Pesticide composition containing sophocarpidine and metarhizium robertii and application of pesticide composition

Abstract

The invention relates to a pesticide composition containing sophocarpidine and metarhizium robertii and application of the pesticide composition, and belongs to the field of biopesticides. The compound composition is a water dispersible granule containing sophocarpidine and metarhizium robertianum, and specifically comprises 15% of metarhizium robertianum MrCb01 pure sporopollen, 5% of sophocarpidine, 5% of a wetting agent NP-10, 4.5% of a dispersing agent Borresse NA, 10.5% of a dispersing agent Prowet WG20, 2% of an anti-ultraviolet agent L-ascorbic acid, 20% of a disintegrating agent ammonium sulfate, 2% of an adhesive sodium carboxymethyl cellulose and 36% of filler diatomite. The granules taking the sophocarpidine and the metarhizium robertianum as the active ingredients have a synergistic effect on prevention and treatment of phyllotreta striolata in a reasonable compounding proportion range, can reduce the use of chemical pesticides, are safe to the environment, and have very good economic, ecological and social benefits.

Description

Technical Field

[0001] This invention relates to a pesticide composition containing matrine and Metarhizium anisopliae and its application, belonging to the field of biological pesticides. Background Technology

[0002] Yellow-striped flea beetle ( Phyllotreta striolata The yellow-striped flea beetle (Fabricius spp.) is one of the most damaging pests of cruciferous vegetables. Both larvae and adults of this beetle can damage crops. The larvae live in the soil, biting off fibrous roots and feeding on rhizomes, leading to missing seedlings and gaps in rows, and easily inducing soft rot in vegetables. Adults feed on leaves, affecting vegetable quality. Flea beetles occur in cruciferous vegetable growing areas worldwide, but cause the most severe damage in North America, Asia, and Europe. Currently, the application of chemical pesticides remains the main method of flea beetle control, but the irrational use of chemical pesticides has led to problems such as pesticide resistance and pesticide residues. Studies have found that the yellow-striped flea beetle has developed varying degrees of resistance to organophosphates, carbamates, pyrethroids, and neonicotinoids.

[0003] Matrine is derived from Sophora flavescens (Sophora flavescens). Sophora flavescens Matrine is an alkaloid isolated from the roots, stems, leaves, and flowers of *Ait.*, belonging to the quinazonidine class of alkaloids. It can control a wide range of pests, including brown planthoppers, citrus parva, Formosan termites, cotton aphids, leek maggots, and yellow-striped flea beetles, exhibiting a very broad insecticidal spectrum. In addition, matrine is safe for natural enemies; it is easily degraded in natural water and soil, exhibiting good environmental safety; and it has the highest annual yield among plant-derived pesticides, thus becoming the most registered plant-derived pesticide in my country.

[0004] Metarhizium anisopliae (Roberts) Metarhizium robertsii It belongs to the kingdom Fungi, phylum Ascomycota, class Pyrenomycetes, order Sphaeriales, family Clavicipitaceae, and genus Metarhizium. Metarhizium Existing research indicates that *Metarhizium anisopliae* has a certain control effect on pests such as bean hawk moth larvae, two-spotted spider mites, American cockroaches, and melon aphids, and has a relatively broad insecticidal spectrum. Currently, there are no pesticide registration certificates in China using *Metarhizium anisopliae* as the active ingredient.

[0005] Currently, there are more than 10 invention patents on biological pesticides or biological pesticide combinations for preventing and treating Phyllotreta striolata. In terms of bacteria, CN201611000946.3 and CN202310211564.9 disclose that Bacillus thuringiensis has good effect on preventing and treating Phyllotreta striolata, and CN202111661439.5 discloses the prevention and treatment effect of a strain of Serratia marcescens. In terms of fungi, CN202011457358.9, CN202010971228.0 and CN202110822046.1 disclose the prevention and treatment effects of Isaria fumosorosea, Cordyceps javanica and Beauveria bassiana on Phyllotreta striolata. In terms of plant source pesticides, CN200910214343.7 discloses a Phyllotreta striolata repellent containing extract of Lantana camara. In addition, CN202310353201.9 discloses the prevention and treatment effect of polyhedrosis virus combined with other effective components on Phyllotreta striolata.

[0006] The existing prevention and treatment of Phyllotreta striolata by microorganisms mainly focuses on the prevention and treatment of Phyllotreta striolata by different microorganisms and single components. There is also research on the prevention and treatment of Phyllotreta striolata by using matrine alone, but there is no report on the use of plant source pesticides and fungi for the prevention and treatment of Phyllotreta striolata. At present, there are three water dispersible granules registered with Beauveria bassiana as the effective component, namely PD20142078, PD20180631 and PD20110965. There is no water dispersible granule registered with matrine and Metarhizium anisopliae, and there is no related literature report. SUMMARY

[0007] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a pesticide composition containing matrine and Roberts Metarhizium anisopliae and its application, by developing a microbial pesticide composition mixed with plant source pesticides and biocontrol fungi and a dispersant containing the pesticide composition, so as to achieve the purpose of preventing and treating Phyllotreta striolata, reduce the use amount of chemical pesticides, improve the quality of crops and environmental safety, and enhance the economic, ecological and social benefits of agricultural production.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows: on the one hand, a pesticide composition containing matrine and Roberts Metarhizium anisopliae, the active ingredients of the composition include matrine and Roberts Metarhizium anisopliae spore powder; the sum of the mass of the matrine and the Roberts Metarhizium anisopliae spore powder is 20% of the total mass of the pesticide composition; the mass ratio of the matrine to the Roberts Metarhizium anisopliae is (1-7) :(2-63.5).

