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Cordyceps sinensis, also known as Cordyceps sinensis, is referred to as Cordyceps sinensis. At present, it has been reported in the world that there are more than 400 kinds of Cordyceps which are parasitic on insects, spiders and other organisms and produce fruiting bodies. 68 species have been recorded in China. In the concepts of some people and scholars at home and abroad, all combinations of fungi parasitic by Cordyceps and capable of producing fruiting bodies are generally called Cordyceps sinensis. However, the traditional Chinese medicine in China, the vast majority of scholars and people in China and the Cordyceps referred to in this paper refer to the combination of insects and bacteria formed by the parasitic fungus Cordyceps sienesis (Berkeley) saccardo (1878), which is only distributed in alpine meadows in the Qinghai-Tibet Plateau and marginal areas of China, on the larvae of Entomorphia, Lepidoptera, Hepialus. ; Other groups are only called Cordyceps sinensis, not authentic Cordyceps sinensis.
First, the medicinal history and development status of Cordyceps sinensis and host insects
Medicinal Cordyceps sinensis, the earliest text can be found in Wang Ang's Compendium of Materia Medica (1694) in Qing Dynasty: "Cordyceps sinensis is sweet, which can protect lung and kidney, stop bleeding and phlegm, and stop fatigue and cough. Those produced in Jiading, Sichuan are the best. Winter is in the soil, shaped like an old silkworm, with hair moving, and when it is unearthed in summer, it turns into grass. If you don't take it, it will turn into a worm in winter. Later, Zhao Xuemin's "Compendium of Materia Medica" (1756) recorded: "Cordyceps sinensis has the same merits as ginseng, which can cure all deficiencies", and the book also described the origin, eating method and dosage of Cordyceps sinensis in detail. 1757 Wu yiluo's book "materia medica from the new" also pointed out: "Cordyceps sinensis protects lung and kidney, stops bleeding and eliminates phlegm." Later, hundreds of ancient Chinese medicine books, such as Qiannang, Textual Research of the Literatures, Sichuan Annals and Materia Medica, recorded Cordyceps sinensis.
The history of exporting Cordyceps sinensis as a medicinal material to foreign countries in China is longer than that recorded in writing. From the middle of Ming Dynasty 1400-1 1465, it spread from Zhejiang to Japan and was widely eaten by nobles. 1723 Cordyceps collected from northwest China was brought to France by the European missionary Jean-Gartley Tzku, and was introduced by Reaumur at the bachelor's conference of French Academy of Sciences, which was recorded in the minutes of the meeting; 1943, Berkeley identified Cordyceps sinensis in China, which was officially named as China Cordyceps sinensis; 1878 was classified as Cordyceps by Saccardo, and the study of Cordyceps sinensis attracted attention abroad, and Cordyceps sinensis in China became famous in the world.
Cordyceps sinensis enjoys a high reputation because it is only distributed in China, and it has been regarded as a precious nourishing Chinese herbal medicine comparable to natural ginseng and velvet antler by Chinese and Asian people for hundreds of years. It has mild medicinal effects, and is more widely used for medicine and food than other kinds of tonics. It has long been a high-grade tonic that can be used for medicine and food in China, and can be eaten all year round. It is suitable for the old, the young, the sick, the weak and the weak without any side effects. More than 70 ancient Chinese medicine documents summarized the functions of Cordyceps sinensis as follows: "It can supplement yin and yang, treat fatigue and cough, and all kinds of deficiency and all kinds of damage;" Its merits are the same as those of ginseng and velvet antler, but its properties are mild, and it is suitable for people of all ages who are sick and weak ... " Since the founding of the People's Republic of China, there are more than 200 drug books in China that record the efficacy and use of Cordyceps sinensis in detail. The Pharmacopoeia of the People's Republic of China records its functions as "tonifying lung and kidney, stopping bleeding and resolving phlegm". It can be used for chronic cough and asthma, cough and hemoptysis, impotence and nocturnal emission, and soreness of waist and knees. " With the development of medicine at home and abroad and the gradual popularization of traditional Chinese medicine in the world, it has been found that the functions and effects of Cordyceps sinensis are more and more extensive in recent years. Studies have proved that it has the functions of calming, stopping bleeding, anticonvulsant, lowering blood pressure, improving myocardial ischemia, resisting platelet coagulation, resisting aging, regulating human immunity and resisting lung cancer, lymphoma and liver cancer. At present, it has attracted more and more attention from researchers in pharmaceutical and biological fields at home and abroad, and the relevant research reports have increased greatly.
