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Waterborne and seedborne Pepino mosaic virus (Pep MV) and Potato spindle tuber viroid ( PSTVd) pose serious threats to tomato production due to seed transmission and mechanical transmission, coupled with their long-term stability outside the host plant. Therefore, rapid and sensitive diagnostic procedures are needed to prevent the spread of these quarantine pathogens. In particular, water and seed contamination are difficult to detect and confirm without efficient concentration methods. This study presents procedures that improve detection of PSTVd from tomato seeds and leaf tissue, and Pep MV from water and tomato leaf tissue. For efficient concentration of Pep MV from water samples, a procedure was optimized using convective interaction media monolithic chromatography columns, which provides concentration by three orders of magnitude. For concentration of PSTVd from seed extracts, an easy-to-use and efficient method was developed based on RNA binding to positively charged anion-exchange resin beads that provides up to 100-fold more sensitive detection in comparison with procedures without a concentration step. This thus allows confirmation of RT- qPCR results with sequencing of RT- PCR products in samples with low viroid levels. In addition, reverse-transcription loop-mediated isothermal amplification assays for detection of PSTVd and Pep MV were optimized and adapted to both laboratory and on-site testing requirements. This allows rapid detection of these pathogens in crude leaf homogenates, in under 30 min. These procedures of concentration and detection are shown to be efficient and to fill the gaps in diagnostics of Pep MV and PSTVd, especially when these pathogens are present at low levels in difficult matrices such as water and seeds. [ABSTRACT FROM AUTHOR]

In Europe the most devastating phytoplasma associated with grapevine yellows ( GY) diseases is a quarantine pest, flavescence dorée ( FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive. [ABSTRACT FROM AUTHOR]

Differences in the early responses of two potato cultivars, Igor and Nadine, to two isolates of Potato virus Y (PVY), the aggressive PVY and the mild PVY, were monitored. Microarray and quantitative real-time PCR analyses were carried out to identify differentially expressed genes after inoculation with each virus isolate. Additionally, symptom severity and development was observed and the amount of virus isolate accumulated in systemically infected leaves was evaluated, where a significantly higher amount of PVY was detected. Microarray analysis revealed 572, 1288 and 1706 differentially expressed genes at 0·5, 12 and 48 h post-inoculation, respectively in cv. Igor, with a similar pattern observed in cv. Nadine. Microarray and quantitative real-time PCR results implied an earlier accumulation of sugars and lower photosynthesis in leaves inoculated with the aggressive isolate than in leaves inoculated with the mild isolate. The PVY isolate did not activate early differential expression of the Fe-superoxide dismutase and pectin methylesterase inhibitor (PMEI) genes, indicating a delay in plant response relative to that following PVY inoculation. Differences in the expression of the β-glucanase-I gene were also observed in early plant responses to inoculation with each virus isolate. [ABSTRACT FROM AUTHOR]

Each season, crops face simultaneous or successive multiple infections. Yet cultivar resistance is often bred against one pest at a time, and screened under single infections. This raises the question of resistance expression bred under mono‐infections when exposed to multiple infections. We tackled this question by measuring the level of quantitative resistance of potato cultivars to either Globodera pallida or Phytophthora infestans under single or multiple infections, using potato virus Y (PVY) or Rhizoctonia solani as the co‐infector. Overall, co‐infection resulted in less multiplication of the target pathogen compared to single infection, although this reduction did not always reach statistical significance. A preliminary assessment of defence gene expression in plants infected with P. infestans alone or together with PVY or R. solani suggested that P. infestans repressed defence genes previously triggered by PVY, but boosted the expression of these genes when primed by R. solani. Together, these data support the view that resistance expression is impacted by co‐infection, and that it should be screened under multiple infection situations to be best assessed and exploited as a component of Integrated Pest Management strategies. [ABSTRACT FROM AUTHOR]

Accurate management practices in crop health and food safety are critical, especially regarding the detection of plant pathogens in the early stages of a disease. To date, specific, fast and sensitive technologies for point‐of‐care diagnosis and simple or grower‐friendly devices are very valuable, as no specialized staff are required for diagnosing a disease in the field. This is especially the case today, when factors such as climate change may cause the appearance of pathogens in areas where years ago they were unexpected. The aim of this research is to review some of the promising techniques that can be applied to in‐field molecular detection of plant pathogens and how these techniques can change the way farmers and pathologists are diagnosing plant diseases. Some of them, like loop‐mediated isothermal amplification and recombinase polymerase amplification, are already being successfully used for routine diagnosis. However, most technologies still need validation in the plant pathology field, where they have a promising future for in‐field diagnosis when combined with simple DNA extraction methods, reagent stabilization techniques and their integration into portable devices. [ABSTRACT FROM AUTHOR]

Virus‐induced systemic necrosis accounts for great yield losses in potato (Solanum tuberosum) cultivars susceptible to the necrotic strain of potato virus Y (PVYNTN). To extend understanding of systemic necrosis, the tissue of healthy and PVYNTN‐infected potato tubers (necrotic and non‐necrotic) during storage was profiled for host gene expression using microarrays while virus concentration was determined using RT‐qPCR. Comparison of non‐necrotic tissue types revealed minor transcriptional differences, while tuber necrosis was characterized by high virus concentration and pronounced expression of defence response‐associated genes. Creation of programmed cell death‐associated gene ontology enabled assessment of expression and regulation of relevant differentially expressed genes implicated in the execution of plant programmed cell death. Vacuolar cell death and unfolded protein response to viral infection were implicated in the necrotic tuber tissue by transcriptional induction of genes possessing caspase‐like activity and genes coding for endoplasmic reticulum chaperones and folding proteins. A meta‐analysis, including all relevant publicly available gene expression datasets differing in genotype, pathogen interaction type and potato organ, revealed common defence responses associated with cell wall fortification, oxidative and biotic stress. The response in necrotic tissues of susceptible tubers resembled the hypersensitive response of resistant primary infected plants, but with hormonal signalling as an important differentiating factor. This study will be useful for in‐depth analysis of programmed cell death regulation in plants by providing gene ontology. It specifically addresses the issue of systemic necrosis formation in PVYNTN‐infected potato tubers during storage. [ABSTRACT FROM AUTHOR]