CN108486008B - Bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, and culture method and application thereof - Google Patents
Bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, and culture method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial insecticides, and particularly relates to a Bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, a culture method and application thereof, wherein the Bacillus thuringiensis YN108 is Bacillus thuringiensis subsp.aizawai, the preservation number of the Bacillus thuringiensis YN108 in the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No.15140, and the preservation date is 2018, month 01 and day 02. The bacillus thuringiensis YN108 disclosed by the invention has high toxicity to lepidoptera pests, and particularly has half-Lethal Concentration (LC) to 3-instar larvae of spodoptera litura50) Only 3.25X 105cfu/mL was smaller than that of the publicly published YN1-1 strain and CAB109 strain. Meanwhile, the indoor pesticide effect and the field control effect of the insecticidal composition are better than commercial T products and SB products of the Bacillus thuringiensis subspecies silurus and SL products of the Bacillus thuringiensis subspecies kustak, and the insecticidal composition shows excellent insecticidal activity. In addition, the bacillus thuringiensis YN108 disclosed by the invention has no residue after being used, has no drug resistance, and is green and environment-friendly.
Description
Technical Field
The invention belongs to the technical field of microbial insecticides, and particularly relates to a bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, a culture method and application thereof.
Background
Bacillus thuringiensis (Bt) is an important factor for biological control as an entomopathogenic bacterium in pollution-free agricultural production. Bt is an aerobic gram-positive bacterium with flagella that forms endogenous spores and endotoxin protein crystals (PICs) when growth conditions deteriorate. The specific endotoxin protein crystals formed in Bt exhibit strong insecticidal activity against larvae of Lepidoptera, Diptera, Coleoptera, etc., and strains toxic to nematodes have been found. The endotoxin proteins can be classified into Cry 1-40 and Cyt 1-2, and can be classified into different types according to the homology of amino acids. Wherein the endotoxin protein Cry I is of the order Lepidoptera, Cry II is of the order Lepidoptera and Diptera, Cry III is of the order Coleoptera, Cry IV is of the order Diptera, and Cry V is of the order Lepidoptera and Coleoptera. Most of endotoxin proteins toxic to lepidopteran larvae have a molecular weight of 130-140 kDa, and are decomposed into active proteins having a molecular weight of 50-70 kDa after treatment with an insect digestive juice. At present, the Bt pesticides are most widely applied in biological pesticides at home and abroad. In developed countries in the western world, pollution-free microorganisms such as Bt have been used for a long time to develop pesticide preparations for agricultural production. Recently, not only studies in molecular genetics for expanding the host range of Bt and improving its toxicity have been widely conducted, but also studies in exploring and isolating and screening of new Bt have been eagerly conducted.
At present, the Bt strain most used at home and abroad is Bacillus thuringiensis subsp. However, the strain is reported to have high toxicity to some insects of the order Lepidoptera, but has low toxicity to important noctuidae pests such as beet armyworm and prodenia litura. Moreover, the strain is relatively easy to induce the drug resistance of pests under laboratory and field conditions, thereby limiting the wide application of the microbial preparation in the prevention and control of crop pests.
In order to solve the limitation of Bt biological control agents, inhibit the drug resistance of pests, expand the host range of pesticides, improve the utilization efficiency of Bt biological pesticides in agricultural production and continuously carry out separation research on high-toxicity Bt new strains of lepidoptera pests.
Disclosure of Invention
The invention provides a bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, a culture method and application thereof, and provides a new strain for preparing a Bt biological control agent, which can inhibit the drug resistance of the pests, enlarge the host range of pesticides and improve the utilization efficiency of the Bt biological control agent in agricultural production.
The invention aims to provide a Bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, wherein the Bacillus thuringiensis YN108 is Bacillus thuringiensis subsp.aizawai, the preservation number of a common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No.15140, and the preservation date is 2018, and is 02 month after 01.
The second purpose of the invention is to provide a culture method of the bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, wherein the bacillus thuringiensis YN108 is cultured for 3-4 days at 27 ℃ by using a nutrient agar culture medium to form rhombic parasporal crystals.
