WO2015003548A1 - 一种在室内培殖虫草的方法 - Google Patents

一种在室内培殖虫草的方法 Download PDF

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Publication number
WO2015003548A1
WO2015003548A1 PCT/CN2014/080072 CN2014080072W WO2015003548A1 WO 2015003548 A1 WO2015003548 A1 WO 2015003548A1 CN 2014080072 W CN2014080072 W CN 2014080072W WO 2015003548 A1 WO2015003548 A1 WO 2015003548A1
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cordyceps
stage
larvae
cultivation
partitioning device
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PCT/CN2014/080072
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French (fr)
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WO2015003548A9 (zh
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刘飞
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重庆市中药研究院
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Publication of WO2015003548A9 publication Critical patent/WO2015003548A9/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms

Definitions

  • the present invention relates to a fungal culture technique, and more particularly to a method of cultivating Cordyceps indoors.
  • Cordyceps sinensis (Berk.) Sacc.] alias Cordyceps, Cordyceps, summer grass insects.
  • Cordyceps sinensis belongs to the fungi community (Fungi), Ascomycota, Ascomycetes, Sordymycetidae, Phaeodonta, Clavicipifaceae, Cordyceps. It is a complex of larvae fungus Cordyceps sinensis (Berk. Sacc.) parasitic on larvae of bat moth insects and larvae. Artificial breeding and the word bat larvae are important links in the artificial cultivation of Cordyceps sinensis.
  • bat moths More than 60 species of bat moths have been reported so far, including 20 species in Yunnan, 17 species in Cambodia, 12 species in Sichuan, 9 species in Qinghai, and 9 species in Gansu.
  • the bat moth larvae are omnivorous insects, and they live in a tunnel-like cave life in the soil, with the roots of the plants as food. Although there are some differences in biology and ecology between different species of bat moths, they are characterized by the need to go through a long larval stage and have obvious generational alternation.
  • Bat moths have typical vertical distribution characteristics, and the lower limit of distribution is The latitude south is 3,000m above sea level, and the northerly latitude is 2500m above sea level; the upper limit is 5100m above sea level; the most suitable growth altitude is 3600_5000m ; the most suitable growing soil is alpine meadow and alpine shrub soil. Since the bat moth completes a life cycle and needs to undergo four stages of egg, larva, pupa and adult, the bat moth larvae are dormant in the soil during the permafrost period from October to April, and the bat moth larvae need to experience 6-8.
  • Age of growth and development can only be phlegm, depending on the species, such as Yushu bat moth larvae need 996 days, Kangding bat moth more than 1000 days, Gongga bat moth 875-1040 days, and can find different ages in the soil all year round Bat moth larva.
  • Some bat moth larvae form ticks in late May each year, and emerge into adult worms from June to August. Adults are more likely to emerge at 17-20 hours during the emergence season.
  • the female moths after mating immediately scatter the eggs in the nearby grass or shrub vegetation. Generally, each female moth lays about 500 eggs.
  • the breeding of bat moth insects in the natural environment is controlled by a combination of ecological geographical distribution, food, vegetation, soil structure, temperature, humidity and natural enemies.
  • the breeding rate of bat moth larvae in the natural environment has become a constraint. Key factors for the production of Cordyceps sinensis in the Qinghai-Tibet Plateau.
  • Cordyc s hawkesi i Gray. is widely distributed in the mountainous and hilly areas of the southern provinces of China.
  • Jiangxi, Anhui, Hunan and other places there are medicinal applications.
  • the host of the Asian scented Cordyceps sinensis was identified by the Chinese Academy of Sciences, Zhu Hongfu and Wang Linyao, and named as Napialus hunanensis Chu et Wang. It is a new genus of Lepidoptera, which is mainly distributed in Hunan and Jiangxi. , Anhui, Hubei, Zhejiang, Fujian, Guangdong and other provinces. Isolation and culture of A.
  • Chinese Patent Application (Publication No.: CN19700734A) discloses a method for producing Cordyceps sinensis.
  • Chinese Patent Application (Publication No.: CN1970733A) discloses a method for cultivating Cordyceps sinensis in a bat moth enclosure field,
  • Chinese Patent Application (publication number: CN1948456A) A method for improving the yield of Cordyceps sinensis in the Qinghai-Tibet Plateau is disclosed.
  • Cisokawa discloses a method for improving the yield of Cordyceps sinensis in the Qinghai-Tibet Plateau, and the four methods disclosed seem to be in the wild environment of the Qinghai-Tibet Plateau.
  • the refurbishment environment establishes a shed to raise bat larvae and increase the number of adults or larvae in the captive area to increase the survival rate of bat moth larvae.
  • the bat moth larvae develop to 6-8 years, by applying the bacterium to the entire captivity site In the way of liquid, the bat moth larvae are infected, and the bat moth larvae are completely infected and the temperature and humidity protection settings are removed to restore the natural temperature and humidity.
  • the growth process of the whole Cordyceps sinensis is carried out in the same area. According to the data disclosed in the patent application documents, the bat larvae of this method only need 365-380 days from the growth of the first to the age of 6-8; the success rate of the infection of Cordyceps can reach 50%.
  • the present invention provides a method for indoor cultivation of Cordyceps sinensis.
  • the method adopts the control of indoor air cleanliness and temperature and humidity conditions, and solves the problem of (man-made mechanical damage) by using a single isolation word of the susceptible larvae.
  • the specific invention content is:
  • a method for cultivating Cordyceps indoors including an egg stage, a larval wording stage, and a weeding stage; a single isolation cultivation is carried out throughout the larval wording stage or after 2-4 years of age.
