阅读说明：本技术 一种农药组合物及其在防治黄曲条跳甲方面的应用 (Pesticide composition and application thereof in aspect of preventing and treating phyllotreta striolata ) 是由 胡琼波 陈纬 袁文静 翁群芳 于 2021-02-03 设计创作，主要内容包括：本发明涉及一种含有金龟子绿僵菌与溴虫腈的农药组合物,还涉及所述农药组合物在防治黄曲条跳甲方面的应用,属于生物农药技术领域。本发明提供一种农药组合物,所述农药组合物的活性成分包括绿僵菌MaGX19S02的纯孢粉与和溴虫腈,所述绿僵菌MaGX19S02的纯孢粉与所述溴虫腈的质量比为1-80:1,优选质量比为15-25:1。该农药组合物能够显著减轻和控制黄曲条跳甲的发生和危害,延缓害虫抗性的产生,还能减少化学农药对土壤和环境的污染,改善土壤中微生物群。(The invention relates to a pesticide composition containing metarhizium anisopliae and chlorfenapyr, and also relates to application of the pesticide composition in the aspect of controlling phyllotreta striolata, belonging to the technical field of biological pesticides. The invention provides a pesticide composition, the active ingredients of the pesticide composition comprise pure sporopollen of metarhizium anisopliae MaGX19S02 and chlorfenapyr, the mass ratio of the pure sporopollen of the metarhizium anisopliae MaGX19S02 to the chlorfenapyr is 1-80:1, and the preferable mass ratio is 15-25: 1. The pesticide composition can obviously reduce and control the occurrence and harm of phyllotreta striolata, delay the generation of pest resistance, reduce the pollution of chemical pesticides to soil and environment and improve the microbial population in the soil.)

1. The pesticide composition is characterized in that active ingredients of the pesticide composition comprise pure sporopollen of Metarrhizium anisopliae MaGX19S02 and chlorfenapyr, and the mass ratio of the pure sporopollen of the Metarrhizium anisopliae MaGX19S02 to the chlorfenapyr is 1-80:1 or other preferable ratios.

2. The pesticide composition as set forth in claim 1, wherein the mass ratio of the pure sporopollen of the metarhizium anisopliae MaGX19S02 to the chlorfenapyr is 15-25: 1.

3. The pesticidal composition according to claim 1 or 2, which is in the form of at least one of a wettable powder, a granule, a dispersible granule, an oil suspension, a dispersible oil suspension, a bait and an ultra-low volume agent.

4. The pesticidal composition according to claim 3, which is in the form of a wettable powder.

5. A wettable powder which comprises the pure sporopollen of the metarhizium anisopliae MaGX19S02 as the active ingredient of claim 1, chlorfenapyr and an auxiliary agent.

6. The wettable powder of claim 5 wherein the adjuvants include fillers, wetting agents, dispersants, stabilizers and UV protectants.

7. The wettable powder of claim 6, which comprises the following components in percentage by mass: 15-40% of green muscardine fungus MaGX19S02 pure spore powder, 1-2% of chlorfenapyr, 1-2% of wetting agent, 12-13% of dispersing agent, 1-2% of stabilizing agent, 0.5-1.5% of ultraviolet protective agent and the balance of filler.

8. The wettable powder of claim 7, which comprises the following components in percentage by mass: 19% of green muscardine fungus MaGX19S02 pure spore powder, 1% of chlorfenapyr, 1.4% of wetting agent, 12.6% of dispersing agent, 2% of stabilizing agent, 1% of ultraviolet protective agent and 63% of filling material.

9. A wettable powder according to any one of claims 6 to 8 wherein the filler is attapulgite; the wetting agent is sodium butylnaphthalenesulfonate; the dispersant is Morwet D-425; the stabilizer is xanthan gum; the ultraviolet protective agent is a fluorescent whitening agent 33.

10. Use of a pesticidal composition according to any one of claims 1 to 4 or a wettable powder according to any one of claims 5 to 9 for controlling the flea beetle, a crucifer crop.

Technical Field

The invention relates to a pesticide composition containing metarhizium anisopliae and chlorfenapyr, and also relates to application of the pesticide composition in the aspect of controlling phyllotreta striolata, belonging to the technical field of pesticides.

Background

Phyllotreta striolata (Fabricius) is a Coleoptera (Coleoptera) phyllotaceae (Chrysometliade) pest, and mainly harms cruciferous vegetables such as cabbage heart, Chinese cabbage, radish and the like. The phyllotreta striolata has strong fertility and complex life habit, eggs, larvae and pupae live in soil, adults live on the ground, and the phyllotreta striolata has strong migration capacity; the larvae and adults can be harmful crops, the adults can eat the whole plant when heavy, and the adults can cause leaf holes to influence the appearance of the vegetables; the larva eats the root of the plant and can cause the death of the whole plant when the harm is serious; meanwhile, the imagoes and the larvae can leave wounds to promote soft rot during feeding.

The phyllotreta striolata has strong stress resistance and high drug resistance, is difficult to prevent and treat, and lacks of efficient medicaments and single effective prevention and treatment means. In order to control the outbreak hazard, chemical pesticides are often used excessively, so that the problems of increased drug resistance, reduced natural enemies, environmental pollution, over-standard pesticide residue of agricultural products and the like are caused. Therefore, the development and improvement of the biological control technology level are urgent needs for controlling the hazards of phyllotreta striolata.