[0009] In some embodiments of the present application, the total amount of the effective component in the pesticide composition is 20% by weight. The mass ratio of matrine to Metarhizium robertsii is 7:2, 7:5, 1:3, 1:5, 1:7, 1:11, 1:17, 1:28, or 1:63.5.

[0010] Preferably, the mass ratio of matrine to Metarhizium robertsii is 1:3.

[0011] In another aspect, the present application provides a water dispersible granule, which comprises the pesticide composition described above and an auxiliary agent; the auxiliary agent comprises a wetting agent, a dispersing agent, an ultraviolet-resistant agent, a binder, a disintegrating agent, and a filler.

[0012] In some embodiments of the present application, the granule comprises the following components by weight: 0.31-15.56% matrine, 4.44-19.69% Metarhizium robertsii spore powder, 5% wetting agent, 10-15% dispersing agent, 2% ultraviolet-resistant agent, 1-4% binder, 10-20% disintegrating agent, and the rest filler.

[0013] In some embodiments of the present application, the granule comprises the following components by weight: 15% Metarhizium robertsii spore powder, 5% matrine, 5% wetting agent, 15% dispersing agent, 2% ultraviolet-resistant agent, 2% binder, 20% disintegrating agent, and 36% filler.

[0014] In some embodiments of the present application, the wetting agent comprises at least one of Morwet-EFW, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, sodium C14-C16 alkenyl sulfate, and NP-10; the dispersing agent comprises at least one of Morwet-D400, GY-D800, Prowet WG20, Borresperse NA, and MF; the ultraviolet-resistant agent is L-ascorbic acid; the binder is one of soybean protein isolate, sodium alginate, xanthan gum, and sodium carboxymethyl cellulose; the disintegrating agent is ammonium sulfate; and the filler comprises at least one of diatomite, kaolin, talc, bentonite, and poplar wood powder.

[0015] In some embodiments of the present application, the wetting agent is NP-10, the dispersing agent is Prowet WG20 and Borresperse NA, the binder is sodium carboxymethyl cellulose, and the filler is diatomite.

[0016] In another aspect, the present application provides the use of the pesticide composition or the water dispersible granule in the prevention and treatment of Phyllotreta striolata.

[0017] Compared with the prior art, the present application has the following advantages: (1) The pesticide composition provided by the application has synergistic effect on the prevention and treatment of Phyllotreta striolata through the compounding of two biological pesticides (matrine and Roberts green muscadine fungus) in a specific mass ratio, thereby improving the efficacy, increasing the crop yield and significantly reducing the amount of chemical pesticides. Meanwhile, the prepared water dispersible granules have the advantages of small dust pollution during production, no use of organic solvents and environmental safety, and have obvious advantages compared with the existing wettable powder and suspension agent, thereby expanding the types of biological pesticide dosage forms.

[0018] (2) Compared with the wettable powder, the water dispersible granule produces less dust pollution during production and use; compared with the suspension agent, the preparation of the water dispersible granule does not need to use organic solvents. Therefore, the preparation of the water dispersible granule compounded by the plant source pesticide and the fungus can expand the biological pesticide resource library of China and provide a reference for the subsequent development and research of biological pesticides. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a Phyllotreta striolata mummy treated by Roberts green muscadine fungus MrCb01 for 5 days.

[0020] Figure 2 It is the mycelial growth of Roberts green muscadine fungus MrCb01 on the plates containing different types of fillers, wherein a is the growth on the plate containing diatomite; b is the growth on the plate containing kaolin; c is the growth on the plate containing talc; d is the growth on the plate containing bentonite; e is the growth on the plate containing poplar wood powder; and f is CK.

[0021] Figure 3 It is the mycelial growth of Roberts green muscadine fungus MrCb01 on the plates containing different types of wetting agents, wherein a is the growth on the plate containing Morwet-EFW; b is the growth on the plate containing sodium dodecyl sulfate; c is the growth on the plate containing sodium dodecyl benzene sulfonate; d is the growth on the plate containing C14-C16 alkenyl sodium sulfate; e is the growth on the plate containing NP-10; and f is CK.

[0022] Figure 4 It is the mycelial growth of Roberts green muscadine fungus MrCb01 on the plates containing different types of dispersants, wherein a is the growth on the plate containing Morwet-D400; b is the growth on the plate containing GY-D800; c is the growth on the plate containing Prowet WG20; d is the growth on the plate containing Borresperse NA; e is the growth on the plate containing MF; and f is CK.

[0023] Figure 5The mycelium growth of Metarhizium robertsii MrCb01 on plates containing different types of binders, wherein a is the growth on plates containing soybean protein isolate; b is the growth on plates containing sodium alginate; c is the growth on plates containing xanthan gum; d is the growth on plates containing sodium carboxymethyl cellulose; and e is CK.

[0024] Figure 6 The prepared 20% Metarhizium · matrine water dispersible granule product.

[0025] Figure 7 The field control effect of 20% Metarhizium · matrine water dispersible granule on Phyllotreta striolata, wherein a is before treatment; b is 20% Metarhizium · matrine water dispersible granule 500 times treatment group 7 days after treatment; c is 20% Metarhizium · matrine water dispersible granule 250 times treatment group 7 days after treatment; d is 80 billion spores / ml Metarhizium chrysopa CQMa421 dispersible oil suspension 500 times treatment group 7 days after treatment; e is 0.5% matrine aqueous solution 300 times treatment group 7 days after treatment; and f is the water treatment group 7 days after treatment. DETAILED DESCRIPTION

[0026] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0027] Example 1: This example is the screening of active ingredients of the pesticide composition of the present application