  • the bacteria are stained with a mixed strain material of any one or several of the ascospores, conidia and fungus of Cordyceps.
  • the Cordyceps is Clavicipitaceae (Kirk et al. 2001) Cordyceps s (Fr.) Link fungus.
  • the single isolation incubation includes the following technical requirements:
  • the incubation temperature is 8-25 ° C;
  • the Cordyceps cultivating tray is composed of a box body with a permeable water tank at the bottom, a partitioning device, a moisturizing material and a lid cover matched with the partitioning device; the moisturizing material is arranged at the bottom of the box body, and the partitioning device is provided with a moisturizing material.
  • the cover covers the upper part of the partition.
  • the seepage tank at the bottom of the box body is matched with the upper edge of the partition device, the seepage tank can be nested in the upper edge of the partition device, and the upper and lower portions of the partition device are closed to form a closed space; the box body and the partition device The combination can be layered over and the lid is covered at the top.
  • the beneficial technical effects of the present invention are as follows: 1) The invention adopts the control of indoor air cleanliness and temperature and humidity conditions, and the single isolation of the susceptible larvae to solve the problem of mutual killing and mutual infection during the growth and development of the susceptible larvae.
  • the invention does not require the larvae to be put into the wild environment, and the whole process of the cultivation of the cordyceps is less interfered by the external environment (to facilitate the observation and management of daily larval living conditions, and to avoid artificial mechanical damage).
  • the stage of the Cordyceps sinensis in the present invention is carried out in a Cordyceps culture tray, and can be carried out under artificial conditions for temperature control and humidity control, and is suitable for large-scale production.
  • Figure 1 is a schematic diagram of the combination of Cordyceps cultivation trays
  • Figure 2 is a schematic view showing the structure of the casing 1;
  • Figure 3 is a schematic view of the blocking device 2
  • FIG. 4 Schematic diagram of multi-layer combination of Cordyceps host larvae cultivation tray 2;
  • Figure 5 is a schematic view of the partition device 2
  • Figure 6 is a schematic view of the partition device 2 inverted;
  • Figure 7 is a schematic structural view of the casing 1;
  • Figure 8 is a flow chart of the indoor cultivation of Cordyceps
  • 1 box 1-1 seepage tank, 1-2 box ribs, 2 partitions, 3 lids.
  • the present invention provides a Cordyceps cultivating tray.
  • the Cordyceps cultivating tray is composed of a box body 1, a moisturizing material, and a partitioning device 2 (see FIGS. 3 and 5).
  • the cover 3 are composed of four partial components; wherein, the casing 1 is an upper open box-like structure, horizontally at the bottom of the casing 1, and a strip-shaped groove is provided on the inner bottom surface.
  • the water tank 1-1, the water permeable tank 1-1 may be composed of parallel strip-shaped grooves, or may be composed of a plurality of grooves intersecting.
  • a moisturizing material is provided on the inner bottom surface, which may be a plant fiber material, a glass fiber material, or a water absorbing material made of cotton cloth or water absorbing foam.
  • the requirement for the above hydrating material is that the water absorbing capacity is 50% of the mass water content. Between -500%; the moisturizing material is applied over the entire inner bottom surface of the casing 1 during use.
  • a partitioning device 2 is further disposed on the bottom surface of the casing 1 covering the moisturizing material, and the partitioning device 2 is composed of one or more barrel-shaped structures which are open at both ends. As shown in FIG. 1, the partitioning device 2 is placed. After the moisturizing material is applied and the lid 3 is capped, the barrel structure in each of the partitioning devices 2 forms an enclosure. Or as shown in FIG. 4, the upper portion and the lower portion of the partitioning device 2 are closed by the bottom of the casing 1 (as shown in FIGS. 4, 5, 6, and 7), the upper portion of the partitioning device 2 and the casing 1 are The bottoms are matched to form an enclosure; and the combination of the blocking device 2 and the casing 1 can be overlapped layer by layer.
  • the moisturizing material is firstly covered on the entire inner bottom surface of the casing 1, and then the partitioning device 2 is placed, and the moisturizing material is sprayed with water to make the moisture content of the moisturizing material reach 50-500%, and then the Cordyceps
  • the host larvae are respectively placed in the upper open box formed by the partition device 2 and the inner bottom surface of the box body 1, and one of each box body is placed, and the larvae of the cordyceps host larvae need to be eaten, and some larvae can be added at the same time.
  • the collection of eggs is carried out under ambient conditions that require no air cleanliness and an air cleanliness of 300,000. Eggs from adult adults of Napialus hunanensis Chu et Wang and adult bat moths were collected.
  • the specific harvesting method is: in the harvesting area, the adult (or the adult collected from the species rejuvenation area) that emerged in each breeding square on the day is placed in a supported mosquito net mat (by gauze or It can be ventilated and ventilated, but it can avoid the escape of insects.)
  • the mosquito net is hung inside or placed in the egg frame, something that can be attached by adult insects, such as green grass, branches with green leaves or bamboo rafts, etc. Placed in the egg box.
  • the spawning temperature is controlled at 8-25 ° C, humidity is 60-70%; natural photoperiod and natural light intensity are carried out. Place the adult bed with the adult mosquito net in the environment of natural photoperiod and natural light intensity.
  • the environment can use the large projector to play the natural environment climate sound and image for the corresponding time period (the main purpose is to imitate its natural environmental conditions), and At 8-25 °C, female adults and male adults are mated, and adults can mate 1-3 times in the 5-15 d life cycle. Spawning begins shortly after mating. The entire process is carried out under conditions of a humidity of 60-70%.
