Application of combination of celangulin and metarhizium anisopliae to control and prevent of anoplophora chinensis

Abstract

This invention discloses the application of a compound combination of cypermethrin and Metarhizium anisopliae for the control of the tea leaf beetle. To screen compound agents with synergistic effects against the tea leaf beetle and evaluate their field efficacy, the co-toxicity coefficients of cypermethrin and Metarhizium anisopliae at different ratios were determined using a combined stomach poison and contact toxicity method. Indoor toxicity tests showed that the co-toxicity coefficients of cypermethrin and Metarhizium anisopliae at ratios of 3:2, 4:1, and 7:3 were 178.10, 160.42, and 134.74, respectively, indicating that the compound agent has a synergistic effect on the tea leaf beetle. Considering both co-toxicity and cost, a cypermethrin:Metarhizium anisopliae ratio of 4:1 was selected for field efficacy trials. The results showed that the control efficacy against the tea leaf beetle was 73.98% and 81.87% at 3 and 7 days after treatment, respectively, which was higher than that of single-agent treatments.

Description

[Technical Field]

[0001] This invention belongs to the field of tea garden pest control technology, and relates to the application of a compound composition of malachite and Metarhizium anisopliae in the control of tea leaf beetle. [Background Technology]

[0002] The tea leaf beetle (Basileptamelanopus Lefevre), a species of beetle in the family Chrysomelidae, is a serious pest that has become a major problem in tea gardens in southern China in recent years. It is distributed in Fujian, Jiangxi, Hunan, Hubei, Guangdong, and Guangxi provinces. The tea-growing areas in northern Fujian, southern Jiangxi, southern Hunan, and northern Guangdong are particularly affected, making it a newly emerging pest causing widespread damage in southern tea-growing regions. In Guangxi tea gardens, adults feed on leaves from April to June each year, causing perforations and rotting of the leaves, resulting in reduced tea yields.

[0003] The tea leaf beetle's damage characteristics: adults feed on the tender shoots and leaves of tea trees, while larvae feed on the roots, which greatly affects the yield and quality of tea.

[0004] Morphological characteristics: Female adults are 3.5-3.8 mm long and 1.8-2 mm wide; males are 3.2-3.4 mm long and 1.5-1.7 mm wide. Body and wings are brownish-yellow. Head and neck are short; head punctures are small and sparse; compound eyes are oval and dark brown. Antennae have 11 segments; the first segment is enlarged, the second segment is short and thick, and the remaining segments are slightly thinner at the base and slightly thicker at the tip. Each segment is densely covered with fine hairs; segments 1-4 are yellowish-brown, and segments 5-11 are dark brown. The pronotum is wider than long; punctures are irregularly arranged, large and dense; the posterior third of the lateral margin bulges outward into a pointed shape, while the anterior third is obtuse; the posterior margin has a raised ridge. The scutellum is nearly trapezoidal, smooth, and without punctures. The elytra have 10-11 rows of small punctures on the dorsal surface, with approximately 24-38 punctures per row, arranged neatly. Hindwings are light brown and membranous. The femora, tibiae, and tarsi of each leg are dark brown at the tips, while the rest are yellowish-brown. The eggs are 0.7 mm long, oblong, with blunt, rounded ends, initially white, turning dark yellow before hatching. The final instar larvae are 4.4-5.2 mm long, C-shaped, with a yellowish-brown head, dark brown mandibles, and a white body with a slight yellow tinge; they have three pairs of thoracic legs. The pupae are 3.9-4.1 mm long, with a light yellow head.

[0005] Life cycle: In Guangxi, Hunan, and Guangdong, there is one generation per year, with larvae overwintering in the soil. Mature larvae overwinter in the soil around the roots. In Guangxi, larvae begin to pupate in batches in the soil in early March, and adults emerge gradually, typically from April to June. The larval stage lasts 280-300 days, the pupal stage 15 days, and the adult stage 40-60 days. Natural enemies include ants, black ground beetles, and hairy ground beetles.

[0006] Currently, the control methods for tea leaf beetle include: (1) strengthening quarantine to prevent its spread; (2) cultivating insect-resistant varieties; (3) using birds, ants, ground beetles, etc. to prey on it, or letting chickens and ducks peck at it. It is recommended to treat the soil with Beauveria bassiana and Bacillus thuringiensis; (4) during the larval and pupal stages, first loosen the soil layer, then open a shallow trench 20cm away from the tea bushes and spray with 20% methomyl or 2.5% deltamethrin EC at 3000 times dilution or 50% phoxim EC at 1000 times dilution, then mix and cover. The effect of applying pesticides during the pupal stage is better than that during the larval stage; (5) 10-15 days after the adults emerge is the appropriate time for control. Spray the above-mentioned insecticides in time, and repeat the control after 10 days.

