Pest-induced plant volatile compound attractant based on Pest-induced plant volatile compound and application of Pest-induced plant volatile compound attractant

Abstract

The invention discloses a composite attractant for inducing plant volatile matters based on insect pests and application of the composite attractant. The attractant is composed of an inducing composition, paraffin and a fiber cotton core, the inducing composition and the paraffin are mixed according to the volume ratio of 1: (5-10) and are filled in the fiber cotton core, and the inducing composition comprises longifolene, trans-nerolidol, (3E, 7E)-4, 8, 12-trimethyltridecane-1, 3, 7, 8-tetramethyltridecane-1, 3, 7, 8-tetramethyltridecane-1, 3, 7, 8-tetramethyltridecane-1, 3, 7, 8-tetramethyltridecane-1, 3, 7, the volume fractions of 1, 11-tetraene, alpha-farnesene, beta-elemene, alpha-ionone and myrcene are 47%, 26%, 12%, 4%, 5%, 2% and 4% respectively. The attractant disclosed by the invention can be used for trapping the eupolyphaga nivalis and improving the searching and positioning capability of the eupolyphaga nivalis on diaphorina citri nymphs, so that the field parasitic efficiency is improved, and the effective biological prevention and control of the citrus liberobacter asiaticum spreading entomophila can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biological control of natural enemies of pests, and particularly to an odor attractant of the natural enemy parasitoid wasp Tamarixia radiata and its application. Background Art

[0002] The Asian citrus psyllid (Diaphorina citri) is an important vector insect for transmitting Huanglongbing (HLB) in nature, and Huanglongbing is the most devastating disease in citrus production. Since there is currently no effective drug for the control of Huanglongbing, the control of the Asian citrus psyllid is one of the effective measures for preventing and controlling the spread of Huanglongbing. At present, the control of the Asian citrus psyllid mainly adopts chemical means, but the large-scale and unreasonable use of chemical agents will not only pollute the environment, but also easily cause the Asian citrus psyllid to develop drug resistance. Therefore, it is very necessary to develop a new, safe and effective control method to control the population of the Asian citrus psyllid.

[0003] Using natural enemy insects to control the damage of the Asian citrus psyllid is an economic, simple, safe and effective method. Tamarixia radiata is a dominant parasitoid wasp of the Asian citrus psyllid, with high host specificity and is an important natural enemy insect in the biological control of the Asian citrus psyllid. However, due to factors such as the openness and complexity of the natural field environment and the short flight distance of Tamarixia radiata, the parasitism rate, residence rate and reproduction rate of artificially released Tamarixia radiata in the field are not high. Therefore, it is necessary to develop a control strategy to improve the field parasitism rate of Tamarixia radiata.

[0004] In the three-level trophic interaction of plant-pest-natural enemy, the feeding of phytophagous insects will induce plants to produce volatile compounds, and plant volatiles will affect the life activities of natural enemy insects such as locating hosts and laying eggs, indirectly defend against pests, increase pest mortality, and thus reduce the damage of phytophagous insects. After citrus is damaged by the feeding of nymphs of the Asian citrus psyllid, it can initiate a plant defense response and release volatile compounds to attract natural enemies, thereby indirectly controlling the damage of the Asian citrus psyllid.

[0005] Based on this, by comparing and measuring the volatile metabolomes of nymphs of the Asian citrus psyllid feeding-induced, mechanically damaged and untreated, the plant volatile metabolites significantly up-regulated by nymph feeding stress were screened out. The content of up-regulated volatile substances was determined and verified by targeted measurement. Then, substances with attractant activity for Tamarixia radiata were screened out through behavioral assays. According to the content ratio in the leaves fed by nymphs, the ratio of active compounds was formulated and slowly released by placing them in a lure core to attract Tamarixia radiata, guiding it to quickly search for and locate hosts, improving the parasitism rate of nymphs of the Asian citrus psyllid, and realizing the green control of pests. Summary of the Invention

