2.1. Chemical and reagents
The acaricides, spiromesifen (Bayer crop science, 3-mesityl-2-oxo-1-oxaspiro[4.4]non-3-en-4-yl 3,3-dimethylbutyrate), Fenazaquin (Dupont, 4-tert-butylphenethyl quinazolin-4-yl ether), Fenpyroximate (Tata Rallis, tert-butyl4-[[(E)-(1,3-dimethyl-5-phenoxypyrazol-4-yl)methylideneamino]oxmethyl]benzoate), Hexythiazox (Biostadt, trans-5-(4-Chlorophenyl)-N-cyclohexyl-4-methyl-2-oxo-3-thiazolidine carboxamide) Abamectin (Abacin, 1'R,2R,3S,4'S,6S,8'R,10'E,12'S,13'S,14'E,16'E,20'R,21'R,24'S)-2-[(2S)-butan-2-yl]-21',24'-dihydroxy-12'-[(2R,4S,5S,6S)-5-[(2S,4S,5S,6S)-5-hydroxy-4-methoxy-6-methyloxan-2-yl]oxy-4-methoxy-6-methyloxan-2-yl]oxy-3,11',13',22'-tetramethylspiro[2,3-dihydropyran-6,6'-3,7,19-trioxatetracyclopentacosa-10,14,16,22-tetraene]-2'-one) and Propargite (, 2-(4-(tert-Butyl) phenoxy) cyclohexyl prop-2-yn-1-yl sulfite) were obtained from local pesticide market.
For residue analysis study, mass-spectrometry grade acetonitrile, magnesium sulphate, formic acid, and anhydrous sodium chloride (analytical grade) were purchased from Merch.India Ltd. Graphitised carbon black and primary secondary amine were supplied by Agilent technologies India Private Ltd. Ultrapure water from the Q3 Merch Millipore unit was utilized for analysis. Certified raw material of spiromesifen was purchased from Sigma Alderich.
2.2. Field experiment
The trial was conducted at a farmers’ field in Coimbatore to evaluate the efficacy of different acaricides against T. urticae, as per the recommendations given by CIBRC (Central Insecticide Board and Registration Committee). Multi star R1 variety was purchased and primarily was sown in protrays and subsequently at their two-leaf stage it was transplanted in the bounds of the polyhouse. Seed rate, spacing and agronomic practices were followed as per the recommendation suggested by Tamil Nadu Agricultural University (TNAU AGRITECH PORTAL). Six different acaricides (Table 1) were selected and sprayed on cucumber under protected conditions. The field trial was laid out using RBD (Randomized Block Design) with replicated thrice during first fortnight of November. Spraying was done in three rounds at fortnightly intervals, commencing at 30 DAS, 45 DAS, and 60 DAS (days after sowing). On 0, 3, 5, 7, 10, and 14 days after each spraying, the population of nymph and adults of T. urticae were counted from the top, middle, and bottom leaves of 10 randomly selected plant from each treatment.
Table 1 Treatment and their concentration
Treatments
|
Formulation
|
Dose
|
Manufacturer
|
T1- Fenazaquin
|
10% EC
|
2.0 ml/ lit
|
Dupont
|
T2-Fenpyroximate
|
5% SC
|
0.8 ml/ lit
|
Tata Rallis
|
T3- Spiromesifen
|
22.90 % SC
|
0.8 ml/ lit
|
Bayer crop science
|
T4- Hexythiazox
|
5.45% EC
|
0.8 ml/ lit
|
Biostadt
|
T5- Abamectin
|
1.9% EC
|
0.5 ml/ lit
|
Crystal
|
T6- Propargite
|
57% EC
|
2.5 ml/ lit
|
Dhanuka Agritech Limited
|
T7- Control
|
-
|
-
|
-
|
2.3. Bioassay studies on field population of T. urticae
A bioassay was carried out to appraise the median lethal dose that causes 50 per cent mortality of mite. To this study, populations were collected from the polyhouses located at kannam palaiyam, Coimbatore, Tamil Nadu. The collected mites were cultured in the laboratory at a temperature of 30± 1°C and a relative humidity of 65-70 percent. In laboratory experiments, leaf dip bioassay was performed. Filter paper was laid over an adsorbent cotton in a petridish, which was maintained wet by adding water. Acaricidal solutions were made at various concentrations such as spiromesifen (4 ppm to 8 ppm), fenazaquin (5 ppm to 9 ppm), fenpyroximate (1.5 ppm to 3ppm), hexythiazox (1.00 ppm to 2.25 ppm), abamectin (0.90 ppm to 1.30 ppm) and propargite (28 ppm to 52 ppm). Mulberry leaves taken from plants that was maintained deprived of spraying any chemical in the Insectary, Department of Agricultural Entomology, Tamil Nadu Agricultural University, Coimbatore, were used for the bioassay. The collected leaves were cut into a 75 mm leaf disc, immersed in the acaricidal solution, and kept upside down on the filter paper, which was then moistened to keep the leaves fresh until an observation was over. The cultured mite was transferred at the rate of 30 mites per Petridish. The treatments were implemented in CRD design with three replications. Mortality count of mite was taken at 6, 12, 24 and 48 hours intervals, by observing under stereo microscope (CETI).