[0028] 1, Toxicity of matrine to Phyllotreta striolata

[0029] The test Phyllotreta striolata (Linnaeus) was collected from the field in the summer of 2018. Phyllotreta striolataCollected from the experimental farm of South China Agricultural University. The collected flea beetles were dressed in insect cages, and the cabbage leaves were replaced every other day. After 48 hours of feeding, healthy and active flea beetles were selected for bioassay. The room temperature was maintained at 25 ± 1 ℃, and the light cycle was 12:12 (L:D). The experiment was conducted in accordance with the Guidelines for Pesticide Indoor Bioassay Test (NY / T 1154.6-2006) of the People's Republic of China. Matrine was prepared into seven concentrations of 25, 50, 100, 200, 300, 400, and 500 mg / L using deionized water. The yellow striped flea beetles were placed in sterilized non-woven fabric filter bags, and then the non-woven fabric bags were immersed in the drug solution for 20-25 s. The excess drug solution was absorbed with filter paper, and the soaked flea beetles were picked into plastic culture jars one by one with a brush, ensuring that all the flea beetles in the jar were alive. Each group contained 10 adult insects, and the experiment was repeated three times. The bottom of the jar was moistened with wet cosmetic cotton, and fresh cabbage leaves were placed in the jar. The cabbage leaves were replaced daily. The culture jars were placed in a light incubator with a temperature of 25 ± 1 ℃ and a light cycle of 12:12 (L:D) for incubation. The mortality of the flea beetles was observed on the 1st, 2nd, and 3rd days after immersion, and the number of dead insects was recorded. The corrected mortality rate, toxicity regression equation, and LC 50 were calculated.

[0030]

[0031] The results showed that the cumulative corrected mortality rate of the yellow striped flea beetles treated with different concentrations of matrine was as shown in Table 1. The toxicity regression equation of matrine on the yellow striped flea beetles after 3 days of treatment was y = 0.0013x + 0.0314 (c 2 = 2.497, p = 0.777), with a determination coefficient R 2 of 0.9684. The LC 50 of matrine was 360.46 mg / L, with a 95% confidence interval of 298.79-455.15 mg / L.

[0032] Table 1 Biological activity of matrine on adult yellow striped flea beetles

[0033] 2. Biological activity of Roberts green muscadine MrCb01 on yellow striped flea beetles Roberts green muscadine MrCb01 was prepared into concentrations of 5 × 10 6 , 1 × 10 7 , 5 × 10 7 , 1 × 108 5×10 8 A spore suspension of conidia / mL was prepared. The flea beetles were placed in a sterilized non-woven fabric filter bag, which was then immersed in the solution for 20-25 seconds. The filter bag was removed, excess solution was absorbed with filter paper, and the flea beetles were carefully picked up one by one with a brush and transferred into a plastic culture jar, ensuring that none of the beetles were dead. Ten adults were used per group, and the process was repeated three times. The bottom of the jar was kept moist with a damp cotton pad, and fresh bok choy leaves were placed inside, replaced daily. The culture jars were placed in a light incubator at 25±1℃ and a photoperiod of 12:12 (L:D). Mortality was observed and recorded on days 3, 7, and 10 after treatment. The corrected mortality rate was calculated using the formula, and the virulence regression equation and LC ratio were calculated. 50 The results are shown in Table 2. The dead worms after treatment are shown in Table 2. Figure 1 As shown.

[0034] Table 2 Bioactivity of MrCb01 against adult yellow-striped flea beetles

[0035] Experimental Results: The regression equation for the toxicity of matrine to flea beetles 10 days after treatment, calculated from the data in Table 2, is y = 0.289lgx – 1.6738 (c 2 =0.496, p =0.920), coefficient of determination R 2 The LC-100 value was 0.9771. (Note: The original text appears to be incomplete and contains several typographical errors. A more accurate translation would require the full context.) 50 =3.33×10 7 conidia / mL, 95% confidence interval is 2.27×10 7 ~4.54×10 7 conidia / mL.

[0036] 3. Safety of matrine against Metarhizium anisopliae strain MrCb01 Effects of matrine on the hyphae of entomopathogenic fungi: A 10000 mg / L stock solution of matrine was prepared using sterilized deionized water. Sterilized PDA was heated to dissolve the matrine, and after cooling to near solidification, an appropriate amount of the matrine stock solution was added to the PDA to achieve a matrine concentration of LC50. 50 and LC 25 Puncture cakes of entomopathogenic fungi were collected using a punch and inoculated into the center of PDA plates. PDA plates without matrine were used as a control. The plates were incubated at 28°C for 7 days. Each treatment was repeated three times. The colony diameters of the treatment groups and the control group were measured, and the mycelial growth inhibition rate was calculated using the formula.

[0037]

[0038] Effect of matrine on spore germination rate of entomopathogenic fungi: Matrine was prepared into 10000 mg / L stock solution with deionized water. Appropriate amount of matrine stock solution was added into 50 mL spore germination solution after sterilization, so that the concentration of matrine in the spore germination solution reached LC 50 and LC 25 . The PDA fungus cake with spores grown out was taken out with a puncher and was connected to 50 mL spore germination solution, and was fully shaken and uniformly distributed. It was placed in a 28℃, 180 r / min shaking bed for 24 h. Blood cell counting plate was used for counting, spore germination was considered as the growth of germ tube or doubling of volume, otherwise it was considered as non-germination. A total of 100 spores were counted each time, and three times were repeated. The spore germination rate was calculated according to the formula, and the experimental results are shown in Table 3.

[0039]

[0040] Experimental results: The mycelial growth inhibition rate and spore germination rate after matrine treatment are shown in Table 3. The mycelial growth of Roberts green muscadine MrCb01 was not affected by matrine. The spore germination rate under different concentrations of matrine was similar, indicating that matrine concentration below 360 mg / L had no effect on the spore germination of MrCb01. The mycelial growth and spore germination of MrCb01 were not affected by matrine, and the mycelial growth inhibition rate and spore germination rate did not change significantly with the change of matrine concentration, indicating that matrine had high safety to MrCb01.