The third purpose of the invention is to provide the application of the bacillus thuringiensis YN108 in preparing a biological control agent for lepidoptera pests.
Preferably, in the application, the cultured bacillus thuringiensis YN108 bacterial liquid is diluted to 106～107The cfu/mL concentration is directly used as a biological control agent.
Preferably, in the above application, the Bacillus thuringiensis YN108 produced endotoxin protein in the preparation of lepidoptera pest biological control agent application.
Preferably, in the above application, the lepidoptera pest is diamond back moth, ramie noctuid, beet noctuid and prodenia litura.
Compared with the prior art, the bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, the culture method and the application thereof have the following beneficial effects:
aiming at lepidoptera pests, particularly noctuidae pests which are difficult to control, and characteristics of Bacillus thuringiensis subsp. When the strain YN108 is subjected to a severe environment of growth conditions, endotoxin protein is produced. The endotoxin protein thus formed was observed by phase contrast microscopy as rhombohedral parasporal crystals, and these parasporal crystals had a considerably high insecticidal effect against lepidopteran pests such as diamond back moth, beet armyworm and prodenia litura.
The results of indoor and outdoor bioassay show that the bacillus thuringiensis YN108 has high toxicity to lepidoptera pests, especially the semilethal concentration (LC) of 3-instar larvae of spodoptera litura50) Only 3.25X 105cfu/mL was smaller than that of the publicly published YN1-1 strain and CAB109 strain, indicating that the activity was higher than that of the latter two strains. Meanwhile, the indoor pesticide effect and the field control effect of the insecticidal composition are better than commercial T products and SB products of the Bacillus thuringiensis subspecies silurus and SL products of the Bacillus thuringiensis subspecies kustak, and the insecticidal composition shows excellent insecticidal activity. In addition, the bacillus thuringiensis YN108 disclosed by the invention has no residue after being used, has no drug resistance, and is green and environment-friendly.
Biological material preservation information description
YN108, called Bacillus thuringiensis YN108 in the application, has been deposited in China general microbiological culture Collection center (CGMCC) in 2018, month 01 and day 02, the deposit number is CGMCC No. 0, the deposit unit address is No. 3 of Beijing Shangyang district North West Lu No.1, zip code 100101, and the classification is Bacillus thuringiensis subsp.
Drawings
FIG. 1 is a phase contrast micrograph (. times.1000) of endotoxin protein (C) and spores (S) of Bacillus thuringiensis YN 108;
FIG. 2 is a SDS-PAGE electrophoresis of endotoxin proteins of Bacillus thuringiensis YN 108;
wherein, M: standard protein molecular weight markers, 1: endotoxin protein of bacillus thuringiensis kurstaki strain HD-1, 2: endotoxin protein of bacillus thuringiensis subsp. Endotoxin proteins of bacillus thuringiensis subsp.catus YN 108;
FIG. 3 is an SDS-PAGE electrophoresis of trypsin-treated endotoxin proteins of Bacillus thuringiensis YN 108;
wherein, M: standard protein molecular weight markers, 1: endotoxin protein of bacillus thuringiensis YN108, 2: endotoxin protein + trypsin of bacillus thuringiensis subsp.catuaensis YN 108;
FIG. 4 is a graph of survival rate for 3-instar larvae of Spodoptera litura treated at different locations;
wherein, the SL product and the contrast have consistent change trend and have no insecticidal action on 3-instar spodoptera litura larvae.
Detailed Description
The present invention is described in detail below with reference to the drawings and the specific embodiments, but it should be understood that the scope of the present invention is not limited by the specific embodiments. In the following examples of the present invention, all reagents used are commercially available unless otherwise specified, and the methods involved are conventional ones unless otherwise specified.
The invention provides a Bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, which is Bacillus thuringiensis subsp.aizawai, wherein the preservation number of the Bacillus thuringiensis subsp.aizawai in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No.15140, and the preservation date is 2018, 01 and 02 days.
