CN115505538A - Metarhizium anisopliae strain CIPPMA0941, application of metarhizium anisopliae strain in preventing and treating red imported fire ants and microbial inoculum - Google Patents
Metarhizium anisopliae strain CIPPMA0941, application of metarhizium anisopliae strain in preventing and treating red imported fire ants and microbial inoculum Download PDFInfo
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Abstract
The invention relates to the field of microorganisms, in particular to a metarhizium anisopliae strain CIPPMA0941, application of the metarhizium anisopliae strain to prevention and treatment of solenopsis invicta and a microbial inoculum. The preservation number of the strain is CGMCC No.40238. The strain has high toxicity to red imported fire ants, and has certain activity to brontispa longissima, scarab larvae and aphids; the indoxacarb and imidacloprid have good compatibility, the germination of spores of a strain is not influenced by the indoxacarb and the imidacloprid with lower concentrations, the invasion of the strain into solenopsis invicta can be facilitated, and the lethality rate to the solenopsis invicta is provided.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a metarhizium anisopliae strain CIPPMA0941, application thereof in preventing and treating solenopsis invicta and a microbial agent.
Background
The Solenopsis invicta Buren is a harmful external invasive species, has strong reproduction destructive power and aggressive property, can eat plant species, prey invertebrates, seriously damage garden farmland industrial grassland and ecological organism diversity, bites and stings livestock and human beings, gnaws and damages a building base layer or an electric insulation layer and the like, and causes serious harm. Seriously threatens human health, and has safe life and production, and the like. At present, chemical agents are mainly used for preventing and controlling, and some physical prevention and biological prevention and control methods are combined.
The biological control of pests is to utilize natural enemies and special pathogens of the pests to restrict the population expansion of the pests, and is a long-acting sustainable control measure with ecological compatibility. In recent years, research and utilization of pathogenic microorganisms of solenopsis invicta has been carried out at home and abroad. Researches show that pathogenic microorganisms of red fire ants include Metarhizium spp, beauveria spp, corynespora Isaria spp and the like of fungi, bacteria mainly comprise bacillus spp, and bacterial strains with bacterial strains can be obtained through strain screening, and the effect of preventing and treating the red fire ants can be enhanced by matching some chemical agents with the pathogenic microorganisms.
Disclosure of Invention
The purpose of the present application is to provide metarhizium anisopliae strain CIPPMa0941.
It is still another object of the present application to provide the use of the metarhizium anisopliae strain CIPPMa0941 described above.
It is still another object of the present application to provide a microbial agent comprising the metarhizium anisopliae strain CIPPMa0941 described above.
Metarhizium anisopliae (Metarhizium anisopliae) according to the present applicationMetarhizium anisopliae) The strain CIPPMA0941 has the preservation number of CGMCC No.40238.
The invention provides the Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) The strain CIPPMA0941 is used for preventing and controlling pests. The bacterial strain is highly pathogenic to red fire ants, and is safe to people and livestock and compatible with the environment.
The application provides a medicine containing the metarhizium anisopliae (Metarhizium anisopliae) ((Metarhizium anisopliae) Bacterial agent of strain CIPPMa0941. The strain is used for developing a fungal insecticide for preventing and controlling the solenopsis invicta, is used as an independent product or a combined product matched with a chemical agent, is suitable for sustainable prevention and control of the solenopsis invicta, and has a good application prospect.
The morbid beetle corpse collected from Jiang Xiji Anjinggan mountain forest land is separated and purified in laboratory to obtain pure strain, and the pure strain is identified by morphology and molecular marker to be Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) The strain was named CIPPMA0941. The toxicity activity test of the strain on several kinds of insects shows that the strain has higher toxicity on red imported fire ants and has certain activity on brontispa longissima, scarab larvae and aphids; the parallel test comparison with a plurality of strains on the solenopsis invicta proves that the strain has the strongest toxicity. The strain has high growth speed on a PDAY culture medium, large spore yield and high spore germination rate; also can be cultured on rice and barley to obtain a large amount of conidia. Spores of different culture modes have pathogenicity on the solenopsis invicta, and the pathogenicity is positively correlated with the concentration of the spores. The strain has good compatibility with indoxacarb and imidacloprid. The indoxacarb and imidacloprid with lower concentration do not affect the germination of the spores of the strains, and can also help the strains to invade the solenopsis invicta so as to provide the lethality rate to the solenopsis invicta.
