CN113396903B - Termite control drug containing alpha-terpineol and application thereof - Google Patents

Termite control drug containing alpha-terpineol and application thereof Download PDF

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CN113396903B
CN113396903B CN202110641623.7A CN202110641623A CN113396903B CN 113396903 B CN113396903 B CN 113396903B CN 202110641623 A CN202110641623 A CN 202110641623A CN 113396903 B CN113396903 B CN 113396903B
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李志强
刘炳荣
曾文慧
张世军
陈勇
陈彤
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Institute of Zoology of Guangdong Academy of Sciences
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Abstract

The invention discloses a termite control drug containing alpha-terpineol and application thereof. It contains alpha-terpineol and entomopathogenic nematodes. The experiment improves the control effect of the entomopathogenic nematodes on the termes formosanus by using the alpha-terpineol to interfere the immune defense system of the white ants and combining the infection of the pathogenic nematodes Steinernema carpocapsae All strain aiming at the most main harmful white ants in China, namely the white ants in Taiwan Coptotermes formosanus Shiraki, and has important significance for further strengthening the application of the pathogenic nematodes in the green control of the white ants.

Description

Termite control drug containing alpha-terpineol and application thereof
The technical field is as follows:
the invention belongs to the field of termite control, and particularly relates to a termite control drug containing alpha-terpineol and application thereof.
The background art comprises the following steps:
termites are listed as one of five worldwide pests by the international center for research on physiological and ecological characteristics of insects, can cause serious damage to various fields such as houses, buildings, reservoir dams, transportation, agricultural production and the like, and are particularly serious in tropical and subtropical regions (yellow regrowth and the like, 2000). Entomopathogenic nematodes (Entomopathogenic nematodes) are one of the important biological control measures, and have the characteristics of being capable of actively searching hosts, having a broad insecticidal spectrum, being difficult to generate drug resistance and the like. Researchers in the 70 th 20 th century have begun to utilize entomopathogenic nematodes to control termites, which have potential as termite control agents, but because of social behaviors, physiological and biochemical defense responses and other factors of the termites, the nematodes have poor effects in practical termite control applications (Chouvenc et Al, 2011; Al-Zaidawi et Al, 2020). However, the current research on the immune defense mechanism of the termites mainly focuses on the research on the bacteria and fungi for defending the termites, and the immune mechanism of the termites for defending the nematodes is not clarified.
The biological control of the termites is environment-friendly and is a trend of future development. Bacteria, viruses, fungi, nematodes, antibiotics and the like can be used for biological control of termites, wherein the application of entomopathogenic nematodes to termite control is reported at home and abroad, but the actual application effect is limited, and the termite behavioral immune defense mechanism is one of important reasons. The plant-derived active substance terpineol is a monocyclic monoterpene tertiary alcohol naturally occurring in plants.
The invention content is as follows:
the first object of the present invention is to provide a termite control agent containing α -terpineol which improves the infestation control effect of entomopathogenic nematodes on termopsis formosana.
The termite control agent containing alpha-terpineol according to the present invention contains alpha-terpineol and entomopathogenic nematodes.
Preferably, the entomopathogenic nematode is Steinernema carpocapsae All.
Preferably, the termites are lactotermite taiwan.
Preferably, the termite control agent containing alpha-terpineol contains more than 13333IJ/mL of entomopathogenic nematodes, and the concentration of the alpha-terpineol is more than 1.163 mg/mL.
Further preferably, the termite control agent containing alpha-terpineol contains 26667IJ/mL of entomopathogenic nematodes, and the concentration of the alpha-terpineol is 1.438 mg/mL.
The second purpose of the invention is to provide the application of alpha-terpineol in combination with entomopathogenic nematodes in controlling termites.
Preferably, said entomopathogenic nematode is Steinernema carpocapsae All.
Preferably, the termites are termes formosanus.
Preferably, the application amount of the alpha-terpineol is more than LD10 of the alpha-terpineol to termites, and the application amount of the entomopathogenic nematode is more than 100 IJ/termite.
Further preferably, the α -terpineol is applied in an amount of LD30 of α -terpineol for termites, and the entomopathogenic nematode is applied in an amount of 200IJ per termite.