Example 1 isolation of Bacillus thuringiensis strain YN108 (hereinafter referred to as Bacillus thuringiensis YN108)
The soil to be tested is from mountains of West cities of Heng of Longbai mountain, and 10g of sample soil is collected at a depth of about 5cm from the surface layer of the soil under the trees for testing. Weighing 1g of the collected soil sample, placing the soil sample into a test tube, adding 9mL of sterilized water, and then stirring the soil sample for 4-5 times. In order to screen and remove the miscellaneous bacteria which cannot form spores, a test tube is treated in a constant-temperature water tank at 65 ℃ for 30min, the test tube is kept stand for 5min after heat treatment, supernatant liquid is taken after soil precipitation, diluted 1000 times and uniformly coated on a Nutrient agar culture medium (Nutrient agar), the culture medium is placed in a constant-temperature incubator at 27 ℃ for 3-4 days to be cultured, strains with the bacterial colony morphology identical to that of bacillus thuringiensis are screened out through visual observation, then the strains are observed through a phase contrast microscope at 1000 times, and finally strains forming parasporal crystals are screened out.
Example 2 serological identification of Bacillus thuringiensis YN108
As a result of identification of flagellar antibodies of Bacillus thuringiensis YN108 obtained in example 1 by a method of agglutination reaction with known antiserum, the strain produced agglutination reaction with antiserum of known Bacillus thuringiensis subspecies silurus, indicating that the YN108 strain belongs to the species Clarias thuringiensis, and the results are shown in Table 1.
TABLE 1 serotype Classification of Bacillus thuringiensis YN108
| Isolation of bacterial strains | Flagellar antigen classification | Subspecies of the species | Agglutination |
| B.t.YN108 | |||
| 7 | Catfish (aizawai) | + |
Example 3 Bacillus thuringiensis YN108 endotoxin protein observation
The endotoxin protein of Bacillus thuringiensis YN108 obtained in example 1 was observed by phase contrast microscope as follows: to obtain the endotoxin protein, it is inoculated on NA medium and incubated at a constant temperature of 27 ℃ for 3-5 days until spores and endotoxin protein (parasporal crystals) are formed and spore segregation occurs. The strain producing the spore isolation is smeared on a glass slide, a drop of sterilized water is dripped, then a cover glass is covered, a drop of cedar oil is dripped, and the observation is carried out under a phase contrast microscope of 1000 times. FIG. 1 shows phase contrast micrographs (. times.1000) of endotoxin protein (C) and spores (S) of Bacillus thuringiensis YN 108. As can be seen from FIG. 1, the parasporal crystals (endotoxin proteins) of Bacillus thuringiensis YN108 are rhomboidal in shape, consistent with the morphology of typical parasporal crystals toxic to lepidopteran insects.
Example 4 electrophoretic analysis of endotoxin protein of Bacillus thuringiensis YN108
Inoculating bacillus thuringiensis YN108 on an NA culture medium, culturing for about 4-5 days at a constant temperature of 27 ℃, observing the formation condition of parasporal crystals by a phase contrast microscope of 1000 times, collecting thalli on the culture medium after the parasporal crystals are formed, putting the thalli into a centrifugal tube, adding PBS buffer solution, and centrifuging for 15min at the speed of 15000rpm and at 4 ℃ by using a centrifugal machine. After completion of the centrifugation, the supernatant was removed, and the precipitate was collected and washed 3 times and 2 times with rinsing buffer I (500mM NaCl, 2% triton X-100) and rinsing buffer II (500mM NaCl), respectively. SDS-PAGE Electrophoresis of crystallins was performed according to the method described by Laemmli, namely 12% partitioning gel (30% acylamide/Bis, 1.5M Tris-HCl, pH 8.8, 10% SDS, TEMED, 10% Ammonium Persulfate) and 4% stacking gel (30% acylamide/Bis, 1M Tris-HCl, pH 6.8, 10% SDS, TEMED, 10% Ammonium Persulfate), Electrophoresis buffer (25mM Tris,192mM Glycine, 0.1% SDS). The concentration of the concentrated gel and the concentration of the separation gel are respectively 4% and 10%. After the electrophoresis is finished, 0.1 percent of R-250 Coomassie brilliant blue is respectively used for dyeing and a decoloring solution is used for decoloring. FIG. 2 is an SDS-PAGE electrophoresis of endotoxin proteins of Bacillus thuringiensis YN108, wherein M: standard protein molecular weight markers, 1: endotoxin protein of bacillus thuringiensis kurstaki strain HD-1, 2: endotoxin protein of bacillus thuringiensis subsp. Endotoxin proteins of bacillus thuringiensis subsp.catus YN 108. As shown in FIG. 2, it was found that the molecular weights of endotoxin proteins of Bacillus thuringiensis Kurstaki strain HD-1, Bacillus thuringiensis Clariana strain and Bacillus thuringiensis Clariana strain YN108 were substantially similar.
