Metarhizium anisopliae CIPPMA154 with high toxicity to migratory locust and application of metarhizium anisopliae CIPPMA154

Abstract

The invention relates to the field of microorganisms, in particular to metarhizium anisopliae CIPPMA154 with high toxicity to migratory locust and application of the metarhizium anisopliae CIPPMA154. Five Metarhizium anisopliae and four Beauveria bassiana are separated, research on pathogenicity of the Beauveria bassiana and the Metarhizium anisopliae on migratory locust shows that nine strains for test can infect third-instar larvae of migratory locust, pathogenicity of different strains is remarkably different, toxicity of the Beauveria bassiana in the strains for test on migratory locust is lower than that of the Metarhizium anisopliae, and pathogenicity of the Metarhizium anisopliae strain CIPPMA154 is superior to that of other strains. In enzyme activity analysis, the chitinase activity of the strain CIPPMA154 is the highest, and the result shows that the green muscardine fungus strain CIPPMA154 has relatively high toxicity to migratory locust when the chitinase activity of the strain CIPPMA154 is above 2.5267 U / mL.

Description

Technical Field

[0001] The present application relates to the field of microorganisms, and in particular to Metarhizium anisopliae CIPPMA154 which is highly toxic to migratory locusts and its application. Background Art

[0002] Locusts (Locusta migratoria), Insecta, Orthoptera, Acrididae, are major agricultural pests. They are commonly known as grasshoppers and mainly feed on plants of the Gramineae family, such as rice, wheat and other important crops. In the adult stage, they eat almost as much food as their own body weight every day. This astonishing appetite has caused great damage to crop production. When a locust plague breaks out, the swarm is so large that they can quickly destroy large areas of crops, causing large areas of farmland to be damaged and crop yields to be severely reduced, thus having a serious impact on the agricultural economy.

[0003] Metarhizium anisopliae is an important insect pathogenic fungus that is pathogenic to more than 20 species of insects. As a new type of microbial insecticide, Metarhizium anisopliae has the advantages of being new, safe and green. It is widely used in the prevention and control of pests in agriculture, forestry, health and other fields, and has a good control effect.

[0004] Beauveria bassiana is the earliest discovered insect pathogenic fungus and is the most widely studied and applied insect pathogenic fungi. Beauveria bassiana has the advantages of broad insecticide spectrum, strong pathogenicity, easy production and storage, low residue and no pollution, and safety to humans, animals and natural enemies. It has become the most widely used biological control agent for agricultural and forestry pests. Summary of the invention

[0005] The purpose of the present invention is to provide a Metarhizium anisopliae strain with high toxicity to migratory locusts.

[0006] Another object of the present invention is to provide an application of the above-mentioned Metarhizium anisopliae strain.

[0007] The Metarhizium anisopliae strain CIPPMA154 with high toxicity to migratory locusts according to the present invention has a preservation number of CGMCC No.40933.

[0008] The present invention isolates 5 strains of Metarhizium and 4 strains of Beauveria. The study on the pathogenicity of Beauveria and Metarhizium to migratory locusts shows that all 9 tested strains can infect the 3rd instar larvae of migratory locusts, and there are significant differences in pathogenicity between different strains. Among the tested strains, the toxicity of Beauveria to migratory locusts is lower than that of Metarhizium, and the pathogenicity of Metarhizium strain CIPPMA154 is better than that of other strains. In the enzyme activity analysis, the chitinase activity of strain CIPPMA154 is the highest, which is 2.5267U / mL. The above results all show that the Metarhizium strain CIPPMA154 has a high toxicity to migratory locusts. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a statistical chart of the growth rate of Metarhizium anisopliae;

[0010] Figure 2 This is a statistical chart of the growth rate of Beauveria bassiana;

[0011] Figure 3 This is a statistical chart of Metarhizium spore content;

[0012] Figure 4 This is a statistical chart of Beauveria bassiana spore content;

[0013] Figure 5 Shows the effect of different strains on the survival rate of locust nymphs;

[0014] Figure 6 Shows the survival rate of locust nymphs treated with different concentrations of strain CIPPMA154.

[0015] The Metarhizium anisopliae strain CIPPMA154 of the present invention was deposited on November 24, 2023 in the General Microbiology Center of the China Culture Collection Administration (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Metarhizium anisopliae, with a collection number of CGMCC No. 40933. DETAILED DESCRIPTION

[0016] The insect source used in the following examples is a long-term reared purified migratory locust population. The preserved locust eggs were incubated in an artificial climate incubator under the following incubation conditions: temperature 30±2°C, relative humidity 60±5%, light:dark = 14h:10h. The first-instar locust nymphs hatched at the same time were transferred to an insect cage of 60cm×50cm×70cm in size and reared with fresh wheat seedlings until the third instar.

