Metarhizium ledebusii culture medium as well as preparation method and application thereof

Abstract

The invention provides a metarhizium ledebusii culture medium as well as a preparation method and application thereof, and belongs to the technical field of microbial culture. The invention provides a Metarhizium anisopliae liquid seed culture medium. The Metarhizium anisopliae liquid seed culture medium comprises the following raw materials in parts by mass: 100-125 parts of potato juice with the mass percentage content of 20%, 3-5 parts of glucose, 0.2-1 part of soybean meal and 100-125 parts of distilled water. The invention also provides a solid fermentation culture medium which comprises the following raw materials in parts by mass: 100-125 parts of wet soybeans and 0.3-0.5 part of inorganic salt, and the inorganic salt is an aqueous solution containing the following components in percentage by mass: 0.04 to 0.06 percent of KH2PO4 and 0.008 to 0.01 percent of MgSO4. 7H2O. The two culture media are adopted for spore production culture of metarhizium anisopliae, the spore production time can be greatly shortened, the spore production quantity can be increased, the prepared spore powder is good in quality, and the germination rate of the spore powder reaches up to 96% or above.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial culture, and in particular relates to a Metarhizium anisopliae culture medium and a preparation method and application thereof. Background Art

[0002] Metarhizium rileyi is a widely distributed entomopathogenic fungus with a broad host range and strong pathogenicity. It is particularly effective against Noctuidae pests, including the beet armyworm, soybean armyworm, Spodoptera litura, and fall armyworm. Compared with traditional chemical pesticides, M. rileyi is more environmentally friendly, with minimal impact on non-target organisms and the environment, and does not cause significant pollution. Furthermore, it can spread naturally in fields and cause multiple infections, resulting in long-term pest control, making it a valuable biocontrol agent for agricultural pests. M. rileyi primarily invades the insect wall by producing specialized structures such as appressorium and germ tubes, disrupting the insect's physiological functions, growing and multiplying within the insect, competing with it for nutrients and ultimately killing it. This suggests that the infectivity of M. rileyi is directly related to the germination capacity of its conidia. Rapid spore germination and a high germination rate naturally increase the lethality of the infection. Therefore, preparing high-quality conidia is the first step in using Metarhizium anisopliae for biological pest control.

[0003] However, the production of conidia of entomopathogenic fungi has been widely studied both domestically and internationally, with liquid-solid biphasic fermentation being the primary method for efficiently producing highly active spores. High-density mycelium and budding spores are obtained through liquid fermentation, which are then inoculated onto solid-state fermentation media for continued cultivation to produce a large number of conidia. However, the current problems with Metarhizium reinhardtii are its slow spore production and low spore production. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a culture medium for Metarhizium anisopliae, in which potato juice, glucose and soybean components are used to prepare a liquid seed culture medium, and soybeans, potassium dihydrogen phosphate and magnesium sulfate are compounded to prepare a solid-phase fermentation culture medium, which can shorten the spore production cycle and increase the spore production amount.

[0005] The present invention provides a Metarhizium anisopliae culture medium, comprising a liquid seed culture medium and a solid fermentation culture medium, wherein the liquid seed culture medium comprises the following raw materials in parts by mass: 100-125 parts of potato juice, 3-5 parts of glucose, 0.2-1 part of soybean powder, and 100-125 parts of distilled water;

[0006] The solid fermentation culture medium comprises the following raw materials in parts by mass: 100-125 parts of wet soybeans and 0.3-0.5 parts of inorganic salts; the inorganic salts are aqueous solutions containing 0.04%-0.06% KH2PO4 and 0.008%-0.01% MgSO4·7H2O in mass percentage.

[0007] Preferably, the liquid seed culture medium comprises the following raw materials in parts by mass: 100-110 parts of potato juice, 4 parts of glucose, 0.6-1 part of soybean powder and 100-110 parts of distilled water;

[0008] The solid fermentation medium comprises the following raw materials in parts by mass: 100 parts of wet soybeans and 0.4 parts of inorganic salts.