Drawings
FIG. 1 shows the mortality rate of Metarhizium anisopliae strain Ma0941 of the present application to Solenopsis invicta;
FIG. 2 shows the virulence of Metarhizium anisopliae strain Ma0941 of the present application against red fire ants at different concentrations.
Metarhizium anisopliae (Metarhizium anisopliae) ((B))Metarhizium anisopliae) The strain CIPPMA0941 is deposited in the general microbiological center of China Committee for culture Collection of microorganisms, address, no. 3 of West Lu No. 1 of North Chen of the Korean-Yang district, institute of microbiology, china academy of sciences, postal code, 100101, date of deposition, 04 days 07 months 2022, and number of deposition CGMCC No.40238.
Detailed Description
Example 1 obtaining and identification of strains
The strain was isolated from a morbid beetle zombie collected from Jinggang mountain, guixi Jian (2005). Cleaning the surface of the stiff insects, transferring the stiff insects into a sterile culture dish, keeping the surface of the stiff insects moist for 5 days at 25 ℃, so that hyphae grow on the surface of the visible body, then generating light green spores, picking a small number of spores by using a sterile inoculating needle, streaking and inoculating the spores on a PDA (personal digital assistant) culture medium, culturing the spores for 5 to 7 days at 25 ℃, picking single colonies, streaking and inoculating the single colonies again for culturing to obtain a plurality of purified single colonies, transferring the single colonies to a test tube, culturing the single colonies for 10 days at 25 ℃, obtaining the strains, and storing the strains at 4 ℃.
Inoculating conidia of the strain on a PDA culture medium plate, culturing at 25 deg.C for 2-4 days, observing the gradual growth and enlargement of colony, wherein the surface is white villous, and continuously culturing to show that green spores are generated on the surface, the color is changed from light green to dark, and the final product is olive green.
Carefully picking a small amount of thallus from the edge of the colony cultured for 3-4 days by using an inoculating needle, placing the thallus on a glass slide, and observing under a microscope, wherein the mycelium is transparent, has partitions and branches and has a width of 2.5-3.5 mu m; conidiophores are unipartite, phialide is unipartite, conidia on phialide grow in series end to end, and some conidia fall off. Conidium is in short cylinder shape or ellipse shape, both ends are blunt round or slightly sharp, size is 4-8 × 2-3.5 μm, and it is identified as Metarhizium anisopliae.
Preparing a hypha culture medium: 20g of cane sugar and eggsBai Dong 5g, dipotassium hydrogen phosphate 0.1g, water to 1000mL, sterilizing for use. Inoculating conidium of the strain into a culture medium, wherein the inoculation amount is 10 7 Spore/100 mL, culturing on a constant temperature shaker at 25 + -1 deg.C for 48 h, filtering to obtain mycelium, and sucking water with filter paper. DNA was extracted using a fungal genomic DNA extraction kit and following the instructions. Amplifying to obtain amplified fragment of ITS1-5.8S-ITS4 rDNA region, detecting nucleotide sequence of the fragment, performing Blast comparison on the sequence in NCBI database, and identifying as Metarhizium anisopliaeMetarhizium anisopliaeThe phylogenetic classification is Ascomycota, pezizomycotina, sordariomycetes, hypocrea, clavicipitaceae.
EXAMPLE 2 insecticidal Activity of the Strain
The bacterial strain of the application has the functions of infecting insects and leading to the death of insect lesions, and the insecticidal activity (pathogenicity and virulence) is evaluated by bioassay.
1. Isolated strains having varying degrees of pathogenicity to several pests
Inoculating the strain on PDAY culture medium, culturing at 25 deg.C for 15d, collecting spore powder, preparing into spore suspension with sterilized 0.1% Tween-80, and adjusting concentration to 1 × 10 7 spores/mL. Respectively treating the green peach aphids by adopting an immersion methodMyzus persicaeAphid of peaAcyrthosiphon pisumNymphs, red fire antsSolenopsis invictaGongant 10sec, processing Brontispa longissimaBrontispa longissimaLarva (Brontispa longissima larva not easy to age) and Holotrichia parallelaHolotrichia parallelaLarvae (2 instar) 20sec, repeated 5 times per treatment, 30 beetles per repetition (25 gill beetle larvae). Sterile water containing 0.01-vol% Tween80 was used as a blank. And (3) placing the treated product in a climatic chamber, wherein the control conditions are 25 +/-1 ℃, the relative humidity is 75%, and the light cycle is L: D = 14. The death number of aphids and red imported fire ants is observed and recorded every day, and the death number is recorded for 7d; brontispa longissima and Holotrichia parallela were observed every other day and recorded at 18 d. Dead individuals were picked up and placed on petri dishes with soaked filter paper for moisture preservation to observe the growth of hyphae. Mortality was calculated and corrected using Abbott's equation.
