CN118435964B - A highly efficient method for using Metarhizium anisopliae Mr006 - Google Patents
A highly efficient method for using Metarhizium anisopliae Mr006 Download PDFInfo
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- CN118435964B CN118435964B CN202410540779.XA CN202410540779A CN118435964B CN 118435964 B CN118435964 B CN 118435964B CN 202410540779 A CN202410540779 A CN 202410540779A CN 118435964 B CN118435964 B CN 118435964B
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- chlorantraniliprole
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- 241000223250 Metarhizium anisopliae Species 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title abstract description 13
- PSOVNZZNOMJUBI-UHFFFAOYSA-N chlorantraniliprole Chemical compound CNC(=O)C1=CC(Cl)=CC(C)=C1NC(=O)C1=CC(Br)=NN1C1=NC=CC=C1Cl PSOVNZZNOMJUBI-UHFFFAOYSA-N 0.000 claims abstract description 76
- 239000005886 Chlorantraniliprole Substances 0.000 claims abstract description 75
- 241000256251 Spodoptera frugiperda Species 0.000 claims abstract description 44
- 239000000203 mixture Substances 0.000 claims description 9
- 241000223201 Metarhizium Species 0.000 claims description 8
- 239000002917 insecticide Substances 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000004563 wettable powder Substances 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims 1
- 239000004562 water dispersible granule Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 27
- 238000011282 treatment Methods 0.000 abstract description 25
- 241000607479 Yersinia pestis Species 0.000 abstract description 19
- 230000000749 insecticidal effect Effects 0.000 abstract description 16
- 239000000575 pesticide Substances 0.000 abstract description 14
- 230000002265 prevention Effects 0.000 abstract description 12
- 230000002195 synergetic effect Effects 0.000 abstract description 9
- 241000255777 Lepidoptera Species 0.000 abstract description 5
- 241000894006 Bacteria Species 0.000 abstract description 4
- 230000002045 lasting effect Effects 0.000 abstract description 2
- 210000004215 spore Anatomy 0.000 description 27
- 241000238631 Hexapoda Species 0.000 description 22
- 239000003814 drug Substances 0.000 description 18
- 238000012360 testing method Methods 0.000 description 15
- 239000003795 chemical substances by application Substances 0.000 description 12
- 239000007788 liquid Substances 0.000 description 12
- 206010059866 Drug resistance Diseases 0.000 description 8
- 241000233866 Fungi Species 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 238000004166 bioassay Methods 0.000 description 6
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 6
- 229920000053 polysorbate 80 Polymers 0.000 description 6
- 230000004763 spore germination Effects 0.000 description 6
- 231100000419 toxicity Toxicity 0.000 description 6
- 230000001988 toxicity Effects 0.000 description 6
- FTQWRYSLUYAIRQ-UHFFFAOYSA-N n-[(octadecanoylamino)methyl]octadecanamide Chemical compound CCCCCCCCCCCCCCCCCC(=O)NCNC(=O)CCCCCCCCCCCCCCCCC FTQWRYSLUYAIRQ-UHFFFAOYSA-N 0.000 description 5
- 240000008042 Zea mays Species 0.000 description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000000967 entomopathogenic effect Effects 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- 241000256259 Noctuidae Species 0.000 description 3
- 239000003905 agrochemical Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000007598 dipping method Methods 0.000 description 3
- 230000002147 killing effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000010413 mother solution Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000028070 sporulation Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000985245 Spodoptera litura Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000013043 chemical agent Substances 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000447 pesticide residue Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- PXBFMLJZNCDSMP-UHFFFAOYSA-N 2-Aminobenzamide Chemical class NC(=O)C1=CC=CC=C1N PXBFMLJZNCDSMP-UHFFFAOYSA-N 0.000 description 1
- OPPWTDFHAFPGOT-UHFFFAOYSA-N 5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(trifluoromethylsulfinyl)pyrazole-3-carboxamide Chemical compound NC1=C(S(=O)C(F)(F)F)C(C(=O)N)=NN1C1=C(Cl)C=C(C(F)(F)F)C=C1Cl OPPWTDFHAFPGOT-UHFFFAOYSA-N 0.000 description 1
- ONILAONOGQYBHW-UHFFFAOYSA-N 5-bromo-n-[2,4-dichloro-6-(methylcarbamoyl)phenyl]-2-(3,5-dichloropyridin-2-yl)pyrazole-3-carboxamide Chemical compound CNC(=O)C1=CC(Cl)=CC(Cl)=C1NC(=O)C1=CC(Br)=NN1C1=NC=C(Cl)C=C1Cl ONILAONOGQYBHW-UHFFFAOYSA-N 0.000 description 1
- 239000005889 Cyantraniliprole Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- 241001303988 Metarhizium rileyi Species 0.000 description 1
- 241001477931 Mythimna unipuncta Species 0.000 description 1
- 241000256247 Spodoptera exigua Species 0.000 description 1
- 101000693622 Spodoptera frugiperda Allatotropin Proteins 0.000 description 1
