Polish penicillium with high pathogenicity to spodoptera litura and application of the same in combination with emamectin benzoate

Abstract

The present application relates to the field of biological control, and discloses screening of entomogenous fungi with high pathogenicity to Spodoptera litura and application of the fungi in combination with emamectin benzoate. The high-toxicity strain of Penicillium polonicum for Spodoptera litura is collected from cotton fields in Qapqal County, Xinjiang Uygur Autonomous Region. Furthermore, an efficient, pollution-free and residue-free insecticide with synergistic effect, emamectin benzoate and Penicillium polonicum, is provided. The present application finds that the insecticide can exhibit significant synergistic effect, improve the control effect, and significantly reduce the dosage of the single chemical pesticide and the environmental pollution by studying the combination effect of emamectin benzoate and Penicillium polonicum.

Description

Technical Field

[0001] This invention relates to the field of plant protection, specifically to the field of biological control, and particularly to the biological control of the beet armyworm. Background Technology

[0002] Since the development of modern agriculture, pesticides have played an extremely important role in agricultural production. However, the correct use of pesticides is also crucial. Improper use of pesticides can cause environmental pollution, and to this day, the environmental pollution problem caused by pesticides is becoming increasingly serious. Pesticide pollution not only occurs in soil, water bodies and the atmosphere, but also leads to the contamination of agricultural products, thereby endangering human health and posing a huge threat to human health (Zhu Cuixia. Problems and countermeasures of pesticide use pollution in the prevention and control of crop diseases and pests [J]. Hebei Agricultural Machinery, 2024, (07): 67-69.).

[0003] Agricultural pests pose a significant threat to agriculture and horticulture worldwide, causing incalculable economic losses. They can directly harm plants by sucking sap or other plant tissues, or indirectly harm crops by spreading plant pathogens. Current pest control heavily relies on the use of chemical pesticides, but this has major drawbacks, such as pesticide resistance and potential toxicity to humans and other non-target organisms. Therefore, there is an urgent need for a new, green, healthy, and sustainable pest control method. Biological control is one promising approach (Wilbert s L, Vuts J, Caulfield JC, Thomas G, Birkett MA, et al. (2022) Impact of endophytic colonization by entomopathogenic fungi on the behavior and life history of the tobacco peach ap hid Myzuspersicae var. nicotianae. PLOS ONE 17(9):e0273791.).

[0004] Entomopathogenic fungi can parasitize the surface and interior of insects. There are over 100 genera and more than 800 species of entomopathogenic fungi recorded worldwide, of which more than 400 are found in China, including 215 species of entomopathogenic fungi. In recent years, due to the unavoidable environmental damage caused by chemical control, people have gradually begun to pay attention to and study biological control. Utilizing entomopathogenic fungi to control pests is one of the important biological control methods (Wang Yudi, Tian Chuanbei. Research progress on the application of entomopathogenic fungi in mite control [J]. Plant Medicine, 2024, 3(01): 8-21.). Entomopathogenic fungi (EPF) are natural enemies affecting insect populations and have long been considered biological control agents for many pests. They are often regarded as effective biological control agents in integrated pest management (IPM) projects. Pests are usually controlled by fungal pathogens through flooding or inoculation of fungal propagules. Some fungal pathogens have been commercialized for use in controlling pests that feed on plants (Sani I, Jamian S, Saad N, Abdullah S, Mohd Hata E, et al. (2023) Inoculation and colonization of the entomopathogenic fungi, Isaria javanica and Purpureocillium lilacinum, in tomato plants, and their effect on seedling growth, mortality and adult emergence of Bemisia tabaci (Gennadius). PLOS ONE 18(5):e0285666.).

[0005] The beet armyworm, belonging to the family Noctuidae in the order Lepidoptera, is also known as the tobacco cutworm. It is native to Southeast Asia. It has spread to India and become a significant pest in the Asia-Pacific region. The beet armyworm is a polyphagous agricultural pest that has a significant economic impact on a wide range of crops worldwide. With over 120 plant species, this species has a great potential to invade new areas due to its ability to adapt to new climates and ecological conditions (Tesari, T., Leksono, AS, & Mustafa, I. (2024). Effectiveness of botanical pesticide combined with Beauveria bassianaon mortality, nutritional index and fecundity of Spodoptera litura L. Cogent Food & Agriculture, 10(1).). The beet armyworm is distributed throughout my country, feeding on 389 host plants including sweet potato, cotton, soybean, and tobacco. The larvae are omnivorous and have a large appetite, exhibiting intermittent outbreaks that cause widespread damage. It is a major pest that causes serious damage to crops and is difficult to control (Zhang Wenmei, Wang Libing, Lu Tian, ​​et al. Study on the control effect of 9 biological pesticides on beet armyworm in tobacco fields [J]. Science of Biological Disasters, 2023, 46(04):438-444.).