[0041] Table 3 Effect of different concentrations of matrine on fungal mycelial growth and spore germination

[0042] 4、Matrine and Roberts green muscadine MrCb01 combined virulence to Phyllotreta striolata Roberts green muscadine MrCb01 and matrine were respectively prepared into LC 50Concentration standby. Matrine and Roberts green muscardine fungus spore powder were respectively compounded according to the ratio of 7:2, 7:5, 1:3, 1:5, 1:7, 1:11, 1:17, 1:28, 1:63 (m:m), 0.02% Tween-80 solution was added to dissolve and configure into a mother liquor, and then the mother liquor was obtained to test the solution. The control group was 0.02% Tween-80 solution, and the total volume of each treatment solution was 10 mL. The yellow striped flea beetles were placed in sterilized non-woven fabric filter bags, and then the non-woven fabric bags were immersed in the solution for 20-25 s. The filter bags were taken out, the excess solution was absorbed with filter paper, and the soaked beetles were picked into plastic culture jars one by one with a brush, ensuring that all the beetles picked into the jar were not dead. There were 10 adult beetles in each group, and each group was repeated three times. The bottom of the jar was kept moist with wet cosmetic cotton, and fresh cabbage leaves were placed in the jar, which were replaced every day. The culture jars were placed in a light incubator with a temperature of 25±1 ℃ and a light cycle of 12:12 (L:D) for incubation. The mortality was observed and recorded on the 5th day after treatment, the corrected mortality was calculated, and the synergistic toxicity index (c.f. value) of MrCb01 and matrine was calculated according to the formula of Yin Fei et al. (Yin Fei et al., 2010). The results are shown in Table 4.

[0043]

[0044] The experimental results: matrine and MrCb01 were compounded according to different ratios (v:v) at LC 50 The biological activity of matrine and MrCb01 compounded at different ratios (v:v) on adult yellow striped flea beetles is shown in Table 4. From the table, it can be seen that when the mass ratio of matrine in the compounded combination is high, the control effect on yellow striped flea beetles is higher. On the 5th day, the cumulative corrected mortality of the combinations with mass ratios of 7:2 and 7:5 was 75.86% and 79.31% respectively, and the c.f. value was 4.76 and 9.52 respectively, showing additive effect; the cumulative corrected mortality of the combination with a volume ratio of 1:3 was 89.66%, and the c.f. value was 23.81, showing synergistic effect; the rest of the combinations showed antagonistic effect. In summary, matrine and MrCb01 were compounded at a ratio of 1:3 (m:m), which showed synergistic effect on yellow striped flea beetles on the 5th day, at this time the concentration of matrine in the solution was 0.252 mg / mL, and the concentration of MrCb01 was 1.00×10 7 conidia / mL.

[0045] Table 4 Biological activity and synergistic toxicity index of matrine and MrCb01 compounded on adult yellow striped flea beetles

[0046] The data in the table are mean ± standard error, and the same column data with different letters indicate significant difference (DMRT method) at P <0.05 level.

[0047] Example 2: Production and quality detection of Metarhizium robertsii MrCb01 conidia powder The Metarhizium robertsii MrCb01 strain was first cultured in PDB medium (containing 1% Tenebrio molitor meal) for 48 h, and then the culture solution was inoculated on rice to perform solid-state fermentation using rice as the substrate. The solid-state fermentation time was 10-14 d. After solid-state fermentation, the conidia were harvested by spore separation, and then the conidia powder was freeze-dried by vacuum freeze-drying agent to obtain high-purity conidia powder (pure spore powder). According to the method in the national standard “GB / T 25864-2010, Beauveria bassiana powder”, the spore content of the pure spore powder was (13.27±0.14)×10 9 / g, the spore germination rate was (91.33±0.93)%, and the dry loss was (2.83±0.10)%.

[0048] Example 3: Formulation screening and processing of 20% Metarhizium • Sophoridine water dispersible granules

[0049] 1. Selection of fillers

[0050] Determination of filler suspension rate: 0.50 g of filler was added to a 50 mL standard hard water beaker, and the hand was moved at a rate of 120 r / min to make a circular motion for 2 min. The suspension was placed in a constant temperature water bath at 30°C for 4 min, and then all of it was transferred to a 250 mL stoppered cylinder with standard hard water. The stopper was covered, and the cylinder was inverted 30 times within 1 min with the cylinder bottom as the axis. The stopper was opened, and the cylinder was placed vertically in the constant temperature water bath to avoid direct sunlight. After 30 min, 9 / 10 (i.e. 225 mL) of the suspension was pumped out with an electric water pump, and the top end of the pump tube was always kept a few millimeters below the liquid surface during pumping, without shaking the cylinder or lifting the sediment in the cylinder. The mass of the petri dish was measured, and the remaining 25 mL of the suspension was transferred to the petri dish. A small amount of standard hard water was used to wash the inner wall of the stoppered cylinder, and the washing solution was also transferred to the petri dish. The petri dish was placed in an oven at 120°C for 6 h, and then the total weight of the dried petri dish was measured again. Each treatment was repeated four times, and the suspension rate was calculated according to the formula.

