Quantitative detection of four pome fruit viruses in apple trees throughout the year

A one-step real-time RT-PCR assay (RT-qPCR) with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed to detect and quantify RNA targets from Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple chlorotic leaf spot virus (ACLSV) in infected apple trees. Single PCR products of 87 bp (ApMV), 70 bp (ASGV), 104 bp (ASPV) and 148 bp (ACLSV) were obtained, and melting curve analyses revealed distinct melting temperature peaks for each virus. A dilution series using in vitro synthesized transcripts containing the target sequences as standards yielded a reproducible quantitative assay, with a wide dynamic range of detection and low coefficients of variance. The content of selected viruses in apple plant tissues was stable throughout the year, and their accumulation did not significantly change between different plant tissues. The only minor exceptions were for ApMV and ACLSV, in which noticeable differences in their concentrations in various biological material were observed within the year. This divergence did not influence their year-round detectability. This one-step RT-qPCR assay is a valuable tool for year-round diagnostics, and molecular studies of the biology of ApMV, ASGV, ASPV and ACLSV

Study of sugar beet viruses transmitted by Polymyxa betae in the Czech Republic

Sugar beet viruses transmitted by Polymyxa betae are very widespread in the Czech Republic. Beet soil-borne virus (BSBV) is present in almost all fields used for sugar beet growing, beet virus Q (BVQ) is present in about 50% of fields but beet necrotic yellow vein virus (BNYVV) is present in some limited regions only. It means that mixed infections of sugar beet by at least two viruses are quite common in the field. P. betae also occurs in almost all fields where sugar beet is now grown. Only two populations of P. betae not transmitting any virus were found. Cystosori of P. betae can harbour viruses without loosing infectivity for a very long time. We were able to detect these viruses in plants grown in soil stored dry for 12 years. BNYVV can cause serious yield losses under mideuropean conditions reaching up to 50% of sugar yield, whereas harmfulness BSBV and BVQ is questionable, because they also occur in fields with no problems concerning sugar beet growing. The host range of these viruses was studied. Both infect all types of beet (sugar fodder, red beet, mangold) and spinach and usually are detectable in root system only. Other chenopodiaceous plants are infected only by some virus strains. These strains are also able to spread into above-ground parts of plants

Population genetic structure of Mycosphaerella graminicola and Quinone Outside Inhibitor (QoI) resistance in the Czech Republic

ISSN:0929-1873ISSN:1573-846

Characterization of the Molecular Mechanisms of Resistance against DMI Fungicides in Cercospora beticola Populations from the Czech Republic

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar pathogen of sugar beet worldwide. Extensive reliance on fungicides to manage CLS has resulted in the evolution of fungicide resistance in C. beticola worldwide, including populations in the Czech Republic. One important class of fungicides used to manage CLS is the sterol demethylation inhibitors (DMI). The aim of our study was to assess DMI resistance in C. beticola from the Czech Republic and elucidate the molecular basis of DMI resistance in this population. A total of 50 isolates were collected in 2018 and 2019 from the major sugar beet growing regions of the Czech Republic and assessed for in vitro sensitivity to the DMI fungicides propiconazole, prochloraz, and epoxiconazole. These analyses identified three strains that exhibited 50% effective concentration (EC50) values > 1.0 μg mL–1 against respective fungicides, which were therefore considered resistant. In contrast, strains that exhibited lowest EC50 values were considered sensitive. To explore the molecular basis of resistance in these three strains, the cytochrome P450-dependent sterol 14α-demethylase (Cyp51) gene was sequenced. Sequence analysis identified a Y464S mutation in all three resistant strains. To assess whether Cyp51 gene expression may play a role in DMI resistance, selected strains were grown in vitro with and without fungicide treatment. These analyses indicated that Cyp51 gene expression was significantly induced after fungicide treatment. Thus, we conclude that Y464S point mutation along with induced Cyp51 gene overexpression is likely responsible for resistance against DMI fungicides in C. beticola from the Czech Republic