Penicillium Polish with high pathogenicity to prodenia litura and application of compound of penicillium Polish and emamectin benzoate

Abstract

The invention relates to the field of biological control, and discloses screening of entomogenous fungi with high pathogenicity to prodenia litura and compound application of the entomogenous fungi and emamectin benzoate. The Penicillium Pomelis strain with high toxicity to prodenia litura is collected from Xinjiang Checkoean cotton land. The invention further provides an efficient, pollution-free and residue-free insecticide combined with emamectin benzoate and Penicillium bauhiniae, which has a synergistic interaction effect and is prepared by combining emamectin benzoate and Penicillium bauhiniae. Research on the compounding effect of emamectin benzoate and Penicillium Polish shows that after compounding, the insecticide can show an obvious synergistic effect, the prevention and treatment effect is improved, the use dosage of a single chemical pesticide can be obviously reduced, the environmental pollution is reduced, and the problems that the existing pesticide is easy to generate resistance, the pesticide effect is not obvious and the like are effectively solved.

Description

Technical Field

[0001] The invention relates to the field of plant protection, specifically to the field of biological control, and in particular to the biological control of Spodoptera litura. Background Art

[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 will cause environmental pollution. Today, the environmental pollution caused by pesticides is becoming increasingly serious. Pesticide pollution not only appears in soil, water and air, but also causes agricultural product pollution, which in turn endangers human health and poses a huge threat to human health (Zhu Cuixia. Pesticide use pollution problems and control measures in crop pest control [J]. Hebei Agricultural Machinery, 2024, (07): 67-69.).

[0003] Agricultural pests pose a huge threat to agriculture and horticulture throughout the world and cause inestimable economic losses. They can directly harm plants by sucking plant sap or other plant tissues, or indirectly harm crops by spreading plant pathogens. Today's pest control relies heavily on the use of chemical pesticides, but this also has a major drawback, such as the development of 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 of the promising control methods (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 lifehistory of the tobacco peach ap hid Myzuspersicae var. nicotianae. PLOS ONE 17(9): e0273791.).

[0004] Insect pathogenic fungi can parasitize on the surface and body of insects. There are 100 genera and more than 800 species of insect pathogenic fungi recorded in the world, of which more than 400 species are found in China, including 215 species of insect pathogenic fungi. In recent years, due to the unavoidable harm of chemical control to the environment, people have gradually begun to pay attention to and study biological control. Using insect pathogenic fungi to control pests is one of the important biological control methods (Wang Yudi, Tian Chuanbei. Research progress on the application of insect pathogenic fungi in mite control [J]. Plant Medicine, 2024, 3(01): 8-21.). Insect pathogenic fungi (EPF) are natural enemies that affect insect populations. They have long been considered as biological control agents for many pests and are often regarded as effective biological control agents in integrated pest management (IPM) projects. Pathogenic fungi are commonly used to control pests by flooding or inoculating fungal propagules. Some pathogenic fungi have been commercially used to control plant-sucking pests (Sani I, Jamian S, Saad N, Abdullah S, Mohd HataE, et al. (2023) Inoculation and colonization of the entomopathogenic fungi, Isaria javanica and Purpureocilliumlilacinum, in tomato plants, and their effect on seedling growth, mortality and adult emergence of Bemisia tabaci (Gennadius). PLOS ONE 18(5): e0285666.).

[0005] Spodoptera litura, belongs to the family Noctuidae of the order Lepidoptera. Also known as the tobacco cutworm, it is a species native to Southeast Asia. It spread to India and became an important pest in the Asia-Pacific region. Spodoptera litura is a polyphagous agricultural pest that has a significant economic impact on a variety of crops in countries around the world. The species has more than 120 plant species and has great potential to invade new areas due to its ability to adapt to new climatic and ecological conditions (Tesari, T., Leksono, AS, & Mustafa, I. (2024). Effectiveness of botanical pesticide combined with Beauveria bassiana on mortality, nutritional index and fecundity of Spodoptera litura L. Cogent Food & Agriculture, 10 (1).). The Spodoptera litura is distributed in all regions of my country, north and south, and feeds on 389 host plants including sweet potato, cotton, soybean, tobacco, etc. The larvae have a omnivorous diet and a large appetite, and they have the characteristics of intermittent outbreaks. They are an important 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 the Spodoptera litura in tobacco fields [J]. Biological Disaster Science, 2023, 46(04): 438-444.).

