Efficacy of Betaproteobacteria-based insecticides for managing whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), on cucumber plants

2.1 Insect colonies and plant culture

Adult B. tabaci specimens were collected from infested cucumber plants in a field in Irbid, Jordan, located at approximately 35.85°N latitude and 35.85°E longitude. These specimens were subsequently maintained for several generations on young cucumber plants cultivated in plastic pots (15 cm in diameter) filled with potting soil. The plants were kept in a controlled growth chamber at a temperature of 25 ± 3°C, with a photoperiod of 16 h of light and 8 h of darkness, at the Department of Plant Production, Jordan University of Science and Technology, Irbid, Jordan. The whitefly colony was housed in a cage measuring 60 cm × 60 cm × 100 cm, which was covered with fine mesh on all sides. To ensure colony sustainability, a continuous supply of newly grown greenhouse plants was provided to replace older plants that deteriorated due to the high feeding pressure exerted by the whiteflies.

Cucumber seeds were sown in 50-cavity plastic seedling trays (50 cm × 30 cm × 0.6 cm) filled with commercial potting soil. After several days of growth under greenhouse conditions, seedlings at the primary leaf stage were individually transplanted into 15 cm diameter plastic pots containing the same potting soil. The experimental plants were maintained under greenhouse conditions at 25 ± 3°C with a photoperiod of 16 h of light and 8 h of darkness. They were fertilized weekly with a 20-9-20 water-soluble fertilizer (N:P:K) and irrigated as needed until they were utilized in the experiments.

2.2 Insecticides

This study employed two commercially formulated microbe-based insecticides: Grandevo WDG (30% Chromobacterium subtsugae strain PRAA4-1 and spent fermentation media) and Venerate XC (94.46% heat-killed Burkholderia spp. strain A396 cells and spent fermentation media), both obtained from Marrone Bio Innovations Inc., Davis, California, USA. Well-established Movento 240 SC (22.4% spirotetramat: cis-3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro [4.5] dec-3-en-4-yl-ethyl carbonate) was used as standard due to its effectiveness against a wide range of pests, its unique mode of action, and its systemic properties. The efficacy of these products was evaluated across three concentrations for their effects on different developmental stages of B. tabaci feeding on cucumber plants under laboratory conditions. The concentrations tested were 0.4, 1.2, and 2.4 g/L for Grandevo; 0.5, 1.0, and 2.0 mL/L for Venerate; and 0.02, 0.04, and 0.08 mL/L for Movento, representing the lower, middle, and upper limits of the recommended field rates for each product.

2.3 Effects of insecticides on whitefly

To assess the impact of the novel insecticides on the survival of adult whiteflies, as well as young and old nymphs and egg hatch rates, leaf disc bioassays were conducted using plastic Petri dishes (5.5 cm diameter) equipped with gauze-covered ventilation holes. The efficacy of direct application was evaluated using cucumber foliage, which was cut into discs (5 cm diameter) with a sharp-edged plastic tube. For each treatment, six plants at the third true-leaf stage were utilized.

2.4 Ovicidal effect of biopesticides

To evaluate egg hatch rates, approximately 30 adult whiteflies were confined to the undersurface of a fully expanded second leaf on each plant using a clip-on cage made from a plastic Petri dish (6 cm diameter). Adults were anesthetized with CO₂ for a few seconds before being transferred with a fine, moist camel-hair brush. These cages were constructed from a plastic Petri dish (6 cm × 1.5 cm) with two side holes covered with fine mesh for ventilation. After 12 h, the cages were removed, the infested leaves were excised, and the ovipositing sites were cut into discs (5 cm diameter) using a sharp-edged plastic tube. The number of eggs laid on each disc was counted using a hand lens at 20× magnification and recorded.

Subsequently, six leaf discs were immersed in each dilution of the test products for 10 s with gentle agitation. The discs were then placed on paper towels, abaxial side up, to air dry before being positioned with the abaxial surface facing upward on a 4 mm layer of 1% agar in a plastic Petri dish (5.5 cm diameter) that had been prepared 1 day prior to testing. The Petri dishes were maintained in an inverted position at 23 ± 3°C under a 16:8 (L) photoperiod. Six replicates were conducted for each treatment, and the proportion of egg hatching was recorded at 24-h intervals over a period of 2 weeks.

2.5 Effect of biopesticides on young and old nymphs

Similar procedures were employed to assess the direct effects of the test insecticides on young nymphs (3 days old) and older nymphs (10 days old). Adult whiteflies were introduced to the plants as described previously for egg hatch assessment. After a 12-h oviposition period, the infested leaves were left intact on the plants for 7 or 14 days to facilitate egg hatching and nymph development. Following these periods, leaves containing the respective immature stages were excised, and the infested areas were cut into 5 cm diameter discs for nymph counting prior to insecticide exposure.

The discs were treated with the appropriate dilution of the insecticides and maintained on agar as previously described. Live and dead nymphs were counted and recorded at 24-h intervals until adult emergence, and the total developmental time for treated young nymphs was documented. Each treatment was replicated six times.

2.6 Effect of biopesticides on survival of adults

The direct and residual effects of the formulated insecticides on adult whiteflies were evaluated using untreated cucumber foliage cut into discs, as previously described. For the assessment of direct application efficacy, leaf discs from each treatment were placed in plastic Petri dishes lined with a layer of agar, abaxial side up. Fifty adult whiteflies were transferred onto each leaf disc using a fine camel-hair brush. The leaf discs containing the insects were then treated with each insecticide at three different concentrations using 500 mL plastic spray bottles until thoroughly saturated. The Petri dishes were immediately inverted to remove excess spray solution and maintained in this inverted position under the aforementioned conditions.