The earliest scientific description and publication of the distribution of the genus Hepialus on the Qinghai-Tibet Plateau in China was that Poujade/Kloc-0 of Britain discovered a species of Hepialus davidi； on the snowy mountain in Baoxing County, Sichuan Province in 886. Later, scholars such as Alpharaky and Staudinger discovered two species, H.nebulosus (homonym: H.luteus) and H.varians, in Tibet in 1889 and 1895 respectively. 1909 Oberthur found an adult bat moth in the medicinal materials and plant specimens brought from Tibet and Sichuan in China on the French Armorica Peninsula, and named it H.armoricanus after the name of the peninsula. However, when the new species was published, it was indicated that the origin of this bat moth may be the Sichuan-Tibet Plateau in China, because it has never been found in the peninsula. Later, until 1959, researchers from the Institute of Zoology, Chinese Academy of Sciences collected a large number of adults and larvae of hepialus in Kangding, Sichuan and other places, which were identified by Zhu Hongfu as the same species as those found in Armorica Peninsula, France, so they were named Cordyceps hepialus and identified as one of the host insects of Cordyceps sinensis, a specialty of China. Up to now, 37 host insects of Cordyceps sinensis endemic to China have been found in the Qinghai-Tibet Plateau and its marginal areas.
Second, the status quo of artificial cultivation and industrialization of Cordyceps sinensis and hepialus insects
(A) the status of artificial cultivation of sexual Cordyceps sinensis
In the past 20 years, more than 60 scientific research units and thousands of people have carried out a lot of research, experiments and explorations in the artificial cultivation of Cordyceps sinensis. Yunnan, Sichuan and other places have achieved successful experience in cultivating sexual types in small areas at high altitudes, but due to many factors such as long cycle, lack of promotion targets, large investment and lower benefits than natural collection in high altitudes, it has not been widely promoted. In the middle and low altitude areas of Yunnan, Sichuan, Zhejiang, Gansu, Qinghai, Beijing, Fujian, Guangdong, Guizhou, Guangxi and other provinces (cities, districts), the research on artificial cultivation of sexual Cordyceps sinensis has also been carried out for many years, although many of them have achieved very gratifying results in some aspects, such as Yunnan and Sichuan provinces, which have successfully propagated the moth insects in Kunming, Chengdu and Chongqing. In Zhejiang and other places, bat moth insects have been successfully propagated indoors; However, it can not be called successful cultivation because it has not solved the difficulties of inoculation of insects and bacteria and batch and repeated growth of daughter bodies. It is still far from the goal of artificially cultivating sexual Cordyceps sinensis in small area, small batch and repeated times at low altitude. Although several companies have claimed to have successfully cultivated sexual Cordyceps sinensis in batches at middle and low altitudes, in fact, the scientific credibility is extremely low. Because:
(1) Up to now, no one has been able to isolate and culture authentic Cordyceps fungi for a long time and stably (that is, repeatedly grow a lot of stroma on the culture medium). In Qinghai, Yunnan, Sichuan, Beijing and other places, the stroma has been cultivated, but its ascospores are few or no, and the growth is unstable, which does not mean that the cultivation technology of sexual artificial in middle and low altitude has been mastered.
(2) The artificial asexual culture technology of Cordyceps sinensis is relatively easy to master, and it can be infected into a stiff stage when inoculated into hepialus larvae indoors, but it is rare to complete sexual forms and grow stroma and ascospores.
(3) Besides altitude, climate, soil, air pressure and other environmental factors, is there a multi-bacterial infection among the bacteria? If it is a compound infection, which bacteria are playing a key role? In what period did it work with Cordyceps sinensis? Wait, the question is not clear.
Therefore, a lot of research and experimental work are needed to cultivate sex Cordyceps sinensis in batches and repeatedly in middle and low altitude areas. Even if large-scale artificial cultivation of Cordyceps sinensis is carried out in high altitude areas, many technical experiments need to be carried out. Therefore, the artificial cultivation of sexual Cordyceps sinensis is still very arduous and far away to achieve large-scale or industrial production.
