Insecticide for preventing and controlling forest and herbivorous defoliating pests
Through the combination of insecticides of the frozen mucor strain and azadirtin, the problem of toxicity degradation of microbial pesticides was solved, and the insecticide effect on yellow-spine bamboo locust was significantly improved, and green and efficient pest control was achieved.
Patent Information
- Application Number
- CN202411508130.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2025-02-14
AI Technical Summary
Existing microbial pesticides are prone to toxicity deterioration during the subculture process, making it difficult to effectively prevent and control forest leaf-eating pests such as yellow-spine bamboo locusts.
Mucor hiemalis DTMM-01 and azadirectin were combined at a mass ratio of 9:1 or 4:1 to form insecticides to improve the insecticide effect on yellow-spine bamboo locust.
The combined insecticides of the frozen mucor strain and azadirtin were significantly improved, especially when the ratio of 9:1, the cotoxicity coefficient was greater than 120, showing obvious Efficacy effect.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of pesticides, and in particular to a pesticide for preventing and controlling forest and grass leaf-feeding pests. Background Art
[0002] The yellow-spine bamboo locust (Ceracris kiangsu Tsai) is a migratory agricultural and forestry pest of the genus Ceracris in the superfamily Acridodea of the order Orthoptera. It mainly feeds on bamboo. When there is a large amount of food shortage, the yellow-spine bamboo locust also feeds on crops such as rice, sugarcane, corn and banana. Its food source is diverse and its damage is widespread. The yellow-spine bamboo locust is mainly distributed in the south of the Yellow River in my country. The annual average temperature is 17℃~23℃, which is the suitable habitat for the yellow-spine bamboo locust. The nymphs of the yellow-spine bamboo locust are called jumping nymphs, and there are 5 age classes. Starting from the third-instar jumping nymphs, the larvae of the yellow-spine bamboo locust all go to bamboo and are voracious eaters, which has a great impact on agricultural and forestry safety production.
[0004] Green control has always been the direction of sustainable locust management advocated. Predatory natural enemies, parasitic natural enemies, locust microsporidia, Metarhizium anisopliae, Buddaria officinalis extract, matrine and azadirachtin are the main ideas for green control of locusts. In order to solve the problem of toxicity degradation of microbial pesticides during subculture and provide rich microbial pesticide resources, this paper isolated a strain of permafrost Mucor from a caterpillar in Dongchuan District, Yunnan Province. The strain was used to determine the toxicity of the yellow-spine bamboo locust nymph indoors, and the strain was combined with azadirachtin for control experiments, aiming to provide a basis for green control of the yellow-spine bamboo locust. Summary of the invention
[0005] The main purpose of the present invention is to provide an insecticide for preventing and controlling forest grass leaf-feeding pests, which can be used for preventing and controlling yellow-spine bamboo grasshopper.
[0006] Specifically, to achieve the above objectives, the present invention provides the following technical solutions:
[0007] A frozen soil Mucor strain (Mucor hiemalis) DTMM-01, with a deposit number of GDMCC No: 65205 and a taxonomic name of Mucor hiemalis.
[0008] Furthermore, the present invention provides an insecticide containing the above-mentioned permafrost Mucor strain.
[0009] Furthermore, the active ingredients in the insecticide are composed of the permafrost Mucor strain and azadirachtin.
[0010] Furthermore, the pesticide contains a spore suspension of the permafrost Mucor strain.
[0011] Furthermore, the concentration of the spore suspension of the permafrost Mucor strain contained in the pesticide is not less than 2.02×10 3 spores / ml.
[0012] Furthermore, the concentration of azadirachtin is not less than 4.87 mg / mL.
[0013] Furthermore, the insecticide is prepared by mixing spores of the frozen soil Mucor strain and azadirachtin in a mass ratio of 9:1 or 4:1.
[0014] Furthermore, the present invention provides the use of the permafrost Mucor strain or any of the insecticides in the control of forest and grass leaf-feeding pests.
[0015] Preferably, the forest grass leaf-feeding pest is the yellow-spine bamboo grasshopper.
[0016] Finally, the present invention provides a method for controlling yellow-striped bamboo locusts. The method comprises applying a compound insecticide of permafrost Mucor strain and azadirachtin during the third-instar stage of the yellow-striped bamboo locusts. The compound insecticide is a compound of permafrost Mucor strain spores and azadirachtin in a mass ratio of 9:1 or 4:1.