[0009] Preferably, the inorganic salt is an aqueous solution containing 0.05% by mass of KH2PO4 and 0.01% by mass of MgSO4·7H2O.

[0010] Preferably, the wet soybeans are prepared by soaking the soybeans in water until saturated and then removing surface moisture.

[0011] Preferably, the particle size of the soybean powder is 0.15 to 0.2 mm.

[0012] The present invention provides the use of the Metarhizium reinhardtii culture medium in culturing Metarhizium reinhardtii and / or preparing Metarhizium reinhardtii spore powder.

[0013] Preferably, the spore powder of Metarhizium anisopliae includes conidia powder of Metarhizium anisopliae.

[0014] The present invention provides a method for preparing Metarhizium reinhardtii conidia based on the Metarhizium reinhardtii culture medium, comprising the following steps:

[0015] inoculating the spore liquid of Metarhizium anisopliae into a liquid seed culture medium for seed culture to obtain a seed liquid;

[0016] The seed liquid is transferred to a solid fermentation medium for solid fermentation to obtain conidia of Metarhizium anisopliae.

[0017] Preferably, the inoculation amount of the Metarhizium anisopliae spore liquid inoculated into the liquid seed culture medium is 1% to 3%; the density of the Metarhizium anisopliae spore liquid is 1.0×10 7 ~1.0×10 8 spores / mL;

[0018] The temperature of the seed culture is 23-27° C., the rotation speed of the seed culture is 190-220 rpm, and the time of the seed culture is 3-5 days.

[0019] Preferably, the inoculation amount of the seed liquid transferred to the solid fermentation medium is 8% to 10%;

[0020] The temperature of the solid fermentation is 25-30° C., and the humidity of the solid fermentation is 60%-80%;

[0021] The paving thickness of the solid fermentation culture medium is 2 to 3 cm.

[0022] The present invention provides a culture medium for Metarhizium reinhardtii, comprising a liquid seed culture medium and a solid fermentation culture medium. The liquid seed culture medium comprises the following raw materials in parts by weight: 100-125 parts of potato juice, 3-5 parts of glucose, 0.2-1 part of soybean powder, and 100-125 parts of distilled water; the solid fermentation culture medium comprises the following raw materials in parts by weight: 100-125 parts of wet soybeans and 0.3-0.5 parts of an inorganic salt; the inorganic salt is an aqueous solution containing 0.04%-0.06% by weight of KH2PO4 and 0.008%-0.01% by weight of MgSO4·7H2O. The liquid seed culture medium for Metarhizium reinhardtii provided by the present invention comprises potato juice, glucose, and soybean powder, wherein the potato juice and glucose provide a rich carbon source for the growth of the strain; and the soybean powder is nutritious and rich in various amino acids, fatty acids, vitamins, and minerals, thereby accelerating the metabolic pathways of Metarhizium reinhardtii and promoting the growth of the strain. At the same time, soybeans are used as the carrier of the solid fermentation medium. Since the soybean particles themselves have a certain particle size, the medium itself forms a certain gap, so no additional ventilation is required during the cultivation process, and no other fillers need to be added to increase the air permeability. At the same time, the seed coat wrapped in soybeans can lock in moisture, stabilize the humidity of the matrix, and facilitate the stable spore production of the strain. In addition, soybeans are rich in ingredients such as protein, carbohydrates, and vitamins, which provide comprehensive nutritional support for the growth of Metarhizium reinhardtii. The inorganic salts added to the solid matrix can regulate the osmotic pressure and pH value of the culture medium, and can also serve as catalysts for enzymes to accelerate the reaction of microorganisms and shorten the spore production time. Experiments have shown that the Metarhizium reinhardtii spore powder produced by culturing Metarhizium reinhardtii using the fermentation medium provided by the present invention has stable quality and high spore germination rate, and can be further processed into various formulations such as wettable powders and dispersible oils to prevent and control ground pests; the soybean culture material can also be directly crushed and applied to the field by broadcasting, furrow application, or hole application, so as to achieve the effect of preventing and controlling underground pests and increasing soil fertility. At the same time, the raw materials required for the fermentation medium of Metarhizium anisopliae provided by the present invention are common and easy to obtain, with low cost, simple preparation method process, simple operation and easy to master, which solves the problems of high cost, low spore production and unstable spore production in the prior art, is easy to realize large-scale batch production, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result of liquid fermentation product in the liquid fermentation stage of Example 1;

