Molecular detection of Entoloma spp. associated with roots of rosaceous woody plants

Seventeen isolates of Entoloma clypeatum, a fungus associated with rosaceous woody plants and suspected to act as a root pathogen, were obtained from fruit-bodies collected at 7 localities in the Czech Republic. The fungus grew best on the medium supplied with cellobiose. The growth of E. clypeatum was inhibited by fungicide preparation Ridomil Gold MZ 68WP. A part of the LSU rRNA gene of 11 selected isolates was sequenced and two reverse primers recognizing the sequence motifs in the LSU rRNA gene of E. clypeatum and other related species associated with rosaceous woody plants were designed. One of these primers, NL4EC2, showed high specificity towards DNA of Entoloma spp. of interest and may be used for detection of these organisms in soil, in roots or in the substrate where the fruit trees are cultivated. The results of in vitro inoculation experiment suggest a resistance of roots of young Prunus domestica plants to colonization by Entoloma clypeatum, and the fungus could be detected only in the surrounding substrate. We were able to detect Entoloma spp. in soil and in roots of Prunus avium but only if the fructification of the fungus occurred at the locality. The method is robust towards the false positive detection caused by nonspecific amplification of DNA of other soil microorganisms.