In recent years, the biological control of phyllotreta striolata has achieved some research results, and some fungi and bacteria are found to be effective on phyllotreta striolata, such as isariafumososea, Metarhizium anisopliae, Bacillus thuringiensis and Serratia marcescens, which can be infected by microorganisms. Patent application No. CN201510084020.6 discloses an application of a metarhizium anisopliae CQMa421 preparation in preventing and treating phyllotreta striolata; the strain Malus sieboldii Ma6 (Fujian agricultural science, 2017, (2): 189-. Patent application number CN2013104943. X discloses a serratia marcescens (with preservation number CGMCC NO.7948) and application thereof, patent application number CN201611253650.2 discloses a bacillus thuringiensis (IPPBiotCo1085) and application thereof, and patent application number CN201611000946.3 discloses a bacillus thuringiensis (with preservation number CCTCC NO: M2015608) and preparation and application thereof; application (patent) No. CN201610276234.8 discloses a microbial pesticide containing Bacillus subtilis and a preparation method thereof. However, microbial insecticides still have many problems in flea beetle control application in general, one is too high cost, for example, the agent cost is about 150 yuan/mu when the agent is applied 5kg per mu for soil treatment by using metarhizium anisopliae granules; secondly, the effect is not ideal, the control effect on the imagoes is low and the effect is slow; and thirdly, the number of the selected microbial insecticide products is small, and the application range is limited.

Based on the above, the inventor considers that the quick-acting property and the low cost of the chemical agent are both considered, and the environment-friendly property and the durability of the fungus insecticide are utilized to research and develop the yellow flea beetle control agent compounded by fungus and chemical pesticide, so that better economic, ecological and social benefits can be obtained.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a pesticide composition compounded by biocontrol fungi and a chemical agent, the pesticide composition has an obvious effect on preventing and treating phyllotreta striolata, the quick-acting property and the low cost of the chemical agent are both considered, the environment friendliness and the durability of the fungal pesticide are utilized, and the pesticide composition has good economic, ecological and social benefits.

The invention also aims to provide a wettable powder, and the active ingredients of the wettable powder comprise pure sporopollen of the metarhizium anisopliae MaGX19S02 and chlorfenapyr.

The invention also aims to provide application of the pesticide composition or the wettable powder in preventing and treating the flea beetle hair of cruciferous crops.

In order to achieve the purpose, the invention adopts the technical scheme that: the active ingredients of the pesticide composition comprise pure sporopollen of Metarrhizium anisopliae MaGX19S02 and chlorfenapyr, and the mass ratio of the pure sporopollen of the Metarrhizium anisopliae MaGX19S02 strain to the chlorfenapyr is 1-80:1 or other preferable ratios.

Preferably, the mass ratio of the pure spore powder of the metarhizium anisopliae MaGX19S02 to the chlorfenapyr is 15-25: 1.

The active ingredients of the pesticide composition of the invention, namely the chlorfenapyr and the metarhizium anisopliae MaGX19S02, have higher activity on phyllotreta striolata and the chlorfenapyr has no influence on hypha growth and spore germination of the metarhizium anisopliae MaGX19S 02. When the pure spore powder of the metarhizium anisopliae MaGX19S02 strain is mixed with the chlorfenapyr in a mass ratio of 1-80:1, the control effect on phyllotreta striolata is obviously higher than that of a single agent, and particularly, when the mass ratio of the two is 19.8:1, the control effect reaches 89.33%.

Preferably, the pesticide composition provided by the invention is at least one of wettable powder, granules, dispersible granules, oil suspending agents, dispersible oil suspending agents, baits and ultra-low volume agents.

More preferably, the pesticide composition is in the form of wettable powder.

In another aspect, the invention provides a wettable powder, which comprises the pure spore powder of the active ingredients of the metarhizium anisopliae MaGX19S02 and chlorfenapyr, and also comprises an auxiliary agent.

Preferably, the auxiliaries include fillers, wetting agents, dispersants, stabilizers and uv-protection agents.

Preferably, the wettable powder provided by the invention comprises the following components in percentage by mass: 15-40% of green muscardine fungus MaGX19S02 pure spore powder, 1-2% of chlorfenapyr, 1-2% of wetting agent, 12-13% of dispersing agent, 1-2% of stabilizing agent, 0.5-1.5% of ultraviolet protective agent and the balance of filler.

Preferably, the wettable powder provided by the invention comprises the following components in percentage by mass: 19% of green muscardine fungus MaGX19S02 pure spore powder, 1% of chlorfenapyr, 1.4% of wetting agent, 12.6% of dispersing agent, 2% of stabilizing agent, 1% of ultraviolet protective agent and 63% of filling material.

Preferably, the filler is attapulgite; the wetting agent is sodium butylnaphthalenesulfonate; the dispersant is Morwet D-425; the stabilizer is xanthan gum; the ultraviolet protective agent is a fluorescent whitening agent 33.

In another aspect, the invention provides an application of the pesticide composition or the wettable powder in preventing and treating striped flea beetle hair of cruciferous crops.