Example 5 protein Change analysis of endotoxin protein of Bacillus thuringiensis YN108 after Trypsin treatment
mu.L of the endotoxin protein obtained in example 4 was treated with 50mM NaOH aqueous solution for 5min, the supernatant was removed by centrifugation, diluted 10 times, mixed with 1. mu.L of trypsin and stirred, and reacted at 37 ℃ for 15 to 30min, and then the change in the protein was analyzed by the Laemmli method. FIG. 3 is an SDS-PAGE electrophoresis of trypsin-treated toxin protein from Bacillus thuringiensis YN 108. Wherein, M: standard protein molecular weight markers, 1: endotoxin protein of bacillus thuringiensis YN108, 2: endotoxin protein of bacillus thuringiensis subsp.catuaensis YN108 + trypsin. As can be seen from FIG. 3, the molecular weight of the endotoxin protein of Bacillus thuringiensis YN108 is 132kDa, and the endotoxin protein is degraded into an active protein having a molecular weight of 65kDa after trypsin treatment.
EXAMPLE 6 insecticidal Effect of Bacillus thuringiensis YN108
Inoculating Bacillus thuringiensis YN108 obtained in example 1 on NA culture medium, culturing at 27 deg.C for 4-5 days, observing formation of parasporal crystal by 1000 times phase contrast microscope, collecting thallus on the culture medium when the parasporal has been separated, placing the thallus in a centrifugal tube, collecting the thallus by centrifugation, adding a certain amount of sterilized water, and counting the thallus to obtain a concentration of 1 × 107cfu/mL, for various toxicity tests.
1) And (3) testing the plutella xylostella: spraying Chinese cabbage leaf with diameter of 2cm to the concentration of 1.0 × 107100 mu L of bacterial liquid of cfu/mL, inoculating 20 heads of 2-instar larvae/culture dish after drying in the shade, and investigating the death rate after standing for 48h at room temperature, wherein the insecticidal effect of 48h, 72h and 120h reaches 100%.
2) Ramie noctuid: collecting larvae of the spodoptera exigua and hosts thereof in the field, shearing host leaves into leaves with the diameter of 3cm, putting the host leaves into a culture dish, spraying 200 mu L of strain stock solution, drying in the shade, inoculating 10 heads of 2-instar larvae of the spodoptera exigua, observing and recording the number of dead and live larvae within 48h, wherein the insecticidal effect reaches 100% in 48h, 72h and 120 h.
3) Beet armyworm: adding 1.0 × 10 of artificial feed into 0.5g of culture dish7Bt (Bacillus thuringiensis culture fluid in the invention) with cfu/mL concentration 100 μ L, drying in shade for 30min, inoculating 2-instar larva 20 heads/culture dish, standing at room temperature for 48h, investigating mortality, and killing pests for 48h, 72h and 120hThe effect reaches 100 percent.
4) Prodenia litura: the method is basically the same as that of beet armyworm, and is characterized in that the 2 nd larva is placed in each culture dish for 30 heads, the 4 th larva is placed in each culture dish for 20 heads, the death rate is investigated after the 2 nd larva is placed at room temperature for 120h, and the insecticidal effect of the 4 th larva for 120h reaches 100%.
All the above experiments were repeated 3 times.
Table 2 shows the toxicity of Bacillus thuringiensis YN108 against several different pests. As can be seen from the table 2, the YN108 strain shows high insecticidal activity to several common but difficult-to-control pests in Lepidoptera, and the pesticide effect reaches 100%.