  • the eggs laid by the adults in the mosquito net are scattered to the bottom of the net bed of the mosquito net - the egg frame, and a dry soft filter paper or cotton cloth or other fibrous material is placed at the bottom of the egg frame to hold the eggs.
  • the mosquito net is separated from the connection with the egg frame, and the end of the net net is collected and opened, and then the egg frame is removed, and the end of the net net is placed in the newly prepared egg box to receive the newly laid egg. Dry the filter paper or cotton or other fibrous material that holds the eggs out of the egg frame, remove the impurities, and concentrate the eggs in the wire mesh that will not leak out.
  • Egg collection time is collected every morning, noon and evening. Egg collection was done at 8-25 °C. The entire process is carried out under conditions of humidity of 60-70%, natural photoperiod and natural light intensity.
  • the eggs are collected, they are transferred to an environment with an air cleanliness requirement of not less than 100,000 for egg washing and egg hatching; workers are required to wear overalls, masks and hats.
  • Eggs that have just been concentrated in the wire mesh are immersed in cold water (or 75% alcohol) heated at 60-100 °C for 5-8 seconds. After 1-3 minutes, the same method is used to clean the second. After the interval of 1-3 minutes, the third method was washed by the same method, and after the washing, the hatching was carried out in stages. The first stage is to clean the eggs until the beginning of the hatching.
  • the eggs are placed on filter paper or cotton cloth or other fiber materials with a certain humidity.
  • the standard of humidity is the degree of humidity when seeing water and not seeing the water when tilting.
  • the second stage is when the eggs start to hatch until all eggs have been hatched, all eggs are transferred to dry conditions to hatch (if transferred to dry filter paper, you can
  • the dry filter paper is placed directly on the mixture of the substrate and the word for the larvae.
  • the larvae can climb into the mixture to protect themselves from light, moisturize and feed. They can also directly put the eggs that are hatching or about to hatch. Into the mixture.).
  • the hatching temperature of the eggs is 5-25 ° C.
  • the light conditions are natural light intensity and photoperiod.
  • the hatched larvae are collected separately, and the larvae of the bat bat moth and the larvae of the bat bat moth are obtained.
  • Collection and inoculation of strain materials The collection and inoculation of Cordyceps sinensis species: Selecting the mature adult Cordyceps sinensis individuals, in order to prevent excessive humidity, choose to clean, disinfect and transfer air cleanliness from the wild after sun harvesting in the sun. Collect and inoculate the susceptibility materials under the environmental conditions; the staff wear appropriate overalls, masks and hats according to the cleanliness requirements.
  • a mixed strain of any one or more of ascospores, conidia, and fungus is collected for use as a host larvae.
  • Mass vaccination of different stages of larvae was carried out in batches using the host infection method mentioned in the patent "Strain materials for host infection of Cordyceps sinensis and host infection methods".
  • the newly hatched larvae are relatively small, and their activity ability is relatively weak. After the susceptibility, they can continue to intensively cultivate the words for 45-60 days and then carry out a single isolation word; other stages of larvae are large due to large individuals, and the amount of activity is large. Killing and killing, after the sense of bacteria is suitable for a single isolation word. In addition to providing regular word usage, the newly hatched larvae need to provide sufficient moisture during the intensive vocabulary.
  • Inoculation by spraying and inoculation by irrigation requires an increase in the amount of subsequent water supply; inoculation by mixing the words or mixing into the substrate requires an increase in the moisture content of the material or substrate. Intensive words are maintained at a temperature of 8-18 °C.
  • the collection and inoculation of the A. philoxeroides species Selecting the mature A. sinensis, in order to prevent excessive humidity, choose to clean, disinfect and transfer to the air cleanliness requirements after harvesting from the wild on sunny days with the sun. Collection and inoculation of susceptible materials under environmental conditions of not less than 100,000; workers wear appropriate overalls, masks and hats according to cleanliness requirements.
  • a mixed strain of any one or more of ascospores, conidia, and fungus is collected for use as a host larvae.
  • the conidia of A. philoxeroides is at 10 ° C ⁇ 3 (TC conditions, preferably 25 ° C, with soluble starch as the carbon source, peptone as the nitrogen source (the mycelial growth is not strict with the nitrogen source requirements,
  • the medium is the best in the range of pH 3. 5 ⁇ 8. 4, and the bacteria can be used for the environment, and the medium can be used in the range of pH 3. 5 ⁇ 8.
  • the pH has certain regulation ability. After the hyphae are cultured for several days under different pH, the pH value of the medium is changed to 7. The amount of conidia is increased to 4 X 10 5 I mL. Large-scale inoculation can be achieved by sand to the surface of the larvae.
  • the wording and germination stage of the susceptible larvae is carried out under the environmental conditions of air cleanliness requirements of not less than 100,000; Personnel wear uniforms, masks and hats according to the requirements of cleanliness.
  • the larvae of the Cordyceps sinensis and the larvae of the A. sinensis which were isolated and cultured in the above steps were placed in the Cordyceps tray provided in Example 1. Each larva lives in a separate space in which a matrix (humus or artificial substrate) for larvae can be added at the same time until the susceptible larvae become rigid and begin to germinate white sub- bodies from the head.
  • a matrix humus or artificial substrate
  • the larvae of the Cordyceps sinensis are mainly fed carrots, chrysanthemum, bead bud, life fruit, etc., supplemented by the rhizome of other highland plants, which can be fed by a single word or a compound word;
  • the larvae host (Hanba bat moth)
  • the larvae are mainly fed carrots, chrysanthemum, bead buds, fruit, etc., supplemented by rhizome of orange, orange, oil, tea, etc., can be fed in a single word.
