Toxicity Test of Different Pesticides on Tobacco Root-knot Nematodes

Fei DAI Peibo LI Quan ZHANG Enfa CHEN

Abstract [Objectives] This study was conducted to quickly screen out effective and safe agents for controlling tobacco root knot nematode disease. [Methods] Six pesticides were tested and screened indoors at five different concentrations. [Results] The six pesticides all had toxic effects on the second-instar larvae of tobacco root-knot nematode， and the corrected mortality was positively correlated with the concentration and time of the pesticides， with the correlation coefficients above 0.8. From the perspective of inhibitory activity， the order was fosthiazate> abamectin>emamectin benzoate>cadusafos>ethoprophos>carbosulfan. Four pesticides that can be used in the field were selected. [Conclusions] This study provides a theoretical basis for field pesticide selection.

Key words Tobacco; Root-knot nematode; Medicament; Virulence determination; Screening

In recent years， tobacco root knot nematode disease has shown an upward trend in Guizhou tobacco areas year by year， which has an impact on tobacco yield and quality， and also has a great impact on the income of tobacco farmers. In order to quickly screen out effective and safe drugs， we have carried out indoor toxicity determination of different drugs on tobacco root knot nematodes to shorten the time of drug screening[1-5]， aiming to provide a basis for field pesticide selection.

Materials and Methods

Materials

Test materials

The tested tobacco root knot nematodes were isolated from the root knot sample of Yunyan 85 tobacco plants in Xixiu District， Anshun.

Tested drugs and preparation methods

90% abamectin powder （produced by North China Pharmaceutical Group Aino Co.， Ltd.）; 92.3% sebufos crude oil （provided by FMC）; 81.7% fosthiazate crude oil （produced by Shouguang Shenda Chemical Industry Co.， Ltd.）; 90% carbosulfan crude oil （provided by Jizhou Kaiming Pesticide Co.， Ltd.）.

The preparation method of mother liquids of test drugs： Two portions of the original drug were dissolved with 90 portions of acetone， and then emulsified with 8 portions of Tween.

Test instruments

A set of sterilization equipment; constant temperature incubator; alcohol lamp; tweezers; petri dishes; pipettes; stereo microscopes; test tubes; needles.

Test methods

Preparation of second-instar larva suspension of root knot nematode

The flue-cured tobacco root system with a large number of root knots was taken and rinsed gently with tap water. The diseased roots were cut into about 5 cm segments， and rinsed in 1% sodium hypochlorite solution for about 4 min. The surface tissue of the tobacco roots was slightly taken with bamboo chips， and the nematode eggs were collected with 200 and 500 mesh nematode sieves. The eggs were put into a small petri dish with a diameter of 8 cm， added with a small amount of sterile water， and incubated in a thermostat at 25 ℃ for 3 to 4 d. The second-instar larvae were collected and added with sterile water， obtaining a suspension with a concentration of about 500 larvae/ml for later use.

Indoor toxicity test method

First， 150 μl of the prepared drug liquids of different concentrations were added to each well of a 96-well cell culture plate， followed by the addition of an equal volume of nematode suspension and culture at 25 ℃ with enough moisture. The numbers of surviving and dead second-instar larvae of root-knot nematode were checked at different times after the treatment. The stiff nematodes were dead insects， and the bent nematodes were live insects. The mortality and adjusted mortality were calculated. The virulence regression equation and LC50 and LC95 confidence limits of the drug were calculated with dps software.

Test treatment method

In the indoor bioassay tests of the inhibitory effects of several drugs on the second-instar larvae of tobacco root-knot nematode， each drug was made into 5 different concentrations to treat the second-instar larvae of tobacco root-knot nematode， and the prepared second-instar larva suspension of tobacco root-knot nematode was taken and mixed with the agent. The death of the second-instar larvae of root-knot nematode was observed at 24 and 48 h， and the corrected 24 and 48 h mortality and the 24 and 48 h LC50 of the agent on the second-instar larvae were calculated.

