KR20020048422A - Inoculating agents, entomogenous fungal beds and process for producing fruit body of pathogenic entomogenous fungus - Google Patents

Abstract

An inoculation agent containing mycelia of insect pathogens such as Cordyceps sinensis is prepared, and the inoculation agent is inoculated into insects to introduce the mycelia of the insect pathogens into the insect body to form the fruiting bodies by making the insects into insects.

Description

Inoculating agents, entomogenous fungal beds and process for producing fruit body of pathogenic entomogenous fungus

Insect pathogens, such as Cordyceps, are used as ingredients for herbal medicine and high-quality food. However, in general, because it is natural to take outdoor things, it is expensive and often uneven quality. The ecology of insect pathogenic fungi is unclear, and it is difficult to obtain large quantities of fruiting bodies of high quality by outdoor harvesting. Therefore, artificial culture of insect pathogens has been attempted.

For example, a method of obtaining a fruiting body obtained by raising the insecticidal fungus artificially inoculated with parasitic insects by controlling the temperature of the host has been reported (Japanese Patent Laid-Open No. 8-75). The inoculation used here is a suspension of follicular spores, and the inoculation is performed by pupae in this suspension.

It has also been reported to obtain a pupa extract of silkworms, to prepare an artificial medium containing the same, and to form a fruiting body using the medium (Japanese Patent Laid-Open No. 10-42691).

In addition to the method of inoculating pupa in the follicle spore suspension or inoculating the hyphae with the pupa, inoculation of the follicle spore turbidity into the pupa has been attempted. It is reported that it is not enough (Journal of Japanese Society of Culture 36: 67-72, 1995, Harada et al.)

The present invention relates to an inoculation agent for producing fruit bodies of insect pathogens, spawned insects, and a method for producing the same, and more particularly, to mass-produce fruiting bodies of insect pathogens such as Cordyceps sinensis. It relates to an inoculant, insect fungus and a production method thereof.

Figure 1 shows approximately the life cycle of the insect pathogens.

As described above, the artificial cultivation of Cordyceps sinensis using spores or mycelium such as follicle spores, conidia spores, etc. has been attempted many times. However, there are the following problems in using the follicle spores as an inoculator.

Asystole spores have a problem that is difficult to collect as an inoculation. Insect pathogens such as Cordyceps sinensis rarely produce fruiting bodies, and there are very few chances of collecting spores outdoors from fruiting bodies. In other words, it is extremely difficult to make the follicle spores an inoculation source and to collect the follicle spores in large quantities outdoors. For example, Cordyceps militaris is known to occur at about 10-year intervals in Tokyo, Japan, but rarely occurs in other years.

Even if it is possible to collect outdoors, it is difficult to collect large amounts of follicle spores for inoculating a large number of insects from small fruiting bodies without incorporating various germs at the same time. Various bacteria are attached to the surface of fruiting bodies growing outdoors. The inoculation source, which cuts off the fruiting bodies from the fruiting bodies and collects them from the fruiting bodies, can not prevent the incorporation of bacteria. When inoculating bacteria and cordyceps spores present together, bacteria often grow faster, and insects as hosts decay and fail to form fruiting bodies.

When isolated cultures are carried out on agar medium such as sabouraud medium using asymptomatic spores or fruiting bodies, insect pathogenic fungi usually grow in mycelial state. However, because mycelia have a filament structure, saliva is easily blocked in the case of inoculation, and mycelium is not suitable as an inoculum for inoculation.

Conidia spores (also called conidia) also have the following problems as inoculators. Mycelia cultured on agar medium form conidia. It is possible to use conidia as the inoculum, but it takes a tremendous amount of time to get the amount for large inoculations. The general method for obtaining conidia is as described below.

First, the mycelium is densely incubated on an agar plate in a petri dish to form conidia. Sterile water and the like are poured into the petri dish and the sterilized glass rod is rubbed with hyphae to separate the conidia from the hyphae. The suspension of the conidia obtained in this manner can be used as an inoculum. However, the sterile water filled with a petri dish should be added, rubbed, and the operation of obtaining the suspension of the conidia should be repeated. The amount of sterilized water to fill the petri dish once is 10-20 ml, but the number of condensed spores remaining in the petri dish is large even when rubbing.

