No abstract

No abstract

Reverse transcription polymerase chain reaction (RT‐PCR) was used for the detection of Citrus exocortis viroid (CEVd) in total nucleic acids from infected citrus indicator hosts or directly from field trees. Grapevine yellow speckle viroid‐1 (GYSVd‐1) and Australian grapevine viroid (AGVd) were also detected by this technique in total nucleic acids from infected grapevine plants. Three protocols for nucleic acid extraction were tested: a phenol‐based method, a protocol using a commercial reagent (Trizol) and a method in which extraction with a high‐salt buffer was combined with partial purification using non‐ionic cellulose. The third protocol, coupled to RT‐PCR, seems to be the most suitable, allowing detection of the three viroids in Tunisian citrus and grapevine samples. This method should be used in a testing programme to produce certified viroid‐free planting material.

Hop stunt viroid (HSVd) variants from cachexia symptomatic citrus tree were subjected to retrotranscription and DNA amplification (RT-PCR), cloning and sequencing. Here we report genetic diversity and phylogenetic analysis of HSVd Tunisian isolate. Our study revealed obvious polymorphism within Tunisian isolates and high similarity with Japanese variants. Neighbor-joining analysis was carried out on the new HSVd-citrus sequences together with 44 previously described HSVd isolates from citrus and one from grapevine. The phylogenetic analysis showed that Tunisian isolates were clustered into 4 different groups (CVd-IIa, b, and c and grapevine group). Furthermore, the predicted secondary structure was scrutinized to be more understanding on how the nucleotide change affects variable (V) and pathogenicity domain (P).