Spread of potato virus Y in potato cultivars (Solanum tuberosum L.) with different levels of sensitivity

The aim of this work was to correlate the appearance of the symptoms, multiplication and spread of virus after mechanical inoculation of potato (Solanum tuberosum L.) cultivars showing different levels of susceptibility and sensitivity to Potato virus Y^NTN (PVY^NTN). The potato cultivars used were the resistant cultivar Sante and susceptible cultivars Igor, Pentland squire and Désirée. The spread of the virus PVY^NTN in infected plants was monitored using different methods: DAS-ELISA, tissue printing, immuno-serological electron microscopy and real-time PCR. In all three susceptible cultivars, the virus was detected in the inoculated leaves 4–5 days after inoculation. From there virus spread rapidly, first into the stem, then more or less simultaneously to the upper leaves and roots. Real-time PCR was shown to be very sensitive and enabled viral RNA to be detected in non-inoculated leaves of susceptible cultivar Igor earlier than other methods. Therefore, for exact studies of plant–virus interaction, a combination of methods which detect viruses on the basis of their different properties (coat protein, morphology or RNA) should be used to monitor the spread of viruses.     

Real-time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

A new real-time PCR detection system was developed for grapevine yellows (GY) using TaqMan minor groove binder probes and including two amplicons for group-specific detection of Flavescence dorée (FD) and Bois noir (BN) phytoplasmas, plus a universal phytoplasma amplicon. FD and BN amplicons were designed to amplify species-specific genomic DNA fragments and the universal amplicon to amplify the 16S ribosomal DNA region. Efficiency of PCR amplification, limit of detection, range of linearity and dynamic range were assessed for all three amplicons. The specificity of detection systems was tested on several other isolates of phytoplasmas and bacteria and on healthy field grapevine and insect samples. No cross-reactivity with other phytoplasma strains, plant or insect DNA was detected. The assay was compared with conventional PCR on more than 150 field grapevine, insect and field bindweed samples. Real-time PCR showed higher sensitivity as phytoplasmas were detected in several PCR-negative and in all PCR-positive samples. A data-mining analysis of results from both detection approaches also favoured real-time PCR over conventional PCR diagnostics. The developed procedure for detection of phytoplasmas in grapevine also included amplification of plant DNA co-extracted with phytoplasmic DNA, providing additional quality control for the DNA extraction and PCR amplification for each sample. The newly developed assay is a reliable, specific and sensitive method easily applicable to high-throughput diagnosis of GY.     

Distribution of Potato virus Y in potato plant organs, tissues, and cells

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.     

LAMP assay and rapid sample preparation method for on‐site detection of flavescence dorée phytoplasma in grapevine

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.     

QBOL: a new EU project focusing on DNA barcoding of Quarantine organisms

In 2009 a new three year EU funded project (QBOL) started on DNA barcoding of important plant pests. An international consortium of 20 partners (universities, research institutes, and phytosanitary organizations) from around the world, coordinated by Plant Research International (Wageningen, the Netherlands), will collect DNA barcodes from many plant pathogenic quarantine organisms, store these sequences in a database accessible over the internet, develop a DNA bank and train end-users. All these activities should help National Plant Protection Services in the correct identification and detection of plant pathogenic quarantine organisms.     

Improved fireblight diagnostics using quantitative real-time PCR detection of Erwinia amylovora chromosomal DNA

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora ( ams C gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick™), the real-time PCR assays reliably detected at least 10³ cells mL^−l ( c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.     

Induced expression of sucrose synthase and alcohol dehydrogenase I genes in phytoplasma-infected grapevine plants grown in the field

Interaction between phytoplasma and grapevine at the physiological level is still poorly understood, as are plant defence mechanisms against the pathogen. This study investigates the level of gene expression of three selected genes in a large number of grapevine plants belonging to six disease/cultivar groups (healthy Chardonnay, Bois noir-infected Chardonnay, Flavescence dorée-infected Barbera and Prosecco, and recovered Barbera and Prosecco). All plants were grown in vineyards in uncontrolled conditions in order to represent the physiology of disease as accurately as possible. Sucrose synthase was significantly upregulated in infected plants of all cultivars with the lowest P -values in cvs Chardonnay and Prosecco ( P  < 0·001) and median fold-change around 2. This clearly indicates that carbohydrate metabolism changed in infected compared to healthy or recovered plants. Alcohol dehydrogenase I was significantly upregulated in infected relative to healthy Chardonnay plants ( P  < 0·05) indicating that alcoholic fermentation, a sign of hypoxic conditions, was induced in infected plants. Heat shock protein 70 was upregulated in infected compared to recovered plants only in cv. Prosecco. Linear discriminant analysis showed that classification of samples into disease status groups based on gene expression was highly accurate (82%), indicating that the response of field-grown plants to phytoplasma infection at the level of expression of selected genes was so intensive and uniform that it was possible to detect it in grapevine plants regardless of natural variables.     

Phytoplasma infection may affect morphology,regeneration and pyrethrin content in pyrethrum shoot culture

The aim of the work was to infect pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz-Bip.), an important secondary metabolite pyrethrin producing plant, with MA phytoplasma (16S rRNA group III, subgroup B) to initiate tissue culture of phytoplasma-infected plants, and to examine possible effects of infection on pyrethrin content, morphology, and regenerative capacity in shoot culture.     

Detection and quantification of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in irrigation waters

A quantitative RT real-time PCR method was developed for the detection and quantification of Tomato mosaic virus (ToMV) in irrigation waters. These have rarely been monitored for the presence of plant pathogenic viruses, mostly due to the lack of efficient and sensitive detection methods. The newly developed method presented here offers a novel approach in monitoring the health status of environmental waters. ToMV was reliably detected at as low as 12 viral particles per real-time PCR reaction, which corresponds to the initial concentration of approximately 4.2 × 10^?10 mg (6,300 viral particles) of ToMV per ml of sample. The sensitivity of the method was further improved by including the Convective Interaction Media^® (CIM) monolithic chromatographic columns for quick and efficient concentration of original water samples. Seven out of nine water sources from different locations in Slovenia tested positive for ToMV, after concentrating the sample. Four samples tested ToMV-positive without the concentrating procedure. The presence and integrity of infective ToMV particles in the original sample, as well as in the chromatographic fraction, was confirmed using different methods from test plants, DAS ELISA to electron microscopy and real-time PCR. In this study, we propose a unique and simple diagnostic scheme for rapid, efficient, and sensitive monitoring of irrigation waters that could also be adopted for other plant, human or animal viruses.