[0051]

[0052] Effect of fillers on mycelial growth: Diatomite, kaolin, talc, bentonite and poplar powder were added to PDA medium at a ratio of 5% (m / m) respectively. After sterilization, pour into the culture dish, after the filler-containing medium solidification, access to the Roberts green muscadine fungus cake punched by 5 mm puncher, placed in 26℃ incubator for 2~3 days, without adding filler as control, measure the colony diameter of control and treatment group, record the data, according to the formula of plate confrontation experiment method to calculate the average inhibition rate. Inhibition rate (%) = [(control group diameter-treatment group diameter) / (control group diameter-fungus cake diameter)] 100. The growth of fungus cake on the plate containing filler is shown in Figure 2

[0053] Effect of fillers on spore germination: The fillers were mixed with pure spore powder at a ratio of 5:1 (m:m) and poured into empty culture dishes, which were incubated in a 28℃ incubator. Each group was repeated three times. After 7 days, about 0.1 g of the mixture of pure spore powder and fillers was added to 50 mL of spore germination solution, and the spore germination rate was measured. The data were analyzed using SPSS 22 software, and the results are shown in Table 5.

[0054] Table 5 Suspension rates of different fillers and their safety to MrCb01

[0055] The data in the table are mean ± standard error, and different letters after the same column data indicate significant differences at the P <0.05 level (DMRT method).

[0056] Experimental results: According to Table 5, among the five fillers tested, diatomite had the highest suspension rate, reaching 28.11%, which was significantly higher than that of the other fillers. Except for bentonite, the mycelial growth inhibition rates of the other four fillers were less than 5%. In terms of spore germination, diatomite and talc had the least effect on spore germination, with no significant difference from the CK group. Considering the suspension rate, mycelial growth inhibition rate, and spore germination rate, diatomite had the best safety to MrCb01 and the best suspension, so it was chosen as the filler for water dispersible granules.

[0057] 2. Selection of wetting agents ​Wetting agent wetting performance determination: First, pure spore powder and diatomite were mixed uniformly at a ratio of 1:3 (m:m), then 5% (m:m) of different types of wetting agent was added to the sample, and mixed again to obtain the sample to be tested. 100 mL of standard hard water was added to a 250 mL beaker, and the beaker was placed in a constant temperature water bath at 25°C, keeping the liquid level in the beaker level with the liquid level of the water bath. 5 g of sample was weighed on the weighing paper, and the sample was poured evenly from the cup opening position onto the liquid surface in the beaker. Timing was started immediately after adding the sample, and stopped until the sample was completely wetted (a small amount of residual fine powder on the liquid surface could be ignored). Record the wetting time, and repeat each treatment three times.

[0058] Effect of wetting agent on mycelial growth: The wetting agent was added to PDA at a ratio of 3% (m / m), sterilized, heated to dissolve and shake evenly, and poured into a culture dish. The puncher was used to punch the fungus cake, inoculated in the central PDA plate containing the wetting agent, and cultured at 28°C for 14 d. The PDA without wetting agent was used as the control group, and 3 replicates were set for each treatment. After 14 d, the colony diameters of the treatment group and the control group were measured, and the growth inhibition rate was calculated. The growth of the fungus cake on the plate containing the wetting agent is shown in Figure 3

[0059] Effect of weting agent on spore germination: The wetting agent was mixed with pure spore powder at a ratio of 3:1 (m:m) and poured into an empty culture dish, and cultured in a 28°C incubator. Each group was repeated three times. After 7 d, about 0.1 g of pure spore powder mixed with wetting agent was added to 50 mL of spore germination solution, and the spore germination rate was measured. The data was analyzed using SPSS 22 software, and the results are shown in Table 6.

[0060] Table 6 Wetting time and safety of different wetting agents on MrCb01

[0061] The data in the table is the mean ± standard error, and the same column data is not the same letter, which means P The difference is significant at the level of 0.05 (DMRT method).

[0062] ​Experimental results: As shown in Table 6, without the addition of wetting agent, the pure spore powder and diatomite mixture cannot be completely wetted. After adding the wetting agent, the mixture can be completely wetted in water, and all wetting agents can shorten the wetting time to within 8 seconds. Among the five wetting agents tested, Morwet-EFW has the best wetting performance, with a wetting time of 2.45 s; followed by NP-10, with a wetting time of 3.57 s. In terms of fungal safety, except for NP-10, the spore germination rate after mixing with the remaining wetting agents is less than 25%; MrCb01 can only grow on plates containing C14-C16 alkyl sodium sulfate and NP-10, and cannot grow on plates containing the remaining wetting agents. In terms of wetting time and fungal safety, NP-10 is relatively safe for MrCb01 and has good wetting performance, so NP-10 is selected as the wetting agent for water dispersible granules.

[0063] 3. Selection of dispersants Performance determination of dispersants: First, mix pure spore powder and diatomite at a ratio of 1:3 (m:m) uniformly, then add 5% wetting agent NP-10, then add 10% (m:m) of different types of dispersants to the sample, and mix again to obtain the sample to be tested. The suspension rate of the sample is determined, and each treatment is repeated four times.

[0064] Effect of dispersants on mycelial growth: Add dispersants to PDA at a ratio of 3% (m / m), sterilize, heat and dissolve, and shake uniformly, then pour into Petri dishes. Use a puncher to punch the fungus cake and inoculate in the center of the PDA plate containing the dispersant, and incubate at 28°C for 14 d. The PDA without dispersant is used as the control group, and each treatment has 3 replicates. Calculate the growth inhibition rate. The growth of the fungus cake on the plate containing the dispersant is shown in Figure 4

[0065] Effect of dispersants on spore germination: Mix the dispersant with the pure spore powder at a ratio of 3:1 (m:m) and pour into empty Petri dishes, and incubate in a 28°C incubator. Each group is repeated three times. After 7 d, add about 0.1 g of the mixture of pure spore powder and dispersant to 50 mL of spore germination solution, and measure the spore germination rate. Use SPSS 22 software to analyze the data, and the results are shown in Table 7.

[0066] Table 7 Suspension rate with dispersants and safety of dispersants to MrCb01

[0067] The data in the table are mean ± standard error, and different letters after the same column data indicate significant differences at the P <0.05 level (DMRT method).