  • Compound corpus can also be given.
  • the bead buds, chrysanthemums and carrots are washed with water and then cleaned with an ultrasonic cleaner, and then processed into small pieces in the clean workshop for the newly hatched larvae to eat; the fruits are washed with water and then ultrasonically cleaned, then Directly for larvae to eat.
  • Bead buds, chrysanthemum, carrots, and fruit of life should be cleaned or smeared before or after processing.
  • the late word temperature is 8-25 °C, and it is protected from light.
  • the culture humidity is the degree of humidity when water is not seen when it is tilted. It is moisturized with cold water that has been sterilized by heating at 60-100 °C.
  • workers should wear finger cots to avoid disease transmission, in addition to conventional anti-microbial contamination, and replace finger cots according to the disease, avoiding the use of sharp tools that can easily damage larvae. Tools that have touched the diseased or pathogenic bacteria during the operation should be replaced in time. 3% ⁇ According to statistics, based on hatching larvae based on the statistics, the incidence of A. sinensis host reached 68.3%. The bacterial infection rate of the host of Cordyceps sinensis reached 81.2%.
  • the humidity condition is that the substrate can be clumped with a force, and the hand can be released without dispersing the humidity standard.
  • the temperature condition of the cultivation is 8-25 ° C, and the illumination intensity and photoperiod of the cultivation are the same as the natural light.
  • the substrate used needs to pass 60-100°. C is heated and disinfected (to remove pathogens and pests, protect germinated fruiting bodies and zombies) to remove pathogens and pests that may invade and feed on germinated fruiting bodies and zombies.
  • Cordyceps Development of fruiting bodies as needed Cordyceps are harvested at different stages. Commercial Cordyceps is suitable for harvesting when the ascus has not begun to develop. When harvesting, hold the fruit body in one hand, pick up the insect in the substrate with a long scorpion, and clean the insect with a plastic toothbrush. The surface of the fungus, dried naturally indoors or dried in an oven at 30-40 ° C to obtain commercial Cordyceps.

