Research experiments were conducted in bio-control laboratory and open field during the years of 2017 and 2018.
Rearing of Bactrocera cucurbitae
Rearing of the melon fruit fly was started with infested bitter gourds, collected from local vegetable markets. Transparent plastic cages (45 × 40 × 40 cm) fitted with muslin cloth were used for fruit fly rearing. Autoclaved sand and soil mixture (5-cm-thick layer) was placed in the cages at the bottom, and infested fruits with emerging maggots were placed on it for pupation. Cages were observed on daily basis, and as soon as the pupation completed, sand and soil mixture was sieved out to separate pupae. Collected pupae were then placed in another cage for adult emergence. Emerged adults of B. cucurbitae were identified (on the basis of morphological characteristics) and separated. B. cucurbitae adults were then fed on artificial diet made-up of banana, egg white, yeast, molasses, nutrients, vitamins and honey. After 2 weeks, males and females were released (♂/♀, 1:1) in rearing cages for mating. After 24 h, females were again separated for experimental studies. Laboratory conditions were maintained at 27 ± 2 °C, 65 ± 5% RH and photoperiod of 14:10 (L/D) hours.
Entomopathogenic culture and formulations
Already established culture of EPF (i.e., B. bassiana and M. anisopliae), which was developed from the commercial products Racer® (NCIM 1216) and Pacer® (NCIM 1311), was obtained from Department of Plant Pathology. Obtained fungal culture was streaked on freshly prepared growth media (PDA). The new culture plates were incubated under dark conditions at 28 ± 2 °C for 15 days. The highest concentration (108 CFU/ml) was made by adding freshly harvested conidia in to distilled water. Hemocytometer was used to determine the required conidial concentration. Surfactant (0.05% Tween®80) was also mixed in each conidial suspension to increase the adhesiveness of fungal spores. Remaining 2 concentrations (i.e., 107 and 106 CFU/ml) were made by adding more distilled water in previously described concentration, and again hemocytometer was used to quantify the required conidia. All these formulations were tested against B. cucurbitae.
Laboratory experiments
Two types of bioassays (i.e., contact and oral) were performed under laboratory conditions.
Contact bioassay
Three concentrations (108, 107 and 106 CFU/ml) of each fungi (B. bassiana and M. anisopliae) were prepared in distilled water (Ain et al. 2021). Thirty randomly selected adult flies (15 days old) were anaesthetized by placing in refrigerator at − 4 °C for 40 s (so that the handling would be easy). These 30 flies were placed in a test tube containing 4 ml conidial suspension from each concentration of both fungi. The test tubes containing flies and conidial suspension were shaken for 30 s until each fly attained some treatment. Two types of controls were maintained, i.e., a positive control with Malathion (3 ml/l of water) and a negative control with distilled water (containing 0.05% Tween®80). After the application of treatment, the tubes were placed vertically to allow the run off of excess suspension. Two hours after treatment, the flies were placed in plastic cages for further studies. Data regarding adult mortality were taken every 2 days after treatment (DAT) and consecutively up to 14 days. Factorial design (3-factors) with 5 replications was followed. Percent mortality was corrected using formula provided by Schneider-Orelli (1947).
$${\text{Corrected mortality}}\,\% = \left( {\frac{{{\text{Mt}} - {\text{Mc}}}}{{100 - {\text{Mc}}}} } \right) \times 100$$
where Mt = mortality (%) in treatment and Mc = mortality (%) in control.
Oral bioassay
Three concentrations of each fungus (2 × 108, 2 × 107 and 2 × 106 CFU/ml) were estimated through hemocytometer. These three fungal concentrations were then mixed with artificial diet in (1:1) ratio resulting in 108, 107 and 106 CFU/ml, respectively (Beris et al. 2013). One milliliter of this (suspension and diet mixture) was placed in small plate (3.5 cm diameter) in cage sized (27 × 25 × 25 cm). Fifteen -day-old 30 fruit flies, which were starved for 36 h prior to use in oral bioassay, were released in the cage for feeding on diet and conidial suspension mixture for 12 h and after this mixture was removed and pure diet was given as described by Beris et al. (2013). Two types of controls were maintained, i.e., a positive control with Malathion (1 ml/3 l of water) and a negative control with distilled water (containing 0.05% Tween®80). Data regarding adult mortality were taken every 2 days after treatment (DAT) and consecutively up to 14 days. After recording of each mortality observation, all dead flies were removed and transferred into sterile Petri dishes and wet filter paper was placed at the bottom of Petri plates. Then, these Petri dishes were sealed by using Parafilm, kept at 25 °C in dark and were examined daily for symptoms of mycosis. Factorial design (3-factors) with 5 replications was followed. Mortality was corrected using Schneider-Orelli (1947) formula (as mentioned above).
Field experiments
During laboratory experiments, the highest mortality causing concentration (i.e., 108 CFU/ml) of each EPF was identified and selected for further trials under field conditions. Each EPF was sprayed directly (Ain et al. 2021) on the crop at rate of 100 l/acre with the help of the knap sack sprayer when bitter gourd fruiting started. Spray was applied on the whole crop, but the soil beneath the plants and bitter gourd fruits were targeted specially. Bitter gourd crop was sown in March at farmers' field, for 2 consecutive years (i.e., 2017 and 2018). ‘Faisalabad Long,’ an indigenous and approved variety, was used. Priming was done by dipping seeds into water for 24 h. Plant-to-plant and row-to-row distances about 2 and 5ft were maintained, respectively. Recommended fertilizers and agronomic practices were applied, following Nawab and Mahmood (2014). When plants started to develop into vine, bamboo poled along with supporting nets were installed. In each unit plot, 10 plants were maintained and each treatment was replicated 5 times with 2 controlled treatments, i.e., positive control and negative control. In positive control, Malathion 57% EC (330 ml/acre), while in negative control only distilled water with 0.05% Tween®80 was used. Yellow sticky traps were hanged in each treatment to note the population of fruit flies before and after treatment. Data regarding fruit infestation (%) and population reduction (%) were taken every 3 days after treatment (DAT) and consecutively up to 30 days. Randomized complete blocked design (RCBD) with 5 replications was followed. Fruit infestation was calculated by the following formula:
$$\text{Fruit infestation }(\%)=\frac{{\text{No of infested fruits}}}{{\text{Total no of fruits}}}\times 100$$
Population reduction was calculated following Henderson and Tilton (1955) equation:
$${\text{Population reduction}}\, \% = \left( {1 - \frac{{{\text{Ta}} \times {\text{Tb}}}}{{{\text{Ca}} \times {\text{Cb}}}} } \right) \times 100$$
where Ta = number of flies stuck on yellow trap after treatment, Tb = number of flies stuck on yellow trap before treatment, Ca = number of flies stuck on yellow trap in control plot after treatment, Cb = number of flies stuck on yellow trap in control plots before treatment.
Statistical analysis
In laboratory experiments, recorded mortality data in each experiment were subjected to analysis of variance (ANOVA), following Tukey–Kramer honest significant difference (HSD) test (α = 0.05). Percent mortality in laboratory experiments was corrected, following Schneider-Orelli (1947) formula, while percent population reduction was calculated in field conditions, following Henderson and Tilton (1955) formula. Analysis of the all the collected data was computed using Statistix 8.1 software (McGraw-Hill 2008), and graphical representations were created on Microsoft Excel (2010).