- 239000005937 Tebufenozide Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 210000004666 bacterial spore Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000443 biocontrol Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- DVBUIBGJRQBEDP-UHFFFAOYSA-N cyantraniliprole Chemical compound CNC(=O)C1=CC(C#N)=CC(C)=C1NC(=O)C1=CC(Br)=NN1C1=NC=CC=C1Cl DVBUIBGJRQBEDP-UHFFFAOYSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000000422 nocturnal effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 102000042094 ryanodine receptor (TC 1.A.3.1) family Human genes 0.000 description 1
- 108091052345 ryanodine receptor (TC 1.A.3.1) family Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- QYPNKSZPJQQLRK-UHFFFAOYSA-N tebufenozide Chemical compound C1=CC(CC)=CC=C1C(=O)NN(C(C)(C)C)C(=O)C1=CC(C)=CC(C)=C1 QYPNKSZPJQQLRK-UHFFFAOYSA-N 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
- A01N43/56—1,2-Diazoles; Hydrogenated 1,2-diazoles
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/04—Insecticides
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- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Chemical & Material Sciences (AREA)
- Insects & Arthropods (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the technical field of pesticides, in particular to a high-efficiency use method of Metarhizium anisopliae Mr006, which is applied to prevention and control of lepidoptera pests such as spodoptera frugiperda. The method has the advantages that the Metarrhizium anisopliae Mr006 and the chlorantraniliprole are mixed, on the basis of clear compatibility of the Metarrhizium anisopliae Mr006 and the chlorantraniliprole, the effects of preventing and controlling spodoptera frugiperda by bacteria before and after the bacteria treatment are found to be excellent, and the combination of the two has excellent synergistic effect, so that the purposes of improving the insecticidal activity of the Metarrhizium anisopliae Mr006 and reducing the dosage of the chlorantraniliprole are achieved, the effect of combining quick action and lasting effect is achieved, the thought is provided for pest resistance treatment, and the basis is provided for long-acting green prevention and control of the population quantity of the Magnonia.
Description
Technical Field
The invention relates to the technical field of pesticides, in particular to a high-efficiency use method of Metarhizium anisopliae Mr006, which is applied to prevention and control of lepidoptera pests such as spodoptera frugiperda.
Background
At present, the damage of pests to crops is one of the important factors causing agricultural yield reduction. The long-term and large-scale use of chemical pesticides brings a series of side effects of exceeding pesticide residues of agricultural products, rising the resistance level of the pests year by year, polluting the environment and the like while killing the pests. In view of this, biological control is increasingly being considered, and the use of entomopathogenic microorganisms for pest control is one of the important means for pest biological control. Compared with other insecticidal microorganisms, the insecticidal fungi have the advantages of being popular, friendly to the environment, not easy to generate drug resistance and the like, but have the defects of being easy to be influenced by environmental conditions, unstable in control effect and the like due to the fact that the control speed is low, and limit the application range and popularization and application of the related insecticidal fungi. In order to overcome the defects of both sides and simultaneously exert the respective advantages, the prevention and control effect is improved, the study on the compatibility of the fungus medicines is started in the 60 th century, and the biocontrol fungi are combined with the low-dose chemical agent, so that the use amount of the chemical pesticide can be reduced, the drug resistance of pests is delayed, the prevention and control duration is prolonged, and the stability of the pesticide is also enhanced.
Chlorantraniliprole is a high-efficiency, broad-spectrum and low-toxicity pesticide which is discovered and developed by DuPont in U.S. from anthranilamide compounds and acts on the insect ryanodine receptor. The strategy mainly comprising chemical control solves the emergency control problem of spodoptera frugiperda, ensures the grain safety and achieves remarkable effect. However, long-term chemical administration has led to spodoptera frugiperda having developed resistance to traditional pesticides, and the resistance of the Guangdong flourishing population to chlorantraniliprole has also reached a moderate level, and the use of chemical agents in large quantities has also caused a series of environmental ecological problems, which have forced the search for alternatives.
