WO2015003546A1

WO2015003546A1 - Culture method for natural species rejuvenation of artificially bred cordyceps host insect

Info

Publication number: WO2015003546A1
Application number: PCT/CN2014/080066
Authority: WO
Inventors: 刘飞
Original assignee: 重庆市中药研究院
Priority date: 2013-07-12
Filing date: 2014-06-17
Publication date: 2015-01-15
Also published as: CN103478080B; CN103478080A

Abstract

Disclosed is a culture method for natural species rejuvenation of an artificially bred cordyceps host insect and a technical solution of recycling a cordyceps host again after rejuvenated nurturing of one generation by placing the host in the wild. The key technique of the culture method for natural species rejuvenation of an artificially bred cordyceps host insect lies in that stage 2 to 4 artificially bred cordyceps host larvae are placed in and cultured in the native environment of the cordyceps host larvae until eclosion. The culture comprises two stages, namely, a transition period for adapting to the wild environment and a natural species exercise period. The present invention can effectively solve the problem of natural species degeneration of multi-generations of artificially bred cordyceps host insects.

Description

Breeding and breeding method for artificially breeding Cordyceps host insects

Technical field

The present invention relates to a method for preventing degradation of biological species (genetic property) in the field of insects, and more particularly to a method for artificially breeding a breeding insect of a Cordyceps host insect, the purpose of which is to solve the large-scale artificialization of Cordyceps host insects. The problem of species degradation in the process of 3-5 generations.

technical background

Cordyceps sinensis (Berk.) Sacc.] alias Cordyceps, Cordyceps, summer grass insects. According to the recent classification system, 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. More than 60 species of bat moths have been reported so far, including 20 species in Yunnan, 17 species in Tibet, 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. In the natural environment, the breeding of bat moth insects is controlled by a combination of ecological geographical distribution, food, vegetation, soil structure, temperature, humidity and natural enemies. The growth rate is slow and the survival rate is low. The bat moth larvae are natural. The breeding rate in the environment has become a key factor restricting the yield 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. In Jiangxi, Anhui, Hunan and other places, there are medicinal applications. The host of A. sinensis was identified by Zhu Hongfu and Wang Linyao of the Institute of Zoology of the Chinese Academy of Sciences, 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. When the host larvae of A. sinensis enters the soil The time is between late October and mid-November. After the fungus infection, he died. In March of the following year, the sub-seat from the head, the sub-seat from the growth of the ascospore and matured in about 1 month; the ascospores began to sporulate the spores to the worms for about 10 days. Generally distributed in a sunny slope of 10 ° -30 °, the lower the latitude, the earlier the time of unearthing; the higher the altitude, the lower the temperature and the later the time of unearthing. The harvesting period is from mid-to-late March to mid-May and mid-May; the lowest altitude is found at 240 meters, the highest is at 1,400 meters, and there are more distributions between 800 and 1000 meters. Because of the close relationship between A. sinensis and Cordyceps sinensis, it also has certain efficacy. Therefore, with the decrease of the resources of Cordyceps sinensis and the sharp increase in price, A. sinensis has gradually been sought after, and its resources are also due to A large number of indiscriminate excavations began to decline sharply. Therefore, the artificial cultivation of A. sinensis is also imperative.

In order to achieve the sustainable cultivation of Cordyceps, artificial breeding of Cordyceps host insects has been widely carried out to increase the number of host larvae and increase the yield of Cordyceps. However, due to the propagation of Cordyceps host insects under full artificial conditions for 3-5 generations, there will be various degradations such as individual reduction, weakening of mating ability and reduction of egg production. For the problem of species degradation of Cordyceps host insects in production, the traditional solution is to continuously collect the species from the native environment every year. Due to the limited resources of wild host insects, uncontrolled indiscriminate excavation not only makes the host insect resources endangered, but also seriously damages the natural ecological environment. Therefore, in order to achieve the sustainable self-production of host insects and the sustainable development of the Cordyceps sinensis industry, it must be adopted. Artificial breeding of host insects.

