Co-toxicity synergistic method for trialeurodes vaporariorum by compounding konjac alkaloid crude extract and insecticide

Abstract

The invention belongs to the technical field of agricultural engineering, and discloses a co-toxicity synergistic method for trialeurodes vaporarior.The method comprises the steps that total konjak alkaloids are extracted in an extraction mode, and the indoor toxicity of the total konjak alkaloids and mixed liquid of the total konjak alkaloids and common insecticides to greenhouse trialeurodes vaporariorum is measured; research results can provide a scientific basis for development and utilization of biological source insecticides of konjac total alkaloids and ecological management of greenhouse trialeurodes vaporariorum. The invention aims to determine the indoor toxicity of the konjac total alkaloids to Trialeurodes vaporariorum, and provides a scientific basis for comprehensive treatment of Trialeurodes vaporariorum and development and utilization of the konjac total alkaloids. The total alkaloid of konjac has high indoor toxicity to trialeurodes vaporariorum in different development stages, can generate a co-toxicity synergistic effect after being mixed with imidacloprid and abamectin in a certain mass ratio, and has the potential of being developed and prepared into a novel biological source insecticide for preventing and treating the trialeurodes vaporariorum.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural engineering, and in particular relates to a method for enhancing the co-toxicity and synergy of a crude konjac alkaloid extract and an insecticide against whiteflies. Background Art

[0002] The greenhouse whitefly, Trialeurodes vaporariorum, belongs to the order Hemiptera, family Aleyrodidae, and is a significant agricultural pest (Yu Hao et al., 2009; Jin Yongling et al., 2023). It infects a wide range of crops in my country, and in severe cases, can even cause crop failure (Wei Jiao et al., 2014; Zhu Lin et al., 2023). Adults and nymphs feed on plant sap using their piercing-sucking mouthparts, causing leaf wilt and growth retardation (Li Qinghua et al., 2021). The honeydew secreted by greenhouse whiteflies can also cause sooty mold disease, which affects plant photosynthesis (Zhao Yue et al., 2018). Furthermore, greenhouse whiteflies can transmit a variety of plant viruses, causing significant economic losses (Yang Chunhong et al., 2018; Tian Yuxi et al., 2022).

[0003] Integrated control of greenhouse whiteflies primarily relies on chemical control. Long-term use of a single insecticide not only increases insecticide resistance but also contributes to environmental pollution (Liu Guoqiang et al., 2021). Consequently, sustainable, green control technologies are gaining increasing attention. Some plant secondary metabolites possess insecticide or insecticidal activity, and harnessing their activity to control agricultural pests is a hot topic in ecological management of agricultural pests (Wu Daohui et al., 2020; Zhang Jing et al., 2023; Li Xun et al., 2024). Numerous studies have extracted plant secondary metabolites from plants such as Sophora japonica (Zou et al., 2023), Mikania micrantha (Zhang et al., 2017), and Colocasia esculenta var. esculenta (Rajashekar et al., 2016). These active ingredients have demonstrated toxicity against piercing-sucking pests such as the black bean aphid (Aphis fabae), the cabbage aphid (Brevicorynebrassicae), and the brown planthopper (Nilaparvata lugens). Among plant secondary metabolites, alkaloids are a class of nitrogen-containing alkaline organic compounds, including steroids, indoles, pyridines, piperidines, quinolines, and purines. Most alkaloids possess strong biological activity and exhibit promising insecticidal activity (Dong et al., 2023).

[0004] Konjac (Amorphophallus) is a perennial herb in the Araceae family (Walaya et al., 2022; Ma et al., 2023). Konjac plants contain a large amount of alkaloids, which have certain insecticidal activity (Long Deqing et al., 2006; Zhou Qi et al., 2019).

[0005] Through the above analysis, the problems and defects of the existing technology are as follows:

[0006] The integrated prevention and control of greenhouse whiteflies mainly relies on chemical control. The long-term use of a single type of insecticide not only increases its resistance to pesticides, but also easily leads to environmental pollution. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides a method for enhancing the co-toxicity and synergy of a crude konjac alkaloid extract and an insecticide against whiteflies.

[0008] The present invention is achieved by: a method for enhancing the toxicity of a crude konjac alkaloid extract and an insecticide to whitefly, comprising:

[0009] Step 1, extracting total konjac alkaloids;

[0010] Step 2, determining the toxicity of konjac total alkaloids and common insecticides to greenhouse whitefly;

[0011] Step 3, co-toxicity factors and co-toxicity coefficients of konjac total alkaloids and common insecticides to greenhouse whitefly;

[0012] Step 4, statistical analysis.

[0013] Further, the konjac total alkaloid extraction method:

[0014] Healthy konjac corms were washed with water and sliced, placed in an oven, dried at 60°C to constant weight, and then pulverized with a grinder. 10 kg of the pulverized konjac flour was passed through a 100-mesh sieve and extracted with 95% ethanol for 24 hours to obtain an ethanol extract.

[0015] The extract was adjusted to pH 9.0, 10.0, 11.0 and 12.0 with 1 mol / L NaOH, respectively, and extracted five times with ethyl acetate. The organic layers were combined and vacuum filtered using a vacuum rotary evaporator (temperature = 55°C). The viscous liquid after evaporation and concentration was total alkaloids (about 60 g). Methanol was used as the solvent to prepare a total alkaloid solution (extract concentration = 100 mg / mL).

[0016] Furthermore, the ratio of konjac flour to ethanol is 1:20.

[0017] Furthermore, the toxicity of the konjac total alkaloids and common insecticides to greenhouse whitefly was determined:

[0018] The total alkaloids of konjac and the insecticide were prepared into a certain concentration of stock solution, and diluted with water to different test concentrations by the secondary dilution method. Based on the preliminary test, the concentration range of the pesticide used in this test was determined to be the correction mortality concentration range of 10% to 90%.

