Use of citrus volatiles to attract Pardosa pseudoannulata

Abstract

The present invention discloses the use of citrus volatile 1,8-cineole to attract Pardosa pseudoannulata, belonging to the technical field of spider attractants. The present invention provides the use of citrus volatiles to attract Pardosa pseudoannulata. Based on this use, the present invention also provides the use of citrus volatiles to prepare a Pardosa pseudoannulata attractant, the use of citrus volatile 1,8-cineole to attract Pardosa pseudoannulata, and the use of citrus volatile 1,8-cineole to prepare a Pardosa pseudoannulata attractant. The present invention utilizes 1,8-cineole, which has an attractant effect on Pardosa pseudoannulata, to develop a spider attractant, promote natural enemies to control citrus pests, meet the needs of green and sustainable development, have great development potential, and are of great significance.

Description

Technical Field

[0001] The invention belongs to the technical field of spider luring, and particularly relates to use of citrus volatiles in luring Pardosa pseudoannulata. Background Art

[0002] The citrus industry plays a vital economic role in southern my country, but its development is primarily constrained by pests and diseases. Currently, citrus pest control relies primarily on chemical methods, which offer rapid effectiveness and widespread protection. However, overreliance on chemical pesticides can cause severe ecological pollution, harm other beneficial organisms, and contribute to the development of pesticide resistance in pests, making future control more difficult.

[0003] At present, chemical methods are still the main means of pest control in my country, but the concept of biological control has gradually been formed, improved, and received attention. Although there are many reports on biological control of citrus pests in my country, the application of parasitic natural enemies and pathogenic microorganisms to control them has not achieved the desired results due to many factors. The research and application of efficient and specific sex pheromones have only been reported on a few citrus pests. It is difficult to achieve the desired control effect by relying on a single control method. The comprehensive use of multiple control methods is necessary to ensure the healthy development of the citrus industry.

[0004] Utilizing natural enemies within ecosystems to regulate the occurrence and development of pest populations is a core component of integrated pest management (IPM). The article "Current Status and Conservation of Spider Resources in Tangerine Orchards in Nanfeng County" reveals that natural predatory spiders account for 62.67% to 82.17% of predatory predators in tangerine orchards. These predators possess numerous advantages over other predators: ① They have a broad diet, specializing in preying on live insects and consuming large quantities; ② They are highly resistant to adversity and starvation; and ③ They can stably inhabit and reproduce in the field, effectively ensuring spider control of pests. Developing attractants or repellents based on volatile compounds is an important strategy for protecting citrus plants from citrus pests and has become an international trend in citrus pest control. Currently, there are few reports in China on the research and development and application of attractants or repellents for citrus pests, and most remain at the theoretical stage.

[0005] Using natural enemies in the ecosystem to control the occurrence of pest populations is one of the core contents of integrated pest management (IPM). Spiders are the main natural enemies in rural ecosystems. Among them, the Pardosa apseudoannulata ( & Strand, 1906)), a member of the family Lycosa (Araneae). Pardosa pseudoannulata preys on large and medium-sized pests such as moths and locusts.

[0006] At present, the development of spider attractants has not been reported, and there is no report in this field that citrus volatiles can be used to attract and regulate the Pardosa pseudoannulata. Summary of the Invention

[0007] In order to solve the technical problem of filling the above-mentioned gap in the prior art in this field, the present invention provides the use of citrus volatiles to attract Pardosa pseudoannulata.

[0008] The technical solutions of the present invention are as follows:

[0009] Use of citrus volatiles to attract Pardosa pseudoannulata.

[0010] The citrus volatiles are selected from: 1,8-cineole.

[0011] Use of citrus volatiles in preparing attractants for Pardosa pseudoannulata.

[0012] The Pardosa pseudoannulata regulating agent contains citrus volatiles as active ingredients;

[0013] Preferably, the citrus volatiles are selected from: 1,8-cineole;

[0014] Preferably, the regulation refers to: positive regulation based on attraction or negative regulation based on repellency.

[0015] Use of citrus volatile 1,8-cineole to attract Pardosa pseudoannulata.

[0016] The effective dosage of citrus volatile 1,8-cineole to attract Pardosa pseudoannulata is 10-100 μL.

[0017] Use of citrus volatile 1,8-cineole in preparing attractant for Pardosa pseudoannulata.

[0018] The attractant for Pardosa pseudoannulata includes an attractant active ingredient; the attractant active ingredient includes citrus volatile 1,8-cineole;

[0019] Preferably, the Pardosa pseudoannulata attractant further comprises: auxiliary materials.

