Bioassay-led isolation of Myrothecium verrucaria and verrucarin A as germination inhibitors of Orobanche crenata

A total of 188 fungal isolates was obtained from the rhizosphere of Vicia faba grown in an Egyptian soil heavily infested with Orobanche species. Agar cultures of 58 isolates inhibited the germination of conditioned seed of Orobanche crenata exposed to the germination stimulant, GR24. Filtrates of inhibitory fungi grown in liquid medium for 9-15 days were also assayed and those of five isolates, which were morphologically similar, inhibited germination even when diluted 16-fold. The fungus was identified as Myrothecium verrucaria (Alb. A Schwein.) Ditmar by its morphology and the nucleotide sequence of the ITS1 and ITS2 regions of the ribosomal repeat unit. Purification of the inhibitor to homogeneity was accomplished by solvent partitioning, flash chromatography on silica gel, semi-preparative HPLC on a reversed phase C18 column, solid phase extraction and tlc on silica gel. The inhibitor was identified as verrucarin A by nuclear magnetic resonance spectroscopy and comparison of the spectra with those of an authentic sample of the compound. A preliminary experiment demonstrated that infection of V. faba by O. crenata could be prevented by addition of spores of the fungus to soil infested by the parasite.