A cucumber mosaic virus (CMV) isolate supporting a natural 390-ribonucleotide satellite was used to reproduce under experimental conditions a disease of processing tomatoes called fruit necrosis. The virus induced incomplete differentiation of the vascular tissue of fruit stalks, which was the likely cause of the disease. On the other hand, the satellite RNA attenuated viral symptoms on tomato leaves reproducing the disease pattern typically observed in the field. The biological properties of this seemingly new variant of cucumoviral satellite RNAs were determined.
Recent technological development of molecular methods has led to the proliferation of new rapid PCR or reverse-transcriptase (RT)-PCR-derived diagnostic tests for plant viruses. Nevertheless, for routine use, the reliability of all these new methods is not widely established and there is still an apprehension to adopt them in official diagnostic for certification of plant material. This is partly because of the lack of confidence in the obtained results and the poor knowledge on the reproducibility and limits of the RT-PCR protocols. There is a lack of information on the adequate risk assessment in the use of this new technology. An interlaboratory evaluation of two RT-PCR duplex protocols for the detection of four different fruit tree viruses was performed to address these questions. Identical samples were sent as crude extract preparation to each of the participant laboratories. Samples were coded to ensure a double-blind test. General principles of result analysis are described, for example calculation of parameters such as specificity, sensitivity, repeatability, reproducibility, likelihood ratios and post-test probabilities. These parameters and the integration of the protocols within official certification scheme are discussed. Finally, guidelines for researchers desirous of validating their new plant virus diagnostic protocols through interlaboratory evaluation are suggested.
A non-radioactive dot-blot hybridization assay for the successful detection of Citrus tristeza virus (CTV) RNA in total nucleic acid extracts of infected citrus was developed. Two digoxigenin (DIG)-labelled minussense riboprobes, complementary to the coat protein gene sequence of a Chinese and an Apulian CTV isolate were synthesized. Several citrus tissues were evaluated as optimal virus source and leaf petioles were found appropriate material for reliable detection. The hybridization assay showed a detection limit corresponding to 0.2 mg of fresh infected tissue. The riboprobes allowed CTV detection in isolates from different geographical areas, grown in the screenhouse or in the field, resulting in similar hybridization patterns. The infected trees were tested during different seasons with positive results, although from July to August most of the samples gave a weaker hybridization signal, compared to other seasons. The high sensitivity and reliability of the molecular hybridization assay described make it a good alternative to serological methods for CTV detection.www.blackwell-synergy.com
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