Furthermore, conidia spores start to form approximately two days after inoculation with agar medium (25 ° C, under light supply conditions for 24 hours). The germination rate of the conidia in the agar medium was 90% in the conidia on the third day after mycelial vaccination, but dropped to 50% on the ninth day after the mycelial vaccination, and the germination rate dropped sharply afterwards. On the 15th, they rarely germinate. The colonies of fungi grow from concentric circles to old conidia. When the conidia are used as inoculum, the old conidia and the new conidia are present in a mixture to inoculate those with uneven germination rates, and as a result of inoculation, for example, mortality of an insect, fruiting body formation rate, etc. are difficult to stabilize. In other words, it is difficult to obtain a large amount of conidia with stable properties.

In addition, as a method of percutaneous infection by contacting fruiting bodies, mycelium, conidia and the like on the surface of an insect body, for example, in case of Militaris cordyceps, it takes 75 to 100 days or more.

It is an object of the present invention to mass-produce fruiting bodies of insect pathogens such as Cordyceps sinensis, which are equipped with quality on the basis of the above-mentioned problems, at a low price in a short period of time and easily.

The present inventors conducted the earnest research in order to achieve the above object, and completed the present invention by finding that the fruiting bodies of the insect pathogens can be easily mass-produced using the hyphal bodies of the insect pathogens.

That is, the present invention is as follows.

(1) An inoculant for producing fruiting bodies of insect pathogens, which contains mycelia of insect pathogens.

(2) A method for producing mycelium by shaking and incubating mycelia and / or conidia of insect pathogenic fungi, and producing an inoculant for producing fruiting bodies of insect pathogenic fungi.

(3) Shaking and culturing the mycelia of the insect pathogen fungi, and the method of producing an inoculant for the production of fruiting bodies of the insect pathogens.

(4) Insect fungus for inoculating the mycelia of insect pathogens in the body to produce fruiting bodies of insect pathogens.

(5) A method of inoculating a mycelia of an insect pathogen into an insect body to produce an fruiting body of an insect pathogen.

(6) A method of producing the fruiting body of the insect pathogenic fungus by injecting the above inoculation.

(7) There is a method for producing the fruiting bodies of the above insect pathogenic fungi, wherein the insects are in the shape of pupa.

In another aspect, the present invention is a fungus belonging to the genus Cordyceps genus Cordyceps , and relates to a method for producing the fruiting body of the insect pathogens.

The life cycle of the insect pathogenic fungus is not necessarily clear, but the approximate life cycle is shown in FIG. 1. For example, Cordyceps militaris , a species of cordyceps, an insect pathogenic fungus, the life cycle is generally considered as follows.

The fruiting body of Cordyceps militaris is a mycelium bundle, and the upper part of the fruiting body forms a club-shaped locus. Many small spores are gathered in the form of half-split, and the small spores are also called asymptomatic (子囊 殼). Breaking the punctate causes a large number of punctal sac, which contains thread-shaped spores. Spores are germ cells in plants and fungi that can become individual reproductive organisms. Spores contained in the capsules are called follicular spores. This follicle is an oily spore. Aspergillus protrudes from the pericardium to the outside, germinates in a desired environment such as under fallen leaves, and forms mycelia from a plurality of cells. In some cases, spores called conidia are formed from a part of mycelia. In nature, mycelia usually infect and infect insects in the soil, such as pupa, through the body surface. Mycelia that have been percutaneously infected and invaded the insect body become mycelium that shows the shape of yeast, and grow in the body. As the proliferation in the body progresses, the insect dies, while the mycelium returns to the form of mycelium, and the mycelium becomes a bundle and leaves the host body to form an fruiting body.

Herein, terms used in the present specification will be described. In the present specification, the term "insect pathogen" refers to a group of eukaryotes such as mushrooms, fungi, yeasts, and slime molds that form fruiting bodies as a host, and does not include bacteria that are prokaryotes. In addition, in the present specification, "fruiting body" refers to the formation of a locus or synnema by a bundle of hyphae. Simply speaking, the fruiting body includes a bundle of hyphae grown from asexual spores and a bundle of hyphae grown from sexual reproduction.

In addition, in the present specification, "Cordyceps sinensis" refers to asymptomatic amunococcus, nucleus fungus, ergot fungus, fungus nucleus and fungi belonging to the genus Cordyceps , and is included in entomopathogenic fungi.