Summary of the invention

In order to solve the above problems, the present invention provides a technical solution for replanting the artificially propagated Cordyceps host in the field after a generation of re-cultivation, and the present invention provides a method for rejuvenating the wild environment of Cordyceps host insects. . The specific content is:

An artificial breeding method for artificially breeding Cordyceps host insects, wherein artificially propagated 2-4 instar Cordyceps host larvae are cultured in the native environment of Cordyceps host insects until emergence; wherein the primary environment culture includes a transition period adapted to the field environment And the two stages of the seed exercise period.

The Cordyceps host insect is a Lepidoptera bat moth insect.

The transition period adapted to the field environment is the first 1-2 months after the 2-4 year old Cordyceps host larvae are placed in the original environment; during this period, the temperature should be controlled at 8-25 ° C, and the soil moisture should be controlled at the mass water content 50. -80%, avoid rodents and birds from killing and killing Cordyceps host larvae, and avoiding animal husbandry trampling on Cordyceps host larvae.

In the transition period to adapt to the wild environment, the buds of the buds, the chrysanthemum, the carrot and the fruit of the fruit should be placed, and the density of the seed is 100-300 plants/m ² .

In the first winter after the Cordyceps host larvae were placed in the native environment of the larvae of the Cordyceps host larvae, the surface was covered with a surface covering thickness of more than 3 cm of hay, and the film was covered with heat preservation measures on the hay; the larvae of the Cordyceps hosted in the larvae of the Cordyceps host larvae In the second winter after the original environment, the surface area is covered with insulation measures for the thickness of the grass exceeding 3 cm; thereafter, no artificial temperature intervention measures are taken for the area.

During the seeding exercise period, the host larvae of the Cordyceps sinensis should avoid the rodents and birds from killing the larvae of the Cordyceps host larvae, so as to squeeze the soil matrix of the larvae of the Cordyceps host larvae in the manner of grazing larvae of the larvae, thereby consolidating the soil matrix. Avoid companions or other natural enemies to harm themselves through the soil matrix; stock livestock and eat them to keep the vegetation base exposed, increase soil oxygen supply and light.

The beneficial technical effect of the invention is that the seed breeding method for artificially breeding Cordyceps host insects provided by the invention can effectively solve the problem of germplasm degradation of the insects of the cordyceps insects which have been artificially propagated for many generations; effectively solving the problem of the species being mined by the insects The problem of destroying the ecological environment of the Cordyceps sinensis; solving the problem of wild resources depletion of Cordyceps host insects. Provides insect protection for the sustainable cultivation of Cordyceps.

detailed description

Example 1 Investigation on the Seed Rejuvenation Conditions of Artificially Breeding Cordyceps Host Insects 1

1. Selection of the original environment for Cordyceps culture

In the Cordyceps sinensis producing area or the area similar to the vegetation, temperature and humidity conditions of the Cordyceps sinensis producing area, two plots were selected as the experimental plot and the experimental experimental plot of the experimental group, in order to prevent large animals such as cattle and sheep from trampling at any time. Barbed wire fence. The slope of the rejuvenation base should be between 10-30 degrees, and the drainage is good; there are other shrubs other than turf plants to avoid high-intensity sunshine and bird damage to adults.

2, seed rejuvenation transition period management

The third generation of the larvae of the genus larvae, which were artificially bred, were collected, and the larvae of the 3rd generation of larvae were 2,000 months old. Each group consists of 1,500, named Xiaojin Group I, Xiaojin Group II, Gongga Group I, and Gongga Group II. Xiaojin group I and Gongga group I were used as the control group; Xiaojin group II and Gongga group II were used as experimental groups.

