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A recently described putative foveavirus, grapevine virus T (GVT), was detected in a Slovak grapevine accession (SK704) using high-throughput sequencing, prompting further studies. Full-length genome sequence of isolate GVT-SK704 was determined. Analyses revealed 86.1% nucleotide identity with the Italian GVT isolate, currently the only available nearly complete sequence of GVT in GenBank. A virus-specific RT-PCR assay was developed, which enabled a survey of GVT incidence in grapevine samples from Slovakia and Czech Republic. Unexpectedly, GVT was present in ~ 30% of tested samples. Analysis of complete CP gene sequences of 20 Slovak and Czech GVT isolates detected in the survey revealed relatively high intra-species variability (up to 11.2% nucleotide divergence), suggesting multiple introductions from different sources, possibly over an extended period of time. [ABSTRACT FROM AUTHOR]

In order to analyse the potential presence of CmEV in Slovakia, additional leaf samples from melons, cucumbers and squashes from four different localities were tested by RT-PCR using the above primers. Keywords: endornavirus; high-hroughput sequencing; Cucurbitaceae; detection EN endornavirus high-hroughput sequencing Cucurbitaceae detection 1179 1180 2 08/23/22 20220801 NES 220801 Supplementary information The online version contains supplementary material available at https://doi.org/10.1007/s42161-022-01149-4. [Extracted from the article]

A portable reconfigurable platform for copper (Cu(II)) determination based on luminescent carbon dot (Cdots) quenching is described. The electronic setup consists of a light-emitting diode (LED) as the carbon dot optical exciter and a photodiode as a light-to-current converter integrated in the same instrument. Moreover, the overall analog conditioning is simply performed with one integrated solution, a field-programmable analog array (FPAA), which makes it possible to reconfigure the filter and gain stages in real time. This feature provides adaptability to use the platform as an analytical probe for carbon dots coming from different batches with some variations in luminescence characteristics. The calibration functions obtained that fit a modified Stern-Volmer equation were obtained using luminescence signals from Cdots quenching by Cu(II). The analytical applicability of the reconfigurable portable instrument for Cu(II) using Cdots has been successfully demonstrated in tap water analysis. [ABSTRACT FROM AUTHOR]

Grapevine Syrah virus-1 (GSyV-1) was identified by small-RNA deep sequencing in Slovak grapevine co-infected by several other viruses. The RT-PCR assays developed in this work substantially improved the virus detection and allowed the identification of GSyV-1 in tested grapevine samples from Slovakia and the Czech Republic at an unexpectedly high rate (ca. 30 %). Subsequently, complete genome sequences of 3 GSyV-1 isolates (2 Slovak and 1 Czech) were determined by Sanger sequencing, showing a typical marafivirus genome organization. Analyses of complete genome sequences showed a higher intra-group diversity among these 3 central European GSyV-1 isolates (differences reaching 7.1 % at the nucleotide level) in comparison to 3 previously characterized North American isolates (only 1.2 % intra-group divergence). A substantially higher divergence among central European isolates and their clustering into two major phylogenetic groups was further confirmed by the partial genome analysis of additional 26 isolates. The CP-centered study did not support the geography-based clustering among central European and American isolates. Nevertheless, the sequence data of the highly variable 5′-proximal portion of the genome obtained for few additional isolates from Slovakia and Czech Republic showed the presence of both, 'European-' and 'north American-like', GSyV-1 isolates in the analyzed grapevine samples. [ABSTRACT FROM AUTHOR]

Keywords: Grapevine; AGVd detection; HTS EN Grapevine AGVd detection HTS 1023 1024 2 08/18/21 20210801 NES 210801 In 2017 and 2018, grapevine leaves and stems were collected from symptomatic and symptomless vines from various cultivars of prefecture of Heraklion and the vineyard of the Greek Institute of Grapevine in Likovrisi (Attica). 10.1002/pca.1078 2 Hajizadeh M, Navarro B, Bashir NS, Torchetti EM, Di Serio F. Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids. Total RNA extractions were performed according to Maliogka et al. ([3]) and multiplex RT-PCR method (Hajizadeh et al. [2]) revealed the presence of Australian grapevine viroid (AGVd) in 13 out of 298 samples tested (11 from Crete and 2 from Attica). [Extracted from the article]

