Method for massively obtaining metarhizium ledebusii conidia

Abstract

The invention provides a method for obtaining a large number of metarhizium anisopliae conidia, and belongs to the technical field of microorganisms. The method comprises the following steps: inoculating 4-instar living larvae of spodoptera frugiperda noctuid with metarhizium anisopliae spore powder to cause infection, carrying out amplification culture on the infected spodoptera frugiperda noctuid, and collecting spores in the 4th day after the spodoptera frugiperda body is dead, so as to obtain a large amount of metarhizium anisopliae conidia. According to the method provided by the invention, the operation process is greatly simplified, the spore production time is shortened, the spore yield is improved, the production cost is reduced, and meanwhile, the problem of strain degeneration caused by continuous culture of metarhizium ledebusii on an artificial culture medium is solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a method for obtaining a large amount of Metarhizium rileyi conidia. BACKGROUND

[0002] Metarhizium rileyi is a widely distributed entomopathogenic fungus, which can infect a variety of lepidopteran pests, especially the Spodoptera exigua, Spodoptera litura, Spodoptera litura, Spodoptera exigua and other noctuid pests, and the spores are easily transmitted by wind to cause field epidemics of pests, and have high development and utilization value. Obtaining a large amount of conidia through propagation is the premise of the application of entomogenous fungi. The most commonly used artificial culture medium for culturing Metarhizium rileyi in the laboratory is the Sas maltose-yeast medium and the potato medium, but there are problems such as low sporulation rate, low spore germination rate and long sporulation cycle. At present, liquid-solid two-phase fermentation is used for Metarhizium rileyi fermentation, and cheap wheat bran, corn powder and rice powder are used as nutrients to obtain the maximum spore yield, but the spore yield is still limited, and the operation steps are complicated, time-consuming and laborious. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a method for obtaining a large amount of Metarhizium rileyi conidia, which uses Spodoptera exigua larvae as a propagation carrier, and finally obtains a large amount of Metarhizium rileyi conidia by screening the dominant instar of Spodoptera exigua.

[0004] The present application provides a method for obtaining a large amount of Metarhizium rileyi conidia, comprising the following steps:

[0005] The Metarhizium rileyi conidia are inoculated into Spodoptera exigua larvae of 4th instar or above for propagation culture, and the conidia are collected when the cadavers are covered with spores and are scattered.

[0006] Preferably, the time for collecting the conidia is 4 days after the cadavers produce spores.

[0007] Preferably, the time for collecting the conidia is 5-6 days after the cadavers produce spores.

[0008] Preferably, the inoculation mode of the Metarhizium rileyi conidia is in the form of Metarhizium rileyi spore powder.

[0009] Preferably, the inoculation method of the Metarhizium rileyi spore powder is that the Spodoptera exigua larvae are allowed to crawl freely in an environment with the conidia powder, and then the Spodoptera exigua larvae are subjected to propagation culture after the conidia powder is distributed on the surface of the Spodoptera exigua larvae.

[0010] Preferably, the inoculation amount of the Metarhizium anisopliae spore powder is 40-50 Helicoverpa armigera larvae placed in the insect-rearing box paved with 0.05-0.1 g of Metarhizium anisopliae spore powder for free crawling for 1-3 min.

[0011] The diameter of the insect-rearing box is 8 cm, and the height is 5 cm.

[0012] Preferably, the Metarhizium anisopliae spore powder is obtained by streak inoculating Metarhizium anisopliae on a culture medium for culture, and collecting Metarhizium anisopliae spore powder.

[0013] Preferably, the culture medium is a Sabouraud maltose agar yeast culture medium; the culture temperature is 24-26℃; the culture humidity is 60%-70%, and the light cycle is 14 h L / 10 h D; and the culture time is 5-6 d.

[0014] Preferably, the temperature of the expanded culture is 24-26℃; the relative humidity of the expanded culture environment is 65%-75%, and the light-dark cycle during the expanded culture is 14 L / 10 D; and the expanded culture time is 9-11 d.

[0015] The application provides application of the method for obtaining a large amount of Metarhizium anisopliae conidia in preparation of an insecticide.