[0006] Penicillium polonicum is a broad-spectrum insecticidal fungus. It mainly reproduces asexually in nature, actively invades the insect body wall and infects different insects. It can reproduce in a yeast-like manner in the insect blood cavity, produce a large number of insect fungi, and produce insecticidal toxins such as destructin, which inhibit insect immunity and accelerate insect killing. Emamectin benzoate has the characteristics of ultra-high efficiency, low toxicity (the preparation is almost non-toxic), low residue, and pollution-free biological pesticides for insects. The use of Penicillium polonicum and Emamectin benzoate in synergistic use to control Spodoptera litura can overcome the problem of slow effect of Penicillium polonicum and at the same time delay the effect of pest resistance, thereby achieving a good insecticidal effect.

[0007] Since Polish Penicillium has strong specialization and the virulence of different strains varies greatly, when conducting experiments on strain-compound pesticides, different regions and different types of pests require corresponding pathogenic fungi and screening of highly toxic strains.

[0008] Since the use of Penicillium alone to control pests such as Spodoptera litura often fails to achieve the desired effect, Penicillium has a slow insecticidal effect, is greatly affected by the environment, and has an unstable effect. The combination of entomogenic fungi and pesticides to control pests has become a hot topic in recent years. Through the combination of fungi and pesticides, the harm of chemical pesticides to the environment and natural enemies can be reduced, the utilization efficiency of biological pesticides can be improved, and suitable chemical agents can be screened for compounding biological control fungi to alleviate the new problem of the rapid increase of resistance to pesticides in Spodoptera litura. Emamectin benzoate has the characteristics of ultra-high efficiency, low toxicity (the preparation is nearly non-toxic), low residue, and pollution-free biological pesticides against insects. The use of Polish Penicillium and Emamectin benzoate in synergistic use to control Spodoptera litura can overcome the problem of slow effect of Polish Penicillium and at the same time delay the resistance of pests, thereby achieving a good insecticidal effect. Summary of the invention

[0009] The purpose of the present invention is to develop biological control of Spodoptera litura in tobacco fields, which is an important demand of agricultural production, and to provide a strain of Penicillium oxysporum with high toxicity to Spodoptera litura collected from cotton fields in Chabuchaer, Xinjiang. At the same time, a highly efficient, pollution-free, and residue-free insecticide for use with emamectin benzoate and Penicillium oxysporum is provided. The present invention studies the compounding effect of emamectin benzoate and Penicillium oxysporum and finds that the insecticide can show a significant synergistic effect after compounding, improves the control effect, and can significantly reduce the dosage of a single agent of chemical pesticides, reduce environmental pollution, and effectively solve the problems of existing pesticides being prone to resistance and having unclear efficacy.

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

[0011] The collected, isolated and purified strains were then subjected to virulence determination and analysis to screen out strains with high virulence to Spodoptera litura. Experiments have found that strains of different genera or the same species may have different pathogenicity to Spodoptera litura, and thus have different degrees of pathogenicity. According to the experimental results of the present invention, combined with the comprehensive consideration of the indicators for screening excellent strains, among which pathogenicity is an important indicator for measuring the biological control potential of fungal strains, and the growth rate of colonies or hyphae is another important evaluation indicator for evaluating excellent insect pathogenic fungi. The strain SCAULS28 has a high virulence rate of 10 8 The strain SCAULS28 had a high pathogenicity to the nymphs of Spodoptera litura at spores / mL. Considering the colony growth rate of the strain, the strain SCAULS28 was an excellent strain for causing disease to the nymphs of Spodoptera litura.

[0012] The fungus was isolated from soil collected from cotton fields in Chabuchaer, Xinjiang. After culturing on PDA plates at 26°C for 10 days, the colony diameter was 63.75 mm and the spore production was 1.237×10 8 spores / ml, and the spore germination rate exceeds 95%. Figure 1As shown, the colony is velvety in the middle, powdery at the edge, green on the surface, red on the back, with a few wrinkles. The spores are oval and nearly spherical. Through molecular biology, it was further verified and identified that it belongs to Penicillium polonicum.

[0013] The present invention is based on the fact that the screened SCAULS28 has a certain biological control effect on the Spodoptera litura. In order to further seek a better control effect, after a large number of explorations and exploratory experiments, it is found that the combination of emamectin benzoate and Polish Penicillium SCAULS28 can produce a significant synergistic effect, has a significant control effect on the Spodoptera litura, and finally completes the present invention.

