30 July 2017

Kukule Kankanamge, 2017

Kukule Kankanamge M. 2017. Analysis of chitinase activity. Master’s thesis, Biological Sciences, Bowling Green State University, Bowling Green, Ohio. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1498843764486231


The oomycete Aphanomyces astaci infects crayfish, which can result in the mass mortality commonly referred to as “crayfish plague.” Additional oomycetes in the genera Aphanomyces and Saprolegnia also infect crayfish. In the present study, two distinct organisms were isolated and identified from infected marbled crayfish Procambarus fallax forma virginalis and two phylogenetic trees based on internal transcribed spacer I (ITSI) were constructed using MEGA 7 software and maximum likelihood method with 1000 bootstraps. It is known that crayfish pathogens that infect crayfish produce chitinases that enable them to penetrate the cuticle of the crayfish. Preliminary testing for chitinase activity of Aphanomyces sp. indicated that in vitro growth in terms of surface area of the plates covered by mycelia and dry weight of mycelia increased with increasing chitin concentration from 1%-3% and leveled off at 4% chitin. The effects of chitin on timing of sporangia formation and zoospore release of Apahanomyces sp. suggested that chitin plays a role in asexual reproduction of the pathogen. The time taken for Aphanomyces sp. to develop sporangia and zoospore release increased with the amount of chitin incorporated in the media. Based on these observations, isolates of Aphanomyces sp. and of Saprolegnia sp. were tested for their chitinase activity. Both isolates could utilize chitin as carbon and nitrogen source in their growth. Additional experiments suggested that the chitinase activity of Aphanomyces sp. and Saprolegnia sp. involved an unidentified acidic substance produced by both organisms. Dinitrosalicylic acid assay (DNS assay) indicated the presence of unidentified secondary metabolites and/or pigment produced by Saprolegnia sp. and Aphanomyces sp.in nutrient deprived media. In DNS assay, the media with chitin and water in which both pathogens were grown showed highest absorbance after 72 hours indicating the possibility of their maximum production of chitinase and other enzymes within 48-72 hours. Based on the average absorbance readings, Aphanomyces sp. could be producing significantly higher amount of enzymes that break down chitinous cuticle compared to Saprolegnia sp. Overall, the observations made in this study could indicate chitinase production in Aphanomyces sp. and Saprolegnia sp.

Keywords: crayfish pathogen • Aphanomyces astaciSaprolegnia sp. • chitinase • dinitro salicylic acid assay

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