The nematodes (H. bacteriophora EUPT-S26) were procured from rhizospheric soil of fruit orchards of district Sirmaur of Himachal Pradesh, India, via soil baiting technique. The isolated nematodes were identified as H. bacteriophora EUPT-S26 on the basis of morphological observations and identification keys. To attain pathogenic and viable infective juveniles (IJs), they were replicated through in vivo using last instar of Galleria mellonella L. (Lepidoptera: Pyralidae) via larvae contagion (Kaya and Stock 1997). Infective juveniles (IJs) were kept at 15 °C in sterilized sponge foams. The seeds of Brassica oleraceae (Brassicales: Brassicaceae) were purchased from local market and grown at polyhouse to establish breeding stock at the University biological control polyhouse (average temperature: 17–20 °C; average relative humidity: 75–81%; light regime: 12 h light/12 h dark). Cabbage plants after 27 days of post-germination were transplanted into the plastic pot (20 × 20 × 14 cm), filled with rice husk and soil. Watering was done daily and plants were fertilized with Agrobium's® (NPK-8:25:25, 2 g/plant). Initiation of breeding stock of P. brassicae was maintained by transferring the P. brassicae larvae on cabbage plant (28 days from seedling stage) 5 larvae/plant, which were then placed in entomological cages (40 cm3) for confinement. The P. brassicae life cycle lasted approximately 27 days on average under polyhouse conditions.
Heterorhabditis bacteriophora EUPT-S26 application
Pieris brassicae susceptibility to H. bacteriophora EUPT-S26 infection was determined by conducting an experiment on cabbage plants at different vegetative stages. The experiment was conducted under the polyhouse (44 × 36 m) where the area of the experiment was conducted in area of 22 × 11 m, further divided into 2 blocks containing 13 plots on each side (2 m2). The cabbage plants were transplanted at 12 plants per plot with a planting distance of 16 cm. To this laboratory reared, 4th instar larvae were stationed in each plant (5 larvae/plant) along with 260 P. brassicae adults, 8 h before the test (experiment). Different treatments containing IJs were suspended in the distilled water with the 0.3% Polysorbate 20. These suspensions with varied juvenile concentrations were sprayed via pressure sprayer over the cabbage leaves (abaxial and adaxial). The result variables were the leaf damage percentage and larvae mortality percentage were assessed at every 24 h for the nine days. Plant damage level was mainly evaluated through graphical method by using scale with 4 levels, namely 1, 2, 3 and 4 with differences in degrees of infestation ranging from 1 to 10%: level 1, 10–40%: 2, 40–70%: 3 and 70–100%: level 4 (Carballo et al. 1989).
Insect pest mortality was determined by yellow-ochre cadavers when placed into the white traps up to 9 days to authenticate the presence of IJs (Kaya and Stock 1997). A completely randomized experimental design was implemented, where nematodes dose factor had 5 levels, IJs (0, 500, 1000, 1500 and 2000 IJs/ml of water). For statistical analyses of damage progress, area under the patches was observed. Results’ variable such as damage percentage of plants and mortality percentage in larvae was calculated. Statistical differences were determined by ANOVA using OPSTAT software. The experiment was repeated twice for the 2 year during the cropping season along with 5 replicates.
Heterorhabditis bacteriophora EUPT-S26 treatment
Field phase was carried out in the agricultural farm, located at (30.7537° N, 77.2965° E, 1900 m altitude), having mean temperature between 15 and 20 °C and a RH between 50 to 64%. For planting a 100 m2 area was prefabricated first, soil was allowed to recover strength and then plowing was done. The planting area was fertilized with Agrobium's NPK (8:25:25). The experimental area was then partitioned into 2 equal blocks (50 m2). Each block was distributed into the 13 plots (3 m2). In the each plot, 26-day post-germinated cabbage plants (16 plants/plot) were transplanted with a planting distance of 18 cm. After 3–4 weeks of cabbage transplantation, 780 pupae and 260 P. brassicae adults were released in the entire field, homogeneously spreading 390 pupae and 130 adults per block. Treatments of nematode juvenile mixed with 0.3% Polysorbate 20 were applied in each plot along with complete control.
Throughout the crop growth or development, 3 applications of EPNs were used, according to the variation in the population dynamic of P. brassicae and cabbage plant’s phenological stage. The 1st application was applied 1 week of post-inoculation of insects containing 5 larvae/ plant at vegetative stage. The 2nd application was sprayed 1 week after the first application (4 larvae/ plant) also at vegetative stage. At the end, the 3rd application was applied 1 week after the 2nd application (during reproductive stage at the completion of head formation). These applications were performed throughout the crop cycle up to harvesting according to P. brassicae population fluctuation.
A completely randomized block experiment was carried out where the result variables, i.e., productivity and damage percentage, were calculated. Percentage of damage was determined according to formula (Carballo et al. 1989) scales. Data were obtained weekly from the time of pest released; the damaged area was also calculated to examine progress and consequently performed statistical analyses. Insect mortality and cabbage productivity was also noted.
For statistical analyses, the used treatments were congregated to perform one-way ANOVA. Each 5 treatments included absolute controls. The treatments correspond to the 4 applications of H. bacteriophora EUPT-S26 along with absolute control. Pearson’s correlation analysis among damage percentage and productivity were also calculated.