[0006] Penicillium polonicum is a broad-spectrum insecticidal fungus. In nature, it primarily reproduces asexually, actively infecting different insects through invasion of the insect's body wall. It multiplies in a yeast-like manner within the insect's hemocoel, producing large quantities of fungal cells and insecticidal toxins such as cytotoxic agents, which suppress insect immunity and accelerate insecticidal activity. Abamectin benzoate is a highly effective, low-toxicity (nearly non-toxic formulation), low-residue, and environmentally friendly biological pesticide for insects. The synergistic use of Penicillium polonicum and abamectin to control the beet armyworm overcomes the slow-acting problem of Penicillium polonicum while simultaneously delaying the development of insecticide resistance, thus achieving excellent insecticidal effects.

[0007] Because Penicillium polens is highly specialized and the virulence varies greatly among different strains, when conducting experiments on compound pesticides, different pathogenic fungi need to be used for different regions and different types of pests, and highly virulent strains need to be screened.

[0008] Because using Penicillium alone to control pests such as the beet armyworm often fails to achieve ideal results—due to its slow insecticidal effect, high susceptibility to environmental influences, and unstable efficacy—the combination of entomopathogenic fungi and pesticides for pest control has become a hot topic in recent years. This combined approach aims to reduce the environmental and natural enemy harm caused by chemical pesticides, improve the utilization efficiency of biological pesticides, and screen suitable chemical agents for biocontrol fungal combinations, thereby alleviating the rapidly increasing pesticide resistance in the beet armyworm. Abamectin benzoate possesses the characteristics of a highly effective, low-toxicity (nearly non-toxic formulation), low-residue, and environmentally friendly biological pesticide for insects. Utilizing Penicillium polonum in synergy with abamectin to control the beet armyworm overcomes the slow-acting problem of Penicillium polonum while simultaneously delaying the development of pesticide resistance, thus achieving excellent insecticidal effects. Summary of the Invention

[0009] The purpose of this invention is to address the critical agricultural need for biological control of the beet armyworm in tobacco fields. It provides a highly toxic *Penicillium polonia* strain collected from cotton fields in Chabuchar County, Xinjiang Uygur Autonomous Region. Simultaneously, it offers a highly effective, pollution-free, and residue-free insecticide combining abamectin and *Penicillium polonia* with synergistic effects. Research on the combined effects of abamectin and *Penicillium polonia* reveals that the insecticide exhibits a significant synergistic effect, improving control efficacy and significantly reducing the dosage of single chemical pesticides, thus minimizing environmental pollution and effectively solving problems such as resistance development and limited efficacy of existing pesticides.

[0010] The technical solution of this invention is as follows:

[0011] The collected and purified strains were subjected to virulence testing and analysis to screen for highly virulent strains against the beet armyworm. Experiments revealed that different genera or even the same species of strains may exhibit varying degrees of pathogenicity against the beet armyworm, thus showing different levels of pathogenicity. Based on the experimental results of this invention, and considering the comprehensive evaluation criteria for selecting superior strains, pathogenicity is an important indicator for assessing the biocontrol potential of fungal strains, while colony or hyphal growth rate is another important evaluation indicator for evaluating superior insect pathogenic fungi. Strain SCAULS28 showed high virulence against the beet armyworm at 10... 8 Strain SCAULS28 exhibits high pathogenicity against Spodoptera litura nymphs at a spore density of 1 / mL. Considering the overall colony growth rate of the strain, strain SCAULS28 is an excellent strain for pathogenicity against Spodoptera litura nymphs.

