I, Cordyceps and host insects of the history of medicinal use and development of the current situation
Medicinal Cordyceps, the earliest text in the Qing Dynasty Wang Ang's "Cordyceps Preparation" (1694) contained: "Cordyceps, sweet and flat, to protect the lungs and kidneys, stopping blood phlegm, stopping the labor cough. The one produced in Jiadingfu, Sichuan Province is good. Winter in the soil, shaped like an old silkworm, hair can move, to the summer is the hair out of the soil, even the body is turned into grass. If you do not take, to the winter again into insects. Later, Zhao Xuemin's "Compendium of Materia Medica Gleanings" (1756) a book recorded: "summer grass, winter worm, work with ginseng, can cure all the deficiency of the hundred losses," the book also on the origin of Cordyceps sinensis, consumption and dosage of a detailed account. 1757 Wu Yiluo's "Materia Medica from the new" book also pointed out that: "Cordyceps to protect the lungs and benefit the kidneys, stopping the blood and resolving phlegm. " Subsequently, "Qian Nang", "Wenfang Wukao", "Sichuan Tongzhi", "Herbal Diagrams" and other hundreds of ancient medicinal books have recorded Cordyceps.
China's export of Cordyceps sinensis as a medicinal herb to foreign countries has a longer history than that recorded in writing. From the middle of the Ming Dynasty 1400-1465 years from Zhejiang to Japan, and widely consumed in the aristocracy. 1723 by the European missionary Shanggat Liziku from China's Northwest harvested Cordyceps to France, by Reaumur in the French Academy of Sciences of the Bachelor's General Assembly made a presentation, and posted in the minutes; 1943, Berkeley identification of Chinese Cordyceps, officially named: Chinese Cordyceps, the Chinese Cordyceps, the Chinese Cordyceps, the Chinese Cordyceps, the Chinese Cordyceps, the Chinese Cordyceps, the Chinese Cordyceps. Formally named: China Cordyceps Sphaeri sienesis; 1878 by Saccardo categorized as Cordyceps Cordyceps, Cordyceps research in foreign countries to attract attention, China Cordyceps also began to be famous.
Cordyceps enjoys a high reputation because it is only distributed in China, and it is a valuable tonic herbal medicine that has been regarded as comparable to natural ginseng and deer antler by the people of China and Asia for centuries. It has mild medicinal effects, more extensive medicinal and edible than other kinds of tonic, and has long been both medicinal and edible high-level tonic in China, and can be eaten throughout the year, for the old, the young, the sick, the weak, the weak and the weak are suitable to take, without any side effects. Ancient more than 70 Chinese medicine literature to the function of Cordyceps is summarized as: "can Yin and Yang and tonic, the treatment of labor cough diaphragm syndrome, all the weakness of the hundred losses; work with ginseng, deer antler, but the nature of the medicine is mild, the old and young, the sick and the weak are suitable for consumption ......." Since the establishment of new China, China has more than 200 medicinal books recorded in detail the efficacy and use of Cordyceps sinensis, the Department of the Chinese People's Republic of China Pharmacopoeia recorded its function as: "tonifying the lungs and kidneys, stopping bleeding and phlegm. Used for chronic cough and asthma, coughing and hemoptysis, impotence and spermatorrhea, lumbar and knee pain." With the development of medicine at home and abroad and the gradual popularization of traditional Chinese medicine all over the world, it has been found in recent years that Cordyceps sinensis has more and more extensive functional effects. Research has proved that it is a drug with sedative, hemostatic, anticonvulsant, antihypertensive, improving myocardial ischemia, antiplatelet coagulation, antiaging, regulating human immunity and with the function of anti-lung cancer, lymphoma and hepatocellular carcinoma. At present, it has attracted more and more attention from domestic and foreign researchers in the field of pharmacology and biology, and the number of related research reports has increased substantially.
