Investigating components of the host-pathogen interaction of Septoria musiva and Populus
Components of host-pathogen interactions in the Septoria musiva-Populus disease system were investigated in an attempt to better understand resistance and susceptiblity to Septoria canker disease. A system for the production and detection of the extracellular proteolytic enzymes produced by Septoria musiva was established using the synthetic medium, casein culture medium (CCM). An isolate of S. musiva (MP1) showed a growth-limiting effect when grown in CCM that contained initial concentrations of <60 mM glucose. SDS PAGE activity gels revealed two putative proteinases, P1 and P2 (M r 24 and 75 kd), in culture filtrates of MP1. Proteinase P1 was isolated and further characterized for optimum pH (8.0) and reaction temperature (60 C). Proteinase inhibitor II (PI II) and phenylmethanesulphonylfluoride (PMSF) reduced proteolytic activity by 55% and 95% respectively. Inclusion of PMSF in SDS PAGE activity gel samples, resulted in inhibition of P1 activity, but not P2 activity. In vitro and in vivo studies were conducted to determine the effect of PI II on the physiology of the fungal pathogen Septoria musiva. PI II reduced in vitro serine proteinase activity in culture filtrates of S. musiva, and reduced conidial germination, mycelial growth, and culture sporulation when the fungus was cultured in medium containing PI II. In vivo tests were conducted with S. musiva and leaf and stem tissues of a transgenic Populus x euramericana clone ('Ogy') that expresses PI II protein. A reduction in lesion size was observed, but there was no significant difference in infection frequency of leaves. Stem inoculation results were inconclusive because of environmental conditions which may have favored host defenses. Expression studies of PI II in transgenic 'Ogy' have revealed an ~24 kd native proteinase inhibitor of Populus that has immunoreactivity to antibodies against PI II. The native inhibitor protein in 'Ogy' has approximately the same biochemical properties, and similar migration patterns, as PI II. A rating system for evaluation of canker disease in Populus clones was developed to standardize ratings of Septoria canker disease. The rating system evaluates both disease incidence and severity and has permitted identification of some possible sources of resistance in hybrid clones of Populus.