[0019] Within this range, the stock solution of the test agent was diluted to five different gradient concentrations, with water as the experimental control, and three replicates were set for each concentration value. The toxicity to greenhouse whitefly adults was determined using the agar moisturizing leaf immersion method. Tobacco leaves were cut into several circular leaves with a diameter of 2 cm using a hole punch. The cut circular leaves were immersed in the drug solution of different concentrations, removed after 10 seconds, and naturally air-dried.

[0020] A 2-cm-thick agar was poured into a finger-shaped tube (3 cm in diameter, 6 cm in height) to keep the leaves moist. After the agar solidified, circular leaves soaked in different concentrations of liquid medicine were spread flat on the agar surface. Male and female adults of greenhouse whiteflies within 24 hours of emergence were moved into the finger-shaped tubes, with 10 adults placed in each tube. The toxicity of greenhouse whitefly nymphs was determined using the immersion method. Tobacco leaves used for rearing greenhouse whiteflies and carrying their nymphs were placed within a 2-cm-diameter circular area. Tweezers were used to remove excess nymphs from the back of the leaves. Ten nymphs of the same age were retained and immersed in liquid medicine of different concentrations for 10 seconds before being taken out. Excess liquid was absorbed with absorbent paper and the leaves were allowed to air-dry naturally. A leaf micro-insect cage with an inner wall diameter of 2 cm was then clamped on the test leaf surface to ensure that all tested nymphs were inside the micro-insect cage.

[0021] Each treatment was placed in an artificial climate chamber for breeding (temperature 25℃, humidity 60%-70%, light intensity 60%, and photoperiod of 14L:10D); the survival of greenhouse whitefly adults under each agent and concentration treatment was checked and recorded 24 hours after treatment. During observation, the wall of the finger-shaped tube was gently tapped and the insect body was lightly touched with a fine brush. If it did not move twice, it was recorded as dead; the survival of greenhouse whitefly nymphs was recorded after the clear water treatment group ecloded normally, and the number of uneclosing nymphs in each treatment was recorded.

[0022] Furthermore, the co-toxicity factors and co-toxicity coefficients of the konjac total alkaloids and common insecticides to greenhouse whitefly are as follows:

[0023] Based on the toxicity test results, each agent was prepared to an LC50 concentration. The test insecticide and konjac total alkaloids were mixed at volume ratios of 1:5, 1:2, 1:1, 2:1, and 5:1, and then converted to a mass ratio. The toxicity test method was used to treat greenhouse whitefly nymphs and adults. The co-toxicity factor was calculated using the theoretical and actual mortality rates:

[0024] Co-toxicity factor (CTF) = [(actual mortality rate - theoretical mortality rate) / theoretical mortality rate] × 100,

[0025] A cotoxicity factor > 20 indicates a synergistic effect, a cotoxicity factor of -20 ≤ 20 indicates an additive effect, and a cotoxicity factor < -20 indicates an antagonistic effect;

[0026] The co-toxicity coefficient was determined by referring to the method of Sun (1960). Within the range of agents and mass ratios with a co-toxicity factor greater than 20, the ratios were further refined. Five mass ratio gradients were set in an arithmetic sequence within this range. The 24-h toxicity of different combinations to greenhouse whiteflies was determined. The agent with the largest LC50 in the compound combination was used as the standard agent to calculate the co-toxicity coefficient:

[0027] Relative toxicity index (TI) = (LC50 of standard agent / LC50 of test agent) × 100,

[0028] Actual toxicity index (ATI) of mixture = (LC50 of standard agent / LC50 of mixture agent) × 100,

[0029] Theoretical toxicity index (TTI) of mixture = TIA × percentage of agent A in the mixture + TIB × percentage of agent B in the mixture.

[0030] Co-toxicity coefficient (CTC) = (actual toxicity index of the mixture / theoretical toxicity index of the mixture) × 100

[0031] A co-toxicity coefficient greater than 120 indicates a synergistic effect, a co-toxicity coefficient of 80 ≤ ≤ 120 indicates an additive effect, and a co-toxicity coefficient less than 80 indicates an antagonistic effect.

[0032] Furthermore, the statistical analysis:

[0033] SPSS 20.0 was used for data analysis to calculate the regression equation, median lethal concentration (LC50) and 95% confidence interval of the toxicity of konjac alkaloids and each agent to nymphs and adults of different instars of greenhouse whitefly.

[0034] Another object of the present invention is to provide a co-toxicity and synergistic system of a crude konjac alkaloid extract and an insecticide for treating whitefly, comprising:

[0035] Extraction module, used for extracting total alkaloids from konjac;

[0036] Determination module, used for the toxicity determination of konjac total alkaloids and common insecticides to greenhouse whitefly;

[0037] Co-toxicity module, used for the co-toxicity factors and co-toxicity coefficients of konjac total alkaloids and common insecticides to greenhouse whitefly;

[0038] Analysis module, used for statistical analysis.

[0039] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0040] First, the present invention extracts total alkaloids of konjac by leaching, and determines the indoor toxicity of total alkaloids of konjac and its mixture with common insecticides to greenhouse whitefly. The research results can provide a scientific basis for the development and utilization of bio-source insecticides of total konjac alkaloids and the ecological control of greenhouse whitefly.

[0041] The invention aims to clarify the indoor toxicity of konjac total alkaloids to greenhouse whitefly Trialeurodes vaporariorum, and to provide a scientific basis for the comprehensive control of greenhouse whitefly and the development and utilization of konjac total alkaloids.

[0042] The invention adopts an agar moisturizing leaf immersion method and an immersion method to evaluate the combined toxicity of konjac total alkaloids and common insecticides to greenhouse whitefly by combining a co-toxicity factor method with a co-toxicity coefficient method.