The earliest scientific description and publication of China's Tibetan Plateau is the distribution of moths of the genus Poujade 1886 in Baoxing County, Sichuan Province, the snowy mountains found a moth, was named Hepialus davidi; later Alphe raky, Staudinger scholars in 1889 and 1895 in Tibet, respectively, found the dark moth Hepialus davidi. Later, Alphe raky, Staudinger and other scholars discovered two species of the dark-colored bat moth H. nebulosus (synonym: mud-colored bat moth H. luteus) and the heterochromatic bat moth H. varians in Tibet in 1889 and 1895, respectively; in 1909, Oberthur discovered an adult bat moth on the Armorica Peninsula in France in a collation of medicinal herbs and botanical specimens brought by China from Tibet and Sichuan, and named it H. armoricanus under the name of the peninsula, but published the name H. armoricanus in his book, which is the most important of all. H. armoricanus, but in the publication of the new species, it is stated that the origin of this species of moth may be the Sichuan-Tibet Plateau of China, because the distribution of this species of moth has not been found in the peninsula; later until 1959, researchers from the Institute of Zoology of the Chinese Academy of Sciences collected a large number of adult and larvae of H. armoricanus in Kangding, Sichuan Province, etc., which was identified by Zhu Hongfu as the same species as that of H. armoricanus in Armorica Peninsula of France, and thus named H. armoricanus as the "Cordyceps". It was identified by Zhu Hongfu as the same kind of bat moth found in Armorica Peninsula of France, and thus named as Cordyceps bat moth and identified as one of the host insects of Cordyceps sinensis which is a specialty of China. So far, 37 species of Chinese endemic Cordyceps sinensis host insects have been found on the Qinghai-Tibet Plateau and its fringe areas.
Two, Cordyceps sinensis and moth insects artificial cultivation and industrialization of the status quo
(A) the status quo of artificial cultivation of sexual type of Cordyceps sinensis
Cordyceps sinensis artificial cultivation of the past 20 years in the country has been more than 60 scientific research units and thousands of people carried out a large number of research, experiments and explorations; Yunnan, Sichuan and other places in the high-altitude areas have been in the cultivation of sexual type of small areas of successful experience, but due to the cycle time of the cultivation of sexual type, it is not possible to find out the results of the cultivation. Successful experience, but due to the long cycle, high-altitude areas without the promotion of the object, the investment is large, the benefit is not as high as the natural collection of a variety of factors, has not been able to promote a large area. In Yunnan, Sichuan, Zhejiang, Gansu, Qinghai, Beijing, Fujian, Guangdong, Guizhou, Guangxi and other provinces (municipalities, districts) in the middle and low altitude areas, to carry out artificial cultivation of sexual type of Cordyceps research for many years, although in some parts of a number of has been a number of very encouraging results or achievements, such as Yunnan and Sichuan provinces have been in Kunming, Chengdu and Chongqing, the indoor artificial batch, repeated reproduction of successful mantis moth insects; Zhejiang Zhejiang and other places in the indoor sporadic reproduction of successful moth insects; but due to the lack of solution to the insect and bacteria inoculation and batch, repeated growth of cysticercus of the difficulties, can not be called cultivation success. From a small area, small batch, many times repeated at low altitude artificial cultivation of sexual type of Cordyceps are still very far away from the goal; although several have claimed in the middle and low altitude batch cultivation of successful sexual type of Cordyceps, in fact, the scientific credibility of the extremely low. Because:
(1) At present, there is no one can be long-term and stable isolation and cultivation to the authentic Cordyceps fungi (i.e., repeatedly in the medium of batch growth of the pedestal). Qingdao, Yunnan, Sichuan, Beijing and other places in the medium have been cultivated in the ascospores, but its ascospores sparse or no ascospores, and the growth of all unstable, can not show that has mastered the sexual type of artificial cultivation technology at low and medium altitudes.
(2) Cordyceps sinensis asexual type artificial cultivation technology is relatively easy to master, inoculated indoors to the manta moth larvae can also be infected to become a stasis stage, but it is indeed rare to have completed the sexual type, the growth of ascospores and ascospores.
(3) At present, in addition to the formation of ascospores, altitude, climate, soil, air pressure and other environmental factors, the bacteria between the bacteria is a multi-bacterial complex infection? If it is a compound infection, and which some bacteria are playing a key role? In what period of time and Cordyceps bacteria play a role? And so on is not clear.
Therefore, to artificial batch and repeated cultivation of sexual type of Cordyceps sinensis in low and medium altitude areas, still need to do a lot of research and experimental work. It is the large-scale artificial cultivation of Cordyceps in high-altitude areas, also need to carry out a lot of technical experimental work. So the artificial cultivation of sexual type Cordyceps to achieve large-scale or industrialized production, research is still very arduous, the journey is still very far away.
