Comparative Effectiveness of ELISA and RT-PCR for Detecting Grapevine Leafroll-Associated Closterovirus-3 in Field Samples

Abstract

We compared the effectiveness of enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) in detecting grapevine leafroll associated virus-3 (GLRaV-3) in infected symptomatic Pinot noir and nonsymptomatic hybrid grapevines throughout the growing season. Tissue samples from leaves, petioles, flowers, fruits, and bark scrapings of canes were tested by nested immunocapture reverse transcriptase PCR (N-IC-PCR) and by double antibody sandwich ELISA. PCR primers were selected from the 55K (heat shock protein 90 homolog) gene region of the GLRaV-3 genome. In symptomatic and nonsymptomatic vines, ELISA and N-IC-PCR detected GLRaV-3 in all samples of bark scrapings of canes that were collected throughout the growing season and from dormant cuttings in storage. Detection of GLRaV-3 in flowers or fruits of symptomatic and nonsymptomatic vines was generally erratic throughout the growing season, regardless of the detection technique. ELISA and N-IC-PCR were similarly effective for detecting GLRaV-3 in petioles of nonsymptomatic and symptomatic vines, with nearly all tested samples being positive from the developing berries stage onward. With lamina of leaves collected from the apical, middle, and basal positions on canes of test vines, GLRaV-3 was detected in many more samples from symptomatic vines than from nonsymptomatic vines. In symptomatic vines, N-IC-PCR and ELISA detected GLRaV-3 in nearly all lamina samples of basal leaves collected throughout the growing season and from the pea-sized berry stage onward, respectively. Both methods detected GLRaV-3 in all lamina samples starting from the veraison stage. In contrast, detection of GLRaV-3 in nonsymptomatic vines was erratic regardless of the detection method that was used. For example, N-IC-PCR detected GLRaV-3 in 100% of samples of middle leaves collected at the pea-sized berry stage, 20% at the veraison stage, and 65% at the berry ripening stage, whereas GLRaV-3 was detected in all similarly collected samples of symptomatic vines. Based on the results, we recommend that bark scrapings of canes from vines or from dormant cuttings be used for reliable detection of GLRaV-3 by ELISA or N-IC-PCR.