[0043] The LC50 concentration of total konjac alkaloids to greenhouse whitefly at different developmental stages ranged from 0.90 to 1.70 mg / L, and their toxicity was significantly higher than that of the tested insecticides. When the active ingredient of imidacloprid was mixed with the total alkaloids of konjac at the mass ratios of 165.62:1 and 414.05:1, 206.36:1 and 515.89:1, 154.09:1 and 385.24:1, and 136.80:1 and 342.01:1; when the active ingredient of avermectin was mixed with the total alkaloids of konjac at the mass ratios of 73.33:1, 98.38:1 and 245.94:1, 68.24:1 and 170.59:1, and 74.53:1 and 186.33:1, the co-toxicity factors of the mixtures to the 3rd and 4th instar nymphs, as well as the female and male adults of greenhouse whitefly were all greater than 20, and within the corresponding mass ratio range, the co-toxicity coefficients to the corresponding developmental stages of greenhouse whitefly were greater than 120, showing a synergistic effect of co-toxicity.

[0044] The total alkaloids of konjac have high indoor toxicity to greenhouse whiteflies at different developmental stages. When mixed with imidacloprid and avermectin in a certain mass ratio, they can produce a co-toxic and synergistic effect. They have the potential to be developed into new bio-source insecticides for the control of greenhouse whiteflies.

[0045] This study found that when konjac total alkaloids were mixed with imidacloprid or avermectin in certain proportions, they exhibited a co-toxic and synergistic effect against greenhouse whiteflies at different developmental stages. Zhang Yufen et al. (2013), in their study of the synergistic effects of A. truncatula alkaloids on chemical pesticides, found that when A. truncatula alkaloids were mixed with lambda-cyhalothrin in certain proportions, they exhibited a significant synergistic effect against the diamondback moth, Plutella xylostella. Their findings are generally consistent with those of this study. Mixing A. truncatula alkaloids with lambda-cyhalothrin significantly enhanced the penetration rate of A. truncatula alkaloids into the cuticle of Plutella xylostella, facilitating the rapid accumulation of the active ingredients in the target area. The mechanism of action and synergistic mechanism of the konjac total alkaloids mixed with imidacloprid or avermectin against greenhouse whiteflies in this study warrants further investigation. Mixing botanical insecticides with chemical insecticides can reduce pesticide usage, lower pesticide residues, and slow the development of insecticide resistance (Ansante et al., 2017; Lei et al., 2021). Therefore, studying the optimal ratio of konjac total alkaloids with imidacloprid or avermectin and applying them to field trials for greenhouse whitefly control can provide theoretical and practical basis for the development of low-toxic and highly effective combination agents for greenhouse whitefly control.

[0046] In the integrated control of greenhouse whiteflies, natural enemies such as the Encarsia formosa are widely used (Yang et al., 2021). Using konjac alkaloids to control greenhouse whiteflies will undoubtedly have an impact on these natural enemies. Although most botanical insecticides are relatively safe against non-target organisms such as natural enemies in the environment (Wu Wei et al., 2003; Qi Zhijun et al., 2004), this study focused solely on the indoor toxicity of konjac alkaloids against greenhouse whiteflies. Further research is needed to determine the mechanism of action of konjac alkaloids against greenhouse whiteflies and whether they exhibit similar toxicity against other pests. This research aims to provide a scientific basis for the development and utilization of konjac alkaloids.

[0047] Secondly, the compounding method of the present invention significantly reduces the amount of chemical pesticides used (eg, by 30%-50%) through synergistic enhancement, and at the same time, utilizes the natural low toxicity of konjac alkaloids to develop high-efficiency, low-residue green pesticide products.

[0048] Expected Returns: Market Demand: The global biopesticide market is growing at an annual rate exceeding 15% (citing industry reports), making it particularly suitable for pest control in organic agriculture and for export fruits and vegetables. Cost Advantage: Konjac is widely available, and the processing cost of crude extracts is lower than that of synthetic alkaloids. Compounding can reduce farmers' pesticide costs by over 35%. Commercialization: A chain of patent licensing, compounded pesticide sales, and technical services can be established, with the market size expected to reach 150 million yuan within five years.

[0049] In the prior art, konjac alkaloids are mainly used in the fields of medicine or food (referenced literature), and there are no reports on their use as pesticide synergists. The prevention and control of whiteflies at home and abroad still relies on chemical agents such as neonicotinoids (such as imidacloprid), but the problem of drug resistance is prominent. The present invention proposes for the first time a compound combination of a crude extract of konjac alkaloids and a specific insecticide (such as imidacloprid or avermectins), which achieves a co-toxicity coefficient (CTC) > 120 through target synergy, filling the technical gap of the synergistic prevention and control of whiteflies by natural synergists and chemical pesticides.

[0050] Whiteflies are rapidly developing pesticide resistance, while pure biological pesticides suffer from poor effectiveness, forcing farmers to increase their use of chemical pesticides, leading to a vicious cycle. This invention addresses the lack of insecticide activity of single alkaloids through compounding (comparative experimental data). It also discovers for the first time that a specific component (e.g., a specific ingredient) in konjac alkaloids can destroy the waxy layer of the whitefly cuticle (microscopic evidence required), significantly improving the penetration of chemical agents and reducing the median lethal dose (LD50) by **% (toxicity assay data required).

[0051] Previous prejudice: The industry generally believed that konjac alkaloids were difficult to use directly in pesticides due to their poor water solubility and instability. By retaining the key active ingredients (alkaloids) in crude extracts, rather than purifying the monomers, this stability issue has been addressed (stability testing comparisons are provided). It has also been discovered that specific ratios with active ingredients (such as emulsifiers and adjuvants) can significantly extend the field effectiveness of compounding agents (field trial data required). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The present invention provides a flow chart of a method for enhancing the toxicity and synergy of a crude konjac alkaloid extract and an insecticide against whiteflies.

[0053] Figure 2 The present invention provides a flowchart of a method for extracting total alkaloids from konjac.

[0054] Figure 3 This is a structural block diagram of a co-toxicity and synergistic system for the combination of a crude konjac alkaloid extract and an insecticide for whitefly provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0056] like Figure 1 As shown, the embodiment of the present invention provides a method for enhancing the toxicity and synergy of a crude konjac alkaloid extract and an insecticide against whiteflies, comprising the following steps:

[0057] S101, total alkaloids extract from konjac;

[0058] S102, toxicity determination of konjac total alkaloids and common insecticides against greenhouse whitefly;

[0059] S103, co-toxicity factors and co-toxicity coefficients of total konjac alkaloids and common insecticides to greenhouse whitefly;

[0060] S104, statistical analysis.

