CN114437936A

CN114437936A - Metarhizium anisopliae IPPMHB614 and application thereof

Info

Publication number: CN114437936A
Application number: CN202111455732.6A
Authority: CN
Inventors: 王广君; 张能; 涂雄兵; 农向群; 张泽华; 李红梅; 蔡霓; 张道刚
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-05-06
Anticipated expiration: 2041-12-02
Also published as: CN114437936B

Abstract

The invention discloses metarhizium anisopliae IPPMHB614 and application thereof. The invention takes 3-instar locust larvae as a test source, measures the growth rate and the sporulation amount of different metarhizium anisopliae strains and the pathogenicity to the locusta migratoria in east Asia, screens out a strain IPPMHB614 with higher pathogenicity to the locust larvae, has the strain preservation number of CGMCC No.19035, and provides reference for the development of a biological control preparation of the locusta migratoria in east Asia.

Description

Metarhizium anisopliae IPPMHB614 and application thereof

Technical Field

The invention belongs to the technical field of locust control, and particularly relates to metarhizium anisopliae IPPMHB614 and application thereof.

Background

China is one of the most frequent countries in the world, and the locust plague has serious influence on the life quality and living conditions of people. Locusta migratoria (Locusta migratoria manilensis) is a fulminant agricultural pest, has large quantity and strong migratory performance, is easy to form locust disasters, and brings serious threat to agricultural production. At present, the locust is mainly controlled by a chemical control method. The use of a large amount of chemical pesticides leads to the gradual improvement of the drug resistance of the locust, increases the treatment cost and difficulty, brings serious pesticide residue and environmental pollution, and leads to a series of serious food and ecological safety problems due to high pesticide residue standard exceeding rate. Therefore, the development of environmentally friendly, sustainable biocontrol agents has become a primary task.

Entomopathogenic fungi are the largest pathogenic microorganisms for controlling insect populations in nature, are safe to crops and human beings, do not pollute the environment, often have epidemic potential, and pests are not easy to generate resistance, so the entomopathogenic fungi are the key points of biological control research at home and abroad. Metarhizium anisopliae is one of the earliest microorganisms used for controlling locusts, has various different strain types and is a broad-spectrum insect pathogen. Metarhizium anisopliae can parasitize various pests, so far, about more than 200 insects can be infected and killed by the metarhizium anisopliae, and the method has the advantages of simple and safe production method, specificity to hosts, contact killing, high mortality and the like. At present, Metarhizium anisopliae plays an important role in pest control of scarab beetles, locust, brontispa longissima, cockroaches, termites and the like. The locusta migratoria manilensis has stronger resistance to common metarhizium anisopliae, the effect of controlling the locusta migratoria manilensis is not obvious, a strain of metarhizium anisopliae which can kill the locusta migratoria manilensis stronger is urgently needed to be screened, the actual application effect is achieved, and a reference is provided for the development of a biological control preparation of the locusta migratoria manilensis.

Disclosure of Invention

The invention aims to provide metarhizium anisopliae IPPMHB614 and application thereof.

The metarhizium anisopliae IPPMHB614 is characterized in that the metarhizium anisopliae IPPMHB614 is preserved in 11.12.2019 in China general microbiological culture collection management center, the preservation number is CGMCC No.19035, and the preservation address is No. 3 of Xilu No.1 Beichen of the Chaojing area facing the Yangyang.

The metarhizium anisopliae IPPMHB614 is applied to prevention and treatment of locusta migratoria in east Asia.

The invention has the beneficial effects that: the method mainly performs the insecticidal activity determination of the metarhizium anisopliae strains on the locusta migratoria in east Asia, determines the enzyme activity of different strains through the enzyme activity analysis of the strains, screens out the metarhizium anisopliae strains which efficiently infect the locusta migratoria in east Asia, and provides reference for the development of a bio-control preparation of the locusta migratoria in east Asia.