In addition, in this specification, the "hyphal body" is a cell in the form of an insect pathogenic fungus that appears when grown in the body of an insect. Mycelium usually consists of one cell, morphologically showing the yeast phase and distinct from mycelium. The yeast phase refers to the state of proliferation by germination in a single cell cylindrical shape. The shape that appears when an insect mutant fungus proliferates in an insect body may be called an injection spore, a blast spore, a cylindrical spore, a short hypha, or a segmented cell. In this specification, all the things which have the form which appears when an insect pathogenic fungus proliferates in an insect body are called mycelium.

In addition, in this specification, an insect bacterium refers to the insect body which becomes a growth place of the fungus which becomes a host of a fungus.

An inoculation agent for producing an fruiting body of an insect pathogenic fungus as defined by the present invention, an insect fungus is provided, and the fruiting body of the insecticidal pathogenic fungus can be mass produced simply by using the same.

Hereinafter, the present invention will be described in detail.

<1> Inoculating Agent of the Present Invention

First, the inoculation agent of this invention is demonstrated. The inoculum of the present invention is characterized by containing a mycelia of the insect pathogen. Insect pathogens are as described above. The present invention with the entomopathogenic fungus used in the present invention, but applicable to entomopathogenic fungi that can form a mycelium preferably Cordyceps genus, Akanthomyces in, Beauveria genus, Gibellula in, Hirsutella in, Hymenostilbe in, Metarhizium in , Fungi belonging to the genus Nomuraea , Paecilomyces , Paraisaria , Stilbella , Tilachlidium , Tolypocladium , and more preferably fungi belonging to the genus Cordyceps , more preferably Cordyceps sinensis and Cordyceps militaris. Etc. are suitable.

The mycelium is as described above. Inoculants containing mycelia can be obtained by shaking the mycelia and / or conidia of insect pathogens and culturing them. Shake the mycelium and culture to easily mass production of mycelium. Once the mycelium is obtained, it may be grown by shaking the mycelium as a raw material to grow the mycelium.

The medium used in the shaking culture is easy to handle, and liquid medium is usually used. In addition, the medium may be suitably made according to the type of insect pathogen, but preferably sabouraud's medium, potato extract broth, silkworm pupa extract broth, and the like. More preferably, savora medium, silkworm pupa extract medium, etc. are mentioned. Savorwood medium is originally agar medium containing agar (Saborowood glucose agar medium), but is usually used as a liquid medium from which agar has been removed for shaking and cultivation when growing mycelium (also referred to as "Saborowood glucose agar." Medium "is described in" Bacterial illustration, High single yarn, p1279, No.25 "). In addition, sucrose, yeast extract, etc. may be used in combination with the liquid medium. Particularly preferred examples of the liquid medium include a yeast extract addition-sucrose liquid medium in which sugar in savora medium is changed from glucose to sucrose, that is, a yeast extract is blended.

The inoculation agent of the present invention should just contain mycelium, but can be used as the inoculation agent of the present invention, for example, by diluting the liquid medium in which the mycelium is grown as described above, and appropriately adjusting the concentration of the mycelium. In addition, for example, a biologically acceptable solution may be used as a liquid that may contain mycelium in the case of an inoculation agent such as an injection, and specifically, the liquid medium used for propagation of the mycelium may be used as it is. It is also good to use a liquid such as sterile water, liquid medium for insect cell culture, saline solution, Carlson's fluid. That is, you may mix | blend components other than mix | blending with the inoculation agent inoculated into the body of an insect in the inoculant of this invention.

The conditions such as temperature, shaking degree, etc. at the time of liquid culture for propagating mycelium can be appropriately adjusted according to the type of insect pathogenic fungus, but preferable conditions are as follows. The temperature at the time of liquid culture is 20-25 degreeC. In addition, it is good to adjust the degree of shaking properly, it is good to shake to about 100 ~ 110 (round trip / min). In addition, shaking and culturing to obtain mycelium is preferably performed in dark conditions.

The conditions for shaking the mycelium culture can be carried out under the same conditions even when starting the culture from the mycelia and conidia, or when starting the culture on the obtained mycelium itself.

Mycelia obtained under the above conditions are tuned and uniform, leading to quality stability of the inoculum. Here, the term "synchronization" means that the rate of growth is constant, so that the mycelia of the same developmental stage can be obtained in large quantities.