[0006] The object of the present invention is to provide an attractant for Asecodes hispinarum and its application. The attractant is a specific component screened from the volatile substances of host plants, and has the characteristics of high selectivity and strong pertinence. By effectively attracting Asecodes hispinarum in the environment or released, the Asecodes hispinarum can be advanced into the target control orchard and enter the search state, improving the parasitism rate of Asecodes hispinarum on the nymphs of Diaphorina citri, so as to achieve the purpose of controlling Diaphorina citri and restricting the spread of citrus huanglongbing. The operation is simple, pollution-free and environmentally friendly, with low cost and good pertinence and effect.

[0007] The first object of the present invention is to provide a composite attractant composition for Asecodes hispinarum, which comprises longifolene, trans - nerolidol, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, α-farnesene, β-elemene, α-ionone and myrcene.

[0008] Preferably, the volume fractions of longifolene, trans - nerolidol, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, α-farnesene, β-elemene, α-ionone and myrcene in the attractant composition are 47%, 26%, 12%, 4%, 5%, 2% and 4% respectively.

[0009] The second object of the present invention is to provide a composite attractant for Asecodes hispinarum, which is composed of the above-mentioned attractant composition, a solvent and a carrier, and the attractant composition and the solvent are mixed and filled in the carrier.

[0010] Preferably, the volume ratio of the attractant composition to the solvent is 1:5 to 10, specifically 1:5 and 1:10. More preferably, the volume ratio of the attractant composition to the solvent is 1:5.

[0011] Preferably, the solvent is paraffin wax.

[0012] Preferably, the carrier is a fiber cotton core.

[0013] Preferably, the fiber cotton core is cylindrical, with a specification of a diameter of 2.0 cm, a height of 3.0 cm and a pore diameter of 500 microns.

[0014] The present invention discovers that using longifolene, trans - nerolidol, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, α-farnesene, β-elemene, α-ionone and myrcene alone can all achieve the attracting effect on Asecodes hispinarum, but when the 7 kinds of attracting compounds are compounded in a specific ratio, they can play a synergistic role and obtain a remarkable attracting effect on Asecodes hispinarum.

[0015] The present invention selects paraffin as the solvent, mainly considering the characteristic that paraffin can dissolve a variety of organic volatile substances. The fiber cotton core selected in the present invention has good liquid permeability and excellent adsorption properties, can fully and evenly adsorb the mixture of the composite attractant composition and paraffin, and can stably and evenly release it during the implementation process, and can reduce the oxidation and decomposition rate of the active ingredients of the attractant compounds, greatly reducing the usage amount and stably and continuously releasing them.

[0016] The third object of the present invention is to provide a method for preparing the above-mentioned composite attractant for Cirrospilus pictus, which is obtained by adding the above-mentioned attractant composition and solvent to the carrier and fully absorbing and preparing.

[0017] Specifically, it includes the following steps:

[0018] S1. Put solid paraffin into a container and heat it to 70 °C until it is completely dissolved to obtain paraffin liquid;

[0019] S2. Mix longifolene, trans-nerolidol, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, α-farnesene, β-elemene, α-ionone and myrcene into the paraffin liquid to obtain a blend of the attractant compound mixture and paraffin liquid;

[0020] S3. Immerse the fiber cotton core into the blend of the attractant compound mixture and paraffin liquid until it is completely absorbed, and then take it out and let it cool and solidify to obtain it.

[0021] The fourth object of the present invention is to provide the application of the above-mentioned attractant composition or attractant in attracting parasitic wasps. Specifically, it is the application in attracting parasitic wasps in the natural environment or artificially released.

[0022] The fifth object of the present invention is to provide the application of the above-mentioned attractant composition or attractant in controlling Diaphorina citri in orchards.

[0023] Preferably, the application method is: put the parasitic wasp attractant into a non-woven fabric bag and hang it in the middle of the fruit tree branches.

