The tritimovirus Wheat streak mosaic virus (WSMV) is widespread throughout the world and represents a severe threat to cereal crop production. To increase knowledge of genetic diversity of WSMV in Europe, until now scarce, capsid protein (CP) sequences of several Czech, French, Italian, Slovak, and Turkish isolates have been determined. A multiple alignment of CP nucleotide sequences using available WSMV sequences revealed only limited sequence variation among 3 previously sequenced European isolates and the 14 European isolates sequenced in this study. Moreover, these isolates were characterized by an identical 3-nucleotide deletion, resulting in the lack of the Gly2761 codon within the CP region of the polyprotein. The results indicate that this monophyletic group of isolates (designated as WSMV-ΔE) is common and widely dispersed throughout the European continent. The close relationship of WSMV-ΔE isolates implies a single common ancestor and, presumably, subsequent dispersal throughout Europe from a single focus. We developed two simple assays for specific and accurate detection of WSMV-ΔE isolates. First, a conserved ClaI restriction site in the core CP gene sequence unique to WSMV-ΔE isolates was used for restriction fragment length polymorphism analysis of amplified polymerase chain reaction (PCR) products. Second, the conserved and specific codon gap in WSMV-ΔE sequences was used as a target to design specific primers functional in one-step reverse-transcription PCR detection of WSMV-ΔE isolates.
A rapid method for detection, discrimination and quantification of wheat and barley strains of wheat dwarf virus (WDV) was successfully developed. The sensitivity of quantification of the wheat and barley strains of WDV ranged from an average of 1.2 × 10(7)-1.2 × 10(2) and from an average of 1.4 × 10(7)-1.4 × 10(4) copies of viral genome, respectively. These standard serial dilutions were applied to plant and vector tissues for virus titer calculations. Both strains of WDV were clearly discriminated by specific probes and melting curve analysis. Both TaqMan(®) and SYBR(®) Green technologies provided accurate and reliable methods for monitoring, detection, discrimination, and quantification of WDV.
Less prevalent viruses of family Poaceae are usually excluded from the focus of interest, even though they represent a possible threat to agricultural production. We designed and validated a set of primer pairs suitable for detection and quantification of five RNA viruses, Lolium latent virus (LoLV), Oat necrosis mottle virus (ONMV), Ryegrass mosaic virus (RgMV), Soil-borne cereal mosaic virus (SBCMV), and Spartina mottle virus (SpMV), by means of one-step RT-qPCR based on SYBR Green I. These primers were used together with primers for Brome mosaic virus (BMV) and Wheat streak mosaic virus (WSMV) described elsewhere to screen grass and cereal samples from the Czech Republic. The results revealed a high prevalence of WSMV and RgMV, which pointed to possible local epidemics. We also make the first report of LoLV presence in the Czech Republic.
The phytopathogenic fungus Sclerotinia sclerotiorum forms dormant structures (termed sclerotia) that germinate myceliogenically under certain environmental conditions. During myceliogenic germination, sclerotia produce hyphae, which can infect leaves or stems of host plants directly from the ground; this is termed basal infection. This study determined which abiotic conditions were most important for promoting myceliogenic germination of sclerotia in vitro. A high sclerotium hydration level and low incubation temperature (158C) improved mycelial growth in the presence of a nutrient source. Sclerotia incubated without a nutrient source on moist sand, vigorously myceliogenically germinated most frequently (63%) when they had been previously imbibed and then conditioned at À208C. By far the most consistent amount of vigorous myceliogenic germination (>75%) was produced when sclerotia were heat-dried before being submerged in water. The hyphae of these sclerotia were shown to infect and proliferate on leaves of intact Brassica napus plants. This research provides a better understanding of the abiotic conditions that are likely to increase the risk of basal infection by S. sclerotiorum.
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