Differentiation of Seiridium species associated with virulent cankers on cypress in the Mediterraneanregion by PCR-SSCP

A rapid and sensitive method was developed to discriminate between Seiridium cardinale and Seiridium cupressi, the fungi causing severe cankers on common cypress in the Mediterranean area. The method amplified sequence variants in the ITS2 region of ribosomal DNA using the polymerase chain reaction (PCR), followed by polyacrylamide gel electrophoresis, to reveal single-strand conformation polymorphism (SSCP) between the two species. The greatest separation pattern was obtained with a gel matrix containing 7-10% formamide and 3-5% glycerol under optimized running conditions, which were found to be 30-40 V at 4-5 degreesC for 4-8 h. Sequence homology among isolates within each of the two species caused no mobility shifts, with all isolates displaying the same migration pattern. A few base differences between S. cardinale and S. cupressi caused markedly different migration patterns, allowing differentiation of the two pathogens. Differences between these fungi at the genetic level are consistent with known data on morphological, physiological and pathogenic characteristics. SSCP analysis constitutes a rapid and easy-to-perform method by which to recognize and distinguish closely related organisms, and has considerable potential for use in diagnosis and taxonomy.