At present, spodoptera frugiperda is mainly controlled by chemical pesticides such as chlorantraniliprole and the like, and meanwhile, indoor control effects of biological pesticides and natural enemy insects are reported. In an open environment, spodoptera frugiperda overlaps generation, various insect states occur simultaneously, and once chemical pesticide loses efficacy, new insect bodies are needed to be repeatedly applied for many times, so that the prevention cost is increased, and meanwhile, the spodoptera frugiperda is easy to generate drug resistance to a prevention and treatment agent. The natural enemy insect prevention and control has long-acting performance, but spodoptera frugiperda has strong migration capability (100 km can fly every night), and is difficult to quickly and synchronously follow.
The Metarhizium anisopliae (Metarhizium rileyi) is an entomopathogenic fungus which is widely distributed in China, can infect various lepidoptera pests, and has extremely high infectivity on spodoptera litura, beet armyworm, soybean armyworm, prodenia litura and other noctuidae pests, and has higher development and utilization values. The Metarhizium anisopliae which takes the noctuidae pests as the target hosts is continuously present in the environment, and has the advantages of safety, sustainable prevention and control, difficult generation of drug resistance and the like.
Metarhizium anisopliae Mr006 is obtained by separating and identifying Spodoptera frugiperda stiff insects infected with entomogenous fungi in the field from investigation of the Taihe corn planting area by a biological control team of a plant protection and agricultural product quality safety institute of Anhui province. The Metarhizium anisopliae Mr006 is disclosed in CN202210572361.8, and is preserved in China general microbiological culture Collection center (China Committee) for 5-9 days, wherein the preservation address is North Star Xili No.1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.40171.
Specific sequencing results were as follows (540 bp):
GCTACCTGATTCGAGGTCACTCTTGGAGAAGTTTGTGCGTTTTACGGCAGTGGCCGCGCCGCGCTCCTGTTGCGAGGTTGTGCTACTACGCAGAGGAGGCCGCGACGGGGCCGCCAATTCATTTCGGGGGCGGCGCCGCAGGGAACCGCCTGAGCGGCCCGGCTGACAATCGCCGGCCCCCAACACCAAACCGCGGGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTCATTTTTGTATGATTCCACTCAGACGTGCCAAAGGCTAAGAGATACAGAGTTTCGGTCCCGCGGCGGGCGCCTGTTTCCGGGCGGGCTCTGGACGAGCCCGGTCCGGGGCAAATGACCCGCCGAGGCAACAGGAAAAGGGTATAAGTTCACATGGGGTTGGGAGTGA
The Metarhizium anisopliae Mr006 is an entomopathogenic fungus with strong pathogenicity to nocturnal moth larvae such as spodoptera frugiperda, and the like, high-strength insect epidemic diseases are often initiated in nature, and pests are not easy to generate resistance to the entomopathogenic fungus, but the Metarhizium anisopliae Mr006 has the defects of unstable control effect, narrow insecticidal spectrum, slow effect and the like in field use. In order to develop and popularize the Metarhizium anisopliae Mr006 and simultaneously delay the drug resistance of spodoptera frugiperda to chlorantraniliprole and reduce pesticide residues. Therefore, different combined application modes of the Metarhizium anisopliae Mr006 and the chlorantraniliprole are explored, and the optimal application sequence of the Metarhizium anisopliae Mr006 and the chlorantraniliprole for preventing and controlling spodoptera frugiperda is excavated, so that the prevention and control efficiency can be improved, the usage amount of the chlorantraniliprole is reduced, the generation of drug resistance of the spodoptera frugiperda is delayed, an important combined medicament is provided for continuously and effectively preventing and controlling the spodoptera frugiperda, and a new means is provided for green prevention and control of pests.
Disclosure of Invention
The invention aims to provide a high-efficiency use method of Metarhizium leigh Mr006, which uses Metarhizium leigh Mr006 and chlorantraniliprole for preventing and controlling lepidoptera pests, and comprises the steps of using Metarhizium leigh Mr006 and then using chlorantraniliprole, or using Metarhizium leigh Mr006 after using chlorantraniliprole, or using Metarhizium leigh Mr006 and chlorantraniliprole in a compounding way.
