Second and 3rd larval instars of L. botrana were used in the following bioassays. Insects were reared, under controlled conditions, on a semisynthetic diet (Standard L. botrana diet) consisting of the following ingredients: distilled water 1.4 l, corn flour 225 g, wheat germs 58 g, agar 32 g, brewing yeast 22 g, sorbic acid 8 g, Nipagin 4 g, Benzoic acid 4 g.
Larvae were initially collected, along with infested grapes, from a vineyard (V. vinifera cv. Savatiano) in the area of Spata (Attica - Greece). Then, infested grapes were placed in cages until first adult insects emerged. Adult female and male insects were placed on plastic cups, covered with filter paper on the top, and the females laid their eggs on the walls of the cups. Then, the cups were half-filled with artificial diet which was previously cut into cubes of about 1 cm length. When the eggs hatched, young larvae fed on diet until they pupated. After the emergence, all adult males and females were placed into plastic cups using a glass tube. The insects were left to lay their eggs for 3–4 days and then removed and killed. Adult insects fed on a solution of 5% honey dissolved into distilled water, which was provided to the insects via a cotton disc placed on the bottom of the cups. The colony of L. botrana was maintained at a temperature of 24 ± 1° C and 60 ± 10% relative humidity. The photoperiod was 16:8 h (light: dark) and the luminosity 500 lx photophase. Insects used in both bioassays remained under starvation for 24 h before the initiation of the experiments.
Bacillus thuringiensis var. kurstaki (Dipel ES, 17,600 International Units (IU)/mg, Abbott, North Chicago, Illinois) was used at both following bioassays. Dipel ES contains Cry1Aa, Cry1Ab, Cry1Ac, Cry2A, and Cry2B endotoxins of Bt along with supplementary components which improve its performance as a microbial insecticide. For the In vitro test, 4 ml of Dipel ES were dissolved in 1.4 l of distilled water, which replaced the total amount of water in an individual block of semisynthetic diet (1650 g). This process resulted in a concentration of ~ 42,700 IU per g of diet. For the latter bioassay, a concentration of 70,400 IU/ml was made by adding 2 ml of Dipel ES into 500 ml of sterile distilled water.
The EPF isolates B. bassiana IMI-391044 and P. fumosoroseus EBAC-01, as well as a commercial bio-insecticide based on B. bassiana strain GHA (BotaniGard® ES), were used at the following bioassays. The two non-commercialised fungi were initially isolated using the “Galleria Bait Method” (Zimmermann 1986). Upon initiation of the experiments, these isolates were sub-cultured and grown on 9 cm Ø Petri dishes with half-strength Sabouraud Dextrose Agar (SDA), at 25° C for 20 days. Spore suspensions of 1 × 108 conidia/ml were made for each isolate, by scraping conidia from the surface of the plates using a microscope slide. Then, a sterile liquid solution of distilled water with 0.1% Tween 80 was poured into the plates to remove, disperse and integrate the conidia. The containing conidia liquid suspension was stirred using a magnetic stirrer and filtered twice using a sterile nylon membrane. The dose of 1 × 108 conidia/ml was selected for all fungal treatments due to the highest efficacy levels that presented in previous bioassays. This concentration has been considered as optimum, especially for single concentration experiments (Galindo-Velasco et al. 2015). In the case of the commercial bio-pesticide (BotaniGard® ES), a spore suspension was adjusted to 1 × 108 conidia/ml by dissolving 6.15 ml of product into 500 ml of sterile distilled water. All spore concentrations were measured, under microscope (400×), using a standard (improved Neubauer) haemocytometer. When needed, extra distilled water was added to adjust the dilution until reaching the 1 × 108 conidia/ml concentration.
Spore viability was calculated by counting the percentage of germinated conidia, which were spread on half-strength SDA plates, under a microscope (400×). B. bassiana IMI-391044 and P. fumosoroseus EBAC-01 presented conidial viability of 99 and 98.3%, respectively while B. bassiana GHA (BotaniGard® ES) showed 96.7% spore viability. A concentration of 1 × 105 conidia/ml was made for each isolate, using the method described above; 100 μl of the suspension was added on each plate using a micropipette and then spread using a microscope slide. After sealing, the plates were incubated at 25° C for 18 h. Three sets of 100 conidia were measured for each isolate. All conidia with visible germ tubes of any length were counted as viable.
Mortality of L. botrana larvae after exposure to Bt diet and subsequent fungal treatment
The 2nd instar L. botrana larvae were used in the present bioassay. For treatments involving Bt consumption, insects were exposed and fed on a diet, which contained Bt var. kurstaki (Dipel ES). Control larvae, as well as insects that were prepared for the single fungal treatments, were fed on standard L. botrana diet (described above). Bt diet was made exactly as the standard, except the addition of 4 ml of the product (Dipel ES) in 1.4 l of distilled water, which produces approximately 1650 g of diet (42,700 IU/g). Insects were fed on either diet in groups of 10 (5 groups for each treatment), inside sterile Petri dishes for 30 h.