[0024] Figure 2 This is the result of Metarhizium anisopliae spore powder produced after solid-state fermentation in Example 1;

[0025] Figure 3 This is a diagram of the spore production effect of Metarhizium anisopliae after fermentation on different solid matrices in comparative example 3, where A. sorghum; B. rice; C. peeled sorghum; D. wheat; E. millet; F. Job's tears; G. corn; H. cornmeal. DETAILED DESCRIPTION

[0026] The present invention provides a Metarhizium anisopliae culture medium, comprising a liquid seed culture medium and a solid fermentation culture medium, wherein the liquid seed culture medium comprises the following raw materials in parts by mass: 100-125 parts of potato juice, 3-5 parts of glucose, 0.2-1 part of soybean powder, and 100-125 parts of distilled water;

[0027] The solid fermentation culture medium comprises the following raw materials in parts by mass: 100-125 parts of wet soybeans and 0.3-0.5 parts of inorganic salts; the inorganic salts are aqueous solutions containing 0.04%-0.06% KH2PO4 and 0.008%-0.01% MgSO4·7H2O in mass percentage.

[0028] In the present invention, the liquid seed culture medium preferably comprises the following raw materials in parts by mass: 100-110 parts potato juice, 4 parts glucose, 0.6-1 part soybean powder, and 100-110 parts distilled water, which may be 100 parts potato juice, 4 parts glucose, 0.6 part soybean powder, and 100 parts distilled water. The potato juice is preferably prepared by washing and peeling the potatoes, chopping them, boiling them in distilled water until soft, filtering, and collecting the filtrate to obtain the potato juice. The material-to-liquid ratio of the potatoes to distilled water is preferably 1:1-6, and may be 1:5. The particle size of the soybean powder is preferably 0.15-0.2 mm, and may be 0.16-0.18 mm. The liquid seed culture medium is preferably prepared by mixing the potato juice, glucose, and soybean powder, adding a fixed volume of distilled water, and sterilizing with high-pressure steam to obtain the liquid seed culture medium. The temperature of the high-pressure steam sterilization is preferably 121-125° C., the pressure is preferably 103-140 Pa, and the time is preferably 25-30 min.

[0029] In the present invention, the solid fermentation medium preferably comprises the following raw materials in parts by weight: 100 parts wet soybeans and 0.4 parts inorganic salts. The wet soybeans are soaked soybean granules. The inorganic salts are preferably an aqueous solution containing 0.05% KH2PO4 and 0.01% MgSO4·7H2O by weight. The wet soybeans are preferably prepared by soaking soybean granules in water until saturated and then removing surface moisture. The soaking time is preferably 10 to 12 hours, and can be 11 hours. The surface moisture removal method is to allow the soaked wet soybeans to dry naturally. The method for preparing the solid fermentation medium comprises mixing the inorganic salts into the wet soybeans and sterilizing them with high-pressure steam to obtain the solid fermentation medium.

[0030] The present invention provides the use of the Metarhizium reinhardtii fermentation culture medium in culturing Metarhizium reinhardtii and / or preparing Metarhizium reinhardtii spore powder.

[0031] In the present invention, the spore powder of Metarhizium reinhardtii preferably includes conidia powder of Metarhizium reinhardtii. The present invention has no special limitation on the source of the Metarhizium reinhardtii, and any Metarhizium reinhardtii known in the art can be used.

[0032] The present invention provides a method for preparing Metarhizium reinhardtii conidia based on the Metarhizium reinhardtii fermentation medium, comprising the following steps:

[0033] inoculating the spore liquid of Metarhizium anisopliae into a liquid seed culture medium for seed culture to obtain a seed liquid;

[0034] The seed liquid is transferred to a solid fermentation medium for solid fermentation to obtain conidia of Metarhizium anisopliae.