Compared with the prior art, the invention has the beneficial effects that:

(1) the pesticide composition provided by the invention is prepared by compounding biocontrol fungi (the pure spore powder of Metarhizium anisopliae MaGX19S 02) and a chemical agent (chlorfenapyr) in a proper proportion, so that the occurrence and harm of phyllotreta striolata can be remarkably reduced and controlled, the generation of pest resistance can be delayed, the pollution of chemical pesticides to soil and environment can be reduced, and the microbial population in the soil can be improved;

(2) the pesticide composition provided by the invention not only gives consideration to the quick-acting property and low cost of chemical agents, but also utilizes the environmental friendliness and durability of the fungal pesticide, is used for preventing and treating phyllotreta striolata, and can obtain better economic, ecological and social benefits.

Drawings

FIG. 1 shows the growth conditions of cabbage heart at 1d, 7d and 14d after spraying 20% metarhizium MaGX19S 02-chlorfenapyr wettable powder 500 times liquid and chlorfenapyr 50 mg/L; wherein, fig. 1a1, fig. 1a2 and fig. 1A3 respectively show the growth conditions of the 1d, 7d and 14d flowering cabbage after the 20% metarhizium MaGX19S 02-chlorfenapyr wettable powder is treated by 500 times of liquid, and fig. 1B1, fig. 1B2 and fig. 1B3 respectively show the growth conditions of the 1d, 7d and 14d flowering cabbage after the chlorfenapyr is treated by 50 mg/L; FIG. 1B1, FIG. 1B2 and FIG. 1B3 show the growth of the 1d, 7d and 14d flowering cabbage after the flowering cabbage is treated with water.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed drawings and examples.

Example 1: screening of active ingredients of the pesticide composition of the invention

(1) Activity measurement of fungus strain on phyllotreta striolata (screening of strain)

The experimental method comprises the following steps: the purple lilac fungus Purpureocillium lilacinum (strains PlSC36C8, PlSC36C1, PlSC41A2, PlSC63C7, PlSC59A3, PlSC57B4, PlSC42B5, PlSC37C2, PlSC55C1), Metarhizium anisopliae (strain MaGX19S 02) and Isaria javanica (strain IjH6102) were selected and subjected to the experiment. Inoculating slant strain to PDA culture medium, culturing at 25 deg.C for 10 days, scraping bacterial colony surface conidia, and preparing into 1.0 × 10 with 0.1% Tween-80 solution⁸spores/mL spore suspension was ready for use. With CO₂Subjecting Aspergillus flavus to stripAnaesthetizing flea beetles, transferring into a 1.5mL centrifuge tube, adding 1mL of the stock solution, quickly covering, turning upside down for 50 times, transferring into a disposable plastic bowl paved with filter paper, and feeding in a climatic chamber with 26 ℃, a photoperiod of 14L:10D and a relative humidity of 95 +/-5%. The soaked phyllotreta striolata is raised by radish, the phyllotreta striolata is replaced every two days, and the death number of the test insects is recorded at the same time. Treatments with 0.1% tween-80 solution were used as a control, repeated three times for each 10 beetles treated. The pathogenicity of the strain to phyllotreta striolata was analyzed by calculation using SPSS25 software, and the results are shown in Table 1.

TABLE 1 pathogenicity of different strains on Phyllotreta striolata adults

Data are mean ± sem of 3 replicates, with 10 heads of striped flea beetle adults treated, and the same column of data followed by an identical lower case letter, indicating no significant difference (Duncan's method, P > 0.05). The same applies below.

The experimental results are as follows: from the results in table 1, it is found that the purple lilac fungus PlSC59a3 strain is most pathogenic to phyllotreta striolata, and the corrected mortality rate of the treated 9d is 96.30%; the corrected mortality rate of the Metarrhizium anisopliae treated by the MaGX19S02 strain for 9 days after the phyllotreta striolata is 80.00%; corrected mortality after 9d treatment with Isaria javanicus IjH6102 strain was 68.33%.

(2) Toxicity determination of chemical agent to phyllotreta striolata (screening of chemical agent)

The experimental method comprises the following steps: 10 chemicals (acetamiprid, tolfenpyrad, chlorfenapyr, thiamethoxam, diafenthiuron, pyridaben, dinotefuran, clothianidin, emamectin benzoate and rotenone) were formulated with 0.1% tween-80 solution to 10mg/L and 100mg/L concentration for use. With CO₂Anaesthetizing phyllotreta striolata, transferring into a 1.5mL centrifuge tube, adding 1mL of the stock solution, quickly covering, turning upside down for 50 times, transferring into a disposable plastic bowl paved with filter paper, and feeding in a climatic chamber with 26 ℃, 14L of photoperiod, 10D of photoperiod and 95 +/-5% of relative humidity. Feeding soaked Phyllotreta striolata with radix Raphani every dayThe test insects are replaced once, and the death number of the test insects is recorded. Treatments with 0.1% tween-80 solution were used as a control, repeated three times for each 10 beetles treated. The toxicity of the chemical agent to phyllotreta striolata was analyzed by calculation using SPSS25 software, and the results are shown in table 2.

