Detection of Australian grapevine viroid in a grapevine more than 100 years old in Xinjiang, China

Australian grapevine viroid (AGVd) was first described in Australia. It contains the entire central conserved region of the apple scar skin viroid group and is a member of the genus Apscaviroid, family Pospiviroidae (Rezaian, 1990). It appears to have originated from extensive RNA recombination involving Grapevine yellow speckle viroid (GYSVd), Citrus exocortis viroid (CEVd), Apple scar skin viroid (ASSVd), and has only been isolated from grapevines, which are frequently also infected by a mixture of Hop stunt viroid (HSVd), GYSVd 1 and 2, CEVd (Rezaian, 1990; Elleuch et al., 2002). To investigate the distribution of AGVd in grapevine in China, in September 2005, 70 grapevine leaf samples were collected from different parts of China. This included one from Turpan, in Xinjiang autonomous region, from a plant of the variety Thompson Seedless, which was more than 100 years old and has never been grafted. Nucleic acids were extracted and tested by northern hybridisation using digoxygenin-labeled riboprobes for Apple fruit crinkle viroid (AFCVd), which has ca. 85% sequence similarity with AGVd (Sano et al, 2004). In these tests only the ‘Thompson Seedless’ sample from Turpan was positive; all the other samples were negative. This positive result was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using the primers F1 5’- TTGGATCCTGGGCACCAACTAGAGGT-3’and R2 5’- TTGGATCCGGGCCTCCAAACAGGGAG-3’. F1 and R2 both contain sites for the restriction endonuclease Bam HI. The amplified products were cloned into the pGEM-T plasmid (Promega, Madison, USA), selected recombinant plasmids were digested with Bam HI and 16 clones were sequenced. The sequence in the primer positions was obtained by direct sequencing of fresh PCR products obtained using a second pair of primers, F3 5’-TTGGATCCTCCAGCGGAGGACTGAAG-3’ and R4 5’-TTGGATCCGGACCCCTAGC GTTCCTG-3’. Eight different sequences were obtained from 16 recombinant plasmids (GenBank Accession Nos DQ362908-15); indicating that AGVd is a quasispecies which contain many slight sequence variants with high overall sequence homology. While not as common in grapevine as HSVd and GYSVd (Li et al., 2006), this report proves that AGVd occurs in China. This is only the third sequence report of ASGVd after Australia and Tunisia (Rezaian, 1990; Elleuch et al., 2002).