[0035] The present invention first inoculates Metarhizium anisopliae spore liquid into a liquid seed culture medium for seed culture to obtain seed liquid.

[0036] In the present invention, the inoculation amount of the Metarhizium anisopliae spore liquid inoculated into the liquid seed culture medium is preferably 1% to 3%, and can be 2%. The density of the Metarhizium anisopliae spore liquid is preferably 1.0×10 7 ~1.0×10 8 Spores / mL, which can be 5.0×10 7Spores / mL. The method for preparing the spore liquid of Metarhizium reinhardtii is preferably to inoculate Metarhizium reinhardtii into SMAY medium and culture it at 24-26°C for 7-10 days, separate the spore powder and add 1% Tween-80 solution, break it up and mix it to obtain the spore liquid of Metarhizium reinhardtii. The temperature of the seed culture is preferably 23-27°C, can be 24-26°C, or can be 25°C. The rotation speed of the seed culture is preferably 190-220rpm, and can be 200-210rpm. The time of the seed culture is preferably 3-5d, and can be wielded for 4d. During the seed culture, full illumination culture is preferred.

[0037] After obtaining the seed liquid, the present invention transfers the seed liquid to a solid fermentation medium for solid fermentation to obtain conidia of Metarhizium reinhardtii.

[0038] In the present invention, the inoculation amount of the seed liquid transferred to the solid fermentation medium is preferably 8% to 10%, and can be 9%. The paving thickness of the solid fermentation medium is preferably 2 to 3 cm, and can be 2.5 cm. The temperature of the solid fermentation is preferably 25 to 30°C, and can be 26 to 29°C, or 27 to 28°C. The humidity of the solid fermentation is preferably 60% to 80%, and can be 70% to 75%. During the solid fermentation, it is preferably accompanied by light. The light cycle L / D is 16h / 8h. The method of the solid culture is preferably to culture for 3 to 4 days under sealing conditions with a sealing film. After the solid culture material is covered with white mycelium, the seal is replaced with a breathable material to tie the mouth, reduce the humidity of the culture material, promote spore production of the strain, and continue to culture for 2-3 days to obtain a large amount of spore-producing Metarhizium rapae.

[0039] In the present invention, the method for collecting the spore powder of Metarhizium reinhardtii preferably comprises drying a large amount of spore-producing Metarhizium reinhardtii, sieving, collecting the sieve underflow, and obtaining the spore powder of Metarhizium reinhardtii. The drying temperature is preferably 34 to 36°C, and may be 35°C. The drying time is preferably 22 to 26 hours, and may be 24 hours. The filtration is preferably sequentially sieved through 20 mesh, 60 mesh, and 100 mesh sieves.

[0040] In the present invention, the above culture medium is used to prepare spore powder of Metarhizium anisopliae. Compared with the traditional culture medium or the control culture medium, the spore production time can be advanced, the spore production cycle can be shortened, and the spore production amount can be greatly increased to 8.25×10 9 Spores / g material. In addition, the spore germination rate of the Metarhizium anisopliae spore powder prepared by the present invention reaches more than 96%, which greatly improves the spore activity compared with the spore powder prepared by traditional culture medium or control culture medium.

[0041] The following is a detailed description of the fermentation medium of Metarhizium anisopliae provided by the present invention, its preparation method and application, in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] A fermentation and culture method of Metarhizium anisopliae

[0044] 1. Culture medium formulation

[0045] Liquid seed culture medium materials: 20% potato juice 500 mL, glucose 20 g, soybean powder 5 g, distilled water 500 ml;

[0046] Solid fermentation medium materials: soybean particles, 0.2 mL of an aqueous solution containing 0.05% KH2PO4 and 0.01% MgSO4·7H2O.

[0047] SMAY medium: 40 g maltose, 10 g peptone, 10 g yeast extract powder, 20 g agar, and 1000 mL distilled water.

[0048] 2. Preparation of Fermentation Medium

[0049] Preparation of liquid seed culture medium: Take the required liquid seed culture medium material and place it in a 300 mL Erlenmeyer flask with a liquid volume of 150 mL. Sterilize it with high-pressure steam at 121°C for 20 minutes and cool it for use.

