Development of a Rapid RT-PCR Test for the Detection of Peach Latent Mosaic Viroid, Pear Blister Canker Viroid, Hop Stunt Viroid and Apple Scar Skin Viroid in Fruit Trees from Tunisia

摘要

A reverse transcription polymerase chain reaction was developed to investigate the occurrence of 'peach latent mosaic viroid' (PLMVd), 'pear blister canker viroid' (PBCVd), 'hop stunt viroid' (HSVd) and 'apple scar skin viroid' (ASSVd) on fruit trees(peach, pear, almond and apple) in Tunisia. The test was initially performed with total RNA preparations from selected isolates and then applied to total RNA preparations from leaf or bark tissues of fruit trees collected in the north of Tunisia and theSahel. PLMVd was found to occur in peach and pear trees, HSVd in pear, peach and almond trees, and PBCVd in pear trees. Mixed PBCVd-HSVd and PLMVd-HSVd infections occurred naturally in pear trees. ASSVd was not detected in any samples from apple trees. The identity of the detected viroids was confirmed by comparing their sequences with those of other previously characterized isolates. The test was then simplified by direct use of diluted crude plant extracts. The results obtained from crude sap extractsof leaves or bark tissues and from total RNA preparations were identical. This improved test is thus quick and useful for large-scale routine analysis. It can be used in a certification programme to contribute to prevention of the occurrence and spreadof PLMVd, HSVd, PBCVd and ASSVd in Tunisia.