(II) Asexual Cordyceps artificial cultivation status
Because of the artificial cultivation of sexual Cordyceps is too difficult, in the past 10 years, most of the artificial cultivation of Cordyceps research units and researchers have turned to the asexual type of research and application. As the asexual type cultivation technology of Cordyceps sinensis is easy to master, and the fungi isolated from the natural collection of Cordyceps sinensis, the vast majority of physicochemical properties and pharmacological functions are similar to the sexual components of Cordyceps sinensis, so all over the world have identified the self-isolated fungi as the asexual stage of the fungus of Cordyceps sinensis and have built factories to produce fungus powder and development and application. At present has been isolated from the natural body of Cordyceps sinensis from I think is the Chinese Cordyceps sinensis asexual type *** there are 9 genera and 31 species, of which there are 16 species have been in Beijing, Jilin, Shanxi, Shanghai, Qinghai, Zhejiang, Fujian, Jiangxi, Guangdong, Yunnan, Guizhou and other 19 provinces (municipalities) more than 40, deep large-scale fermentation cultivation and solid culture of mycelium and conidiophores as a sexually oriented type of Cordyceps sinensis Substitute products, some manufacturers have entered into large-scale industrialized production, such as Zhi Ling capsule, Jin Shui Bao capsule, Ning Xin Bao capsule, Heart and Liver Bao, Cordyceps sinensis chicken essence, Cordyceps sinensis capsule, Cordyceps sinensis wine, Cordyceps sinensis ginseng tonic wine and so on, more than 20 kinds of products annual output value reached ten million to hundreds of millions of yuan. Moreover, due to the short cycle and high yield of artificial reproduction with mycelium and hyphal gowns, the potential of development and application as a substitute of Cordyceps sinensis sexual type in the future is still great.
Three, Cordyceps sinensis and bat moth insect artificial cultivation technology
(a) Cordyceps sinensis Yin host insect feeding and breeding technology to carry out the artificial cultivation of Cordyceps sinensis, first of all, must be a large number of artificial feeding of the host of a bat moth insects. Moths live 98% of their life in the soil, and only 2.5-13d of their adult life on the ground. Therefore, the artificial feeding management of these insects is mainly carried out in the soil.
1. Bat moth rearing tools
(1) Bat moth egg rearing tools. There are two ways to raise bat moth eggs, one is to use a glass Petri dish with a diameter of 15-20cm, padded with 2-3 layers of filter paper, and put a piece of absorbent cotton inside the dish to keep the humidity, and each Petri dish can be used to raise 200-500 eggs. This method is conducive to observation, but it is difficult to grasp the humidity. The second is to use l0cm x XX5-8cm wide-mouth bottle or 25cm x 30cm glass feeding cylinder, built-in 3-5cm thick sifted fine humus, planted with 1 or 2 plants, put the eggs in the soil surface to the lcm depth of the soil layer, each bottle can be raised 300-1000 eggs; feeding as long as the plants do not wither, you can not add humidity. This method of egg hatching rate is high, but observation and transplantation of larvae on the more difficult.
(2) There are four kinds of larval rearing tools as follows.
a. Glass rearing jar: 18 a 25cmX30 a 35cm glass jar, can also be used for specimen immersion tank, placed in the soil 15 a 25cm, planted with larvae to feed on the plant, the newly hatched larvae moved into the tank, each tank can be reared 10 a 30 head of larvae.
b. Underground bug box: the box is generally 30-50cm high, 20-25cm wide, 3-5cm thick. the skeleton of the feeding box is welded with rebar, above the rebar made into a handle, easy to lift; wide on both sides of the 3-5mm glass, easy to observe the activities of the bugs; the bottom and the two sides of the copper gauze mesh fitted with 16 holes to facilitate the water infiltration. Then loaded with soil, planting fodder plants, each box stocked with 20 to 50 head of larvae; in the more solid ground dug a slightly larger than the size of the feeding box and deep groove, put the box into the groove, observation of the box will be raised to the ground, wipe off the appearance of the glass on the soil, you can clearly see the activities of the larvae.
c. Large-scale insect cage: in a semi-natural situation, choose a well-drained, loose soil site, digging a long 12 l5m, 2-3m wide, 4Ocm deep groove, the bottom and around the nylon mesh pads (preferably with a double-layer, small insects are not easy to drill), and then shoveled into the loose humus to fill in the loose humus, planted with larval food, into the larvae rearing, per square meter can be stocked with 30 to 50 heads! The method is favorable for large-scale rearing, but observation is difficult.
d. Use all kinds of pots and pans for naked rearing: wash the plant feed into the pots and put the larvae into rearing, with the mouth of the pots and pans covered with a layer of black cloth or black paper to cover the light. This method is good for life history observation, but attention should be paid to changing feed and removing feces.
