Filling the gaps in diagnostics of Pepino mosaic virus and Potato spindle tuber viroid in water and tomato seeds and leaves
| Autor: | Nataša Mehle, Maja Ravnikar, Tjaša Jakomin, Petra Kramberger, Nejc Rački, Ion Gutiérrez-Aguirre, Polona Kogovšek |
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| Rok vydání: | 2017 |
| Předmět: |
0106 biological sciences
0301 basic medicine Mechanical transmission biology Viroid Loop-mediated isothermal amplification food and beverages Plant Science Horticulture Contamination biology.organism_classification 01 natural sciences 03 medical and health sciences 030104 developmental biology Plant virus Seed contamination Botany Genetics Pepino mosaic virus Agronomy and Crop Science Potato spindle tuber viroid 010606 plant biology & botany |
| Zdroj: | Plant Pathology. 66:1191-1201 |
| ISSN: | 0032-0862 |
| Popis: | Waterborne and seedborne Pepino mosaic virus (PepMV) 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 PepMV from water and tomato leaf tissue. For efficient concentration of PepMV 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 PepMV 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 PepMV and PSTVd, especially when these pathogens are present at low levels in difficult matrices such as water and seeds. |
| Databáze: | OpenAIRE |
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