Mortality (%) = (number of dead insects/number of test insects) × 100,
corrected mortality (%) = [ (treatment mortality-control mortality)/(1-control mortality) ] × 100.
TABLE 1 virulence of the strains on different test insects
As shown in table 1, the strains of the present application were somewhat pathogenic to several insects tested, but were significantly different in pathogenicity. The separated strain has the highest toxicity to the solenopsis invicta, reaches 94.2 percent, has the second toxicity to Holotrichia parallela and has lower toxicity to Brontispa longissima and aphid.
2. Comparing the virulence of different strains against Solenopsis invicta
Metarhizium anisopliae has evolved into many strains, and different strains have large differences in the pathogenic ability of insects.
The pathogenicity of the strain of the present application was compared with the other 10 strains. The strain CIPMa 0011 CIPMa 0024 CIPMa 0035 CIPMa 0054 CIPMa 0065 IPPMa0077 CIPMa 0082 CIPMa 0088 CIPMa 0125 CIPMa 0127 CIPMa 0941 is provided by the plant protection institute of Chinese academy of agricultural sciences, and is stored at 4 ℃ in a laboratory.
Inoculating the strain on PDAY culture medium, culturing at 25 deg.C for 15 days, collecting spore powder, preparing into spore suspension with sterilized 0.1% Tween80, and adjusting concentration to 1 × 10 7 spores/mL. The method comprises respectively treating the ergates of Solenopsis invicta with soaking method for 10sec, and repeating for 5 times and 30 worms. A blank was prepared by using sterile 0.01% Tween-80 aqueous solution as a blank, and the blank was placed in a climatic chamber under conditions of 25. + -. 1 ℃ and 75% relative humidity with a photoperiod of L: D = 14. The number of deaths was recorded daily and 9 days were recorded. Dead individuals were picked up and placed on petri dishes with soaked filter paper for moisture preservation to observe the growth of hyphae. Mortality was calculated and corrected using Abbott's equation.
Mortality (%) = (number of dead insects/number of test insects) × 100,
corrected mortality (%) = [ (treatment mortality-control mortality)/(1-control mortality) ] × 100
And (6) obtaining the result.
TABLE 2 pathogenicity of different strains to Solenopsis invicta
As shown in Table 2, the strain Ma0941 of the present application showed high virulence to Solenopsis invicta, and the cumulative mortality of 9d reached 94.6%, while the other test strains were only low or non-virulent to Solenopsis invicta, and the corrected mortality was below 25.4%. The mortality course of the Ma0941 strain against Solenopsis invicta is shown in FIG. 1, according to the regression equation y = -1.2778x 2 + 25.522x-28.556 (R = 0.9775) calculation shows high pathogenicity to solenopsis invicta, 9d cumulative mortality reaches 94.6%, and half death time LT under test conditions 50 3.802 days.
3. Pathogenic force of strains with different dosages to Solenopsis invicta
Inoculating the strain Ma0941 of the present application on PDAY culture medium, culturing at 25 deg.C for 15d, collecting spore powder, preparing into spore suspension with sterilized 0.1% Tween80, and adjusting the concentration to 5 concentration gradients, i.e. 10 4 、10 5 、10 6 、10 7 、10 8 spores/mL. The Solenopsis invicta ergate is treated by soaking for 10sec, and each treatment is repeated for 5 times and 30 worms. A blank control was carried out using sterile 0.01% Tween-80 aqueous solution, and the blank was placed in a climatic chamber under conditions of 25. + -. 1 ℃ relative humidity of 75% and photoperiod L:: D = 14. The number of deaths was recorded daily and 9 days. Dead individuals were picked up and placed on petri dishes with soaked filter paper for moisture preservation to observe the growth of hyphae. Daily mortality was calculated and the time to half-death at each concentration was calculated.
