Plant material and C. medinalis mass rearing
Fine rice Basmati-515 variety was used for the evaluation of EPF against C. medinalis at 28 ± 2 °C and 65 ± 5% RH, at the Rice Research Institute, Kala Shah Kaku, Punjab, Pakistan, during the year 2018. Larvae of C. medinalis were collected from grown rice plants in nursery trays, seedlings were transferred from the nursery after 40 days to pots (30 cm height and 9 cm diameter). Stock culture of the insect was maintained, following the method of Fujiyoshi et al. (1980), by releasing the newly emerged adults in ovipositional cages (120 × 80 × 50 cm). Moths were provided with a 10% honey solution as food and left for mating and egg laying. Ovipositional cages were observed daily till the hatched larvae reached the third instar (slightly dark green in color and a brownish patch on either side of pronotum) and then chosen for the bioassay tests. For the bioassay test, the third instar larvae were kept unfed for 3 h before each test, then transferred into Petri dishes, containing moistened filter paper at their bottom, to maintain the freshness and turgidity of clipped leaves.
Entomopathogenic fungi
Three fungi, B. bassiana, V. lecanii, and M. anisopliae, in talc forms, were obtained from AgriLife SOM Phytopharma (India) Limited (www.agrilife.in). All fungi were tested against C. medinalis, at a feasible conidial concentration (1 × 108 CFU/ml) (Dal Bello et al. 2018). The quality of the treatments was checked by a heamocytometer. Potato dextrose agar (PDA) was used to determine the conidial germination. The conidial germination was measured, based on the counts of 200 random conidia per plate, 18 h post-incubation at 25 ± 2 °C (Ayala-Zermeňo et al. 2015).
Fungal pathogenicity against C. medinalis larvae
In vitro assay
Available concentration [1 × 108 colony-forming unit/gram (CFU/g)] of fungi was used in the laboratory in order to study the pathogenicity of the tested fungi against the third instar larvae of C. medinalis. Twenty larvae were used in each replication. The tested larvae were collected from the potted plants in vials, starved for 3 h, then dipped in each tested fungi solution at the chosen concentration for 10 s, as described by Negasi et al. (1998). Sterilized distilled water was used as a control treatment. Mortality counts of the larvae were recorded for 10 days (Riasat et al. 2011).
Greenhouse assay
A rice nursery was grown in plastic trays with 60 plugs in each. The nursery was transplanted in a greenhouse after 30 days. Four plants were grown at (22.5 cm row × row and plant × plant distance) and considered for each treatment replication. At the age of 60 days, the plants were sprayed by the help of a Pump Pressure Sprayer (Hommold, Lahore) with the fungal talc formulation. The third instar of C. medinalis larvae were collected, kept starved for 3 h, and then shifted on the treated rice plants. Three replications, of 12 larvae, were used in each case. The mortality rate of the larvae was recorded after 10 days. The cadavers of C. medinalis were used to record the mycosis percentage. The cadavers were collected and preserved in sterile Petri plates. The cadavers were washed three times in sterile distilled water, and then the surface was sterilized for 2–3 min by a 0.05% sodium hypochlorite solution. Then, these cadavers were placed on Sabouraud dextrose agar (SDA) plates and incubated at 25 ± 2 °C, 75 ± 5 RH for 7 days to observe the external white fungal growth under a stereomicroscope (Cole-Parmer 625 East Bunker Court Vernon Hills, IL, 60061, USA) (Riasat et al. 2011). Sporulation data were determined by mixing mycosed cadavers from each replicate in a beaker with a drop of Tween 80 with 20 ml of distilled water (Tefera and Pringle 2003). Treatments were replicated three times independently. The solution was carefully stirred and the number of conidia was counted by using a hemocytometer under the microscope (Riasat et al. 2011).
Statistical analysis
All statistical analyses were conducted using Statistix software (version 8.1) (Tallahassee, FL). One-way ANOVA was applied in CRD to understand the mortality of C. medinalis and mycosis and sporulation from cadavers of tested EPF in in vitro and greenhouse assays. The means were separated, using the Tukey’s HSD test at P = 0.05.