[0024] Preferably, the application method is: hang the parasitic wasp attractant 7-10 days before the shoot stage of citrus plants (including spring shoots, summer shoots, autumn shoots, late autumn shoots and winter shoots).

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) Based on the chemical communication relationship among plants, pests, and natural enemies, the present invention qualitatively and quantitatively analyzes herbivore-induced plant volatiles using a volatile metabolomics system, and rationally compound them, achieving a good effect of attracting *Acerophagus notatus*. The compounded attractant has good attracting effects both in laboratory and field cage experiments, with good repeatability and high stability.

[0027] (2) The present invention is mainly used to trap and guide the artificially released *Acerophagus notatus* to the target prevention and control orchard, effectively improving the parasitism rate of *Acerophagus notatus* on the nymphs of *Diaphorina citri*, and using the parasitic wasps of *Diaphorina citri* natural enemies to control its population number, with the aim of restricting the spread of citrus huanglongbing.

[0028] (3) The present invention can be regularly hung according to the occurrence law of the *Diaphorina citri* population, that is, during the citrus shoot growth period. After use, it can reduce the application amount of chemical insecticides in the orchard, effectively reduce the pesticide residues in the orchard, and conform to the development direction of green agriculture. Description of the Drawings

[0029] Figure 1 Results of relative quantitative determination of key volatile metabolites in citrus young leaves under feeding stress, mechanical damage, and untreated conditions by *Diaphorina citri* nymphs.

[0030] Figure 2 Results of targeted determination of key volatile metabolites in citrus young leaves under feeding stress, mechanical damage, and untreated conditions by *Diaphorina citri* nymphs.

[0031] Figure 3 Schematic diagram of the experimental layout for attracting effect test in the cage.

[0032] Figure 4 Results of the attracting effect test in the cage. Detailed Description of the Invention

[0033] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention.

[0034] In the following examples, solid paraffin and fiber cotton cores are selected as the slow-release lure cores to explain the present invention. It should be noted that based on the disclosed content of the present invention, those skilled in the art can also process the lure cores of the present invention into natural rubber, synthetic rubber, polyethylene, polyvinyl chloride, paper materials, resins, or fiber matrices in combination with the conventional preparation process of lure cores for conventional attractants.

[0035] Example 1

[0036] Use a gas chromatography-mass spectrometry (GC-MS) to determine the volatile metabolome of citrus young leaves under feeding by *Diaphorina citri* nymphs, mechanical damage, and untreated conditions:

[0037] Using the citrus psyllid preference host plant - Citrus reticulata 'NianJu' (1-year-old tree) as the experimental material. Pest treatment: Transfer 3-5 instar nymphs of the citrus psyllid to the young leaves of the plant for feeding and damage; Mechanical damage treatment: Select the young leaves of the plant and prick them with a sterilized fine needle; Control group: Plants without any treatment, with 5 replicates set for each treatment. After 24 h of treatment, collect the leaves of the treatment group and the control group. Transfer the leaves to 1.5 mL centrifuge tubes and store them in a -80 °C refrigerator after quick freezing in liquid nitrogen for later use. Take out the samples from the -80 °C refrigerator, grind them in liquid nitrogen, vortex and mix evenly. Weigh about 500 mg (1 mL of liquid) of each sample into a headspace vial; Add 2 mL of saturated NaCl solution and 20 μL (10 μg / mL) of internal standard solution respectively; Use automatic headspace solid-phase microextraction (HS-SPME) to extract the samples, and then use gas chromatography-mass spectrometry (GC-MS) to qualitatively and relatively quantitatively analyze the volatiles.