For the control group, the same culture mode as that of the larvae cultured in the semi-wild culture Cordyceps sinensis technique was used, that is, at the time of operation, the host larvae of the above-mentioned artificial words for 1-2 months were ordered together with the substrate and the corpus to the rejuvenation base. When sowing, use a digging tool such as a taro to dig a pit with a length, width and height of about 10-15 cm, put the larva into the pit, and return the turf to cover. The row spacing and nest spacing of the seeding are mainly based on the density of the larvae. When 2-5 larvae are placed, the row spacing and the nest spacing can be 50-60 cm; when 5-10 larvae are placed, the row spacing and the nest spacing can be 60. -lOOcm; other densities and so on. The time for larvae should be selected on cloudy or sunny days. On the day of larva release, a certain amount of water should be sprayed on the area where the larvae are placed. The humidity is held in groups by hand, and the hands are not released as standard to keep the larvae growing properly. humidity. To avoid disease Propagation, larvae should be replaced according to the disease during the operation of the larvae. During the transition period, large animals such as cattle and sheep should be strictly prohibited from entering the base; physical or chemical measures should be taken to prevent the harm of rodents and birds to larvae. During the transition period management process, light, temperature and humidity are not managed; in the transition period to adapt to the wild environment, bead buds, chrysanthemum, carrots and human fruit should be used for the larvae of the insects, and the density of the seeds is 100-300. /m ² .

For the experimental group, in addition to the same method as the control group, the following management measures were added: After the larvae were placed in the cordyceps to cultivate the native land, the sunny weather should be properly sprayed according to the humidity of the environment in which the larvae are located. When encountering extreme or low extreme temperatures, appropriate precautions should be taken. When the ground temperature is 10 °C higher than the growth temperature of the larvae and the time is more than 5-10d, the water can be cooled by spraying or covering the sunshade. The maximum temperature of the ground is not higher than 15 °C. When the ground temperature is 10 °C lower than the larval growth temperature and the time is more than 5-10d, it can be heated by covering the straw, plastic film or building a greenhouse; the minimum temperature of the ground is controlled at not less than 5 °C. . In the transition period to adapt to the wild environment, the buds of the buds, the chrysanthemum, the carrot and the fruit of the fruit should be placed, and the density of the seed is 100-300 plants/m ² .

After 45 days of transitional management, the survival rates of the control and experimental groups were counted separately. The details are shown in Table 1.

3, seed rejuvenation exercise management

At the end of the transition period, the larvae that were placed gradually adapted to the living environment in the wild, and began to crawl from the area to the surrounding native humus. Entering the exercise period; the larvae at this stage will adapt to the feeding of native grass roots, adapt and enhance the ability to fight against pathogens and natural enemies in the ground, and accept natural exercise of temperature, humidity and light in nature until the larva develops into an adult. At this stage, for the control plots, large animals such as cattle and sheep are strictly prohibited from trampling at any time, and no artificial intervention is made on the temperature and humidity in the plot and the growth of vegetation.

For the experimental group plot, in the first winter after the Cordyceps host larvae were placed in the native environment of the larvae of the Cordyceps host larvae, the ground cover was used to cover the hay with a thickness of more than 3 cm, and the film was covered with moisture on the hay; In the second winter after the larvae were placed in the native environment of the larvae of the larvae, the surface was covered with a surface covering thickness of more than 3 cm of hay. After that, no artificial temperature intervention was taken for the area. In addition, when the base grass begins to grow and gradually covers the surface, the herbivores such as cattle and sheep are arranged to enter the base to feed the grass, so that the base of the turf is exposed to the outside. At the same time, physical or chemical measures should be taken to prevent the harm of rodents and birds to the larvae of the experimental and control plots. When the larvae grew to a level of about 6-8 in the native environment, the survival rates of the control and experimental larvae were counted separately. The details are shown in Table 1. Table 1 Effect of species rejuvenation treatment on growth and development of host larvae