[0050] Preparation of solid fermentation medium: Soak soybeans in advance for 10 hours, filter out the water, and air-dry for 30 minutes. Add an aqueous solution containing 0.05% KH2PO4 and 0.01% MgSO4·7H2O at a rate of 0.3% by weight of the wet material, mix well, and place in a conical flask. Sterilize with high-pressure steam at 121°C for 30 minutes, and cool for later use.

[0051] Preparation of SMAY medium: Mix 40 g of maltose, 10 g of peptone, 10 g of yeast extract powder, and 20 g of agar, add distilled water to make up to 1000 mL, sterilize with high-pressure steam at 121°C for 30 min, and cool for later use.

[0052] 3. Preparation method of conidia of Metarhizium anisopliae

[0053] 3.1 Liquid fermentation stage

[0054] Inoculate Metarhizium anisopliae into SMAY medium and culture in a 25°C constant temperature incubator for 7 to 10 days until conidia mature. Take an appropriate amount of spore powder and add sterile 1% Tween-80 solution, break it up and mix it to prepare 1×10 8The spore suspension was inoculated into the liquid seed culture medium at a 1% inoculum rate and cultured for 5 days in a shaker at 200 r / min and 25°C under full illumination to obtain a liquid fermentation product (such as Figure 1 shown).

[0055] 3.2 Solid fermentation stage

[0056] In a sterile operating table, the liquid fermentation product of Metarhizium anisopliae was added to the solid fermentation medium at a ratio of 10% (v / w) and shaken. The thickness of the solid matrix was about 2 to 3 cm and should not be too thick. After the sealing film was tied, it was placed in a culture condition with a temperature of 25°C and a light intensity of L / D = 16h / 8h for 3 to 4 days. After the solid culture medium was covered with white mycelium, the sealing film was replaced with a newspaper to reduce the humidity of the culture medium and promote the spore production of the strain. The culture was continued for 2-3 days, and a large number of spore-producing Metarhizium anisopliae were cultured (see Figure 2 shown).

[0057] 3.3 Collection of M. anisopliae spore powder

[0058] The fermented solid fermentation medium is placed in an oven at 35°C and dried for 24 hours. It is first filtered through a 20-mesh sieve, then through a 60-mesh sieve, and finally through a 100-mesh sieve to collect the conidia of Metarhizium reinhardtii; the collected spores are dried twice to prepare Metarhizium reinhardtii spore powder.

[0059] 4 Determination of spore production and spore germination rate of Metarhizium anisopliae

[0060] Conidia production determination method: Mix equal volumes of solid fermentation medium and sterile 0.5% Tween-80 water at a 1:1 (m:v) ratio. Oscillate at 200 rpm for 1 hour until the spores are completely dispersed. Filter through a 100-mesh screen to obtain a spore suspension. Observe and count the spores using a hemocytometer under an optical microscope. Calculate the concentration of the spore suspension and convert it to the number of spores produced per gram of substrate (spores / g).

[0061] Spore germination rate determination method: Spores of Metarhizium anisopliae FJMR2 strain scraped from each treatment medium plate were prepared at a concentration of 5×10 7 200 μL of the spore suspension at spores / mL was evenly spread on the surface of SMAY medium, and then the plate was placed in a constant temperature incubator for culture. The temperature and environmental conditions of the incubator were consistent with those of the previous experiment.

[0062] Take out a small amount of spore powder and make 5×10 7200 μL of a spore suspension at spores / mL was evenly spread on SMAY medium and incubated in a 25°C incubator for germination. The germination criterion was that the length of the germ tube was greater than half the spore diameter. The spore germination rate at 72 hours was calculated according to Formula I.

[0063] Spore germination rate (%) = number of germinated spores in the field of view / total number of spores in the field of view × 100% Formula I.