The second object of the invention is to provide an insecticidal composition, which is characterized in that the active ingredients consist of Metarhizium anisopliae Mr006 and chlorantraniliprole, wherein the concentration of the Metarhizium anisopliae Mr006 is 1-9 multiplied by 10 8 spore mL -1, and the mass ratio of the Metarhizium anisopliae Mr006 to the chlorantraniliprole is 1000:0.1-10. The mass ratio of the preferred Metarhizium anisopliae Mr006 to the chlorantraniliprole is 1000:0.1-5.
Preferably, the concentration of the Metarhizium anisopliae Mr006 is 1-9×10 8 spores mL -1, the concentration of the chlorantraniliprole is 1-5mg mL -1, and the volume ratio of the two is 10:1-1:10.
Further preferably, the chlorantraniliprole is 2.29 mg.mL -1, the Metarhizium anisopliae Mr006 is 1.84×10 8 spore.mL -1, and the volume ratio of the chlorantraniliprole to the Metarhizium anisopliae is 10:1-1:1.
The insecticidal composition also comprises agricultural auxiliary materials, and the insecticidal composition is in the form of suspending agent, wettable powder and water granules.
The insecticidal composition is used for controlling lepidoptera pests, namely spodoptera frugiperda.
Compared with the prior art, the invention has the following beneficial technical effects:
Experiments show that the Metarhizium anisopliae Mr006 obtained by separation in the laboratory has a good effect on spodoptera frugiperda, and the biological test result shows that the chlorantraniliprole also has high toxicity on spodoptera frugiperda. The method has the advantages that the Metarrhizium anisopliae Mr006 and the chlorantraniliprole are mixed, on the basis of clear compatibility of the Metarrhizium anisopliae Mr006 and the chlorantraniliprole, the effects of preventing and controlling spodoptera frugiperda by bacteria before and after the bacteria treatment are found to be excellent, and the combination of the two has excellent synergistic effect, so that the purposes of improving the insecticidal activity of the Metarrhizium anisopliae Mr006 and reducing the dosage of the chlorantraniliprole are achieved, the effect of combining quick action and lasting effect is achieved, the thought is provided for pest resistance treatment, and the basis is provided for long-acting green prevention and control of the population quantity of the Magnonia.
Detailed Description
All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention is further illustrated with reference to specific embodiments.
EXAMPLE 1 Metarhizium leigh Mr006 compatibility test with pesticide
1. Materials and methods
1. Insect source for test
Spodoptera frugiperda lines 2019 for indoor bioassays were collected from Huang Shanchun corn fields in Anhui province, laboratory populations fed indoors with artificial feed for long periods of time without any insecticide exposure during feeding. The feeding condition is 27+/-1 ℃, the photoperiod L is D=14:10 h, and the relative humidity is 75+/-10%. Selecting 3-year-old primary larvae with consistent bodies and healthy larvae for test.
2. Test agent
Tetrazolium tebufenozide, 90%, bayer corporation; tetrachlorantraniliprole 95%, shenyang chemical Co., ltd., cyantraniliprole 94%, shanghai Du Bangnong chemical Co., ltd., chlorantraniliprole 95.3%, fumei Co., U.S. Fu Mei, flurobendiamide 95%, bayer Co.
Preparing each raw medicament into mother solution with a certain concentration by using acetone, diluting the mother solution into 5-7 treatment solutions with serial concentration gradients by using 0.1% Tween 80 aqueous solution, and taking 0.1% Tween 80 aqueous solution without chemical medicament as a control, wherein the liquid medicine amount per concentration is not less than 50mL.
3. Determination of toxicity of bisamide insecticide to spodoptera frugiperda
The measurement was performed with reference to NY/T1154.6-2006, guidelines for bioassay tests in agricultural chemical chambers (section 6, insect dipping method). After the tested insects are immersed in the liquid medicine for 10 seconds, the excessive liquid medicine is sucked by filter paper, and the tested insects are transferred to normal conditions and are fed by fresh corn leaves. The total number of insects was 30 per treatment, and the treatment was repeated 3 times, with tween 80 aqueous solution without the agent as a control. The death of the test insects was investigated 24, 48 and 72 hours after the treatment, the total number of insects and the number of dead insects were recorded, and the death rate was counted.