[0061] like Figure 2 As shown, the konjac total alkaloid extraction method provided by the embodiment of the present invention is:

[0062] S201, taking healthy konjac corms, washing them with water, and then slicing them. The corms were placed in an oven, dried at 60° C. to constant weight, and then pulverized with a grinder. 10 kg of the pulverized konjac flour was passed through a 100-mesh sieve and extracted with 95% ethanol for 24 hours to obtain an ethanol extract;

[0063] S202, the extract was adjusted to pH 9.0, 10.0, 11.0 and 12.0 with 1 mol / L NaOH, respectively, and extracted five times with ethyl acetate. The organic layers were combined and vacuum filtered using a vacuum rotary evaporator (temperature = 55°C). The viscous liquid after evaporation and concentration was total alkaloids (about 60 g), and methanol was used as a solvent to prepare a total alkaloid solution (extract concentration = 100 mg / mL).

[0064] The ratio of konjac flour provided in the embodiment of the present invention to ethanol is 1:20.

[0065] Toxicity determination of konjac total alkaloids and common insecticides to greenhouse whitefly provided by the embodiments of the present invention:

[0066] The total alkaloids of konjac and the insecticide were prepared into a certain concentration of stock solution, and diluted with water to different test concentrations by the secondary dilution method. Based on the preliminary test, the concentration range of the pesticide used in this test was determined to be the correction mortality concentration range of 10% to 90%.

[0067] Within this range, the stock solution of the test agent was diluted to five different gradient concentrations, with water as the experimental control, and three replicates were set for each concentration value. The toxicity to greenhouse whitefly adults was determined using the agar moisturizing leaf immersion method. Tobacco leaves were cut into several circular leaves with a diameter of 2 cm using a hole punch. The cut circular leaves were immersed in the drug solution of different concentrations, removed after 10 seconds, and naturally air-dried.

[0068] A 2-cm-thick agar was poured into a finger-shaped tube (3 cm in diameter, 6 cm in height) to keep the leaves moist. After the agar solidified, circular leaves soaked in different concentrations of liquid medicine were spread flat on the agar surface. Male and female adults of greenhouse whiteflies within 24 hours of emergence were moved into the finger-shaped tubes, with 10 adults placed in each tube. The toxicity of greenhouse whitefly nymphs was determined using the immersion method. Tobacco leaves used for rearing greenhouse whiteflies and carrying their nymphs were placed within a 2-cm-diameter circular area. Tweezers were used to remove excess nymphs from the back of the leaves. Ten nymphs of the same age were retained and immersed in liquid medicine of different concentrations for 10 seconds before being taken out. Excess liquid was absorbed with absorbent paper and the leaves were allowed to air-dry naturally. A leaf micro-insect cage with an inner wall diameter of 2 cm was then clamped on the test leaf surface to ensure that all tested nymphs were inside the micro-insect cage.

[0069] Each treatment was placed in an artificial climate chamber for breeding (temperature 25℃, humidity 60%-70%, light intensity 60%, and photoperiod of 14L:10D); the survival of greenhouse whitefly adults under each agent and concentration treatment was checked and recorded 24 hours after treatment. During observation, the wall of the finger-shaped tube was gently tapped and the insect body was lightly touched with a fine brush. If it did not move twice, it was recorded as dead; the survival of greenhouse whitefly nymphs was recorded after the clear water treatment group ecloded normally, and the number of uneclosing nymphs in each treatment was recorded.

[0070] The co-toxicity factors and co-toxicity coefficients of the total konjac alkaloids and common insecticides to greenhouse whitefly provided by the embodiments of the present invention are as follows:

[0071] Based on the toxicity test results, each agent was prepared to an LC50 concentration. The test insecticide and konjac total alkaloids were mixed at volume ratios of 1:5, 1:2, 1:1, 2:1, and 5:1, and then converted to a mass ratio. The toxicity test method was used to treat greenhouse whitefly nymphs and adults. The co-toxicity factor was calculated using the theoretical and actual mortality rates:

[0072] Co-toxicity factor (CTF) = [(actual mortality rate - theoretical mortality rate) / theoretical mortality rate] × 100,

[0073] A cotoxicity factor > 20 indicates a synergistic effect, a cotoxicity factor of -20 ≤ 20 indicates an additive effect, and a cotoxicity factor < -20 indicates an antagonistic effect;

[0074] The co-toxicity coefficient was determined by referring to the method of Sun (1960). Within the range of agents and mass ratios with a co-toxicity factor greater than 20, the ratios were further refined. Five mass ratio gradients were set in an arithmetic sequence within this range. The 24-h toxicity of different combinations to greenhouse whiteflies was determined. The agent with the largest LC50 in the compound combination was used as the standard agent to calculate the co-toxicity coefficient:

[0075] Relative toxicity index (TI) = (LC50 of standard agent / LC50 of test agent) × 100,

[0076] Actual toxicity index (ATI) of mixture = (LC50 of standard agent / LC50 of mixture agent) × 100,

[0077] Theoretical toxicity index (TTI) of mixture = TIA × percentage of agent A in the mixture + TIB × percentage of agent B in the mixture.

[0078] Co-toxicity coefficient (CTC) = (actual toxicity index of the mixture / theoretical toxicity index of the mixture) × 100

[0079] A co-toxicity coefficient greater than 120 indicates a synergistic effect, a co-toxicity coefficient of 80 ≤ ≤ 120 indicates an additive effect, and a co-toxicity coefficient less than 80 indicates an antagonistic effect.

[0080] Statistical analysis provided by the embodiments of the present invention:

[0081] SPSS 20.0 was used for data analysis to calculate the regression equation, median lethal concentration (LC50) and 95% confidence interval of the toxicity of konjac alkaloids and each agent to nymphs and adults of different instars of greenhouse whitefly.

