CN115505538B - Metarhizium anisopliae strain CIPPMA0941, application thereof in preventing and treating solenopsis invicta and microbial inoculum - Google Patents
Metarhizium anisopliae strain CIPPMA0941, application thereof in preventing and treating solenopsis invicta 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, and application and a microbial inoculum for controlling solenopsis invicta. The preservation number of the strain is CGMCC No.40238. The strain has high toxicity to solenopsis invicta, and has certain activity to brothers, chafer larva and aphid; the indoxacarb and the imidacloprid have better compatibility, the germination of spores of the strain is not influenced by the indoxacarb and the imidacloprid with lower concentration, the invasion of the strain into the solenopsis invicta can be facilitated, and the mortality rate of the solenopsis invicta is provided.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a Metarhizium anisopliae strain CIPPMA0941, and application and a microbial inoculum for controlling solenopsis invicta.
Background
The solenopsis invicta Solenopsis invicta Buren is a harmful foreign invasive species, has strong destructive power of reproduction, has aggressiveness, can eat plant seeds, predate invertebrates, seriously damages grasslands and ecological biodiversity of garden farmland, bites stings livestock and poultry and human beings, and damages a building base layer or an electric insulating layer by biting, and the like, so that the solenopsis invicta is seriously damaged. Seriously threatens the health of human beings, and is safe in life and production, etc. At present, chemical agent control is mainly adopted, and some physical control and biological control methods are combined.
The biological pest control is to restrict the population expansion of pests by utilizing natural enemies and special pathogens of the pests, and is an ecologically compatible and long-acting sustainable prevention and control measure. In recent years, research and utilization of red fire ant pathogenic microorganisms are being carried out at home and abroad. Researches show that the pathogenic microorganisms of the solenopsis invicta comprise Metarhizium spp, beauveria spp, isaria spp, etc. of fungi, and the bacteria mainly comprise bacillus spp, bacterial strain screening can be used for obtaining virulent strain, and the cooperation of chemical agents and pathogenic microorganisms can enhance the effect of preventing and treating the solenopsis invicta.
Disclosure of Invention
The purpose of the application is to provide metarhizium anisopliae strain CIPPMA0941.
It is a further object of the present application to provide the use of the Metarhizium anisopliae strain CIPPMA0941 as described above.
It is still another object of the present application to provide a microbial inoculum comprising the metarhizium anisopliae strain CIPPMa0941 as described above.
Metarhizium anisopliae according to the present applicationMetarhizium anisopliae) The preservation number of the bacterial strain CIPPMA0941 is CGMCC No.40238.
The invention provides the metarhizium anisopliaeMetarhizium anisopliae) Application of the strain CIPPMA0941 in pest control. The strain of the application is highly pathogenic to solenopsis invicta, and is safe to people and livestock and compatible with the environment.
The application provides a preparation method comprising the above scarab beetleMetarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) Bacterial agent of the strain CIPPMA0941. The strain is used for developing a fungal pesticide for preventing and controlling solenopsis invicta, is used as a single product or a combined product matched with chemical agents, is suitable for sustainable prevention and control of solenopsis invicta, and has good application prospect.
The method is characterized in that a morbid beetle zombie collected from Jiangxi Jian Jinggang mountain forest lands is separated and purified in a laboratory to obtain pure strains, and the pure strains are identified by morphology and molecular marker identification to be metarhizium anisopliaeMetarhizium anisopliae) The strain was named CIPPMA0941. The strain is subjected to toxicity activity test on a plurality of insects, which shows that the strain has higher toxicity on solenopsis invicta and also has certain activity on brothers, chafer larvae and aphids; compared with parallel tests of a plurality of strains on the solenopsis invicta, the strain has the strongest toxicity. The strain grows fast on a PDAY culture medium, the spore yield is large, and the spore germination rate is high; or culturing on rice and barley to obtain a large amount of conidium. Spores of different culture modes have pathogenicity on solenopsis invicta, and pathogenicity is positively related to spore concentration. The strain has better compatibility with indoxacarb and imidacloprid. The indoxacarb and imidacloprid with lower concentration do not influence the germination of spores of the strain, and can also help the strain to invade the solenopsis invicta and provide the mortality rate for the solenopsis invicta.