(2) Current situation of artificial culture of asexual Cordyceps sinensis
Because the artificial cultivation of sexual Cordyceps sinensis is too difficult, in recent years, most units and researchers who have carried out artificial cultivation of Cordyceps sinensis have turned to the research and application of asexuality. Because the asexual culture technology of Cordyceps sinensis is easy to master, and most of the fungi isolated from Cordyceps sinensis collected in nature have similar physical and chemical properties and pharmacological functions to the sexual components of Cordyceps sinensis, so all localities have identified the self-isolated fungi as the asexual stage of Cordyceps sinensis, and have set up factories to produce bacterial powder and develop and apply them. At present, there are 9 genera, 3 1 species isolated from Cordyceps sinensis in nature, among which 16 species have been found in Beijing, Jilin, Shanxi, Shanghai, Qinghai, Zhejiang, Fujian, Jiangxi, Guangdong, Yunnan, Guizhou and other provinces (kloc-0/9) Submerged large-scale fermentation culture and solid culture of mycelium and conidia are used as substitutes for sexual Cordyceps sinensis, and some manufacturers have entered large-scale industrial production, such as Zhiling capsule, Jinshuibao capsule, Ningxinbao capsule, Xinganbao, Cordyceps sinensis chicken essence, Cordyceps sinensis capsule, Cordyceps sinensis wine, Cordyceps sinensis ginseng tonic wine and other more than 20 kinds of products with annual output value reaching 10 million yuan to several hundred million yuan. Moreover, because of the short artificial propagation cycle and high yield, mycelium and meristem robe have great potential for development and application as sexual substitutes of Cordyceps sinensis in the future.
Three, Cordyceps sinensis and hepialus insect artificial cultivation technology
(I) Host Insect Feeding and Propagation Techniques of Cordyceps sinensis To carry out artificial cultivation of Cordyceps sinensis, a large number of hosts-hepialus insects must be artificially raised first. Hepialidae lives in the soil for 98% of its life, and only 2.5 13d adults live on the ground. Therefore, the artificial feeding management of such insects is mainly carried out in the soil.
1. Feeding tools for bat moths
(1) Feeding tools for hepialus eggs. There are two ways to raise hepialus eggs. One is to use a glass culture dish with a diameter of 15-20cm, pad it with 2-3 layers of filter paper, and put a piece of absorbent cotton in the dish to keep the humidity. Each dish can raise 200-500 eggs. This Faliu observation, but it is difficult to grasp the humidity. Second, use l0cm xX5-8cm jar or 25cm x 30cm glass feeding jar, put 3-5cm thick sifted fine humus soil, plant 1 1 2 plants with few bulbils, and put the eggs in the soil layer from the soil surface to the depth of lcm, each bottle can feed 300 eggs 1000; When feeding, as long as the plants are kept from wilting, no humidity can be added. This method has a high hatching rate, but it is difficult to observe and transplant larva pairs.
(2) There are four kinds of feeding tools for larvae.
A. Glass insect tank: use a glass tank of 18-25cmX30-35cm, or use a specimen soaking tank, put soil of 15-25cm, plant the larvae to feed on, and move the newly hatched larvae into the tank. Each tank can raise 10-30 larvae.
B. Underground insect rearing box: The box is generally 30-50cm high, 20-25cm wide and 3-5cm thick. The frame of the feeding box is welded with steel bars, and the steel bars on it are made into handles for easy lifting; The wide two sides are made of 3-5mm glass, which is convenient for observing insect activities; The bottom and two sides are fitted with 16 mesh copper gauze to facilitate moisture infiltration. Then loading soil, planting feed plants, and stocking 20 to 50 larvae in each box; Dig a trough slightly larger and deeper than the rearing box on the solid ground, put the rearing box into the trough, lift the rearing box out of the ground during observation, and wipe off the soil on the outer glass, so as to clearly see the larval activity.
C. Large-scale insect cage: under semi-natural conditions, select a site with good drainage and loose soil, dig a deep groove with a length of 12-15m, a width of 2-3m and a depth of 4Ocm, and pad the bottom and the periphery with nylon gauze (it is best to use double layers, so that insects are difficult to get out), then shovel the loose humus soil to fill it up, plant larval food, and put it into larval breeding. This method is beneficial to large-scale feeding, but it is difficult to observe.
D, bare feeding with various pots: the plant feed is washed and put into the pot, and larvae are put into the pot for feeding, and the mouth of the pot is covered with a layer of black cloth or black paper for shading. Observe the life history of Faliu, but pay attention to changing feed frequently and removing feces.