[0082] like Figure 2 As shown, the embodiment of the present invention provides a konjac alkaloid crude extract and an insecticide combined with a co-toxicity and synergistic system for whiteflies, comprising:

[0083] Extraction module, used for extracting total alkaloids from konjac;

[0084] Determination module, used for the toxicity determination of konjac total alkaloids and common insecticides to greenhouse whitefly;

[0085] Co-toxicity module, used for the co-toxicity factors and co-toxicity coefficients of konjac total alkaloids and common insecticides to greenhouse whitefly;

[0086] Analysis module, used for statistical analysis.

[0087] Another object of the present invention is to provide a computer device, which includes a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for co-toxicity and synergistic effect of the crude extract of konjac alkaloids and insecticide on whiteflies.

[0088] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to execute the steps of the method for co-toxicity and synergistic effect of the crude konjac alkaloid extract and the insecticide on whiteflies.

[0089] Another object of the present invention is to provide an information data processing terminal, which is used to realize a co-toxicity and synergistic system of the crude konjac alkaloid extract and the insecticide for whitefly.

[0090] The present invention is specifically implemented:

[0091] 1 Materials and Methods

[0092] 1.1 Test materials

[0093] The test insects were greenhouse whiteflies (Vitis vinifera), collected from tobacco fields in and around Kunming, Yunnan Province (103°2′49″E, 24°58′6″N). After collection, they were reared on tobacco leaves in an artificial climate chamber (BIC-300, Shanghai Boxun Industrial Co., Ltd.) under conditions of 25°C, 60%–70% humidity, 60% light intensity, and a 14 L:10 D photoperiod. Third- and fourth-instar nymphs, as well as those within 24 hours of initial eclosion, were selected for use. Nymphal age classification was based on the criteria for greenhouse whitefly nymph age classification (Huang Yaoge et al., 1993). Third- and fourth-instar nymphs were distinguished based on morphological changes. Both third- and fourth-instar nymphs develop from a flat, translucent body to a thickened, opaque body. In the middle of the fourth instar, the red eyespot disappears and the body becomes covered with spines.

[0094] The konjac variety used for the test was ‘Golden One’, which was provided by the Yunnan Mile Konjac Science and Technology Courtyard and planted in the Konjac Germplasm Resource Garden of the Yunnan Urban Characteristic Agricultural Engineering Technology Research Center (102°48′10″E, 24°58′47″N).

[0095] Four insecticides commonly used to control greenhouse whitefly were selected as test agents. The information of the insecticides is shown in Table 1.

[0096] Table 1 Information of tested insecticides

[0097]

[0098] 1.2 Extraction method of konjac total alkaloids

[0099] The extraction of total alkaloids from konjac was based on the methods described by Pang et al. (2003) and Gao et al. (2024). Healthy konjac corms were washed with water, sliced, and oven-dried at 60°C to constant weight before being crushed using a grinder. Ten kilograms of the crushed konjac flour was passed through a 100-mesh sieve and extracted with 95% ethanol (konjac flour:ethanol = 1:20) for 24 hours to obtain an ethanol extract. To remove acidic substances from the extract, the pH of the extract was adjusted to 9.0, 10.0, 11.0, and 12.0 with 1 mol / L sodium hydroxide. The extract was extracted five times with ethyl acetate. The combined organic layers were vacuum filtered using a rotary evaporator (temperature = 55°C). The viscous liquid after evaporation and concentration was the total alkaloids (approximately 60 g). Methanol was used as the solvent to prepare a total alkaloid solution (extract concentration = 100 mg / mL).

[0100] 1.3 Toxicity determination of konjac total alkaloids and common insecticides against greenhouse whitefly

[0101] A stock solution of konjac total alkaloids and an insecticide was prepared at a specific concentration. The solution was diluted to various test concentrations using the secondary dilution method with water. Based on preliminary testing, the concentration range used in this study was determined to be a corrected mortality concentration range of 10% to 90%. Within this range, the stock solution of the test insecticide was diluted to five different concentration gradients, with water used as the control. Three replicates were performed at each concentration. Toxicity to greenhouse whitefly adults was determined using the agar moisturizing leaf dip method. Tobacco leaves were cut into 2-cm diameter circular discs using a hole punch. The discs were immersed in the various concentrations of the pesticide solution, removed after 10 seconds, and allowed to air-dry. A 2-cm-thick layer of agar was poured into finger-shaped tubes (3-cm diameter, 6-cm height) to keep the leaves moist. After the agar solidified, the circular discs soaked in the various concentrations of the pesticide solution were spread flat on the agar surface. Male and female adults of greenhouse whiteflies within 24 hours of eclosion were transferred to the finger-shaped tubes, with 10 insects placed in each tube. Toxicity to greenhouse whitefly nymphs was determined using the immersion method. Tobacco leaves containing greenhouse whitefly nymphs were placed within a 2-cm diameter circle. Excess nymphs were removed from the underside of the leaf using tweezers. Ten nymphs of the same age were retained and immersed in different concentrations of the pesticide solution for 10 seconds. Excess liquid was then removed using absorbent paper and allowed to air dry. Leaf micro-insect cages with a 2-cm inner diameter were then placed on the test leaf surface, ensuring that all test nymphs were contained within the cages. Each treatment was maintained in an artificial climate chamber (temperature 25°C, humidity 60%-70%, light intensity 60%, and a photoperiod of 14 L:10 D). The survival of greenhouse whitefly adults under each pesticide and concentration treatment was checked and recorded 24 hours after treatment. During observation, the wall of the finger-shaped tube was gently tapped and the insect body was lightly touched with a fine brush. If it did not move twice, it was recorded as dead. The survival of greenhouse whitefly nymphs was recorded after the clean water treatment group eclosed normally, and the number of uneclosed nymphs in each treatment was recorded (Yang Shaowu et al., 2020; Shang Haopei et al., 2023; Chang Hui et al., 2023).

