Additional Factors Affecting dsRNA Analysis of Citrus Tristeza Virus

We have investigated factors that may have an effect on dsRNA analysis of citrus tristeza virus (CTV). Storage of bark tissue and purified dsRNA at -20 C for up to at least 4 yr and repeated freezing and thawing of each of them appeared to have no detrimental effect on the quality of dsRNA detected by polyacrylamide gel eletrophoresis. The aqueous phase following phenol extraction could be stored at room temperature or at 4 C with or without ethanol (adjusted to 16.5%) for at least 2 weeks. Buffer saturated phenol which has been kept at room temperature for 2.5 yr was still useful for CTV ds RNA extraction. DsRNA extraction could be done without adding nuclease inhibitors, i.e. bentonite or macaloid. Precipitated dsRNA could be recovered by centrifugation immediately after addition of ethanol and sodium acetate. Hosts which yielded good dsRNAs were Pineapple, Navel, and Valencia sweet orange and the scions of Madam Vinous sweet orange on sour orange rootstock. Shoots from the sour orange rootstock gave poor results. Green bark tissue from the older part of the current shoot usually gave a better result than tissue from the young tender shoot tip and the old brown stem from previous flushes. DsRNA results were least acceptable in the summer from various well infected screenhouse grown citrus species. Citrus virologists have attempted to develop several techniques for the identification of strains of citrus tristeza virus (CTV) and these may now be used to complement established methods which rely on biological indexing in seedlings of C i t m s spp. (9). The newer methods include the use of monoclonal antibodies (10,15,20), nucleic acid hybridization probes (6,7,16, 17,18), and double-stranded (ds) RNA analysis (3, 4, 5, 6, 8, 13). DsRNA analysis has been used to discriminate between multiple strains since each is often clearly distinguished from all others. The other techniques are better suited for grouping strains into broad categories based on their reactivity or not with a single reagent. Correlations with biological properties have been obtained with all methods, but much more work will be needed before they can be used with confidence to type any strain for virulence without resort to biological indexing. Dodds et al. (6,8) and Lee (13) have demonstrated the value of dsRNA analysis for CTV detection, diagnosis and for identification of strains. In subsequent studies, the value of the technique for investigating host-virus interactions including host passage effects, seasonal variation, and field surveys has been demonstrated (5,7,11). In the course of doing these various experiments we have gained additional insight into factors that need or do not need critical attention in order to obtain reliable results. We have also had numerous requests to provide information and share our experiences in order to solve problems with dsRNA analysis. This article summarizes several experiments which were designed to answer questions about dsRNA analysis of CTV using cellulose chromatography purification and polyacrylamide gel electrophoresis for analysis. Some of the experiments (PAGE) selected for inclusion in this report will be of special interest to those with limited resources who would like to adopt the technique or simplify the process while reducing overall costs without sacrificing the quality of the results. In addition, experiments which are extensions or continuations of previous studies (more strains, additional hosts, other growth conditions, etc.) are also