2.1 Insect and insecticide
In a screenhouse, the colonies of B. tabaci were kept alive on untreated plants of the brinjal variety Hisar Shyamal (HAU, Hisar). Whitefly adults were removed from the field using an aspirator (30 cm) and released onto untreated brinjal plants housed inside the screenhouse to start the culture. F1 generation of these adults was employed to maintain healthy whitefly colonies, and fresh plants were introduced instead of the old ones whenever needed. Different insecticides used in experiment were diafenthiuron (50% Wettable powder, NACL, India) at 150, 210 and 300 g active ingredient/ha, fenpropathrin (30% Emulsifiable Concentrate, Sumitomo, Japan) at 50,70 and 100 g active ingredient/ha, thiamethoxam (25% Wettable granule, Syngenta, India) at 25,35 and 50 g active ingredient/ha and deltamethrin (2.8% Emulsifiable Concentrate, Isagro Asia, Italy) at 7.5,10.5 and 15 g active ingredient/ha.
2.2 Research Area
The studies were carried out in a screenhouse on Brinjal var. Hisar Shyamal, at Entomological Research Farm (HAU), Hisar, during the 2018 and 2019 seasons. The pots were filled with the sandy loam soil used in the studies. A 1:1 combination of soil and thoroughly decomposed FYM (Farm Yard Manure) was created. The soil mixture was divided evenly across the pots to ensure that each pot contained a soil mixture with the same characteristics. The combination was identified as sandy loam with a pH of 7.40, an electrical conductivity of 0.27 m mhos/cm, 0.78 percent organic carbon, and 26.0 kg/ha available phosphorous and more than 196 kg/ha potassium.
2.3 Raising of test plants
The Hisar Shyamal variety of brinjal seedlings was utilized and was purchased from the nursery at the Botanical Garden Chaudhary Charan Singh Haryana Agricultural University, Hisar Haryana. Forty days old Brinjal seedling was planted in a pot (30 cm x 23 cm). These pots were kept in a whitefly-free screenhouse (mesh size: 12.5 mm x 3 mm). The plants in screenhouse were protected from the outside infestation of whitefly by spraying the walls, ceiling, and floor of the screenhouse with 0.3% formalin (Isochem Angamaly, Kochi, India). The entryway was also covered with a muslin cloth to keep whiteflies out of the screenhouse.
2.4 Leaf cages
The leaf cage was used for confining the adults of the whitefly. The cylindrical leaf cage was made from the cylindrical transparent plastic container by cutting a hole on its bottom with a sharp edge blade. Fevicol was used to cover the top of the glass with a muslin cloth to allow for ventilation, and a cotton swab plug was placed over the opening on the bottom side to allow for the release of adult whiteflies into the cage.
2.5 Ovipositional preference
The test plants were grown in pots and housed in a screenhouse without whitefly. The plants, which were 40 days old, split into five sets, with the first set given one spray, the second set given two sprays, the third set given three sprays, the fourth set given four sprays, and the fifth set given five sprays. Subsequent sprays were spaced apart by seven days. A total of 196 plants from all the treatments were distributed randomly in 12 rows across the open field after 72 hours of one, two, three, four, and five sprays. Treated plants were put in rows number 2 to 11. Each row had 20 plants, except the 10th and 11th rows had 18 plants, each keeping 67.5 and 30 cm distance between rows and plants, respectively. The experimental plants were surrounded by one border row of untreated plants. Double layered insect-proof nets were placed over an experimental area. The plant's leaves were removed, leaving only the top two fully developed leaves. Twenty males and twenty females, collected from the main colony, were released at equal distances from each of the four plants (10 adults per plant). By scanning the whole leaf surface with a 60X magnification lens, the total number of eggs placed on leaves was counted, and the mean number of eggs was computed. Newly hatched nymphs were included in the egg count. The experiment was repeated in 2019.
2.6 Biochemical analysis
Chemical analysis of treated brinjal leaves was carried out in Biochemistry Laboratory, Cotton Section, Department of Genetics and Plant Breeding, Chaudhary Charan Singh Haryana Agricultural University, Hisar. Third fully formed leaf from the top of the treated and untreated brinjal plants were plucked up 3 days after the treatment in each set of the spray. The leaves were kept in an oven at 65 + 1°C for three days. Later, each dried leaf was powdered and kept in a small air-tight plastic vial for further analysis. 200 mg of leaf sample were refluxed with 80% ethanol for 20 minutes to create the extract to determine total soluble sugar and total free amino acid. The filtrate was collected, and the residue was refluxed twice with 70 percent ethanol, keeping the tubes with a water condenser in a boiling water bath for 20 minutes. The supernatants were collected and pooled. Ethanol from pooled extracts was removed at 50˚C in a flash evaporator under a vacuum. Then the extract was diluted with water to make a 10 ml final volume. The same aqueous extract was then used for the estimation of total soluble sugars and total amino acids. Total sugar content and total free amino acid content were estimated by Dubois et al and by Lee and Takahashi, respectively (Dubios et al. 1951; Lee and Takahashi 1966)
Total phenol content was estimated by the method of Swain and Hillis (Swain and Hills 1959). Extraction was done by refluxing a 200 mg leaf sample with 5 ml of 80 percent aqueous methanol for one hour. The refluxed material was then filtered, and the volume was made to 5 ml by washing with 80 percent hot methanol. This extract was used for the estimation of total phenols. It was estimated by evaporating the 0.5 ml of methanol extract to dryness & the residue was dissolved in 6.5 ml of distilled water. To this, 0.5 ml of Folin-phenol reagent was added and shaken. One ml of saturated solution of sodium carbonate was added after 5 minutes. The blue color thus formed was read at 760 nm after one hour against the blank. The standard curve was prepared by using gallic acid in the range of 10–50 µg.
Crude protein content was estimated by the method of John Kjeldahl (Kjeldahl 1883). For digestion, 0.2 g sample was taken in a micro-Kjeldahl flask. 10 ml of concentrated H2SO4 and 2 g digestion mixture was added to it. The micro-Kjeldahl flasks were placed on digestion bench and heated till the solution became clear blue. The flasks were removed, cooled, and volume was made to 100 ml with distilled water. An aliquat of 10 ml was transferred to micro-Kjeldahl assembly, and 10 ml of 40 percent NaOH was added to it. 10 ml of N/100 H2SO4 was taken in a conical flask. To that, 2–3 drops of methyl red indicator were added. This conical flask was set under a condenser. The distillation was carried out for 10–15 min till the solution turned blue. The conical flask was removed after washing the tip of a condenser with distilled water into the flask. The flask's content was titrated against N/100 NaOH until the endpoint reached when the red column solution changed into pink. The volume of alkali used for neutralization of H2SO4 was recorded. The amount of nitrogen and hence protein in the sample was calculated using the following relationship:
Where,
V = Volume of N/100 H2SO4 – Volume of N/100 NaOH
D = Dilution factor (Volume made in volumetric flask)
W = Weight of the sample (g)
A = Aliquot taken for distillation
2.7 Data Analysis
The data on the ovipositional preference of B. tabaci eggs were subjected to two-way ANOVA after checking normality by Levene Test in IBM SPSS Statistic 23 Software. Different treatment means were separated by posthoc comparison using Tukey’s Honestly Significance Difference (HSD). The data on biochemical components were analyzed from an experiment with a completely randomized design (CRD) using ANOVA followed by a Tukey’s Honestly Significance Difference (HSD).