The experiment improves the control effect of the entomopathogenic nematodes on the termes formosanus by using the alpha-terpineol to interfere the immune defense system of the white ants and combining the infection of the pathogenic nematodes Steinernema carpocapsae All strain aiming at the most main harmful white ants in China, namely the white ants in Taiwan Coptotermes formosanus Shiraki, and has important significance for further strengthening the application of the pathogenic nematodes in the green control of the white ants.
Drawings
FIG. 1 is a graph of the effect of various concentrations of alpha-terpineol on Steinernema carpocapsae All survival;
FIG. 2 is the combing time duration of termites under different treatment conditions (CK: control; All: nematode treatment; Tp: alpha-terpineol treatment; Tpm: mixed treatment);
FIG. 3 is the combing frequency of termites under different treatment conditions (CK: control; All: nematode treatment; Tp: alpha-terpineol treatment; Tpm: mixed treatment);
FIG. 4 is the termite shift under different treatment conditions (CK: control; All: nematode treatment; Tp: alpha-terpineol treatment; Tpm: mixed treatment);
FIG. 5 is the rate of termites under different treatment conditions (CK: control; All: nematode treatment; Tp: alpha-terpineol treatment; Tpm: mixed treatment);
FIG. 6 is a graph showing the changes in expression amounts of lactotermite ter (left) and lys (right) genes at different treatment times (CK: control; All: nematode treatment; Tp: alpha-terpineol treatment; Tpm: mixed treatment);
FIG. 7 is the tyrosinase activity change in the foregut of Lactotermes under different treatment times (CK: control; All: nematode treatment; Tp: alpha-terpineol treatment; Tpm: mixed treatment);
FIG. 8 is the tyrosinase activity change in the middle intestine of Lactotermes formosanus at different treatment times (CK: control; All: nematode treatment; Tp: alpha-terpineol treatment; Tpm: mixed treatment);
FIG. 9 is the tyrosinase activity change in the cuticle of Lactotermes formosanus at different treatment times (CK: control; All: nematode treatment; Tp: alpha-terpineol treatment; Tpm: mixed treatment);
FIG. 10 shows the antibacterial activity of different interstitial fluids of Oplophorus japonicus (CK: control; All: nematode treatment; Tp: alpha-terpineol treatment; Tpm: mixed treatment).
Detailed Description
In order to specifically explain the means and the implementation effect of the present invention, the present invention will be further explained with reference to the following embodiments and the accompanying drawings.
1. The entomopathogenic nematode used in the examples below was Steinernema carpocapsae All and the termites were Latotermes formosanus.
2. The following solutions or suspensions are not specifically illustrated, and the solvent is water.
3. Virulence determination of alpha-terpineol
Preparing a mother solution: dissolving 600mg of alpha-terpineol in 10mL of ethanol to prepare a mother solution with the concentration of 60mg/mL for later use, and storing at 4 ℃. The alpha-terpineol mother liquor is diluted into 5 concentrations of 0, 1.5, 2.0, 2.5 and 3.0mg/mL by ethanol. 0.5mL of drug diluent is sucked from the filter paper by a pipette
Figure BDA0003108081730000041
The outer edge is uniformly dotted inwards, and the filter paper is placed in a culture dish after being dried
Figure BDA0003108081730000042
Cover in the middle for standby, and add 300. mu.L ddH2O before use. 40 healthy workers of the species Oplopanax formosanus were present in each dish, 3 biological replicates. Ethanol self-filtering paper for control group
Figure BDA0003108081730000043
The outer edge is uniformly and inwardly spotted on the upper part, and the filter paper is put in a culture dish after being dried slightly
Figure BDA0003108081730000044
And covering the container for later use. Before use, 300. mu.L of ddH2O was added and placed in a dark incubator at 27. + -. 1 ℃ and 86% humidity. Dead termites were removed and the number of dead termites was recorded 1 time per day for 3 consecutive days.
The regression equation for virulence is shown in table 1 as y-8.121 x-1.820 and R-0.795. LD10 is 1.163mg/mL, and the confidence interval of the concentration of 95% is 0.996-1.287 mg/mL; LD20 is 1.316mg/mL, and the confidence interval of the concentration of 95% is 1.167-1.430 mg/mL; LD30 is 1.438mg/mL, and the confidence interval of the concentration of 95% is 1.303-1.548 mg/mL; LD50 is 1.666mg/mL, with a 95% confidence interval of 1.547-1.785 mg/mL.