Transition period

Survival rate (%) Body length and body weight (g) Survival rate (%) Body length and body weight (g) Small gold group I 71.2 11.3±2.05 0.09±0.01 21.5 50.3±2.4 0.71±0.11 Gongga group I 56.8 12.1±1.03 0.09±0.01 19.3 48.9±3.1 0.67±0.09 Xiaojin Group II 89.4 12.3± 1.13 0.10±0.01 54.3 50.9±4.2 0.89±0.12 Gongga Group II 91.3 11.9±1.04 0.09±0.01 61.5 50.7±2.8 0.94±0.13

4, adult management

When the host insects of the control group and the experimental group grow to the adult stage, like the wild host insects, the adult will climb to the surface of the turf. When the time is ripe, the female adult will hang on the blades or leaves, and Male adults, including wild male adults, are attracted to fly over female adults. When the male adult subducts into the turf, it can track the flight path of the male adult to find male and female adults that are mating or mating. Male and female adults are collected into the netting bed of the net or in the petri dish with filter paper to form the host insect source after wild exercise. In this way, the seed of the host insects is revived. During adult collection, birds should be strictly controlled for adult feeding. The number of adults eventually growing into adult worms was counted.

Table 2 Effect of species rejuvenation treatment on morphological characteristics of adult moth

Female moth

Average body length Average body weight (g) Average wingspan (mm) Mean body length (mm) Average body weight (g) Average wingspan (mm) Small gold group I 2.34±0.14 0.40±0.09 4.13±0.07 1.81±0.13 0.19±0.03 3.51 ±0.11 Gongga group I 2.51±0.17 0.49±0.11 3.99±0.09 1.89±0.11 0.21±0.05 3.61±0.12 Xiaojin group II 3.89±0.42 0.67±0.12 5.12±0.10 1.98±0.16 0.20±0.04 3.98±0.15 Gongga group II 3.21 ±0.19 0.68±0.16 4.38±0.08 2.21±0.15 0.24±0.05 4.02±0.13 The results of Table 1 and Table 2 demonstrate that the rejuvenation base can squash the soil matrix of the larvae after grazing. The compact soil matrix is a protective wall that prevents companions or other natural enemies from damaging themselves through the soil matrix; proper grazing can also prevent overgrowth of vegetation, and the grazing of the vegetation can not be used for the growth and development of the bas Provide adequate oxygen conditions, while light will also have a positive impact on larval growth and development.

Example 2 Investigation on the conditions of seed rejuvenation of Cordyceps host insects 2

The 1000 larvae of the 3rd generation of the small golden bat moth and the Gongga bat moth were respectively named as Xiaojin Formation III and Gonggao group III, and the two groups of larvae were divided into 10 groups and transferred to the main mixed bead buds. , chrysanthemum ginger, carrots and fruit of life and other plateau humus of a few plateau plants (also can be replaced by artificial matrices). The humus soil can be clumped by hand and the hand can be released without the humidity standard. The word temperature condition is 8-18 °C. The word density is the amount of no more than 30 larvae in the unit word box (length 30cmX width 20cm height X 15cm). Because the newly hatched larvae have weak activity, the range of activities is small, and the damage between individuals can be neglected, so the newly hatched larvae can be suitable for intensive words. The newly hatched larvae intensive words are raised for 30-60 days. Time (depending on the temperature of the word and the occurrence of the disease to determine the specific time. In the word temperature range of 8-18 °C, the lower the word temperature, the longer the intensive wording; the more serious the disease, the intensive words The time is relatively shorter). During the period, the word and matrix can be replaced or added according to the degree of decay of the word and the presence or absence of the disease. When replacing or adding larval word materials or matrices, the staff (according to the cleanliness requirements) dress according to the conventional anti-microbial contamination, and wear finger cots to avoid disease transmission. The tools that touch the diseased worms or pathogens during the operation should be replaced in time. . During the processing of the lexical materials, the bead buds, chrysanthemums and carrots are washed with water and then cleaned with an ultrasonic cleaner. After that, they are 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, chrysanthemums, carrots, and fruit of life should be rotted or undersized before or after processing.

