title: Application of direct PCR in rapid rDNA ITS haplotype determination of the hyperparasitic fungus Sphaeropsis visci (Botryosphaeriaceae).
abstract: BACKGROUND
  The plant pathogenic fungus, Sphaeropsis visci a dark-spored species of Botryosphaeriaceae, which causes the leaf spot disease of the European mistletoe (Viscum album). This species seems to have potential as a tool for biological control of the hemiparasite. For the rapid detection of S. visci haplotypes we tested a direct PCR assay without prior DNA purification. This approach was based on a polymerase enzyme from the crenarchaeon Sulfolobus solfataricus engineered by fusion protein technology, which linked the polymerase domain to a sequence non-specific DNA binding protein (Sso7d).
  
  
  RESULTS
  Most isolates of Sphaeropsis visci grouped together in our phylogenetic analyses, indicating that isolates had a previously reported haplotype sequence, which is commonly found in the analyzed Hungarian population. This haplotype was also reported from diseased mistletoe bushes from other European countries. We further identified unique single nucleotide polymorphisms (SNPs) in the ITS region, which were specific to the only well resolved clade in the phylogenetic analysis.
  
  
  CONCLUSIONS
  The diPCR approach allowed amplification of ITS rRNA gene directly from small amounts of fungal samples without prior DNA extraction. This simple bioassay in plant disease management enables collection of genomic data from fungal plant pathogen populations.
authors.affiliation: Plant Biology, Department of Biosciences, University of Helsinki, PO Box 65, Helsinki, FI-00014 Finland.
authors.forename: Ildikó
authors.initials: I
authors.lastname: Varga
authors.affiliation: Plant Biology, Department of Biosciences, University of Helsinki, PO Box 65, Helsinki, FI-00014 Finland ; Botanical Museum, University of Helsinki, PO Box 7, Helsinki, FI-00014 Finland.
authors.forename: Péter
authors.initials: P
authors.lastname: Poczai
authors.affiliation: Potato Research Centre, Centre of Agricultural Sciences, University of Pannonia, Festetics u. 7, Keszthely, H-8360 Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Cernák
authors.affiliation: Plant Biology, Department of Biosciences, University of Helsinki, PO Box 65, Helsinki, FI-00014 Finland.
authors.forename: Jaakko
authors.initials: J
authors.lastname: Hyvönen
keywords: Biological control; Botryosphaeriaceae; European mistletoe (Viscum album); Genotyping; Polymerase inhibitors; Sphaeropsis visci; diPCR