[0017] The technical effects achieved by the present invention are:
[0018] The present invention adopts the leaf dipping method to determine the indoor toxicity and combined effects of different concentrations of frozen soil Mucor strain DTMM-01, azadirachtin, and a mixture of frozen soil Mucor and azadirachtin on the third instar larvae of yellow-spine bamboo grasshopper. The results show that the mortality rate of yellow-spine bamboo grasshopper larvae is proportional to the concentration of frozen soil Mucor spore suspension. The concentration of spore suspension is 1.0×10 8 Spores·mL -1 The 7-day mortality rate of the yellow-spine locust larvae was the highest at 76.67% and the concentration was 1.0×10 4 Spores·mL -1 The 7-day mortality rate of the yellow-spine bamboo grasshopper larvae was the highest and the lowest at 46.67%. The mortality rate of the yellow-spine bamboo grasshopper larvae was positively correlated with the concentration of azadirachtin, and the concentration of azadirachtin was 500.00 mg·mL -1 The 7-day mortality rate of the yellow-spine locust larvae was the highest at 80.00% at a concentration of 0.5 mg·mL -1 The 7-day mortality rate of the yellow-spine bamboo grasshopper larvae was the highest and the lowest, with a mortality rate of 33.33%; after the frozen soil Mucor and azadirachtin were mixed in a volume ratio of 9:1, 4:1, 1:1, 1:4 and 1:9, the LC of the yellow-spine bamboo grasshopper larvae 50 3.50, 9.42, 12.63, 25.52 and 14.39 g·L respectively -1,The co-toxicity coefficients of different mixed combinations are different. The co-toxicity coefficients of the mixtures of Mucor and azadirachtin at 9:1 and 4:1 are greater than 120, and the mixed scheme has a significant synergistic effect. Although azadirachtin has a certain bactericidal effect, when the number of Mucor spores in the mixed scheme reaches a certain amount, the mixture of azadirachtin and Mucor can show a significant synergistic effect, indicating that microbial pesticides and botanical pesticides can achieve insecticidal effects through mixing. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Statistical chart of toxicity test of frozen soil Mucor strain DTMM-01 (different concentrations) on the third-instar nymph of yellow-spine bamboo grasshopper;
[0020] Figure 2 Statistical chart of the toxicity test of azadirachtin to the third-instar nymphs of the yellow-spine bamboo locust;
[0021] Figure 3 Statistical chart of the toxicity test of different mixing modes of permafrost Mucor DTMM-01 and azadirachtin on the third-instar nymphs of the yellow-striped bamboo locust. DETAILED DESCRIPTION
[0022] The present invention will be further described below by means of the accompanying drawings and embodiments.
[0023] Example 1
[0024] 1 Materials and methods
[0025] 1.1 Materials
[0026] Insect source: Yellow-striped bamboo grasshopper nymphs were collected from Mengla County, Jinghong City, Xishuangbanna Prefecture, Yunnan Province. They were reared in a greenhouse with awnless brome (awnless brome seeds were purchased from Beijing Zhengdao Seed Co., Ltd.) under the conditions of (25±2)℃, relative humidity (80±5)%, and photoperiod of 16L:8D. The 3rd instar nymphs of similar size were selected for the experiment.
[0027] Test strains and agents: The frozen soil Mucor strain (DTMM-01) in this article was derived from Trichoderma heterotrophicum in Dongchuan District, Kunming City, and was stored in a -80°C refrigerator in a test tube. When the strain was used, it was activated in PDA culture medium; azadirachtin was purchased from Kunming Baoteng Biochemical Technology Co., Ltd.
[0028] Preservation information of the frozen soil Mucor strain (DTMM-01): the preservation name is: Mucor hiemalis DTMM-01, the preservation unit is: Guangdong Provincial Microbiological Culture Collection Center; the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the preservation time is September 24, 2024, the preservation number is: GDMCC No: 65205, the taxonomic name is: Mucor hiemalis, the biological material was received by the preservation center on September 24, 2024, and registered. According to the request, it will be preserved for thirty years from that date. The viability of the biological material was tested by the preservation center on September 24, 2024, and the result was viable.