[0016] The application provides a method for obtaining a large amount of Metarhizium anisopliae conidia, which comprises the following steps: inoculating Metarhizium anisopliae conidia on Helicoverpa armigera larvae of more than 4 instars for expanded culture, collecting the conidia when the larvae are covered with spores and in a scattered state. 9 Compared with larvae of 2-3 instars, the larvae of more than 4 instars can obtain larger larvae, higher spore yield (9.44×10 10 The application overcomes the technical problems of low spore yield, unstable spore production, fast strain degeneration, complicated operation and high cost caused by artificial culture medium expansion of Metarhizium anisopliae, and is easy to realize large-scale batch production and has good application prospect.

[0017] The application further limits the inoculation mode of Metarhizium anisopliae conidia in the method, specifically, the conidia are inoculated on Helicoverpa armigera larvae in the form of spore powder. The results show that different inoculation modes have differences in larval mortality, spore production time and spore yield. Compared with immersion treatment and spraying treatment, the mortality of spore powder treatment is the highest, reaching 91.7%, the spore production time is shortened to 6-7 d, and the spore yield is the highest, reaching 2.04×10 10Spores / g. The method of inoculating larvae with visible spore powder is superior to insect immersion treatment and spraying treatment in terms of insect mortality, sporulation time and sporulation amount. The mummified insects collected after spores are obtained can still produce spores after being moisturized, and the insect bodies themselves can be used as organic matter and processed into microbial fertilizer for pest control and crop growth.

[0018] The present application further provides a method for inoculating L. lecanicum conidium powder. The present application inoculates by means of block inoculation and streaking inoculation, and under the same culture conditions, the results show that the streaking inoculation is beneficial to early conidium production, and the conidium production is advanced to the 5th day, which is 4 days earlier than that of the block inoculation, and the conidium production amount is greatly increased, the whole culture period is shortened, and the production efficiency of conidium is improved.

[0019] The present application further limits the time for collecting conidium, specifically, 4 days after sporulation. In order to further improve the spore amount, the present application optimizes the time for collecting conidium (2-6 days after sporulation), and the results show that collecting conidium 4 days after sporulation and later is beneficial to improving the conidium production amount and quality, and the collection operation is easier.

[0020] The present application also provides application of the method for obtaining a large amount of L. lecanicum conidium in preparing insecticides. The L. lecanicum conidium powder produced by the method has stable quality, and can be further processed into wettable powder, dispersible oil and other various dosage forms for controlling ground pests. In addition, the insect body itself is an organic matter, which can be crushed and applied to the field by scattering, trenching or hole application, so as to achieve the effects of controlling underground pests and promoting crop growth. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 1 is a growth chart of a L. lecanicum strain inoculated by block inoculation;

[0022] Figure 2 Figure 2 is a growth chart of a L. lecanicum strain inoculated by streaking inoculation;

[0023] Figure 3 Figure 3 is a conidium chart obtained by inoculating L. lecanicum into 4th instar C. geolepia larvae for propagation. DETAILED DESCRIPTION

[0024] The present application provides a method for obtaining a large amount of L. lecanicum conidium, which comprises the following steps:

[0025] The L. lecanicum conidium is inoculated into C. geolepia larvae of 4th instar or above for propagation, and the conidium is collected when the insect body is covered with spores and in a scattered state.

[0026] The method of the present application has no special limitation on the strain of Metarhizium anisopliae, and the Metarhizium anisopliae strain known in the art can be used.

[0027] In the present application, the inoculation of the conidia of Metarhizium anisopliae is preferably in the form of Metarhizium anisopliae spore powder. The inoculation method of the Metarhizium anisopliae spore powder is preferably to let the larvae of Spodoptera frugiperda freely crawl in an environment paved with conidia, and then enter the expansion culture after the larvae of Spodoptera frugiperda are distributed with the conidia. The inoculation amount of the Metarhizium anisopliae spore powder is preferably to place 40-50 larvae of Spodoptera frugiperda in a rearing box paved with 0.05-0.1 g of Metarhizium anisopliae spore powder and let them freely crawl for 1-3 min; it can be to place 45 larvae of Spodoptera frugiperda in a rearing box paved with 0.08 g of Metarhizium anisopliae spore powder and let them freely crawl for 2 min. The diameter of the rearing box is preferably 8 cm, and the height is preferably 5 cm.

[0028] In the present application, the method for obtaining the Metarhizium anisopliae spore powder is preferably to inoculate Metarhizium anisopliae in the culture medium, and collect the Metarhizium anisopliae spore powder. The culture medium is preferably SMAY (Sauter's malt agar yeast medium). The formula of the SMAY culture is preferably to include 10 g / mL of proteose peptone, 40 g / mL of maltose, 2 g / mL of yeast extract powder, and 20 g / mL of agar.