[0012] This bacterium was isolated from soil collected from cotton fields in Chabuchar County, Xinjiang Uygur Autonomous Region. Incubated on PDA plates at 26°C for 10 days, the colony diameter was 63.75 mm, and the spore yield was 1.237 × 10⁻⁶. 8 Spores / ml, spore germination rate exceeding 95%. Figure 1As shown, the colonies are fluffy in the center and powdery at the edges, green on the surface and red on the back, with a few wrinkles. The spores are oval and nearly spherical. Further molecular biological verification and identification confirmed that it belongs to *Penicillium porconicum*.

[0013] Based on the fact that SCAULS28, which was screened out, has a certain biological control effect on beet armyworm, this invention, in order to further seek better control effects, after a large number of research and exploratory experiments, found that the combination of abamectin and Penicillium polonum SCAULS28 can produce a significant synergistic effect and has a significant control effect on beet armyworm, thus completing this invention.

[0014] Therefore, this invention provides a highly pathogenic entomopathogenic fungal strain against the beet armyworm, namely *Penicillium polonicum*, which was deposited on November 22, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, Guangdong Institute of Microbiology), with accession number GDMCC NO: 65532.

[0015] The present invention further provides the application of the strain in the biological control of Spodoptera litura.

[0016] The present invention also provides an insecticide containing abamectin and Penicillium polonum as active ingredients; the insecticide is used to control the beet armyworm.

[0017] Specifically, the Polish Penicillium is a suspension or spore powder of Polish Penicillium spores.

[0018] Preferably, the ratio of *Penicillium polonum* to abamectin is: 1 × 10⁻⁶ for the final concentration of *Penicillium polonum*. 4 ~1×10 9 Conidia / mL, preferably 1×10⁻⁶ 7 ~1×10 8 The concentration of conidia / mL and the final concentration of abamectin is 0.001–0.05 mg / L, preferably 0.01–0.05 mg / L.

[0019] More preferably, the ratio of *Penicillium polonum* to abamectin is 1 × 10⁻⁶. 4 ~1×10 9 Conidia / mL: 0.005–0.025 mg / L, preferably a ratio of Penicillium polonide to abamectin of 1 × 10⁻⁶. 7 ~1.0×10 8 Conidia / mL: 0.01~0.02mg / L.

[0020] More preferably, the ratio of *Penicillium polonum* to abamectin is 1 × 10⁻⁶. 8 Conidia / mL: 0.01 mg / L.

[0021] The present invention further provides the application of the aforementioned insecticide in the control of beet armyworm.

[0022] This invention also provides a method for controlling the beet armyworm, which involves applying the aforementioned bacterial strain or insecticide to the crops or environment where beet armyworm control is needed. Specifically, the application is performed at the initial stage of beet armyworm appearance.

[0023] This invention utilizes a highly toxic Penicillium polonia strain against the beet armyworm, collected from cotton fields in Chabuchar County, Xinjiang Uygur Autonomous Region. Further research on the combined effects of abamectin and Penicillium polonia revealed a significant synergistic effect, enhancing control efficacy and significantly reducing the dosage of single-agent chemical pesticides, thus minimizing environmental pollution and effectively addressing the problems of resistance development and limited efficacy associated with existing pesticides. Attached Figure Description

[0024] Figure 1 This is a colony morphology diagram of SCAULS28.

[0025] Figure 2 This is a morphological diagram of SCAULS28 spores.

[0026] Figure 3 This is an electrophoresis image of PCR amplification products used in molecular identification.

[0027] Figure 4 Phylogenetic tree. Detailed Implementation

[0028] The following description of the research and development process and specific implementation methods of this invention does not constitute a limitation of this invention.

[0029] Example 1: Initial Screening of Strains

[0030] 1. Test insects

[0031] Spodoptera litura eggs were purchased from Keyun Biotechnology Co., Ltd., and were reared using artificial feed in an artificial climate chamber at the Engineering Research Center for Biological Control of the Ministry of Education to establish a population of Spodoptera litura. The rearing conditions were: temperature (27±1)℃, photoperiod L14:D10, and relative humidity (75±5)%.

