Varga Ildikó, Poczai Péter, Cernák István, Hyvönen Jaakko
Plant Biology, Department of Biosciences, University of Helsinki, PO Box 65, Helsinki, FI-00014 Finland.
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.
Springerplus. 2014 Sep 30;3:569. doi: 10.1186/2193-1801-3-569. eCollection 2014.
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).
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.
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.
植物病原真菌葡萄座腔菌属的深色孢子种——槲寄生球座菌，可引发欧洲槲寄生（白果槲寄生）的叶斑病。该物种似乎有潜力成为控制这种半寄生植物的生物防治工具。为了快速检测槲寄生球座菌的单倍型，我们测试了一种无需事先纯化DNA的直接PCR检测方法。这种方法基于通过融合蛋白技术改造的嗜热栖热放线菌的聚合酶，该技术将聚合酶结构域与一种序列非特异性DNA结合蛋白（Sso7d）相连。
在我们的系统发育分析中，大多数槲寄生球座菌分离株聚集在一起，这表明这些分离株具有先前报道的单倍型序列，该序列在分析的匈牙利种群中普遍存在。其他欧洲国家患病的槲寄生灌木丛中也报道过这种单倍型。我们还在ITS区域鉴定出独特的单核苷酸多态性（SNP），这些多态性是系统发育分析中唯一解析良好的分支所特有的。
直接PCR方法能够直接从少量真菌样本中扩增ITS rRNA基因，无需事先提取DNA。这种植物病害管理中的简单生物测定法能够从真菌植物病原体种群中收集基因组数据。