Drawings
FIG. 1 shows mortality of Metarhizium anisopliae strain Ma0941 of the present application to Formica Fusca;
FIG. 2 shows the pathogenicity of Metarhizium anisopliae strain Ma0941 of the present application against solenopsis invicta at different concentrations.
Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) The strain CIPP MA0941 is preserved in China general microbiological culture Collection center, address, north West Lu No. 1, 3 of the Korean area of Beijing, national academy of sciences microbiological institute, postal code, 100101, preservation date, 2022, 07 month 04, and preservation number CGMCC No.40238.
Detailed Description
Example 1 obtaining and identification of strains
The strain was isolated from a comic beetle zombie harvested from jingan Jinggan mountain in Jiangxi (2005). Cleaning the surface of stiff worms, transferring into a sterile culture dish, moisturizing at 25 ℃ for 5 days, allowing hyphae to grow on the surface of the stiff worms, generating light green spores, picking a small amount of spores by using a sterile inoculating needle, streaking and inoculating on a PDA (personal digital assistant) culture medium, culturing at 25 ℃ for 5-7 days, picking single bacterial colony, streaking and inoculating again for culturing to obtain a plurality of purified single bacterial colonies, transferring to a test tube for culturing at 25 ℃ for 10 days, and obtaining bacterial strains, and storing at 4 ℃.
Inoculating the bacterial conidium on a PDA culture medium plate, culturing for 2-4d at 25 ℃, observing that bacterial colony grows and expands gradually, and continuously culturing until green spores are generated on the surface, wherein the color of the bacterial colony turns from light green to dark, and finally the bacterial colony turns into olive green.
Carefully picking a small amount of thalli from the edge of a colony for 3-4d, placing the thalli on a glass slide, and observing the thalli under a microscope, wherein the mycelia are transparent, have a partition and branches, and have a width of 2.5-3.5 mu m; conidiophores are single, bottle peduncles are columnar single, conidiophores on bottle peduncles are connected end to grow and are arranged in series, and some conidiophores are already shed. The conidium is in a short column shape or an oval shape, both ends of the conidium are rounded or slightly pointed, and the conidium is 4-8 multiplied by 2-3.5 mu m in size, and the conidium is identified as metarhizium anisopliae.
Preparing a mycelium culture medium: 20g of sucrose, 5g of peptone, 0.1g of dipotassium hydrogen phosphate, and adding water to 1000mL, and sterilizing for later use. Inoculating conidium of strain into culture medium with an inoculum size of 10 7 Spores/100 mL were incubated on a 25.+ -. 1 ℃ constant temperature shaker for 48 h, filtered to obtain mycelia and the water was blotted off with filter paper. DNA was extracted using the 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 of the sequence in NCBI database, and identifying Metarhizium anisopliaeMetarhizium anisopliaeThe system is classified into Ascomycota, pezizomycetin, sordariomycetes, hypocrea, clavicpicitaceae, hypocrea.
Example 2 insecticidal Activity of strains
The strain of the application has the effect of infecting insects and leading to the death of lesions of the insects, and the insecticidal activity (pathogenicity and virulence) is evaluated through bioassay.
1. Isolated strains are pathogenic to several pests to varying degrees
Inoculating the strain on PDAY medium, culturing at 25deg.C for 15d, collecting spore powder, preparing spore suspension with sterilized 0.1% Tween-80, and adjusting concentration to 1×10 7 spores/mL. Adopting dipping method to treat the green peach aphids respectivelyMyzus persicaeAleurites pisiformis (L. Pisum)Acyrthosiphon pisumNymphs and solenopsis invictaSolenopsis invictaTreating coconut leaf beetles 10secBrontispa longissimaLarvae (coconut leaf beetle larvae are not easy to age) and Holotrichia parallelaHolotrichia parallelaLarvae (2 years) for 20sec, 5 replicates per treatment, 30 replicates per larva (25 heads of gill scarab larvae). Sterile water 0.01% Tween80 was used as a blank. After treatment, the mixture was placed in a climatic chamber under control of 25.+ -. 1 ℃ and 75% relative humidity and photoperiod of L: D=14:10. Aphids and solenopsis invicta were observed daily to record the number of deaths, record 7d; the coconut leaf beetles and Holotrichia parallela were recorded every other day, record 18 d. After picking up dead individuals, the mycelia were observed by placing the individuals in a petri dish with soaked filter paper and keeping the dishes moist. Mortality was calculated and corrected with Abbott's formula.
