Banding of tree trunk
The experiment was conducted in the 2 consecutive year 2017 and 2018 in 4 different apple orchards of Kargil District viz., Slikchey, Shanigund, Bagh-e-Khomini, and Hardas located at 34° 33′ 27.54′′ N and 76° 07′ 34.39′′ E of Ladakh Region, India. By the end of August, in each year (2017 and 2018), 10 tree trunks of each orchard were banded with gunny bags up to 1 m height from the ground level. The banding was performed in order to provide shelter to overwintering third generation larvae of Codling moth.
Application of entomopathogenic nematode
The freshly prepared clay formulation of local EPN strain, Heterorhabditis pakistanensis NBAIR H-05, used in the study, was obtained from ICAR-National Bureau of Agricultural Insect Resources (NBAIR), Bengaluru, India. One gram of clay formulation contained approximately 50,000 live infective juveniles (IJs) of H. pakistanensis.
The clay powder formulation of EPN was evaluated at 3 different concentrations, 15 g (7.5 × 105 IJs), 20 g (1.0 × 106 IJs), and 25 g (1.25 × 106 IJs). The treatments were accompanied with and without post wetting of tree trunk. Besides, these 6 treatments (T1–T6) and 1 treatment (T7) was included as untreated control. All the 10 banded tree trunks were made thoroughly wet (1 l of water/ tree trunk) with their respective treatments, using rose can sprinkler provided with a nozzle having small holes to break up the stream of water into small droplets. Application was performed in evening hours in order to allow the bands to remain moist for longer period unlike during sunshine hours for survival ability and aggressive foraging of EPN against over wintering larvae, during the last week of August, which marked termination of larval overwintering of Codling moth. Five trees of the treatment T2, T4, and T6 were provided post wetting by fresh water, after 12 h of EPN treatment and marked as “post wet.”
Collection and storage of dead larvae
Thirty-six hours after EPN treatment, the trunk bands of all 10 trees were opened for collection of larvae present under the band, and/or under the bark of each tree, for post treatment count. Larvae collected from each treatment (with or without post wet) were kept separately in plastic container (250 ml) half filled with moist soil. The container was brought safely to laboratory, placed in BOD (Bio-Oxygen Demand) incubator, maintained at 27 ± 1 °C.
Data regarding larval density per tree trunk, larval mortality after 48 and 72 h, treatment wise and year wise was duly recorded for subsequent analysis.
Confirmation of nematode killed larvae
Change in larval color from original light pink to brick red indicated specifically Heterorhabditis induced mortality (Fig. 1). But for further confirmation, the dead insect larvae (cadavers) were placed on a White trap (White, 1927) for the release of infective juveniles.
Statistical analysis
Minitab 11.12 (Minitab LLC) was used to analyze the data for ANOVA. Percent larval mortality was determined by dividing the number of dead larvae from total number of larvae in a sample. Percent reduction over control was calculated by using Abbott’s (1925) formula: T−C/100−C*100 (where T = mortality in treated condition and C = mortality in untreated control condition)