Abstract

一种在室内培殖虫草的方法，包括卵阶段、幼虫饲养阶段和出草阶段；其技术关键为在幼虫饲养阶段的全程或2—4龄之后进行单条隔离培育；在幼虫饲养阶段用虫草的子囊孢子、分生孢子和菌皮中的任意一种或几种的混合菌种材料进行染菌；本方法采用控制室内空气洁净度和温湿度条件，将感菌幼虫单条隔离饲养以解决感菌幼虫生长发育过程中互相残杀和互相传染病害的问题；该方法不用将感菌幼虫投入到野生环境，整个虫草培殖的过程受外界环境干扰少；本方法中虫草发菌阶段在虫草培养盘中进行，对于控温、控湿等均可以在人工条件下进行，适合大规模生产。

Description

一种在室内培殖虫草的方法
技术领域
本发明涉及一种真菌培殖技术， 更具体的说涉及一种在室内培殖虫草的方法。
技术背景
冬虫夏草 [Cordyc印 s sinensis (Berk. ) Sacc. ]别名虫草、 冬虫草、 夏草冬虫。 根据最近的 分类系统，冬虫夏草隶属于真菌界 (Fungi)、子囊菌门（Ascomycota)、子囊菌纲 (Ascomycetes)、 粪壳菌亚纲（Sordariomycetidae)、 肉座菌目、 麦角菌科 (Clavicipifaceae)、 虫草属 ( Cordyceps)。 是麦角菌科真菌冬虫夏草菌 [Cordyc印 ssinensis (Berk. ) Sacc. ]寄生在蝙蝠蛾 科昆虫幼虫上的子座及幼虫尸体的复合体。人工繁殖和词养蝙蝠娥幼虫是冬虫夏草人工培育的 重要环节。 迄今报道的蝙蝠蛾昆虫超过 60种， 其中云南 20种， 西藏 17种， 四川 12种， 青海 9种， 甘肃 9种。 蝙蝠蛾幼虫为杂食昆虫， 在土壤中营隧道式穴居生活， 以植物嫩根为食物。 不同种 类的蝙蝠蛾在生物学和生态学上虽然有一定的差异， 但其特点是需要经历较漫长的幼虫期，并 具有明显的世代交替现象， 蝙蝠蛾具有典型的垂直分布特征， 其分布下限在纬度偏南地区海拔 3000m,而在纬度偏北地区海拔 2500m; 分布上限为海拔 5100m; 最适合的生长海拔 3600_5000m; 最适宜的生长土壤为高寒草甸和高寒灌木丛土。由于蝙蝠蛾完成一个生命周期需经历卵、幼虫、 蛹、 成虫四个阶段， 在每年 10月至次年 4月冻土期蝙蝠蛾幼虫在土壤中处于休眠状态， 蝙蝠蛾 幼虫需要经历 6-8龄的生长发育期才能化蛹， 具体因种类而异， 如玉树蝙蝠蛾幼虫需要 996天， 康定蝙蝠蛾越 1000天， 贡嘎蝙蝠蛾 875-1040天， 而且终年可以在土中找到不同龄期的蝙蝠蛾幼 虫。 每年 5月下旬部分蝙蝠蛾幼虫形成蛹， 6-8月羽化成成虫。 成虫多在羽化季节期间 17-20时 羽化，交配后的雌蛾立即边扑边动将卵散产于附近草丛中或灌丛植被中，一般每只雌蛾产卵 500 粒左右。 自然环境中蝙蝠蛾属昆虫的繁育受到生态地理分布、 食物、 植被、 土壤结构、 温度、 湿度和自然天敌等多种生态因子的综合制约控制，蝙蝠蛾幼虫在自然环境中的繁育速率已成为 制约青藏高原冬虫夏草产量的关键因素。
亚香棒虫草（Cordyc印 s hawkesi i Gray. )广泛分布于我国南方各省山区、 丘陵地带。 在江 西、 安徽、 湖南等地民间有药用。 亚香棒虫草的寄主经过经中科院动物所朱弘复、 王林瑶先生 鉴定， 定名为湖南棒蝠蛾（Napialus hunanensis Chu etWang) , 为鳞翅目蝙蝠蛾科的一新属新 种， 主要分布于湖南、 江西、 安徽、 湖北、 浙江、 福建、 广东等省。 亚香棒虫草组织及子囊孢 子进行分离培养； 记述了分离物的形态和培养性状， 并将分离物鉴定为霍克斯拟青霉菌 (Paeci lamyces hawkesi iXiao, T B Li et Q T Chen)或称之为 （古尼拟青霉） 。 寄主幼虫入 土时间约在 10月下旬至 11月中旬。 受到真菌感染后死亡， 至次年 3月份子座从头部长出， 子座 从长出到子囊果形成并成熟约需 1个月； 子囊果开始散发孢子到虫体枯萎约 10d。一般都分布在 10 ° -30 ° 的向阳坡地， 纬度越低， 出土时间也越早； 海拔越高， 气温也越低， 出土时间就越 迟。采收期从 3月中、下旬一直到 5月上、中旬；海拔最低见于 240米，最高在 1400米，而以 800-1000 米之间分布较多。 由于亚香棒虫草与冬虫夏草亲缘关系较近， 同时也具有一定的药效， 因此， 随着冬虫夏草资源量的减少和价格急剧攀升， 亚香棒虫草也逐渐收到追捧， 并且其资源量也由 于大量的滥采滥挖而开始急剧的减少。 所以， 亚香棒虫草的人工培养也势在必行。
中国专利申请 （公开号： CN19700734A) 公开了一种冬虫夏草的生产方法， 中国专利申请 (公开号： CN1970733A)公开了一种在蝠蛾圈养场培育冬虫夏草的方法， 中国专利申请（公开 号： CN1948456A) 公开了一种提高青藏高原冬虫夏草产量的方法， 中国专利申请 （公开号： CN19484757A) 公开了一种提高青藏高原冬虫夏草产量的方法， 所述四种方法公开的就似乎内 容均为在青藏高原野生环境下改造环境建立温棚圈养蝙蝠娥幼虫，增加圈养区域成虫或幼虫的 数量， 以提高蝙蝠蛾幼虫的存活率， 在蝙蝠蛾幼虫发育至 6-8龄时， 通过向整个圈养场地施用 染菌孢液的方式， 对蝙蝠蛾幼虫进行感染，蝙蝠蛾幼虫完全感染去掉温湿保护设置让其恢复到 自然温湿。整个冬虫夏草的生长过程都在同一地域中进行。据专利申请文件公开的数据， 该方 法蝙蝠蛾幼虫从 1龄生长发育至 6-8龄仅需要 365-380天；最终冬虫夏草的感染成功率能够达 到 50%左右。