As shown in Table 3 and FIG. 2, the pathogenicity of the strain Ma0941 to red imported fire ant increased with increasing concentration of the bacterial liquid, and 10 4 、10 5 、10 6 、10 7 、10 8 The cumulative mortality for 9d at spore/mL concentration was 10.67%, 17.33%, 26.00%, 62.67%, 95.33%, and 97.33%, respectively; high concentration (10) 7 、10 8 SporemL) showed a significant increase in mortality already in the first 3 days; the rise is in the form of a quadratic curve by regression calculation, 10 6 、10 7 、10 8 Time to semilethality at concentration (LT) 50 ) 11.49d, 3.72d and 3.19d, respectively, the higher the concentration, the more lethal and the higher the concentration (10) 7 、10 8 spores/mL) to 90% Lethality (LT) 90 ) 7.16d and 6.09d, respectively.
TABLE 3 pathogenicity of different doses of the strains to Solenopsis invicta
Regression calculations were performed based on the number of deaths recorded daily at each concentration to achieve half-lethal bacterial fluid concentrations (LC) at 3d, 4d, 5d, 6d, 7d, 8d or 9d 50 ) Are respectively 2.40 multiplied by 10 6 、1.60×10 5 、5.12×10 4 、1.86×10 4 、9.34×10 3 、5.90×10 3 、4.11×10 3 spores/mL (see table 4 below):
TABLE 4
4. Biological Properties of Strain Ma0941
4.1 Culture characteristics of the Strain
The test compares the growth rate and sporulation yield of 5 strains on PDAY medium.
Strain: ma0011, ma0035, ma0082, ma0327 and Ma0941 are all provided by plant protection research institute of Chinese academy of agricultural sciences and stored at 4 ℃ in a laboratory.
Culture medium: PDAY culture medium (200 g of fresh potatoes, filtrate obtained after boiling, 20g of glucose, 10g of yeast extract powder, 18g of agar and distilled water to 1000 mL); czapek's medium (sodium nitrate 3g, dipotassium hydrogen phosphate 1g magnesium sulfate (MgSO4.7H2O) 0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g, sucrose 30g, agar 20g, distilled water to 1000 mL). The plates were prepared by sterilizing 15 lbs of steam at 121 ℃ for 20 minutes.
Respectively taking spore powder of each test strain, preparing into uniform spore suspension with sterilized 0.1% Tween water, counting, adjusting concentration to 1 × 10 7 spores/mL. And (3) sucking 20 mul of spore liquid by a micropipette, dripping and inoculating the spore liquid into the center of a culture medium plate, and culturing in an incubator at 25 ℃. Each strain was inoculated in 10 replicate plates per medium. The colony growth speed and the sporulation quantity are observed and measured.
As a result: on day 3 after inoculation, white microcolonies were visible on most plates, and from day 5, the diameters in both the vertical and horizontal directions of the colonies were measured with a vernier caliper every day for 5 consecutive days, and the growth rates were calculated and the differences between the strains were compared. Culturing is continued until day 15, 4 fungus cakes are punched at 1/2 radius of the longitudinal and transverse diameters of the colony by a puncher (the inner diameter is 7.0mm, and the area = 0.3848cm2), the fungus cakes are placed in a test tube with 10mL sterile warm water, the fungus cakes are fully shaken and dispersed on a vortex shaker to prepare uniform suspension, after the uniform suspension is diluted by 1:1, the number of the produced spores per unit area is calculated by a blood counting plate under an optical microscope, and the difference between the strains is compared. Meanwhile, the seeds are inoculated on 100 mu L to 1/2 reduced PADY plates, evenly coated, cultured at 25 ℃, and subjected to microscopic observation at 12h and 20h to detect the spore germination condition. Each strain was checked 5 replicates per plate, each plate was observed for at least 3 fields, and the number of spores was counted for at least 100, and germination rate was calculated.
TABLE 5 growth and sporulation Properties of the different strains on PDAY Medium
4.2 Compatibility of Strain Ma0941 with several insecticides
The influence of 5 chemical pesticides on conidiophore germination and colony growth was examined. Source of pesticide and conventional application concentration (recommended dilution factor): 10% imidacloprid wettable powder (shandong korean agro-chemical limited), 1500 ×;15% indoxacarb suspension concentrate 5000 × (Su Ke agro-chemical llc, jiangsu province); 0.5% matrine solubles (beijing agon biological pharmaceuticals, ltd.) 500 ×;1.5% pyrethrin aqueous emulsion (inner Mongolia Qingyuan biological science and technology limited) 1500 ×;1% osthole emulsion in water Inmunogu Qingyuan Baoshi Co., ltd.), 500X.