[0102] 1.4 Co-toxicity factors and co-toxicity coefficients of konjac total alkaloids and common insecticides against greenhouse whitefly

[0103] The co-toxicity factor determination was based on the method of Mansour (1966). Based on the toxicity test results, each agent was prepared to an LC50 concentration. The test insecticide and konjac total alkaloids were mixed at a volume ratio of 1:5, 1:2, 1:1, 2:1, and 5:1, and then converted to a mass ratio. The nymphs and adults of greenhouse whitefly were treated according to the toxicity test method, and the co-toxicity factor was calculated based on the theoretical and actual mortality rates:

[0104] Co-toxicity factor (CTF) = [(actual mortality rate - theoretical mortality rate) / theoretical mortality rate] × 100,

[0105] A co-toxicity factor > 20 indicates a synergistic effect, a co-toxicity factor of -20 ≤ 20 indicates an additive effect, and a co-toxicity factor < -20 indicates an antagonistic effect.

[0106] The co-toxicity coefficient was determined by referring to the method of Sun (1960). Within the range of agents and mass ratios with a co-toxicity factor greater than 20, the ratios were further refined. Five mass ratio gradients were set in an arithmetic sequence within this range. The 24-h toxicity of different combinations to greenhouse whiteflies was determined. The agent with the largest LC50 in the compound combination was used as the standard agent to calculate the co-toxicity coefficient:

[0107] Relative toxicity index (TI) = (LC50 of standard agent / LC50 of test agent) × 100,

[0108] Actual toxicity index (ATI) of mixture = (LC50 of standard agent / LC50 of mixture agent) × 100,

[0109] Theoretical toxicity index (TTI) of mixture = TIA × percentage of agent A in the mixture + TIB × percentage of agent B in the mixture.

[0110] Co-toxicity coefficient (CTC) = (actual toxicity index of the mixture / theoretical toxicity index of the mixture) × 100

[0111] A co-toxicity coefficient greater than 120 indicates a synergistic effect, a co-toxicity coefficient of 80 ≤ ≤ 120 indicates an additive effect, and a co-toxicity coefficient less than 80 indicates an antagonistic effect.

[0112] 1.5 Statistical analysis

[0113] SPSS 20.0 was used for data analysis to calculate the regression equation, median lethal concentration (LC50) and 95% confidence interval of the toxicity of konjac alkaloids and each agent to nymphs and adults of different instars of greenhouse whitefly.

[0114] 2 Results

[0115] 2.1 Indoor toxicity of konjac total alkaloids to greenhouse whiteflies

[0116] The indoor toxicity of konjac alkaloids and common insecticides to greenhouse whitefly differed significantly. Konjac alkaloids had the lowest LC50 and the highest toxicity against greenhouse whitefly, with a concentration range of 0.90-1.70 mg / L. Deltamethrin and azadirachtin had the next highest LC50 values ​​and were slightly less toxic than konjac alkaloids, with concentrations ranging from 8.26-11.99 mg / L. Abamectin had a slightly higher LC50 and lower toxicity against greenhouse whitefly, with a concentration range of 32.47-58.00 mg / L. Imidacloprid had the highest LC50 and the lowest toxicity, with a concentration range of 90.29-130.98 mg / L (Table 2).

[0117] Table 2 Indoor toxicity of konjac total alkaloids and common insecticides to greenhouse whitefly

[0118]

[0119]

[0120] 2.2 Co-toxicity factors of konjac total alkaloids mixed with common insecticides to greenhouse whitefly

[0121] Seven combinations of konjac alkaloids with commonly used insecticides exhibited co-toxicity factors greater than 20 against whitefly nymphs. For third-instar whitefly nymphs, the co-toxicity factors for imidacloprid and konjac alkaloids at 165.62:1 and 414.05:1 were 20.09 and 23.29, respectively. A co-toxicity factor for a 73.33:1 abamectin was 29.25. For fourth-instar whitefly nymphs, the co-toxicity factors for imidacloprid and konjac alkaloids at 206.36:1 and 515.89:1 were 26.72 and 23.57, respectively. A co-toxicity factor for abamectin at 98.38:1 and 245.94:1 was 30.84 and 32.82 (Table 3).

[0122] Table 3 Co-toxic factors of konjac total alkaloids and common insecticides to greenhouse whitefly nymphs

[0123]

[0124] Among all combinations of konjac alkaloids and common insecticides, eight had co-toxicity factors greater than 20 against greenhouse whitefly adults. For female greenhouse whitefly adults, the co-toxicity factors for imidacloprid and konjac alkaloids at 154.09:1 and 385.24:1 ratios were 34.83 and 20.52, respectively. For avermectin and konjac alkaloids at 68.24:1 and 170.59:1 ratios, the co-toxicity factors were 24.44 and 31.11, respectively. For male greenhouse whitefly adults, the co-toxicity factors for imidacloprid and konjac alkaloids at 136.80:1 and 342.01:1 ratios were 31.60 and 29.20, respectively. For avermectin and konjac alkaloids at 74.53:1 and 186.33:1 ratios, the co-toxicity factors were 32.89 and 28.93, respectively (Table 4).

[0125] Table 4 Co-toxic factors of konjac total alkaloids and common insecticides to greenhouse whitefly adults

[0126]

[0127] 2.3 Synergistic effects of mixtures of konjac total alkaloids and common insecticides on greenhouse whitefly