When the larva grows into the forehead stage in the cultivation tray, it is transferred to a plastic dish with a relatively large space. The culture dish is padded with moisturizing cotton cloth or filter paper or other fibrous material, and each culture dish is placed on a front sputum. . The incubation temperature of the front mites is 8-18 ° C, and it is protected from light; the culture humidity is the degree of humidity when the water is not seen when it is tilted, and it is moisturized with cold water sterilized at 60-100 ° C. In order to avoid the spread of the disease, the fingertips should be replaced according to the disease during the operation. The cockroach that turns white will continue to be incubated under the same conditions. When the color of the cockroach is changed to dark brown, the cockroach will be transferred to the feathering tray in the feathering chamber to prepare for emergence.

The feathering of the dragonfly is done in the feathering tray. The entire feathering tray has a plurality of feathering holes. The bottom of the feathering hole is padded with a cotton cloth or filter paper or other fiber material that can be moisturized. The humidity is the degree of humidity when water is not seen when tilting, and is sterilized by 60-100 ° C high temperature. Cold water moisturizing. The feathering disc is covered with a gauze, which not only prevents the emergence of adult feathers, but also becomes an attachment to adults. The mature cockroach breaks through the head first in the feathering tray, then slowly unshells, and the successful adult husks are attached to the gauze to complete the relaxation of the wings. The temperature of the feathering is 8-18 ° C, the light conditions are natural light intensity and photoperiod; the air humidity of the feather is 60-70%. The feathered adults are transferred to the mating bed of the nets for mating and spawning. A species of artificially propagated or artificially cultivated Cordyceps is obtained. The mating rate of adult male adults and the egg laying rate of female adults in Xiaojin group III, Gongga group III and Xiaojin group II and Gongga group II in Example 1 were counted. The details are shown in Table 3.

Table 3 Investigation of reproductive ability of adults

Quantity Average number of crossings Average number of eggs laid (pieces) Small gold group III 111 1. 16

Male adult

Gongga Group III 96 0. 94

Xiaojin Group II 621 2. 35

Gongga Group II 483 2. 53

Xiaojin Formation III 107 324. 25

Female adult

Gongga Group III 102 309. 88

Xiaojin Formation II 704 445. 42

Gongga Group II 473 435. 91

1000 eggs of Xiaojin group III and Gongga group III were collected as the hatching control group, and 1000 eggs of the adults of Xiaojin group II and Gongga group II in Example 1 were collected as the hatching experiment group; the wild environment was collected separately. 1000 granules of the golden bat moth and the tribute bat moth of the bat moth spawned as the Xiaojin wild group and the Gongga wild group. Remove the bracts and other debris that broke during spawning in each group, and wash them with water to complete the collection of fertilized eggs. Eggs are placed in a petri dish that always keeps the filter paper moist, each dish

200 capsules, covered with a lid, according to the larval embryo development can be divided into three stages of pre-, middle, and late temperature-changing culture, about 15 days before, during the day (8 am to 8 pm, the same below), temperature 6 ~12 °C, night (8 pm to 8:00 am, the same as below), temperature 6~8 °C; mid-20 days, daytime temperature 11~13 °C, night temperature 7~9 °C, late About 15 days, the daytime temperature is in the range of 12 to 16 °C, and the nighttime temperature is around 10 °C. At the end of each stage, the eggs should be cleaned with clean water (60-100 °C high temperature disinfected cold water), cleaned (60-100 °C dry heat sterilized filter paper, cooled) clean filter paper to absorb dry eggs The moisture on the surface of the grain is placed in a freshly placed dish covered with moist filter paper to moisturize the culture.