[0029] The temperature of the culture is preferably 24-26℃, which can be 24.5-25.5℃, and can be 25℃. The humidity of the culture is preferably 60%-70%, which can be 62%-68%, and can be 65%. The light cycle is preferably 14 h L / 10 h D. The time of the culture is preferably 5-6 d.

[0030] In an embodiment of the present application, in order to obtain a large amount of Metarhizium anisopliae spore powder early, the research on different inoculation methods for obtaining Metarhizium anisopliae spore powder was carried out, and experiments were carried out by using traditional inoculation methods of mycelium block and streaking method respectively. Under the same culture conditions, the results showed that the spores were produced on the 9th day by using the traditional mycelium block method; the spores were produced on the 5th day by using the streaking method, which was 4 days earlier than the mycelium block method. Compared with the traditional mycelium block method, the amount of spores produced by using the streaking method was also greatly improved. Therefore, the streaking method is selected to culture the initial strain of Metarhizium anisopliae to obtain the spore powder.

[0031] In the present invention, the age of the larvae of the fall armyworm is also an important factor affecting the conidia yield. The larvae of the fall armyworm can be the 5th instar, but the production cycle will be prolonged. In one embodiment of the present invention, experiments were carried out with the 2nd, 3rd, 4th, 5th and 6th instar larvae as test larvae. The results showed that the Metarhizium anisopliae had infectious activity on the test larvae, but the mortality rate, spore production and spore quality of larvae of different instars were different. The 2nd to 3rd instar larvae had a high mortality rate and a relatively short half-lethal time, but the insect corpses were small, resulting in a low spore production, which was not suitable as a spore production carrier of Metarhizium anisopliae; while the 5th instar larvae had a low mortality rate and a long half-lethal time, which resulted in a prolonged spore production cycle, and were not suitable as a spore production carrier of Metarhizium anisopliae; the 4th instar larvae had a high mortality rate, a short half-lethal time, and a large insect corpse. A single insect body obtained more spores, and the spores were easily scattered, which was conducive to the reproduction of Metarhizium anisopliae. It can be seen from this that compared with the 2nd, 3rd and 5th instar larvae, the 4th instar larvae are the best age for the reproduction of Metarhizium muscardinei.

[0032] In the present invention, the temperature of the expanded culture is preferably 24-26°C, and may be 25°C. The relative humidity of the expanded culture environment is preferably 65%-75%, and may be 70%. The light-dark cycle during the expanded culture is preferably 14 L / 10 D. The expanded culture duration is preferably 9-11 days, and may be 10 days. During the expanded culture, the fall armyworm larvae are preferably fed with artificial feed until they die or pupate.

[0033] In the present invention, the time for collecting conidia is preferably the 4th day after the insect corpse produces conidia, and can be the 5th to 6th day after the insect corpse produces conidia. The time after the insect corpse produces conidia refers to the 1st to 2nd day after the Metarhizium anisopliae infects the fall armyworm larvae and kills them. The surface of the insect corpse is covered with white hyphae. 2-3 days after death, spores begin to grow on the white hyphae. The insect corpse produces conidia on the first day, which is called the first day after conidia production. In one embodiment of the present invention, the effect of the time for collecting conidia (2 to 6 days after conidia production) on the amount of conidia was studied. The results showed that the amount of conidia produced showed a trend of first increasing and then stabilizing with the extension of the conidia production time. The amount of conidia produced on the 4th day after conidia production was 2.56×101 0 spores / g, and there was no significant difference in the conidia production on the 6th and 7th days. On the 3rd day after conidia production, the insect corpse was covered with spores, the spores were light green, the spores were dense and tightly wrapped around the insect corpse, and the spore powder was not easy to scatter; on the 4th day after conidia production, the spores were light green, and a small amount of spores were scattered; on the 4th, 5th, and 6th days after production, the spore morphology tended to be stable with little change, the spore color was dark green, and the spore powder was very easy to scatter and easy to obtain. This shows that the conidia production of Metarhizium anisopliae on the 4th day after conidia production is high and the spore powder is easy to obtain. This shows that the time of collecting conidia not only affects the conidia production but also affects the quality of the conidia and the difficulty of harvesting.

