PATHOTYPE IDENTIFICATION OF LEPTOSPHAERIA-MACULANS WITH PCR AND OLIGONUCLEOTIDE PRIMERS FROM RIBOSOMAL INTERNAL TRANSCRIBED SPACER SEQUENCES

In order to develop a pathotype-specific assay, the internal transcribed spacer region 1 (ITS 1) of ribosomal RNA genes of highly and weakly virulent isolates of Leptosphaeria maculans were sequenced using primers from the flanking 17S and 5·8S ribosomal DNA (rDNA). The sequenced ITS 1 region had approximately 67% similarity among highly and weakly virulent isolates, and 37-46% similarity between L. maculans and other species of fungi. From the ITS 1 sequences, two pairs of oligonucleotide primers were synthesized which specifically amplified DNA from either the highly or weakly virulent pathotype of L. maculans using the polymerase chain reaction (PCR). Primer pair HV17S/5·8S was based on sequences from the highly virulent isolates, Leroy and LM26, and only amplified highly virulent isolates. Primer pair WV17S/5·8S was based on sequences from the weakly virulent isolate, Unity, and only amplified weakly virulent isolates. This PCR-based assay utilizing pathotype-specific primers was employed to detect an isolate of the highly virulent pathotype of L. maculans in infected plant tissue and shows promise as a diagnostic tool. © 1992.