(3) Pupa and adult rearing tools. Make a 50cm long, 50cm wide, 55-60cm high insect rearing box. Bottom with a thin iron plate or double-layer copper gauze mesh bottom; top and around the iron or nylon gauze cover, the front can be made into a movable gauze door. In the bottom of the box into 10-20cm of soil, planting plants, the pupa and adult insects in the soil, this method can be densely planted rearing.
2. Technical points of rearing
(1) Soil selection. Soil is the most important place for manta moth insects to move around in their lives. Whether the soil conditions are suitable for the life of the insect is one of the keys to the survival rate of artificial rearing. The most suitable for the growth of the moth is the alpine meadow soil; the second is the mixed soil of the driftstone beach and the meadow; the second is the alpine brown, dark brown forest soil, and other soils are not good for the growth of this insect.
(2) Selection of food. The larvae like to feed on the young roots and buds of plants such as round spikenard, pearl bud polygonum, astragalus, small rhubarb, and so on. As the moth insect is a class of omnivorous insects, in the absence of the above plants, available grass (such as barley, wheat, grain buds, etc.); cruciferous, salvia young root feeding. In the middle and low altitude (suitable for living temperature) with soil rhubarb, carrots, carrots, white potatoes (sweet potatoes), potatoes, apples and other feeding can also be normal growth and development, the best for the soil rhubarb, carrots and white potatoes, three types.
(3) Selection of temperature and humidity. Moth insects are a class of low-temperature resistant and afraid of high temperature insects. Best suited to live and grow in 15 a 19C; this insect is best suited to air humidity 75% a 85%, soil moisture content between 40% a 45%.
3. Management techniques
(1) Adult stage. Adult moths do not take any food, as long as the moisture and suitable temperature can maintain normal survival and egg laying. However, the moth habitat is related to the amount of eggs laid. Adult moths like to choose the roots of Polygonum and other plants that have azalea and bat moth larvae to feed on to lay eggs, so it is best to plant a few related plants before the moths mate and lay eggs; in addition, there should not be strong light effects.
(2) Egg stage. To make the eggs can hatch normally, must master the appropriate temperature and humidity.
(3) Larval stage. Larvae due to the long growth period, and the body wall is thin and easy to break, rearing must be careful, in addition to the necessary observation, change the soil and change the food, minimize turning. The key to larval rearing is the first hatchling period, when the first hatchlings should be given enough fine and tender food with low humidity; the soil should preferably be sifted so as not to bruise the main body with coarse and hard objects. In addition, in order to prolong the food and soil not moldy, before replacing with ultraviolet light irradiation sterilization l5min, soil can also be exposed to the sun 1 a 2h before use.
(4) Pupal stage. When raising pupae, the soil in the pupae raising vessel is compacted, and then a soil chamber is artificially created with a stick with the thickness of the pupa, the soil chamber is slightly slanted, about 2-3cm deep, and one pupa is placed in each chamber, and when placing the pupa, the pupa's head is directed to the surface of the soil, and the upper part is covered with a fine layer of soil. The temperature and humidity of the pupa can be controlled in the atmospheric humidity of 80% 85%, soil moisture content of 42-45%, the temperature between 18-22 ℃.
[Two] Cordyceps sinensis bacteria artificial culture technology
To obtain pure winter early summer grass strains must be artificial separation, purification, rejuvenation, preservation and other work.
1.Technology and conditions for isolation of Cordyceps sinensis bacteria
(1) Material requirements. Obtaining pure strains of bacteria has strict requirements for materials; generally speaking, if it is to carry out the method of isolation of Cordyceps sinensis tissue block, the best isolation material is the end of October to November every year, the Tibetan Plateau alpine meadow soil just began to freeze the period of the material, this period of time Cordyceps sinensis has just infected the moth larvae of bats to enter the rigor mortis stage for a short period of time, the young small cyst buds have just grown out of the head of the worm 0.2-0.5cm, the body other stray bacteria are less, and the worm is most likely to be infected. The other stray bacteria in the body are less, it is the easiest to separate. If the material is taken in May-June of the following year, there are many kinds of stray bacteria **** or putrefactive on the body and the ascospores of the stiffworm, it is difficult to obtain pure strains. If the ascospores for isolation culture, the material is best in the middle and late July each year, the ascospores just have part of the maturity of the collection of isolation culture.
