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Grapevine leafroll-associated virus 3 (GLRaV-3; Ampelovirus, Closteroviridae) isolates from Galicia in northwestern Spain were selected to characterize their genetic diversity according to different factors (age, origin, location, variety, etc.). The vines belonged either to local white and red varieties autochthonous to Galicia or to varieties from other Spanish regions but widely used in Galicia. These GLRaV-3 isolates came from different vineyards in Galicia located in coastal or inner areas. Multiplex RT-PCR allowed the detection of isolates belonging to groups I, II, III-V and VI. Two genomic regions were studied in the isolates, the HSP70h and the capsid protein, using specific primers that allow the detection of variants from groups I to V. Some possible recombinants could be detected; however, multiple infections with different variants indicated that they were not genuine recombinants. No differences were found in the population structure considering variety or geographical factors. Isolates belonging to four groups were found in the distinct areas surveyed: groups I and II were the most common, followed by groups VI and III, as is the case in the rest of the world. In the same surveys, the presence of insect vectors for GLRaV-3 was investigated and found lacking in inland areas but present in those with milder climate. Genetic analysis did not support isolation of the GLRaV-3 isolates in Galicia, suggesting that the uncontrolled exchange of infected vines and/or rootstocks has been a major agent of virus spread.

Grapevine leafroll-associated virus 3 (GLRaV-3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV-3; however, there is no PCR-based assay available to quantify virus populations within plant tissues. A real-time RT-PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV-3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA-dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV-3 in field-grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV-3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV-3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV-3 diagnosis, disease monitoring and population ecology studies.

Multiple detection of the phytoplasmas associated with Flavescence doree (FD) and Bois noir (BN) diseases and of the viruses Grapevine leafroll associated virus -1 and -3 (Ampelovirus) and Grapevine virus A (Vitivirus) is described, using the same crude extract as template. Sap was prepared by semi-automatic maceration requiring minimal time and effort, consisting of a tissue grinding step in carbonate buffer and a boiling step in glycine buffer; two microlitres were used as template in each pathogen-specific assay. RealTime reverse transcription (RT)-PCR for FD phytoplasma detection was found to be five orders of magnitude more sensitive than the RT-PCR method described previously. However, the RealTime RT-PCR assay for the detection of BN phytoplasma needed a nested step to achieve high sensitivity, suggesting low concentration of template in the host. The viruses were detected by RealTime nested-PCR, which was more sensitive than the ELISA and RealTime RT-PCR assays previously described. The methods presented here have been successfully used to monitor infections in field and nursery samples during the 2008 grapevine growing season.

The transmission of two closteroviruses associated with grapevine leafroll, GLRaV-1 and GLRaV-3, from grapevine to grapevine by the mealybugs, Pseudococcus longispinus and P. calceolariae (Homoptera: Pseudococcidae) was studied. Controlled transmission experiments using the first and third instars of each insect were conducted twice during the 1993–94 growing season to investigate the consequence of virus accumulation within the donor vine leaf tissue on the incidence of virus transmission to healthy recipient vines. Transmission of GLRaV-1 and GLRaV-3 was determined by ELISA testing recipient vines in July 1994 and March 1995. GLRaV-3 was transmitted to recipient vines by P. longispinus and P. calceolariae first instars only. An increase in virus titre within the season did not significantly alter the transmission rate of GLRaV-3 by either P. longispinus or P. calceolariae first instars. P. longispinus and P. calceolariae failed to transmit GLRaV-1 to recipient vines.