[0034] The application can obtain a large amount of Metarhizium rileyi conidia with stable quality by screening and optimizing the preparation of Metarhizium rileyi conidia, the larval stage of Spodoptera frugiperda, the inoculation method and the collection time, and has the advantages of simple and rapid operation, high preparation efficiency, low preparation cost, realization of large-scale batch production and high application prospect.

[0035] In view of the fact that the preparation method of the application can obtain a large amount of Metarhizium rileyi conidia with stable quality, the application provides the application of the method for obtaining a large amount of Metarhizium rileyi conidia in the preparation of insecticides.

[0036] In the application, the application object of the insecticide preferably includes at least one of the following: Spodoptera exigua, Spodoptera litura, Spodoptera litoralis and Spodoptera frugiperda and other noctuid pests. The dosage form of the insecticide is preferably wettable powder and / or dispersible oil. The preparation method of the insecticide is not particularly limited in the application, and the insecticide preparation method known in the art can be used.

[0037] The method for obtaining a large amount of Metarhizium rileyi conidia provided by the application will be described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.

[0038] Example 1

[0039] Screening of inoculation method of Metarhizium rileyi initial strain

[0040] The mycelial block method is as follows: Metarhizium rileyi mycelial blocks are inoculated in the center of a plate containing SMAY medium, and are cultured at a temperature of 25±1℃, a humidity of 65±5% RH and a light-dark cycle (L:D) of 14h:10h for 14 days to obtain Metarhizium rileyi conidia.

[0041] The streaking method is as follows: Metarhizium rileyi is streaked and inoculated in SMAY medium, and is cultured at a temperature of 25±1℃, a humidity of 65±5% RH and a light-dark cycle (L:D) of 14h:10h for 6 days to obtain Metarhizium rileyi conidia.

[0042] The traditional mycelial block method is used, and the conidia are produced on the 9th day; the streaking method is used, and the conidia are produced on the 5th day, which is 4 days earlier than the mycelial block method. Compared with the traditional mycelial block method, the amount of conidia produced by the streaking method is also greatly increased. Therefore, the streaking method is selected to culture the Metarhizium rileyi initial strain to obtain spore powder.

[0043] Example 2

[0044] Screening of dominant test insects of Spodoptera frugiperda

[0045] The Spodoptera frugiperda was bred indoors with artificial feed under the conditions of temperature 25±1℃, relative humidity 65±5% RH, light-dark cycle L:D=14h:10h, and 2nd, 3rd, 4th, and 5th instar larvae were selected for infection test. The Metarhizium anisopliae spores were obtained by using SMAY medium, and a spore suspension with a concentration of 5×10 7 Spores / mL was prepared. The 2nd to 5th instar larvae of the Spodoptera frugiperda were immersed in the prepared spore suspension for 10 seconds, taken out and dried, and then moved into the insect breeding box. Three repeats were set for each instar treatment, with 40 larvae in each repeat, and the breeding conditions were the same as the above method. The death of the test insects was observed and recorded every day, the dead insects were continuously cultured, the spore production of the insects was observed, and the spore production amount was measured on the 3rd day after spore production.

[0046] As shown in Table 1, the Metarhizium anisopliae had infection activity on the 2nd to 5th instar larvae of the Spodoptera frugiperda. When the concentration of the spore suspension was 5×10 7 Spores / mL, the infection rates after 10 days were 93.3%, 85.0%, 71.7%, and 43.3%, respectively, and the lethal times were 4.3d, 5.7d, 6.5d, and 8.5d, respectively. Although the 2nd and 3rd instar larvae had high mortality and relatively short median lethal time, the dead insects were small, resulting in low spore production, and were not suitable as spore production carriers of the Metarhizium anisopliae. The 5th instar larvae had low mortality and long median lethal time, resulting in prolonged spore production period, and were not suitable as spore production carriers of the Metarhizium anisopliae. The 4th instar larvae had high mortality, short median lethal time, and large dead insects, and the number of spores obtained from a single insect was more, and the spores were easy to scatter, which was beneficial to the reproduction of the Metarhizium anisopliae. Therefore, compared with the 2nd, 3rd, and 5th instar larvae, the 4th instar larvae were the best insect instar for the reproduction of the Metarhizium anisopliae. Therefore, the 4th instar larvae of the Spodoptera frugiperda were selected as the dominant test insects for the subsequent spore propagation test.