Although it does not restrict | limit especially in the formulation of the inoculation agent of this invention, The formulation which can introduce | transduce directly into the body of an insect is preferable, Specifically, it is preferable to set it as injection or the like. As described below, the use of an injection can improve the infection rate and the like and contribute to stable fruiting production. Suspensions containing mycelium cultured in liquid medium can be prepared at an appropriate concentration and used as an injection as it is.

<2> insect fungus of the present invention

Next, the insect fungus of the present invention will be described. Insect fungus of the present invention is characterized in that the mycelia of the insect pathogens are inoculated directly into the body, the mycelium is retained in the body.

Insect-like insects depend on the type of insect pathogen, such as inoculation, but preferably Mamestra brassicae, Tenebrio molitor (also called mealworm) , Bombyx mori, Galleria mellonella, Spodoptera litura, etc. However, particularly preferably Mamestra brassicae, Tenebrio molitor, Bombyx mori and the like can be mentioned. However, insects that become insect germs can be selected by limiting them to pair with the original host and insect pathogens in nature, and insects other than the original host can also be used as insect germs.

In addition, insects that become insect fungus use a pupa form, which is suitable in that it does not need food and can be contained at a high density. In addition, insects that become insect fungus are good for both adults and larvae, and they can be used for dead, if there is no corrosion, and they do not necessarily need to be dormant. That is, for example, a chrysalis stored in a frozen state can be used.

Inoculating mycelium in the body of an insect can be carried out by using the inoculation agent of the present invention described above as an injection and injecting it into an insect. After the mycelium is introduced into the insect body, the fruiting body can be formed under conditions suitable for fruiting body formation. Insect fungi of the present invention also depend on the inoculated insect pathogens and the types of insects that are hosts, etc., but can be stored at a low temperature (about 4 to 5 ° C) for at least one month.

<3> fruiting body production method of the present invention

Next, the fruiting body production method of the insect pathogen of the present invention will be described. The fruiting body production method of the present invention is characterized by inoculating a mycelium of an insect pathogenic fungus into the body of an insect. In the method of the present invention, the mycelia of the insect pathogen may be introduced directly into the body of the insect. For example, the method can be carried out by injecting the inoculation agent of the present invention as an injection into an insect that is a host. . Inoculation of insects into the body by injection leads to a high infection rate and stable production of fruiting bodies in a short period of time. When injecting the inoculation agent of the present invention into the body of an insect, the concentration of the mycelia in the inoculation agent is preferably 10 ⁴ to 10 ⁷ cells / ml, more preferably 10 ⁶ , depending on the type of insect pathogen or the like. ˜10 ⁷ cells / ml. It is preferable to keep the concentration in such a range from the point of increasing the infection rate and shortening the period from the inoculation to the fruiting body formation. In addition, the concentration in such a range also reduces work inconvenience, such as clogging the needle.

In addition, the inoculation amount of the mycelium can be appropriately controlled by the size of the insect, the concentration of the inoculation agent, and the like. It is usually desirable to inoculate the mycelia as much as possible in such an amount that the inoculant is not pushed out of the insect body. As the amount of mycelium introduced into the body is large, insects that are host tend to die early and shorten the period until fruiting body formation.

Preferred types and forms of the insects inoculated with the mycelium are the same as those exemplified as preferred in the above description of the insect flora.

In addition, the types of insect pathogens which are preferably used in the fruiting body production method of the present invention are the same as those of the insect pathogens given as the preferred inoculation agent of the present invention.

Insects inoculated with mycelium (insect fungus) can be placed under appropriate environmental conditions such as temperature and humidity to produce fruiting bodies. Depending on the type of fungus inoculated and the type of insect to be a host, the following conditions are preferable. As a temperature for generating and growing fruiting bodies in the inoculated mycelium, 20-25 degreeC is preferable. Within this range, the period until the fruiting body is formed can be shortened. On the other hand, below this range, it is difficult to form fruiting bodies or takes a long time to form fruiting bodies. In addition, the insect fungus is preferably kept under moisturizing conditions, specifically, a humidity of 90 to 100% is appropriate. In order to manage insect fungus under such temperature and humidity conditions, it is easy to use water moss. In addition, the degree of illumination is preferably 50 to 350 lx, the length of the light can be carried out by adjusting appropriately.