[0007] There are currently no registered pesticides for controlling the tea leaf beetle. Numerous studies have shown that combining different types of pesticides can enhance efficacy. For example, Liu Yuqing et al. found that a combination of natural citric acid and natural vitexin has a synergistic effect, and the resulting mixture showed significant field control efficacy against wheat aphids and locust aphids. Li Wanli et al. found that a mixture of Metarhizium anisopliae Ma1775 + 3.0% abamectin and Beauveria bassiana BLK + 1.5% pyrethroids significantly reduced the population and increased the control effect of the false-eyed green leafhopper compared to other treatments. Sun Xingxing et al. found that rhamnolipin and matrine at effective ratios of 1:10 and 1:20 both showed strong synergistic effects in controlling aphids. It is necessary to explore a highly efficient, green, and low-cost biological agent for controlling the tea leaf beetle. [Summary of the Invention]

[0008] To address the lack of an efficient, green, and low-cost biological agent for the control of the tea leaf beetle in existing technologies, this invention provides the application of a compound composition of styrax and Metarhizium anisopliae for the control of the tea leaf beetle. This invention selects two biological pesticides, styrax and Metarhizium anisopliae, to study their control effect on the tea leaf beetle, providing a reference for the green control of the tea leaf beetle in tea gardens.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] The application of a compound composition of styrax chinensis and Metarhizium anisopliae for the control of tea leaf beetle, wherein the compound composition of styrax chinensis and Metarhizium anisopliae is mixed evenly at a volume ratio of (3-7):(1-3); wherein the styrax chinensis is selected as 1wt% styrax chinensis water emulsion; wherein the Metarhizium anisopliae is selected as 8 billion spores / mL Metarhizium anisopliae CQMa421 dispersible oil suspension.

[0011] Furthermore, the compound composition of quisicerin and Metarhizium anisopliae is mixed evenly at a volume ratio of 3:2, 4:1 or 7:3.

[0012] Furthermore, the compound composition of quisicerin and Metarhizium anisopliae is mixed evenly at a volume ratio of 4:1.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] 1. The application of the compound composition of styrax and Metarhizium anisopliae described in this invention for the control of the tea leaf beetle. In the early stage, the inventors selected 12 single agents and used a combination of stomach poison and contact killing to test the toxicity of adult tea leaf beetles in the laboratory. It was found that Metarhizium anisopliae, styrax, chlorfenapyr, and high-efficiency cypermethrin had good toxic effects. Further research showed that the compound composition of styrax and Metarhizium anisopliae, when mixed evenly in a volume ratio of (3-7):(1-3), has a good control effect on the tea leaf beetle.

[0015] 2. The application of the compound composition of cypermethrin and Metarhizium anisopliae in the control of the tea leaf beetle was investigated. To screen compound agents with synergistic effects against the tea leaf beetle and evaluate their field efficacy, the co-toxicity coefficients of cypermethrin and Metarhizium anisopliae at different ratios were determined using a combined stomach poison and contact toxicity method. Indoor toxicity tests showed that the co-toxicity coefficients of cypermethrin and Metarhizium anisopliae at ratios of 3:2, 4:1, and 7:3 were 178.10, 160.42, and 134.74, respectively, indicating that the compound agent had a synergistic effect on the tea leaf beetle. Considering both co-toxicity and cost, a ratio of cypermethrin to Metarhizium anisopliae of 4:1 was selected for field efficacy trials. The results showed that the control efficacy against the tea leaf beetle was 73.98% and 81.87% at 3 and 7 days after treatment, respectively, which was higher than that of single-agent treatments.

Detailed Implementation Methods

[0016] The specific embodiments of the present invention will be further described below with reference to examples.

[0017] Example 1:

[0018] Application of the compound composition of malachite extract and Metarhizium anisopliae in the control of tea leaf beetle, wherein the compound composition of malachite extract and Metarhizium anisopliae is mixed evenly at a volume ratio of 3:2.

[0019] The test reagents were 1 wt% quistigmata extract emulsion (Shandong Huimin Zhonglian Biotechnology Co., Ltd.) and 8 billion spores / mL Metarhizium anisopliae CQMa421 dispersible oil suspension (Chongqing Julixin Bioengineering Co., Ltd.).

[0020] Example 2:

[0021] Application of the compound composition of malachite extract and Metarhizium anisopliae in the control of tea leaf beetle, wherein the compound composition of malachite extract and Metarhizium anisopliae is mixed evenly at a volume ratio of 4:1.