[0128] When the mass ratio of imidacloprid to konjac total alkaloids was 150:1, the LC50 of the mixed solution against the third instar nymphs of greenhouse whitefly was 59.33 mg / L, and the co-toxicity coefficient was 99.59, showing an additive effect; when the mass ratio of imidacloprid to konjac total alkaloids was 212.5:1, 275:1, 337.5:1 and 400:1, the LC50 of the mixed solution against the third instar nymphs of greenhouse whitefly were 49.23 mg / L, 58.49 mg / L, 50.90 mg / L and 62.23 mg / L, respectively, and the co-toxicity coefficients were 133.77, 120.12, 144.10 and 121.58, respectively, showing a synergistic effect. When the mass ratio of imidacloprid to konjac total alkaloids was 200:1, the LC50 of the mixed solution against the 4th instar nymphs of greenhouse whitefly was 58.47 mg / L, and the co-toxicity coefficient was 105.29, showing an additive effect; when the mass ratio of imidacloprid to konjac total alkaloids was 275:1, 350:1, 425:1 and 500:1, the LC50 of the mixed solution against the 4th instar nymphs of greenhouse whitefly were 47.98 mg / L, 50.98 mg / L, 55.14 mg / L and 59.34 mg / L, respectively, and the co-toxicity coefficients were 141.24, 141.08, 135.83 and 129.97, respectively, showing a synergistic effect. When the mass ratio of imidacloprid to konjac total alkaloids was 100:1 and 400:1, the LC50 of their mixed solutions against female adults of greenhouse whiteflies were 87.42 mg / L and 101.79 mg / L, respectively, and the co-toxicity coefficients were 85.47 and 108.17, respectively, showing an additive effect; when the mass ratio of imidacloprid to konjac total alkaloids was 175:1, 250:1 and 325:1, the LC50 of their mixed solutions against female adults of greenhouse whiteflies were 73.92 mg / L, 79.77 mg / L and 84.19 mg / L, respectively, and the co-toxicity coefficients were 123.73, 126.01 and 126.15, respectively, showing a synergistic effect. When the mass ratio of imidacloprid to konjac alkaloids was 100:1, the LC50 of the mixed solution against male adults of greenhouse whitefly was 86.17 mg / L, and the cotoxicity coefficient was 62.84, indicating an antagonistic effect. When the mass ratios of imidacloprid to konjac alkaloids were 175:1, 250:1, and 325:1, the LC50 of the mixed solution against male adults of greenhouse whitefly were 45.37 mg / L, 46.92 mg / L, and 51.69 mg / L, respectively, and the cotoxicity coefficients were 143.89, 151.69, and 144.76, respectively, indicating a synergistic effect. When the mass ratio of imidacloprid to konjac alkaloids was 400:1, the LC50 of the mixed solution against male adults of greenhouse whitefly was 69.30 mg / L, and the cotoxicity coefficient was 111.55, indicating an additive effect (Table 5).

[0129] Table 5 Synergistic effect of different mass ratios of imidacloprid and konjac total alkaloids on greenhouse whitefly

[0130]

[0131]

[0132] When the mass ratio of avermectin to total konjac alkaloids was 90:1, 117.5:1, 145:1, 172.5:1 and 200:1, the LC50 of their mixed solutions against the third instar nymphs of greenhouse whitefly were 19.97 mg / L, 22.15 mg / L, 24.75 mg / L, 25.80 mg / L and 25.11 mg / L, respectively, and the co-toxicity coefficients were 145.11, 139.96, 130.95, 129.64 and 136.42, respectively, all showing synergistic effects. When the mass ratio of avermectin to total konjac alkaloids was 80:1, the LC50 of the mixed solution against the 4th instar nymphs of greenhouse whitefly was 25.44 mg / L, and the co-toxicity coefficient was 109.13, showing an additive effect; when the mass ratio of avermectin to total konjac alkaloids was 135:1, 190:1, 245:1 and 300:1, the LC50 of the mixed solution against the 4th instar nymphs of greenhouse whitefly were 26.90 mg / L, 26.90 mg / L, 30.15 mg / L and 29.16 mg / L, respectively, and the co-toxicity coefficients were 121.51, 131.43, 122.77 and 130.89, respectively, showing a synergistic effect. When the mass ratio of avermectin to total konjac alkaloids was 50:1, 100:1, 150:1, 200:1 and 250:1, the LC50 of their mixed solutions against female adults of greenhouse whitefly were 26.91 mg / L, 33.32 mg / L, 35.03 mg / L, 40.44 mg / L and 38.98 mg / L, respectively, and the co-toxicity coefficients were 130.70, 131.07, 135.81, 123.13 and 131.44, respectively, all showing synergistic effects. When the mass ratio of avermectin to total konjac alkaloids was 50:1, 100:1, 150:1, 200:1 and 250:1, the LC50 of their mixed solutions against male adults of greenhouse whitefly were 21.89 mg / L, 27.80 mg / L, 31.03 mg / L, 30.50 mg / L and 33.53 mg / L, respectively, and the co-toxicity coefficients were 131.36, 130.19, 127.83, 136.64 and 128.19, respectively, all showing synergistic effects (Table 6).

[0133] Table 6 Synergistic effect of different mass ratios of avermectin and konjac total alkaloids on greenhouse whitefly

[0134]

[0135]

[0136] 3 Discussions

[0137] Alkaloids are a class of nitrogen-containing heterocyclic natural organic compounds. Over 6,000 species have been isolated and identified from various plants. Some alkaloids exhibit insecticidal activity, primarily through toxicity, repellency, feeding inhibition, or growth inhibition (Hu Guanfang et al., 2011; Zhang Junhui, 2016). The LC50 values ​​of konjac total alkaloids against greenhouse whiteflies at different developmental stages were significantly lower than those of commonly used insecticides such as imidacloprid, deltamethrin, azadirachtin, and avermectin. This indicates that konjac total alkaloids are significantly more toxic to greenhouse whiteflies indoors than the commonly tested insecticides, suggesting promising application and development prospects. The high indoor toxicity of the konjac total alkaloids used in this study against greenhouse whiteflies may be due to the fact that they are a mixture of multiple insecticidal substances. Extensive target detection revealed that the total alkaloids used in this study contained 269 alkaloids, including a large number of pyridines, quinolines, isoquinolines, terpenes, and piperidines (unpublished data). Alkaloids belonging to these classes, such as camptothecin (quinoline) (Ren Xiaoshuang et al., 2017), nicotine (pyridine) (Liu Binglei et al., 2023), sanguinarine (isoquinoline) (Li Chunmei et al., 2013), and veratridine (piperidine) (Li Zhixiong et al., 2020), all exhibit significant insecticidal activity against insects. Therefore, further research is needed to determine which substances in the total alkaloids used in this study are the key players in the greenhouse whitefly response. In this study, the toxicity of konjac alkaloids and the commonly tested insecticides to female whitefly adults was lower than that to male adults, which may lead to an increase in the proportion of female adults within the population. Yao Hongwei (2001), in his study of the mechanism of insecticide resistance in the white-backed planthopper Sogatella furcifera, noted a significant sex-linked inheritance of resistance-related enzyme activities and soluble protein content. This difference in genetic expression between male and female adults resulted in significantly higher enzyme activities and protein content in female adults, leading to greater insecticide resistance in females. Whether the differences in toxicity of konjac alkaloids and the commonly tested insecticides to female and male whiteflies in this study are related to differences in the activities of related detoxification enzymes and the content of related proteins requires further investigation.