[0038] In this study, GC-MS was used to detect the content levels of volatile metabolites in the young leaves of citrus psyllid nymphs under feeding stress, mechanical damage, and the untreated control group, and to explore and compare the change characteristics of key terpenoids in the leaves, with a focus on the significant increase in feeding stress of nymphs compared to mechanical damage and the untreated group, and mechanical damage being higher than or close to the untreated group. The results showed that 6 sesquiterpenes (longifolene, trans-nerylidol, ledol, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, α-farnesene, β-elemene) and 5 monoterpenes (6-methylionone, α-ionone, carvyl acetate, β-damascenone, myrcene) were detected in the young leaves and met the screening criteria for the above volatile changes ( Figure 1 ), among which the feeding stress of nymphs was upregulated by 2-5 times compared to mechanical damage and the untreated group.

[0039] Example 2

[0040] Using gas chromatography-mass spectrometry (GC-MS) to determine the targeted volatile metabolome of citrus psyllid nymphs feeding on, mechanically damaging, and untreated citrus young leaves:

[0041] Weigh appropriate amounts of each standard product precisely, dissolve them with chloroform, and prepare a standard stock solution of 1 mg / mL; Subsequently, dilute it with chloroform to standard series concentration solutions of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, 10 μg / mL, and 20 μg / mL, and perform on-machine detection.

[0042] Accurately weigh about 0.2 g of the sample, add 5.0 mL of chloroform-methanol solution (1:1, v / v), vortex for 30 s, extract ultrasonically in a water bath for 30 min, add 1 mL of water, let it stand for stratification, take the chloroform layer, repeat the extraction of the upper layer solution with chloroform twice, combine the chloroform, dry it under nitrogen, then dissolve it with 1 mL of chloroform, filter it through a membrane filter and measure it on a machine.

[0043] In this study, based on the results of volatile metabolomics, 11 terpene volatile substances were screened out. Standard curves were established by purchasing standard products, and then the samples were extracted by automatic headspace solid-phase microextraction (HS-SPME). The absolute content levels of volatile metabolites in the young leaves of the nymph feeding stress, mechanical damage, and untreated control groups were detected by gas chromatography-tandem mass spectrometry. The results showed that 11 terpene substances could be detected in the targeted determination of young leaves, and the changing trends of the 11 terpene substances were consistent with the results of volatile metabolomics ( Figure 2 ). After detection, in the nymph feeding stress group, the average weight ratio of longifolene was 623.8921 μg / g, the average weight ratio of trans-nerolidol was 346.3445 μg / g, the average weight ratio of ledol was 224.3300 μg / g, the average weight ratio of (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene was 162.1988 μg / g, the average weight ratio of farnesene was 46.5804 μg / g, the average weight ratio of β-elemene was 60.6256 μg / g, the average weight ratio of methyl ionone was 21.5955 μg / g, the average weight ratio of α-ionone was 24.7746 μg / g, the average weight ratio of carvyl acetate was 52.2601 μg / g, the average weight ratio of damascenone was 16.8980 μg / g, and the average weight ratio of myrcene was 51.7437 μg / g (see Table 1).

[0044] Table 1. Targeted determination of specific terpene compounds in the leaves of Diaphorina citri nymphs, mechanically damaged, and untreated citrus

[0045]

[0046] Note: The above data are average weight ratios, and each replicate has 5.

[0047] Example 3

[0048] Determination of the behavioral response of Chrysocharis parksi to single terpene compounds by Y-tube olfactometer:

[0049] Using paraffin oil as a solvent, prepare test solutions of each standard product with concentrations of 0.01 g / mL (1%) and 0.1 g / mL (10%). Respectively take 10 μL of the experimental reagent and paraffin oil and place them in two odor source bottles. Use a Y-tube olfactometer to measure the orientation behavior response of female Asecodes hispinarum to 11 single terpenoids. The experimental method refers to CN117461638A. One female Asecodes hispinarum is used for each treatment, and it is repeated 60 times. Only one female Asecodes hispinarum is placed each time for the chemotaxis behavior selection. Introduce the female Asecodes hispinarum from the straight wall of the Y-tube and start timing. Within 5 minutes, if the insect can cross 1 / 3 of the two arms of the Y-tube and stay for 5 seconds, it is recorded as having a choice; otherwise, there is no response. After each experiment, swap the positions of the two test arms, and clean the experimental instrument after each group of experiments.