[0022] The test reagents were 1 wt% quistigmata extract emulsion (Shandong Huimin Zhonglian Biotechnology Co., Ltd.) and 8 billion spores / mL Metarhizium anisopliae CQMa421 dispersible oil suspension (Chongqing Julixin Bioengineering Co., Ltd.).

[0023] Example 3:

[0024] The application of the compound composition of malachite extract and Metarhizium anisopliae in the control of tea leaf beetle, wherein the compound composition of malachite extract and Metarhizium anisopliae is mixed evenly at a volume ratio of 7:3.

[0025] The test reagents were 1 wt% quistigmata extract emulsion (Shandong Huimin Zhonglian Biotechnology Co., Ltd.) and 8 billion spores / mL Metarhizium anisopliae CQMa421 dispersible oil suspension (Chongqing Julixin Bioengineering Co., Ltd.).

[0026] Experimental example:

[0027] The toxicity and field control efficacy of the combination of styrax and Metarhizium anisopliae against the tea leaf beetle.

[0028] 1. Materials and Methods

[0029] 1.1 Test reagents

[0030] The test reagents were 1 wt% quistigmata extract emulsion (Shandong Huimin Zhonglian Biotechnology Co., Ltd.) and 8 billion spores / mL Metarhizium anisopliae CQMa421 dispersible oil suspension (Chongqing Julixin Bioengineering Co., Ltd.).

[0031] 1.2 Test insect source

[0032] The tea leaf beetle, the insect source for the experiment, was collected from an organic tea garden in Dachun Village, Liubao Town, Cangwu County, Wuzhou City, Guangxi Zhuang Autonomous Region. This tea garden has experienced severe infestations of the tea leaf beetle in the past three years, and no chemical pesticides were used in the field management. Adult beetles were captured using a film-laying and shaking method and brought back indoors to be reared in insect cages with fresh tea shoots. After one day, healthy and active adults with uniform body color and size were selected for the experiment.

[0033] 1.3 Experimental Design

[0034] 1.3.1 Indoor toxicity determination of mixed formulations

[0035] Since the cost of using Metarhizium anisopliae per acre is 5-6 times that of styrax chinensis extract, a higher proportion of styrax chinensis extract was chosen to account for cost considerations. Styrax chinensis extract (A) and Metarhizium anisopliae (B) were mixed in volume ratios of 9:1, 4:1 (8:2), 7:3, 3:2 (6:4), and 1:1 (5:5), respectively, to prepare compound formulations (total concentration 750 mL / L). The experiment used a combined stomach poison and contact toxicity method: filter paper was laid flat in a 9 cm diameter petri dish, and 1.2 mL of the drug solution was added. Fresh tea leaves were soaked in different treatment agents for 10 seconds, then removed, air-dried, and placed in petri dishes lined with filter paper treated with the same drug solution. Ten test insects were inoculated into each dish, with each concentration repeated three times, using water as a control. The petri dishes were kept in an artificial climate chamber at 25℃ and 80% relative humidity (L:D = 14:10). If the insect remains motionless when touched with a brush, it is considered dead. The mortality rate and corrected mortality rate are calculated after 24 hours. A control group mortality rate of less than 10% is considered a valid test. Five concentration gradients are set up using compound agents with a corrected mortality rate of over 75%, and their co-toxicity coefficients (CTC) are calculated.

[0036] 1.3.2 Field efficacy trial

[0037] The experiment was conducted in May 2023 in a tea garden where the pest source was collected. The initial population of the tea leaf beetle was investigated before spraying. Four treatments were selected for field efficacy testing: a 1000-fold dilution of a compound agent with high co-toxicity and low cost; a 1000-fold dilution of 1% malachite extract; a 1000-fold dilution of 8 billion spores / mL Metarhizium anisopliae; and a control (CK) solution of water. Each treatment was replicated three times in a randomized block design, with each plot measuring 30 m². At 1, 3, and 7 days after spraying, six parallel sampling points were taken from each plot. At each point, a 33 cm diameter white plastic basin was used to collect the number of live beetles shaken off the tea canopy by tapping. The control effect was then calculated.

[0038] 1.4 Data Processing

[0039] Statistical analysis was performed using SPSS 19.0 software, and multiple comparisons were conducted using the Duncan method. The co-toxicity coefficient (CTC) of each compound was calculated using the Sun-Johnson method based on the indoor toxicity test data. A CTC ≥ 120 indicates a synergistic effect; 80 < CTC < 120 indicates an additive effect; and CTC ≤ 80 indicates an antagonistic effect.