TABLE 2 bioassay results of Bacillus thuringiensis YN108 against several pests
Note: +++: high activity (mortality rate 100%); ++: medium activity (mortality rate 80-99%); +: low activity (mortality rate 50-79%); -: no activity (mortality rate 0-49%).
Example 7 comparative experiment of Bacillus thuringiensis YN108 with products already commercially available
To compare Bacillus thuringiensis YN108 with commercial Bacillus thuringiensis T products, SB products and Kustak species SL products, the procedure of example 6 was used to collect Bacillus thuringiensis T products, SB products and Kustak species SL products. Meanwhile, for the purpose of toxicity comparison, 3 rd larvae of spodoptera litura which are difficult to control are selected for testing. Table 3 shows that 3 kinds of commercial biopesticides registered as Spodoptera litura were tested for comparison of biological activities with the isolated and screened Bacillus thuringiensis YN108 in order to confirm the feasibility as a biopesticide.
TABLE 3 comparison of insecticidal efficiency of Bacillus thuringiensis YN108 with 3 commercially available strains
| Bacterial strains | Concentration (cfu/mL) | Insecticidal ratio (%) |
| Bacillus thuringiensis YN108 | 1.0×107 | 100 |
| Bacillus thuringiensis T product of subspecies catzeae | 1.0×107 | 80 |
| Bacillus thuringiensis SB product | 3.0×107 | 14 |
| Bacillus thuringiensis subspecies custark SL product | 2.5×107 | 0 |
In order to obtain accurate test results, the concentrations of the above 4 strains were uniformly adjusted to a × 10 by colony counting method7cfu/mL (guaranteed at 10)7An order of magnitude). Table 3 and FIG. 4 show that the concentrations are substantially the same (10)7cfu/mL), wherein it can be seen from Table 3 that the activity of the Bacillus thuringiensis strain YN108 is much higher than that of the other commercial 2 strains, while it can be seen from FIG. 4 that the survival rate of YN108 is much lower than that of other commercial biopesticides from day 1 after the treatment,shows strong quick-acting insecticidal effect. FIG. 4 is a graph of the survival rate of various strains treated with 3 rd larvae of Spodoptera litura; wherein the SL product and the control have the same change trend and are represented by a trend line, and the SL product and the control have no insecticidal effect on 3-instar spodoptera litura larvae. From the results shown in table 3 and fig. 4, it can be seen that bacillus thuringiensis YN108 exhibited an extremely high control effect on 3 rd larvae of spodoptera litura, and also had a quick-acting property that is incomparable with other commercial biopesticides.
EXAMPLE 8 toxicity assay of 3 Bacillus thuringiensis isolated and screened from soil against Spodoptera litura
A plurality of high-activity bacillus thuringiensis are separated and screened in soil in sequence, wherein strains YN108 and YN1-1 of the bacillus thuringiensis (the protein characteristic and the field control effect of high-toxicity bacillus thuringiensis YN1-1, Lizhen, agricultural science and technology) and strains CAB109 (the protein characteristic and the field control effect of high-toxicity bacillus thuringiensis CAB109, Jinda warong, Yanbian university agronomy report) have the potential of developing biological pesticides, and therefore toxicity comparison tests are carried out on the 3 strains.
Through preliminary experiments, the concentration of the bacillus thuringiensis capable of killing 0-100% of 3-instar larvae of prodenia litura is prepared, namely 5.0 multiplied by 103cfu/ml、5.0×104cfu/ml、5.0×105cfu/ml、5.0×106cfu/ml、5.0×107cfu/ml of the 5 concentration gradients, respectively adding different strains and Bt 200 muL with different concentrations into 1g of artificial feed in a culture dish, inoculating 30 heads of 3-age prodenia litura larvae/the culture dish after drying in the shade for 30min, changing fresh artificial feed after 2d, investigating mortality after standing at room temperature for 120h, repeating the experiment for 4 times, taking clear water as a contrast, respectively calculating the semi-Lethal Concentration (LC) by using PC software according to the probit calculation method of Finney (1971) and according to the mortality and the corrected mortality when different concentrations are used, taking the concentration of bacteria liquid and the corrected mortality as parameters50) And selecting Bt strains with the strongest toxicity by comparison.