Mortality (%) = (number of dead insects/number of test insects) ×100,
corrected mortality (%) = [ (treatment mortality-control mortality)/(1-control mortality) ]x100.
TABLE 1 pathogenicity of strains against different test insects
As shown in Table 1, the strains of the present application were pathogenic to several insects tested, but the pathogenicity was greatly different. The separated strain has the highest toxicity to solenopsis invicta reaching 94.2%, has lower toxicity to holotrichia parallela and to brontispa longissima and aphid.
2. Comparing the toxicity of different strains to Formica Fusca
The metarhizium anisopliae species evolves to form a plurality of strains, and the pathogenic ability of different strains to insects is greatly different.
The strains of the present application were compared for pathogenicity with the other 10 strains. The strain CIPPMa0011 CIPPMa0024 CIPPMa0035 CIPPMa0054 CIPPMa0065 IPPMa0077 CIPPMa0082 CIPPMa0088 CIPPMa0125 CIPPMa0127 CIPPMa0941, all supplied by plant protection institute of national academy of agricultural sciences, was stored at 4 ℃ in laboratory.
Inoculating the strain on PDAY medium, culturing at 25deg.C for 15d, collecting spore powder, preparing spore suspension with sterilized 0.1% Tween80, and adjusting concentration to 1×10 7 spores/mL. The solenopsis invicta workers were treated with the dipping method for 10sec, 5 times per treatment and 30 worms per repetition. A sterile 0.01% Tween-80 aqueous solution was used 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=14:10. Daily observations record the number of deaths, record 9d. After picking up dead individuals, the mycelia were observed by placing the individuals in a petri dish with soaked filter paper and keeping the dishes moist. Mortality was calculated and corrected with Abbott's formula.
Mortality (%) = (number of dead insects/number of test insects) ×100,
corrected mortality (%) = [ (treatment mortality-control mortality)/(1-control mortality) ]x100
As a result.
TABLE 2 pathogenicity of different strains on Formica Fusca
As shown in Table 2, the strain Ma0941 of the application shows high pathogenicity to the solenopsis invicta, the accumulated death rate of 9d reaches 94.6%, other tested strains have low toxicity or no toxicity to the solenopsis invicta, and the corrected death rate is below 25.4%. The mortality rate process of Ma0941 strain on solenopsis invicta is shown in figure 1, according to regression equation y= -1.2778x 2 Calculated + 25.522x-28.556 (R, r= 0.9775), shows high pathogenicity to solenopsis invicta, an accumulated mortality rate of 9d up to 94.6%,semi-lethal time LT under test conditions 50 For 3.802 days.
3. Pathogenicity of different doses of strains against solenopsis invicta
Inoculating strain Ma0941 of the application on PDAY medium, culturing at 25deg.C for 15d, collecting spore powder, preparing spore suspension with sterilized 0.1% Tween80, and adjusting concentration to 5 concentration gradients, namely 10 4 、10 5 、10 6 、10 7 、10 8 spores/mL. The solenopsis invicta workers were treated with the dipping method for 10sec, 5 times per treatment and 30 worms per repetition. A sterile 0.01% Tween-80 aqueous solution was used 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=14:10. Daily observations record the number of deaths, record 9d. After picking up dead individuals, the mycelia were observed by placing the individuals in a petri dish with soaked filter paper and keeping the dishes moist. 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 against solenopsis invicta at different concentrations increased with increasing concentration of the bacterial liquid, at 10 4 、10 5 、10 6 、10 7 、10 8 Cumulative mortality at spore/mL concentrations for 9d was 10.67%, 17.33%, 26.00%, 62.67%, 95.33% and 97.33%, respectively; high concentration (10) 7 、10 8 spore/mL) mortality has shown to rise significantly over the first 3 days; rising in a quadratic curve mode, through regression calculation, 10 6 、10 7 、10 8 Time to half-death at concentration (LT 50 ) 11.49d, 3.72d and 3.19d, respectively, the higher the concentration, the faster the death, the higher the concentration (10 7 、10 8 spore/mL) to 90% lethal time (LT 90 ) 7.16d and 6.09d, respectively.