申请人自 1970年代末就立项开展冬虫夏草人工培殖的研究。 在承担的 "七五"、 "八五" 国家重点科技攻关项目 "冬虫夏草人工培殖研究"课题中， 确立了室内人工培殖冬虫夏草的模 式后， 继而又承担了 "九五"国家重点科技攻关项目 "西藏那曲冬虫夏草半野生抚育及开发研 究"， 且获得了重大突破——首次在西藏那曲海拔 4700多米的藏北高原， 培殖出了半野生抚育 的冬虫夏草， 从而确立了冬虫夏草半野生抚育的新模式。 "十一五"期间承担国家科技支撑计 划项目 "冬虫夏草人工培殖关键技术优化研究"， 在四川康定又对半野生抚育新模式的关键技 术进行了优化， 进一步提高了产量， 简化了操作环节， 且降低了成本。
发明内容
针对当前技术存在的问题， 本发明提供了一种室内培殖虫草的方法， 本方法采用控制室内 空气洁净度和温湿度条件，将感菌幼虫单条隔离词养以解决 (人为的机械损伤)感菌幼虫生长发 育过程中互相残杀和容易互相传染病害的问题。 具体发明内容为：
一种在室内培殖虫草的方法， 包括卵阶段、幼虫词养阶段和出草阶段； 在幼虫词养阶段的 全程或 2-4龄之后进行单条隔离培育。 在幼虫词养阶段用虫草的子囊孢子、分生孢子和菌皮中的任意一种或几种的混合菌种材料 进行染菌。
所述虫草为麦角菌科 Clavicipitaceae (Kirk et al. 2001)虫草属 Cordyc印 s (Fr. ) Link 真菌。
所述单条隔离培育包括以下技术要求：
1 ) 培育空气洁净度要求不低于 10万级；
2 ) 幼虫被放置在质量含水量为 50-500%的保湿材料上， 其中保湿材料上不能出现流动的 水流。
所述培育的温度是 8-25 °C ;
单条隔离培育采用虫草培育盘进行培育；
所述虫草培育盘由一个底部设置渗水槽的盒体、隔断装置、保湿材料和与隔断装置相匹配 的盒盖四部分组成； 所述保湿材料设置在盒体的底部， 隔断装置设置保湿材料上， 盒盖覆盖在 隔断装置上部。
所述盒体的底部的渗水槽与隔断装置的上沿相匹配， 渗水槽能嵌套到隔断装置的上沿中， 隔断装置的上下部被封闭， 形成一个封闭空间； 盒体和与隔断装置的组合能逐层重叠， 盒盖覆 盖在最上部。
本发明的有益技术效果是： 1 ) 本发明采用控制室内空气洁净度和温湿度条件， 将感菌幼 虫单条隔离词养以解决感菌幼虫生长发育过程中互相残杀和互相传染病害的问题。该发明不用 将感菌幼虫投入到野生环境， 整个虫草培殖的过程受外界环境干扰少（便于日常幼虫生活状况 的观察和管理，避免人为的机械损伤）。
2 ) 本发明中虫草发菌阶段在虫草培养盘中进行， 对于控温、 控湿等均可以在人工条件下 进行， 适合大规模生产。
附图说明
图 1 虫草培育盘的组合示意图；
图 2 盒体 1的结构示意图；
图 3隔断装置 2的示意图；
图 4 虫草寄主幼虫培育盘多层组合示意图 2;
图 5 隔断装置 2的示意图；
图 6 隔断装置 2倒置的示意图； 图 7 盒体 1的结构示意图；
图 8 室内培殖虫草的流程框图；
其中， 1盒体， 1-1渗水槽， 1-2盒体加强筋， 2隔断装置， 3盒盖。
具体实施方式
实施例 1 虫草培育盘的结构及使用方式
为解决虫草培殖过程中感菌幼虫 （包括冬虫夏草和亚香棒虫草的寄主幼虫） 的词喂问题； 本发明提供了一种虫草培育盘。所述虫草培育盘的方案由两种， 其中一种如附图 1所示或有附 图 4所示； 所述虫草培育盘由盒体 1、 保湿材料、 隔断装置 2 (参见图 3、 5、 6 ) 和盒盖 3 四个部分组件组成； 其中， 所述盒体 1为一个上部开口的盒状结构， 在盒体 1的底部水平， 并且在内底面上设置有条状凹槽的渗水槽 1-1， 渗水槽 1-1可以是平行的条状凹槽组成， 也可 以是多条凹槽交叉组成。
在内底面上设有一层保湿材料， 该材料可以是植物纤维材料， 玻璃纤维材料； 也可以是棉 布、 吸水泡沫制成的吸水材料， 对于上述保湿材料的要求是吸水能力在质量含水量 50%-500% 之间； 在使用时将保湿材料覆盖在盒体 1的整个内底面上。
在覆盖保湿材料的盒体 1内底面还设置有隔断装置 2， 所述隔断装置 2 是由一个或多个 两端开口的桶状结构排列组成， 如附图 1所示， 将隔断装置 2放置在保湿材料上后， 且加盖盒 盖 3后， 每一个隔断装置 2中的桶状结构形成一个封闭体。 或如附图 4所示， 隔断装置 2的 上部和下部均被盒体 1的底部封闭 （如图 4、 5、 6、 7所示的技术方案）， 隔断装置 2的上部 与盒体 1的底部相匹配， 形成一个封闭体； 且隔断装置 2与盒体 1的组合可以逐层重叠。
在使用虫草培育盘时先将保湿材料覆盖在盒体 1的整个内底面上， 然后安放上隔断装置 2 , 向保湿材料上喷水使保湿材料的质量含水量达到 50-500%， 然后将虫草寄主幼虫分别投放 到隔断装置 2与盒体 1的内底面形成的上部开口的盒体内， 每个盒体内投放一只， 并投入虫 草寄主幼虫需要食用的词料， 也可以同时添加一些供幼虫躲避的基质 （腐殖土或人工基质）， 然后加盖盒盖 3; 使每一只虫草寄主幼虫在一个独立封闭的空间内培养， 每隔一段时间列行检 查时将逐层取出盒体 1与隔断装置 2的组合， 或将盒盖 3打开， 当需要更换培育盘时， 取出隔 断装置 2将虫草寄主幼虫放养到另外一个虫草培育盘内，放养的方法相同， 然后将使用过的虫 草培育盘清洗消毒再次使用； 其中室内培殖虫草的流程可以参照附图 8所示的流程。
