Molecular detection of ascomycetes associated with Fucus serratus

The association of ascomycetes with Fucus serratus was investigated by comparing the broad-based molecular and cultural diversities of healthy and dead fronds. Four PCR primer pairs were used to amplify the 18S (primers NS1-FR1; NS1-EF3) or 28S rRNA (primers NL209-NL912; NL359-NL912) genes directly from the DNA of algal thalli. Two novel primer pairs, NL209-NL912 and NL359-NL912 giving product sizes of 700 and 559 bp respectively, were designed to amplify the 28S rDNA from ascomycetes specifically. All primer combinations amplified DNA from 33 reference taxa isolated from Fucus serratus, and the products generated by primers NS1-FR1GC and NL359-NL912GC were separated in 18-38% and 38-60% denaturant gradients respectively after DGGE. The 18S rDNA DGGE system resolved eight bands from algal DNA, but many of the sequences separated were not fungal, whereas the 28S rDNA system resolved seven bands that were all identified as ascomycetes. Phylogenetic analysis and BLAST search results of environmental sequences revealed the presence of four main ascomycete groups: (1) the Halosphaeriales, (2) the Hypocreales, (3) an unidentified Lulworthiales complex, and (4) the Pleosporales. Few fungal isolates were detected molecularly suggesting that fungal colonisation of fronds was limited, mainly to species in dead casts.