[0064] Example 2

[0065] A fermentation and culture method of Metarhizium anisopliae

[0066] The difference from Example 1 is that the liquid seed culture medium materials are: 500 mL of 20% potato juice, 20 g of glucose, 3 g of soybean powder, and 500 ml of distilled water; the solid fermentation medium is prepared by soaking soybeans for 10 hours, filtering out the water, and airing them in the sun for 30 minutes. 0.05% of KH2PO4 and 0.01% of MgSO4·7H2O are added to the mixture at a ratio of 0.5% by weight of the wet material. The mixture is then mixed thoroughly and placed in a conical flask. The mixture is then sterilized with high-pressure steam at 121°C for 30 minutes and cooled for later use. All other steps are the same as in Example 1.

[0067] Example 3

[0068] A fermentation and culture method of Metarhizium anisopliae

[0069] The difference from Example 1 is that the liquid seed culture medium materials are: 500 mL of 20% potato juice, 20 g of glucose, 1 g of soybean powder, and 500 ml of distilled water; the solid fermentation medium is prepared by soaking soybeans for 10 hours, filtering out the water, and airing them in the sun for 30 minutes. 0.05% KH2PO4 and 0.01% MgSO4·7H2O are added at a ratio of 0.4% by weight of the wet material, mixed thoroughly, placed in a conical flask, and sterilized by high-pressure steam at 121°C for 30 minutes. The mixture is then cooled and used. All other steps are the same as in Example 1.

[0070] Comparative Example 1

[0071] A fermentation and culture method of Metarhizium anisopliae

[0072] The difference from Example 1 is that soybean powder is not added to the liquid seed culture medium, and the remaining steps are the same as Example 1.

[0073] Comparative Example 2

[0074] A fermentation and culture method of Metarhizium anisopliae

[0075] The difference from Example 1 is that 0.05% KH2PO4 and 0.01% MgSO4·7H2O are not added to the solid fermentation medium. The remaining steps are the same as Example 1.

[0076] Comparative Example 3

[0077] A fermentation and culture method of Metarhizium anisopliae

[0078] The difference from Example 1 is that soybean powder is not added to the liquid seed culture medium, and 0.05% KH2PO4 and 0.01% MgSO4·7H2O are not added to the solid fermentation culture medium. The remaining steps are the same as Example 1.

[0079] Comparative Example 4

[0080] A fermentation and culture method of Metarhizium anisopliae

[0081] The difference from Example 1 is that 0.05% KH2PO4 in the solid fermentation medium is replaced by 0.05% CaCl2, and 0.01% MgSO4·7H2O is replaced by 0.01% NaNO3. The remaining steps are the same as Example 1.

[0082] To further illustrate that the fermentation medium has a large spore production amount, a short spore production cycle and good spore quality, the fermentation levels of Metarhizium anisopliae in the fermentation medium of the present invention, conventional fungal culture media SMAY and PDA and comparative examples 1 to 4 were compared. The results are shown in Table 1.

[0083] Table 1 Results of sporulation time, sporulation amount and spore germination rate in different culture media

[0084] Test example Sporulation start time <![CDATA[Sporulation amount (10 9 spores / g of material)]]> Spore germination rate (%) Example 1 4～5d 7.36 97.1 Example 2 4～5d 8.25 96.5 Example 3 4～5d 7.84 97.3 Comparative Example 1 >9d 0.77 79.6 Comparative Example 2 >9d 1.54 82.4 Comparative Example 3 >10d 0.39 80.7 Comparative Example 4 >8d 2.83 87.2 PDA medium Mycelium grows too long (15 days) -- -- SMAY medium 7～8d 0.33 85.2

[0085] As shown in Table 1, the time for the fermentation medium to start spore production in Examples 1 to 3 of the present invention is shortened to 4 to 5 days, and the spore production is increased. The addition of soybean powder to the liquid seed culture medium significantly increases the spore production of Metarhizium rapae. At the same time, the spore powder obtained by using the fermentation medium provided by the present invention is of good quality, and the germination rate of the spore powder is as high as 96% or more. However, in Comparative Examples 1 to 4 and SMAY culture medium, the spore production time is 7 to 10 days or even longer, and the spore production is also not ideal, only 0.33 to 2.83 × 10 9 spores / g material, and the spore germination rate was 79.6% to 85.2%.

