Evidence for the involvement of ascochitine in phoma leafspot-wilt disease of Clematis

Ascochitine, a phytotoxic metabolite, was purified from cultures ofPhoma clematidina and identified by 'H and r3C nuclear magnetic resonance spectroscopy and electron impact and chemical ionization mass spectroscopy. This toxin-induced electrolyte leakage from leaf discs of Clematis cultivars that were susceptible to fungal infection, while there was no significant electrolyte leakage from leaf discs of the cultivar most resistant to fungal infection. The level of ascochitine production in vitro by P. clemafidina isolates was related to isolate virulence. The fungal isolates could be characterized into two groups: (1)high virulence, high ascochitine. production, and (2)low virulence, low ascochitine production. Ascochitine was isolated from P. clemafidinn-infected leaf discs, indicating the toxin is produced in ho. Leaf tissues exposed to ascochitine solutions showed black flecking in proportion to the concentration of ascochitine. Scanning electron and light microscopy of infected leaves indicated that the fungal hyphae were well behind the necrotic zone in leaf spots, while transmission electron and light microscopy suggested that mitochondria and chloroplasts were the major organelles affected by ascochitine, although extensive cellular damage was evident. These observations suggest that ascochitine may be involved in the pathogenesis of P. clemnlidina against Clemafis, by killing plant cells prior to hyphae ramification through the necrotic tissue.