Two fungal isolates viz M. rileyi MTCC 4254 and MTCC 10395, procured from the Institute of Microbial Technology (IMTECH), Chandigarh, India, and one M. rileyi NIPHM isolate procured from the National Institute of Plant Health Management (NIPHM), Hyderabad, India, were used in the present study. The three isolates were grown and maintained on Sabouraud maltose agar media with yeast extract (SMAY). These were then stored at refrigeration temperature for further use.
Production of mycoformulations
M. rileyi isolates were individually inoculated and incubated on sterilized broken sorghum grains for 14 days at 25 ± 2 °C and formulated to talc formulation according to the methodology of Kaur and Joshi (2014) with some modification. The formulations had a viable colony count of 1 × 108 cfu/g.
Rearing of Spodoptera litura in laboratory
Larvae and egg masses of S. litura were collected from cabbage and cauliflower field crops and reared on cabbage leaves in glass battery jars in the laboratory at suitable environmental conditions of 25 ± 2 °C. The open end of the glass jars was covered by a piece of muslin cloth and tied with a rubber band. The leaves were changed daily to provide adequate food. The 2nd instar larvae obtained from the 2nd generation of S. litura were used in the present experiment.
Pathogenicity against Spodoptera litura larvae
Talc-based formulations of M. rileyi at 3 different concentrations (8, 10 and 12 g/l) were tested for their efficacy against 2nd instar larvae of S. litura under laboratory conditions. The bioassay was carried out according to the methodology of Devi et al. (2003) with some modifications. The bio-formulation was sprayed on cabbage leaves to be tested. Ten treatments and 3 replications per treatment were used. Each replication had 20 larvae of S. litura, which were released onto the sprayed cabbage leaves kept in plastic insect rearing vials. The control treatment was maintained on leaves sprayed with distilled water. The larvae were kept at room temperature, and the mortality rate was recorded up to 10 days post-treatment.
Preparation of conidial suspension of M. rileyi
M. rileyi isolates were inoculated and incubated on SMAY media for 14 days at 25 ± 2 °C and 80% RH. The conidia were collected by scraping the conidia off the media plates. The volume of conidial suspension was made up to 100 ml with distilled water and to it Tween80 @ 0.1% was added. The suspension was vortexed for 5 min to obtain a uniform conidial suspension. The sample of this suspension was quantified in the Neubauer chamber. A suspension containing 108 conidia/ml was used for the analysis of enzymatic activity.
Production of cuticle-degrading enzymes
Production and activity of extracellular cuticle-degrading enzymes (CDEs) viz chitinase, protease and lipase productions were investigated from the isolates of M. rileyi according to the methodology of Dhawan and Joshi (2017) with some modifications. Each isolate of M. rileyi (having 108 spores/ml) was inoculated in 100 ml of chitin media. The composition of media (g/l) was KH2PO4, 3.0; K2HPO4, 1.0; (NH4)2SO4, 1.4; NaCl, 0.5; CaCl2, 0.5; MgSO4, 0.7; bacto-peptone, 0.5; yeast extract, 0.5; chitin, 5.0; and olive oil, 5 ml/l. The flasks were incubated at 25 ± 2 °C at 150 rpm in an orbital shaking incubator. The enzyme assays were carried out on every alternate day up to 10 days. Flasks were removed from incubation on successive days, and the broth was centrifuged at 8000 rpm for 25 min to extract clear supernatant. This supernatant was used to determine the enzymatic activities.
Chitinase activity in the extracted supernatant was determined, using acid-swollen chitin as substrate (Nahar et al. 2004). Acid-swollen chitin was prepared by suspending 10 g chitin flakes (HiMedia) in 300 ml chilled o-phosphoric acid (88% w/v) and kept at 4 °C for 1 h stirring occasionally. The mixture was poured into ice-cold distilled water and then filtered through Whatman filter paper. This was followed by washing with 1% (w/v) sodium bicarbonate solution and adjusting the pH to 7. The solution was then homogenized in a blender for 1 min, and the concentration of acid-swollen chitin was adjusted to 7 mg/ml by adding 50 mM acetate buffer (pH 5.0). The reaction mixture for the assay containing 1 ml of 0.7% acid-swollen chitin, 1 ml 50 mM acetate buffer (pH 5.0) and 1 ml crude enzyme extract was incubated at 50 °C for 1 h. The product formed and colour so developed was measured calorimetrically at 520 nm according to the methodology of Somogyi (1952) to estimate the N-acetylglucosamine residues produced. One unit of enzyme activity was expressed as 1 μg of N-acetylglucosamine produced per min per ml.
Protease activity was determined using casein as substrate (Nahar et al. 2004). Casein substrate contained 10 g casein in 100 ml of 0.2 mM sodium carbonate buffer (pH 9.7). The reaction mixture contained 1 ml casein substrate, 1 ml sodium carbonate buffer (pH 9.7) and 1 ml crude enzyme solution. This reaction mixture was incubated at 35 °C for 20 min and the reaction was then terminated by adding 3 ml of trichloroacetic acid (2.6 ml 5% TCA + 0.4 ml 3.3 N HCl). The absorbance of TCA soluble fraction was read at 280 nm. One unit of enzyme activity was expressed as 1 μg of tyrosine produced per min per ml.
Lipase enzyme activity was measured by using olive oil and gum acacia as substrate according to the methodology of Pignede et al. (2000). The substrate was prepared by mixing 50 ml of olive oil and 50 ml of gum acacia (10% w/v, Hi Media) in the ratio 1:1. The reaction mixture for lipase assay contained 5 ml of substrate emulsion, 2 ml of 50 mM phosphate buffer (pH 6.8) and 1 ml of crude enzyme extract. The reaction mixture was incubated at 37 °C for 1 h with intermittent shaking, followed by the addition of 4 ml acetone-ethanol (1:1 v/v), containing 0.09% phenolphthalein as an indicator to terminate the reaction. Enzyme activity was determined by titration of the above reaction mixture against 50 mM sodium hydroxide solution for the estimation of the free fatty acids released during the reaction. One unit of lipase activity was expressed as 1 μmol of fatty acids released per min per ml. All enzyme assays were carried out in duplicates at alternate days.
The data was subjected to one-way analysis of variance (ANOVA) in SPSS 16.0 statistical software, and means were compared (at 0.05 level of significance) by Tukey’s post hoc test. Means with P < 0.05 were considered to be significantly different from each other. The transformed mean values were obtained by applying one-way ANOVA under CRD in CPCS1 software.