TABLE 1 lethal conditions of alpha-terpineol for termites
Figure BDA0003108081730000045
Example 1
1. Effect of alpha-terpineol on nematode survival and infectivity
Adding alpha-terpineol into the nematode suspension to ensure that the concentration of nematodes in the suspension mixed by the alpha-terpineol and nematode liquid is 200 IJ/nematode (26667IJ/mL), the concentration of the alpha-terpineol is 1.163mg/mL, 1.316mg/mL and 1.438mg/mL (respectively marked as LD10, LD20 and LD30), placing the suspension in a dark incubator at 25 +/-1 ℃ for 7d, observing every day, and randomly taking 100 mu L of nematode suspension in a counting plate under a stereoscope to check the survival condition of the nematodes. Nematode suspensions were used as controls. And (3) calculating: survival% (% live nematode ÷ bust worm count) × 100%. Each treatment was 3 replicates.
10 larvae of the greater wax moth are picked into a filter paper of a 9cm culture dish, 1.5mL of the alpha-terpineol treated nematode suspension is dripped into each dish, the greater wax moth is checked for death after being placed in a dark incubator at the temperature of 25 +/-1 ℃ for 48h and 72h, and the incubation is continued to observe whether the offspring of the greater wax moth is generated. Nematode suspension, alpha-terpineol treatment with different dosages, and clear water treatment of greater wax moth are used as controls.
2. Bioassay of lethal effect of alpha-terpineol and nematode mixture on white ants
Blank control group: add 300. mu.L of ddH2O was dropped on a 6 cm-diameter filter paper in a petri dish, and 40 healthy workers were further added.
Nematode infection treatment: 300. mu.L of each of the nematode suspensions at a concentration of 3333IJ/mL (25 IJ/round), 6667IJ/mL (50 IJ/round), 13333IJ/mL (100 IJ/round) and 26667IJ/mL (200 IJ/round) was added dropwise to a 6cm filter paper petri dish, followed by addition of 40 healthy workers.
Alpha-terpineol treatment: 500. mu.L of alpha-terpineol (1.163 mg/mL, 1.316mg/mL, 1.438mg/mL) was added dropwise to 6cm of filter paper, and 300. mu.L of ddH was added thereto2O, and a further 40 healthy termites.
Mixing treatment: the nematode solution is prepared into the concentrations of 3333IJ/mL (25 IJ/mL), 6667IJ/mL (50 IJ/mL), 13333IJ/mL (100 IJ/mL) and 26667IJ/mL (200 IJ/mL), 500 mu L of alpha-terpineol with different concentrations is dripped on a 6cm filter paper, 300 mu L of nematode solution with different concentrations is dripped on the filter paper, and then 40 healthy termites are added.
The termites were placed in an incubator at 27. + -. 1 ℃ and RH 86% for 10 days in the dark and the mortality of the termites was recorded every day. Each treatment was 3 replicates.
Note: in the bioassay experiment, each container of 40 termites is filled with 0.3mL of nematode suspension, and the concentration is 200 IJ/container, (200 IJ/container is multiplied by 40/container) ÷ 0.3mL is 26667IJ/mL, namely 200 IJ/container is converted into 26667 IJ/mL.
3. Termite behaviour assay
A. Termite combing activity.
Blank Control (CK): add 300. mu.L of ddH2And dripping O into a culture dish with 6cm of filter paper, adding 40 healthy workers, and treating for 6 h.
α -terpineol treatment group: mu.L of alpha-terpineol (1.438mg/mL) solution was added dropwise to a 6cm filter paper petri dish, and 300. mu.L of ddH was added2O, adding 40 healthy termites and treating for 6 h.
And (3) a nematode treatment group: 300 μ L of a nematode suspension at a concentration of 26667IJ/mL (200 IJ/individual) was added dropwise to a 6cm filter paper petri dish, followed by addition of 40 healthy termites and treatment for 6 h.
Mixing group: the nematode solution was prepared as a suspension at a concentration of 26667IJ/mL (200 IJ/individual). mu.L of alpha-terpineol (1.438mg/mL) solution was dropped on a petri dish with a 6cm diameter filter paper, 300. mu.L of 26667IJ/mL (200 IJ/termite) nematode suspension was dropped on the filter paper, and 40 healthy termites were added and treated for 6 hours.
Each termite treated was placed in a 24-well plate, 2 per well, and repeated 6 times. Video recording is carried out for 15min by a video camera, and the frequency and the duration of the combing action of the termites are recorded.