[0014] Therefore, the present invention provides an entomogenous fungus strain with high pathogenicity to Spodoptera litura, which is Penicillium polonicum, deposited with Guangdong Microbiological Culture Collection Center (GDMCC for short, the address of the depository is Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China) on November 22, 2024, with a deposit number of GDMCC NO: 65532.

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

[0016] The invention also provides an insecticide containing emamectin benzoate and penicillium polishi as effective ingredients; the insecticide is used for preventing and controlling Spodoptera litura.

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

[0018] Preferably, the usage ratio of the said Penicillium polishii and emamectin benzoate is: the final concentration of the said Penicillium polishii is 1×10 4 ~1×10 9 Conidia / mL, preferably 1×10 7 ~1×10 8 conidia / mL, the final concentration of emamectin benzoate is 0.001-0.05 mg / L, preferably 0.01-0.05 mg / L.

[0019] More preferably, the ratio of the Penicillium polishi and emamectin benzoate is 1×10 4 ~1×10 9 Conidia / mL: 0.005-0.025 mg / L, preferably the ratio of Penicillium policum to emamectin benzoate is 1×10 7 ~1.0×10 8 Conidia / mL: 0.01~0.02mg / L.

[0020] More preferably, the ratio of the polish mold to the emamectin benzoate is 1×10 8 Conidia / mL: 0.01 mg / L.

[0021] The present invention further provides application of the insecticide in preventing and controlling Spodoptera litura.

[0022] The present invention also provides a method for controlling Spodoptera litura, which comprises applying the strain or the insecticide to crops or environments where Spodoptera litura needs to be controlled, specifically, applying the strain or the insecticide at the early stage of the emergence of Spodoptera litura.

[0023] The highly toxic Polish Penicillium strain to Spodoptera litura was collected from cotton fields in Chabuchaer, Xinjiang. Further, by studying the compounding effect of emamectin benzoate and Polish Penicillium, it was found that the compounded insecticide can show significant synergistic effect, improve the control effect, and significantly reduce the dosage of chemical pesticides, reduce environmental pollution, and effectively solve the problems of easy resistance and unclear efficacy of existing pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 This is the spore morphology of SCAULS28.

[0026] Figure 3 This is the electrophoresis diagram of PCR amplification products in molecular identification.

[0027] Figure 4 Phylogenetic tree. DETAILED DESCRIPTION

[0028] The following is an introduction through the research and development process and specific implementation methods of the present invention, but does not constitute a limitation of the present invention.

[0029] Example 1: Initial screening of strains

[0030] 1. Test insects

[0031] The eggs of Spodoptera litura were purchased from Keyun Biological Co., Ltd. and were reared with artificial diet in an artificial climate chamber of the Engineering Research Center of Ministry of Education for Biological Control to form 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) Collection of soil samples and isolation and purification of fungi

[0034] Soil samples were collected. When sampling, about 100g of soil was taken from 10 to 15cm below the surface soil, which was sealed in a plastic bag and taken back to the laboratory for processing. The soil samples were sieved to remove stones and debris, and then 10g of clean soil was suspended in 90mL of 0.1% Tween 80 solution, shaken, and allowed to stand for 15min. 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 on a Bengal red agar medium plate, and the suspension was evenly pushed under the surface of the plate with a triangular glass scraper. It was cultured at 25℃ for 3 to 7d, and then a single colony was cut with an inoculation loop and inoculated on a PDA plate for further culture to obtain the strain SCAULS28. The mycelium block was cut and transplanted to the PDA slope, and cultured continuously and stored in a refrigerator at 4℃.

[0035] 2) Observation of biological characteristics of strains

[0036] Preparation of potato glucose medium: 200g potatoes, 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 patterns, edges, exudates, etc. on the front and back of the colony and take pictures.

[0037] Determination of colony growth rate: Inoculate the purified strain SCAULS28 onto a PDA plate, place it in a 26°C incubator and culture it for 7-10 days. Use a 5mm hole puncher to punch holes in the cultured medium, then inoculate it onto a new PDA plate, seal it with a sealing film, and place it in a 26°C constant temperature incubator for culture. Repeat 5 times. Use the cross method to measure the colony diameter and record it, and calculate the average daily growth.