Drawings

FIG. 1 shows the growth rates of different strains.

FIG. 2 shows the sporulation yield of different strains.

FIG. 3 shows the control effect of different strains on locusta migratoria in east Asia.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Example 1

1. Feeding locusta migratoria manilensis

1.1 incubation of locusta migratoria (L.) Okawachii

When hatching, the egg blocks stored in a refrigerator at 4 ℃ are taken out, and the egg granules are placed in a plastic hatching box containing 2/3 sterilized vermiculite (with 55% of water, preferably no water is discharged when being pinched by hands, no block is formed, and sand is loose) for hatching. The incubation temperature is 29 ℃, the illumination is more than 2000Lux, and the humidity is controlled within the range of 60 percent.

1.2 raising of locusta nymphs

And (4) picking out the locust nymphs after hatching with a writing brush, and putting the locust nymphs in a cage for breeding. Fresh wheat was fed daily. When the locusta nymphs grow to 3 years old, healthy and active individuals are selected as test insects for testing.

2. Culture of metarhizium anisopliae

2.1 Medium formulation

PDAY medium: 200g of potato, 20g of cane sugar, 5g of yeast powder, 18g of agar and 1000ml of water.

2.2 culture of Metarrhizium anisopliae strains

The strains used in the experiments included: IPPM2028E05, IPPM2028E37, IPPM2028E13, IPPM2028E24, IPPM2028E33, IPPMHB614, IPPM3003, IPPM0027, IPPM3013-3, IPPMB084

Spore powder of the strain is picked up in Tween water, and the spores are scattered by a shaking instrument. Then, 100. mu.l of spore solution was pipetted onto the plate using a pipette, spread evenly on a spreading bar, and cultured in an incubator at 25 ℃. 7. Taking out the culture dish from the incubator after 8 days, placing the culture dish at a low temperature to evaporate water in the culture medium so as to facilitate scraping spore powder, and placing the scraped spore powder in a refrigerator at a low temperature of 4 ℃ for preservation.

After selecting a single colony and inoculating the single colony on a PDAY plate, from the 7 th largest after inoculation, the diameters of the colony in the longitudinal direction and the transverse direction are measured by a vernier caliper for 1 time every day, the average value of the diameters is taken, the growth diameter of the colony every day is calculated, the growth speed is calculated, and the difference is compared. Three dishes were inoculated for each strain and repeated three times.

TABLE 1 growth rates of different strains

Bacterial strains	Daily average growth (mm/d)
		IPPM2028E05	3.44±0.01
IPPM2028E37	3.54±0.04
		IPPM2028E13	3.34±0.03
IPPM2028E24	3.53±0.04
		IPPM2028E33	3.62±0.04
IPPMHB614	4.16±0.07
		IPPM3003	3.84±0.22
IPPM0027	3.83±0.02
		IPPM3013-3	3.69±0.01
IPPMB084	3.80±0.07

The growth speed test results of different strains on the fungus culture medium PDAY show that the production speed of IPPMHB614 is fastest, is obviously higher than that of other strains, and is 4.16 mm/d; secondly, IPPM3003 and IPPM0027 are adopted, and the growth speed is respectively 3.84mm/d and 3.83 mm/d; the growth rate of IPPM2028E13 was the slowest, 3.34mm/d (Table 1, FIG. 1).

A piece of the colony growing 15 days was picked up by a punch with an inner diameter of 5.00mm, and the piece was placed in a test tube containing 5ml of 0.1% Tween-80 solution, sufficiently shaken and broken up by a vortex shaker, diluted 10 times, counted by a blood counting plate under an optical microscope, the spore concentration was measured, and the difference between the strains was compared. Three dishes were inoculated for each strain and repeated three times.