Analysis of complete genome sequences of three Slovak isolates of grapevine Pinot gris virus (GPGV) showed their low heterogeneity (reaching 1.7 %) and a close relationship to the Italian NC_015782 isolate (4.2-4.5 % divergence). Comparison of Slovak and Italian isolates revealed an unusual accumulation of 21 indel mutations in ORF1, resulting in a localized high divergence in the encoded amino acid sequences. An elevated divergence in the 5′ extremity of the GPGV genomes is suggestive of a recombination between Slovak isolates and grapevine berry inner necrosis virus. RT-PCR allowed the frequent detection of closely related GPGV isolates in grapevines from Slovakia and the Czech Republic. [ABSTRACT FROM AUTHOR]

Lamium mild mosaic virus (LMMV) is the only one of the five members of the genus Fabavirus for which there are no nucleotide sequence data. In this study, the complete genome sequence of LMMV was determined and compared with the available complete genome sequences of other members of the genus Fabavirus. The genome was the largest of the genus but maintained the typical organization, with RNA 1 of 6080 nucleotides (nt), RNA 2 of 4065 nt, and an unusually long 3′ untranslated region in RNA 2 of 603 nt. Phylogenetic analysis of the amino acid sequences of the protease-polymerase (Pro-Pol) region and the two coat proteins confirmed that LMMV belongs to a distinct species within the genus Fabavirus. [ABSTRACT FROM AUTHOR]

multi-analyte platform based on a portable instrument is presented that enables oxygen and carbon dioxide to be determined in sample gases. The use of four sensing channels (two channels for each analyte to provide redundancy) warrants high system reliability. The sensing scheme in case of oxygen is based on the quenching of the phosphorescence of the platinum octaethylporphyrin complex. In case of carbon dioxide, a secondary inner-filter effect is exploited that is caused by a pH indicator whose color is reversibly changed. The sensing membranes were placed directly on the detectors and on the light sources so to make additional optical element dispensable, reduce system costs, avoid problems related to optical alignment, optimize the efficiency of data acquisition, and enable facile replacement of sensors. The resulting microcontroller-based system is immune against optical and electrical interferences, contains simple digital signal processing circuitry, and has low power consumption. The response of the system to the two gases was modeled, and calibration curves are corrected for effects of temperature. The instrument was characterized in terms of cross-sensitivity and dynamic response. It can determine oxygen and carbon dioxide in terms of volume percentage. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Table grapes from one of the most important growing area in Spain (Vinalopó, Alicante) protected by the Designation of Origin 'Vinalopó bagged table grape', were surveyed and analysed to determine the prevalence of the five viruses included in the Spanish certification program: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV), Grapevine leafroll associated virus-1 (GLRaV-1) and Grapevine leafroll associated virus-3 (GLRaV-3). Ninety five sampling points were selected and the position of grapevine plants georeferenced. Samples were collected in two different vegetative periods and analyses were performed by ELISA and real-time RT-PCR. Purified RNA and immobilized viral targets from plant extracts on nylon membranes were used in parallel assays as templates for PCR assays. In order to analyse these five viral species by real-time RT-PCR, new specific primers and TaqMan probes were designed for detection of ArMV and GFkV. Real time RT-PCR from purified RNA was more sensitive than spot version and ELISA tests. The most prevalent virus was GFLV (95.8%) followed by GLRaV-3 (94.7%), GLRaV-1 (66.3%) and GFkV (65.3%). ArMV was not detected in any sample. The high level of viral infections and the presence of mixed infections suggest that initial infected plant material and uncontrolled traffic of propagation material have played an important role in the spread of viruses. [ABSTRACT FROM AUTHOR]

RT-PCR from the original pomegranate plant using the newly designed primers SPeSV-6F (5'-GGCTAGAAACGGTGGGATGA-3') and SPeSV-6R (5'-ACCACCTGGCTCATGGCGA-3') generated the expected 165 nt amplicon, as confirmed by Sanger sequencing (100% nt identity with the HTS sequence). PeSV has also been reported in Turkey, China and India infecting pomegranate (Caglayan et al. [1]). Keywords: PeSV; pomegranate; HTS EN PeSV pomegranate HTS 1375 1375 1 11/29/21 20211101 NES 211101 Passiflora edulis symptomless virus (PeSV), family I Potyviridae i , genus I Roymovirus i , was first identified in Israel infecting I Passiflora edulis i plants (Jover-Gil et al. [2]). [Extracted from the article]

Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 ± 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for large-scale analyses. [ABSTRACT FROM AUTHOR]

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.