Evidence for a primary role of active oxygen species in induction of host cell death during infection of bean leaves with Botrytis cinerea

Abstract

Bean genotypes (Phaseolus vulgaris) selected for high or low sensitivity to oxidants showed similar stress responses to ozone, paraquat and to infection with Botrytis cinerea. The potentially oxidative nature of the fungal attack was investigated on bean leaf discs floated in diluted malt broth medium and inoculated with conidia of B. cinerea isolates differing in aggressiveness. Hydrogen peroxide and OH* radicals were measured in situ during infection. Additionally, peroxidases, cellulases, xylanases and polygalacturonases (PG) diffusing into the incubation medium during infection were analysed. An assay based on the oxidative degradation of 2-deoxyribose enabled the specific detection of in situ generated OH* radicals. The aggressiveness of six isolated of Botrytis was closely correlated with the amount of H2O2 and OH* radicals detected in the leaf tissue during infection. Aggressive isolates induced peak signals of OH* radical formation within 24 h and strongly elevated levels of H2O2 within 48 h post-inoculation (hpi), in a phase of most rapidly progressing infection. No such increases in active oxygen species (AOS) were detected in non-aggressive interactions. All fungal isolates tested suppressed plant peroxidase activity as compared to non-inoculated leaf discs. However, aggressive isolates were significantly more suppressive than non-aggressive ones, suggesting a role for peroxidases in plant resistance as scavengers of harmful AOS. Hydrogen peroxide production measured in infected leaf discs was stronger than its accumulation in the incubation medium, pointing to sources of H2O2 inside the plant. The scavengers of AOS, catalase and D-mannitol significantly reduced severity of infection. Secretion of cellulases and xylanases occurred later than the start of infection, suggesting a role of these cell-wall degrading enzymes in the digestion of dead rather than the killing of living host tissue. PGs were produced by all fungal isolates much earlier (12 hpi) but the production did not reflect the isolate aggressiveness. This is the first study to directly detect toxic AOS in situ during infection of plant tissue by B. cinerea. It is concluded that AOS, besides their probable role in plant resistance, might be instrumental for necrotrophs like B. cinerea to kill the host tissue in initial stages of infection.