Culture technique of spider mite
The two-spotted spider mite, Tetranychaus urticae (Koch) (Acarina: Tetranitride), was reared according to the Dittrich method [21]. The colonies were obtained from castor bean plants from Kafrelsheikh Governorate and reared under laboratory conditions on the castor bean plant to avoid contamination with pesticide residues. Before starting, the prey culture was kept at 25 ± 2 °C under a 16 h photoperiod and 70 ± 5% relative humidity to encourage plant growth.
Materials
Abamectin
Technical grade abamectin (95% w/w) is used as a recommended acaricide by the Agricultural Pesticide Committee, Ministry of Agriculture and Land Reclamation, Egypt [22] and was kindly supplied by KZ Pesticides and Chemicals Company, Egypt.
Preparation of abamectin emulsion and nanoemulsion
Nanoemulsion of abamectin (10% w/w) was prepared using the O/W emulsion high-energy method. Briefly, abamectin dissolved in methanol was added to Tween 80 (a non-ionic surfactant) at a ratio of 3:1 w/w using a magnetic stirrer at 400 rpm for 30 min to form an organic phase. Abamectin nanoemulsion was prepared through the organic phase being added dropwise under ultrasonic and high shearing physical emulsification processes to distilled water at a ratio of 1:4 w/w at 100 W for 15 min. Abamectin emulsion was formed using the same method described above using a magnetic stirrer instead of an ultrasonic high shear mixer.
Extraction of neem oil
Fresh leaves of neem, Azadirachta indica, were collected from trees that had not been treated with pesticides (Agricultural Research Center, Egypt). Dried at 50 °C for 48 h, neem leaves were ground into a fine powder. Oil extraction from 50 g of neem powder was performed using Soxhlet apparatus and 300 ml ethanol at a temperature of 68 °C. Neem oil was obtained after solvent evaporation according to a previously described method [23].
Extraction of garlic oil
Fresh cloves of garlic, Allium sativa L., were collected from garlic plants that had not been treated with pesticides (Agricultural Research Center, Egypt). Cloves were cut and dried in an oven at 60 °C for 48 h and a powder was formed by milling. Fifty grams of garlic powder was used for extraction with a Soxhlet apparatus using ethanol (20:1) at a temperature of 50 °C. Garlic oil was obtained according to a previously described method [24].
Preparation of neem and garlic oil nanoemulsions
Both neem and garlic oils (considered as 100% w/w) were prepared as nanoemulsion formulations according to the methods described above for the preparation of abamectin nanoemulsion.
Characterization of prepared nanoemulsions
Morphology and droplet size were characterized by transmission electron microscopy (TEM; JOEL 1400 Plus, Japan) at 80 keV. The polydispersity index (PDI) was measured using a Brookhaven zeta potential analyzer. The stability of tested nanoemulsions was confirmed by testing under varying heating, cooling, and freezing conditions based on previously described methods [25]. The stability of prepared nanoemulsions was tested by centrifugation at 10,000 rpm for 30 min at 25 °C. Centrifuged nanoemulsions were then stored for 4 weeks at room temperature to monitor for separation or creaming.
Bioassay techniques
Acaricidal activity of prepared nanoemulsions against adult females of the two-spotted spider mite T. urticae. The leaf disc dip technique was used to evaluate the acaricidal activity of formulated nanoemulsions compared with abamectin in emulsion form against the two-spotted spider mite T. urticae [26]. Based on the results of preliminary tests, a range of concentrations that caused 10 to 90% mortality were selected for bioassay tests. Formulated compounds were diluted in distilled water to prepare a series of concentrations of the active ingredient (a.i). The tested concentrations of abamectin in emulsion and nanoemulsion forms were 0.001, 0.01, 0.1, 1, and 10 µg/ml, while the tested concentrations of neem and garlic oils nanoemulsions were 0.1, 1, 10, 100, and 1000 µg/ml. Four discs (35 mm in diameter) of castor bean leaves were dipped in each concentration for 5 s and left to dry. Then, ten adult female mites were transferred to each disc. Discs were placed on moist philtre paper resting on moist cotton wool pads in Petri dishes (90 × 105 mm) and maintained in the same conditions as the breeding room. Mite mortality was measured at 24 h after treatment with abamectin and 48 h after treatment with neem and garlic oils. With data being plotted on log concentration probit paper and LC50 values being calculated according to previously described methods [28], each treatment was replicated 4 times, with mortality rates being corrected against controls as previously decribed [27].