B. Termite displacement and velocity measurements.
Blank Control (CK): add 300. mu.L of ddH2And dripping O into a culture dish with 6cm of filter paper, adding 40 healthy workers, and treating for 6 h.
α -terpineol treatment group: mu.L of alpha-terpineol (1.438mg/mL) solution was added dropwise to a 6cm filter paper petri dish, and 300. mu.L of ddH was added2O, adding 40 healthy termites and treating for 6 hours.
And (3) a nematode treatment group: 300 μ L of a nematode suspension at a concentration of 26667IJ/mL (200 IJ/individual) was added dropwise to a 6cm filter paper petri dish, followed by addition of 40 healthy termites and treatment for 6 h.
Mixing group: the nematode solution was prepared as a suspension at a concentration of 26667IJ/mL (200 IJ/individual). mu.L of alpha-terpineol (1.438mg/mL) solution was dropped on a petri dish with a 6cm diameter filter paper, 300. mu.L of 26667IJ/mL (200 IJ/termite) nematode suspension was dropped on the filter paper, and 40 healthy termites were added and treated for 6 hours.
A20 cm straight line is drawn on the paperboard, scales are marked (one scale is marked every 0.5 cm), and the paperboard is fixed. The termites were removed and placed on one end of the glass tube and the displacement and speed of the termites was recorded by a video recorder for two minutes. There were 6 replicates.
4、
A. Measurement of expression level of defense-related Gene
Distilled water, alpha-terpineol (1.438mg/mL), 26667IJ/mL (200 IJ/single) nematode suspension, mixed treatment, namely 1.438mg/mL alpha-terpineol solution and 26667IJ/mL (200 IJ/single) nematode suspension are subjected to combined treatment. The method specifically comprises the following steps:
blank control group: add 300. mu.L of ddH2O was dropped into a 6cm petri dish of filter paper, and then 40 healthy workers were added.
Group of nematode infections: 300 μ L of a nematode suspension at a concentration of 26667IJ/mL (200 IJ/individual) was added dropwise to a 6cm filter paper petri dish, followed by an additional 40 healthy termites.
α -terpineol treatment group: 500 μ L of α -terpineol (1.438mg/mL) solution was added dropwise to a 6cm filter paper petri dish, and 300 μL ddH2O, and a further 40 healthy termites.
And (3) mixing treatment group: the nematode solution was prepared as a suspension at a concentration of 26667IJ/mL (200 IJ/individual). mu.L of α -terpineol (1.438mg/mL) solution was dropped on a petri dish with a 6cm diameter filter paper, and 300. mu.L of 26667IJ/mL (200 IJ/individual) nematode suspension was dropped on the filter paper, and 40 healthy termites were added.
The treatments were placed in a incubator at 27. + -. 1 ℃ and RH 86% for continuous culture in the dark, and samples were collected at 0.5d, 1d, 5d and 9 d. And the RNA is extracted and stored in a refrigerator at-80 ℃.
Reverse transcription (One-Step gDNA Removal and cDNA Synthesis SuperMIX), sterile ddH for all template cDNAs2Diluting O by 10 times, mixing uniformly and using in an RT-qPCR reaction system (see table 2), using an internal reference gene HSP70, and using a primer sequence as shown in table 3.
TABLE 2 RT-qPCR reaction System
Figure BDA0003108081730000081
TABLE 3 immune defense genes and reference gene primer sequences
Figure BDA0003108081730000082
B. Tyrosinase Activity assay
Using a Solarbio tyrosinase activity detection kit (BC4055), each termite is dissected into a head, a foregut, a midgut and a cuticle, and is respectively put into 300 mu L of tyrosinase extracting solution, and each tube is dissected with 15 termites. When the enzyme label is used for measurement, the volume of the crude enzyme solution is 20 mu L, and the volume of the color developing agent is 100 mu L, and the crude enzyme solution and the color developing agent are added into a 96-hole enzyme label plate. Wherein head + foregut, epidermis: reacting at 30 ℃, measuring the absorbance at 475nm after reacting for 0.5h, and then measuring for 1 time every half hour until the reaction is detected for 2.5 h. Middle intestine: reaction at 30 deg.C, measuring absorbance at 475nm after 1 hr, and measuring 1 time every half hour until reaction time is 3 hr. Protein concentration determination: solarbio BCA protein concentration assay kit (PC0020) samples were diluted 8-fold and assayed. And (3) calculation of tyrosinase activity: enzyme activity (U/mg. min) 270 XKmaxCpr (see kit formula and parameters of experiment) Kmax: making time and absorbance change trend lines, and selecting the maximum slope in a determination time interval; cpr: the sample protein concentration is mg/mL.