TABLE 3 pathogenicity of different doses of strains against Formica Fusca
Processing mortality based on daily recorded individual concentrationsCalculated, the bacterial concentration (LC) at 3d, 4d, 5d, 6d, 7d, 8d or 9d was calculated to achieve half the mortality 50 ) 2.40×10 respectively 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 spore/mL (see table 4 below):
TABLE 4 Table 4
4. Biological Properties of Strain Ma0941
4.1 Culture characteristics of strains
The test compares the growth rate and sporulation yield of 5 strains on PDAY medium.
Strains: ma0011, ma0035, ma0082, ma0327, ma0941, all from Protect of plant institute of the national academy of agricultural sciences, stored at 4℃in the laboratory.
Culture medium: PDAY culture medium (200 g of fresh potato, 20g of glucose, 10g of yeast extract powder, 18g of agar, and distilled water to 1000 mL) is obtained after boiling; czapek's medium (sodium nitrate 3g, dipotassium hydrogen carbonate 1g magnesium sulfate (MgSO4.7H2O) 0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g, sucrose 30g, agar 20g, distilled water to 1000 mL). 15 lbs. of steam was sterilized at 121℃for 20 minutes and then plated.
Respectively collecting spore powder of each test strain, preparing into uniform spore suspension with sterilized 0.1% Tween water, counting, and adjusting concentration to 1×10 7 spores/mL. 20. Mu.l of spore liquid was pipetted by a micropipette and inoculated in the center of the medium plate and placed in a 25℃incubator for cultivation. Each strain was inoculated in 10 plates on each medium. The colony growth rate and spore yield were measured by observation.
Results: on day 3 post inoculation, white microcolonies were seen on most plates, and from day 5, the diameters of the colonies in both the longitudinal and transverse directions were measured daily with a vernier caliper for 5 consecutive days, the growth rate was calculated, and the differences between the strains were compared. Continuously culturing until 15 days, punching 4 bacterial cakes at 1/2 radius of longitudinal and transverse diameters of bacterial colonies by using a puncher (with an inner diameter of 7.0mm and an area of 0.3848cm < 2 >), placing the bacterial colonies in a test tube with 10mL of sterile warm water, fully oscillating and dispersing the bacterial colonies on a vortex oscillator to prepare uniform suspension, counting the bacterial colonies by using a blood cell counting plate under an optical microscope after diluting the bacterial colonies by 1:1, calculating the spore yield per unit area, and comparing the differences among bacterial strains. Simultaneously inoculating 100 mu L to 1/2 decrement of PADY plate, uniformly coating, culturing at 25 ℃, and carrying out microscopic observation at 12h and 20h to detect spore germination. Each strain was examined 5 plate replicates at a time, at least 3 fields were observed per plate, the number of spores was counted at least 100, and germination rates were calculated.
TABLE 5 growth and sporulation Properties of different strains on PDAY Medium
4.2 Compatibility of the Strain Ma0941 with several pesticides
The effect of 5 chemical pesticides on conidium germination and colony growth was examined. Pesticide source and normal application concentration (recommended dilution): 10% imidacloprid wettable powder (Shandong Han Nong chemical Co., ltd.) 1500×;15% indoxacarb suspension 5000X (Su Kenong of Jiangsu province, inc.); 0.5% matrine soluble (Beijing Asia Gonong biopharmaceutical Co., ltd.) 500×;1.5% pyrethrin aqueous emulsion (inner Mongolian Qing Source Biotechnology Co., ltd.) 1500X; 1% osthole aqueous emulsion inner mongolian definition source biosciences limited), 500×.
The chemical insecticide is prepared into dilution factors of 1X, 2X and 4X according to recommended dilution factors of the specification, and then the solution with the concentration of conventional, 1/2 conventional and 1/4 conventional decreasing is obtained.