[0017] Example 1 Strain Isolation and Purification

[0018] The insect carcasses collected from the vegetable fields around Beidagang, Huanghua City, Hebei Province were immersed in 70% ethanol by volume for disinfection. After 30 seconds, they were placed in 0.1% mercuric chloride for disinfection for another 1.5 minutes. They were rinsed with sterile water for 3 times, and the water on the insect bodies was absorbed with sterilized filter paper. Then, the dead insects were cut into small pieces with high-temperature sterilized scissors. Then, each group of 3 carcasses was inoculated in an equilateral triangle shape on a potato glucose PDA medium containing 0.1g / L chloramphenicol. After being cultured in a 27℃ incubator for 7 days, the edge hyphae were picked up with an inoculation loop and streaked on the PDA medium for purification; after the colonies grew out, colonies of different shapes, colors, and sizes were picked up and transferred to a new PDA medium by the plate streaking method. Repeat the above operation until pure colonies are obtained, and 9 strains CIPPMA154, CIPPMA024, CIPPMA025, CIPPMA149, CIPPMA156, CIPPBb105, CIPPBb106, CIPPBb107, and CIPPBb049 are obtained, which are stored in a refrigerator at 4°C for use. When in use, the preserved strain is inoculated on the sterilized PDAY medium, cultured at 28°C for 7 days, and then placed at room temperature to wait for the conidia to dry, then scrape off the spore powder to make a suspension for use, or store the spore powder in a refrigerator at 4°C for use.

[0019] The above strains were prepared to a concentration of 1×10 8 / mL spore suspension. Take 1mL of spore suspension and inoculate it into 200mL mycelium production medium, place it at 28℃ and 200r / min shaking in a shaker for 3 days until the logarithmic phase, and extract genomic DNA. Use genomic DNA as a template and use ITS primers to amplify the ITS sequence. After PCR product detection, it was sent for sequencing, and the determined sequence was subjected to BLAST analysis in GenBank. After multiple sequence alignment, the phylogenetic analysis software MEGAversion 7.0 was used to construct a phylogenetic tree using the neighbor-joining method (NJ). The phylogenetic status of the strains was determined. The results showed that 5 strains were Metarhizium anisopliae (CIPPMA154, CIPPMA024, CIPPMA025, CIPPMA149, CIPPMA156), and 4 strains were Beauveria bassiana (CIPPBb105, CIPPBb106, CIPPBb107, CIPPBb049).

[0020] Example 2 Strain growth characteristics, spore production, and enzyme activity determination

[0021] Weigh 0.1 g of spore powder and add it to 5 mL of 0.05% Tween-80. Mix well with an oscillator for 2 min. After shaking several times, dilute it 100 times immediately. Take 10 μL and drop it onto a hemocytometer. Cover it with a coverslip and observe it under a microscope. Adjust the concentration of the spore suspension to 2.5 × 108 Pieces / mL.

[0022] Concentration calculation formula of spore suspension:

[0023] Concentration of spore suspension (spores / mL) = (number of spores in 5 grids / 5) × 25 × 10 4 × dilution factor.

[0024] 2.1 Determination of strain growth rate

[0025] After 7 days of transfer and activation, the nine strains were prepared with 0.05% Tween80 solution at 1×10 6 The concentration of spores / mL was dropped on a filter paper with a diameter of 0.8 cm, and the spore growth diameter was measured every day for 10 consecutive days.

[0026] The growth rate of 9 strains was measured, and the diameter of the strains was measured every day for 10 consecutive days. The results showed that CIPPMA154 had the fastest growth rate of 0.44 cm / day ( Figure 1 ), the fastest growth rate of CIPPBb107 in Beauveria bassiana was 0.41 cm / day ( Figure 2 ).

[0027] 2.2 Determination of spore production of strains

[0028] After 7 days of transfer and activation, the nine strains were prepared with 0.05% Tween80 solution at 1×10 6 The concentration of spores / mL was applied to PDAY medium. After 7 days, the strain produced spores and 5 points were taken from the same position to measure the spore content. The spore production per square centimeter was calculated. The spore production of 9 strains was measured. The results showed that the highest spore production of CIPPMA154 in Metarhizium was 3.53×10 7 Spores / cm 2 ( Figure 3 ), the highest conidia production of Beauveria bassiana was 4.63×10 7 Spores / cm 2 ( Figure 4 ).

[0029] 2.3 Determination of enzyme activity of strains

[0030] Each strain was prepared at a concentration of 1×10 8 Inoculate 100uL of the spore suspension of 100 / mL into 5mL of liquid fermentation medium, repeat three times, culture at 28℃, 200r / min shaking for about 72h until the logarithmic phase, centrifuge at 4℃, 12000rpm for 10min, carefully pipette the supernatant into a 1ml centrifuge tube, and store at -20℃ for later use. Determine chitinase activity according to the chitinase kit method.