C. Termite tissue fluid antibacterial activity determination
Inoculating Escherichia coli (Trans1-TI competent cells) in LB culture medium at 37 deg.C and 220rpm, shaking overnight, recovering Escherichia coli, inoculating Escherichia coli into new LB culture medium the next day, and shaking at 37 deg.C and 220rpm for 8 hr; the sample treated for 1d was dissected into three parts, foregut, midgut and epidermis, which were filled with 300. mu.L of Tris-NaCl solution, respectively, and 10 termites per tube were dissected. Extracting tissue fluid: adding the sterilized steel balls into a centrifuge tube, grinding at 40Hz for 90s, centrifuging for 15min, sucking the supernatant into a new sterilized centrifuge tube, and placing on ice for later use. 150 mu L of LB culture medium is added into the ELISA plate, 5 mu L of tissue fluid with uniform concentration is obtained, the inoculation amount of Escherichia coli is 0.5%, the temperature is 37 ℃, the rpm is 220, and the absorbance is measured at 600nm after shaking the bacteria for 4 h. Tissue fluid antibacterial activity ═ (Ack-a) ÷ Ack × 100. Where A represents the absorbance value and Ack represents the control absorbance value.
Third, experimental results
1. Effect of alpha-terpineol on nematode survival and infectivity
The survival rate of the nematodes after the nematodes are cultured in a mixture of alpha-terpineol and alpha-terpineol for 7 days (figure 1) is about 90 percent, which shows that the alpha-terpineol solution with 3 concentrations has little influence on the survival of the nematodes. The remaining nematode suspension is used for infecting the greater wax moth, and the greater wax moth is dissected after two weeks of infection to observe the occurrence of the greater mother worm, which shows that the alpha-terpineol solution with 3 concentrations has no influence on the infection capacity of the nematode.
2. Bioassay of lethal effect of alpha-terpineol and nematodes on termites
The half knock-down time KT50 of termites was calculated by mixing a low concentration nematode suspension with a low concentration alpha-terpineol solution as shown in Table 4. When the nematode concentration is 26667IJ/mL (200 IJ/single) and the alpha-terpineol concentration is 1.438mg/mL, KT50 is minimum, namely KT50 is 17 d. When the concentration of the nematode is 26667IJ/mL (200 IJ/single) and the concentration of the alpha-terpineol is 1.438mg/mL, the KT50 is more than 30 d. It can be seen that the mixing effect is much higher than the single treatment effect.
TABLE 4 half knockdown time after Mixed treatment of alpha-terpineol nematodes
Figure BDA0003108081730000101
Figure BDA0003108081730000111
And Tp injection: alpha-terpineol; an All: treatment with carbopapsae All; \: shows KT50>30 and no insecticidal effect. The number of blank knocks was 0 and is not included.
3. Response of termite behavior
The duration of termite combing under different treatment conditions (fig. 2) and frequency of combing (fig. 3), the duration of termite combing among nematode groups was significantly longer than the control and other treatment groups (P <0.05), and the combing behavior of the alpha-terpineol group and the mixed group was not significantly different from the control group. The combing frequency of the alpha-terpineol group and the mixed group is not significantly different from that of the control group, and the combing frequency of the termites in the nematode group is significantly higher than that of the termites in the mixed group (P & ltgt 0.002) and the control group (P & ltgt 0.006). Indicating that the alpha-terpineol can inhibit the cleaning behavior caused by the nematodes to a certain extent.
The displacement (fig. 4) and speed (fig. 5) of termites under different treatment conditions, the displacement distance of nematode-treated termites within 2min was significantly greater than the control group (P ═ 0.001), while the displacement of α -terpineol-treated (P <0.001) and mixed-treated (P <0.001) termites was significantly shorter than the control group. From the termite movement rate, the nematode treatment rate was significantly higher than the control group (P <0.001), and the rate of the α -terpineol and the mixed treatment was opposite. The change of termite displacement and speed shows that the nematode can enhance the movement ability of the termite, and the alpha-terpineol has the inhibition effect on the movement ability of the termite and can effectively inhibit the increase of the movement ability induced by the nematode.