[0029] 1.2 Determination of the toxicity of Mucor to the yellow-spine bamboo grasshopper
[0030] 1.2.1 Preparation of frozen soil Mucor suspension
[0031] The frozen soil Mucor strains stored at -80℃ were picked up and placed on PDA medium with antibiotics added, and then cultured in a 25℃ incubator for 5-7 days. Then, the vigorous colonies were picked up and cultured on new medium. The above process was repeated 2-3 times to activate the strains. The spores on the activated PDA plate were washed with sterile water containing 0.1% Tween-80 and the concentration was 1.0×10 8 Spores mL -1 The mother liquid of frozen soil Mucor spore suspension was diluted to 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 Spores·mL -1 of frozen soil Mucor spore suspension.
[0032] 1.2.2 Toxicity determination of different concentrations of frozen soil Mucor to the third instar nymphs of the yellow-spine bamboo grasshopper
[0033] The toxicity of the third-instar hoppers of the yellow-spine bamboo locust was determined by leaf dipping. The test hoppers were starved for 12 hours, and different concentrations of frozen soil Mucor spore suspensions were sprayed on the awnless brome with a handheld pressure sprayer, with a spraying amount of 100 mL each time. The awnless brome sprayed with different concentrations of frozen soil Mucor spore suspensions was fed to the starved hoppers, and the number of dead insects was recorded once at the same time every day for 7 consecutive days to calculate the half-lethal concentration LC 50 Each treatment was repeated 3 times with 10 test insects, and Bromus sibiricus sprayed with 0.1% Tween-80 solution was used as a blank control.
[0034] 1.3 Toxicity determination of different concentrations of azadirachtin to the third instar nymphs of the yellow-striped bamboo locust
[0035] Dilute the azadirachtin stock solution with sterile distilled water to prepare concentrations of 0.5, 1, 5, 50, and 500 mg mL -1 The method for determining the toxicity of azadirachtin to yellow-spine bamboo locust hoppers is the same as above.
[0036] 1.4 Toxicity test of the mixture of DTMM-01 and azadirachtin to the third-instar nymphs of the yellow-striped bamboo grasshopper
[0037] The frozen soil Mucor spore suspension and azadirachtin were prepared into LC 50 The concentration was then mixed in 5 volume ratios of 9:1, 4:1, 1:1, 1:4, and 1:9. The toxicity of the mixture of DTMM-01 and azadirachtin to the third-instar nymphs of the yellow-spine bamboo locust was determined by the leaf immersion method. The method was the same as above. The number of dead insects was recorded once at the same time every day for 7 consecutive days. Each treatment had 10 test insects, and the results were repeated 3 times. A 0.1% Tween-80 solution was set as the blank control. The toxicity regression equation, LC 50 And the co-toxicity efficiency (CTC) of the mixed agents.
[0038] 1.5 Data Analysis
[0039] Calculation of the corrected mortality, toxicity regression equation, and LC of the third-instar nymphs of the yellow-striped bamboo grasshopper using oringin 2022 50 and co-toxicity coefficient.
[0040]
[0041] The calculation method of co-toxicity coefficient refers to the method of Sun et al., and the CTC value is used to judge the joint effect: 100<CTC<120 means that the mixture has an additive effect; CTC≥120 means that the mixture has a significant synergistic effect; CTC≤100 means that the mixture has an antagonistic effect.
[0042]
[0043] 2 Results and analysis
[0044] 2.1 Virulence of Mucor frostbite to the yellow-spine grasshopper
[0045] The results of toxicity test of different concentrations of frozen soil Mucor on the third instar nymphs of the yellow-spine bamboo grasshopper are as follows: Figure 1 The mortality rate of the larvae of the yellow-spine bamboo grasshopper is positively correlated with the concentration of the spore suspension of Mucor in frozen soil. The mortality rate of the larvae of the yellow-spine bamboo grasshopper increases with the increase of the time of spraying Mucor in frozen soil. The concentration of the spore suspension is 1.0×10 8 Spores·mL -1The 7-day mortality rate of the yellow-spine locust larvae was the highest at 76.67% and the concentration was 1.0×10 4 Spores·mL -1 The 7-day mortality rate of the yellow-striped bamboo grasshopper larvae was the highest and the lowest, with a mortality rate of 46.67%, which was 2.80 times that of the control treatment.