[0032] 2. Experimental Methods

[0033] 1) Soil sample collection and fungal isolation and purification

[0034] Soil samples were collected, with approximately 100g of soil taken from a depth of 10-15cm below the topsoil. The samples were sealed in plastic bags and brought back to the laboratory for processing. The soil samples were sieved to remove stones and impurities. Then, 10g of the clean soil was suspended in 90mL of 0.1% Tween 80 solution, shaken well, and allowed to stand for 15 minutes. 2mL of the supernatant was diluted in 8mL of 0.05% Tween 80 to prepare a soil suspension. 0.1mL of the soil suspension was inoculated onto a Bengal red agar plate, and the suspension was spread evenly on the surface of the plate using a triangular glass scraper. The plate was incubated at 25℃ for 3-7 days. Single colonies were then excised using an inoculation loop and inoculated onto PDA plates for further culture to obtain strain SCAULS28. Mycelial blocks were excised and transplanted onto PDA slant plates for further culture and stored at 4℃ in a refrigerator.

[0035] 2) Observation of the biological characteristics of the strain

[0036] Preparation of potato glucose medium: 200g potato, 20g glucose, 20g agar, 1000ml water. Inoculate the purified strain onto PDA medium and culture for 7-10 days. Observe the color, texture, size, height, surface ornamentation, edge, exudate, etc. of the front and back of the colony and take pictures.

[0037] Colony growth rate determination: The purified strain SCAULS28 was inoculated onto PDA plates and incubated at 26℃ for 7-10 days. Using a 5mm punch, holes were made in the cultured medium, and the resulting plates were inoculated into new PDA plates. The plates were sealed with sealing film and incubated at 26℃. This process was repeated 5 times. The colony diameter was measured and recorded using the cross-hatching method, and the daily average growth rate was calculated.

[0038] Determination of sporulation yield: In a clean bench, use a pipette to transfer 500 μL of a solution with a concentration of 1×10⁻⁶. 4 A spore suspension of spores / mL was inoculated onto a 90mm Petri dish. The dish was rotated at a constant speed to ensure even distribution of the liquid on the surface of the culture medium. The dish was then sealed with sealing film and incubated at 26℃ for five replicates. After 15 days of incubation, five uniformly grown colonies were collected from the culture medium using a 10mm punch. Each colony was added to 20mL of 0.5‰ Tween-80 solution and stirred at medium speed with a magnetic stirrer for 20 minutes to prepare a spore suspension. The spore content was determined using a hemocytometer.

[0039] Determination of spore germination rate: Take 1 mL of 1×10⁻⁶ spores. 5Spore suspensions at a concentration of spores / mL were added to 10 mL of SDY medium and cultured at 26℃ and 220 rpm for 24 h, with 5 replicates. A suitable amount of bacterial suspension was placed on a glass slide and observed and recorded under a microscope. Spores with a germ tube length greater than or equal to the spore diameter were considered to have germinated. Each treatment should have at least 50 spores examined per microscopic examination, with 5 repeated examinations.

[0040] The calculation formula is: Spore germination percentage (%) = (Number of germinating spores in the field of view / Total number of spores in the field of view) × 100

[0041] 3) Molecular biological identification

[0042] Molecular biological identification of the purified strains was performed.

[0043] DNA extraction using the CTAB method includes the following steps:

[0044] (a) After culturing strain SCAULS28 on a PDA plate for one week, carefully scrape the mycelium into a mortar and add liquid nitrogen to quickly and thoroughly grind it.

[0045] (b) Take an appropriate amount of crushed mycelium and quickly transfer it to a centrifuge tube. Add 300 μL of DNA extraction lysis buffer preheated at 65°C, mix thoroughly, add an equal volume of chloroform / isoamyl alcohol (24 / 1) mixture, mix well, incubate in a water bath at 65°C for 1 h, gently shake once every 10 min, and centrifuge at 12000 rpm at room temperature for 5 min.

[0046] (c) Slowly aspirate the supernatant into another new centrifuge tube, slowly add an equal volume of chloroform / isoamyl alcohol (24 / 1) mixture for extraction again, and centrifuge at 12000 rpm for 5 min at room temperature.

[0047] (d) Slowly aspirate the supernatant into another new centrifuge tube, add 1 / 10 volume of sodium acetate and an equal volume of pre-cooled isopropanol, and let stand at room temperature for 15 min;

[0048] (e) Wash the precipitate twice with 70% ethanol;

[0049] (f) Slowly aspirate ethanol with the pipette close to the tube wall and place it in a clean bench to dry;

[0050] (g) The resulting precipitate was dissolved in 50 μL of TE;

[0051] (h) Electrophoresis was performed using 1% agarose gel, and the remaining extracts were stored at -20°C.