[0068] ​Experimental results: As shown in Table 7, the suspension rate of the mixture after adding the dispersant was greatly improved compared with the suspension rate of the mixture of pure spore powder and diatomite. The suspension rate of all treatment groups except GY-D800 was more than 50%. Among them, the suspension rate of each treatment group using Morwet-D400, Prowet WG20 and Borresperse NA as dispersants was relatively high. Morwet-D400, Prowet WG20, Borresperse NA and MF four kinds of dispersants had little effect on the spore germination rate of MrCb01. In terms of growth inhibition, the inhibition rate of GY-D800, Prowet WG20 and Borresperse NA on mycelial growth was less than 10%. In terms of spore germination rate and growth inhibition rate, Prowet WG20 and Borresperse NA both had good safety to MrCb01. In terms of suspension rate and safety to fungi, Borresperse NA was selected as the dispersant of water dispersible granules.

[0069] 4. Preliminary screening of binders Effect of binders on mycelial growth: The binder was added to PDA at a proportion of 1% (m / m), sterilized, heated and dissolved, and then shaken uniformly and poured into a culture dish. The puncher was used to take the fungus cake and inoculate in the center of the PDA plate containing the dispersant. The culture was incubated at 28°C for 14 d. The PDA without dispersant was used as the control group, and 3 replicates were set for each treatment. The growth inhibition rate was calculated. The growth of the fungus cake on the plates containing different binders is shown in Figure 5 .

[0070] Effect of binders on spore germination: The binder was mixed with pure spore powder at a proportion of 1:5 (m:m) and then poured into an empty culture dish and incubated in a 28°C incubator. Each group was repeated three times. After 7 d, about 0.1 g of the mixture of pure spore powder and binder was added to 50 mL spore germination solution, and the spore germination rate was measured. The data were analyzed using SPSS 22 software, and the results are shown in Table 8.

[0071] Granulation effect of binders: 1-4% (m:m) of the binder was dissolved in water to prepare a binder aqueous solution, which was mixed with the filler to prepare water dispersible granules in a granulator. The granulation of the granules, whether it was easy to stick during granulation, and the strength of the granules after drying were recorded, and the results are shown in Table 9.

[0072] Table 8 Effect of binders on mycelial growth of MrCb01

[0073] The data in the table are mean ± standard error. Different letters after the same column data indicate that the difference is significant at the 0.05 level. P<0.05 level difference significant (DMRT method).

[0074] Table 9 Granulation of different binders

[0075] Experimental results: As shown in Table 8, the four binders are all good for the safety of MrCb01, and have no obvious inhibitory effect on spore germination and mycelium growth. As shown in Table 9, xanthan gum, sodium alginate, soybean protein isolate and sodium carboxymethyl cellulose can be used to prepare granules with strength under a certain amount. Since the binder will affect the disintegration performance of the granules, subsequent tests are needed to determine the disintegration of the granules after adding disintegrants to determine the selected binder.

[0076] 5. Preparation and formula optimization of Metarhizium anisopliae • matrine water dispersible granules Binder re-screening: 5% wetting agent NP-10, 10% disintegrant ammonium sulfate and sufficient diatomite (remaining percentage after subtracting the amount of binder) are mixed evenly, and four kinds of binders are prepared into aqueous solution according to their minimum granulation amount, and then mixed with the above wetting agent, disintegrant and filler to granulate. 0.5 g of dried granules is weighed and added to a 250 mL graduated cylinder containing 100 mL of distilled water, the stopper is inserted and placed for 1 min, and then inverted 180° up and down with the middle of the cylinder as the axis until the granules are completely disintegrated in water. The number of inversions is recorded, and the test is repeated three times. The results are shown in Table 10.

[0077] Table 10 Disintegration times of different binders

[0078] The data in the table are mean ± standard error, and the same column data with different letters indicate P <0.05 level difference significant (DMRT method).

[0079] Experimental results: As shown in Table 10, among all the treatments, the treatment using sodium carboxymethyl cellulose as the binder has the least disintegration times, indicating that among the four disintegrants, sodium carboxymethyl cellulose has the least effect on the disintegration performance of the preparation. Therefore, sodium carboxymethyl cellulose is selected as the binder of the preparation, and the addition amount is 2%.

[0080] Optimization of formulation suspension rate: all treatments keep the types and addition amounts of active ingredients (20% Roberts Metarhizium anisopliae pure spore powder: matrine 3:1 (m:m)), wetting agent (5% NP-10), anti-ultraviolet agent (2% ascorbic acid), binder (2% sodium carboxymethyl cellulose) and disintegrant (10% ammonium sulfate) unchanged, change the type and ratio of dispersing agent (addition amount fixed at 15%), and the remaining mass is supplemented with diatomite. The suspension rate of different formula combinations is determined, and each treatment is repeated three times. The results are shown in Table 11.

[0081] Table 11 Suspension rate of different dispersant types and ratios

[0082] The data in the table are mean ± standard error, and the same letter after the data in the same column indicates that there is no significant difference between P The difference is significant at the level of <0.05 (DMRT method).

[0083] Experimental results: As shown in Table 11, under the premise of adding the same amount of dispersant, the suspension rate of the complex dispersant treatment group is higher than that of the single dispersant treatment. When Borresperse NA or Prowet WG20 is used alone as a dispersant, the suspension rate of the preparation cannot reach more than 80%, so these two dispersants with better safety to Metarhizium anisopliae are used in combination. Among the 9 combinations, the combination of Borresperse NA: Prowet WG20 at a ratio of 3:7 (m:m) has the highest suspension rate, reaching 82.74%. Therefore, the dispersant in the preparation is selected as Borresperse NA and Prowet WG20 in a combination of 3:7 (m:m), and the total amount of dispersant added is 15%.