(3) Feeding tools for pupae and adults. Make an insect box with a length of 50cm, a width of 50cm and a height of 55-60cm. The bottom is sealed with thin iron plate or double-layer copper gauze; The top and periphery are covered with iron yarn or nylon yarn, and a movable gauze screen door can be made in front. Put 10-20cm soil at the bottom of the box, plant and put pupae and adults in the soil. This method can be used for close planting and feeding.
2. Key points of feeding technology
(1) Selection of soil. Soil is the most important activity place of hepialus in its life. Whether the soil conditions are suitable for the life of the insect is one of the keys to the survival rate of artificial feeding. Alpine meadow soil is the most suitable for the growth of hepialus; Followed by mixed soil of flowing stone beach and meadow; The third is the brown and dark brown forest soil in the mountains, and other soils are not good for this insect.
(2) the choice of food. Larvae like to eat tender roots and buds of Polygonum viviparum, Polygonum viviparum, Astragalus membranaceus and Rhubarb. Because hepialus is an omnivorous insect, Gramineae (such as highland barley, wheat, rice sprouts, etc.) can be used in places where there are no such plants. Tender root breeding of Cruciferae and Cyperaceae. In the middle and low altitude areas (at the temperature suitable for living), it can also grow normally when fed with rhubarb, carrot, radish, sweet potato (sweet potato), potato, apple, etc., and it is best to use rhubarb, carrot and sweet potato.
(3) Selection of temperature and humidity. The hepialid insect is a kind of insect that is resistant to low temperature and afraid of high temperature. It is most suitable for living and growing in 15-1 19C; This insect is most suitable for air humidity of 75%-85% and soil water content of 40%-45%.
3. Management technology
(1) Adult stage. Adults do not eat any food, but can survive and lay eggs normally as long as they keep moisture and appropriate temperature. But the moth habitat is related to the amount of eggs laid. Adults prefer to lay eggs by the roots of plants such as Polygonaceae, which are fed by rhododendrons and hepialus larvae, so it is best to plant several related plants before moths mate and lay eggs. In addition, there should be no strong light.
(2) the egg stage. In order to hatch eggs normally, we must master the appropriate temperature and humidity.
(3) larval stage. Because of the long growth period and the thin wall of the larva, it must be carefully raised, except necessary observation, soil change and food change, and try to reduce the turnover. The key to the rearing of larvae is the young period of initial hatching, and the newly hatched young worms should be given enough fine and tender food with low humidity; It is best to screen the soil so as not to scratch the main body with coarse and hard objects. In addition, in order to prolong the food and soil from mildew, ultraviolet lamp should be used for sterilization for l5min before replacement, and the soil can also be exposed to the sun 1 2h before reuse.
(4) pupal stage. When raising pupae, the soil in the pupa-raising vessel is compacted, and then a soil chamber is artificially built with a wooden stick the thickness of the pupa. The soil chamber is slightly inclined and about 2-3cm deep, and a pupa is placed in each chamber. When putting the pupa, the pupa head faces the soil surface and the top is covered with fine soil. The temperature and humidity of pupa can be controlled between 80%-85% of atmospheric humidity, 42-45% of soil moisture and 18-22℃.
[2] Artificial culture technology of Cordyceps sinensis
In order to obtain pure strains of winter, early summer and grass, manual separation, purification, rejuvenation and preservation must be carried out.
1. Techniques and conditions for isolating Cordyceps sinensis.
(1) Material requirements. Obtaining pure strains requires strict materials; Generally speaking, if the tissue block separation method of Cordyceps sinensis is used, the best separation material is the material collected from the end of1October to1month every year, when the soil of alpine meadow in Qinghai-Tibet Plateau just started to freeze. In this period, Cordyceps sinensis has just infected hepialus larvae and entered the period of zombie for a short time, and the tender daughter bud just grew 0.2-0.5cm in the head of the insect. If the material is collected in May and June of the following year, there are many kinds of miscellaneous bacteria living or saprophytic in the body and sub-seat of the stiff worm, so it is difficult to obtain pure strains. If the ascospores are used for isolation and culture, it is best to collect and isolate the materials in the middle and late July of each year when the ascospores have just partially matured.
(2) Operation method. There are three steps.