[0052] Universal primers were used for PCR of fungal DNA. Amplification conditions were as follows: PCR products were subjected to 1% agarose gel electrophoresis (containing EB) with 1×TAE buffer, voltage 150V, current 220mA, and marker DL2000 was used as a molecular weight standard reference. After electrophoresis, the gel was detected and photographed using a gel imaging system.

[0053] Specific PCR products were sequenced by Shanghai Meiji Biotechnology Co., Ltd. Sequence alignment and phylogenetic tree construction: Sequence results were used to search for highly homologous related sequences using BLAST, and sequence alignment and multiple sequence alignment were performed using Cluster W in MEGA 5.1 software. A Neighbour-joining (NJ) phylogenetic tree was then constructed using this software.

[0054] 4) Toxicity determination and analysis of second-instar beet armyworm larvae

[0055] a. Preparation of SCAULS28 spore suspension

[0056] Take strain SCAULS28 cultured for approximately 10-14 days, add distilled water containing 0.05% Tween-80 in a clean bench, scrape off the colonies, wash out the spores from the petri dish, transfer to a sterile Erlenmeyer flask, and stir at medium speed with a thermostatic magnetic stirrer for 20 minutes to ensure uniform dispersion, thus preparing a stock solution. Dilute the bacterial solution 100-fold according to the 10-fold dilution method, and count the conidia under a hemocytometer using a microscope, with 5 replicates. Then dilute the conidia concentration to 1×10⁻⁶. 8 Spores / mL for later use.

[0057] b. Toxicity determination of strain SCAULS28 against Spodoptera litura

[0058] Using the feeding method, the feed is picked up and placed at a concentration of 1×10⁻⁶. 8 Soak the larvae in a spore suspension of 1 spore / mL for about 30 seconds, then feed them to the beet armyworm. Sterilized 0.05% Tween-80 water was used as a control. Each treatment contained 30 larvae, with three replicates per treatment. After inoculation, all petri dishes (12cm in diameter) were ventilated by sealing them with an insect-proof net and placed in a biochemical incubator. Observations were recorded daily for 5 consecutive days, and dead larvae were promptly removed and kept in a humidified environment at 26℃. Infected larvae were confirmed to be lethal based on the characteristics of growths on the body surface of second-instar beet armyworm larvae. Infected larvae were termed "mutilated worms."

[0059] c. Concentration gradient analysis of highly virulent strains

[0060] The spores of the selected strains with good virulence were collected and prepared into a solution with a concentration of 1×10⁻⁶. 8A spore suspension of 1 spore / mL was prepared and diluted in a gradient manner, with a final concentration of 1×10⁻⁶. 8 1 spores / mL, 1×10 7 1 spores / mL, 1×10 6 1 spores / mL, 1×10 5 1 spores / mL, 1×10 4 The mortality rate of second-instar larvae of the beet armyworm was determined by feeding a spore suspension of 1 spore / mL at different concentrations.

[0061] Experimental results

[0062] 1. Biological characteristics of strain SCAULS28

[0063] On PDA plates, at 26°C for 10 days, the colony diameter was 64.62 mm, and the sporulation yield was 1.478 × 10⁻⁶. 8 Spores / ml, spore germination rate exceeding 95%. Figure 1 As shown, the colonies are fluffy in the center and powdery at the edges, green on the surface and red on the back, with a few wrinkles. The spores are oval and nearly spherical.

[0064] 2. Molecular biological identification

[0065] (1) As Figure 3 As shown, the electrophoretic bands in each lane are relatively clear, and the gene fragment of the strain is approximately 600 bp based on the migration distance of the PCR product.

[0066] (2) The ITS sequence of the obtained strain genotype was combined with the relevant sequences of four Penicillium strains downloaded from GenBank to construct a molecular phylogenetic tree. The results are as follows: Figure 4 As shown, strain SCAULS28 belongs to (Penicillium polonicum), and the results of comprehensive morphological and physiological biochemical identification of Penicillium polonicum show that strain SCAULS28 belongs to the genus Penicillium polonicum.

[0067] 3. Screening and virulence analysis of highly virulent strains of *Spodoptera litura*.

