Identification of Portuguese Armillaria Isolates by Classic Mating-Tests and RFLP-PCR Analysis of the ITS1 Region of Ribosomal DNA

The diagnosis of Armillaria, a genus including distinct species of highly woody plantpathogenic root-infecting fungi with worldwide distribution, is usually based on morphological characteristics and mating-tests, although the PCR-based restriction fragment length polymorphism (RFLP-PCR), specifically in nuclear rDNA spacers, have also been applied. In the present study, mating-tests and restriction analysis of Internal Transcribed Spacer 1 (ITS1) were used to identify 20 isolates of a Portuguese Armillaria collection. Although the majority of the diploid isolates (80%) could be identified in diploid-haploid pairings, the method is laborious, takes too much time (up to 2 months), and presents a high rate of inconclusive results. The ITS1 region showed to be a reliable molecular marker for A. mellea, in particular when HinfI restriction analysis is applied, since two fragments with 245 bp and 125 bp have been obtained for this most aggressive species whereas 290 bp and 70 bp were produced from isolates of the other European species. As simple molecular techniques are involved and the whole procedure can be performed in one day, A. mellea identification by ITS1 analysis is a clearly accessible and more advantageous tool to plant pathology laboratories, mainly those involved on the control and preservation of forest trees