TABLE 2 Activity of chemical Agents on Phyllotreta striolata adults

The experimental results are as follows: the results in table 2 show that most of the 10 chemical agents have certain toxicity to phyllotreta striolata, wherein the 100mg/L bromothalonil solution has good toxicity to phyllotreta striolata, and the lethality reaches 100% after 48 hours.

(3) Effect of chemical Agents on fungal growth

Influence of chemical agents on hypha growth of three fungi (lilac violet spore bacteria PlSC59A3, Metarrhizium anisopliae MaGX19S02 and Isaria javanicus IjH6102)

The experimental method comprises the following steps: the effect of 10 chemicals on the growth of three fungal hyphae was tested by adding each chemical to autoclaved PDA medium (potato 200g, glucose 20g, agar 10-15g, water 1000mL), preparing 10mg/L and 100mg/L drug-containing media plates, inserting the cake cultured for 2 days with a 5mm punch, repeating 3 times, placing in an incubator for 7 days and 14 days, measuring the growth diameter of the colony (average value in both vertical and horizontal directions), comparing with no chemical, and the results are shown in Table 3, Table 4 and Table 5.

TABLE 3 Effect of chemical Agents on hyphal growth of the Strain of purple lilac PlSC59A3

TABLE 4 Effect of chemical Agents on the hyphal growth of Metarrhizium anisopliae MaGX19S02 Strain

TABLE 5 Effect of Chemicals on hyphal growth of Isaria javanicus IjH6102 strain

The experimental results are as follows: from the results shown in tables 3 to 5, it was found that tolfenpyrad had an adverse effect on the growth of hyphae of all 3 test strains (violonyea lilac PlSC59a3, metarhizium anisopliae MaGX19S02 and clavulans IjH6102), diafenthiuron and pyridaben had an adverse effect on the growth of hyphae of violonyea lilac, and other agents had no significant effect on the growth of hyphae of all 3 test strains.

② the influence of chemical agents on the spore germination of three fungi (purple lilac spore bacteria PlSC59A3, Metarrhizium anisopliae MaGX19S02 and Isaria javanicus IjH6102)

The experimental method comprises the following steps: testing the influence of each chemical agent on the germination of three fungal spores, adding the chemical agent and fungal spore powder into 100mL sterilized nutrient solution (2% glucose, 1% peptone) to prepare chemical agent with final concentration of 10mg/L and 100mg/L and bacterial solution of 1 × 10⁶Culturing in a shaker at 25+ -1 deg.C for 12h at 170r/min for 3 times, counting spores with a blood counting plate, recording the number of germinated and ungerminated spores, and calculating the spore germination rate by using the culture solution without chemical reagent as a control, the results are shown in Table 6.

TABLE 6 Effect of chemical Agents on spore germination of 3 fungal strains

The experimental results are as follows: from the results in Table 6, it is understood that the spore germination rates of Isaria javanicus and Viola lilacina strains were greatly affected by 10 chemical agents, while the influence of Metarrhizium anisopliae was small.

From the results of the experiment in example 1, it was found that the strain metarhizium anisopliae MaGX19S02 and chlorfenapyr were selected as the active ingredients of the pesticidal composition of the present invention.

Example 2: metarrhizium anisopliae MaGX19S02 strain conidium production and quality detection

Conidium production: taking a preserved slant strain, scraping a small amount of conidia, inoculating the conidia onto a PDA plate, culturing for 7 days in an incubator at the temperature of 27 +/-1 ℃, inoculating the conidia into a PD seed culture medium for culturing, inoculating a seed solution into bagged rice for solid fermentation, collecting conidia powder by using a spore separator, freeze-drying, and packaging for later use after quality detection.

And (3) water content determination: drying the cleaned weighing bottle in a drying oven at 120 ℃ for 10min, cooling and weighing to obtain the weight m1 of the weighing bottle; weighing about 1g of spore powder in a weighing bottle, weighing the total weight m2 (accurate to 0.0001g), drying in a drying oven at 120 ℃ for 2h, covering a bottle cap, and weighing as m 3. The procedure was repeated 3 times, and the water content of spores was calculated and averaged, and the results are shown in Table 7. It is known that pure sporopouenin has a water content of 2.15%.

Water content (%) - (m2-m3)/(m2-m1) × 100

And (3) measuring the spore content: accurately weighing 10mg of dried spore powder in a ten-thousandth balance, dissolving the spore powder in a centrifuge tube uniformly by using 1mL of 0.1% Tween-80 solution, fully and uniformly mixing the spore powder on a vortex mixer, diluting the spore suspension by 100 times, measuring the spore concentration by using a blood counting plate, repeating the steps for 3 times, taking the average value of the values, calculating the spore content of the spore powder, and obtaining the result shown in Table 7. As can be seen, the pure sporopollen contained 2.36X 10 of spores¹⁰Conidia/g.

And (3) measuring the spore germination rate: 10mg of the collected spore powder is taken and put into a 1mL sterilized centrifuge tube, and 1mL of autoclaved 0.05 percent Tween-80 solution is taken to be dissolved and mixed evenly. Placing the spore suspension in a 250mL triangular flask containing 100mL sterilized germination solution (2% glucose, 1% peptone), placing in a shaker at 25+ -1 deg.C for 72h at 170 r/min; the number of germinated and ungerminated spores (spores are considered to germinate when the expansion of the spores is 1 time larger than that of original spores or the length of the buds is larger than the radius of the spores) is recorded under a microscope, the germination rate of the spores is calculated by 3 times, and the result is shown in table 7, and the germination rate of pure spore powder of the metarhizium anisopliae strain MaGX19S02 is 93.69%.