[0040] Actual Toxicity Index (ATI) of Compound Preparation = (LC50 of Standard Preparation) 50 / Compound Agent LC 50 )×100; Theoretical toxicity index (TTI) of compound preparation (A+B) = ATIA×A percentage of A in the compound preparation + ATIB×B percentage of B in the compound preparation; CTC = ATI / TTI×100.

[0041] Mortality rate (%) = Number of dead insects / Total number of insects × 100; Corrected mortality rate (%) = (Morality rate of treatment group - Mortality rate of control group) / (100 - Mortality rate of control group) × 100;

[0042] Insect population reduction rate (%) = (number of live insects before treatment - number of live insects after treatment) / number of live insects before treatment × 100;

[0043] Control efficacy (%) = [1 - (number of live insects in the control area before treatment × number of live insects in the treatment area after treatment) / (number of live insects in the control area after treatment × number of live insects in the treatment area before treatment)] × 100.

[0044] 2 Results and Analysis

[0045] 2.1 Comparison of Indoor Toxicity of Compound Formulations

[0046] Five different ratios of styrax chinensis and Metarhizium anisopliae were combined, and the mortality rate of the combined formulations on adult tea leaf beetles is shown in Table 1. The results showed that, 24 hours after application, the mortality rate of tea leaf beetles, from highest to lowest, was 3:2 > 4:1 > 7:3 > 9:1 > 1:1. Three ratios, 3:2, 4:1, and 7:3, had a corrected mortality rate above 75%. The corrected mortality rate of ratio 3:2 was the highest, at 96.55%, with no significant difference between it and ratio 4:1, but a significant difference between it and ratio 7:3 (p < 0.05).

[0047] Table 1. Effects of different ratios of citrus twig extract and Metarhizium anisopliae on *Tea horn beetle*.

[0048]

[0049] Note: The data in the table are the average of three replicates. Different lowercase letters in the same column indicate significant differences (p<0.05).

[0050] Table 2. Toxicity and co-toxicity coefficient of different formulations against the tea leaf beetle.

[0051]

[0052] Toxicity analysis was conducted on three compound formulations (3:2, 4:1, and 7:3) with significant effects against *Tetracentron sinense* and mortality rates exceeding 75%, compared with single-agent formulations. Table 2 shows that the 3:2 formulation exhibited the highest toxicity against *Tetracentron sinense*, with an LC50 of [missing value]. 50 The concentration was 279.426 mL / L, and the CTC was 178.10; the second best was a 4:1 ratio, with LC... 50 The concentration was 287.733 mL / L, and the CTC was 160.42; the 7:3 ratio had the lowest toxicity. 50The concentration was 365.399 mL / L, and the CTC was 134.74. The co-toxicity coefficient (CTC) of the three formulations was greater than 120, indicating that the toxicity of styrax chinensis was enhanced after being combined with Metarhizium anisopliae, and the combination effect was synergistic.

[0053] 2.3 Field efficacy

[0054] Based on the co-toxicity coefficient results, and following the principle of relatively better efficacy and lower application cost, a compound formulation with a ratio of 4:1 of *Metarhizium anisopliae* and *Metarhizium spp.* was selected for field efficacy trials. Table 3 shows that the efficacy of the compound formulation and *Metarhizium anisopliae* extract showed an increasing trend and high persistence, while the efficacy of *Metarhizium anisopliae* showed a decreasing trend and low persistence. One day after application, the efficacy of *Metarhizium anisopliae* single-agent reached 88.13%, indicating good rapid-acting properties. *Metarhizium anisopliae* single-agent efficacy was 39.06%, indicating poor rapid-acting properties, while the compound formulation's efficacy was 67.03%, indicating moderate rapid-acting properties. At 3 and 7 days after application, the efficacy of the compound formulation was higher than that of the single-agent formulation. On day 3, there was a significant difference between the compound formulation and *Metarhizium anisopliae* efficacy, but after day 7, the difference between the compound formulation and the single-agent formulation was not significant (p < 0.05).

[0055] Are there significant differences in preventive efficacy?

[0056] Table 3. Field control efficacy of single agents and compound agents against the tea leaf beetle.

[0057]

[0058] Note: The data in the table are the average of three replicates. Different lowercase letters in the vertical column indicate significant differences (p<0.05).

