Abstract
BackgroundThe 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).FindingsMost 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.ConclusionsThe 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.
Highlights
The hyperparasitic fungal plant pathogen [Sphaeropsis visci (Alb. & Schwein.) Sacc.], which causes leaf spot disease of European mistletoe (Viscum album L.) seems to have potential as a tool for biological control of this hemiparasite (Varga et al 2012a; Karadžić et al 2004; Fischl 1996; Stojanović 1989)
The connection between the asexual and sexual morph of the fungus was established by Phillips et al (Phillips et al 2008) by the discovery that the ascomycete Phaeobotryosphaeria visci occurring on Viscum album produces conidia typical of Sphaeropsis visci
The analytical sensitivity of direct PCR (diPCR) was proved to be high with lower concentrations of DNA that are close to the content of a single fungal cell that is reported to be around ca. 0.15 pg per cell (Kim et al 2000)
Summary
The diPCR approach allowed amplification of ITS rRNA gene directly from small amounts of fungal samples without prior DNA extraction.
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