Proceedings of the 18th Congress of ICVG, Ankara, TURKEY | 7-11 September 2015

OP 26 - Detection and characterization of Chilean isolates of grapevine viroids.

Alan Zamorano, Ximena González, Nicolás Quiroga, Nicola Fiore*
Universidad de Chile, Facultad de Ciencias Agronómicas, Departamento de Sanidad Vegetal. Santa Rosa 11315,
Santiago, Chile.
*
Corresponding author: nfiore@uchile.cl

INTRODUCTION

Grapevine is the most important fruit crop in Chile, associated with pisco, wine and table grape production. Sanitary
status has been previously reviewed in relation with viral and phytoplasmic diseases (Fiore et al., 2008; Gajardo et al.,
2009) but none of them considered viroidal diseases. Viroids are the smallest pathogens that can replicate autonomously in
plants. They are circular single stranded RNAs, with no coding sequence and completely dependent of host plant replication
machinery (Owens and Hammond, 2009). In grapevine, five viroids have been reported in different grapevine producing
regions worldwide, Grapevine yellow speckle viroid 1 and 2 (GYSVd-1, -2), Hop stunt viroid (HSVd), Australian grapevine
viroid (AGVd) and Citrus exocortis viroid (CEVd), but in South America there is not information about viroids infecting
grapevine. Thus the objective of this research was to detect and characterize the viroids present in the main grape producing
areas in Chile (Koltunow y Rezaian, 1988; Koltunow and Rezaian 1989; Rezaian, 1990; Sano et al., 2001).

MATERIALS AND METHODS

One hundred and ten samples were collected mainly among the regions Atacama (with a high planted area of table grape
vines) and Maule (known for its wide distribution of wine production). Total nucleic acid extraction was performed using silica
capture method (Mackenzie et al., 1997). Specific detection of each viroid was performed according previously described
primers (Astruc et al., 1996; Eiras et al., 2006). PCR fragments were purified and cloned in pGEM-T Easy kit (Promega).
Five clones per isolate were sequenced to avoid problems in the sequences due to multiple infections. Molecular analyses
were performed using MEGA6.0 tools (Tamura et al., 2013).

RESULTS AND DISCUSSION

Four of the five viroids analyzed were successfully detected. HSVd was widely detected in Chilean grapevine with no
distinction about cultivar or geographic distribution. Only CEVd was not detected in all samples tested. HSVd was detected
in 91.0% of the samples, followed by GYSVd-1 (20%), GYSVd-2 (10.9%), and AGVd (9.1%) (Table 1).

Table1: Viroids detection in Chilean grapevines. aNumber of positives versus number of tested samples. Total: ratio of
positives versus all analysed samples for each viroids.

Viroidsa
Grapevine variety
HSVd GYSVd-1 GYSVd-2 AGVd CEVd
Cabernet Sauvignon 11/12 2/12 0/12 0/12 0/12
Merlot 6/6 0/6 0/6 0/6 0/6
Carménère 5/6 0/6 0/6 0/6 0/6
Pinot noir 14/14 4/14 2/14 1/14 0/14
Syrah 15/15 10/15 1/15 1/15 0/15
Sauvignon blanc 9/9 0/9 1/9 1/9 0/9
Chardonnay 4/5 0/5 0/5 0/5 0/5
Thompson Seedless 9/10 1/10 2/10 3/10 0/10
Flame Seedless 5/5 1/5 1/5 2/5 0/5
Crimson Seedless 9/10 1/10 2/10 1/10 0/10
Superior 4/5 1/5 1/5 0/5 0/5
Red Globe 4/5 0/5 2/5 1/5 0/5
Other varieties 5/8 2/8 0/8 0/8 0/8
TOTAL 100/110 22/110 12/110 10/110 0/110

76 ICVG 2015 Abstracts

 Proceedings of the 18th Congress of ICVG, Ankara, TURKEY | 7-11 September 2015

HSVd was previously described in several crops, but in grapevine was usually detected with high prevalence (Sano et
al., 2001). Thus, a high number of infected plants were expected. Sequence analyses gave phylogenetic association with
two groups, according to classification proposed by Amari et al., (2001): P-H/Cit3 and Hop, both associated with grapevine
isolates of HSVd. Another remarkable aspect is the high association of GYSVd-1 with variety Syrah, being detected in 10
of 15 samples analyzed (67%), all of them showing decline symptoms. Phylogenetic analyses of GYSVd-1 isolates gave
an association of Chilean isolates to variant 1 and variant 3. Particularly, variant 3 was previously described as pathogenic
variant in contrast with variant 1 described as asymptomatic (Szychowski et al., 1998). Seven of nine sequenced isolates
were clustered in variant 3 but only one plant showed symptoms of vein banding. GYSVd-2 and AGVd were more prevalent
in table grape varieties with 8 out of 12 detections and 7 out of 10 detections, respectively. Phylogenetic analysis of
GYSVd-2 clustered all isolates in one group, closely related with Chinese and Australian isolates. Both, GYSVd-1 and
GYSVd-2 phylogenetic analyses do not showed geographic origin association of different isolates, in agreement with
information described by Jiang et al. (2009b). In the same way, Chilean isolates of AGVd clustered indistinctly in both
reported phylogenetic groups, even when the isolates shared cultivar and geographic origin. This is not in concordance with
previous reports, where it was proposed a geographic influence on the viroid variability (Jiang et al., 2009a).

The information obtained in this work represents the first report of grapevine viroids in Chile.

ACKNOWLEDGEMENTS

This work was supported by CONICYT grant Nº 21090139, Ministerio de Educación, Gobierno de Chile.

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