Characterization of major hydrolytic enzymes secreted by Pythium myriotylum, causative agent for soft rot disease.

Abstract

Pythium myriotylum, an oomycetous necrotroph is the causal agent of soft rot disease affecting several crops. Successful colonization by necrotrophs depends on their secretion of a diverse array of plant cell wall degrading enzymes (CWDEs). The induction dynamics of CWDEs secreted by P. myriotylum was analysed as little information is available for this pathogen. Activities of CWDEs that included pectinase, cellulase, xylanase and protease were detected using radial diffusion assay and differential staining. In Czapek Dox minimal medium supplemented with respective substrates as carbon source, the increase in CWDE activities was observed till 8days of incubation after which a gradual decline in enzymatic activities was observed. With sucrose as sole carbon source, all the enzymes studied showed increase in activity with fungal growth while with cell wall material derived from ginger rhizome as sole carbon source, an initial spurt in cellulase, xylanase and pectinase activities was observed 3days post incubation while protease activity increased from three days of incubation and reached maximum at 13days of incubation. To further evaluate the role of CWDEs in pathogenicity, UV-induced mutants (pmN14uv1) were generated wherein significant reduction in cellulase, pectinase and protease activities were observed while that of xylanase remained unchanged compared to wild type isolate (RGCBN14). Bioassays indicated changes in infection potential of pmN14uv1 thereby suggesting the crucial role played by P. myriotylum CWDEs in initiating the rotting process. Hence appropriate strategies that target the production/activity of these secretory hydrolytic enzymes will help in reducing disease incidence/pathogen virulence.