实施例 2单条隔离培育方法的考察
1、 虫卵的收集 虫卵的收集在无需空气洁净度要求到空气洁净度为 30万级之间的环境条件下进行。 分别 采集湖南棒蝠蛾（Napialus hunanensis Chu et Wang)成虫和贡嘎蝙蝠蛾成虫产的卵。 具体采 收方法是， 在采收地域， 将当天在每一个培育方格内羽化的成虫（或从种性复壮区域内收集到 的成虫）放置到一个被支撑的蚊帐交尾床内（由纱布或可以通风透气但又可以避免成虫逃逸的 材料制成）， 蚊帐内部悬挂或在盛卵框内放置一些成虫可以附着的东西， 比如绿草、 有绿色树 叶的树枝或竹丫等， 蚊帐悬挂的末端置于盛卵框内。产卵温度控制在 8-25°C、湿度为 60-70%; 自然光周期和自然光强度的条件下进行。将放有成虫的蚊帐交尾床置于自然光周期和自然光强 度的环境内， 环境内可以用大型投影仪播放相应时间段的自然环境气候声音和影像（主要目的 是模仿其自然环境条件）， 并于 8-25°C的条件下任由雌成虫和雄成虫交配， 成虫在 5-15 d的 生活周期内可以交配 1-3次。 交配后不久就开始产卵。 整个过程在湿度为 60-70%的条件下进 行。 成虫在蚊帐内产下的卵散落到蚊帐交尾床的底部 -盛卵框内， 盛卵框底部放置一张干燥柔 软的滤纸或棉布或其它纤维材料用于盛接产下的卵。收集时将蚊帐从其与盛卵框连接处分开后 收集蚊帐末端并掀开， 然后挪走盛卵框， 同时将蚊帐末端置于新准备的盛卵框内盛接新产下的 卵。将盛接卵的干燥滤纸或棉布或其它纤维材料拿出盛卵框， 去掉杂质， 并将卵集中到不会漏 出的钢丝网内。 卵收集的时间是每天早上、 中午和晚上各收集一次。 卵的收集在 8-25°C的条 件下完成。 整个过程在湿度为 60-70%、 自然光周期和自然光强度的条件下进行。
卵收集完后， 转移到空气洁净度要求不低于 10万级的环境条件下进行卵的清洗和卵的孵 化； 工作人员要求穿戴工作服、 口罩和帽子。将刚刚集中到钢丝网内的卵没入到经 60-100°C加 温消毒的冷水中流水 （或 75%的酒精） 冲洗 5-8秒钟， 间隔 1-3分钟后采用相同方法清洗第 2次， 间隔 1-3分钟后采用相同方法清洗第 3次， 清洗完后分阶段进行孵化。第一阶段是清洗完至卵刚 开始孵化时止， 卵置于具有一定湿度的滤纸或棉布或其它纤维材料上孵化，湿度多少的标准是 见水而在倾斜时不见流水的湿度程度， 用经 60-100°C加温消毒的冷水保湿； 第二阶段为卵开始 孵化时至所有卵全部孵化完毕止， 将所有卵转移到无水的条件下孵化（如果转移到干燥的滤纸 上， 可以将干燥滤纸直接放在用于词养初孵幼虫的基质和词料的混合物上面， 幼虫孵化后可以 自己爬到混合物里避光、 保湿和取食； 也可以直接将正在孵化或即将孵化的卵投放到混合物 里。）。 卵孵化的第一阶段期间每隔 2-3 d用经 60-100°C加温消毒的冷水流水冲洗 10秒钟从而达 到卵的清洁工作。 卵的孵化温度是 5-25°C， 光照条件是自然光强度和光周期， 分别收集孵化出 的幼虫， 得到湖南棒蝠蛾的幼虫和贡嘎蝙蝠蛾的幼虫。
2、 菌种材料的收集及接种 冬虫夏草菌种的收集与接种为： 选择子座成熟的冬虫夏草个体， 为防止湿度过大， 选择在 有太阳的晴天从野外采挖后清洗、消毒并转入空气洁净度要求不低于 10万级的环境条件下进行 感菌材料的收集和接种； 工作人员按洁净度要求穿戴相应的工作服、 口罩和帽子。
收集子囊孢子、 分生孢子和菌皮中的任意一种或几种的混合菌种作为寄主幼虫感菌时用。 采用专利《一种用于冬虫夏草寄主感染的菌种材料及寄主感染方法》中提及的寄主感染方法对 不同阶段的幼虫进行批量大规模接种。初孵幼虫由于个体小， 活动能力相对较弱， 感菌后可以 继续集约化词养 45-60天后再进行单条隔离词养； 其它阶段的幼虫由于个体大， 活动量较大， 相互之间容易残杀致死， 感菌后适合单条隔离词养。初孵幼虫集约化词养期间除了提供常规的 词料用量外， 需要提供足够的水分。通过喷洒方式接种的和通过灌喂方式接种的需要增加后续 供水量； 通过拌入词料或混合入基质方式接种的需要增加词料或基质的湿度。 集约化词养在 8-18 °C的温度条件下完成。
亚香棒虫草菌种的收集与接种为： 选择子座成熟的亚香棒虫草个体， 为防止湿度过大， 选择在有太阳的晴天从野外采挖后清洗、消毒并转入空气洁净度要求不低于 10万级的环境条件 下进行感菌材料的收集和接种； 工作人员按洁净度要求穿戴相应的工作服、 口罩和帽子。
收集子囊孢子、分生孢子和菌皮中的任意一种或几种的混合菌种作为寄主幼虫感菌时用。 亚香棒虫草分生孢子是在 10°C〜3(TC条件， 最好是 25°C条件下， 以可溶性淀粉为碳源， 以蛋白 胨为氮源（菌丝生长对氮源要求不严格， 硝态氮、 铵态氮及有机氮均可被利用， 其中以有机氮 效果最好）的培养基最佳， 菌丝在 pH3. 5〜8. 4范围内均能生长， 且该菌对环境酸碱度有一定的 调控能力， 菌丝在不同酸碱度下培养数天后， 培养基的 pH值均转为 7。 分生孢子产生量增加到 4 X 105个 I mL。 在接种时将菌液直接喷沙到幼虫体表上就能实现规模化接种。