Spore germination rate (%) ═ number of germinated spores/total number of spores × 100

TABLE 7 quality test results of pure spore powder of Metarrhizium anisopliae MaGX19S02 strain

Example 3: screening the effective components of Metarrhizium anisopliae MaGX19S02 and chlorfenapyr

Experimental materials: chlorfenapyr was prepared into 10mg/L, 20mg/L, 40mg/L, 60mg/L, 80mg/L and 100mg/L solutions using 0.1% Tween-80 solution, and the pure powder of Metarrhizium anisopliae prepared in example 2 was prepared into 1X 10⁹、1×10⁸、1×10⁷、1×10⁶、1×10⁵spore suspension of spores/mL is ready for use.

(1) Toxicity determination of metarhizium anisopliae MaGX19S02 and chlorfenapyr on phyllotreta striolata

The experimental method comprises the following steps: testing LC of chlorfenapyr and metarhizium anisopliae pure spore powder on phyllotreta striolata by using an immersion method₅₀By CO₂Anaesthetizing phyllotreta striolata, transferring into a 1.5mL centrifuge tube, adding 1mL of the stock solution, quickly covering, turning upside down for 50 times, transferring into a disposable plastic bowl paved with filter paper, and feeding in a climatic chamber with 26 ℃, 14L of photoperiod, 10D of photoperiod and 95 +/-5% of relative humidity. The soaked phyllotreta striolata is raised by radish, the phyllotreta striolata is replaced every two days, and the death number of the test insects is recorded at the same time. Treatments with 0.1% tween-80 solution were used as a control, repeated three times for each 10 beetles treated. Mortality was calculated using SPSS25 software and the results are shown in tables 8 and 9.

TABLE 8 toxicity of Chlorfenapyr at various concentrations against Phyllotreta striolata adults

TABLE 9 pathogenicity of Metarrhizium anisopliae MaGX19S02 to Phyllotreta striolata adults at different concentrations

The experimental results are as follows: the results in tables 8 and 9 show that when the chlorfenapyr concentration is higher than 60mg/L, the toxicity to phyllotreta striolata adults is good, and after the treatment for 36 hours, the mortality rate reaches 100%; suspension of Metarrhizium anisopliae MaGX19S02 spore at 1X 10⁸And 1X 10⁹The phyllotreta striolata imagoes treated by spores/mL has good pathogenicity to the phyllotreta striolata, and the fatality rate reaches more than 60% on the ninth day. The SPSS software analyzes to obtain that the chlorfenapyr is used for treating the phyllotreta striolata adults LC when the phyllotreta striolata adults are treated for 2 days₅₀52.08mg/L, the metarhizium anisopliae MaGX19S02 treats the striped flea beetle adults with LC when the striped flea beetle adults are treated for 9 days₅₀Is 3.37 multiplied by 10⁷spores/mL。

(2) Activity determination of mixing metarhizium anisopliae MaGX19S02 and chlorfenapyr on phyllotreta striolata

The experimental method comprises the following steps: compounding Metarrhizium anisopliae 1X 10⁷Spore suspension of spore/mL and chlorfenapyr 50mg/L solution are mixed according to a certain volume ratio to obtain 10mL of mixed liquid medicine, and the striped flea beetle imagoes are treated according to the method. The results are shown in Table 10.

TABLE 10 Activity of Metarrhizium anisopliae and Chlorfenapyr in combination on Phyllotreta striolata

The experimental results are as follows: from the results in table 10, at 7d after the treatment, when the conidia of the metarhizium anisopliae and the chlorfenapyr are mixed in different proportions, the lethality rate to the phyllotreta striolata is remarkably higher than that of the blank Control (CK) and the single treatment, particularly when the pure sporopollen and the chlorfenapyr are mixed in a ratio of 19.8:1(m/m), and the lethality rate to the phyllotreta striolata is highest and reaches 89.33%.

Example 4: formula screening and processing of green muscardine fungus pure spore powder and chlorfenapyr mixed wettable powder

(1) Screening of fillers

Influence of the filler on the germination of metarhizium anisopliae spores: uniformly mixing a filler and active ingredients (metarhizium anisopliae pure spore powder and chlorfenapyr) according to a proportion of 1/1, placing the mixture in an incubator at a temperature of 25+1 ℃ for 14 days, then taking 10mg of the mixture, adding sterile 1mL of 0.05 percent Tween-80 solution, shaking and uniformly mixing the mixture, placing the mixture into 100mL of sterilized nutrient solution (2 percent glucose and 1 percent peptone), placing the mixture in a shaking table at a temperature of 25+1 ℃ for 170r/min, performing microscopic examination after 72 hours, observing the germination rate of spores, repeating the steps for 3 times, and calculating the average number, wherein the results are shown in Table 11.

