Etiology and Detection of the Cause of Syrah Decline in Syrah grapevine

Syrah decline disease is a well documented problem in California and France. It is characterized by swelling and cracking of the graft union, stem pitting and grooving, and premature leaf reddening. French scientist have been studying this problem since 1999 and have failed to find any correlation with genetic incompatibility, known pathogens (viruses, bacteria, fungi, viroids), or environmental conditions. The potential of the Syrah decline disease to be associated with a virus is suspicious and the symptoms observed support the hypothesis. In a search for viruses associated with decline symptoms of Syrah grapevines, we have undertaken an analysis of total plant RNA sequences using dsRNA as template and the Life Sciences 454 high-throughput sequencing. In phase 1 (year 1) of this project we selected FPS Syrah clone 6 which was showing sever pitting, grooving and wood necrosis on the woody cylinder including die back and declining. For control a healthy looking Syrah clone 8 was selected. The Life Science 454 sequencer produced 67.5 megabases of quality sequence data, and screened for sequences of viral or viroid origin. The data revealed that the Syrah clone 6 supported a mixed infection that included seven different RNA genomes including 4 viruses and 3 viroids. In the second phase (year 2) of the project, 5 more syrah clones were selected and dsRNAs were produced and sequenced by the high-throughput sequencing facilities. The clones selected included three which are reported to produce high incidence of Syrah decline syndrome (clons B0 and B1 obtained from France and clone 99) and 2 with moderate incidence (clones 525 and 877). Total of 76.6 megabases of sequence information, from 371,906 fragment reads (each approximately 200 bases long) were initially produced from these five source vines in which 354,441 were high quality reads. Among high quality reads, 253,867 showed sequences which identified mostly as viral nucleic acids, but plant, bacterial and fungal sequences (and unassigned sequences) were also detected. Further analysis of the remaining unassigned reads (100,574) is in progress. Up to date segments of RNA sequences have been identified which belongs to members of two virus family, Betaflexiviridae and Tymoviridae, each includes number of viruses infecting grapevines. For example, Grapevine rupestris stem pitting associated virus, Grapevine virus A, -B, -D and -E are members of Betaflexiviridae and Grapevine fleck virus, Grapevine syrah virus-1 and Grapevine rupestris vein feathering virus are members of Tymoviridae. Research is in progress to identify the viruses present in each of these 5 Syrah clones by further analysis of the available sequences; to investigate the correlation between each virus found and the disease syndrome; and finally to develop molecular methodologies for their detection.