(2) Operation method. There are 3 steps as follows.
a. Isolation of the stipe body (i.e. nucleus): before isolation, brush the surface of the main body clean with water, and then wash it with sterile water for 2 or 3 times; use 0.1% and 0.2% of mercuric chloride solution to disinfect the surface of the isolation material for about 3 or 5 min; and then wash it with sterile water. The anterior part of the segment bounded by the thoracic peduncle was selected. Cut off the epidermis with a scalpel, avoiding the digestive tract, take the blood cavity mycelium and cut it into the size of sesame grains, and press it into the plate culture medium, 1 or 2 grains per dish. Placed in 15 a 19 ℃ in the culture, until the colony grows 0.2 a 0.5 cm, select a small number of mycelium repeatedly in the plate medium purification 2 a 4 times, to determine no other stray bacteria, moved into the test tube preservation and culture.
b. Separation of ascospore buds: cut the washed ascospore buds from the top of the head of the stiffworm, put them into 0.1% mercury solution to disinfect for 2-3min, wash them with sterile water, and then cut the middle part of the tissue block and press it into the medium; the culture conditions are the same as those of a.
c. Separation of ascospores: use a transparent paper bag to put over the ripening ascospores and let the ascospores bounce and paste on the paper bag, then the paper bag with ascospores was immersed into a paper bag with a clear paper bag. A slide was placed underneath to maintain the humidity of the main body at all times; daily microscopic examination was made, and when the ascospores were seen to pop onto the slide, they were sucked with a micropipette into the medium of the flat dish for cultivation; the cultivation conditions were the same as those of a.
(3) Selection of culture medium.
a. Potato dextrose agar medium (PDA), this formula is a common medium for all Cordyceps spp. fungi, which can be used in the early stage of isolation culture, but the growth of strains is not very vigorous, and it is easy to aging and degradation.
b. Gafu medium (1): polysexual protein umbilical cord 10g, dextrose 50g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, 30g of live silkworm pupa, growth hormone 0.5μg, 20g of agar, add 1000mL of water, pH: 5.0.
Gafu medium (Ⅱ): protein proteins 40g, 40g of dextrose, peeled fresh potato 100g, 1g of dipotassium hydrogen phosphate, 1g of potassium sulfate, 1g of potassium sulfate, 1g of potassium sulfate, 1g of potassium sulfate, 0.5μg of growth hormone, agar 20g, add 1000mL of water, pH: 5.0. 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 10g of beef paste, 0.5μg of growth hormone, 30g of live larvae of bat moth (grinded), 20g of agar, 10OOmL of alpine meadow soil leaching solution, pH: 5.0.
On the Gafu medium, the colonies grew more vigorously and rapidly than on the PDA medium; the Gafu Ⅱ was better than the Ⅰ.
2. Growth characteristics of Cordyceps sinensis in the isolation of culture must grasp the temperature, pH, light, carbon and nitrogen sources and the utilization of inorganic salts.
(1) The effect of different temperatures on mycelial growth. Cordyceps fungus likes low temperature. In 0 a 4 ℃ can grow slowly; 5 a 8 ℃ when the growth rate accelerated; 10 a 19 ℃ is the more suitable growth and development of this fungus range; the optimal growth temperature of 15 a l9 ℃. More than 20 ℃ mycelium fierce growth, colonies from white to gray-black or brown-yellow, began to appear mutation, so it is not suitable for high-temperature culture.
(2) the requirements of pH value. Cordyceps sinensis is a kind of acidic fungi, the optimal pH value of 5.0 a 6.0, when the pH value of 4.5 or less and 6 or more, with the increase or decrease, the growth is slow to no growth.
(3) the effect of light. The early stage of ascospore germination and mycelial growth of this fungus is adapted to low light and short light, and the later stage is adapted to stronger light. In artificial culture, mycelium, conidia and ascospores have obvious phototropism, the sunny side of the growth of more and dense, the backlit side of the growth of sparse, all-black environment under the cultivation of a variety of mycorrhizae are weak, slender, sparse.
(4) The utilization of carbon source. Cordyceps sinensis can utilize a variety of carbon sources, but the fastest growth when glucose and maltose are combined, and good growth when glucose alone is used, followed by starch such as potato, and again sucrose.
Cordyceps sinensis is a kind of fungi by parasitic living host, and then by the putrefactive stage of development and maturity, so the nitrogen source of a variety of organic nitrogen can be better utilized. And the best to live worm body; peptone and yeast paste combined growth is also very ideal, followed by the two alone, and again the beef paste and so on. And inorganic nitrogen such as ammonium sulfate, potassium nitrate and other poor utilization.
There is a certain demand for ash nutrients, there are traces of magnesium sulfate, dipotassium hydrogen phosphate in the medium of growth, followed by potassium dihydrogen phosphate, other sodium, calcium, iron, copper and other inorganic salts can also be used.