所有菌种材料准备好后， 采用申请人提交的中国专利申请 "一种用于冬虫夏草寄主感染 的菌种材料及寄主感染方法" 中提及的寄主感染方法对不同阶段的幼虫进行批量大规模接种。 初孵幼虫由于个体小， 活动能力相对较弱， 感菌后可以继续集约化词养 45-60天后再进行单条 隔离词养； 其它阶段的幼虫由于个体大， 活动量较大， 相互之间容易残杀致死， 感菌后适合单 条隔离词养。 初孵幼虫集约化词养期间除了提供常规的词料用量外， 需要提供足够的水分。通 过喷洒方式接种的和通过灌喂方式接种的需要增加后续供水量；通过拌入词料或混合入基质方 式接种的需要增加词料或基质的湿度。 集约化词养在 15-25°C的温度条件下完成。
3、 染菌后的虫草寄主的饲养及发菌
感菌幼虫的词养及发菌阶段是在空气洁净度要求不低于 10万级的环境条件下进行的；工作 人员按洁净度要求穿戴相应要求穿戴工作服、 口罩和帽子。
把上述步骤中接种后需要单条隔离培养的冬虫夏草寄主幼虫和亚香棒虫草寄主幼虫置于 实施例 1提供的虫草培育盘中。 每条幼虫生活在独立的一个空间内， 空间内可以同时添加一些 供幼虫躲避的基质 （腐殖土或人工基质）， 一直到感菌幼虫僵化并开始从头部萌发白色的子实 体。 冬虫夏草寄主 （贡嘎蝙蝠蛾）幼虫词养主要投喂胡萝卜， 菊姜、 珠芽蓼， 人生果等， 辅以 其它高原植物的根茎，可以单一词料投喂，也可给与复合词料； 亚香棒虫草寄主（湖南棒蝠蛾) 幼虫词养主要投喂胡萝卜， 菊姜、 珠芽蓼， 人生果等， 辅以花皮樟、 橙木、 油茶、 茶树等的根 茎， 可以单一词料投喂， 也可给与复合词料。 词料加工过程中， 珠芽蓼、 菊姜和胡萝卜用清水 清洗后再用超声波清洗仪清洗， 之后在洁净车间内加工成小块供初孵幼虫食用； 人生果用清水 清洗后用超声波清洗， 然后直接供幼虫食用。 珠芽蓼、 菊姜、 胡萝卜和人生果在加工之前或加 工之后都要将腐烂的或过小的清理干净。
定期检查幼虫生活情况， 根据幼虫取食情况和病害发生情况对词料、保湿材料以及词养盘 进行更换。后期词养温度是 8-25 °C， 避光词养； 培养湿度是见水而在倾斜时不见流水的湿度程 度， 用经过 60-100°C加温消毒的冷水保湿。更换幼虫词料或基质时， 工作人员除常规防杂菌污 染着装外， 须佩戴避免病害传染的指套， 根据病害有无更换指套， 避免使用容易伤害幼虫的尖 硬工具。操作过程中触碰过病害虫体或病原菌的工具要及时更换。 经过统计， 以孵化的幼虫为 基数统计， 亚香棒虫草寄主的发菌率达到 68. 3%。 冬虫夏草寄主的发菌率达到 81. 2%。
4、 虫草出草和采收
开始萌发白色的子实体后， 将其集中移栽到事先布置好基质的子实体培育盘内， 白色子实 体向上， 整个埋入基质并距基质表面1. 0〜1. 7(^。 培育的湿度条件为基质用力握可成团， 手放 开不会散的湿度标准。 培育的温度条件是 8-25°C， 培育的光照强度和光照周期与自然光相同。 所用基质需要经过 60-100°C的加温消毒， （从而除去病原菌和害虫， 保护已萌发的子实体及僵 虫）从而除去可能侵害和取食已萌发的子实体及僵虫的病原菌和害虫。根据需要， 在子实体发 育的不同阶段采收虫草。 商品虫草适合在未开始发育子囊壳的时候采收。采收时， 一手捏着子 实体， 一手用长镊子撬起基质中的僵虫， 用塑料牙刷刷干净僵虫表面的菌皮， 在室内自然晾干 或在 30-40°C烘箱内烘干即得商品虫草。

Claims

1. 一种在室内培殖虫草的方法， 包括卵阶段、 幼虫词养阶段和出草阶段； 其特征在于： 在幼虫词养阶段的全程或 2-4龄之后进行单条隔离培育。
2. 根据权利要求 1所述的在室内培殖虫草的方法， 其特征在于： 在幼虫词 养阶段用虫草的子囊孢子、分生孢子和菌皮中的任意一种或几种的混合菌种材料 进行染菌。
3. 根据权利要求 2所述的在室内培殖虫草的方法，其特征在于： 所述虫草为 麦角菌科 Clavicipitaceae(Kirk et al. 2001)虫草属 Cordyc印 s (Fr. ) Link真 菌。
4.根据权利要求 3所述的在室内培殖虫草的方法，其特征在于： 所述单条隔 离培育包括以下技术要求：
1) 培育空气洁净度要求不低于 10万级；
2) 幼虫被放置在质量含水量为 50-500%的保湿材料上， 其中保湿材料上不 能出现流动的水流。
5. 根据权利要求 4所述的室内培殖虫草的方法， 其特征在于： 所述培育的 温度是 8-25 °C。
6. 根据权利要求 1-5所述的室内培殖虫草的方法， 其特征在于： 单条隔离 培育采用虫草培育盘进行培育；
所述虫草培育盘由一个底部设置渗水槽（1-1) 的盒体（1)、 隔断装置 （2)、 保湿材料和与隔断装置相匹配的盒盖 （3) 四部分组成； 所述保湿材料设置在盒 体（1) 的底部， 隔断装置（2)设置保湿材料上， 盒盖（3)覆盖在隔断装置（2) 上部。
7.根据权利要求 6所述的室内培殖虫草的方法， 其特征在于： 所述盒体（1) 的底部的渗水槽 （1-1) 与隔断装置 （2) 的上沿相匹配， 渗水槽 （1-1) 能嵌套 到隔断装置（2)的上沿中， 隔断装置（2)的上下部被封闭， 形成一个封闭空间； 盒体 （1) 和与隔断装置 （2) 的组合能逐层重叠， 盒盖 （3) 覆盖在最上部。
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