Acaricidal activity of prepared nanoemulsions against T. urticae egg deposition and egg hatching
To measure the residual effect of each tested nanoemulsions on adult female mites, five adult females T. urticae of known age were placed on each disc after dipping in one-fifth of the LC50 concentration of each tested chemical. Each treatment was replicated 4 times. The numbers of eggs laid were assessed individually on all discs after 24, 48, 72, 96, and 120 h. The number of hatched eggs was also counted 4 days after eggs were laid. This experiment was conducted at 25 ± 2 °C with a 16 h photoperiod.
Field experiments
Performed at the experimental farm of the Faculty of Agriculture, Kafrelsheikh University, Egypt (31º–07 ′N latitude and 30º–57 ′E longitude at 6 m above sea level), field experiments were conducted to evaluate the pesticidal activity of tested formulations against spider mites, Tetranychs urticae, infesting soybean plants under field conditions. The experiment was conducted in a randomized block design with four replicates. Plot areas were 10 m2. All tested nanoemulsions of abamectin, neem, and garlic oils, as well as a conventional emulsion of abamectin, were applied using a knapsack sprayer with one nozzle. Plant oil nanoemulsions were applied at concentrations equal to 100 times respective LC50 values, while abamectin formulations were applied at concentrations equal to 10 times respective LC50 values. The applied concentrations of plant oil nanoemulsions were selected based on the results of preliminary trials. Samples of 10 soybean leaves were randomly collected from each plot before treatment and after 2 days and 1 week of treatment. Reductions in mite numbers were calculated for each treatment according to the method of Henderson and Tilton [29].
Statistical analyses and equations
The Henderson and Tilton formula [29] was used to evaluate population changes as follows:
$${\text{Corrected}}\,\,\left[ \% \right] = \frac{{{\text{N}}\,{\text{in}}\,{\text{Co}}\,{\text{before}}\,{\text{treatment}} \times {\text{N}}\,{\text{in}}\,{\text{T}}\,{\text{after}}\,{\text{treatment}}}}{{{\text{N in}}\,{\text{Co}}\,{\text{after}}\,{\text{treatment}} \times {\text{N}}\,{\text{in}}\,{\text{T}}\,{\text{before}}\,{\text{treatment}}}} \times 100$$
Where N = Insect population, T = treated, \(\mathrm{Co}\)=control.
Abbott’s formula [27] was used to correct the observed mortality for natural mortality:
$${\text{Corrected}}\,\,\left[ \% \right] = 1 - \frac{{{\text{N}}\,{\text{in}}\,{\text{T}}\,{\text{after}}\,{\text{treatment}}}}{{{\text{N}}\,{\text{in}}\,{\text{Co}}\,{\text{after}}\,{\text{treatment}}}} \times 100$$
Where N = Insect population, T = treated, Co = control.
Toxicity indexes of tested compounds were determined according to a previously described method [30] as follows:
$${\text{Toxicity}}\,{\text{index}} = \frac{{{\text{LC}}50\,{\text{of}}\,{\text{the}}\,{\text{most}}\,{\text{effective}}\,{\text{compound}}}}{{{\text{LC}}50\,{\text{of}}\,{\text{the}}\,{\text{tested}}\,{\text{compound}}}} \times 100$$
ANOVA and the post-hoc Tukey test at the 5% level were used to compare treatments. All statistical analyses were performed using SPSS v.18 (SPSS Inc, Chicago, USA).