Differentiation of Phoma foveata from P-exigua using a RAPD generated PCR-RFLP marker

Phoma foveata and P. exigua variety exigua both infect potatoes and are mor phologically very similar. P. foveata produces a pigment which allows diffe rentiation from P. exigua in culture. Discrimination of the two species bas ed on the production of a secondary metabolite, which is dependent on the g rowth conditions, is not reliable. Therefore, there is a need to develop nu cleic acid based identification markers. A 482 bp random amplified polymorp hic DNA (RAPD) fragment from P. foveata was isolated and sequenced. Polymer ase chain reaction (PCR) primers, developed from the sequence of the RAPD p roduct, amplified a 474 bp fragment for P. foveata and P. exigua varieties exigua, diversispora, inoxydibilis and sambuci-nigrae. The similarity of th e PCR fragments was demonstrated by sequence analysis and by using the rest riction enzymes DdeI and DpnII. P. foveata was distinguished from the four varieties of P. exigua on the basis of the RFLP patterns of the PCR fragmen t. Ten isolates of P. foveata and nine of P. exigua var. exigua from differ ent geographic locations were tested and all isolates but one showed the re striction digest pattern of the PCR fragment (PCR-RFLP) specific to each sp ecies. One isolate of P. foveata demonstrated a PCR-RFLP pattern similar to P. exigua var. exigua leading to the conclusion that the isolate had been previously misidentified as a strain of P. foveata lacking the ability to p roduce pigment.