[0084] Disintegration time optimization: All treatments keep the types and amounts of active ingredients (20% Metarhizium anisopliae pure spore powder: sophoridine 3:1 (m:m)), wetting agent (5% NP-10), anti-ultraviolet agent (2% ascorbic acid), and adhesive (2% carboxymethyl cellulose sodium) unchanged, only change the amount of disintegrating agent, and the remaining mass is supplemented with diatomite. The suspension rate and disintegration time of each treatment are measured, and the results are shown in Table 12.

[0085] Table 12 Suspension rate of different dispersant types and ratios

[0086] The data in the table are mean ± standard error, and the same letter after the data in the same column indicates that there is no significant difference between P The difference is significant at the level of <0.05 (DMRT method).

[0087] Experimental results: From the data in the table, with the increase of the amount of disintegrating ammonium sulfate, the suspension rate of the preparation is improved, and the disintegration time is decreased. The suspension rate of all treatment groups is greater than 80%, meeting the requirements of national standards. The disintegration time decreases with the increase of the amount of disintegrating agent. When the amount of disintegrating agent is 20%, the disintegration time is 206.67 s. When 20% ammonium sulfate is used as the disintegrating agent, the disintegration time is close to the index requirement (≤180 s). To further shorten the disintegration time, the suspension rate and disintegration time of the dried preparation passing through different mesh sieves are measured to determine the most suitable mesh sieve aperture.

[0088] Effect of different mesh sieves on the suspension rate and disintegration time of the preparation: 20% (m:m) active ingredients (the mass ratio of pure spore powder to matrine is 7:3), 15% (m:m) dispersant (Borresperse NA / WG20=3:7), 5% (m:m) wetting agent NP-10, 2% (m:m) anti-ultraviolet agent L-ascorbic acid, 20% (m:m) disintegrating agent ammonium sulfate, and 36% (m:m) filler diatomite are mixed uniformly. In addition, 2% (m:m) sodium carboxymethyl cellulose is dissolved in warm water and mixed again. After granulation and drying, pass through 18, 24, and 30 mesh sieves, with each treatment repeated three times. The suspension rate and disintegration time after sieving are measured. The results are shown in Table 13.

[0089] Table 13 Suspension rate of different dispersant types and ratios

[0090] The data in the table are mean ± standard error. The same letter after the data in the same column indicates that there is no significant difference (DMRT method). P <0.05 level (DMRT method).

[0091] Experimental results: By using smaller mesh sieves to reduce the volume of the preparation, the disintegration time required by the preparation can be further shortened. When using a 24 mesh sieve, the disintegration time and suspension rate of the preparation meet the requirements. Although using a smaller 30 mesh sieve can further reduce the disintegration time, the suspension rate of the preparation is also reduced, and the particles are more easily broken. In summary, the best mesh size for sieving after drying the water dispersible granules is 24 mesh.

[0092] Preparation of 20% Metarhizium · Sophora water dispersible granules: According to the amount in Table 14, mix the active ingredients and adjuvants except the binder uniformly, then add the aqueous solution of the binder, and pass through a 24 mesh sieve after rotary extrusion granulation and oven drying at 28°C for 24 h to obtain 20% Metarhizium · Sophora water dispersible granules. The water dispersible granules obtained after drying are shown in Figure 6 .

[0093] Table 14 Formula of 20% Metarhizium · Sophora water dispersible granules

[0094] Quality detection of 20% Metarhizium • Sophoridine water dispersible granules: The prepared water dispersible granules were measured for wetting time, suspension rate, disintegration time and persistent foaming property. The persistent foaming property was measured according to GB / T 28137-2011 “Pesticide Persistent Foaming Property Determination Method”. Each index was measured repeatedly for 3 times. The results are shown in Table 15.

[0095] Table 15 Indexes of 20% Metarhizium • Sophoridine Water Dispersible Granules

[0096] Example 4: Application of 20% Metarhizium • Sophoridine Water Dispersible Granules in the Prevention and Control of Phyllotreta striolata The test crop was 120-day-old leafy oilseed rape, and the test treatments were as follows: A. 20% Metarhizium • Sophoridine water dispersible granules diluted 500 times; B. 20% Metarhizium • Sophoridine water dispersible granules diluted 250 times; C. 80 billion spores / mL Metarhizium anisopliae CQMa421 dispersible oil suspension (PD20171744) diluted 500 times; D. 0.5% Sophoridine aqueous solution (PD20141711) diluted 300 times; E. water treatment. A total of five treatments were set up, with an area of 25 m² per plot, 4 repetitions for each treatment, a total of 20 plots, and a randomized block arrangement. An electric sprayer (HM-16A, Zhongshan Hemei Electrical Appliance Co., Ltd.) was used to uniformly spray the test pesticides, with a water volume of 40 L / mu. Before treatment, 10 plants per plot were selected to record the injury index, and 2 days and 7 days after treatment, the injury index of the marked plants was investigated to calculate the control effect. The results are shown in Table 16. Figure 7 0 level: no injury; 1 level: sporadic injury on leaves; 3 level: less than one-third of the area of the leaves was injured; 5 level: one-third or one-half of the area of the leaves was injured; 7 level: one-half to two-thirds of the area of the leaves was injured; 9 level: more than two-thirds of the area of the leaves was injured.

[0097]

[0098]

[0099] Table 16 Field control effect of 20% Metarhizium • Sophoridine water dispersible granules on Phyllotreta striolata

[0100] ​The data in the table are mean ± standard error, and the same column data with different letters represent significant differences (DMRT method). P <0.05 level difference (DMRT method).

