Insecticides
Seven insecticides belong to different new generations as avermectins (Emperor 0.5% EC, Shandong Jingbo Agrochemical Co., China; Vapcomic 1.8% EC, Vapco, Jordan), chlorfenapyr (Challenger 24% SC, BASF LTD., Egypt), diamide (Coragen 20% SC, Dupont, Canada), neonicotinoids (Actara 25% WG, Syngenta, Agro, Egypt; Imaxi 35% SC, Rotam agrochemical, Hong Kong), and spinosyns (Radiant 12% SC, Dow Agrosciences, England), along with 3 insecticides affiliated to conventional groups of organophosphates (Pestban 48% EC, Agrochem, Egypt), Organophosphates + pyrethroids (Action Phos 50% EC, Agrochem, Egypt) and pyrethroids (Delta Plus 50% EC, Delta Plus Co., Egypt) were evaluated against T. absoluta.
Insect
The populations of T. absoluta were established using larvae collected from untreated tomato fields, in Elfashn Village, Bani Sweif Governorate. The stock was maintained under laboratory conditions, and leaves of tomato were used to feed the insect larvae during the experiment.
The physico-chemical properties
Emulsion stability test
The emulsion stability test was carried out according to WHO specifications (WHO 1979). Into a 250 ml beaker, 75–80 ml of tested water (distilled, soft, and hard water) was poured. Five milliliters of the emulsifiable concentrate formulation was added by a pipette, while stirring with a glass rod. The beaker contents were stirred with a glass rod then completed to 100 ml by addition of the tested water, while the stirring was continuous. The beaker contents are poured immediately into a clean, dry, graduated 100 ml cylinder. The cylinder was kept at 30–31 °C for 1 h and examined for any creaming or separation.
Foam test
The emulsion stability test was carried out to measure the foam amounts formed on the emulsion surface in the cylinder after 5 min.
Wettability test
The test was carried out according to CIPAC specifications (Dobrat and Martijn 1995). Into a 250 ml beaker having an internal diameter of 6–6.5 cm, 100 ml of the tested water (distilled, soft, and hard water) were poured. Five grams of Actara insecticide were weighted and added at once by dropping them on the water, from a position level with the rim of the beaker, without undue agitation of the liquid surface. The time taken from the powder added until completely wetted was calculated to the nearest second.
pH test
The test was carried out according to CIBAC specifications (Dobrat and Martijn 1995). One gram of the tested formulation was weighed and transferred to a measuring cylinder (100 ml) containing about 50 ml distilled water. The cylinder was made up to 100 ml and shook vigorously for 1 min then it was allowed to settle. The pH of the supernatant liquid was measured.
The insecticidal efficiency against 2nd instar larvae of Tuta absoluta
Laboratory experiments
The biological efficacy of the tested insecticides was evaluated against 2nd larval instar of T. absoluta by leaf dipping method (IRAC 2017). Leaves of tomato plants (from un-infested green house) were immersed individually in a series of concentrations of each tested compound for 3 s. The leaves were placed in Petri dishes with 10 larvae. Four replicates were carried out for each treatment. Larvae in control treatment were fed on leaves treated only with water. The mortality percentages were recorded after 48 h of treatment. Data were corrected for mortality from control by Abbott (Abbott, 1925) The mortality data were subjected to Probit analysis to obtain the LC90 (Finney 1971).
Abbott formula
$$ \mathrm{Corrected}\%=1-\frac{\mathrm{Insect}\ \mathrm{population}\ \mathrm{in}\ \mathrm{treatment}\ \mathrm{after}\ \mathrm{treatment}}{\mathrm{Insect}\ \mathrm{population}\ \mathrm{in}\ \mathrm{control}\ \mathrm{after}\ \mathrm{treatment}}\times 100 $$
Field experiment
Field experiments were carried out on infested tomato plants with T. absoluta cultivated in Elfashn Village, Bani Sweif Governorate, from January to May 2016. A known area was cultivated with tomato plants and divided into plots; each one is 42 m2. Tomato plants were sprayed by three folds of LC90 values that previously estimated for the most promising insecticides (Coragen, Emperor, Challenger, Radiant, and Vapcomic). Samples of treated plants were taken before application and after periods of (1, 3, 5, and 7 days). Another sample of non-treated infested plants were also taken after the same previously tested periods and used as control. The reduction percentages were calculated according to Henderson and Tilton (1955). Larvae were considered dead if they were unable to move.
$$ \%\mathrm{Reduction}=1-\frac{\mathrm{Ta}\ \mathrm{X}\ \mathrm{Cb}}{\mathrm{Tb}\ \mathrm{X}\ \mathrm{Ca}}\times 100 $$
Where
Ta is the number of larvae/10 leaflets in the treatment after application.
Tb is the number of larvae/10 leaflets in the treatment before application.
Ca is the number of larvae/10 leaflets in check after application.
Cb is the number of larvae/10 leaflets in check before application.
The side effects of the most promising insecticides on some biochemical parameters of tomato plants
The activity of the enzymatic and non-enzymatic antioxidants of tomato-treated plants was evaluated. Leave samples were taken before spraying and after periods of 1, 3, 5, and 7 days. Another sample from non-treated infested plants was taken after the same previously tested periods and used as a control.