[0020] The beneficial effects of the present invention are as follows:

[0021] The present invention discovered the attractiveness of the citrus volatile 1,8-cineole to the spider P. pseudoannulata. The spider's preference for 1,8-cineole was tested through a behavioral choice experiment. The electrophysiological response of the spider's sensory organs to 1,8-cineole was also tested using EAG (insect antennal potential), confirming its attractiveness. The behavioral test results showed that 1,8-cineole exhibited a highly significant (P < 0.001) positive attraction to P. pseudoannulata, indicating that the volatile 1,8-cineole has a strong attractant effect on the spider. In the EAG experiment, the peak stimulation of the volatile 1,8-cineole on the spider was greater than that of the positive control, dichloromethane, and significantly (P < 0.05) higher than that of the air control.

[0022] The present invention further tested the attractant effect of 1,8-cineole, a citrus extract, in the wild. Using indoor behavioral choice experiments and electroantennographic experiments, the present invention demonstrated the tropism of the Pardosa pseudoannulata spider to the citrus volatile 1,8-cineole, demonstrating its significant indoor attractant effect on the spider.

[0023] This invention can provide a reference for the subsequent development of spider attractants and is of great significance for the biological control of citrus pests. The invention utilizes 1,8-cineole, which has an attractant effect on the Pardosa pseudoannulata, to develop a spider attractant, promoting the control of citrus pests by natural enemies. This approach meets the needs of green and sustainable development, has great development potential, and is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram of the physical device of the two-way selection box system used in Section 1.2.1 of Experimental Example 1 of the present invention.

[0025] Figure 2 This is a diagram of the actual device of the glass electrode used in Section 1.2.2 of Experimental Example 1 of the present invention.

[0026] Figure 3 This is a bar graph showing the selectivity of Pardosa pseudoannulata to the citrus volatile 1,8-cineole in Section 2.1 of Experimental Example 1 of the present invention.

[0027] Figure 4 This is a bar graph of the potential values ​​of the antennal potential response of the Pardosa pseudoannulata to citrus volatiles in Section 2.2 of Experimental Example 1 of the present invention.

[0028] Figure 5 This is a bar graph showing the attractant effect of different concentrations of 1,8-cineole on Pardosa pseudoannulata in Experimental Example 2 of the present invention.

[0029] Figure 6 This is a bar graph of the field attraction experiment of the citrus volatile 1,8-cineole to the Pardosa pseudoannulata in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below with reference to specific embodiments and experimental examples, but the scope of protection of the present invention is not limited thereto.

[0031] Group 1 Example: Use of Citrus Volatiles in Regulating Pardosa pseudoannulata

[0032] This group of embodiments provides the use of citrus volatiles to attract the Pardosa pseudoannulata.

[0033] In a specific embodiment, the citrus volatiles are selected from: 1,8-cineole.

[0034] Example 2: Use of Citrus Volatiles in the Preparation of Pardosa Pseudoannulata Attractants

[0035] This group of examples provides the use of citrus volatiles to prepare an attractant for the Pardosa pseudoannulata.

[0036] In a specific embodiment, the Pardosa pseudoannulata regulating agent contains citrus volatiles as active ingredients;

[0037] Preferably, the citrus volatile is: 1,8-cineole.

[0038] The third group of examples: Use of citrus volatile 1,8-cineole to attract Pardosa pseudoannulata

[0039] This group of embodiments provides the use of the citrus volatile 1,8-cineole to attract the Pardosa pseudoannulata.

[0040] In a specific embodiment, the effective amount of the citrus volatile 1,8-cineole for attracting the Pardosa pseudoannulata is 10-100 μL.

[0041] Example 4: Use of citrus volatile 1,8-cineole in preparing attractants for Pardosa pseudoannulata

[0042] This group of examples provides the use of the citrus volatile 1,8-cineole in preparing an attractant for the Pardosa pseudoannulata.

[0043] In a specific embodiment, the attractant of Pardosa pseudoannulata comprises an attractant active ingredient; the attractant active ingredient comprises citrus volatile 1,8-cineole;

[0044] Preferably, the Pardosa pseudoannulata attractant further comprises: auxiliary materials.

[0045] Experimental Example 1

[0046] 1 Materials and Methods

[0047] 1.1 Experimental materials and treatments

[0048] Spider Collection and Rearing: Pardosa pseudoannulata (Lycodidae, Pardosa genus) were collected from lawns throughout the Jinggangshan University campus and housed individually in transparent plastic boxes (L×W×H = 16.5 cm × 11.5 cm × 5.8 cm). Moisturized sterile cotton balls were placed inside the boxes to provide moisture. The laboratory temperature was set at 25 ± 2°C, with a photoperiod of 14 L:10 D (i.e., 14 h daylight, 10 h nightlight). Advanced-aged spiderlings or adults were selected for the experiments and were starved for two days prior to the experiment.