Effect of fillers on the growth of Metarhizium anisopliae hyphae: each filler was added to PDA medium (potato 200g, glucose 20g, agar 10-15g, water 1000mL) to a final concentration of 0.03g/mL, autoclaved, poured into a petri dish, coagulated, inoculated with a cake of 2 days old by a 5mm punch, repeated 3 times, placed in an incubator for 7 and 14 days, and the colony growth diameter (average value in both vertical and horizontal directions) was measured to control without filler, and the results are shown in Table 11.

Screening the proportion of the filler: the filler/active ingredient/sodium dodecyl benzene sulfonate is evenly mixed according to the following mass fractions of 54/40/6, 64/30/6 and 74/20/6, 1g of sample is accurately weighed, the sample is evenly poured onto the liquid surface of a 250mL beaker filled with 100mL of water from the flush position of the opening of the beaker, the time is counted until the sample is completely wetted, the wetting time is recorded, the operation is repeated for 3 times, and the average value is calculated. The control was made without the wetting agent sodium dodecylbenzene sulfonate and the results are shown in Table 12.

TABLE 11 Effect of fillers on the growth and spore germination of Metarrhizium anisopliae MaGX19S02 Strain

TABLE 12 wetting times for different filler contents

The experimental results are as follows: from the results in tables 11 and 12, it is known that both attapulgite and kaolin have no significant inhibitory effect on spore germination and hypha growth of metarhizium anisopliae, but the wetting time is shorter when the filler is attapulgite; therefore, the filler in the wettable powder of the present invention is preferably attapulgite.

(2) Wetting agent screening

The experimental method comprises the following steps: the various humectants tested were added at a rate of 6% to a sample containing 20% active ingredient, with the remainder of the filler being replenished. Taking 1g of the uniformly mixed sample, adding the uniformly mixed sample into 100mL of sterilized nutrient solution (2% glucose, 1% peptone), culturing for 72h in a shaking table at 25+1 ℃ and 170r/min, repeating for 3 times, using a blood counting plate, recording the number of germinated spores and ungerminated spores, and calculating the spore germination rate by taking the sample without the wetting agent as a control, wherein the result is shown in Table 12.

Each wetting agent was added to a 20% sample of spore powder at a ratio of 6%, the remainder was supplemented with filler and mixed thoroughly. Weighing 1g of sample by adopting a ten-thousandth balance, weighing 100mL of tap water, pouring the tap water into a 250mL beaker, placing the beaker into a constant-temperature water bath kettle at 25+1 ℃, weighing a proper amount of wettable powder sample after the temperature is constant, pouring the sample on the liquid surface of the beaker uniformly at one time from the position flush with the opening of the beaker, keeping the liquid surface undisturbed, simultaneously timing by using a stopwatch immediately until the sample is completely wetted, recording the wetting time, repeating the steps for 5 times, taking the average value of the values as the wetting time of the sample, taking the sample without adding a wetting agent as a reference, measuring the wetting time, comparing the wetting property, and finding the result in a table 13.

TABLE 13 wetting time of wetting agent and its effect on germination rate of conidia

The experimental results are as follows: the experimental results in table 13 show that the wetting agents have no significant difference in germination of metarhizium anisopliae spores, and the sodium butylnaphthalenesulfonate has the best wettability; therefore, the wetting agent in the wettable powder of the invention is preferably sodium butylnaphthalenesulfonate.

(3) Screening of dispersants

The experimental method comprises the following steps: each dispersant was added at a rate of 6% to a sample containing 6% sodium butylnaphthalenesulfonate and 20% active ingredient, with the remainder being supplemented by filler. 1g of the uniformly mixed sample is taken and added into 100mL of sterilized nutrient solution (2% glucose, 1% peptone), cultured for 72h in a shaking table at the temperature of 25+1 ℃ and at the speed of 170r/min, repeated for 3 times, a blood counting plate is used, the number of germinated spores and ungerminated spores is recorded, the sample without the dispersant is used as a control, and the spore germination rate is calculated, and the result is shown in Table 14.

Adding a dispersing agent into a sample containing 6% of sodium butylnaphthalenesulfonate and 20% of active ingredients according to the proportion of 6%, supplementing the rest part with a filler, and fully and uniformly mixing to prepare a sample to be detected for later use. Weighing 500mg of sample, putting the sample in a measuring cylinder containing 250mL of tap water at 30+1 ℃, slightly shaking to disperse the sample, then covering a plug, turning the sample upside down for about 30 times within 1min, opening the plug, standing the sample in a water bath at normal temperature for 30min, sucking out the solution from a 225mL (9/10) position in a straw within 10-15s, in the operation process, not shaking or stirring sediment in the measuring cylinder, ensuring that the top end of the straw is always below the liquid level by a few millimeters, wherein the remainder in the measuring cylinder is a lower suspension, pouring the lower suspension into a 100mL beaker which is dried and weighed, washing the measuring cylinder with a small amount of distilled water, pouring the washing solution into the beaker, putting the beaker with the lower suspension into a 100C oven, drying the beaker to constant weight, taking out and cooling the beaker to room temperature for weighing, repeating the weighing for 3 times, and calculating the suspension rate, wherein the result is shown in Table 13.

Suspension rate (%) ((m1-m2)/m1) × (10/9) × 100

m 1: preparing the mass/g of the effective components in the sample of the suspension;

m 2: the mass of the active ingredient per g was retained in 25mL of the suspension remaining at the bottom of the cylinder.