[0046] According to the regression relationship between the mortality rate of the third-instar nymphs of the yellow-spine bamboo locust and the concentration of the spore suspension during the 7th day of the highest mortality period, the median lethal concentration (LC) of Mucor frost on the third-instar nymphs of the yellow-spine bamboo locust was obtained. 50 2.02×10 3 Spores·mL -1 (Table 1).
[0047] Table 1 LC of different concentrations of frozen soil Mucor DTMM-01 spore suspensions infecting the third instar nymphs of the yellow-spine locust for 7 days 50
[0048]
[0049] 2.2 Toxicity of azadirachtin to Pseudocoptera: Azadirachta
[0050] The results of toxicity test of different concentrations of azadirachtin on the third instar nymphs of the yellow-spine locust are as follows: Figure 2 The mortality rate of the larvae of the yellow-spine bamboo grasshopper is positively correlated with the concentration of azadirachtin, and the mortality rate of the larvae of the yellow-spine bamboo grasshopper increases with the increase of treatment time. The concentration of azadirachtin is 500.00 mg·mL -1 The 7-day mortality rate of the yellow-spine grasshopper larvae was the highest at 80.00%, which was 4.80 times that of the control treatment; the concentration was 0.50 mg·mL -1 The mortality rate of the larvae of the yellow-spine bamboo locust was the highest at 7 days and the lowest at 33.33%, which was twice that of the control treatment. According to the regression relationship between the mortality rate of the third-instar larvae of the yellow-spine bamboo locust at 7 days during the period with the highest mortality rate and the concentration of azadirachtin, the median lethal concentration (LC) of azadirachtin on the third-instar larvae of the yellow-spine bamboo locust was 1.34%. 50 4.87 mg·mL -1 (Table 2).
[0051] Table 2 LC values of different concentrations of azadirachtin solution on the third instar nymphs of the yellow-spine locust at 7 days 50
[0052]
[0053] 2.3 Toxicity of different mixtures of permafrost Mucor and azadirachtin to P. fulvidraco
[0054] The four formulations had different lethality against yellow-spine bamboo grasshopper larvae, but all had toxic effects ( Figure 3). Among them, the mortality rates of the yellow-striped bamboo grasshopper larvae were 63.33% and 40% when the volume ratios of Mucor and azadirachtin were 9:1 and 4:1, respectively, which were higher than the 36.67% of the control treatment. The mortality rates of the yellow-striped bamboo grasshopper larvae when the volume ratios of Mucor and azadirachtin were 1:1, 1:4 and 1:9 were lower, which were 23.33%, 26.67% and 33.33%, respectively.
[0055] The LC of DTMM-01 mixed with azadirachtin in the volume ratio of 9:1, 4:1, 1:1, 1:4 and 1:9 against the larvae of yellow-striped bamboo grasshopper 50 3.50, 9.42, 12.63, 25.52 and 14.39 g·L respectively -1 . The co-toxicity coefficients of different mixing combinations were different. Among them, the co-toxicity coefficients of the mixtures of Mucor and azadirachtin at 9:1 and 4:1 were greater than 120, indicating a significant synergistic effect; when Mucor and azadirachtin were mixed at 1:1, 100<CTC<120 indicated that the mixture had an additive effect; the co-toxicity coefficients of the mixtures of Mucor and azadirachtin at 1:4 and 1:9 were less than 100, indicating that the mixture had an antagonistic effect at this ratio (Table 3).
[0056] Table 3 Toxicity and co-toxicity coefficient of different compound modes to the third instar nymph of yellow-striped bamboo grasshopper (7 days)
[0057]
[0058] 3 Conclusion
[0059] In this study, the indoor toxicity experiment found that different concentrations of frozen soil Mucor suspension and different concentrations of azadirachtin had insecticidal effects on the larvae of yellow-spine bamboo grasshopper, among which 1.0×10 8 Spores·mL -1 Mucor suspension and 500.00 mg mL -1 Azadirachtin had the highest insecticidal rate against the larvae of yellow-spine bamboo locust, and the mortality rates of the larvae of yellow-spine bamboo locust were 76.76% and 80.00% after 7 days, respectively. This study mixed permafrost Mucor and azadirachtin for the first time to explore green and efficient biological pesticides. The results showed that the combined effect of the mixture was affected by the mixing volume. When permafrost Mucor and azadirachtin were mixed at a ratio of 9:1, the co-toxicity coefficient was 366.72, showing a significant synergistic effect. When permafrost Mucor and azadirachtin were mixed at a ratio of 1:4, the co-toxicity coefficient was 50.22, and the mixing scheme showed antagonism.