[0059] 3 Discussion

[0060] 3.1 To screen for synergistic effects of compound pesticides against the tea leaf beetle and evaluate their field efficacy, the co-toxicity coefficients of cypermethrin and Metarhizium anisopliae at different ratios were determined using a combined stomach poison and contact toxicity method. Indoor toxicity tests showed that the co-toxicity coefficients of cypermethrin and Metarhizium anisopliae at ratios of 3:2, 4:1, and 7:3 were 178.10, 160.42, and 134.74, respectively, indicating that the compound pesticides had a synergistic effect on the tea leaf beetle. Considering both co-toxicity and cost, a 4:1 ratio of cypermethrin to Metarhizium anisopliae was selected for field efficacy trials. The results showed that the control efficacy against the tea leaf beetle was 73.98% and 81.87% at 3 and 7 days after treatment, respectively, which was higher than that of single-agent treatments.

[0061] 3.2 Currently, many studies on the combined toxicity of compound pesticides on test insects employ both the co-toxicity factor method and the co-toxicity coefficient method for screening. However, some studies have shown that combinations with a co-toxicity factor greater than 20 do not exhibit synergistic effects. This study initially screened different ratios of the mixture based on the mortality rate of the test insects. Ratios with high mortality rates were then quantitatively screened using the co-toxicity coefficient method, yielding the optimal ratio and degree of synergistic effect, thus reducing workload. In actual field application, dosage and cost must be considered. In this study, the mortality rates of ratios 3:2 and 4:1 were not significantly different, and the co-toxicity coefficients were similar. The 4:1 ratio was more cost-effective; therefore, this ratio was selected for field efficacy trials.

[0062] 3.3 The inventors and their research team had previously discovered in the laboratory that both maltodextrin and Metarhizium anisopliae, among other biological agents, exhibited high toxicity against the tea leaf beetle. While Metarhizium anisopliae had a higher cost per acre, maltodextrin, although cheaper per acre, required a larger dosage. Investigations revealed that both were rarely used in actual tea garden production. Combining the two in a 3:2 or 4:1 ratio showed a synergistic effect, resulting in better field control. Furthermore, the combination reduced the dosage, lowering costs and achieving the goal of reduced dosage and increased efficacy. The research results can provide data support for the screening and control of tea leaf beetles using compound agents.

[0063] References

[0064] 1. Huang Liyun, Lian Nana, Jiang Zhiyi, et al. Screening experiment on highly effective pesticides for controlling tea leaf beetle [J]. Tea, 2023, 49(02):70-72.

[0065] 2. Liu Yuqing, Dong Jianjun, Fan Yi, et al. Synergistic effect of natural citric acid and natural vitexin in combination on four aphid species [J]. China Plant Protection Guide, 2014, 34(02):13-17.

[0066] 3. Li Wanli, Bao Yaxing, Lin Xiaoting, et al. Application of Beauveria bassiana and Metarhizium anisopliae and their compound agents in the control of tea false-eyed green leafhopper [J]. Journal of Jiangxi Agricultural University, 2017, 39(04): 699-705.

[0067] 4. Sun Xingxing, Wang Kai, Li Hongyang, et al. Control effect of novel biological pesticide rhamnolipin and its compound on cabbage aphids [J]. Zhejiang Agricultural Sciences, 2016, 57(12):2071-2073.

[0068] 5. Wu Haibin, Geng Hairong, Gong Qingtao, et al. Toxicity and field control effects of eight insecticides on adult leaf beetles [J]. Acta Entomologica Sinica, 2016, 53(01):200-206.

[0069] 6.Sun YP,Johnnson E R.Analysis of joint action of pesticidesagainsthouse files[J].Ecological Entomology,1960,53:887-891.

[0070] 7. Yu Haoyong, He Falin, Qiao Zhihua, et al. Screening of pesticide compound synergistic formulations for controlling wireworm [J]. Chinese Agricultural Science Bulletin, 2019, 35(34):115-121.

[0071] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. The application of a compound composition of malachite extract and Metarhizium anisopliae in the control of the tea leaf beetle, characterized in that: The aforementioned compound composition of quisicerin and Metarhizium anisopliae is mixed evenly at a volume ratio of (3-7):(1-3); The cucurbitacin used is a 1wt% cucurbitacin water-in-oil emulsion; the Metarhizium anisopliae used is an 8 billion spores / mL Metarhizium anisopliae CQMa421 dispersible oil suspension.

2. The application of the compound composition of malachite and Metarhizium anisopliae according to claim 1 in the control of tea leaf beetle, characterized in that: The compound composition of quisicerin and Metarhizium anisopliae is mixed evenly at a volume ratio of 3:2, 4:1 or 7:

3.

3. The application of the compound composition of malachite and Metarhizium anisopliae according to claim 2 in the control of tea leaf beetle, characterized in that: The compound composition of quisicerin and Metarhizium anisopliae is mixed evenly at a volume ratio of 4:1.

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