[0068] The virulence of eight strains was determined using the dip-dip method, in which larvae were picked up with a brush and immersed in a solution of 1×10⁻⁶ ppm. 8Soak the larvae in a spore suspension of 1 spore / mL for approximately 5 seconds, then transfer them to petri dishes containing feed for observation. Sterilized 0.05% Tween-80 water was used as a control. Each treatment contained 30 larvae, with three replicates per treatment. After inoculation, all petri dishes (12cm in diameter) were sealed with plastic wrap, punctured for ventilation, and placed in a biochemical incubator for rearing. Observations were recorded daily for 5 consecutive days, and dead larvae were promptly removed. The larvae were then cultured at 26℃ under humid conditions, and effective lethality was confirmed based on the growth characteristics on the body surface of second-instar larvae of the beet armyworm. Infected larvae were termed "mute worms." The experimental results showed that the pathogenicity of the eight entomopathogenic fungi varied at the same concentration. At a spore suspension concentration of 1×10⁻⁶, the pathogenicity was significantly higher. 8 At spores / ml, after 5 days of treatment, the corrected mortality rates of the eight strains against second-instar larvae of the beet armyworm ranged from 17.31% to 57.84%. The strain SCAULS28 had the highest corrected mortality rate of 57.84%, which was significantly higher than that of the other strains, indicating that strain SCAULS28 had stronger pathogenicity than the other strains.

[0069] The results showed that strain SCAULS28 had a good lethal effect on second-instar larvae of the beet armyworm, and the pathogenicity of strain SCAULS28 was significantly better than that of other strains, making it a potentially excellent biocontrol bacterium.

[0070] Example 2: Compatibility study of highly virulent strains and abamectin, and screening of combined dosages.

[0071] 1. Effect of abamectin on the spore germination rate of strain SCAULS28

[0072] In the experiment, the test strain was first transferred to a PDA plate and cultured at 26±1℃. After conidia were produced, the conidia were washed away with 0.05% Tween-80 sterile water, stirred with a magnetic stirrer, filtered, and counted using the hemocytometer method to prepare a 1×10⁻⁶ plate. 6 Prepare a spore suspension at spore / mL for later use. Dissolve the emamectin benzoate technical grade in acetone to prepare a 10 mg / L solution for later use. Add emamectin benzoate to 50 mL of SDA liquid medium to prepare SDA liquid medium with emamectin benzoate concentrations of 0.005, 0.015, and 0.01 mg / L. Add 2 mL of 1×10 6 Spore suspensions ( / mL) were added to culture media containing different concentrations of abamectin and incubated on a shaker at 180 rpm and 25°C. Samples were taken using a pipette at 24 h, 48 h, and 72 h, and spore germination was examined under a hemocytometer to determine the spore germination rate. Each treatment was repeated three times, with liquid culture medium containing sterile water serving as a blank control.

[0073] 2. Effects of abamectin on mycelial growth of strain SCAULS28

[0074] In the experiment, 2.5.1 concentrations of abamectin were added to each PDA culture dish (7.5 cm in diameter). The solution was evenly spread onto the culture medium surface using a sterilized triangular glass rod to prepare PDA culture plates with different concentrations of drug. A *Penicillium polonide* culture dish with evenly distributed hyphae was used. Mycelial cakes were punched using a 5 mm diameter punch on a clean bench and placed in the center of the drug-containing PDA plate for inoculation. The plates were then incubated in a 26±2℃ light incubator. Colony diameters were measured in mm on days 3, 5, and 7 using the cross-cross method. Each treatment was repeated in 5 replicates, with PDA culture medium containing sterile water serving as a control.

[0075] 3. Toxicity determination of strain SCAULS28 and the combination of abamectin against Spodoptera litura.

[0076] a. Toxicity determination of abamectin against second instar larvae of the beet armyworm

[0077] Prepare abamectin solutions with concentrations of 0.005, 0.01, and 0.01 mg / mL using sterile water, and follow the same steps as above.

[0078] b. Toxicity determination of the combination of strain SCAULS28 and abamectin against second instar larvae of the beet armyworm.

[0079] Indoor toxicity determination was performed according to the combinations in Table 1, using the same experimental methods and procedures as above.

