RT-PCR Based Detection of Rupestris stem pitting associated virus Within Field

ABSTRACT
Stewart, S., and Nassuth, A. 2001. RT-PCR based detection of Rupestris stem pitting associated virus within field-grown grapevines throughout the year. Plant Dis. 85:617-620.
The presence of Rupestris stem pitting associated virus (RSPaV) can go unnoticed since symptoms appear only if additional viruses are present. Detection by reverse transcription–polymerase chain reaction (RT-PCR) is possible; however, this assay could be unreliable if the tissue that is being tested has detection-interfering compounds, or if the virus has a low titer.
This paper reports on (i) use of a recently developed extraction method and internal control to determine which tissues from field-grown grapevines yield extracts that are reliable for virus detection by RT-PCR, and (ii) a survey for RSPaV of different tissues from the Vitis vinifera varieties Riesling, Chardonnay, Cabernet Franc, Merlot, Sauvignon Blanc, Pinot Noir, and Gamay,as well as from the rootstocks 3309 and Riparia, which were harvested in Ontario, Canada,
at different times of the year. Amplifiable extracts were obtained from virtually all bud, shoot tip, seed, and cane samples tested. Detectable amounts of RSPaV were generally found in all tissues of infected plants except young buds collected in the summer. A combination of three single buds from dormant canes, less time-consuming than the preparation of cane shavings,was a reliable source for RSPaV detection.
Additional keywords: Rupestris stem pitting, virus distribution Rupestris stem pitting, corky bark,Kober stem grooving, and LN33 stem grooving, collectively referred to as Rugose wood, are a group of grafttransmitted diseases of grapevines characterized by distortions of the woody trunk which can be found in all viticultural regions of the world (5). These diseases are responsible for graft incompatibility problems,
delayed budburst, severe decline, and even death of vines (2,8). A combination of viruses and possibly phytoplasmas is
thought to be the causative agent for the Rugose wood diseases (2). However, exactly which combinations of viruses and phytoplasmas are present in diseased plants, and which combinations of those pathogens cause which symptoms still need to be determined.
Rupestris stem pitting (RSP) appears to be the most widespread disease of the Rugose wood complex (10). Rupestris
stem pitting associated virus (RSPaV), a foveavirus, is consistently detected in RSPinfected grapevines (9,22); however,RSPaV alone generally does not produce any symptoms and has no major impact on grapevine growth and yield (2,15). The presence of another virus, such as Grapevine virus A (GVA), Grapevine fleck virus,or Grapevine leafroll associated virus-1, in addition to RSPaV, is required for Rugose wood-type symptoms to occur (2). These
viruses can be transmitted from infected vines to an RSPaV-infected vine via grafting (2,8) or, at least for GVA, via the vine mealybug Planococcus ficus (3,11). This means that cultivating symptomless but RSPaV-infected vines carries a risk for the development of RSP, and it is therefore of interest to know if a vine contains RSPaV.
The presence of RSPaV can be determined by reverse transcription–polymerase chain reaction (RT-PCR) using primers based on its genomic sequence (9,22) with apparent greater reliability than biological indexing on St. George (9). It is not known if the RT-PCR assay is reliable for all tissues and at all times of the year. Grapevines contain high amounts of compounds such as phenolics and polysaccharides, believed to interfere with RT-PCR (11,16,17,21), and it is likely that these amounts differ among different tissues at different times of the year. Also, several grapevine viruses appear to have an irregular distribution and/or low virus titer at certain times of the year (11–13,17,19,20), and such may also be the case for RSPaV.As a result, it is possible that its presence is not detected even though a plant is infected.……………………………

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