Inoculating strain Ma0941 on PDAY medium, culturing at 25deg.C for 15d, collecting spore powder, preparing spore suspension with sterilized 0.1% Tween80, and adjusting concentration to 10 7 spores/mL.
(1) Effect of chemical pesticides on Ma0941 conidium germination
200 μl of the spore suspension was added to a test tube containing 1.8mL of pesticide dilution, and 3 tubes were repeated for each treatment, with sterile water as a control. Placing in a shaker at 160 rpm and 25 ℃, respectively inoculating 200 mu L of the mixture to PDAY culture medium plates after 2h and 8h, uniformly coating, repeating 3 plates, culturing at 25 ℃ for 24 hours, observing spore germination conditions under a microscope, observing at least 3 fields of view of each plate, counting at least 100 spores, and calculating spore germination inhibition rate.
Results: as shown in tables 6 and 7, several chemical pesticides have a certain inhibition effect on bacterial spore germination at conventional concentrations, the inhibition rates after 2h and 8h treatment are respectively 6.37-10.13% and 18.9-32.7% at conventional concentrations, the inhibition rate after 8h treatment is 3.57-13.75% at 1/4 conventional concentrations, and 2h treatment has no inhibition effect basically compared with a control.
TABLE 6 influence of different concentrations of chemical pesticides on spore germination of strain 0941 (treatment 2 h)
TABLE 7 influence of different concentrations of chemical pesticides on spore germination of strain 0941 (treatment 8 h)
(2) Effect of chemical pesticides on Ma0941 colony growth
Inoculating 20 μl of the treatment solution to the center of the PDAY medium plates, repeating 3 plates each, culturing in a 25℃incubator, measuring the diameters of the colonies in the longitudinal and transverse directions at 4d, 7d and 10d with vernier calipers, and calculating the growth inhibition ratio.
Results: as shown in tables 8 and 9, several chemical pesticides have a certain inhibition effect on colony growth rate at conventional concentrations, and the inhibition effect is stronger at higher concentrations and stronger at initial colony growth stages in relation to the concentration of the pesticide and the treatment time. The inhibition rate of 4d culture on a flat plate after 2h and 8h treatment under the conventional concentration is 8.45-50.23%, and the inhibition rate under the conventional concentration of 1/4 is very low and is only 0-10%; after 7d and 10d incubation, the inhibition at different concentrations of each agent gradually decreased to be substantially no difference from the control.
TABLE 8 influence of different concentrations of chemical pesticides on bacterial colony growth of strain 0941 (treatment 2 h)
TABLE 9 influence of different concentrations of chemical pesticides on bacterial colony growth of strain 0941 (treatment 8 h)
In conclusion, the strain Ma0941 has certain compatibility with 5 chemical pesticides at conventional dosages, has better compatibility at conventional concentration dilution of 1/2 and 1/4 dosages, has better compatibility at a short time of 2 hours, and the performance A is favorable for being matched with the use of controlling the solenopsis invicta.
The above embodiments are only used for explaining the technical solution of the present application, and do not limit the protection scope of the present application.
Claims (7)
1. Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) The bacterial strain CIPPMA0941 is characterized in that the metarhizium anisopliae is prepared byMetarhizium anisopliae) The preservation number of the bacterial strain CIPPMA0941 is CGMCC No.40238.
2. The metarhizium anisopliae of claim 1Metarhizium anisopliae) Use of the strain CIPPMa0941 for controlling pests.
3. The use according to claim 2, wherein the pest is solenopsis invicta, brontispa longissima or scarlet beetle.
4. The use according to claim 3, wherein the scarab beetle is a Holotrichia parallela.
5. A microbial inoculum for preventing and controlling pests, which is characterized by comprising the metarhizium anisopliae strain according to claim 1Metarhizium anisopliae) Bacterial strain CIPPMa0941.
6. A microbial agent for controlling pests according to claim 5, wherein said microbial agent comprises a chemical pesticide.
7. The pest-controlling microbial agent of claim 6, wherein the chemical pesticide is imidacloprid, indoxacarb, matrine, pyrethrin, osthole.
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