MSc

The investigation entitled “Pathogenicity and biochemical properties of entomopathogenic fungus Lecanicillium saksenae (Kushwaha) Kurihara and Sukarno” was conducted at College of Agriculture, Vellayani during 2014 -2016. The objective was to study the pathogenicity and biochemical properties of L. saksenae in order to establish its virulence to insect, mite and nematode pests.
L. saksenae is an indigenous isolate from the soils of Vellayani. Pathogenicity studies revealed infectivity of the fungus to the hemipteran pests, Aphis craccivora Koch, Coccidohysterix insolitus Green, Amrasca biguttula biguttula Ishida, Bemisia tabaci Gennadius, Lecanium sp, Leptocorisa acuta Thunberg and Riptortus pedestris F. Apart from this it was pathogenic to the plant parasitic mites, Tetranychus sp. and Polyphagotarsonemus latus Banks . On the other hand, neither the lepidopteran nor the coleopteran pests tested were found to be susceptible to the fungus. It was non parasitic on eggs of root knot nematode, Meloidogyne incognita (Kofoid and White) Chit. The effective dose for pathogenicity was determined as 107 spores mL-l for all the above pests except for R. pedestris, where it was 108 spores mL-l.
The fungus was non pathogenic at a spore concentration of 108 sporesmL-1 to crop plantsviz., cowpea, bhindi, brinjal and tomato in which Verticillium diseases were reported. It was safe to the coccinellid predators, Chilomenes sexmaculata F. and Coccinella septumpunctata L., the syrphids, Ischiodon scutellare F. and Xanthogrammascutellare F. and spiders, Tetragnatha maxillosa Thorell. andOxyopus sp., the parasitoids Trichogramma. japonicum Ashmead , T. chilonis, Bracon brevicornis F., Goniozus nephantidis Muesebeck as well as to the pollinators Xylocopa sp. and Vespula sp. The beneficial insects Apis cerana indica F.. A. mellifera and Tetragonula iridipennis Smith were found to be unaffected by the fungus.
Among the various substrates tested chitosan (five per cent) enriched rice bran was the best solid substrate for mass multiplication with a maximum spore yield (7.27 x 108 spores mL-1), number of viable colonies (9.10 x 107 cfu mL-1) and mortality to C. insolitus (95.66 per cent) at 14 DAI . Of the liquid substrates, though RW + chitosan (0.3 per cent) was superior with 9.22 x 107spores mL-1 and 5.32 x 107 cfu mL-1, considering the cost factor and yield, Rice bran or Wheat bran enriched with chitosan 0.3 per cent is the best that supports maximum spore yield and number of viable colonies.
Qualitative assay revealed the presence of cuticle degrading enzymes chitinase, protease and lipase, the activity of which was maximum in Sabouraud Dextose Broth + chitosan, the indices being 5.57, 8.03 and 4.07 respectively. Quantitative assay indicated the dominance of protease (6.52 mL-1) followed by lipase (1.55 U mL-1) and chitinase (0.36 mL-1). The experiment conducted to study the bioefficacy of enzymes on the pathogenicity of the fungus revealed a strong positive correlation (0.978, 0.889 and 0.874) between all the cuticle degrading enzymes viz., chitinase, protease and lipase and the pathogenicity of the fungus.
L. saksenae produced crude toxin @ 2 g L -1 which resulted in 91.33 per cent mortality of C. insolitus at 1000 ppm after 24 h. The chromatographic separation of crude toxin revealed the presence of dipicolinic acid on a thin silica plate with Rf value 0.26. Confirmation and quantification using HPTLC detected dipicolinic acid at a concentration of 0. 044 per cent along with eight other higher peaks indicating the presence of an array of strong unknown metabolites.
The investigation concludes that L. saksenae is an efficient entomopathogen to hemipteran pests @ 107spores mL-1, and it is safe to crop plants and non target organisms. It multiplies well in rice bran with chitosan (0.3 per cent) as a sporulation enhancer. Among the cuticle degrading enzymes protease is the dominant one. Dipicolinic acid present in crude toxin resulted in 91.33 per cent mortality of test insect. A detailed metabolite profile of the fungus and identification of genes responsible for hike in protease can tap the potential of L. saksenae to its maximum.