[0047] Table 1 Lethal effect of Metarhizium anisopliae on 2nd to 5th instar larvae of Spodoptera frugiperda

[0048]

[0049] Example 3

[0050] Screening of Metarhizium anisopliae inoculation method for test insects

[0051] The Spodoptera frugiperda was bred indoors with artificial feed under the conditions of temperature 25±1℃, relative humidity 65±5% RH, light-dark cycle L:D=14h:10h, and 4th instar larvae were selected for infection test. The Metarhizium anisopliae spores were obtained by using SMAY medium. The spore powder was obtained by scraping the conidia, and was prepared as a spore suspension with a concentration of 5×10 7 Spores / mL.

[0052] Insect immersion treatment: 120 fourth-instar larvae of Spodoptera frugiperda were selected, grouped into groups of 40, and immersed in a prepared spore suspension of Metarhizium anisopliae for 10 seconds. After drying, the larvae were transferred to an insect box and placed in an artificial climate chamber maintained at a temperature of (25±1)°C, a relative humidity of (65±5)%, and a photoperiod of 14 L:10 D. The insects were then reared on an artificial diet. Infestation and sporulation were observed. Spores were collected and sporulation was measured five days after death.

[0053] Spraying treatment: 120 fourth-instar larvae of Spodoptera frugiperda were selected, grouped in groups of 40, and sprayed with the above-prepared Metarhizium anisopliae spore suspension. The insects were then placed in an insectary incubator at a temperature of (25±1)°C, a relative humidity of (65±5)%, and a photoperiod of 14 L:10 D. The insects were then fed an artificial diet. Infestation and sporulation were observed. Spores were collected on the fifth day after sporulation and the spore count was measured.

[0054] Spore treatment: 120 fifth-instar larvae of Spodoptera frugiperda were selected, grouped into groups of 40. They were placed in a box containing a certain amount of spores and allowed to crawl for approximately 2 minutes to coat their body walls with spores. The larvae were then collected in the box and placed in an artificial climate chamber at a temperature of (25±1)°C, a relative humidity of (70±5)%, and a photoperiod of 14 L:10 D. The larvae were then fed an artificial diet until they died. The infestation status of the larvae was observed, and the time of sporulation was recorded. Spores were collected five days after death, and sporulation was measured.

[0055] As shown in Table 2, all three inoculation methods were suitable for propagating M. anisopliae spores. However, larval mortality, sporulation time, and sporulation yield varied among the different inoculation methods. The mortality rate in the immersion group was 73.3%, while that in the spraying group was 70.0%. The spore powder treatment group had the highest mortality rate, reaching 91.7%. Sporulation lasted 8–10 days for both the immersion and spraying treatments, while it was shortened to 6–7 days for the spore powder treatment. The sporulation yield in the immersion group was 0.92 × 10 10 spores / g, the spore production of the spraying treatment group was 0.75×10 10 spores / g, and the spore powder treatment group had the highest spore production, reaching 2.04×10 10 spores / g. This indicates that, among the three inoculation methods, spore powder treatment reduced larval mortality, increased spore production time, and increased spore production compared to insect immersion and spraying. Furthermore, after spores are collected, the dead insects can continue to produce spores after being moisturized. The insect bodies themselves can also serve as organic matter and, after being crushed, can be processed into bacterial fertilizer for pest control and crop growth.

[0056] Table 2 Infection effects of different inoculation methods of Metarhizium rapae on the 5th instar larvae of Spodoptera frugiperda

[0057]

[0058]

[0059] Example 4

[0060] Screening of the collection time of conidia of Metarhizium anisopliae

[0061] Fall armyworms were reared indoors on an artificial diet at 25 ± 1°C, 65 ± 5% relative humidity, and a photoperiod of 14 h:10 h. Fourth-instar larvae were selected for infestation testing. Conidia of Metarhizium anisopliae were obtained using SMAY medium and set aside.

[0062] Fourth-instar larvae of the fall armyworm were placed in an insect box containing a certain amount of spores and allowed to crawl for approximately 2 minutes to adhere to their body walls. They were then collected in the insect box and placed in an artificial climate chamber at a temperature of (25±1)°C, a relative humidity of (70±5)%, and a photoperiod of 14 L:10 D. They were fed an artificial diet until they died or pupated. The insects were observed for infection, and spores were collected on days 2, 3, 4, 5, and 6 after sporulation, and sporulation production was measured. Three replicates were used for each treatment, with 20 larvae per replicate.