<4> Advantages of Using Mycelium

The biggest advantage of using mycelium as inoculum is that it can be mass produced in a short time. In addition, since the mycelium can be cultured in a liquid medium, the culture solution containing the mycelium can be adjusted at an appropriate concentration and used immediately as an injection, and is easy to handle.

For example, when a mycelium is multiplied and cultured once in a 200 ml liquid medium using a 500 ml flask (25 ° C., savored sucrose sucrose liquid medium with yeast extract), 10 to 20 Mycelium is injected into two petri dishes, and sterile water must be added and rubbed to prepare a suspension, which takes a long time in practical use.

Mycelia are a form of proliferation of insect pathogens in nature. In the present invention, the insect can be killed in a short period of time by forcibly introduced into the body of the insect in that it is prepared in a proliferative form in advance. The outdoor captured by mycelia of Cordyceps classified characters have been determined to start the dermal infection, and proliferation is attached to the body surfaces of the insects, the process for Cordyceps, usually known it takes about 40 days from which dead insects in the pupa form . However, mycelial injections can kill them in two to three days. Short-term death of insects is considered to occur by injecting high concentrations of mycelium directly into insect blood. Significantly reducing the time from inoculation to fruiting body formation is the biggest advantage of using mycelium as inoculum.

In other words, the mycelium can be stored for 1 month or more when the low temperature management (about 5 ℃) in the liquid medium suspension state. In terms of preservative capacity, mycelium is an easy inoculum.

In addition, the mycelia can be grown in large quantities of uniform quality, and can increase the production efficiency of fruiting bodies. In addition, the mycelium can be grown at a low price after it has obtained follicle spores and mycelium.

Although the present invention will be described in detail through embodiments of the present invention, the present invention is not limited only to the following examples.

Example 1 Preparation of Inoculant

Inoculation containing mycelia of Millilis cordyceps was prepared by the following method. Militerless Cordyceps sinensis was harvested from Mt. Iwaki (Aomori Prefecture) of Japan in July 1995 and used to be preserved in agar medium at the Forest Research Institute of Forestry and Fisheries. No. F-1176-21). The mycelia were collected from the conserved strains of this miterless Cordyceps sinensis, and the mycelia were added to a yeast extract-added savoraoud-sucrose liquid medium, and the liquid was incubated for 5 to 7 days by stirring culture (at 25 ° C. under dark conditions). Yeast extract added Savoraoud sucrose liquid medium is a peptone, yeast extract, sucrose is added to the water in a compounding amount as shown in Table 1. Yeast extract is also BACTO from DIFCO YEAST EXTRACT was used.

After 7 days, it was confirmed that the mycelia of Millilis cordyceps were growing about 10 ⁷ cells / ml in the liquid medium.

Inoculating agents of 2.1 × 10 ⁷ cells / ml, 1.7 × 10 ⁷ cells / ml, 2.3 × 10 ⁷ cells / ml were prepared by adjusting the concentration of the mycelia in the liquid medium.

Composition of Sucrose Liquid Medium with Yeast Extract (pH: 6.5) ingredient Compounding amount Peptone Yeast Extract Sucrose Distilled Water 10g10g20g1 ℓ

Example 2 Production of Fruiting Body

[Example 2-1] A fruiting body was formed under three different temperature conditions by injecting an inoculation agent containing a mycelium to a Memestra brassicae pupa. Specifically, it is as described below.

The inoculation agent of the mycelium concentration of 2.1 × 10 ⁷ cells / ml prepared in Example 1 was injected into the Memestra brassicae chrysalis with a microdispenser with a thinned tubule and a sharply improved tip. Memestra brassicae chrysalis inoculated with mycelium are stuffed and moisturized in spagnum moss moistened with water. Inoculated to 90 pupa, 30 were separated separately and managed under the temperature conditions of 15 ℃, 20 ℃, 25 ℃ respectively. In addition, the illumination degree was 100 to 300 lx, and the light was radiated for 14 hours and dark for 10 hours. Tables up to 47 days after inoculation are shown in Table 2.