TABLE 14 suspension percentage of dispersants and their effect on germination rate of conidia

The experimental results are as follows: as can be seen from the results in Table 14, the dispersion of Morwet D-425 was the best without any significant difference in spore germination of Metarrhizium anisopliae; therefore, the dispersant in the wettable powder of the invention is preferably Morwet D-425.

(4) Determination of the proportion of wetting agent and dispersant

The experimental method comprises the following steps: the auxiliary agent with better suspension property and the auxiliary agent with higher wettability which are screened from the auxiliary agents are uniformly mixed according to the proportion of 1/9, 3/7, 5/5, 7/3 and 9/1, the sample lg is taken, the suspension rate and the wetting time are respectively measured, and the optimal proportion of the compound auxiliary agent is screened, and the result is shown in table 15.

TABLE 15 Effect of wetting agent to dispersant ratio on suspension percentage and wetting time

The experimental results are as follows: from the results in Table 15, it is understood that the suspension percentage and the wettability were good when the wetting agent and the dispersant ratio was 1/9. Therefore, the wetting agent/dispersing agent (sodium butylnaphthalenesulfonate/Morewet D-425) ratio in the wettable powder of the present invention is preferably 1/9.

(5) Determination of content of wetting agent and dispersant

The experimental method comprises the following steps: the dispersing agent and the wetting agent are mixed according to the optimal ratio, the ratio of the wetting agent/the dispersing agent is 4%, 6%, 8%, 10%, 12%, 14% and 16%, 20% of active ingredients, and the rest part of the filler is filled into 1g of mixed preparation, and suspension rate and wetting time determination experiments are respectively carried out. The mixed formulation with better suspension and wetting time was selected as the content of the final aid and the results are shown in table 16.

TABLE 16 Effect of wetting agent + dispersant dosage on suspension percentage and wetting time

And (3) test results: from the results in table 16, it is understood that when the wetting agent + dispersing agent use ratio is 14%, the wettability and dispersibility are good; therefore, the total content of the wetting agent and the dispersing agent in the wettable powder of the invention is preferably 14%.

(6) Determination of the stabilizer

The experimental method comprises the following steps: the test stabilizer 2.0% was added to the wetting agent and dispersant (1/9 for wetting agent/dispersant, 14% for total) respectively, the filler was made up to 100%, after 7 days of storage at room temperature, the preparation without stabilizer was repeated 3 times for each treatment with the control, and the germination and suspension rates of the spores were determined, the results are shown in table 17.

The optimum proportion and dosage of the screened wetting agent and the dispersant are respectively added with 2.0 percent of the tested stabilizer into the preparation, the filler is used for complementing 100 percent, after the preparation is subjected to heat storage at 54 ℃ for 14 days, the preparation without the stabilizer is used as a control, and the treatment is repeated for 3 times. The change in spore germination and suspension rates was determined and the best stabilizer was selected for the results shown in Table 17.

The selected stabilizers are added into the initial preparation according to the optimal proportion and dosage of the selected wetting agents and the selected dispersants, the proportion of the selected stabilizers is 1.0%, 1.5%, 2.0%, 2.5% and 3.0%, the white carbon black filler is used for complementing 100% to prepare 1g of sample, the wetting time, the suspension rate and the change of fluidity of different preparations are comprehensively considered, the dosage of the selected stabilizers is selected, and the result is shown in table 18.

TABLE 17 Effect of stabilizers on germination and suspension rates of conidia

TABLE 18 Effect of xanthan gum dosage on suspension and wetting time

The experimental results are as follows: from the results in table 17, it is understood that xanthan gum is excellent in stability to the system after 14 days of storage at room temperature and 14 days of storage at heat. Therefore, the stabilizer in the wettable powder of the invention is preferably xanthan gum.

The results in table 18 show that the difference between the wetting time and the suspension rate is not significant, and the suspension rate is better when the xanthan gum is used in an amount of 2%, thus integrating the practical situation; therefore, the amount of xanthan gum used as a stabilizer in the wettable powder of the present invention is preferably 2%.

(7) Determination of UV protectant

Placing spore powder 10mg in autoclave sterilized centrifuge tube, shaking with 0.05% Tween water, and diluting to 10 × 10⁶conidia/mL conidia suspension, the conidia suspension is divided into 5 beakers by 30mL, the first four beakers are respectively added with 0.01g of each of fluorescein sodium, 1-hydroxybenzotriazole, fluorescent brightener 33 and 2- (3, 5-di-tert-amyl-2-hydroxyphenyl) benzotriazole, the 5 th beaker is used as a blank control, then shaking and shaking are carried out, 0.1mL of diluent is removed and coated on a PDA plate, and each treatment is set to be 3 times. All treated plates were placed in a 25+1C incubator for 2d and the spore germination rate was calculated for each plate and the results are shown in Table 19.

