جداسازی و شناسائی مولکولی گونه Ectophoma multirostrata از گیاه زینتی Catharanthus roseus

Periwinkle is an ornamental plant belonging to Apocynaceae family. Three diseased Periwinkle plants showing small, asymmetric, brown-colored leaf spot with dark border were collected from greenspaces of Ahvaz. To isolate probable pathogens, the small pieces of leaf were taken from the interface between healthy and diseased tissue and surface-sterilized using 2% sodium hypochlorite (2 min), and then washed with sterile distilled water. Leaf pieces were dried on sterile filter paper and plated on petri dishes containing potato dextrose agar (PDA). Three obtained fungal isolates were purified by single-spore method. Primary morphological study showed that these isolates belong to family Didymellaceae. To molecular identification, fungal isolates were grown in potato-dextrose-broth (PDB) medium. After ten days incubating in darkness at 28 °C on shaker, mycelial biomass were collected by passing through sterile filter paper and then freeze-dried. Total DNA were extracted and then, ITS and tub2 regions were amplified using primer pairs ITS1/NL4 and Btub2Fd/Btub4Rd, respectively. In the BLASTn analysis of the tub2 sequence, these isolates showed the highest identity to different strains of Ectophoma multirostrata. A phylogenetic analysis based on both ITS and tub2 regions was carried out, and on which all three isolates were clustered with three known strains of the species Ectophoma multirostrata (including CBS 110.79, CBS 274.60 and CBS 368.65) and formed a monophyletic clade with strong BS 99% support. The proper morphology of the isolates under study (including the shape and size of pycnidia, pycnidiospores and the number of ostioles) showed that these isolates are E. multirostrata species. This is the first report of E. multirostrata on periwinkle plant.