4. Effect of Chlorantraniliprole on Mr006 spore germination
The spores of Metarhizium anisopliae Mr006 cultured for 14d on SMAY plates were washed off with sterile water containing 0.05% Tween-80, poured into a beaker, diluted to a certain concentration after being sufficiently stirred on a magnetic stirrer, and the concentration of the spores was checked under a microscope by a hemocytometer and adjusted to 1.0X10 8 spores/mL as a mother liquor. 20mL of SMY culture solution, 2mL of spore liquid mother solution and corresponding amounts of chlorantraniliprole stock solution are sequentially added into a 50mL centrifuge tube, the spore final concentration is 1.0X10 7 spores, and the chlorantraniliprole (24 h) final concentration is 33.57mg/L (LC 90)、2.29mg/L(LC50)、0.56mg/L(LC25). Binding the centrifuge tube with rubber band, placing into a shaking table, setting the temperature in the shaking table to be (28+ -1) deg.C, and rotating at 150r/min. After 18h of incubation, the spores were removed and observed under a microscope for germination. Every 3 replicates of treatment, every 100 spores were observed, the number of spores germinated was recorded, and the spore germination rate was calculated. The spore liquid without pesticide is used as a control.
5. Effect of Chlorantraniliprole on Mr006 hyphae growth
3 Drug-containing plates (90 mm in diameter) were prepared at 33.57mg/L (LC 90)、2.29mg/L(LC50)、0.56mg/L(LC25) by adding the corresponding amounts of the drug to SMAY media. mu.L of spore suspension at a concentration of 1.0X10 7 spores/mL was inoculated in the center of the medicated plate with a pipette. The petri dish was sealed and placed in an incubator at (27.+ -. 1 ℃ C.) and a relative humidity of 85% for cultivation. After 15d, the colonies were taken out and the colony diameters were measured by the crisscross method. Each treatment was repeated 3 times. The control was a plate without drug.
6. Influence of chlorantraniliprole on sporulation of Mr006
After the colony diameter measurement is completed, 30mL of sterile water containing 0.05% Tween-80 is poured into SMAY plates for 15d of culture, spores of the Metarhizium anisopliae Mr006 are scraped off by a glass coating rod, spore liquid is poured into a beaker, and after shaking and mixing uniformly on a magnetic stirrer, quantitative dilution is carried out, spore concentration is checked by a blood cell counting plate under a microscope, and the spore yield of each treatment is reduced.
7. Effect of sequence of use of Metarhizium anisopliae Mr006 and Chlorantraniliprole on synergistic action of Spodoptera frugiperda
Preparing liquid medicine concentration LC 50 dilution of chlorantraniliprole, wherein the concentration of Mr006 spore suspension is 1×10 7 spores/mL, and the concentration of chlorantraniliprole agent is 0.88mg/L (48 h). Two groups of tests are set, a soil-leaching method is adopted to measure the toxicity of spodoptera frugiperda, ① is firstly treated with Mr006 bacterial spore suspension (soaking for 10 s) at intervals of 12h, 24h, 36h and 48h, then chlorantraniliprole normal bioassay is carried out, ② is firstly treated with chlorantraniliprole LC 50 medicament (soaking for 10 s), then Mr006 is used at intervals of 12h, 24h, 36h and 48h, and then Mr006 is used for normal bioassay at intervals of 12h, 24h, 36h and 48 h.
8. Data statistics method
According to the toxicity measurement result, using a SAS 8.0 statistical analysis system, calculating LC 25、LC50 and LC 90 values of a toxicity regression line, and 95% confidence limit, b value and standard error (SD) of the values, and evaluating insecticidal activity on spodoptera frugiperda.
2. Results and analysis
1. Comparison of the contact killing Activity of bisamide Agents against Spodoptera frugiperda (Legensis method)
TABLE 1 contact killing Activity of bisamide type Agents against 3-instar Spodoptera frugiperda larvae (Leaching method)
Bioassays were performed using the insect dipping method, and after 24, 48, and 72 hours post-dose, the lethal medium concentration, 95% confidence interval, and virulence regression equations of 5 bisamide-based agents on spodoptera frugiperda 3-year larvae are shown in table 1. As can be seen from the table, the contact activity of the 5 medicaments on 3-instar larvae of spodoptera frugiperda is gradually enhanced along with the extension of the action time, the contact activity of chlorantraniliprole is highest after 24 hours, 48 hours and 72 hours after the medicaments, and the LC 50 values are 2.29, 0.88 and 0.74 mg.L -1 respectively. The contact activity of the fipronil amide is the lowest, and LC 50 of 24h, 48h and 72h after the medicine is 303.45, 179.78 and 114.92 mg.L -1 respectively. Therefore, the subsequent test selects chlorantraniliprole as a test agent, and performs a compatibility test and a combined toxicity test with the Metarhizium anisopliae Mr 006.
