosa strains; the obtained high-purity Alternaria albuminosa strains are then inoculated into a special liquid culture medium at a weight ratio of 1:10 to 1:100 under sterile conditions, and cultivated in a low O2 and high CO2 gas atmosphere for 3 to 30 days to obtain a liquid culture of Alternaria albuminosa with a mycelium content of 0.001 to 0.1 g / mL, which is inoculated by horizontal subculture method; The special solid culture medium comprises 0.001-1 wt% of synergistic microbial flora, 0.001-0.1 wt% of essential synergistic trace element components and the balance of acidic PDA or improved Martin solid culture medium with a pH of 3.0-7.0; The special liquid culture medium comprises 0.001-0.1 wt% of essential synergistic trace element components and the balance of acidic PDA or modified Martin solid culture medium with a pH of 3.0-7.0; (4) Preparing a feed matrix: The feed matrix comprises component A and component B, wherein component A is a raw material of lignin, hemi-lignin and hemicellulose that can be efficiently utilized by Albizia albuminosa, and component B is a raw material of cellulose and hemicellulose that is suitable for carbon and water energy supply to termites; (5) preparing inoculated feed; mixing the intestinal microorganisms with quantitative bacterial count concentration, the synergistic microorganisms, the purified cultured liquid strain of Alternaria albuminosa with quantitative mycelium content, and the sterilized feed matrix in a specific proportion under aseptic conditions to prepare an inoculated feed with a stable bacterial population, and storing it in a low-oxygen sealed environment; The specific ratio means that the intestinal microbial flora and the synergistic microbial flora are mixed at a ratio of 1:10 in terms of bacterial species count, and then mixed with the liquid bacterial species of Alternaria albuminosa at a volume ratio of 1:10 to form a bacterial flora mixture, and finally inoculated into the feed matrix at a volume dry weight ratio of 10% mL / g; (6) Inoculation of ant seedlings: Select the ant seedling cup where the first generation of termite workers have been active for one day as the ant nest to be inoculated. Under sterile conditions, take 1-20 g of inoculation feed and place it on the ant tunnel of the ant nest to be inoculated for ant seedling inoculation; (7) Management after inoculation: Use a semi-permeable membrane with small holes to seal the inoculated ant nest. Control the moisture content of the ant nest to 8-20%, the oxygen content to 10-25%, and the temperature to 16-32°C. After cultivation, a fungus bed will be formed in the ant nest.

2. The method for producing highly viable termite and Alternaria albuminosa symbiotic nests by scalable inoculation according to claim 1, characterized in that: The method for obtaining the intestinal microorganism culture matrix described in step (1) is as follows: fresh cow stomach or fresh healthy cow dung fed with clean grass is extracted with sterile water to retain the extract, and the extract is filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained and rapidly pasteurized at 60-90°C for 5-20 seconds.

3. The method for producing highly viable termite and Alternaria albuminosa symbiotic nests by scalable inoculation according to claim 1, characterized in that: The intestinal microbial flora described in step (1) can also be obtained from fresh stomachs of cattle fed clean grass or fresh healthy cow dung, specifically: fresh stomachs of cattle fed clean grass or fresh healthy cow dung are taken, extracted with sterile water, and the extract is retained, and then filtered with a bacterial filter membrane after coarse filtration, and the filtrate is retained, and sterile water is added to adjust the bacterial count concentration to 10 5 ~10 9 / mL, fill with O2 / CO2 / N2 mixed gas to replace it into a low oxygen environment, and store it at low temperature.

4. The method for producing highly viable termite and Alternaria albuminosa symbiotic nests by scalable inoculation according to claim 1, characterized in that: In step (3), the surface of the freshly opened Alpinia terrestris fruiting body is disinfected, and then, in a sterile or natural environment, a sterile water-diluted bacterial solution of the synergistic microbial flora is added at a ratio of 0.001 to 1 wt% of the fresh weight of Alpinia terrestris to prepare an Alpinia terrestris tissue suspension, and the pH is adjusted to 3.0 to 7.0 by buffering, and the suspension is directly used for inoculation.

5. The method for producing highly viable termite and Alternaria albuminosa symbiotic nests by scalable inoculation according to claim 1, characterized in that: In step (4), component A is derived from fresh cow dung from clean grass feed pasteurized within 0 to 3 days at 60 to 90°C for 10 to 40 seconds or dead plant material fermented with brown rot fungi sterilized at 100 to 120°C for 1 to 5 hours; Ingredient B comes from fresh cow dung fed with clean grass, which has been sterilized under high temperature and high pressure at 100-120°C for 1-5 hours, or dead plant materials fermented by other fungi.

6. The method for producing highly viable termite and Alternaria albuminosa symbiotic nests by scalable inoculation according to claim 1, characterized in that: The inoculated feed in step (5) may not be inoculated with the liquid strain of Alternaria albuminosa. Instead, the liquid strain of Alternaria albuminosa may be placed on the ant tunnels where termites are active and inoculated separately during the inoculation of termite seedlings in step (6).

7. The method for producing highly viable termite and Alternaria albuminosa symbiotic nests by scalable inoculation according to claim 1, characterized in that: In step (5), the inoculated feed may not be inoculated with liquid strains of Alternaria albuminosa, but may instead be inoculated using strains carried by adult termites for vertical subculture.

8. The method for producing highly viable termite and Alternaria albuminosa symbiotic nests by scalable inoculation according to claim 1, characterized in that: Step (6) can also be performed in a sterile workshop by using an inoculation feed assembly line to add a certain amount of inoculation feed and liquid strains of Alternaria albuminosa to the ant seedling cup.

9. The method for producing highly viable termite and Alternaria albuminosa symbiotic nests by scalable inoculation according to claim 1, characterized in that: The oxygen semipermeable membrane described in step (7) is a PE, PVC, PVDC, or PMP membrane made of non-toxic material with a thickness of 2 to 50 µm, and the ratio of the pore diameter to the ant nest volume is 0.2 to 1.0 mm / 50 mL.