[0038] Determination of spore production: Use a pipette to transfer 500 μL of a 1×10 4 The spore suspension of spores / mL was inoculated on a 90mm culture dish. The culture dish was rotated at a constant speed to make the liquid evenly distributed on the surface of the culture medium, and then sealed with a sealing film and placed in a 26°C incubator for constant temperature culture. Repeat 5 times. After 15 days of culture, 5 colonies with uniform growth in the culture medium were taken with a 10mm puncher, added to 20mL of 0.5‰ Tween-80 solution, and stirred at medium speed for 20 minutes using a constant temperature magnetic stirrer to prepare a spore suspension. The content was determined using a hemocytometer.

[0039] Determination of spore germination rate: Take 1 mL of spores with a concentration of 1×10 5Spore suspensions with a concentration of spores / mL were added to 10 mL of SDY medium and cultured at 26°C and 220 r / min for 24 h, repeated 5 times. An appropriate amount of bacterial liquid was dropped on a glass slide, placed under a microscope, and the germination of spores was observed and recorded. Spores with a germ tube length greater than or equal to the spore diameter were considered to have germinated. No less than 50 spores were examined under the microscope for each treatment, and the examination was repeated 5 times.

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

[0041] 3) Molecular biological identification

[0042] The purified strains were identified by molecular biology.

[0043] DNA was extracted using the CTAB method, which included the following steps:

[0044] (a) After culturing strain SCAULS28 on a PDA plate for one week, the mycelium was carefully scraped into a mortar and quickly and thoroughly ground by adding liquid nitrogen;

[0045] (b) Take an appropriate amount of ground mycelium and quickly transfer it to a centrifuge tube, add 300 μL of DNA extraction solution 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, shake gently once every 10 min, and centrifuge at 12,000 rpm for 5 min at room temperature;

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

[0047] (d) Slowly pipette 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) adding 70% ethanol to wash the precipitate twice;

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

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

[0051] (h) The extract was detected by 1% agarose gel electrophoresis and the remaining extract was stored at -20°C.

[0052] The primers used in the PCR of fungal DNA were universal primers, and the amplification conditions were as follows. The PCR products were subjected to 1% agarose gel electrophoresis (containing EB), the buffer was 1×TAE, the voltage was 150V, the current was 220mA, and Marker DL2000 was used as a molecular weight standard reference. After the electrophoresis, the gel was detected on a gel imager and photographed and recorded.

[0053] The specific PCR products were sequenced by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. Sequence alignment and phylogenetic tree construction: The sequencing results were searched for related sequences with high homology using BLAST, and the Cluster W in MEGA5.1 software was used for sequence alignment and multiple sequence alignment. A Neighbour-joining (NJ) phylogenetic tree was constructed in the software.

[0054] 4) Toxicity determination and analysis on second-instar larvae of Spodoptera litura

[0055] a. Preparation of spore suspension of strain SCAULS28

[0056] Take the strain SCAULS28 that has been cultured for about 10-14 days, add distilled water containing 0.05% Tween-80 in the clean bench, scrape off the colony, wash out the spores in the culture dish, transfer to a sterilized triangular flask, use a constant temperature magnetic stirrer to stir at medium speed for 20 minutes to make it evenly dispersed, and prepare a mother solution. Dilute the bacterial solution 100 times according to the 10-fold dilution method, count the number of conidia with a hemocytometer under a microscope, and repeat 5 plates. Then dilute the conidia concentration to 1×10 8 Spores / mL for later use.

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

[0058] The feed was fed at a concentration of 1×10 8 The larvae were soaked in a spore suspension of spores / mL for about 30 seconds, and then fed with it. Sterilized 0.05% Tween-80 water was used as a control, 30 larvae were treated in each treatment, and each treatment was repeated 3 times. After the inoculation was completed, all culture dishes (with a diameter of 12 cm) were ventilated with insect-proof nets after opening holes and placed in a biochemical incubator for breeding. Regular observations and records were made every day for 5 consecutive days, and the dead insects were removed in time. The dead insects were placed in a moisturizing culture at 26°C and confirmed to be effectively lethal based on the characteristics of the growths on the body surface of the 2nd instar larvae of the dead insects. The infected dead insects were dead insects.