2. Effect of Chlorantraniliprole on Mr006 spore germination
TABLE 2 Effect of Chlorantraniliprole on the germination of Metarhizium anisopliae Mr006 spores
| Treatment of | Spore germination percentage (%) |
| CK | 83.90±2.89a |
| 0.56mg/L(LC25) | 87.32±1.53a |
| 2.29mg/L(LC50) | 88.80±2.35a |
| 33.57mg/L(LC90) | 90.76±2.16a |
As can be seen from the results in Table 2, the germination rate of the Mr006 spores of the Metarhizium anisopliae treated with 3 concentrations of chlorantraniliprole is higher than that of the control, wherein the germination rate of the Mr006 spores measured by LC 90 treatment is highest and is 90.76%, but no significant difference exists between the treatments.
3. Effect of Chlorantraniliprole on Mr006 hyphae growth
TABLE 3 Effect of Chlorantraniliprole on growth Rate of Metarhizium anisopliae Mr006
| Treatment of | Colony diameter (cm) |
| CK | 1.70±0.05a |
| 0.56mg/L(LC25) | 1.63±0.04a |
| 2.29mg/L(LC50) | 1.60±0.05a |
| 33.57mg/L(LC90) | 1.70±0.03a |
As can be seen from the results in Table 3, the colony diameters of Metarhizium anisopliae Mr006 treated with 3 concentrations of chlorantraniliprole were 1.63cm,1.60cm and 1.70cm, respectively, and there was no significant difference from the control.
4. Influence of chlorantraniliprole on sporulation of Mr006
TABLE 4 Effect of Chlorantraniliprole on the sporulation of Metarhizium anisopliae Mr006
As can be seen from the results in Table 4, the yield of the 3 concentrations of chlorantraniliprole-treated Metarhizium anisopliae Mr006 was 1.55X10 7/cm 2,1.54×107/cm 2 and 1.95X10 7/cm 2, respectively, which were not significantly different from the control.
5. Effect of sequence of use of Metarhizium anisopliae Mr006 and Chlorantraniliprole on synergistic action of Spodoptera frugiperda
The test results show that 3 types of chlorantraniliprole (raw material) with different concentrations have no influence on hypha growth, spore germination and spore yield of the Metarhizium anisopliae Mr006, so that the chlorantraniliprole (raw material) has good compatibility with the Metarhizium anisopliae Mr 006.
TABLE 5 toxicity of Metarhizium anisopliae Mr006 on Spodoptera frugiperda at various times after Chlorantraniliprole treatment
The results in Table 5 show that, after the spodoptera frugiperda larvae are treated by chlorantraniliprole for 12 hours, 24 hours, 36 hours and 48 hours respectively, the spodoptera frugiperda larvae are treated by the Metarhizium anisopliae Mr006, the LC 50 values are respectively 2.49 multiplied by 10 6、3.19×106、3.35×106、1.88×106 spores-mL -1, the LC 50 values of the contrast Metarhizium anisopliae Mr006 which is not treated by the chlorantraniliprole at the same time are respectively 1.58 multiplied by 10 8、1.92×108、1.49×108、1.51×108 spores-mL -1, the Metarhizium anisopliae Mr006 LC 50 values after the chlorantraniliprole treatment for 4 time periods are all obviously lower than that of the contrast, and the LC 50 value of the Metarhizium anisopliae Mr006 is reduced by nearly hundred times. From this, it can be seen that chlorantraniliprole shows excellent synergistic effect on Metarhizium anisopliae Mr 006.