[0049] Citrus Volatile Extraction: Citrus volatiles were extracted using organic solvents for testing. Five freshly harvested citrus leaves were picked with alcohol-wiped tweezers and placed in a clean 10 mL glass vial. Methylene chloride, an organic solvent, was added and the leaves were removed after 5 minutes of extraction. The solution in the vial was then concentrated using a nitrogen blowdown apparatus until approximately 1 mL of concentrate remained for use in EAG and behavioral testing.

[0050] Reagents and instruments: 1,8-cineole (purity ≥98%), TCI (Shanghai) Chemical Industry Development Co., Ltd., Japan).

[0051] The electroantennography (EAG) instrument consisted of a stimulus airflow controller (CS-55, Syntech, the Netherlands) and a signal recording controller (Keysight 34461A, Tangshan Dinggan Technology Co., Ltd.).

[0052] 1.2 Research Methods

[0053] 1.2.1 Electroantennogram of Pardosa pseudoannulata to the citrus volatile 1,8-cineole

[0054] Studies have confirmed that spiders possess odor-sensing hairs on their bodies that can sense chemicals in the environment, typically located on the tarsus and metatarsus of their legs. Xiao Yonghong et al. successfully recorded the electrical responses of female Beijing ghost spiders (Arachnoptera: A ...

[0055] During the experiment, the tarsal segment of the first step of the Pardosa pseudoannulata was selected and the claw at the end was cut off. The two ends of the walking leg were quickly connected to two homemade electrodes (glass capillaries were made into a pointed shape with a wire drawer, and conductive silver wire and physiological saline were filled into the electrodes, see Figure 2 The other ends of the two glass electrodes were connected to antennal potential electrodes. The potential responses of the receptors on the legs were amplified by a DG3FA-1 high-impedance microelectrode amplifier (Tangshan Dinggan Technology Co., Ltd.) and displayed on a computer. The test volatiles were dropped onto clean filter paper (length × width = 2 cm × 1 cm, Taizhou Jinao Paper Co., Ltd.) and placed at the airflow outlet. During the experiment, stimulation was repeated every 3 minutes. After each substance was stimulated, a new filter paper was replaced for the next substance. Air tests were performed 6 minutes before the start, after each substance test, and 6 minutes before the end of the test for that leg. Each experiment lasted 33 minutes, with 10 replicates for each volatile. The continuous humidification gas flow rate was set at 250 mL / min, the stimulation gas flow rate was 200 mL / min, and the stimulation method was foot-operated pulse delivery.

[0056] 1.2.2 Experiment on behavioral response of Pardosa pseudoannulata to citrus volatile 1,8-cineole

[0057] The spider behavior selection test device uses a two-way choice box system ( Figure 1 This system has been used to test the preference behavior of the dark-spotted spider Pireneitega luctuosa and the funnel-web spider Hololena curta. The two-way choice box system consists of three transparent polypropylene plastic boxes placed side by side (top: 16.5 cm × 11.5 cm, bottom: 14 cm × 8.5 cm, height: 5.8 cm). Two choice boxes are located on the left and right sides of the box, and a release box is located in the middle. The release box is connected to the left and right choice boxes by circular holes approximately 4 cm in diameter. The test spiders can freely enter and exit the release box. During the experiment, the left and right choice boxes were randomly assigned as the treatment box (with the test agent) and the control box (without the test agent). To facilitate climbing within the boxes, the interior walls of the three boxes were manually scratched to create roughness. The two-way choice box system was used to test the preference of the Pardosa pseudoannulata spider for the citrus volatile 1,8-cineole, thereby identifying compounds with a strong attractant effect on the spider. The test was conducted in a closed behavioral chamber with low airflow, an ambient temperature of 24-27°C, and a relative humidity of 50-60%. The test substance was the citrus volatile 1,8-cineole. Each volatile was diluted 10-fold with dichloromethane before use.

[0058] Before the experiment, the spiders were introduced into the release box, and the two choice box connections were sealed. They were allowed to acclimate for 20 minutes. During the experiment, a 10 μl drop of reagent (treatment box: citrus volatile 1,8-cineole, control box: dichloromethane) was placed onto qualitative filter paper (length × width = 3 cm × 3 cm, Taizhou Jinao Paper Co., Ltd.). After evaporation for 2 minutes, the paper was placed into the choice box. The two choice box connections were opened, and the experiment began. Recordings were taken every 20 minutes for the experiment, which lasted for 2 hours. After each volatile was tested, the filter paper was replaced, and the two-way choice box was wiped with 95% anhydrous ethanol. After air drying, the next volatile test was performed. Each experimental group consisted of 40 spiders.