The chemical insecticide is prepared into 1 x, 2x and 4 x dilution times according to the recommended dilution times of the instruction book, and the solution with the concentration decreasing in a conventional manner, a 1/2 conventional manner and a 1/4 conventional manner is obtained.
Inoculating Ma0941 strain to PDAY culture medium, culturing at 25 deg.C for 15d, collecting spore powder, preparing into spore suspension with sterilized 0.1% Tween80, and adjusting concentration to 10 7 spores/mL.
(1) Effect of chemical pesticides on the Germination of Ma0941 conidia
200 μ l of the above spore suspension was added to a test tube containing 1.8mL of a dilution of the pesticide, repeated for 3 tubes per treatment, with sterile water as a control. Placing in a shaker at 160 rpm and 25 ℃, respectively inoculating 200 mu L of the mixed solution on PDAY medium plates after 2h and 8h, uniformly coating, repeating 3 plates, culturing for 24h at 25 ℃, observing the spore germination condition under a microscope, observing at least 3 visual fields on each plate, counting at least 100 spores, and calculating the spore germination inhibition rate.
As a result: as shown in tables 6 and 7, several chemical insecticides have certain inhibitory effect on the germination of the spores of the strains at the conventional concentration, and related to the concentration of the chemical agent and the treatment time, the inhibitory rates after 2h and 8h treatment at the conventional concentration are respectively 6.37-10.13% and 18.9-32.7%, the inhibitory rate after 8h treatment at 1/4 conventional concentration is 3.57-13.75%, and the inhibitory effect is basically not generated after 2h treatment compared with the control.
TABLE 6 Effect of different concentrations of chemical pesticides on spore germination of Strain 0941 (treatment 2 h)
TABLE 7 Effect of different concentrations of chemical pesticides on spore germination of Strain 0941 (treatment 8 h)
(2) Effect of chemical pesticides on the growth of Ma0941 colonies
20 μ L of the treatment solution was inoculated into the center of a PDAY medium plate, 3 plates were repeated, and the plate was incubated at 25 ℃ in an incubator at 4d, 7d, and 10d, and the diameters of the colonies in both the vertical and horizontal directions were measured with a vernier caliper, and the growth inhibition ratio was calculated.
As a result: as shown in tables 8 and 9, several chemical insecticides had a certain inhibitory effect on colony growth rate at the conventional concentration, and the inhibitory effect was strong at a higher concentration and strong at the initial growth stage of colonies, in relation to the concentration of the chemical agent and the treatment time. The inhibition rate of 4d cultured on the flat plate after 2h and 8h of treatment under the conventional concentration is 8.45-50.23%, while the inhibition rate under the 1/4 conventional concentration is only 0-10% lower; after the culture for 7d and 10d, the inhibition effect of different concentrations of each medicament is gradually reduced to be basically not different from that of the control.
TABLE 8 Effect of different concentrations of chemical insecticides on the growth of the colony of Strain 0941 (treatment 2 h)
TABLE 9 Effect of different concentrations of chemical insecticides on the growth of the colony of Strain 0941 (treatment 8 h)
In conclusion, the strain Ma0941 has certain compatibility with 5 chemical insecticides under the conventional dosage, has better compatibility under the dosage of 1/2 and 1/4 diluted by the conventional concentration, and has better compatibility under the short time of 2h, and the performance A is favorable for preventing and controlling the red imported fire ants.
The above examples are only for explaining the technical solutions of the present application, and do not limit the scope of protection of the present application.
Claims (7)
1. Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) Bacterial strainsClpmpa 0941, characterized in that said metarhizium anisopliae (Metarhizium anisopliae)) (Metarhizium anisopliae) The preservation number of the strain CIPPMA0941 is CGMCC No.40238.
2. The metarhizium anisopliae (Metarhizium anisopliae) (of claim 1)Metarhizium anisopliae) Application of the strain CIPPMA0941 in pest control.
3. Use according to claim 2, wherein the pest is solenopsis invicta, brontispa longissima or chafer.
4. The use of claim 3, wherein the chafer is Holotrichia parallela.
5. A fungicide for controlling pests, comprising the metarhizium anisopliae (Metarhizium anisopliae) of claim 1Metarhizium anisopliae) Strain CIPPMa0941.
6. The pest controlling agent according to claim 5, wherein said agent comprises a chemical pesticide.
7. The pesticidal agent according to claim 6, wherein the chemical insecticide is imidacloprid, indoxacarb, matrine, pyrethrin, osthole.
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