2.4. Pot culture and field experiment
The pot culture and field experiment were conducted in cucumber under protected conditions to evaluate the efficacy of selected six acaricides against two spotted spider mite (T. urticae) in the Insectary polyhouse (latitude: 11.0162913˚N and longitude: 76.9286577˚E), Department of Agricultural Entomology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore. Cucumber variety of Multistar Rizwan F1 was employed for the field and pot culture experiment and the plants of 25 days old were inoculated artificially by the mite culture, which was collected from polyhouse and cultured in the laboratory. As the preliminary laboratory bioassay studies shown resistance of mite to the dose recommended by the CIB&RC (Central Insecticide Board and Registration Committee), the effective dose obtained from the bioassay study was selected sprayed (Table 2) and the efficacy of the dose was observed in both pot culture and field experiment. Two rounds of chemicals spray were given at fortnightly intervals. During second fortnight of February, the trial was conducted in a CRD and RBD design with three replications for field and pot culture experiment respectively. The population count of nymph and adult of T. urticae was recorded on the top, middle, and bottom leaves of both potted and field plants in each treatment at 0,3, 5, 7, 10, and 14 days after each spray.
Table 2 Treatment with resulted concentration
Treatments
|
Concentration
|
Fenazaquin 10EC
|
2.99ml/lit
|
Fenpyroximate 5EC
|
3.3 ml/lit
|
Spiromesifen 22.90SC
|
2.93 ml/lit
|
Hexythiazox 5.45SC
|
3.90 ml/lit
|
Abamectin 1.9EC
|
1.4 ml/lit
|
Propargite 57EC
|
3.1 ml/lit
|
control
|
-
|
2.5. Harvest time residues in cucumber
2.5.1. Sampling of fruits
The acaricide, spiromesifen was applied at the dose of 2.93 ml/lit against two spotted spider mites in cucumber under poly house condition. Fruit sample from treated plants were collected during first three harvest and subjected for residue studies. Sample of 1 kg were collected and stored at -4˚C for residues analysis.
2.5.2. Sample extraction
A 50 mL Polypropylene centrifuge tube was filled with approximately 10 g of homogenised cucumber fruits sample. The solution was then vortexed for 10 minutes with 10 ml ultrapure water and 20 ml of acetonitrile in a vortex mixer. The homogenised solution was then mixed with 4 g of anhydrous MgSO4 and 1 g of NaCl in a vortexer and centrifuged for 10 minutes at 6000 rpm. A six ml of the supernatant extract was then placed in a 15 ml centrifuge tube containing 100 mg florisil, 100 mg PSA, 25 mg GCB, and 600 mg anhydrous Magnesium sulphate (MgSO4). It was vortexed for 1 minute and then centrifuged for 10 minutes at 3000 rpm. A four ml of extract of the supernatant was transferred to a glass tube and dried in a turbovap LV at 40°C using nitrogen gas. Following optimal settings, the dried extract was redissolved in 1 ml acetonitrile and placed into an autosampler vial for Liquid Chromatography-Mass Spectrometry-Mass Spectrometry (LC-MS/MS) analysis.
2.4.3. Instrument conditions for chromatography optimization
A Waters Alliance 2695 Separations Module and an ACQUITY tandem triple quadrupole mass spectrometer with electrospray ionisation interface in positive mode constitutes the LC-MS/MS system. The LC analysis was carried out on a 5µm (4.8 250 mm) Xterra analytical column C18 (Waters, Milford, MA, USA). The temperature of the column was fixed at 30℃. Water with 0.1 percent formic acid (A) and acetonitrile with 0.1 percent formic acid (B) (70:30 ratio) were used as mobile phases, with a flow rate of 0.5 ml min-1. The injection volume was 10 µl, and the analyte was eluted in 7.05 minutes using an isocratic mode. The optimal MS/MS settings were set to be 3.5 kV voltage, 150°C ion source temperature, and 500°C desolvation temperature. The cone gas and desolvation gas flow rates were 50 and 1100 l h-1, respectively. Working standard solutions of spiromesifen at 0.5 µg ml-1 in acetonitrile containing 0.1 percent (v/v) formic acid was directly fed into the mass spectrometer and chromatogram was acquired in the full scan mode to identify the parent and daughter ions.
2.4.4. Data analysis
The following formula was used to calculate insecticide residue levels using data from the LC-MS/MS chromatogram.
2.5. Data aanalysis
The data recorded from pot culture and field experiment was analysed using IBM SPSS software. Finney's (1971) approach was used to compute LC50 and LC95 values of bioassay studies with help of IBM SPSS software.