Semi-lethal concentration of Table 43 strains on 3-instar larvae of Prodenia litura
| Bacterial strains | Number of young insects | LC50(cfu/mL) |
| YN108 | 120 | 3.25×105 |
| YN1-1 | 120 | 3.31×105 |
| CAB109 | 120 | 6.87×106 |
As can be seen from Table 4, the semi-Lethal Concentration (LC) of YN108 strain50) The minimum value indicates that the YN108 strain has higher toxicity to prodenia litura than the YN1-1 strain and the CAB109 strain, has better control effect and can be used for a microorganism strain for controlling lepidoptera pests.
Example 9 field test of Bacillus thuringiensis YN108
In order to prove the field control effect of the bacillus thuringiensis YN108, a series of experiments are carried out on the onion field by taking beet armyworm larvae as objects. The basic situation of the onion field at that time is that the pest density is 0.2 head/plant, the pest ratio is 20 percent, and the area of each test cell is 5m2(5 m.times.1 m). The Bacillus thuringiensis YN108 obtained in example 1 was diluted to a concentration of 2X 10 strains in comparison with the commercial T product of the species T of Bacillus thuringiensis6After cfu/mL, the cells were kept on onion leaves at 1 week (7 day) intervalsThe medicine was sprayed 3 times. Before and 1 week after each spraying, the number of pests and the plant damage rate on the leaves of 30 scallion plants are randomly investigated in the cell, and all the tests are repeated for 3 times.
TABLE 5 field control of Bacillus thuringiensis YN108 and T products
Table 5 shows the results of comparative field control tests of commercial T products of Bacillus thuringiensis YN108 and Bacillus thuringiensis subsp.catus on larvae of beet armyworm in onion fields, and Table 4 shows that the control effect of the YN108 strain in 1 time and 2 times is obviously higher than that of the T product, although no obvious difference is generated between the YN108 strain and the T product in 3 times, the average control effect of the YN108 strain reaches 76.6 percent and is 16 percent higher than that of the T product, and the YN108 strain shows extremely high insecticidal activity. Table 6 shows the results of comparative experiments on the pest damage reduction rate of commercial Bacillus thuringiensis YN108 and commercial Bacillus thuringiensis T products on the beet armyworm larvae in onion fields, and as can be seen from Table 6, the pest damage rate of onion leaves after spraying with YN108 strain is reduced by 27.2% on average and is improved by more than 7% compared with 20.1% of T products.
TABLE 6 reduction of pests in Bacillus thuringiensis YN108 and T products
As can be seen from tables 5 and 6, the YN108 strain is superior to the commercial T product in terms of field control effect and damage reduction rate.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (6)
1. Bacillus thuringiensis (B) with high toxicity to lepidoptera pestsBacillus thuringiensis subsp.aizawai) YN108, which is characterized in that the preservation number of the China general microbiological culture Collection center is CGMCC number 15140, and the preservation date is 2018, 01 and 02 days.
2. The method for culturing the bacillus thuringiensis YN108 with high toxicity to lepidoptera pests according to claim 1, wherein the bacillus thuringiensis YN108 forms rhomboid parasporal crystals after being cultured in a nutrient agar medium at 27 ℃ for 3-4 days.
3. Use of the bacillus thuringiensis YN108 with high virulence against lepidopteran pests according to claim 1 in the preparation of a biological control agent for lepidopteran pests.
4. The use according to claim 3, wherein the cultured Bacillus thuringiensis YN108 strain is diluted to 106 ~ 107The cfu/mL concentration is directly used as a biological control agent.
5. Use of the endotoxin protein produced by Bacillus thuringiensis YN108 having high virulence against lepidoptera pests according to claim 1 in the preparation of a biological control agent for lepidoptera pests.
6. The use according to any one of claims 3 to 5, wherein the lepidopteran pest is diamondback moth, ramie looper, beet armyworm and prodenia litura.
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