[0080] Table 1. Combinations of strain SCAULS28 and abamectin

[0081]

[0082]

[0083] c. Data Statistics and Analysis

[0084] SPSS 19.0 software was used for experimental data processing and analysis. One-way ANOVA was employed to analyze the mortality rate of second-instar larvae of the beet armyworm, and Tukey's test was used to assess the significance of differences. The data calculation formulas were based on those of Huang et al. (2013), and the specific formulas are as follows:

[0085] Me = Ma + Mb × (1 - Ma)

[0086]

[0087] In the formula, the meanings of each term are as follows:

[0088] Ma: Actual corrected mortality rate of second-instar larvae of Spodoptera litura alone;

[0089] Mb: Actual corrected mortality rate of second-instar larvae of the beet armyworm alone by a highly virulent strain;

[0090] Mab: Actual corrected mortality rate of second-instar larvae of the beet armyworm using a fungicide-fungicide compound.

[0091] Experimental results:

[0092] 1. Virulence analysis of strain SCAULS28

[0093] Table 2 shows the mortality rate of second-instar larvae of the beet armyworm 5 days after infection with spore suspensions of strain SCAULS28 at different concentrations. The results indicate that spore concentrations below 1×10⁻⁶ significantly reduced mortality. 6 At a spore concentration of 1×10⁻⁶ / ml, strain SCAULS28 showed a mortality rate of less than 25% against second-instar larvae of the beet armyworm. 7 The lethality rate was 51.24% at a spore / ml concentration of 1×10⁻⁶. 8 The mortality rate of spores / ml was 61.2%, and the mortality rate against the beet armyworm increased with increasing spore concentration.

[0094] Table 2 Corrected mortality rate (%) of second instar larvae of the beet armyworm at different concentrations of spore suspension.

[0095] Treatment (spore concentration) / ml Corrected mortality rate (%) <![CDATA[1×10 8 ]]> 61.2±0.72 <![CDATA[1×10 7 ]]> 51.34±3.55 <![CDATA[1×10 6 ]]> 20.42±1.66 <![CDATA[1×10 5 ]]> 13.76±1.53 <![CDATA[1×10 4 ]]> 4.81±1.24 TW-80 Sterile Water 0.00

[0096] 2. Effects of abamectin on spore germination rate and mycelial growth of strain SCAULS28

[0097] Table 3 shows the experimental results. After 24 hours of treatment, the spore germination rates of strain SCAULS28 at concentrations of 0.005, 0.01, and 0.015 mg / L were 96.43%, 95.62%, and 95.36%, respectively, significantly different from the blank control (98.88%). After 48 hours of treatment, the spore germination rates of strain SCAULS28 at concentrations of 0.005, 0.01, and 0.015 mg / L were 98.68%, 98.45%, and 98.11%, respectively, not significantly different from the sterile water blank control (98.90%). After 72 hours of treatment, the effect of different concentrations of abamectin on the spore germination rate of strain SCAULS28 was not significantly different from the sterile water blank control. In treatments with 0.005, 0.01, and 0.015 mg / L abamectin, the spore germination rates of strain SCAULS28 were 98.84%, 98.62%, and 98.32%, respectively, which were not significantly different from the blank control (98.98%).

[0098] Table 3 shows the experimental results. When the concentration of abamectin was 0.005, 0.01, and 0.015 mg / L, the colony diameter of strain SCAULS28 on day 7 was 43.85 mm, 43.42 mm, and 43.54 mm, respectively, which was not significantly different from the blank control (43.60 mm).

[0099] The results showed that abamectin has good compatibility with strain SCAULS28 and can be compounded in subsequent experiments and practical applications.

[0100] Table 3 Effects of abamectin on spore germination rate and mycelial growth of strain SCAULS28

[0101]

[0102] Note: According to Tukey's test, different lowercase letters in the same column indicate significant differences in pathogenicity among different strains (P < 0.05).

[0103] 3. Toxicity determination of strain SCAULS28 and the combination of abamectin against Spodoptera litura.

[0104] 1) Results of toxicity test of abamectin against second instar larvae of the beet armyworm.

[0105] The corrected mortality rates of second-instar larvae of the beet armyworm varied significantly with different concentrations of abamectin. The corrected mortality rate of second-instar larvae increased with increasing abamectin concentration; when the abamectin concentration was 0.005–0.15 mg / L, the corrected mortality rate of second-instar larvae after treatment ranged from 47.21% to 100%.

[0106] Table 4. Results of toxicity assay of different concentrations of abamectin on second-instar larvae of Spodoptera litura.

