Application of direct PCR in rapid rDNA ITS haplotype determination of the hyperparasitic fungus Sphaeropsis visci (Botryosphaeriaceae)

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.

In vitro cultivation and biocontrol potential of Botryosphaeria visci against European mistletoe (Viscum album L.)

“…Identification was based solely on morphological characteristics of conidia from infected host material, but molecular analysis of the fungus is also required. This fungus is an aggressive pathogen on mistletoe in various countries, for example in Romania (Sutton 1980), Croatia (Idžojtić et al 2008), Ukraine (Phillips et al 2008), Austria, the Czech Republic, Egypt (Phillips et al 2013), Hungary (Varga et al 2014, Poczai et al 2015, Serbia, (Zlatković et al 2016, Zlatković et al 2015, China (Chen et al 2018), and Poland (Tkaczyk and Sikora 2020).…”

“…The choice of media for cultivation was based on the information available in the literature about the growth of the fungus; for a more effective comparison and reproduction of the results, the synthetic media that are most often used were chosen (Varga et al 2012b(Varga et al , 2013(Varga et al , 2014.…”

“…According to the formation of pycnidia or of their absence on leaves, shoots or berries of mistletoe, pathogenicity was evaluated as positive or negative, respectively. Re-isolations were performed from infected leaves and shoots (Varga et al 2014), and the resulting isolates were identified as previously described.…”

“…There are several genera of pathogenic fungi associated with mistletoe diseases, among which Botryosphaeria visci is relatively more aggressive as it causes mistletoe dieback (Kotan et al 2013, Karadžić et al 2004, Kahle-Zuber 2008. Traditional research on B. visci to date has focused on taxonomic profiling, particularly based on molecular, macro-and microscopic studies (Phillips et al 2008, Poczai et al 2015, Varga et al 2014). It should be noted that studies of the growth of this pathogenic fungus in vitro can reveal some important physiological features.…”

Consuming and consumed: Biotic interactions of African mistletoes across different trophic levels

“…In addition, the use of plant pathogens (such as fungi and bacteria) in biological control of pest mistletoes is increasingly gaining attention as an environmentally beneficial method applicable to agroecosystems (Shamoun et al, 2003). Given the potential success and major challenges of this method as outlined by the recent efforts of its implementation against Viscum album in Europe (Kotan et al, 2013; Poczai et al, 2015; Varga et al, 2014), designing targeted studies on the identification and use of specific mistletoe pathogens in Africa would be crucial for controlling mistletoes in areas where they threaten crop production.…”