[0060] Mucor is considered to be a weak aquatic parasite. The toxicity experiment of Mucor on yellow-spine grasshopper using Mucor showed that the pathogenicity of Mucor increased with the increase of the concentration of the Mucor spore suspension. 8 Spores·mL -1The 7-day mortality rate of the yellow-spine bamboo locust larvae reached 76.67%. The outbreak of the yellow-spine bamboo locust is affected by temperature and relative humidity. Heavy rainfall in September of the previous year is conducive to the laying of eggs by the bamboo locust. Indoor feeding of the nymphs also requires a relative humidity of 60-70%. In other words, the ecological niche of the yellow-spine bamboo locust nymphs overlaps with the environmental niche of the permafrost mold, providing favorable habitat conditions for the permafrost mold to control the yellow-spine bamboo locust.
[0061] Azadirachtin is a plant-derived biological pesticide. It is a tetracyclic triterpenoid active substance extracted from neem seeds, bark and leaves. It is currently recognized as an insecticide that is completely inactive on vertebrates and has growth and development inhibitory effects on most arthropods. Azadirachtin has effects on the insect digestive system, nervous system, endocrine system, etc. It can inhibit the activity of multiple enzymes such as esterase in the midgut and protease and amylase in the fat body of insects, affecting the insects' feeding rate and food conversion and utilization rate; azadirachtin can also affect the normal respiratory rhythm of insects, the growth and development of reproductive systems such as ovaries and oviducts, and cause lesions in the insects' brains, prothoracic glands, and other organs, affecting the hormone balance in the insects and interfering with the growth and development of insects. Azadirachtin is toxic to more than 400 species of insects from 10 orders, especially to invasive pests such as fall armyworm, East Asian migratory locust, and American white moth, and can achieve fast and high prevention effects. However, the stability of azadirachtin during use is still insufficient. It is easily affected by sunlight, temperature, and pH, and generally decomposes and becomes ineffective in 5-8 days. Mixing azadirachtin with long-acting insecticides can not only solve the defects of azadirachtin's light and heat instability, but also achieve the effect of green and efficient prevention and control.
[0062] In order to develop green, environmentally friendly and long-lasting biological insecticides, this paper mixed Mucor and azadirachtin in different proportions. Although azadirachtin has a certain bactericidal effect, it was found through mixing that when the number of Mucor spores is large enough, the mixture of azadirachtin and Mucor (9:1) showed obvious synergistic effect, indicating that microbial pesticides and botanical pesticides can achieve insecticidal effects through mixing, which provides new ideas for the future development of green and efficient pesticides.
Claims
1. A strain of frozen soil Mucor ( Mucor hiemalis ) DTMM-01 ,characterized in that, The deposit number is: GDMCC No:65205, and the taxonomic name is: Mucor hiemalis.
2. An insecticide, characterized in that: Containing the frozen soil Mucor strain according to claim 1.
3. The insecticide according to claim 2, characterized in that The active ingredients in the insecticide are composed of Mucor solani strains and azadirachtin.
4. The insecticide according to claim 2, characterized in that The pesticide contains a spore suspension of the permafrost Mucor strain.
5. The insecticide according to claim 4, characterized in that The concentration of the spore suspension of the permafrost Mucor strain contained in the pesticide is not less than 2.02×10 3 spores / ml.
6. The insecticide according to claim 3, characterized in that The concentration of azadirachtin is not less than 4.87 mg / mL.
7. The insecticide according to claim 3, characterized in that The insecticide is prepared by mixing spores of permafrost Mucor strain and azadirachtin in a mass ratio of 9:1 or 4:
1.
8. Use of the permafrost Mucor strain according to claim 1 or the insecticide according to any one of claims 2 to 7 in the control of forest and grass leaf-feeding pests.
9. The use according to claim 8, characterized in that: The forest grass leaf-eating pest is the yellow-spine bamboo locust.
10. A method for controlling yellow-spine bamboo locust, characterized in that: During the third instar stage of the yellow-striped bamboo locust, a compound insecticide of permafrost Mucor strain and azadirachtin is applied. The compound insecticide is prepared by compounding permafrost Mucor strain spores and azadirachtin in a mass ratio of 9:1 or 4:1.