[0031] The results of chitinase activity determination showed that CIPPMA154, CIPPMA025, CIPPMA149, and CIPPMA024 had relatively high chitinase activities (Table 1), which were 2.5267, 2.3133, 2.2733, and 2.2920 U / ml, respectively, which were positively correlated with the corrected mortality rate.

[0032] Table 1 Analysis of enzyme activities of different strains

[0033]

[0034] Note: The data in the table are mean ± standard error, and different letters indicate significant differences (p<0.05).

[0035] Example 3 Determination of the toxicity of locusts

[0036] 3.1 Determination of toxicity to third-instar larvae of locusts

[0037] Select the third-instar nymphs of locusts with consistent growth and development, treat 15 in each group, put them into a sterilized plastic basket (30cm×25cm×7.5cm), repeat 3 times, and starve them for 12 hours in advance; weigh 1g of sterilized wheat bran, add 50μL of vegetable oil and 1ml of spore suspension, add 1mL of 0.05% Tween-80 to the control group, put the bait into a sterile culture dish (60mm×15mm) after preparation, and then put it into a plastic basket. After 24 hours, take out the culture dish and feed it with fresh wheat seedlings until the end of the experiment. Record the number of dead locusts in each treatment group every day, and finally calculate the mortality rate of locusts in each treatment group.

[0038] Mortality rate (%) = (number of dead insects / number of test insects) × 100%

[0039] Corrected mortality rate (%) = [(treatment mortality rate - control mortality rate) / (1 - control mortality rate)] × 100%

[0040] The toxicity of different strains to locusts was determined by feeding method. Figure 5 , Table 1). The results showed that all the tested strains were virulent to migratory locusts, and the virulence of Beauveria bassiana to migratory locusts was lower than that of Metarhizium anisopliae among the tested strains; the Metarhizium anisopliae strain CIPPMA154 with good pathogenicity to migratory locusts was preliminarily screened out as the test material for further research.

[0041] Table 2 Control effect of different strains on 3rd instar locust nymphs

[0042]

[0043]

[0044] Note: The data in the table are mean ± standard error. Multiple comparisons were performed using the LSD method. Different letters indicate significant differences among different treatments (P ≤ 0.05).

[0045] 3.2 Further determination of the toxicity of locusts

[0046] For strains with good initial screening results, different spore concentrations were set to test their pathogenicity to locusts. The specific method is: the concentrations of spore suspensions are 1×10 7 / mL, 5×10 7 / mL, 2.5×10 8 The insecticidal activity against locusts was also determined by feeding method.

[0047] The pathogenicity of CIPPMA154 strain to host locusts at different concentrations was studied. Figure 6 ). The results showed that when the spore concentration was 1×10 7 , 5×10 7 , 2.5×10 8 / mL, after 12 days of treatment, the survival rates of the 3rd instar nymphs were 54%, 17% and 0, respectively, proving that the strain has a good pathogenicity to locusts, and the higher the concentration of green muscardine, the greater its toxicity to locusts.

[0048] The results showed that different strains of entomogenous fungi had very significant differences in virulence to different host insects, and the virulence also had a very significant linear relationship with the spore concentration. With the increase of spore concentration, the virulence of the strain had a significant increasing trend. The study on the pathogenicity of Beauveria bassiana and Metarhizium anisopliae to locusts showed that all 9 strains tested could infect the third-instar larvae of locusts, and there were significant differences in pathogenicity between different strains. Among the tested strains, the virulence of Beauveria bassiana to locusts was lower than that of Metarhizium anisopliae, among which the pathogenicity of Metarhizium anisopliae strain CIPPMA154 was better than that of other strains. In the enzyme activity analysis, the chitinase activity of strain CIPPMA154 was the highest, which was 2.5267U / ml. The above results all showed that the Metarhizium anisopliae strain CIPPMA154 had high virulence to locusts.

[0049] The above embodiments are only used to explain the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. A strain of Metarhizium anisopliae CIPPMA154 which is highly toxic to locusts, characterized in that: The deposit number of the Metarhizium anisopliae strain CIPPMA154, which is highly toxic to locusts, is CGMCC No.40933.

2. A preparation for controlling locusts, characterized in that: The locust control preparation comprises the spore suspension of Metarhizium anisopliae strain CIPPMA154 which is highly toxic to migratory locusts as claimed in claim 1.

3. The preparation for controlling migratory locusts according to claim 2, characterized in that The concentration of the spore suspension is 1×10 7 ~2.5×10 8 Pieces / mL.

4. Use of the Metarhizium anisopliae strain CIPPMA154, which is highly toxic to migratory locusts, as claimed in claim 1, for preventing and controlling migratory locusts.

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