Establishment of an in vitro model system for the study of Pinus pinaster Ait. interactions with pathogenic fungi

Abstract

Maritime pine (Pinus pinaster Ait.) is the most widely distributed conifer in the portuguese territory, assuming a preponderant role in both ecological and industrial terms. Pine forests have, nonetheless, been the target of several infestations. The pathogenic fungi Lophodermium seditiosum and Botryotinia fuckeliana (Botrytis cinerea), by atacking young seedlings, have been responsible for the lack of natural regeneration in portuguese pine forests, and the devastation of pine nurseries. Here we report the establishment of a P. pinaster cell suspension culture as an in vitro system for studying pine-pathogen interactions. Development of the culture along time was analysed by the monitoring of growth (dry weight), pH, carbon source (sucrose, frutose and glucose), nitrogen source (amonia and nitrate), phosphate and cell viability. To validate the in vitro model system, we studied the production of Reactive Oxygen Species (ROS) after eliciation with the pathogenic fungi, since an oxidative burst occurs when pathogen recognition induces the plant’s Hypersensitive Response (HR). The tetrazolium dye XTT (sodium,3’-(1-[phenylamino-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate) was used to moniter superoxide ion (02-.) formation. The HR was observed after eliciting with a B. fuckeliana spore suspencion, but didn’t occur when micelium extracts of both fungi were used.