[0086] Comparative Example 5

[0087] A fermentation and culture method of Metarhizium anisopliae

[0088] The difference from Example 1 is that the soybeans in the solid fermentation medium material are replaced by rice, millet, sorghum, peeled sorghum, wheat, coix seed, corn and corn flakes, respectively, and the remaining steps are the same as Example 1.

[0089] Table 2 Sporulation of Metarhizium anisopliae under different solid substrate conditions

[0090]

[0091]

[0092] The results are shown in Table 2. Common food crops were used instead of soybeans as the fermentation matrix for fermentation. The results showed that the spore production time was shorter than that of soybeans, generally after 8 days, and some groups after 9 days. At the same time, the spore production amount and spore germination rate were also lower than those of soybeans. The spore production amount was reduced by 1000 to 3000 times (4 orders of magnitude) compared with the soybean group, and the spore germination rate was significantly reduced.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A culture medium for Metarhizium anisopliae, comprising a liquid seed culture medium and a solid fermentation culture medium, characterized in that: The liquid seed culture medium comprises the following raw materials in parts by mass: 100-125 parts of potato juice, 3-5 parts of glucose, 0.2-1 part of soybean powder and 100-125 parts of distilled water; The solid fermentation culture medium comprises the following raw materials in parts by mass: 100-125 parts of wet soybeans and 0.3-0.5 parts of inorganic salts; the inorganic salts are aqueous solutions containing 0.04%-0.06% KH2PO4 and 0.008%-0.01% MgSO4·7H2O in mass percentage.

2. The culture medium of Metarhizium anisopliae according to claim 1, wherein The liquid seed culture medium comprises the following raw materials in parts by weight: 100-110 parts of potato juice, 4 parts of glucose, 0.6-1 part of soybean powder and 100-110 parts of distilled water; The solid fermentation medium comprises the following raw materials in parts by mass: 100 parts of wet soybeans and 0.4 parts of inorganic salts.

3. The culture medium of Metarhizium anisopliae according to claim 1, wherein The inorganic salt is an aqueous solution containing 0.05% by mass of KH2PO4 and 0.01% by mass of MgSO4·7H2O.

4. The culture medium of Metarhizium anisopliae according to claim 1, wherein The wet soybeans are prepared by soaking the soybeans in water until saturated and then removing surface moisture.

5. The culture medium of Metarhizium anisopliae according to any one of items 1 to 4 of claim 1, characterized in that The particle size of the soybean powder is 0.15-0.2 mm.

6. Use of the Metarhizium reinhardtii culture medium according to any one of claims 1 to 5 in culturing Metarhizium reinhardtii and / or preparing Metarhizium reinhardtii spore powder.

7. The application according to claim 6, characterized in that The spore powder of Metarhizium anisopliae includes conidia powder of Metarhizium anisopliae.

8. A method for preparing conidia of Metarhizium reinhardtii based on the Metarhizium reinhardtii culture medium according to any one of claims 1 to 5, characterized in that: The following steps are involved: inoculating the spore liquid of Metarhizium anisopliae into a liquid seed culture medium for seed culture to obtain a seed liquid; The seed liquid is transferred to a solid fermentation medium for solid fermentation to obtain conidia of Metarhizium anisopliae.

9. The method according to claim 8, characterized in that The inoculation amount of the spore liquid of Metarhizium anisopliae inoculated into the liquid seed culture medium is 1% to 3%; the density of the spore liquid of Metarhizium anisopliae is 1.0×10 7 ~1.0×10 8 spores / mL; The temperature of the seed culture is 23-27° C., the rotation speed of the seed culture is 190-220 rpm, and the time of the seed culture is 3-5 days.

10. The method according to claim 8 or 9, characterized in that: The inoculation amount of the seed liquid transferred to the solid fermentation medium is 8% to 10%; the humidity of the solid fermentation is 60% to 80%; The thickness of the solid fermentation medium is 2 to 3 cm; the temperature of the solid fermentation is 25 to 30°C.

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