[0059] c. Perform concentration gradient analysis on highly virulent strains

[0060] The spore powder of the strain with better virulence effect was taken and prepared into a concentration of 1×10 8The spore suspension was diluted in a certain gradient, and the final dilution concentration was 1×10 8 spores / mL, 1×10 7 spores / mL, 1×10 6 spores / mL, 1×10 5 spores / mL, 1×10 4 The mortality rate of the strain SCAULS28 on the second-instar larvae of Spodoptera litura at different concentrations was determined by feeding method.

[0061] Experimental Results

[0062] 1. Biological characteristics of strain SCAULS28

[0063] On the PDA plate, at 26°C, the colony diameter was 64.62 mm and the spore production was 1.478×10 8 spores / ml, and the spore germination rate exceeds 95%. Figure 1 As shown, the colony is velvety in the middle, powdery at the edge, green on the surface, red on the back, with a few wrinkles. The spores are oval and nearly spherical.

[0064] 2. Molecular Biology Identification

[0065] (1) Figure 3 As shown, the electrophoresis bands in each lane are relatively clear, and the gene fragment of the strain can be accurately judged to be about 600 bp based on the migration distance of the PCR product.

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

[0067] 3. Screening and toxicity analysis of highly toxic strains of Spodoptera litura

[0068] The virulence of the eight strains was tested by dipping the larvae into a concentration of 1×10 8The larvae were immersed in a spore suspension of spores / mL for about 5 seconds, and then moved into a culture dish containing feed for observation. Sterile 0.05% Tween-80 water was used as a control, 30 larvae were treated in each treatment, and each treatment was repeated 3 times. After the inoculation was completed, all culture dishes (12 cm in diameter) were sealed with plastic wrap and punctured for ventilation, and then placed in a biochemical incubator for breeding. Regular observations and records were made every day for 5 consecutive days, and the insect corpses were removed in time. The insects were placed in a moisturizing culture at 26°C and confirmed to be effectively killed based on the characteristics of the growths on the body surface of the second-instar larvae of the Spodoptera litura. The infected insect corpses were dead insects. The experimental results showed that at the same concentration, the pathogenicity of the eight entomogenous fungi varied. When the spore suspension concentration was 1×10 8 spores / ml, after 5 days of treatment, the corrected mortality rates of the eight strains against the second-instar larvae of Spodoptera litura were between 17.31% and 57.84%, among which the strain SCAULS28 had the highest corrected mortality rate of 57.84%, which was significantly higher than that of the other strains. This also showed that the strain SCAULS28 had a stronger pathogenicity than the other strains.

[0069] The results showed that strain SCAULS28 had a good lethal effect on the second-instar larvae of Spodoptera litura, and the pathogenic effect of strain SCAULS28 was significantly better than other strains, indicating that it is a potential excellent biocontrol bacterium.

[0070] Example 2: Compatibility study of highly toxic strains and emamectin benzoate and screening of combined doses

[0071] 1. Effect of Emamectin Benzoate on Spore Germination Rate of Strain SCAULS28

[0072] During the test, the strain to be tested was first transferred to a PDA plate and cultured at 26±1°C. After conidia were produced, the spores were washed with 0.05% Tween-80 sterile water, stirred with a magnetic stirrer, filtered, and counted by hemocytometer. The spores were then prepared into 1×10 6 The spore suspension of 1×10 spores / mL was prepared for later use. Emamectin benzoate was dissolved in acetone to prepare a 10 mg / L agent for later use. Emamectin benzoate was added to 50 mL of SDA liquid culture medium to prepare SDA liquid culture medium with emamectin benzoate concentrations of 0.005, 0.015, and 0.01 mg / L. 2 mL of 1×10 6 Spores / mL spore suspension was added to the culture medium with different concentrations of emamectin benzoate and placed in a shaker at 180rpm and 25℃ for shaking culture. Samples were taken with a pipette at 24h, 48h and 72h, and the spore germination of each treatment was examined under a microscope on a hemocytometer, and the spore germination rate was calculated. Each treatment was repeated 3 times, and the liquid culture medium with sterile water was used as a blank control.

[0073] 2. Effect of Emamectin Acid on Mycelial Growth of Strain SCAULS28

[0074] During the test, add emamectin benzoate of the corresponding concentration of 2.5.1 to each PDA culture dish (7.5cm in diameter), and use a high-temperature sterilized triangular glass rod to evenly spread the liquid on the surface of the culture medium to make PDA culture medium plates with different concentrations of drugs. Take a Polish Penicillium culture dish with uniform mycelium distribution, punch the bacterial cake with a 5mm diameter puncher on the clean bench, place the bacterial cake in the center of the drug-containing PDA plate for inoculation, and culture it in a light incubator at 26±2℃. Use the cross method to measure the colony diameter on the 3rd, 5th and 7th days, respectively, in mm. Each treatment is repeated 5 times, and the PDA culture medium with sterile water is used as a control.