Plant tissue preparation
Enzyme extracts were prepared according to the method described by Chen and Wang (2006). Leaf tissues were homogenized in ice-cold phosphate buffer (50 mM, pH 7.8), followed by centrifugation at 8000 rpm and 4 °C for 15 min. The supernatant was used immediately to determine the activities of the tested enzymes.
Super oxide dismutase (SOD)
SOD (EC 1.12.1.1) activity was spectro-photometrically assayed at 560 nm by nitro-blue-tetrazolium (NBT) reduction method (Chen and Wang 2006). The reaction mixture (3 ml) contained 150 μl riboflavin (13 μM), 2.5 ml methionine (13 μM), 250 μL NBT (63 μM), 50 μl phosphate buffer (50 mM, pH 7.8), and 50 μl enzyme extract. One unit of SOD activity was defined as the amount of enzyme protein required for inhibition of 50% reduction of NBT.
Catalase (CAT)
CAT (EC 1.11.1.6) activity was determined spectro-photometrically by following the decrease in absorbance at 240 nm (Chen and Wang 2006). The mixture (3 ml) contained 1.9 ml phosphate buffer (50 mM, pH7.0), 100 μl enzyme extract, and 1 ml of 0.3% H2O2. The reaction was initiated by adding enzyme extract. One unit of CAT activity was defined as 0.01 deductions in absorbance at 240 nm per minute. The enzyme activity was calculated by Kong et al. (1999).
Poly phenol oxidase (PPO)
PPO (EC 1.10.3.1) activity was determined using a spectrophotometric method based on an initial rate of increase in absorbance at 410 nm (Soliva et al. 2000). Phosphate buffer solution pH 7 (0.1 M, 1.95 ml), 1 ml of 0.1 M pyrogallol as a substrate and 50 μl of the enzyme extract were pipetted into a test tube and mixed thoroughly. Then, the mixture was rapidly transferred to cuvette. The absorbance at 410 nm was recorded continuously at 25 °C for 5 min.
Glutathione S-transferase (GST)
The activity of GST was determined in supernatant (as enzyme solution) and measured spectrophotometrically by measuring the conjugation of CDNB (1-chloro-2,4-dinitrobenzene) with glutathione according to Habig et al. (1974). The conjugation is accompanied by an increase in absorbance at 340 nm. The rate of increase is directly proportional to the GST in the sample.
Total phenolic compounds content
A known weight of the fresh leaf tissue samples was extracted with 85% cold methanol (v/v) for three times at 0 °C. The combined extracts were collected, dried under vacuum, and made up to a known volume with distilled water. Total phenols were determined using Folin-Ciocalteu reagent, and then 0.5 ml extract was added to 0.5 ml Folin reagent, shaken, and allowed to stand for 3 min. Then, 1 ml of saturated sodium carbonate was added to each tube followed by distilled water, shaken, and allowed to stand for 60 min. The optical density was determined at 725 nm using spectrophotometer as described by (Diaz and Martin 1972).
Photosynthetic pigments content
Chlorophyll a, chlorophyll b, and total carotenoids contents were extracted from 1 g of longitudinal sections of fresh leaves and grounded in a mortar in 85% acetone. The optical density of the solution was recorded at 663, 644, and 452.5 nm for chlorophyll a, b, and carotenoids, respectively, measuring spectrophotometrically according to Metzner et al. 1965, and their levels were calculated according to the formula of Harmut and Lichtenthaler (1987). Values of photosynthetic pigments were expressed in mg/g fresh weight.
Yield production
At the harvest, yield of marketable healthy fruits was recorded. All fruits harvested from each plot throughout the harvest period (about five successive harvests) were weighted to calculate the total yield per feddan (ton/fed.), as well as the total cost and the net profit for feddan and for tons of tomato yield were also calculated. The income statement was used to estimate the profit of tomato production.
$$ \mathrm{Increase}\ \mathrm{of}\ \mathrm{yield}\ \left(\%\right)=\frac{\mathrm{Yield}\ \mathrm{of}\ \mathrm{treated}\ \mathrm{tomato}-\mathrm{Yield}\ \mathrm{of}\ \mathrm{untreated}\ \mathrm{tomato}}{\mathrm{Yield}\ \mathrm{of}\ \mathrm{untreated}\ \mathrm{tomato}} $$
Cost of treatments
Cost of various insecticides was taken as premarket price and labor charges were calculated. The total cost of spraying was calculated on the basis labor requirement per feddan per day. Market price of insecticides and labor charges were summed up to work out the total cost of application of each treatment. This cost was taken as an additional cost required for treatment against the pest.
Additional income over control
Additional income over control was calculated by multiplying the additional yield over untreated control with prevailing average local market price of tomato fruits.
Net profit
This was calculated by subtracting the additional cost required for treatment from the monetary benefit for each treatment.
Cost benefit ratio
The cost-benefit ratio (CBR) was calculated by dividing the net monetary return by total additional cost due to treatment.
Statistical analysis
Experimental design was a randomized complete block design. Data were subjected to one-way analysis of variance followed by Student–Newman–Keuls test to determine significant differences among mean values at the probability level of 0.05. The concentration–mortality data were subjected to Probit analysis to obtain the LC90 values using the Statistical Package for the Social Sciences (SPSS) 25.0 software program (SPSS 2017). The values of LC50 were considered significantly different if the 95% confidence limits did not overlap.