In the late stage of the egg, close attention should be paid to the hatching of the larvae. Once the hatching is discovered, the egg filter paper should be taken out of the culture dish in time, and the absorption part is cleaned with a clean dry filter paper (60-100 ° C dry heat sterilized filter paper, cooling). Moisture, keeping it semi-moist, making it easy for newly hatched larvae to crawl freely. At the same time, the worm room should be cleaned and dried. After disinfecting with 300~800ppm of sodium hypochlorite solution, close the doors and windows, and open the door and window for 1~3 hours before the insects are kept. The words used in the indoor word nursery are the natural cultivar bead bud, the underground rhizome of the ginseng and the ginseng fruit, and the artificial pronouns, sweet potato, carrot, etc.; at the end of the bat moth, the previously prepared vocabulary and substrate are loaded into the worm Inside the box, see the day when the larvae start to hatch, and then disinfect and dry the surface moisture, spread it evenly in the box, put it into the larvae, and let it feed after the larvae hatch. Each box was given 100 eggs of bat eggs and raised to the 4th instar larvae, and the survival rate was counted. The results are shown in Table 4 below. Table 4: Table of survival of larvae in the indoor wording stage of bat moth larvae

Number of eggs administered, larval hatching rate, percentage of larval survival

(grain) (%) (%)

Xiaojin Formation III 1000 43. 5 31. 65 Gongga Group III 1000 51. 6 51. 49 Xiaojin Formation II 1000 81. 5 79. 46 Gongga Group II 1000 69. 1

Xiaojin Wild Group 1000 79. 4

Gongga wild group 1000 71. 5 91. 35 The data in Tables 3 and 4 show that the regenerative treatment of host Kunming is superior to that of artificial words, and the hatching rate and survival rate of the rehabilitated bat moth adult spawning and wild The bat moth eggs are close. The above data indicate that the species degradation of the host bat moth under the artificial wording condition can be effectively solved by the seed rejuvenation treatment of the host insect.

Claims

The invention relates to a seed breeding method for artificially breeding Cordyceps host insects, characterized in that: the artificially propagated 2-4 instar Cordyceps host larvae are placed in the original environment of the Cordyceps host larvae until they are feathered; wherein the cultivation includes adapting to the field The transition period of the environment and the two stages of the seeding exercise period.

The method according to claim 1, wherein the insect-carrying host insect is a Lepidoptera bat moth insect.

The method according to claim 1, wherein the transition period of adapting to the field environment is the first stage after the 2-4 year old stage of the Cordyceps host larva is placed in the native environment. 2 months; during this period, the temperature should be controlled at 8-25 °C, and the soil moisture should be controlled at 50-80% of the mass water content.

The method for cultivating the insect breeding of the artificially propagated Cordyceps host according to claim 3, characterized in that: in the transition period adapted to the wild environment, the buds of the buds of the buds of the buds, the turmeric, the carrots and the fruit of the living should be placed, The density of the delivery is 100-300 strains/m ² .

The method for cultivating an insect breeding species of an artificially propagated Cordyceps host according to claim 1, characterized in that: in the first winter after the Cordyceps host larvae are placed in the native environment of the larvae of the Cordyceps sinensis, surface coverage is applied to the putting area. Insulation measures for covering more than 3 cm of hay and covering the hay on the hay; in the second winter after the Cordyceps host larvae are placed in the native environment of the larvae of the Cordyceps larvae, the surface covering the ground cover is covered with a thickness of more than 3 cm of hay; No artificial temperature interventions are taken for the area to be placed.

The method for cultivating the breeding of the insect-infested host insect according to claim 1, characterized in that: during the seed-type exercise period, the soil of the larvae of the cordyceps host larvae is squeezed in the manner of grazing in the larvae of the Cordyceps host larvae. The substrate, the compact soil matrix is a protective wall that prevents the companion or other natural enemies from damaging themselves through the soil matrix; the livestock eats the vegetation, keeps the base of the vegetation exposed, and increases the soil supply.