TABLE 6 toxicity of Metarhizium anisopliae Mr006 on Spodoptera frugiperda with chlorantraniliprole at various times after treatment
The results in Table 6 show that, after the spodoptera frugiperda larvae are treated by the Metarhizium anisopliae Mr006 for 12 hours, 24 hours, 36 hours and 48 hours respectively, the chlorantraniliprole is treated by the Metarhizium anisopliae larvae, the LC 50 values are respectively 0.094, 0.045, 0.016 and 0.039 mg/mL -1, the LC 50 values of the control chlorantraniliprole which is not treated by the Metarhizium anisopliae Mr006 are respectively 0.957, 0.589, 0.248 and 0.646 mg/mL -1, the LC 50 values of the chlorantraniliprole after the Metarhizium anisopliae Mr006 treatment are respectively lower than that of the control, and the LC 50 value of the Metarhizium anisopliae Mr006 is reduced by more than 10 times. From this, the Metarhizium anisopliae Mr006 also shows excellent synergy to chlorantraniliprole.
It is concluded that the diamide pesticide is the main chemical pesticide for preventing and controlling noctuid pests, such as spodoptera frugiperda, etc. The chlorantraniliprole exhibits excellent insecticidal activity against spodoptera frugiperda control relative to the other several bisamide agents. And the growth of the mycelium of the Metarhizium anisopliae Mr006, the spore germination and the spore yield are not influenced, and the compatibility of the two is good.
The Metarhizium anisopliae Mr006 and the chlorantraniliprole are combined, the LC 50 value of the Metarhizium anisopliae Mr006 can be obviously reduced by more than hundred times by the pretreatment of the chlorantraniliprole, the LC 50 value of the chlorantraniliprole can also be obviously reduced by more than 10 times by the pretreatment of the Metarhizium anisopliae Mr006, the use sequence is not affected, and the two mutually show excellent synergistic effect. In addition, the LC 50 value of chlorantraniliprole can be obviously reduced by the prior treatment of the Metarrhizium anisopliae Mr006, and data support can be provided for the subsequent treatment of the Metarrhizium anisopliae Mr006 to solve the problem of drug resistance of spodoptera frugiperda. The above test results have not been reported in the literature.
Example 2 test of the Combined action of Metarhizium anisopliae Mr006 and Chlorantraniliprole on Spodoptera frugiperda
The test method is to measure the content of the agricultural chemical by referring to NY/T1154.6-2006, guidelines for biological assay in agricultural chemical room (part 6, insect dipping method). After the tested insects are immersed in the liquid medicine for 10 seconds, the excessive liquid medicine is sucked by filter paper, and the tested insects are transferred to normal conditions and are fed by fresh corn leaves. The total number of insects was 30 per treatment, and the treatment was repeated 3 times, with tween 80 aqueous solution without the agent as a control. And (5) investigating the death condition of the test insects 24 hours after treatment, recording the total number of insects and the death number, and counting the death rate.
Based on a single-dose LC50, a chlorantraniliprole liquid medicine and Mr006 bacterial liquid (the chlorantraniliprole is 2.29 mg.mL -1, the Metarrhizium anisopliae Mr006 is 1.84 multiplied by 10 8 spore.mL -1) are prepared, and the Mr006 bacterial liquid and the chlorantraniliprole are respectively compounded according to the volume ratio of 0:10, 1:9, 3:7, 1:1, 7:3, 9:1 and 10:0.
Calculation of combined virulence synergy:
Eth=I1×V1+I2×V2, wherein Eth is the theoretical control effect of the compound agent, namely the theoretical insecticidal activity of the compound agent on spodoptera frugiperda, I1 is the insecticidal activity of Mr006 on spodoptera frugiperda, V1 is the volume ratio of Mr006 in the mixture, I2 is the insecticidal activity of chlorantraniliprole on spodoptera frugiperda, and V2 is the volume ratio of chlorantraniliprole in the mixture
I R = Eab/Eth, wherein IR is the synergistic ratio, eab is the actual control effect of the compound, i.e. the insecticidal activity of the compound on spodoptera frugiperda.
And judging the synergistic effect of different formulas according to the IR value. IR >1 indicates synergistic effect, I R =1 indicates additive effect, and IR <1 indicates antagonistic effect.
The test results show that the Metarhizium anisopliae Mr006 and chlorantraniliprole have remarkable synergism on spodoptera frugiperda, the dosage of the active ingredients can be remarkably reduced, and the generation of drug resistance of pests is slowed down as shown in Table 7.
TABLE 7 Combined action of Metarhizium anisopliae Mr006 and Chlorantraniliprole on Spodoptera frugiperda
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting.
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