[0075] 3. Toxicity test of the combination of strain SCAULS28 and emamectin benzoate against Spodoptera litura

[0076] a. Toxicity test of emamectin benzoate to the second instar larvae of Spodoptera litura

[0077] Use sterile water to prepare emamectin benzoate with concentrations of 0.005, 0.01, and 0.01 mg / mL, and the remaining steps are the same as above.

[0078] b. Toxicity test of the combination of strain SCAULS28 and emamectin benzoate against the second-instar larvae of Spodoptera litura

[0079] According to the combination in Table 1, indoor toxicity test was carried out, and the experimental method and steps were the same as above.

[0080] Table 1. Combination of strain SCAULS28 and emamectin benzoate

[0081]

[0082]

[0083] c.Data statistics and analysis

[0084] SPSS 19.0 software was used to process and analyze the experimental data. One-way ANOVA was used to analyze the results of the mortality rate of the second-instar larvae of Spodoptera litura, and Tukey's test was used to test the significance of the difference. The data calculation formula was based on Huang et al. (2013), and the specific formula is as follows:

[0085] Me=Ma+Mb×(1-Ma)

[0086]

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

[0088] Ma: actual corrected mortality of emamectin benzoate alone against the second instar larvae of Spodoptera litura;

[0089] Mb: actual corrected mortality of the highly virulent strain against the second-instar larvae of Spodoptera litura alone;

[0090] Mab: Actual corrected mortality of fungicide combination against second instar larvae of Spodoptera litura.

[0091] Experimental results:

[0092] 1. Virulence analysis of strain SCAULS28

[0093] The mortality of the second-instar larvae of Spodoptera litura infected with spore suspensions of strain SCAULS28 at 5 days after infection is shown in Table 2. The results showed that the spore concentration was lower than 1×10 6 spores / ml, the lethality of strain SCAULS28 to the second-instar larvae of Spodoptera litura was less than 25%, and the spore concentration was 1×10 7 The lethality rate of spores / ml was 51.24% and the spore concentration was 1×10 8 The mortality rate of spores / ml was 61.2%. As the spore concentration increased, the mortality rate to Spodoptera litura also increased.

[0094] Table 2 Corrected mortality of spore suspensions of different concentrations against second-instar larvae of Spodoptera litura (%)

[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 Emamectin Benzoate on Spore Germination Rate and Mycelial Growth of Strain SCAULS28

[0097] The test results in Table 3 show that when treated for 24 hours, the spore germination rates of strain SCAULS28 were 96.43%, 95.62%, and 95.36% respectively at 0.005, 0.01, and 0.015 mg / L concentrations, which were significantly different from the blank control (98.88%). When treated for 48 hours, the spore germination rates of strain SCAULS28 were 98.68%, 98.45%, and 98.11% respectively at 0.005, 0.01, and 0.015 mg / L concentrations of emamectin benzoate, which were not significantly different from the sterile water blank control (98.90%). When treated for 72 hours, the effects of different concentrations of emamectin benzoate on the spore germination rate of strain SCAULS28 were not significantly different from the sterile water blank control. In the treatments of 0.005, 0.01, and 0.015 mg / L emamectin benzoate, 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] The test results in Table 3 show that when the concentrations of emamectin benzoate were 0.005, 0.01, and 0.015 mg / L, the colony diameters of strain SCAULS28 on the 7th day were 43.85 mm, 43.42 mm, and 43.54 mm, which were not significantly different from the blank control (43.60 mm).

[0099] The results showed that emamectin benzoate had good compatibility with strain SCAULS28 and could be compounded in subsequent experiments and practical applications.

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

[0101]

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

[0103] 3. Toxicity test of the combination of strain SCAULS28 and emamectin benzoate against Spodoptera litura

[0104] 1) Results of toxicity test of emamectin benzoate against second-instar larvae of Spodoptera litura

[0105] The corrected mortality rates of the second-instar larvae of Spodoptera litura treated with different concentrations of emamectin benzoate were different and the differences were significant. With the increase of emamectin benzoate concentration, the corrected mortality rate of the second-instar larvae of Spodoptera litura also increased. When the concentration of emamectin benzoate was 0.005-0.15 mg / L, the corrected mortality rate of the second-instar larvae of Spodoptera litura after treatment was 47.21%-100%.

