MODE OF ACTION OF THE ANILINO-PYRIMIDINE FUNGICIDE PYRIMETHANIL .2. EFFECTS ON ENZYME-SECRETION IN BOTRYTIS-CINEREA

The effect of pyrimethanil on the levels of cell wall degrading enzymes secreted by Botrytis cinerea Pers. was investigated in diseased plant tissues and in liquid B. cinerea cultures. Total proteinase activity isolated from infected carrot slices which were treated with 5 . 0 mu M pyrimethanil was decreased by 76%, 3 d after inoculation. Polygalacturonase, cellulase, proteinase and laccase activities were all decreased in the medium of three day-old cultures grown in the presence of pyrimethanil. The pyrimethanil concentrations resulting in 50% reduction in total enzyme activities (IC50) were approximately 0 . 25 mu M for polygalacturonase, cellulase and proteinase, and approximately 1 . 0 mu M for laccase. No significant growth inhibition was observed at these pyrimethanil concentrations. Pyrimethanil did not inhibit the enzymes directly, nor did it inhibit the synthesis of cytosolic proteins. Therefore, it was proposed that the fungicide inhibits protein secretion at a post-translational stage in the secretory pathway. Large differences were found in the effects of pyrimethanil on the growth of B. cinerea in liquid cultures and on agar plates, depending on the composition of the medium. In liquid media containing cellulose and protein as carbon and nitrogen sources, growth inhibition occurred at 5 . 0 mu M pyrimethanil, whilst no growth inhibition was observed with 50 mu M pyrimethanil in malt extract. Similarly, growth occurred on potato/dextrose agar (PDA) at 0 . 5 mu M pyrimethanil, but no growth was seen at this concentration on agars containing cellulose and protein. Thus it appears that pyrimethanil is most active in media where the fungus has to utilise extracellular enzymes to mobilise the nutrients it requires for growth.