Results and Analysis

Toxicity of emamectin benzoate in difference concentrations on the second-instar larvae of tobacco root-knot nematode

The second-instar larvae of tobacco root-knot nematode were treated with emamectin benzoate at 25， 50， 100， 200 and 400 mg/L， respectively. The results showed （Table 1） no matter it was at 24 h or 48 h， the corrected mortality of the second-instar larvae of root-knot nematode showed a certain law， that is： as the concentration increased， the corrected mortality was on the increase. After 48 h， except for the 400 mg/L liquid， the corrected mortality values were 28.52%， 38.14%， 65.36% and 98.54%， respectively. The 48 h mortality of the 25 and 50 mg/L treatments was lower than the 24 h， which was because some nematodes were in a suspended animation state and revived at 48 h. After 24 and 48 h of treatment， the toxicity equations were y=0.162 3x+38.86 and y=0.403 4x+19.819， respectively， and the LC95 and LC50 confidence limits were 345.9， 186.37 and 68.63， 74.81 mg/L， respectively. From the perspective of correlation coefficient， the correlation degree was greater than 0.9 at both 24 and 48 h. The concentration of 200-400 mg/L achieved a better effect.

Toxicity of abamectin in difference concentrations on the second-instar larvae of tobacco root-knot nematode

The second-instar larvae of tobacco root-knot nematode were treated with abamectin at 25， 50， 100， 200 and 400 mg/L （Table 2）， respectively. The results showed that the corrected mortality of the second-instar larvae of tobacco root-knot nematode increased with the increase of the concentration at both 24 or 48 h， and the corrected mortality showed an upward trend[6-7]. The reason why the corrected mortality of 25 and 50 mg/L at 48 h was lower than that at 24 h was still due to suspended animation of nematodes. After 24 and 48 h of treatment， the toxicity equations were y=0.157 1x+41.328 and y=0.366 5x+26.487， respectively; the LC95 and LC50 confidence limits were 341.64， 186.94 and 55.20， 64.16 mg/L， respectively; and from the perspective of correlation coefficient， the correlation degree was greater than 0.9 at both 24 and 48 h. The concentration of 200-400 mg/L achieved a better effect.

Toxicity of fosthiazate in difference concentrations on the second-instar larvae of tobacco root-knot nematode

The second-instar larvae of tobacco root-knot nematodes were treated with fosthiazate at 10， 20， 40， 80， and 160 mg/L， respectively. The results are shown in Table 3， and the corrected mortality also exhibited an increasing trend with the increase of concentration whether at 24 or 48 h， showing a positive correlation. After 24 and 48 h of treatment， the toxicity equations were y=0.393 2x+38.665 and y=0.396 8x+41.782， respectively. The LC95 and LC50 confidence limits were 143.27， 134.12 and 28.83， 20.71 mg/L respectively. From the perspective of correlation coefficient， the correlation degree exceeded 0.8 after 24 and 48 h. The concentration of 160 mg/L achieved the best effect.

Toxicity of carbosulfan in difference concentrations on the second-instar larvae of tobacco root-knot nematode

The second-instar larvae of tobacco root-knot nematode were treated with carbofuran at 100， 200， 400， 800 and 1 600 mg/L （Table 4）， respectively. The corrected mortality increased with the concentration increasing at both 24 and 48 h， showing a positive correlation with the correlation coefficients all above 0.9. After 24 and 48 h of treatment， the toxicity equations were y=0.043 5x+29.678 and y=0.044 1x+30.898， respectively; and the LC95 and LC50 confidence limits were 1 501.66， 1 453.56 and 467.17， 433.15 mg/L， respectively. The effect was best at 1 600 mg/L.

