PRODUCTION OF A PROTEINACEOUS PHYTOTOXIN BY ASCOCHYTA-RABIEI GROWN IN EXPRESSED CHICKPEA SAP

Production of the solanapyrone toxins by Ascochyta rabiei is nutrient dependent. When grown on a medium consisting entirely of expressed sap from the aerial parts of young chickpea plants (PSM), only low concentrations of the solanapyrones were produced (<24 mu M). However, toxicity of 4-day-old culture filtrates to isolated cells of chickpea leaflets was comparable with that obtained from 12-day-old culture filtrates on Czapek Dox nutrients supplemented with chickpea seed extract or cations-media that are conducive to solanapyrone production. The additional toxic component which peaked at 4 days in culture was heat labile, losing about 50% of its activity on boiling for 10 min. Affinity for solid-phase extraction media, precipitation with ammonium sulphate and acetone, ionization properties and UV absorption characteristics suggested that the toxin was a polypeptide. The toxin was purified by solid-phase extraction, acetone precipitation and high performance liquid chromatography (HPLC) on a C2 column. Hydrolysis of the purified toxin yielded 14 amino acids, and calculation of the numbers of residues of each amino acid suggested a molecular mass of 7,551 Da. A band corresponding to this molecular mass was present on SDS-PAGE gels. However, both the native peptide and its hydrolysate contained a compound that reacted with p-anisaldehyde suggesting the possibility of a glycosidic moiety.