4. Analysis of defense levels
(1) Measurement of expression level of defense-related Gene
Alpha-terpineol can inhibit the expression of key defense response genes of termites. FIG. 6, at 0.5d to 5d after treatment, the expression level of the ter gene of each treatment group is significantly lower than that of the control group, and the expression level of the mixed group is lower than that of the nematode single treatment group (P < 0.05). In 5 d-9 d, the expression level of lys in each treatment group is significantly lower than that in the control group (P <0.05), and the expression level of lys in the mixed group of 0.5 d-1 d is significantly lower than that in the single treatment group of nematodes.
(2) Tyrosinase Activity assay
Alpha-terpineol inhibits the activity of enzymes essential to the termite respiratory process. Tyrosinase activity in foregut was changed (fig. 7), and α -terpineol (P ═ 0.004) and mixed group (P <0.001) were all significantly higher than control group at 5 d. Tyrosinase activity was varied in the middle intestine (fig. 8), at 9d, the tyrosinase activity was significantly lower in the mixed group (P ═ 0.001) than the control group, and the tyrosinase activity was significantly lower in the mixed group than in the single treatment group (P < 0.05). Tyrosinase activity was varied in the epidermis (fig. 9), and at 9d, α -terpineol (P ═ 0.017) and the mixed group (P ═ 0.011) both had significantly lower tyrosinase activity in the epidermis than the control group. The results show that the alpha-terpineol has an induction effect on the tyrosinase activity of the termite foregut in the period of 5 days to 9 days, and the mixed treatment has an induction effect on the tyrosinase activity; at 9d, the combined treatment showed a synergistic effect of α -terpineol on tyrosinase activity inhibition in the midgut of termites; alpha-terpineol has an inhibitory effect on tyrosinase activity in the termite epidermis at 9 days.
(3) Termite tissue fluid antibacterial activity determination method
The alpha-terpineol can inhibit nematode from inducing the midgut tissue fluid of the termites to generate certain antibacterial activity. Crude enzyme protein concentration: 0.19 plus or minus 0.09mg/mL of foregut, 0.27 plus or minus 0.02mg/mL of midgut and 4.72 plus or minus 0.16mg/mL of hindgut. The crude enzyme solution concentration of the homogenized foregut and midgut is 1ug/mL, and the crude enzyme solution concentration of the epidermis is 3 ug/mL. The antibacterial activity of different tissues of the lactotermite is shown in fig. 10, and the antibacterial activity of the alpha-terpineol group and mixed foregut tissue fluid is lower than that of the control group. The antibacterial activity of the midgut tissue fluid of the alpha-terpineol group (P is 0.002) and the mixed group (P is 0.001) is obviously lower than that of the control group. The antibacterial activity of the epidermal tissue fluid of each treatment group is lower than that of the control group. The results show that the alpha-terpineol has an inhibiting effect on the antibacterial activity of the three interstitial fluids of the termites in the treatment period of 1d, wherein the antibacterial inhibiting activity of the mixed treatment of the alpha-terpineol and the nematodes in the midgut tissue is obviously enhanced compared with the antibacterial inhibiting activity of the nematodes in the single treatment of the nematodes.

Claims (4)

1. The termite control drug containing alpha-terpineol is characterized by containing more than 13333IJ/mL of entomopathogenic nematodes, wherein the concentration of the alpha-terpineol is more than 1.163mg/mL, and the entomopathogenic nematodes areSteinernema carpocapsaeAll; the termite is the Laurencia formosana.
2. The termite control agent set forth in claim 1 wherein the termite control agent containing α -terpineol comprises entomopathogenic nematode 26667IJ/mL and the concentration of α -terpineol is 1.438 mg/mL.
3. Use of a termite control agent containing α -terpineol according to claim 1 for controlling termites, wherein the entomopathogenic nematode isSteinernema carpocapsaeAll, the termite is the Laptotermes formosanus, the application amount of the alpha-terpineol is more than LD10 of the alpha-terpineol to the termite, and the application amount of the entomopathogenic nematode is more than 100 IJ/termite.
4. The use of claim 3, wherein said α -terpineol is applied in an amount of LD30 α -terpineol for termites and said entomopathogenic nematode is applied in an amount of 200IJ per termite.
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