A. Separation of stiff worms (i.e. sclerotia): Before separation, wash the surface of the main body with water, and then wash it with sterile water for 2-3 times; Disinfect the surface of the separated materials with 0. 1%-0.2% mercuric chloride solution for about 3-5min minutes; Then clean with sterile water. Select the front part bounded by chest and foot. Cut off the epidermis with a scalpel, avoid the digestive tract, take hyphae from the blood cavity and cut them into sesame seeds, and press them into a flat culture medium, with 2 grains per dish 1 1. Culture in 15-19℃, when the colony grows 0.2-0.5cm, select a small amount of hyphae and repeatedly purify them on the plate medium for 214 times, and then transfer them into test tubes for preservation and culture after confirming that there are no other miscellaneous bacteria.
B, daughter bud separation: cutting off the washed daughter bud from the top of the stiff worm, putting it into 0. 1% mercuric chloride solution for disinfection for 2-3min, washing it with sterile water, cutting off the tissue block in the middle part and pressing it into the culture medium; The culture conditions are the same as a.
C. Ascomycete spore separation: cover the mature stroma of the ascospores with a transparent paper bag, and let the ascospores bounce and stick on the paper bag. Then, immerse the paper bag with the ascospores in 25% glucose solution, wash off the spores, put them in 15-1 19℃ for culture, and perform microscopic examination every day. When the robe germinates, suck a single spore with a micropipe. The culture conditions are the same as a.
Alternatively, the whole mature Cordyceps sinensis can be brought indoors, and the sub-seats of stiff worms and even worms are wrapped with cotton paper, leaving only the pregnant part, and a glass slide is placed horizontally in the sterile room to keep the main body humidity at any time; Microscopic examination every day, when ascospores bounce onto the glass slide, they are sucked into the culture medium of the dish with a micropipette for culture; The culture conditions are the same as a.
(3) Selection of culture medium.
A. Potato glucose agar medium (PDA), which is a universal medium for all Cordyceps fungi, can be used in the initial stage of isolation and culture, but the growth of the strain is not very vigorous, and it is easy to age and degenerate.
B. Enrichment medium (1): pluripotent protein umbilical cord 10g, glucose 50g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, live silkworm chrysalis 30g, auxin 0.5μg, agar 20g, water 1000mL, and pH：5.0.
Enriched medium (Ⅱ): preserved protein 40g, glucose 40g, peeled fresh potato 100g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, beef paste 10g, auxin 0.5μg, hepialus moth live larva (ground) 30g, agar 20g, alpine meadow soil leachate/kloc.
On enriched medium, colonies grew more vigorously and rapidly than on PDA medium. Jiafu Ⅱ is superior to Ⅰ.
2. Growth characteristics In the separation and culture of Cordyceps sinensis, it is necessary to master the utilization of temperature, pH value, light, carbon and nitrogen sources and inorganic salts.
(1) Effects of different temperatures on mycelium growth. Cordyceps sinensis likes low temperature. It can grow slowly at 0-4℃; The growth rate is accelerated at 5-8℃; 10-19℃ is the suitable growth and development range of this strain; The optimum growth temperature is 15-l9℃. When the temperature exceeds 20℃, the hyphae grow rapidly, and the colonies change from white to grayish black or brownish yellow, and begin to mutate, so it is not suitable for high temperature culture.
(2) Requirements for pH value. Cordyceps sinensis is a kind of acid fungus, and its optimum pH value is 5.0-6.0. When the pH value is below 4.5 and above 6, it will grow slowly or not with the increase or decrease.
(3) the influence of light. In the early stage of ascospore germination and mycelium growth, this strain was adapted to weak light and short light, and in the later stage it was adapted to strong light. In artificial culture, hyphae, conidia and stroma have obvious phototaxis, and they grow densely on the sunny side, sparsely on the backlight side, and all kinds of bacteria cultured in the all-black environment are delicate, slender and sparse.
(4) Utilization of carbon sources. Cordyceps sinensis can use a variety of carbon sources, but when glucose and maltose are used together, it grows fastest, and when glucose is used alone, it can grow well, followed by starch such as potato and sucrose.
Cordyceps sinensis is a kind of fungus that grows from parasitic living host to saprophytic stage, so it can make good use of various organic nitrogen sources. And live worms are the best; The combination of peptone and yeast extract is also very ideal for growth, followed by their separate use, followed by beef paste and so on. However, the utilization of inorganic nitrogen such as ammonium sulfate and potassium nitrate is poor.
There is a certain demand for ash nutrients, which grow vigorously in the medium with trace amounts of magnesium sulfate and dipotassium hydrogen phosphate, followed by potassium dihydrogen phosphate, and other inorganic salts such as sodium, calcium, iron and copper can also be used.