[0050] In this study, a Y-tube olfactometer was used to measure the selection behavior of Asecodes hispinarum for 11 terpenoid compounds as shown in Table 2. The results showed that 7 terpenoid volatile metabolites (longifolene, trans-nerolidol, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, α-farnesene, β-elemene, α-ionone, and myrcene) could attract Asecodes hispinarum to varying degrees. Among them, longifolene, trans-nerolidol, and (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene were closely related to the concentration of the compounds, while α-farnesene, β-elemene, α-ionone, and myrcene showed obvious attracting effects on Asecodes hispinarum at both 1% and 10%.

[0051] Table 2. Selectivity of Asecodes hispinarum for 11 terpenoid compounds

[0052]

[0053]

[0054] Example 4

[0055] Effect of the compound attractant on the selectivity of Asecodes hispinarum:

[0056] According to the weight ratio of terpenoid volatiles determined by targeted assay of citrus young leaves treated with the feeding stress of Diaphorina citri nymphs, 7 terpenoid volatile metabolites (longifolene, trans - nerolidol, (3E,7E)-4,8,12 - trimethyltrideca - 1,3,7,11 - tetraene, α - farnesene, β - elemene, α - ionone, and myrcene) were measured and mixed (Table 3), dropped into paraffin oil and dissolved, and mixed evenly to prepare a composite attractant with a total mass - volume concentration of 0.1 g / mL; meanwhile, a 0.1 g / mL attractant of a single terpenoid compound was prepared. The results showed that compared with single compounds, the composite formula had a more obvious attracting activity on Asecodes hispinarum, indicating that after these compounds were compounded, there was a synergistic effect among the compounds.

[0057] Table 3. Selectivity analysis of compound attractants and single terpenoid compounds on Asecodes hispinarum

[0058]

[0059]

[0060] Note: This table shows volume fractions.

[0061] Example 5

[0062] Evaluation of the net - room effect of the compound attractant:

[0063] In this experiment, the main components of the configured attractant were longifolene, trans - nerolidol, (3E,7E)-4,8,12 - trimethyltrideca - 1,3,7,11 - tetraene, α - farnesene, β - elemene, α - ionone, and myrcene, and paraffin was used as the solvent. The mixed formula of the measured terpenoids and paraffin were prepared according to the volume ratios of 1:5, 1:10, 1:20, and 1:30.

[0064] The preparation steps of the slow - release attractant are as follows:

[0065] S1. Put solid paraffin into a beaker, heat it to 70 °C and stir until the paraffin is completely dissolved to obtain paraffin liquid;

[0066] S2. Mix longifolene, trans - nerolidol, (3E,7E)-4,8,12 - trimethyltrideca - 1,3,7,11 - tetraene, α - farnesene, β - elemene, α - ionone, and myrcene into the paraffin liquid according to the verified composite formula ratio (volume ratio 47:26:12:4:5:2:4), heat it to 70 °C and stir evenly to obtain a co - blend liquid of the terpenoid mixture formula and paraffin;

[0067] S3. Immerse the fiber cotton core column into the co - blend liquid until it is completely absorbed (no bubbles emerge), and then take it out and let it cool and solidify.

[0068] The test site was arranged indoors in the experimental net room of Zhongkai University of Agriculture and Engineering. The length × width × height of the net room was 80 cm × 80 cm × 80 cm. Then, the samples and the control were divided diagonally and hung at a height of 50 cm from the ground ( Figure 3 ). Five replicates were set for each formulation. Different formulations of attractants and sticky boards were used in combination. The attractant was placed in the middle of the sticky board to facilitate separately recording the number of Apoanagyrus lucasii captured. The paraffin cotton wick column without attractant was used as the blank control. Each time, 50 female Apoanagyrus lucasii were released, and the release time was 10:00 in the morning. The number of Apoanagyrus lucasii captured on the sticky board was recorded at 10:00 the next morning. The parasitoid trapping rate = (the number of parasitoids trapped on the sticky board / the total number of released parasitoids) × 100%.

