ISOLATION AND CHARACTERIZATION OF DSRNA FROM GRAPEVINES SHOWING SYMPTOMS OF RUPESTRIS STEM PITTING DEASEASE AND DETECTION OF DSRNA FROM UNCINULA NECATOR (SCHW.) BURR., THE POWDERY MILDEW PATHOGEN

Ossmat  I.  AZZAM

In the United States, rupestris stem pitting (rSP), a graft transmissible disease, was first recognized by Austin Goheen at the University of California, Davis in 1976. The disease has not been mechanically transmitted, and it is identified only by its reaction (stem pitting) on inoculated vitis rupestris cv St. George. The causal agent of rSP is as yet undetermine; however, a virus or virus‑like pathogen is suspected of being involved . The etiology of rSP was examined using a dSRNA analysis technique. In this study, I showed the presence of two dsRNA species (B and C) of molecular weight (MW) 5.3 and 4.4 x 10⁶ in the stem phloem tissue of one positive and one negative rSP‑indexed vines in New York. However, species B and C were consistently detected in rSP positive vines from California and Canada. The dsRNA nature of these species was confirmed by DNase and RNase treatments. I concluded that species B and C are most likely associated with rSP based on dsRNA results from California and Canada. In addition, I detected eight different dsRNA species (species A, D-F of MW 0.95 ‑ 6.3 x 10⁶ ) in the grape leaf samples collected from New York and which were indexed positive and negative on St. George for rSP. In this study I showed that these species are not associated with the disease. Instead they are associated with the grape powdery mildew pathogen, Uncinula necator (Schw.) Burr. Isometric and rod‑like particles were seen in an extract from a single cleistothecium under transmission electron microscopy.

An attempt was made to clone the rSP‑associated dsRNA species B and C that were isolated from agarose gels, and 62 clones containing inserts ranging in size from 200‑1200 bp were identified. However, Northern hybridization showed that none of the tested clones hybridized with the denatured rSP‑associated dsRNA species. Instead, they hybridized to contaminating low molecular weight SsRNA species. Therefore, I evaluated different dsRNA isolation techniques that might improve the quality and quantity of dsRNA extracted from grape tissue for cloning and labeling purposes.

An attempt was made to clone the rSP‑associated dsRNA species B and C that were isolated from agarose gels, and 62 clones containing inserts ranging in size from 200‑1200 bp were identified. However, Northern hybridization showed that none of the tested clones hybridized with the denatured rSP‑associated dsRNA species. Instead, they hybridized to contaminating low molecular weight SsRNA species. Therefore, I evaluated different dsRNA isolation techniques that might improve the quality and quantity of dsRNA extracted from grape tissue for cloning and labeling purposes