[0059] 1.2.3 Orchard attraction experiment of the citrus volatile 1,8-cineole to the Pardosa pseudoannulata

[0060] A field baiting experiment was conducted in a pomelo orchard in Tongping Town, Jizhou District, Ji'an City, Jiangxi Province. Sticky traps were placed among the weeds in the orchard. Two treatment and control groups were set up, with five traps in each group spaced 5-10 meters apart. The treatment group traps contained a rubber lure in the center, containing 40 μL of dodecanal, while the control group traps contained only 40 μL of dichloromethane. The experiment lasted 48 hours, and after two days, the number of Pardosa pseudoannulata caught by the sticky traps was checked.

[0061] 1.3 Data Analysis

[0062] Chi-square tests were used to analyze differences between treatment and control groups in the behavioral choice experiment. One-way analysis of variance (ANOVA) was used to analyze differences in peak EAG responses between the various reagents. T-tests were used to compare the attractant efficacy between the treatment and control groups in the orchard attractant experiment. A P value < 0.05 was considered significant, and a P value < 0.001 was considered extremely significant. All statistical analyses were performed using Excel and SPSS 19.0 (IBM), and plotting was performed using Origin 2018 (OringinLab).

[0063] 2 Results and Analysis

[0064] 2.1 Behavioral selective responses of Pardosa pseudoannulata to the citrus volatile 1,8-cineole

[0065] In this study, a two-way choice box system was used to study the preference of Pardosa pseudoannulata for 1,8-cineole, a citrus extract. Statistical analysis showed that the number of times Pardosa pseudoannulata entered the treatment box containing 1,8-cineole was significantly higher (χ 2 =12.126, df=1, P<0.001, Figure 3 ) more than the number of times they entered the control box. This indicates that 1,8-cineole has a very significant attractant effect on Pardosa pseudoannulata.

[0066] 2.2 Electroantennographic responses of Pardosa pseudoannulata to citrus volatiles

[0067] The peak EAG potential responses of Pardosa pseudoannulata to eight volatiles are shown in Table 1. The data were compared between groups using one-way analysis of variance (ANOVA) and the least significant difference (LSD) method. Figure 4 ) showed that the potential response value of the receptors on the legs of the Pardosa pseudoannulata to the volatile 1,8-cineole was higher than that of other volatiles and dichloromethane controls, and was significantly (P < 0.05) higher than that of the air control.

[0068] Table 1. Peak antennal potentials of Pardosa pseudoannulata in response to citrus volatiles

[0069]

[0070] Experimental Example 2: Concentration Gradient and Field Attraction Experiment of Citrus Volatile 1,8-Cineole on Pardosa pseudoannulata I. Concentration Gradient Experiment of Citrus Volatile 1,8-Cineole on Pardosa pseudoannulata

[0071] The two-way selection system was used to test the attractant effect of different concentrations of 1,8-cineole on the spider Pardosa pseudoannulata. The results showed that 20-50 μl of 1,8-cineole had a significant attractant effect on the spider Pardosa pseudoannulata (P<0.05, Figure 5 ).

[0072] 2. Field Experiment on the Attraction of Pardosa pseudoannulata by the Citrus Volatile 1,8-Cineole

[0073] The results of the luring test showed that ( Figure 6 ): On the weedy ground of the pomelo orchard, a single sticky trap treated with 1,8-cineole attracted a maximum of 30 and a minimum of 14 Pardosa pseudoannulata spiders, with an average of 19.6±6.43. The control group attracted less than 10 Pardosa pseudoannulata spiders, with an average of 6.40±1.14. The number of Pardosa pseudoannulata spiders attracted by the treatment group was significantly higher than that of the control group, and the difference between the groups was extremely significant (t=7.835, df=8, P<0.001), indicating that 1,8-cineole has a certain attractant effect on Pardosa pseudoannulata spiders.

Claims

1. Use of citrus volatile 1,8-cineole to attract Pardosa pseudoannulata.

2. The use of the citrus volatile 1,8-cineole for attracting the Pardosa pseudoannulata according to claim 1, characterized in that: The effective dosage of citrus volatile 1,8-cineole to attract Pardosa pseudoannulata is 10-100 μL.

3. Use of citrus volatile 1,8-cineole in the preparation of attractants for Pardosa pseudoannulata.

4. The use of the citrus volatile 1,8-cineole in preparing an attractant for the Pardosa pseudoannulata according to claim 3, characterized in that: The attractant for Pardosa pseudoannulata includes an attractant active ingredient; the attractant active ingredient includes citrus volatile 1,8-cineole; 5. The use of the citrus volatile 1,8-cineole in preparing an attractant for the Pardosa pseudoannulata according to claim 3, characterized in that: The attractant for the Pardosa pseudoannulata further comprises auxiliary materials.

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