[0069] The results showed that under the net room conditions, the selection rates of the parasitoids for Formulations 1, 2, 3, and 4 were 63.6%, 60.8%, 53.2%, and 51.6% respectively, indicating that Formulation 1 and Formulation 2 had a strong attracting effect on Apoanagyrus lucasii ( Figure 4 ).

[0070] In summary, through non-targeted comparative analysis of the volatile metabolomes of citrus psyllid nymphs under feeding stress, mechanical damage, and untreated citrus young leaves, the present invention screened out terpene volatile metabolites with significantly higher feeding stress of nymphs than mechanical damage and untreated ones, and further clarified the contents of these up-regulated terpene compounds in young leaves under different treatments by using targeted determination methods. Through indoor behavioral experiments, individual terpene substances that could significantly attract Apoanagyrus lucasii were screened out. At the same time, a compound formulation was developed according to the proportion of terpenes with significant attracting effects in the feeding induction treatment of citrus psyllid nymphs. That is, it was compounded at a volume ratio of 47% longifolene, 26% trans-nerolidol, 12% (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, 4% α-farnesene, 5% β-elemene, 2% α-ionone, and 4% myrcene, with the best selectivity and a good attracting effect on Apoanagyrus lucasii. Using fiber cotton wick as the carrier, the compound formulation and paraffin had a volume ratio of 1:5 attractant, showing a significant field attracting effect.

[0071] The present invention is an attractant for the natural enemy Apoanagyrus lucasii applicable to field applications. The attractant of the present invention is used in cooperation with the release of artificially reared Apoanagyrus lucasii, which can develop attracting technology to guide and continuously trap the released parasitoids in the field, and can largely make up for the deficiencies of the artificial release of parasitoids technology in biological control. Therefore, the formulation of the present invention is greener and safer during use and has a synergistic effect on the biological control of citrus psyllids.

[0072] Finally, it should also be noted that the above are only several specific embodiments of the present invention. Therefore, the present invention is not limited to the above embodiments and can have many variations. For those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A composite attractant composition for the bright-bellied enameled wasp, characterized in that: Contains longifolene, trans-nerolidol, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, α-farnesene, β-elemene, α-ionone and myrcene.

2. The composite attractant composition of the bright-bellied enameled wasp according to claim 1, characterized in that: The volume fractions of longifolene, trans-nerolidol, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, α-farnesene, β-elemene, α-ionone and myrcene in the composition are 47%, 26%, 12%, 4%, 5%, 2% and 4%.

3. A composite attractant for the bright-bellied enameled wasp, characterized in that: The invention comprises the attractant composition according to claim 1 and a solvent, wherein the volume ratio of the attractant composition to the solvent is 1:5-10.

4. The composite attractant of the bright-bellied enameled wasp according to claim 3, characterized in that The solvent is paraffin.

5. The composite attractant of the bright-bellied enameled wasp according to claim 3, characterized in that: Also included is a carrier.

6. The composite attractant of the bright-bellied enameled wasp according to claim 5, characterized in that The carrier is a fiber cotton core.

7. A method for preparing the composite attractant of the bright-bellied enameled wasp according to any one of claims 3 to 6, characterized in that: The attractant composition and solvent described in claim 1 are added to the carrier and fully absorbed to obtain the product.

8. Use of the attractant composition according to any one of claims 1 to 2 or the attractant according to any one of claims 3 to 6 in attracting parasitic wasps.

9. Use of the attractant composition according to any one of claims 1 to 2 or the attractant according to any one of claims 3 to 6 in controlling citrus psyllids in orchards.

10. The use according to claim 9, characterized in that: The attractant is put into a non-woven bag and then hung on the middle part of the tree trunk of the fruit tree.

Images