[0106] Table 4 The results of toxicity test of different concentrations of emamectin benzoate on the second instar larvae of Pseudomonas obliquus

[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 test, different lowercase letters in the same column indicate significant differences in pathogenicity between different strains (P < 0.05)

[0109] 2) Results of toxicity test of strain SCAULS28 and emamectin benzoate against the second-instar larvae of Spodoptera litura

[0110] The toxicity of strain SCAULS28 and emamectin benzoate to the second-instar larvae of Spodoptera litura was as follows: when emamectin benzoate (0.005, 0.01, 0.015 mg / L) was used alone to treat the second-instar larvae of Spodoptera litura, the corrected mortality rates after the 5th day were 65.19%, 90.52%, and 100%, respectively; when strain SCAULS28 (1×10 7 , 1×108 spores / mL) were used to treat the 2nd instar larvae of Spodoptera litura, the corrected mortality rates on the 3rd day were 45.32% and 54.79%, and the corrected mortality rates on the 5th day were 51.32% and 61.2%, respectively.

[0111] The mortality rate of the combination of different concentrations of emamectin benzoate and different concentrations of strain SCAULS28 on the second instar larvae of Spodoptera litura on the 5th day was significantly higher than that of each single agent. In the toxicity test of the combined action of emamectin benzoate and strain SCAULS28 on the second instar larvae of Spodoptera litura, the combination T8 (1×10 7 +0.01), T9(1×10 7 +0.015), T10(1×10 8 +0.01), T11(1×10 8 +0.015) showed synergistic effects 1 to 5 days after treatment, with the highest corrected mortality rates of 93.34%, 100%, 100%, and 100%, respectively, and the differences were not significant. These four combinations all had good synergistic effects. Combined with the dosage and actual lethal effect, 1×108 spores / ml strain SCAULS28 and 0.01mg / L emamectin benzoate combination was the best combination.

[0112] Table 5 Corrected mortality of the synergistic effect of strain SCAULS28 and emamectin benzoate on the second instar larvae of Spodoptera litura

[0113]

[0114] Note: The data in the table are mean ± standard error. After Tukey test, different lowercase letters indicate significant differences among 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 their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An entomogenous fungus strain with high pathogenicity to Spodoptera litura, characterized in that: It is Penicillium polishii ( Penicillium polonicum ), the deposit number is: GDMCC No:65532.

2. Use of the strain as claimed in claim 1 in biological control of Spodoptera litura.

3. An insecticide containing emamectin benzoate and Penicillium polishii as effective ingredients, wherein the deposit number of the Penicillium polishii is GDMCC No: 65532; the insecticide is used for controlling Spodoptera litura.

4. The insecticide according to claim 3, characterized in that The Penicillium polishii is in the form of a suspension or spore powder of Penicillium polishii spores.

5. The insecticide according to claim 4, characterized in that The dosage ratio of the Polish Penicillium and emamectin benzoate is: the final concentration of the Polish Penicillium is 1×10 4 ~1×10 9 Conidia / mL, preferably 1×10 7 ~1×10 8 conidia / mL, the final concentration of emamectin benzoate is 0.005~0.05mg / L, preferably 0.01~0.02mg / L.

6. The insecticide according to claim 4, characterized in that The ratio of the Polish Penicillium to the emamectin benzoate is 1×10 4 ~1×10 9 Conidia / mL: 0.005~0.05mg / L, preferably the ratio of Polish Penicillium to Emamectin Benzoate is 1×10 7 ~1.0×10 8 Conidia / mL: 0.01~0.02mg / L.

7. The insecticide according to claim 6, characterized in that The ratio of the Polish Penicillium to the emamectin benzoate is 1×10 8 Conidia / mL: 0.01 mg / L.

8. Use of the insecticide according to any one of claims 3 to 7 in controlling Spodoptera litura.

9. A method for controlling Spodoptera litura, characterized in that: Apply the strain according to claim 1 or the insecticide according to any one of claims 3 to 7 to crops or environments where Spodoptera litura needs to be controlled.

10. The method according to claim 9, characterized in that Apply at the early stage of Spodoptera litura emergence.

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