Toxicity of ethoprophos in difference concentrations on the second-instar larvae of tobacco root-knot nematode

The second-instar larvae of tobacco root-knot nematode were treated with ethoprophos at 25， 50， 100， 200 and 400 mg/L （Table 5）， respectively. The corrected mortality increased with the concentration increasing at both 24 and 48 h， showing a positive correlation with the correlation coefficients of 0.917 5 and 0.900 7， respectively. After 24 and 48 h of treatment， the toxicity equations were y=0.171 7x+31.796 and y=0.172 8x+33.233， respectively; and the LC95 and LC50 confidence limits were 368.11， 357.45 and 106.02， 97.03 mg/L， respectively. The effect was best at 400 mg/L.

Toxicity of cadusafos in difference concentrations on the second-instar larvae of tobacco root-knot nematode

The second-instar larvae of tobacco root-knot nematode were treated with cadusafos at 5 concentrations （Table 6）， respectively. The corrected mortality was positively correlated with the concentration at both 24 and 48 h， with the correlation coefficients of 0.917 5 and 0.900 7， respectively. After 24 and 48 h of treatment， the toxicity equations were y=0.157 2x+38.395 and y=0.154 9x+41.351， respectively; and the LC95 and LC50 confidence limits were 360.08， 346.35 and 55.84， 73.82 mg/L， respectively. The concentration of 400 mg/L achieved the best effect.

Analysis and screening of comprehensive effects of different drugs

The test results of the 6 drugs （Table 7） showed that the 6 drugs had certain toxic effects on the second-instar larvae of root-knot nematode[8-11]. The LC50 values of the test agents 24 h after the treatment from high to low ranked as carbosulfan>ethoprophos>cadusafos>emamectin benzoate>abamectin>fosthiazate. The ability to kill the second-instar larvae was improved at 48 h after the drug treatment. From the point of view of the toxicity of the pesticides to the second-instar larvae of root-knot nematode， fosthiazate was the most toxic to the second-instar larvae of root-knot nematode. The LC50 was 28.83 and 20.71 mg/L at 24 and 48 h， respectively， and the LC95 was 143.27 and 134.12 mg/L， respectively. The LC50 of ethoprophos was 106.62 and 97.03 mg/L at 24 and 48 h， respectively， and the LC95 was 368.11 and 357.45 mg/L， respectively. The LC50 of cadusafos was 73.82 and 55.84 mg/L at 24 and 48 h， respectively， and the LC95 was 360.08 and 346.35 mg/L， respectively. The toxicity of abamectin and emamectin benzoate to the second-instar larvae of tobacco root-knot nematode was lower than that of fosthiazate， but better than other tested agents， and their LC50 values were slightly different. The LC50 of abamectin was 55.20 and 64.16 mg/L at 24 and 48 h， respectively， and the LC95 was 341.64 and 186.94 mg/L， respectively. The LC50 of emamectin benzoate was 68.63 and 74.81 mg/L at 24 and 48 h， respectively， and the LC95 was 345.9 and 186.37 mg/L， respectively. Meanwhile， in accordance with the prohibition of tobacco pesticides， the two banned pesticides， ethoprophos and cadusafos were eliminated， and 4 suitable pesticides were screened， namely fosthiazate， emamectin benzoate， abamectin and carbosulfan.

Conclusions and Discussion

The toxicity of 6 different pesticides on the second-instar larvae of tobacco root-knot nematode was determined. The results showed that the 6 pesticides had toxic effects on the second-instar larvae of tobacco root-knot nematode. The mortality showed an upward trend with the increase of concentration and time[12-14].

Due to time constraints and limited drugs， there are still some pesticides that are available for testing in the future. We hope that there will be an opportunity to conduct more tests in the future to screen out better drugs.

Among the 6 pesticides tested， for safety reasons， 2 were excluded as they are banned tobacco pesticides. Therefore， 4 pesticides were actually screened out. It is recommended to use the selected pesticides in rotation or in combination to achieve economic and safe purposes[15-17].

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