And (3) testing the protective effect of the ultraviolet protective agent on spore powder: mixing 0.5-1.5% of ultraviolet protective agent with the selected filler, wetting agent, dispersant and stabilizer to prepare wettable powder, respectively treating under the following four ultraviolet irradiation conditions (treatment 1: 254nm,10cm,30 min), (treatment 2: 254nm,10cm,1h), (treatment 3: 365nm,10cm,30min) and (treatment 4: 365nm,10cm,1h), culturing at 25+ -1 ℃ for 48h, observing spore germination rate, and calculating protective performance of the protective agent on spores. The reaction was repeated 3 times without any protective agent, and the results are shown in Table 20.

TABLE 19 influence of UV protectant on spore germination Rate

TABLE 20 protective Effect of UV protectant on conidia

The experimental results are as follows: from the results in table 19, it can be seen that none of the four uv protectants significantly inhibited the germination of metarhizium anisopliae spores.

As is clear from the results in table 20, the fluorescent whitening agent 33 has a good spore-protecting property, and therefore, the ultraviolet protectant for the wettable powder of the present invention is preferably the fluorescent whitening agent 33.

(8) Preparation of 20% metarhizium anisopliae MaGX19S02 conidium and chlorfenapyr wettable powder

Preparing 20% metarhizium anisopliae MaGX19S 02. chlorfenapyr wettable powder: according to the mass fraction, 63% of attapulgite, 19% of pure spore powder of a metarhizium anisopliae MaGX19S02 strain, 1% of chlorfenapyr, 1.4% of butyl naphthalene sodium sulfonate, 12.6% of Morwet D-425, 2% of xanthan gum and 1% of fluorescent whitening agent 33 are taken, the components in the formula are accurately weighed, and the mixture is put into a stirrer to be fully and uniformly stirred. The quality of the preparation is qualified according to the relevant pesticide preparation standard for standby.

Example 5: application of 20% metarhizium anisopliae MaGX19S02 conidium and chlorfenapyr wettable powder in preventing and treating phyllotreta striolata

(1) Indoor biological activity assay

The experimental method comprises the following steps: the 20% metarhizium anisopliae MaGX19S 02-chlorfenapyr wettable powder prepared in the example 3 is prepared into suspensions with different concentrations, the phyllotreta striolata is soaked and treated under laboratory conditions, the treatment is repeated three times, the number of treated test insects is 10, and the mortality of the phyllotreta striolata is investigated for 7 days continuously after the treatment. The corrected mortality for phyllotreta striolata was found for wettable powder suspensions of different concentrations in table 21.

Metarhizium anisopliae MaGX19S 02-chlorfenapyr wettable powder with surface 2120% pathogenicity to phyllotreta striolata

The experimental results are as follows: from the results in table 21, it is known that the 20% metarhizium anisopliae MaGX19S 02-chlorfenapyr wettable powder with 500 times liquid has the best pathogenicity to phyllotreta striolata, the fatality rate to phyllotreta striolata adults after 7d reaches 93.33%, and the fatality rates of phyllotreta striolata adults with 500 times liquid, 1000 times liquid and 1500 times liquid diluted wettable powder are respectively 93.33%, 50% and 40% which are respectively superior to 33.33% of the metarhizium anisopliae pure spore powder and 26.67% of the chlorfenapyr technical product.

(2) Test of field drug effect

The test method comprises the following steps: the test is carried out on a farm in the school of southern China agricultural university, the test object is a flowering cabbage in the growing period, the test treatment is respectively 500 times of the 20% metarhizium anisopliae MaGX19S 02. chlorfenapyr wettable powder prepared in the example 3, chlorfenapyr (50mg/L) is compared with clear water, three treatments are carried out in total, each treatment is carried out on a 12 square meter per treatment cell, each treatment is repeated for three times, and a test medicament is sprayed by an electric sprayer (equal amount of clear water is sprayed in blank comparison). At 1, 7 and 14 days after treatment, 20 seedlings are randomly selected in each repetition to investigate the damage condition of the leaves. Leaf damage index was recorded and calculated as follows:

level 0: no harm is caused;

level 1: the leaves are damaged sporadically;

and 3, level: less than one third of the area of the blade is damaged;

and 5, stage: one third or one half of the area of the blade is damaged;

and 7, stage: one-half to two-thirds of the area of the blade is damaged;

and 9, stage: over two thirds of the area of the blade is damaged.

The leaf damage index is shown in Table 22, and the growth of flowering cabbage at 1d, 7d and 14d is shown in FIG. 1.

TABLE 22 seedling protecting effects of different treatments on flowering cabbage

And (3) test results: the results in Table 22 show that the flowering cabbage damage index after the 7 th and 14 th days of the 500-fold liquid of 20% metarhizium MaGX19S 02-chlorfenapyr wettable powder is 38.89 and 48.89 respectively, and the flowering cabbage damage index after the 7 th and 14 th days of the chlorfenapyr (50mg/L) respectively is 91.11 and 171.11, so that the seedling protection effect of the 500-fold liquid of 20% metarhizium MaGX19S 02-chlorfenapyr wettable powder on the flowering cabbage after the 7 th and 14 th days of the pesticide is far better than that of the chlorfenapyr alone,

FIG. 1 shows that the flowering cabbage growth conditions of 1d, 7d and 14d after application show that the pesticide composition (20% metarhizium anisopliae MaGX19S 02. chlorfenapyr wettable powder) provided by the invention has a remarkable effect of controlling phyllotreta striolata.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.