Subsequently, fungal treatments were applied by spraying the insects, using Mist trigger micro-sprayers “Hozelock 4120 Spray 0.5 l”, inside sterile Petri dishes with filter paper covering the bottom of the plates. Ten larvae were placed into each dish. Insects were sprayed until the entire area of the paper was wet but not saturated. Single and combination B. bassiana and P. fumosoroseus treatments were sprayed with the respective suspensions (1 × 108 conidia/ml of sterile distilled water with 0.1% Tween 80). Control and single Bt insects were sprayed with a solution of sterile distilled water with 0.1% Tween 80.
Therefore, six (6) groups of differently treated insects were evaluated:
Control: Larvae fed on standard L. botrana diet for 30 h and then, sprayed with sterile distilled water.
Bt: Larvae fed on Bt diet for 30 h and then, sprayed with sterile distilled water.
B. bassiana: Larvae fed on standard L. botrana diet for 30 h and then, sprayed with a spore suspension of 1 × 108 conidia B. bassiana/ml.
P. fumosoroseus: Larvae fed on standard L. botrana diet for 30 h and then, sprayed with a spore suspension of 1 × 108 conidia P. fumosoroseus/ml.
Bt + B. bassiana: Larvae fed on Bt diet for 30 h and then, sprayed with a spore suspension of 1 × 108 conidia B. bassiana/ml.
Bt + P. fumosoroseus: Larvae fed on Bt diet for 30 h and then, sprayed with a spore suspension of 1 × 108 conidia P. fumosoroseus/ml.
Post treatment, larvae were provided with a standard L. botrana diet inside Petri dishes (10 larvae per dish). Four cubes of about one cubic centimetre each were added on each plate. Then, plates were sealed and placed into incubators at 25 °C, 60 ± 1%RH and 14:10 (L:D) photoperiod (Sanyo - MLR-351H). Each treatment consisted of 50 replicates (5 dishes × 10 larvae each) and the entire experiment was repeated twice.
Mortality measurements took place 3 and 6 days after the spraying treatments. Any larva which either remained still after contact with the pincers, or was rotten or disintegrated, or presented symptoms of mycosis was recorded as dead. After each mortality measurement, all dead insects were removed from the plates.
Combined effect of Bt and B. bassiana for the control of L. botrana on grapes
Young 3rd instar L. botrana larvae were exposed to grapes that were previously sprayed by either B. bassiana strain GHA (BotaniGard® ES), or Bt var. kurstaki (Dipel-ES) or a combination of both entomopathogens. Control insects were exposed to grapes, sprayed with sterile distilled water. The B. bassiana suspension was adjusted to 1 × 108 conidia/ml of sterile distilled water and 0.5 l of such suspension was made. The Bt treatment was made by dissolving 2 ml of Dipel ES into 500 ml of sterile distilled water, resulting in a final concentration of 70,400 IU/ml. Equal parts of both liquids (250 ml) were mixed to prepare the combination treatment.
Ripe grapes of Vitis vinifera cv. Sauvignon Blanc were used in the bioassay (11.8–14.1 Baumé degrees at 20° C). Small grape clusters or parts of clusters (6–15 berries each) were totally covered by the spraying liquid. Then, grapes were rested to dry for 2 h. Subsequently, grape clusters were placed into plastic jars (9.5 cm Ø and 14 cm height). The lids of the jars were pierced multiple times so air was allowed in the jars. A piece of wet filter paper was placed to cover the bottom of each jar. Grapes of the respective treatment (one small cluster) as well as 10 L. botrana larvae were placed into each jar. All jars were incubated at 25° C, 60 ± 1%RH and 12:12 h (L:D) photoperiod for 48 h and larvae were observed to feed on the grapes. Then, insects from each jar were transferred into a sterile Petri dish, using entomological pincers. Standard L. botrana artificial diet was added into the dishes and insects were incubated under the same conditions as above for 5 more days. Twenty replicates of 10 insects were made for each treatment and the experiment was repeated twice. Mortality measurements were conducted 3, 5 and 7 days after the initial placement of the insects into the jars. Any larva that was rotten, covered by mycelium, or remained still was counted as dead. Dead larvae were removed from the plates during each measurement.
One-way analysis of variance (ANOVA) was conducted to compare different treatments in terms of larval mortality (SPSS, 2008). In both bioassays, Levene’s test was used to estimate if variances were assumed homogenous or not. According to those estimations, respective Post-Hoc tests were used to indicate significant differences between particular treatments (Hilton and Armstrong 2006). In the latter bioassay, Probit analysis was used to estimate the median lethal time (LT50) (Reddy et al. 2016). The synergism factor (SF) was calculated by dividing the predicted theoretical mortality value for each treatment by the observed value. According to Tabashnik (1992), an SF ratio equal to 1 was additive, lower than 1 was antagonistic, and a ratio over 1 was synergistic.