3. Culture methods After separation and purification, Cordyceps sinensis can be used as various experiments and expanded production applications.
(1) There are usually three methods to expand its production: solid static culture, shaking culture and large tank aeration fermentation culture.
A. Static culture: mainly used in solid culture, such as tube slant, triangular flask culture, rice culture and so on. In static culture, as long as the temperature and light are well mastered, the bacteria can grow normally. When the conidia on the inclined plane are mature, they can be stored in the refrigerator at 1-2℃ for 8-1 14 months, and can also be directly used as seeds for production.
B. Oscillatory culture: both liquid culture and small-scale propagation culture can be used. Shake the culture medium and subtract agar from the solid culture medium. It is best to choose a constant temperature oscillation culture machine, put liquid culture medium in a triangular bottle, and inoculate the test tube solid strain to culture. After continuous oscillation, all components in the liquid culture medium are mixed evenly without precipitation, and at the same time, the contact and exchange between gas and liquid are promoted, so that oxygen enters the liquid culture medium, which is beneficial to mycelium growth and conidia formation.
C. aeration fermentation culture in large tanks: When producing mycelia, conidia and other bacterial powders on a large scale, aeration fermentation culture in large tanks must be used. In this method, aeration is carried out by suction or vacuum pump decompression, and bacteria are removed through a filter, which is sent to a liquid culture medium in a tank for the growth and development of Cordyceps sinensis.
(2) Fermentation process. Separation and purification of natural Cordyceps sinensis → slant seed in test tube → primary seed of oscillator liquid → secondary seed → small seed fermentation tank → production fermentor → concentration → powder spraying and drying → finished bacterial powder.
The fermentation of Cordyceps sinensis in a tank requires a temperature of 20-25℃, a tank pressure of 392.3-686.5kPa(0.4-0.7kg/cmz) and a ventilation rate of 0.5-11.ovvm *; The liquid culture medium injected into the fermentor should be 65%-75% of the tank capacity. When the inoculation amount is 10%, the stirring speed is 180r/min, and the culture time is 72-96h, it can be put into a jar for concentration and powder spraying.
(3) Multi-purpose fermentation medium. Fresh potato (peeled) 8%, sucrose 2%, corn starch 0.5%, silkworm chrysalis powder 1%, peptone 0.4%, ammonium sulfate 0.2%, pH value 5.5-6.0.
(4) The standard of fermentation culture products. Conidia are almost completely detached female spore stalks. When the number of spore stalks does not increase obviously, they are counted under the microscope, and each milliliter contains conidia1800 million to 250 million. Residual sugar is less than1%; When the amino nitrogen is lower than 0.2mg/mL, it can become the finished bacterial liquid.
4. Key points of Cordyceps sinensis inoculation: Inoculating sexual or asexual Cordyceps sinensis spores to host insects is a key measure for artificially cultivating sexual Cordyceps sinensis and rejuvenating asexual strains.
(1) tieback period. The key to the reconnection of Cordyceps sinensis is to choose the weak antibacterial period of host insects, one is molting period; The second is the period when the larval feeding activity is intense and the friction damage rate is high.
(2) tieback method. There are two kinds.
A. Spraying method: when the larvae of hepialus hepialus reach the age of 4-6, and more than13 of the larvae shed their skin, the larvae are concentrated, and 5% 1 10% glucose solution is sprayed on the insect body or food with ascospores or conidia, and the bacterial solution is slightly dried on the insect body about 30 minutes after inoculation, and the larvae are returned to the soil for any purpose. This method has a high infection rate, but the disadvantage is that the larvae are not easy to be taken out of the soil and concentrated, and when concentrated, the larvae have the habit of killing each other. After being bitten, the worms are easily infected by miscellaneous bacteria and die. When the hyphae of Cordyceps sinensis are not full of body cavities, the injured worms will rot and cannot form rigid worms (sclerotia).
B. Natural contact method: raise hepialus moth larvae in a large area of semi-natural soil, and regularly take small samples to observe the growth status of the larvae. When most of the larvae are suitable for being infected, plant them with feed plant soaking solution and inoculate them with fine soil, evenly spread them on the surface where the insects are raised, and then spray water to make the bacteria penetrate into the soil, so that the larvae are exposed to infection during feeding activities. The infection rate of this method is not too high, but once infected, Cordyceps sinensis grows well, and the uninfected hepialus larvae can grow normally and reproduce.