[0101] The experimental results: 2 days after treatment, the control agent 20% Metarhizium anisopliae and Sophora flavescens water dispersible granules had a control effect of 56.73%~61.06% on Phyllotreta striolata, and the control agent 80 billion spores / ml Metarhizium anisopliae CQMa421 dispersible oil suspension had a control effect of 55.29% at the same period, and 0.5% Sophora flavescens water agent had a control effect of 46.63% at the same period. 7 days after treatment, 20% Metarhizium anisopliae and Sophora flavescens water dispersible granules had a control effect of 61.29%~68.55% on Phyllotreta striolata, and the control agent 80 billion spores / ml Metarhizium anisopliae CQMa421 dispersible oil suspension had a control effect of 57.26% at the same period, and 0.5% Sophora flavescens water agent had a control effect of 37.90% at the same period.

[0102] In summary, 20% Metarhizium anisopliae and Sophora flavescens water dispersible granules diluted 500 times had no significant difference in control effect on Phyllotreta striolata 2 days and 7 days after treatment compared with the control agent 80 billion spores / ml Metarhizium anisopliae CQMa421 dispersible oil suspension diluted 500 times, and the control effect 7 days after treatment diluted 250 times was significantly better than that of 80 billion spores / ml Metarhizium anisopliae CQMa421 dispersible oil suspension diluted 500 times, and the control effect of two concentrations 2 days and 7 days after treatment was significantly better than that of 0.5% Sophora flavescens water agent at the same period. In addition, compared with the control effect 2 days after treatment and 7 days after treatment of the same treatment, the control effect of three treatments except 0.5% Sophora flavescens water agent increased. The control effect of 20% Metarhizium anisopliae and Sophora flavescens water dispersible granules increased more than that of 80 billion spores / ml Metarhizium anisopliae CQMa421 dispersible oil suspension, which was speculated to be due to the synergistic effect of Sophora flavescens and Roberts Metarhizium anisopliae in the complex pesticide, which made the control effect of single effective component Metarhizium anisopliae more obvious. Under the condition of water consumption of 40 liters / mu, it is recommended that 20% Metarhizium anisopliae and Sophora flavescens water dispersible granules be diluted 250~500 times for field preparation to control Phyllotreta striolata. In addition, the test agent 20% Metarhizium anisopliae and Sophora flavescens water dispersible granules did not cause phytotoxicity to Brassica parachinensis in the test dose range, and had good safety to Brassica parachinensis.

[0103] The control agent 8 billion spores per milliliter Metarhizium anisopliae CQMa421 dispersible oil suspension is the only fungal insecticide registered on Phyllotreta striolata at present, and the 20% Metarhizium anisopliae · Sophora flavescens water dispersible granule has no significant difference in the control effect with the control agent at the same dilution multiple, and the control effect is higher than that of the control agent after spraying, which not only shows the effectiveness and synergistic effect of the effective component sophoridine and Roberts Metarhizium anisopliae, but also increases the available pesticide in organic planting cruciferous vegetables. It can be seen that the pesticide composition provided by the present application provides a new reference for biological pesticides in the prevention and control of Phyllotreta striolata.

Claims

1. A pesticide composition containing matrine and Metarhizium anisopliae, characterized in that, The active ingredients of the pesticide composition include matrine and pure spores of Metarhizium anisopliae MrCb01. The sum of the masses of matrine and Metarhizium anisopliae MrCb01 pure spores is 20% of the total mass of the pesticide composition; The mass ratio of matrine to pure spores of Metarhizium anisopliae MrCb01 is (1~7):(2~63.5).

2. The pesticide composition according to claim 1, characterized in that, The mass ratio of matrine to the pure spores of Metarhizium anisopliae MrCb01 is 7:2, 7:5, 1:3, 1:5, 1:7, 1:11, 1:17, 1:28, and 1:63.

5.

3. The pesticide composition according to claim 1, characterized in that, The mass ratio of matrine to the pure spores of Metarhizium anisopliae MrCb01 is 1:

3.

4. A water-dispersible granule, characterized in that, The water-dispersible granules comprise the pesticide composition according to any one of claims 1-3, and further comprise adjuvants; the adjuvants include wetting agents, dispersants, binders, UV stabilizers, disintegrants, and fillers.

5. The water-dispersible granule as described in claim 4, characterized in that, The water-dispersible granules, by weight percentage, comprise the following components: 0.31-15.56% matrine, 4.44-19.69% Metarhizium anisopliae pure spore powder, 5% wetting agent, 15% dispersant, 2% UV stabilizer, 1-4% binder, 10-20% disintegrant, and filler balance.

6. The water-dispersible granule as described in claim 5, characterized in that, The water-dispersible granules, by weight percentage, comprise the following components: 5% matrine, 15% Metarhizium anisopliae pure spore powder, 5% wetting agent, 15% dispersant, 2% UV stabilizer, 2% binder, 20% disintegrant, and 36% filler.

7. The water-dispersible granule as described in claim 5, characterized in that, The wetting agent includes at least one of Morwet-EFW, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, C14-C16 alkenyl sulfate, and NP-10; the dispersant includes at least one of Morwet-D400, GY-D800, Prowet WG20, Borresperse NA, and MF; the binder is one of soy protein isolate, sodium alginate, xanthan gum, and sodium carboxymethyl cellulose; and the filler includes at least one of diatomaceous earth, kaolin, talc, bentonite, and poplar wood powder.

8. The water-dispersible granule as described in claim 7, characterized in that, The wetting agent is NP-10, the dispersant is ProwetWG20 and Borresperse NA, and the filler is diatomaceous earth.

9. The water-dispersible granule as described in claim 5, characterized in that, The UV protectant is L-ascorbic acid, the binder is sodium carboxymethyl cellulose, and the disintegrant is ammonium sulfate.

10. The application of the pesticide composition according to claims 1 to 3 or the water-dispersible granules according to any one of claims 4 to 8 in the control of the Chinese cabbage flea beetle.

Images