[0063] As shown in Table 3, the spore production and spore morphology of Metarhizium anisopliae vary greatly with the extension of spore production time. The spore production shows a trend of increasing first and then stabilizing with the extension of spore production time. The spore production on the 4th day after spore production is 2.56×10 10 spores / g, showing no significant difference compared to the sporulation yield on days 5 and 6. On day 2 after sporulation, the insect carcass was covered with spores, which were light green in color and tightly wrapped around the insect carcass, making it difficult for spore powder to scatter. On day 3 after sporulation, the spores were light green with a small amount of spores scattered. On days 4, 5, and 6 after sporulation, the spore morphology remained stable with little change. The spores were dark green in color, and the spore powder was easily scattered and easy to obtain. This indicates that the sporulation yield of M. anisopliae on day 4 after sporulation is high and the spore powder is easy to obtain. Therefore, day 4 after sporulation was selected as the time for spore powder collection.

[0064] Table 3 Conidia production of Metarhizium anisopliae conidia at different collection times

[0065] Collection time Spore production (10 10 Spores / g) Spore production description 2 days after sporulation 1.26 The insect corpse is covered with spores. The spores are light green and dense, making them difficult to scatter. 3 days after sporulation 1.98 The insect corpse is covered with spores, the spores are light green, and a small amount of spores are scattered 4 days after sporulation 2.56 The insect corpse is covered with spores, the spores are dark green, and the spore powder is easy to scatter. 5 days after sporulation 2.54 The insect corpse is covered with spores, the spores are dark green, and the spore powder is easy to scatter. 6 days after sporulation 2.57 The insect corpse is covered with spores, the spores are dark green, and the spore powder is easy to scatter.

[0066] 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 method for obtaining a large number of conidia of Metarhizium anisopliae, characterized in that: The following steps are involved: The conidia of Metarhizium anisopliae were inoculated into larvae of Spodoptera frugiperda at the fourth instar and above for expansion culture. The conidia were collected when the insect corpses were covered with spores and were scattered.

2. The method for obtaining a large number of conidia of Metarhizium anisopliae according to claim 1, characterized in that: The time for collecting conidia is 4 days after the insect corpse produces conidia.

3. The method for obtaining a large number of conidia of Metarhizium anisopliae according to claim 2, characterized in that: The time for collecting conidia is 5 to 6 days after the insect corpse produces conidia.

4. The method for obtaining a large number of conidia of Metarhizium anisopliae according to claim 1, characterized in that: The inoculation method of the conidia of Metarhizium anisopliae is to inoculate in the form of spore powder of Metarhizium anisopliae.

5. The method for obtaining a large number of conidia of Metarhizium anisopliae according to claim 4, characterized in that: The inoculation method of the Metarhizium anisopliae spore powder is to allow the larvae of the fall armyworm to crawl freely in an environment covered with conidia powder, and then enter the expanded culture after the larvae of the fall armyworm are distributed on the body surface of the larvae.

6. The method for obtaining a large number of conidia of Metarhizium anisopliae according to claim 5, characterized in that: The inoculation amount of the Metarhizium anisopliae spore powder is as follows: 40 to 50 larvae of Spodoptera frugiperda are placed in an insect box paved with 0.05 to 0.1 g of Metarhizium anisopliae spore powder and allowed to crawl freely for 1 to 3 minutes; The diameter of the insect raising box is 8 cm and the height is 5 cm.

7. The method for obtaining a large number of conidia of Metarhizium anisopliae according to claim 4, characterized in that: The method for obtaining the spore powder of Metarhizium reinhardtii comprises streaking and inoculating the Metarhizium reinhardtii into a culture medium for cultivation, and collecting the spore powder of Metarhizium reinhardtii.

8. The method for obtaining a large number of conidia of Metarhizium anisopliae according to claim 6, characterized in that: The culture medium is a Sabouraud maltose agar yeast culture medium; the culture temperature is 24-26° C.; the culture humidity is 60%-70%, the photoperiod is 14 hL / 10 hD; and the culture time is 5-6 days.

9. The method for obtaining a large number of conidia of Metarhizium anisopliae according to any one of claims 1 to 8, characterized in that: The temperature of the expanded culture is 24-26° C.; the relative humidity of the expanded culture environment is 65%-75%; the light-dark cycle during the expanded culture is 14L / 10D; and the time of the expanded culture is 9-11d.

10. Use of the method for obtaining a large amount of conidia of Metarhizium anisopliae according to any one of claims 1 to 9 in the preparation of insecticides.

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