Similar procedures were employed to quantify the residual effects of Grandevo, Venerate, and Movento on adult whiteflies, utilizing three concentrations for each insecticide. In this assessment, six leaf discs (5 cm diameter) were first dipped in each test product dilution for 10 s and then air-dried before being placed in agar in a plastic Petri dish. The leaf discs were subsequently infested with whiteflies at a density of 50 adults per dish, with each treatment replicated six times. Mortality was monitored in both tests at 24-h intervals until no further increase in mortality was observed. Control treatments for all bioassay tests consisted of leaf discs treated with tap water.

2.7 Statistical analysis

Mortality data were adjusted for control mortality using Abbott’s formula [30]. Data were analyzed using analysis of variance (ANOVA) with SAS software version 9.2 [31], and mean values were compared using the least significant differences test at P ≤ 0.05.

3.1 Effect of novel insecticides on egg mortality

The results indicate that different life stages of B. tabaci exhibit varying susceptibility to the tested Betaproteobacteria-based insecticides, influenced by the specific product, concentration, and target life stage. In leaf-dip bioassays, these novel insecticides significantly reduced eclosion rates in a concentration-dependent manner (F = 68.90, df = 8, 45, P < 0.0001) (Table 1). Among the products evaluated, Grandevo demonstrated superior efficacy against eggs across all concentrations, achieving up to 74.5% egg mortality at the highest concentration. Venerate exhibited moderate ovicidal activity, resulting in egg hatching rate reductions of 40.2–63.3%, which correlated significantly with increasing concentration. In contrast, the acute contact toxicity of spirotetramat (Movento) to B. tabaci eggs was minimal, with only approximately 42.9% mortality observed at the highest labelled rate (0.8 mL/L) (Table 1).

3.2 Effect of novel insecticides on nymph mortality

A similar pattern in the efficacy of the novel insecticides against whitefly nymphs was observed, though with varying magnitudes of effect (Table 1). The insecticides exhibited significant toxicity toward both young (3 days old) and older (14 days old) nymphs in a concentration-dependent manner (F = 46.44, df = 8, 45, P < 0.0001 for young nymphs; F = 61.38, df = 8, 45, P < 0.0001 for older nymphs). All products demonstrated comparable efficacy against young nymphs, resulting in survival reductions of 47.7–72.2% when applied at the lowest and highest labelled rates, respectively. However, no significant differences in efficacy were observed between the highest and lowest concentrations for young nymphs. Grandevo at an intermediate concentration induced a marked decrease in nymph survival compared to the corresponding concentrations of other products (Table 1).

For late instar nymphs, the response to the application of insecticides mirrored that of young nymphs but with differing degrees of effectiveness (Table 1). The acute contact toxicity of the novel insecticides against older nymphs, when applied at the highest labelled rates, was notably pronounced, achieving up to 94.8% mortality depending on the specific insecticide. In contrast, older nymphs exhibited less vulnerability to the lowest labelled rates, with mortality not exceeding 31.4%, compared to the 47.7% mortality observed in young nymphs exposed to the corresponding concentrations.

3.3 Effect of novel insecticides on adult mortality

In terms of direct toxicity, the novel insecticides elicited comparable mortality levels in adult whiteflies when applied directly at relevant concentrations (Table 2). A significant increase in adult mortality was observed as a function of concentration (F = 43.32, df = 8, 45, P < 0.0001). All test insecticides applied at the lowest recommended field rates resulted in a maximum of 15.2% mortality. Increasing the concentration to the highest recommended field dosage produced a substantial suppressive effect, with maximum adult mortality reaching 76.5%.

The residual toxicity of the insecticides on adult whiteflies feeding on leaves previously treated with an aqueous solution is presented in Table 2. The adults responded to the residual activity similar to that observed for direct sprays, though with differing magnitudes. Exposure to treated leaves resulted in a significant reduction in adult survival in a concentration-dependent manner (F = 76.63, df = 8, 45, P < 0.0001). Generally, the residual effect of Venerate was less effective in suppressing pest populations compared to the other insecticides. Even at the highest recommended field dosage, Venerate achieved a maximum mortality of only 21%, indicating lower susceptibility of adult whiteflies to its residual activity. In contrast, Grandevo effectively reduced adult survival by up to 67.4% when applied to foliage at the highest labelled rate. However, spirotetramat demonstrated the highest residual efficacy, suppressing adult populations by up to 93.7% compared to the corresponding concentrations of other insecticides.

Overall, the residual activities of Grandevo and spirotetramat demonstrated greater effectiveness against adult whiteflies than their acute contact toxicity. In contrast, Venerate exhibited a reduced capacity to decrease adult survival compared to direct application.

3.4 Effect of novel insecticides on development and adult emergence

The sublethal effects of the insecticides on the development time of 3-day-old nymphs that survived insecticide application are presented in Table 3. Grandevo did not significantly impact the development period of nymphs at any concentration compared to the control group. In contrast, both Venerate and spirotetramat resulted in significant increases in development time (F = 16.01, df = 9, 40, P < 0.0001). Specifically, spirotetramat extended the development period by 4.1–4.9 days relative to the control, while Venerate prolonged nymph development by 2.1–3.1 days. However, the effects of spirotetramat and Venerate were not significantly correlated with the concentrations of the insecticides.

As indicated in Table 3, the proportion of adult emergence was significantly decreased following the exposure of young nymphs to insecticides, with both product type and concentration exerting an influence (F = 15.32, df = 9, 40, P < 0.0001). Adult emergence rates were measured at 51.8% and 30.4% for the lowest and highest recommended field doses, respectively, compared to 86.7% in the control.