TABLE 2 sporulation amounts of the different strains

Bacterial strains	Sporulation yield (10)⁸sp/cm²)
		IPPM2028E05	1.41±0.23
IPPM2028E37	1.45±0.13
		IPPM2028E13	1.44±0.21
IPPM2028E24	1.48±0.12
		IPPM2028E33	1.53±0.21
IPPMHB614	2.05±0.13
		IPPM3003	2.13±0.12
IPPM0027	1.91±0.31
		IPPM3013-3	1.58±0.11
IPPMB084	1.72±0.20

Spore yield of the above 10 strainsThe result of the determination of (1) shows that the spore yield of each strain is 1.41-2.13 × 10⁸sp/cm²In the meantime. Wherein IPPM3003 has highest spore yield of 2.13 × 10⁸sp/cm²Followed by IPPMHB614, 2.05X 10⁸sp/cm²But the difference between the two is not significant. The sporulation quantity of the strain IPPM2028E05 is the lowest and is 1.41 multiplied by 10⁸sp/cm²(Table 2, FIG. 2).

Example 2 determination of insecticidal Activity of different Metarrhizium anisopliae strains on migratory locust in east Asia

Preparing a bait agent: 2g of wheat bran (dried heat sterilization at 100 ℃ for 1h), 0.1g of strain spore powder and 200 mul of soybean oil; the wheat bran and the soybean oil are put into a sterilized culture dish and are stirred uniformly, then the spore powder is added and is stirred uniformly, and the mixture is divided into 3 equal parts and is respectively put into the culture dish for standby.

The toxicity of different strains to the locusta migratoria in east Asia is determined by bait feeding method, and each strain is repeated for 3 times.

The high-toxicity strain is screened by performing bioassay by using 3 rd larva of locusta migratoria in east Asia. The raw test baskets (37X 25X 16cm) were treated with 84 disinfectant and air-dried. A layer of newspaper is laid on the bottom of each bioassay basket, 15 larvae of 3 years old with the same size are placed in each bioassay basket, and the bioassay basket is covered by a sterilized glass plate. And (3) respectively putting the separately packaged bait agents into a bioassay basket filled with 3-year-old locust nymphs, repeating each strain for 3 times, marking, and culturing at room temperature. And taking out the bait agent after the locust nymphs eat the bait for 24 hours, and supplementing the wheat seedlings in time. And (4) continuously recording the dead number and the live number of the locusts in each basket for 12 days, taking out the dead locusts for moisturizing, recording the dead rate of the locusts, and performing data analysis.

The 3 rd larva of locusta migratoria manilensis is taken as a test object, and the insecticidal activity of 10 strains is tested. After 3 days of inoculation, the tested larvae begin to die, the dead larva has typical stiff worm symptoms (larva bodies turn red), white hyphae penetrate out of the surfaces of the dead larva bodies, and finally green spores grow out. The data of 12 days of locusts infected were counted and the regression equation of the lethal time and mortality and the median lethal time were calculated by the BASIC program of LD50 (LT50 Table 3). The cumulative mortality of different strains (table 4, fig. 3) was analyzed in combination, and it was found that there was a significant difference in virulence between the different strains. Strains IPPMHB614, IPPM3003, IPPM0027 and IPPM2028E13 have high toxicity. Wherein the strain IPPMHB614 has the highest toxicity, and the cumulative mortality rate in 12 days reaches 96.43 percent; next are strains IPPM3003 and IPPM 0027.

TABLE 3 lethal time of different strains on locusta migratoria (LT50)

TABLE 4 cumulative mortality of different strains at different times on Acridia migratoria larvae (%)

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The metarhizium anisopliae IPPMHB614 is characterized in that the metarhizium anisopliae IPPMHB614 is preserved in 11.12.2019 in China general microbiological culture collection management center, the preservation number is CGMCC No.19035, and the preservation address is No. 3 of Xilu No.1 Beichen of the Chaojing area facing the Yangyang.

2. The use of metarhizium anisopliae IPPMHB614 as claimed in claim 1 for controlling locusta migratoria in east asia.