PhD

ABSTRACT
Lecanicillium saksenae an indigenous isolate from soils of Vellayani isolated by Rani et al. (2015) was reported to have high speed of kill in major hemipteran pests. Its quick insecticidal action prompted to profile its toxic metabolites. Therefore the study entitled ‘Mycotoxins and enzymes of entomopathogenic fungus Lecanicillium saksenae (Kushwaha) Kurihara and Sukarno and their bioefficacy on crop pests” was carried out in the Department of Agrl. Entomology, College of Agriculture, Vellayani and Department of Agrl. Entomology, College of Horticulture, Vellanikkara during2016-2019. The objective of the study was to characterize the cuticle degrading enzymes and mycotoxins produced by L. saksenae and explore their potential for safer pest management. Qualitative assay revealed the presence of different cuticle degrading enzymes viz. chitinase, lipase, protease, chitosanase and amylase, with the Enzyme Indices 1.08, 4.3, 1.1, 1.36, and 1.57 respectively. Qualitative assay using pH indicator medium revealed high activity of chitin deacetylase. Quantitative assay carried out at two day interval for a period of 14 days revealed the peak activity of chitinase to the tune of 1.35 U mL-1 min-1, lipase 0.7 U mL-1 min-1, protease 4.89 U mL-1 min-1, chitosanase 0.26 U mL-1 min-1, amylase 23.42 U mL-1 min-1 and keratinase 73.61 U mL-1 min-1. The activity of Pr1 and Pr2, the key proteolytic enzymes were also detected to the tune of 0.22 U mL-1 min-1 and 0.32 U mL-1 min-1respectively. The peak activity period of these enzymes varied from 4th to 10th day of incubation, whereas the peak activity of Pr2 was on the 2nd day and that of keratinase was on the 40th day, in their respective minimal media with inducing substrates. The biomass reduction of 80.2 per cent in poultry feather substrate due to keratinase activity is notable. Bioefficacy studies of partially purified major cuticle degrading enzymes revealed that protease @ 100 μg mL-1 exhibited 71.79 per cent mortality of brinjal mealybug, Coccidohystrix insolita nymphs, 96 hours after treatment (HAT). The corresponding mortality with chitinase and lipase was 58.10 and 54.23 per cent respectively. These enzymes when applied in combination resulted in 78.79 per cent mortality. However, the efficacy of individual enzymes on adults was meagre and the combined application caused only 40.88 per cent mortality. Bioefficacy studies on the root knot nematode Meloidogyne incognita revealed its inhibitory action in the hatching of eggs. The hatching percentage was drastically reduced in chitinase (18.8%), protease (10.4%) and its combination (7.8 %), as against 70.80 per cent in absolute control. Scanning electron microscopy of the treated eggs exhibited uneven and shrunken surface with distinct lines of breakage on the outer vitelline layer, leading to disintegration of egg mass within five days of treatment. The predominant enzyme, protease was purified through Sephacryl G-100 column and DEAE-cellulose and was characterised with molecular weight 71 kDa by SDS - PAGE analysis. Spectral analysis of crude toxin extracted through standard procedures could detect an array of metabolites, of which the dominant one was purified and characterised. Ethyl acetate extraction of culture filtrate followed by vacuum evaporation and purification yielded 100 mg L-1 of the purified compound. Structural characterisation and identification of the metabolite through UV spectrometry, Fourier Transform Infrared spectrometry (FTIR), High Resolution Liquid Chromatography Mass spectrometry (HR-LCMS) and Nuclear Magnetic Resonance Spectrometry (NMR) unambiguously identified the metabolite as oosporein, the empirical formula of which was confirmed to be C14H10O8 and molecular weight, m/z 306.22. Dipicolinic acid (DPA), another volatile insecticidal metabolite was identified through LC-MS. Bioassay guided fractionation of crude toxin could identify a fraction that caused 64. 23 per cent mortality of C. insolita nymphs at 96 HAT. Spectral data of LC-ESI-MS analysis of this insecticidal fraction showed a strong signal for the ion of m/z 485.6 which was identified as beauverolide. One Strain Many Compounds (OSMAC) technology was carried out to detect multiple metabolites produced by L. saksenae. HR-LCMS and GC-MS analysis of the toxin extracts from various media, identified multiple metabolites whose bioactivity was determined using Dukes phytochemical and ethno botanical and PubChem databases. Of the bioactive metabolites, 22 were insecticidal, seven were nematicidal and 20 were antimicrobial. The dominant insecticidal metabolites other than oosporien, DPA and beauverolide were picolinic acid, cordycepin, anthranilic acid, dibutyl phthalate, hexadecanoic acid, harmine etc. while the major nematicidal metabolites were 2,6, pyridine dicarboxylic acid, 8-hydroxyl quinone, nicotinic acid, hexadecanoic acid, harmine etc. The predominant antimicrobial metabolites were vanillic acid, aminophenol, anthranilic acid, salisylamide, oleamide, terephthalic acid 4-H pyran-4-one, 2, 3, dihydro 3, 5 dihydroxyl 6- methyl, and and Dl-mevalonic acid lactone etc. The volatile compounds detected were hexadecanoic acid, octadecanoic acid, harmine. 2,6, pyridine dicarboxylic acid, 4H pyran-4-one, 2, 3, dihydro 3, 5 dihydroxyl 6- methyl, and Dl-mevalonic acid lactone. Bioefficacy studies of the purified toxin revealed a dose dependent mortality in C. insolita. Toxin at 1000 ppm caused 60.25 per cent mortality in nymphs, 96 HAT, whereas the corresponding mortality in adults was 51.00 per cent. However, the crude toxin caused 100 per cent mortality with the same concentration, justifying the multifaceted properties of the fungus in causing insect diseases. Probit analysis revealed LC50 value of 87.14 ppm in nymphs at 48 h whereas it was higher (193.61 ppm) in adults. Bioefficacy studies on J2 of M. incognita revealed the nematicidal potential of secondary metabolites of L. saksenae. At highest concentration of 250 ppm, mortality of 95.22 per cent was observed at 24 HAT whereas at 48 HAT, mortality was 100 per cent with 50 ppm and above. The study revealed the safety of crude toxin to different life stages of the coccinellid predator Cheilomenes sexmaculata . LC50 value for ovicidal activity was 69 fold higher than that of the pest (51.06 ppm at 96 h) while for grubs it was 79 times higher than that of pest. In silico molecular docking of the metabolites detected from L. saksenae revealed that 18 of them were interacted with acetylcholinesterase (AChE). Among these, 3-Hydroxy-2-methylpyridine exhibited the highest binding energy (-133.24 kcal mol-1) and therefore highest interaction with AChE. Metabolite profiling of L. saksenae revealed its potential entomopathogenic, nematicidal and keratin degrading properties. The study paves way to the possibilities of tapping the potential of the bioactive metabolites for safer pest management. It could be made use in leather industry and keratinous waste management as well.