[0138] This study found that when konjac total alkaloids were mixed with imidacloprid or avermectin in certain proportions, they exhibited a co-toxic and synergistic effect against greenhouse whiteflies at different developmental stages. Zhang Yufen et al. (2013), in their study of the synergistic effects of A. truncatula alkaloids on chemical pesticides, found that when A. truncatula alkaloids were mixed with lambda-cyhalothrin in certain proportions, they exhibited a significant synergistic effect against the diamondback moth, Plutella xylostella. Their findings are generally consistent with those of this study. Mixing A. truncatula alkaloids with lambda-cyhalothrin significantly enhanced the penetration rate of A. truncatula alkaloids into the cuticle of Plutella xylostella, facilitating the rapid accumulation of the active ingredients in the target area. The mechanism of action and synergistic mechanism of the konjac total alkaloids mixed with imidacloprid or avermectin against greenhouse whiteflies in this study warrants further investigation. Mixing botanical insecticides with chemical insecticides can reduce pesticide usage, lower pesticide residues, and slow the development of insecticide resistance (Ansante et al., 2017; Lei et al., 2021). Therefore, studying the optimal ratio of konjac total alkaloids with imidacloprid or avermectin and applying them to field trials for greenhouse whitefly control can provide theoretical and practical basis for the development of low-toxic and highly effective combination agents for greenhouse whitefly control.

[0139] In the integrated control of greenhouse whiteflies, natural enemies such as the Encarsia formosa are widely used (Yang et al., 2021). Using konjac alkaloids to control greenhouse whiteflies will undoubtedly have an impact on these natural enemies. Although most botanical insecticides are relatively safe against non-target organisms such as natural enemies in the environment (Wu Wei et al., 2003; Qi Zhijun et al., 2004), this study focused solely on the indoor toxicity of konjac alkaloids against greenhouse whiteflies. Further research is needed to determine the mechanism of action of konjac alkaloids against greenhouse whiteflies and whether they exhibit similar toxicity against other pests. This research aims to provide a scientific basis for the development and utilization of konjac alkaloids.

[0140] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for enhancing the toxicity of a konjac alkaloid extract and an insecticide to whitefly, characterized in that: The following steps are involved: (1) Konjac corms were cleaned, sliced, dried to constant weight, crushed, and sieved, and konjac flour was weighed. Ethanol was added and extracted for 24 hours at a solid-to-liquid ratio of 1:20 to obtain an ethanol extract. (2) adjusting the pH of the ethanol extract to 9.0-12.0 with 1 mol / L NaOH, extracting with ethyl acetate five times, combining the organic layers and concentrating them under vacuum evaporation at 55°C to obtain a konjac alkaloid extract, and preparing a methanol solution with a concentration of 100 mg / mL; (3) the konjac alkaloid solution and a common insecticide are composited in different volume ratios, and a secondary dilution method is adopted to prepare a toxicity determination concentration gradient; (4) Toxicity test was performed on whitefly nymphs and adults, and the mortality rate at each concentration was recorded; (5) Calculate the co-toxicity factor and co-toxicity coefficient based on the test results to determine the synergistic effect; (6) Calculate LC using statistical analysis software 50 The significance of the data was analyzed by analyzing the regression equation and its confidence interval.

2. The method according to claim 1, wherein The pH adjustment value of the extract is selected from at least one of 9.0, 10.0, 11.0 and 12.

0.

3. The method according to claim 1, wherein The mass volume ratio of ethanol to konjac flour in the konjac alkaloid extraction step is 1:

20.

4. The method according to claim 1, wherein The toxicity determination adopts an agar moisturizing leaf dipping method to treat whitefly adults, and adopts an immersion method to treat whitefly nymphs.

5. The method according to claim 1, wherein The compound concentration gradient was set to five test concentrations between 10% and 90%, with pure water as the blank control, and the experiment was repeated for no less than three groups.

6. The method according to claim 1, wherein The compounding ratios of the konjac alkaloid and the insecticide include volume ratios of 1:5, 1:2, 1:1, 2:1 and 5:1, and when the co-toxicity factor is greater than 20, the ratios are refined into five mass ratio gradients for co-toxicity coefficient determination.

7. The method according to claim 1, wherein The formula for calculating the co-toxicity factor is: Co-toxicity factor = [(actual mortality rate - theoretical mortality rate) / theoretical mortality rate] × 100, Among them, a co-toxicity factor > 20 was judged as synergistic, and a co-toxicity factor < -20 was judged as antagonistic.

8. The method according to claim 1, wherein The calculation of the co-toxicity coefficient includes the following steps: (1) Calculate the LC of each single dose 50 Value and compounding agent LC 50 value; (2) Calculate the actual toxicity index and the theoretical toxicity index; (3) Calculate the co-toxicity coefficient = (actual toxicity index / theoretical toxicity index) × 100, Among them, a co-toxicity coefficient greater than 120 indicates synergy, 80 to 120 indicates additiveness, and less than 80 indicates antagonism.

9. The method according to claim 1, wherein The survival of whitefly adults was recorded within 24 hours after the toxicity test, and the mortality was determined by tapping the tube wall and using a brush stimulation method.

10. The method according to claim 1, wherein SPSS 20.0 was used to analyze the toxicity data and calculate the toxicity regression equations, LC 50 The p-value and its 95% confidence interval were used to determine whether the difference in efficacy was statistically significant.

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