[0107] Treatment concentration (mg / L) mortality rate(%) 0.005 65.19±17.98bc 0.01 90.52±1.97a 0.015 100±0a CK 0±0.00d

[0108] Note: According to Tukey's test, different lowercase letters in the same column indicate significant differences in pathogenicity among different strains (P < 0.05).

[0109] 2) Results of toxicity assay of strain SCAULS28 and abamectin synergistic effect on second instar larvae of the beet armyworm.

[0110] The toxicity of strain SCAULS28 synergistically with abamectin against second-instar larvae of the beet armyworm: Treatment of second-instar larvae of the beet armyworm with different concentrations of abamectin alone (0.005, 0.01, 0.015 mg / L) resulted in corrected mortality rates of 65.19%, 90.52%, and 100% after day 5, respectively. Treatment of second-instar larvae of the beet armyworm with different concentrations of abamectin alone (1×10⁻⁶ mg / L) resulted in toxicity rates of 65.19%, 90.52%, and 100%, respectively. 7 1×108 Treatment of second-instar larvae of the beet armyworm with spores / mL resulted in corrected mortality rates of 45.32% and 54.79% on day 3, and 51.32% and 61.2% on day 5, respectively.

[0111] The lethality of different concentrations of abamectin combined with different concentrations of strain SCAULS28 on the 5th day of second instar larvae of the beet armyworm was significantly higher than that of any single agent. In the toxicity test of the combined action of abamectin and strain SCAULS28 on second instar larvae of the beet armyworm, the combination T8 (1×10⁻⁶) showed the highest mortality rate. 7 +0.01), T9 (1×10 7 +0.015), T10(1×10 8 +0.01), T11(1×10 8 The compound formulations (+0.015%) all exhibited synergistic effects 1–5 days after treatment, with the highest corrected mortality rates being 93.34%, 100%, 100%, and 100%, respectively, and the differences were not significant. All four compound formulations showed good synergistic effects. Considering the dosage and actual lethality, the 1×10⁸ spores / ml strain SCAULS28 and the 0.01 mg / L abamectin compound formulation were the optimal combination.

[0112] Table 5. Corrected mortality rate of second instar larvae of the beet armyworm due to the synergistic effect of strain SCAULS28 and abamectin.

[0113]

[0114] Note: The data in the table are mean ± standard error. According to Tukey's test, different lowercase letters indicate significant differences between different combination concentrations at the same time (P < 0.05).

[0115] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An insecticide containing abamectin and an entomopathogenic fungal strain as active ingredients, wherein the entomopathogenic fungal strain is *Penicillium polonum* (…). Penicillium polonicum )SCAULS28, accession number: GDMCC No:65532; the insecticide described is used to control beet armyworm.

2. The insecticide according to claim 1, characterized in that, The Polish Penicillium is a suspension or spore powder of Polish Penicillium spores.

3. The insecticide as described in claim 2, characterized in that, The dosage of *Penicillium polonum* and abamectin was as follows: the final concentration of *Penicillium polonum* was 1 × 10⁻⁶. 4 ~1×10 9 Conidia / mL, with a final concentration of 0.005~0.05 mg / L for abamectin.

4. The insecticide according to claim 3, characterized in that, The dosage of *Penicillium polonum* and abamectin was as follows: the final concentration of *Penicillium polonum* was 1 × 10⁻⁶. 7 ~1×10 8 Conidia / mL, with a final concentration of 0.01~0.02 mg / L for abamectin.

5. The insecticide as described in claim 2, characterized in that, The ratio of *Penicillium polioense* to abamectin was 1 × 10⁻⁶. 4 ~1×10 9 Conidia / mL: 0.005~0.05 mg / L.

6. The insecticide as described in claim 5, characterized in that, The ratio of *Penicillium polioense* to abamectin was 1 × 10⁻⁶. 7 ~1.0×10 8 Conidia / mL: 0.01~0.02 mg / L.

7. The insecticide according to claim 6, characterized in that, The ratio of *Penicillium polioense* to abamectin was 1 × 10⁻⁶. 8 Conidia / mL: 0.01 mg / L.

8. The use of the insecticide as described in any one of claims 1 to 7 in the control of beet armyworm.

9. A method for controlling the beet armyworm, characterized in that, Apply the insecticide according to any one of claims 1 to 7 to crops or environments where control of beet armyworm is required.

10. The method as described in claim 9, characterized in that, Apply at the initial stage of the appearance of the beet armyworm.

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