RT-PCR DETECTION OF ASPARAGUS 2 ILARVIRUS

Asparagus 2 ilarvirus (AV-2) is a major viral disease of asparagus that is transmitted through seed, sap and pollen. Therefore it is important for breeders to produce seed that is free of AV-2. In order to develop a more sensitive test for detection of AV-2, we designed PCR primers to amplify the coat protein gene and 3’ untranslated region of RNA3 of AV-2. RT-PCR produced a fragment of the expected size from template RNA isolated from a plant material known to be infected with AV-2. Cloning and sequencing showed that the RT-PCR product was more than 99% identical to the published sequence of RNA3 of AV-2. Double-stranded RNA analysis of asparagus samples revealed dsRNA molecules differing in size from those expected for AV-2 or other viruses known to infect asparagus in California. AV-2 could be detected by Northern blot analysis of dsRNA gels using the cloned RT-PCR product as probe.