Results of Example 2-1 Temperature Number of injection Developmental stage Days Fruiting Mature fruiting body Fruiting Mature fruiting body Early Maturity Early Maturity 25 ℃ 20 ℃ 15 ℃ 30 30 30 30 items0 items 29 Pieces15 Pieces0 Pieces 29 items0 items 21st 21st- 30 Days 43 Days 0 34 days46 days

As shown in Table 1, fruiting bodies mature, at least 34 days at 25 ° C., at least 46 days at 20 ° C., and no fruiting bodies at 15 ° C.

In a known report using Memestra brassicae (JPA 36: 67-72, 1995, Harada et al.), Mature fruiting body formation takes 75 days after inoculation, but in the present invention matures more than one month earlier. Fruiting bodies could be recovered.

[Example 2-2] Tenebrio molitor pupa was injected with an inoculator containing mycelium and fruiting bodies were formed under three types of temperature conditions in the same manner as in Example 2-1. As the inoculant, the mycelium concentration was 1.7 × 10 ⁷ cells / ml, and the total number of injections was 135, and 45 were maintained under each temperature condition. Other conditions were the same as Example 2-1. The progress until 47 days after inoculation is shown in Table 3.

Results of Example 2-2 Temperature Number of injection Developmental stage Days Fruiting Mature fruiting body Fruiting Mature fruiting body Early Maturity Early Maturity 25 ℃ 20 ℃ 15 ℃ 45 pieces 45 pieces 32 20 20 5 12 4 4 0 7 4 4 0 19 days 19 days 26 days 26 Days 32 Days 29 days and 40 days

Tenebrio molitor belongs to the order Coleoptera, and is very taxonomically different from Mamestra brassicae belonging to the order Lepidoptera, and in nature, the miterous cordyceps is not host. However, according to the method of the present invention, it was found that the fruiting body can be formed even by using Tenebrio molitor which is not normally a host in nature. In addition, it was found that the fruiting body of the miteris cordyceps was formed in a short time even with Tenebrio molitor as a host.

[Example 2-3] Bombyx mori pupa was injected with 100 µl of inoculating agent having a mycelium concentration of 2.3 × 10 ⁷ cells / ml, and the pupa was filled with water moss soaked in water and moisturized. Managed indoors. Formation of mature fruiting bodies was observed at least 47 days.

In addition, Bombyx mori pupae is larger than the insect pupae used in Examples 2-1 and 2-2, so it can be easily inoculated with a human tuberculin syringe, and fruiting bodies can be easily produced. Human tuberculin injectors are readily available sterile and require some experience to perform inoculation with the microdispenser described in Example 2-1, but once human tuberculin injectors are available, Even beginners can easily inoculate.

In addition, Bombyx mori pupa was not in the dormant state, it was found that the fruiting body can be formed even from insects that are not dormant according to the method of the present invention.

According to the present invention, it is possible to mass-produce an inoculant for producing fruiting bodies of insect pathogens such as Cordyceps sinensis at a low cost. Such inoculum of the present invention is uniform in quality, easy to handle, and can be stored. Furthermore, according to the present invention, the fruiting body of insect pathogens such as Cordyceps sinensis using the above inoculation agent can be mass-produced easily and in a short period of time with good efficiency. According to the present invention, the fruiting body can be produced without depending on the season.

Insect pathogens such as Cordyceps sinensis can be used as herbal medicine and high-quality food. Insect pathogens can also be developed for use as biopesticides. The present invention contributes to the stable and mass supply of the fruiting bodies of the insect pathogens in the field of using the insect pathogens.

Claims (8)

An inoculation agent for producing fruiting bodies of insect pathogenic fungi containing mycelia of insect pathogenic fungi. Method of producing an inoculation agent for the production of fruiting bodies of insect pathogens by shaking and culturing the mycelia and conidia of insect pathogens or mycelia or conidia. A method of producing an inoculum for the production of fruiting bodies of insect pathogens by proliferating the mycelia by shaking and incubating the mycelia of insect pathogens. Insect fungus for inoculating the mycelia of insect pathogens into the body of insects to produce fruiting bodies of insect pathogens. A method for producing fruiting bodies of insect pathogens by inoculating the mycelia of insect pathogens into the bodies of insects. The method of producing fruiting bodies of insect pathogens according to claim 5, wherein the inoculation is injection. 7. The method according to claim 5 or 6, wherein the insects are in the form of pupa. The method according to claim 5, wherein the insect pathogenic fungus belongs to genus Cordyceps.