Helicoverpa armigera (Hub.) rearing
The larvae of H. armigera were collected from tomato, chickpea and pigeon pea crop is sown in the university field and brought to the laboratory for the 2nd generation larvae rearing. It was reared on an artificial diet, as per standard procedure given by Abbasi et al. (2007) at a temperature of 27 ± 2°C, 75 ± 5% RH. Freshly emerged 2nd instar larvae from the culture were used for experiment. In each treatment, 40 larvae were tested and the process was repeated 4 times. Control treatment consisted of spraying with water.
Identification and Isolation of Metarhizium anisopliae isolates
The indigenous M. anisopliae isolates M3 and M7 were isolated from cadavers of Spodoptera litura and H. armigera collected from farms of Jawaharlal Nehru Krishi Vishwa Vidayalaya, Jabalpur, Madhya Pradesh, India. In addition, ten more isolates of M. anisopliae (M1, M2, M4, M5, M6, M8, M9, M10, M11 and M12) were included in the study which was obtained from National Bureau of Agriculturally Important Microorganisms (NBAIM) Mau, Uttar Pradesh, India. To obtain the pure cultures, the infected cadavers were surface disinfected with 5% Sodium hypochlorite and were placed on a thin film of potato dextrose agar (PDA) medium and incubated at 270C ± 20C and 75 ± 5% RH and were kept for 7 to 14 days in Biological Oxygen Demand (BOD). The cadavers with hyphae were transferred to potato dextrose agar medium for the isolation of M. anisopliae spores. The spores were stained with cotton blue and hyphae germination was identified under the microscope by the presence of color, shape and size of phialides (Fig. 1). Slants were prepared from purified culture of PDA and the isolates were passed through the host insect at monthly intervals to maintain virulence.
Preparation and assessment of spore suspension for M. anisopliae
All isolates of M. anisopliae were grown on potato dextrose agar (PDA) medium in 250 ml Erlenmeyer flasks and Petri dishes incubated at 27 ± 2°C and 75 ± 5% relative humidity for 14 days after inoculation. The spores were harvested by scraping the surface of 15 days old culture and suspended in the solution of 0.02% Tween 80 in distilled water. The mixture was stirred with a magnetic stirrer for one minute. The spores concentration of the final suspension was determined by the haemocytometer. Initially, the highest required concentration (1×1010 spore ml-1) of the fungal suspension was prepared and filtered through double-layered muslin cloth. It was maintained as the stock solution, and further lower concentrations (1×109, 1×108 and 1×107 spore ml-1) were prepared from it by serial dilution technique (BenDavid and Davidson 2014). Harvested spores were air-dried under laminar airflow and stored in small airtight screw cap vials at 40C until further use. The viability of spores was evaluated according to Oliveira et al. (2015).
Larval dip methodology for virulence study of M. anisopliae
Larvae dip method as described by Tupe (2017) was followed for virulence studies against H. armigera larvae. Second instar H. armigera larvae were dipped for 10 seconds individually in M. anisopliae spore suspension at each concentration which contained 0.02% Tween 80. In control, the larvae were dipped in distilled water with 0.02% Tween 80. Before placing the treated larvae in Petri dishes, they were allowed to crawl independently on filter paper individually to remove excess moisture. Tomato leaves and soaked chickpea seeds were added as a food source for the larvae, which were regularly replaced by fresh ones. The Petri dishes were kept in BOD incubator at 27 ± 20C, and 75 ± 5% RH and the larval mortality was observed at 24 hours intervals after treatment.
Statistical analysis
The log-dose probit (LDP) were analyzed to median lethal concentration (LC50) and median lethal time (LT50). The mortality data were subjected to analysis as per the procedure of Finney (1971). Moreover, the mortality data observed at different concentrations and time interval were analyzed using one-way ANOVA after transforming the per-cent mortality data into arcsin transformation\(\sqrt{x}+0.5\). The corrected mortality was worked out using the formula of Abbott's (Abbott 1925).