UBC Theses and Dissertations
Detection of nepoviruses by ELISA in tissue-cultured and field-grown grapevines Johnson, Raymond Camille Joseph
The detection by serology of nematode-transmitted polyhedral viruses (nepoviruses) in grapevines is often unreliable. Nepoviruses were detected by enzyme-linked immunosorbent assay (ELISA) in tissue-cultured and field-grown grapevines. Nepovirus detection in in vitro plants (plantlets) was affected by virus distribution and growth room temperature. The reliability of virus detection in field-grown grapevines was improved when modified grinding buffers were used. Arabis mosaic virus (AMV) was detected by ELISA, for the first time, in in vitro grapevines initiated from field-and screenhouse-grown plants throughout the summer. The virus was not reliably and repeatedly detected in in vitro plantlets grown at 25°C. AMV and grapevine fanleaf virus (GFLV) distribution was not uniform throughout the plantlets. This distribution was affected by the culture room temperature. The best plant parts to sample for virus detection came from the zones of rapidly proliferating shoots. The viruses were sometimes not detected in samples taken from other tissues. Growth room temperature had an important effect on virus detection in plantlets. The highest virus titres were found in plants growing at 15°C. Temperature increases in 5°C steps to 30°C reduced virus titre. AMV became undetectable in nearly all plantlets growing at 30°C for as little as 30 days. Growth at 30°C reduced ELISA absorbance values by 76% after 8 days and after 21 days the values were at 4% of pre-treatment levels. The virus titre dropped below detectable levels in most plantlets. AMV could not be detected in plantlets or rooted explants after being placed in a 30°C treatment for 2 months. Tomato ringspot virus was detected by ELISA, for the first time, in in vitro grapevine plants. The virus was repeatedly detected in in vitro plants growing at 20°C. Under the typical summer conditions experienced at Sidney, B.C., modifying the standard ELISA grinding buffer (0.01 M phosphate buffered saline, pH 7.4, 0.05% Tween-20, 0.2% ovalbumin, 2% polyvinylpyrrolidone) was essential for reliable detection of AMV or GFLV. AMV was reliably detected by ELISA in foliar samples from field or screenhouse plants throughout the summer when the grinding buffer was modified by increasing the pH to at least 8.2 and adding 1% nicotine or 0.15 M phosphate buffered saline. The most reliable results with GFLV were obtained with the nicotine enhanced buffers. In comparison, because of the increased workload associated with growing plants in vitro and the unreliable detection of viruses in these plants, it remained preferable to detect nepoviruses in field plants by ELISA.
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