3. Cultivation mode After the isolation and purification of Cordyceps sinensis bacteria, it can be used as a variety of experimental and expanded production applications.
(1) There are often three methods of its expanded production: solid static culture method, oscillation culture method and large tank aeration fermentation culture method.
a. Static culture: mainly used in solid culture, such as test tube slant, triangular bottle culture, rice culture and so on. In the static culture, as long as the temperature and light can be mastered to make the normal growth of bacteria. When the conidia on the slant are mature, they can be stored in the refrigerator at 1-2℃ for 8-14 months, and can also be used as seeds directly for production.
b. Oscillatory culture: the use of liquid culture strains or small-scale propagation culture, can be used for oscillatory culture. Oscillatory culture medium, the solid medium minus agar can be. The best choice of equipment for constant temperature oscillation culture machine, with a triangular bottle of liquid culture medium, the test tube of solid strains can be accessed to culture. After constant oscillation so that the liquid medium of various components mixed uniformly without precipitation, while promoting gas and liquid contact and exchange, so that oxygen into the liquid medium, conducive to the growth of mycelium and conidia formation.
c. Large tank aeration fermentation culture: in the large-scale production of mycelium and conidia and other bacterial powder, must be used in large tank aeration fermentation culture method. The method of aeration is the use of suction or vacuum pump depressurization and other methods, after the filter in addition to impurities, sent into the tank liquid medium for the growth and development of Cordyceps sinensis.
(2) Fermentation process. Natural Cordyceps isolation and purification → test tube slant seed → oscillator liquid primary seed → secondary seed → small seed fermentation seed tank → production fermentation tank → concentration → spray powder drying → product powder.
Cordyceps mushroom tank fermentation requirements temperature at 20-25 ℃, tank pressure 392.3 686.5kPa (0.4 0.7kg/cmz), the aeration amount of 0.5 1.OVVm *; injected into the fermentation tank for the liquid medium of 65% of the tank capacity of 75% of the appropriate. Inoculation amount of 10%, stirring speed of 180r/min, culture time of 72 a 96h, you can release the tank concentration and spray powder.
(3)Fermentation medium multi-use. Fresh potato (peeled) 8%, sucrose 2%, corn starch 0.5%, silkworm pupa powder 1%, peptone 0.4%, ammonium sulfate 0.2%, pH 5.5 a 6.0.
(4) fermentation culture finished product standards. Conidia that a few full shedding parent spore peduncle, spore peduncle number increase is not obvious, counted under the microscope check, each milliliter contains 1.8 billion 2.5 billion conidia; residual sugar is less than 1%; amino nitrogen is less than 0.2mg/mL, can be a finished product of the bacterial liquid.
4. Cordyceps sinensis back to the main points of the sexual type or asexual type of Cordyceps sinensis spores back to the host insect body, is the artificial cultivation of sexual type of Cordyceps sinensis and rejuvenation of asexual strains of key measures.
(1) Rejoining period. Cordyceps sinensis back to the key in the selection of host insects antibacterial weak period, one is the molt period; the second is the larvae feeding activity is intense, friction damage rate is high period.
(2) Rejoining method. It is further divided into two kinds.
a. Spray method: when the rearing of bat moth larvae up to 4-6 instars, more than 1/3 of the larvae molt, the larvae are concentrated, with the ascospores or conidia into the 5% 10% glucose solution sprayed to the body or food, inoculated about 30min, the fungal liquid in the body of the insect is slightly dry, the larvae back to the soil to let the natural life. This method has a high susceptibility rate, but the disadvantage is that the larvae are not easy to take out of the soil concentration, and concentration of the larvae have the habit of killing each other, the insect body is bitten, easy to be infected by the death of the fungus, Cordyceps sinensis mycelium is not full of body cavities, the injured insects will rot, can not form the stalemate (nucleus).
b. Natural contact method: in a large area of semi-natural soil rearing bat moth larvae, regularly take small samples to observe the growth of larvae, when most of the larvae are suitable for disease susceptibility, planting with fodder plants dipped in bacterial solution and mixing bacteria with fine soil inoculation, evenly sprinkled into the surface of the insect, and then sprayed with water, so that the bacteria seeped into the soil, and the larvae are infected by contact with the feeding activities. This method of host infection rate is not too high, but once infected with disease, Cordyceps sinensis fungus grows well, and the uninfected bat moth larvae can grow normally and reproduce offspring.