First report of grapevine Flavescence dorée phytoplasma in the Czech Republic.

Viticulture is a traditional branch of agriculture in the Czech Republic. Grapevines (Vitis vinifera L.) are cultivated on more than 18,000 hectares in the wine-growing regions of Bohemia and South Moravia. South Moravia alone accounts for more than 90 % of the total wine-growing area in the country. Grapevine yellows are a complex of diseases associated with the phytoplasma presence. Phytoplasmas of at least five different groups can cause similar symptoms in grapevines, and they can be distinguished only on a molecular basis (EPPO 2016). One of them, the grapevine Flavescence dorée phytoplasma (GFDP), which belongs to the 16SrV group, is listed in Annex II, Part B, of the Commission Implementing Regulation (EU) 2019/2072 of 28 November 2019 as a Union quarantine pest known to occur in the Union territory. Official surveys for GFDP in the Czech Republic have been carried out since 2007. In 2016, the first occurrence of Scaphoideus titanus Ball, 1932, the main vector of GFDP, was reported in the South Moravian Region (EPPO Reporting Service 2016). This is a matter of concern because it indicates that there is a risk of disease dissemination to other geographical locations. In September 2021, a total of 250 samples of V. vinifera (preferentially focused on symptomatic plants) and four samples of the wild plant host Clematis vitalba L. were collected from 50 vineyards in South Moravia. Total DNA was extracted using High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland). For phytoplasma screening, a real-time PCR test for generic detection of phytoplasmas was used (Christensen et al. 2004). Samples evaluated as positive were further tested by PCR using phytoplasma universal P1 and P7 primers (Deng and Hiruki 1991; Schneider et al. 1995), followed by nested PCR using the 16SrV group-specific primers fB1 and rULWS1 (Smart et al. 1996). For identification of 16SrV phytoplasma, sequence analysis of the secY-map genetic locus was performed. Two sets of primers were used: FD9f5/MAPr1 primers for the first PCR and FD9f6/MAPr2 for the nested PCR (Arnaud et al. 2007) with PCRBIO TaqMix (PCR Biosystems, London, UK). The nested PCR products were purified and sequenced (Eurofins Genomics, Ebersberg, Germany). The sequences were compared with sequences from the GenBank database. Phytoplasma of the 16SrV group was detected in three samples: V. vinifera cv. Gewürztraminer with symptoms of leaf reddening with no rolling and no other typical symptoms; C. vitalba L. with leaf curling (Fig. 1A); symptomless C. vitalba. The obtained sequences of the secY-map locus of all three 16SrV-positive samples were identical to the sequence of GFDP, isolate Vv-SI257 (Acc. No. FN811141), detected in grapevine in Tuscany (Italy), which belongs to 16SrV group. The sequence of the V. vinifera cv. Gewürztraminer isolate was submitted to GenBank under Acc. No. OQ185203. This isolate belongs to the Map-FD3 cluster (Fig. 1B), and the genotype identified is M51 (corresponding to FD-C), which has already been found in C. vitalba and outbreaks of Flavescence dorée in grapevines in some other European countries (Malembic-Maher et al. 2020). Based on the abovementioned results, this is the first report of the GFDP in the Czech Republic.

Evaluation of Methods and Processes for Robust Monitoring of SARS-CoV-2 in Wastewater.

The SARS-CoV-2 pandemic has accelerated the development of virus concentration and molecular-based virus detection methods, monitoring systems and overall approach to epidemiology. Early into the pandemic, wastewater-based epidemiology started to be employed as a tool for tracking the virus transmission dynamics in a given area. The complexity of wastewater coupled with a lack of standardized methods led us to evaluate each step of the analysis individually and see which approach gave the most robust results for SARS-CoV-2 monitoring in wastewater. In this article, we present a step-by-step, retrospective view on the method development and implementation for the case of a pilot monitoring performed in Slovenia. We specifically address points regarding the thermal stability of the samples during storage, screening for the appropriate sample concentration and RNA extraction procedures and real-time PCR assay selection. Here, we show that the temperature and duration of the storage of the wastewater sample can have a varying impact on the detection depending on the structural form in which the SARS-CoV-2 target is present. We found that concentration and RNA extraction using Centricon filtration units coupled with Qiagen RNA extraction kit or direct RNA capture and extraction using semi-automated kit from Promega give the most optimal results out of the seven methods tested. Lastly, we confirm the use of N1 and N2 assays developed by the CDC (USA) as the best performing assays among four tested in combination with Fast Virus 1-mastermix. Data show a realistic overall process for method implementation as well as provide valuable information in regards to how different approaches in the analysis compare to one another under the specific conditions present in Slovenia during a pilot monitoring running from the beginning of the pandemic.

Development and Validation of a New TaqMan Real-Time PCR for Detection of 'Candidatus Phytoplasma pruni'.

Phytoplasmas of the 16SrIII group are wide spread, and have a broad plant host range. Among these, 'Candidatus phytoplasma pruni' ('Ca. P. pruni'; phytoplasmas of 16SrIII subgroup A) can cause serious diseases in Prunus species and 'Ca. P. pruni'-related strains can infect other plant species, including grapevines. In this study, a new real-time PCR detection system was developed for 'Ca. P. pruni' using TaqMan chemistry. This test was designed to detect 'Ca. P. pruni', by amplifying the species-specific secY gene. In addition, a test to amplify the group-specific 16S rRNA gene region was also developed. The performances of both tests were evaluated. The test that amplifies the secY gene provided reliable and quick detection of 'Ca. P. pruni'. Using the newly developed and validated test, 'Ca. P. pruni' was not found in any of the 434 field samples collected from different plants species grown in different regions of Slovenia.

Viral Fitness Determines the Magnitude of Transcriptomic and Epigenomic Reprograming of Defense Responses in Plants.

Although epigenetic factors may influence the expression of defense genes in plants, their role in antiviral responses and the impact of viral adaptation and evolution in shaping these interactions are still poorly explored. We used two isolates of turnip mosaic potyvirus with varying degrees of adaptation to Arabidopsis thaliana to address these issues. One of the isolates was experimentally evolved in the plant and presented increased load and virulence relative to the ancestral isolate. The magnitude of the transcriptomic responses was larger for the evolved isolate and indicated a role of innate immunity systems triggered by molecular patterns and effectors in the infection process. Several transposable elements located in different chromatin contexts and epigenetic-related genes were also affected. Correspondingly, mutant plants having loss or gain of repressive marks were, respectively, more tolerant and susceptible to turnip mosaic potyvirus, with a more efficient response against the ancestral isolate. In wild-type plants, both isolates induced similar levels of cytosine methylation changes, including in and around transposable elements and stress-related genes. Results collectively suggested that apart from RNA silencing and basal immunity systems, DNA methylation and histone modification pathways may also be required for mounting proper antiviral defenses and that the effectiveness of this type of regulation strongly depends on the degree of viral adaptation to the host.