Download sequence variability of grapevine rupestris stem pitting

Genetic diversity of grapevine leafroll-associated virus-2 in Washington state vineyards
S. Jarugula, M.J. Soule, P.S. Rajakaruna, and R.A. Naidu*
Department of Plant Pathology, Washington State University, Irrigated Agriculture Research and
Extension Center, 24106 N. Bunn Road, Prosser, WA 99350.
Introduction
Grapevine leafroll disease (GLD) is the most economically important viral disease of grapevines
worldwide. Nine serologically distinct viruses, designated as Grapevine leafroll-associated virus (GLRaV)
-1 to -9 in the order of their discovery, have so far been found associated with the disease (Gugerli,
2003). All GLRaVs belong to the family Closteroviridae (Karasev, 2000). GLRaVs -1, -3, -4, -5, -6, -8 and
-9 belong to the genus Ampelovirus, whereas GLRaV-2 belongs to the genus Closterovirus. GLRaV-7
has not yet been assigned to any of the genera in the family Closteroviridae.
GLD is a serious constraint to the sustainability of viticulture industry in Washington State (WA). Six of
the nine GLRaVs (GLRaV-1, -2, -3, -4, -5, and -9) have so far been documented in WA vineyards (Martin
et al., 2005; Naidu et.al., 2006). Among them, GLRaV-2 was found to be the second most predominant
virus, after GLRaV-3, in several vineyards. GLRaV-2 was also found as a mixed infection with other
GLRaVs in several GLD-infected vines. The mixed infection of GLRaV-2 was found to be more frequent
with GLRaV-3 than with other viruses. In this study, we have conducted genetic diversity studies among
GLRaV-2 isolates collected from WA vineyards.
Materials and Methods
A total of forty seven isolates were collected from four red-fruited cultivars showing GLD symptoms in
seven different vineyards. Selected isolates were tested by double antibody sandwich (DAS)-ELISA
using a commercial kit (Agritest, Valenzano-Bari, Italy). A 332 nucleotide (nt) fragment of the heat shock
protein homologue (HSP70h) gene was amplified by one-tube RT-PCR method (Rowhani et al., 2000).
The forward primer (5’-ATAATTCGGCGTACATCCCCACTT-3’) and reverse primer: (5’GCCCTCCGCGCAACTAATGACAG-3’) used in RT-PCR were identical to nt 9136-9159 and
complementary to nt 9445-9467, respectively, in the sequence of a New York isolate of GLRaV-2
(GenBank Accession # AF039204). The amplicons were cloned into pCR2.1 vector (Invitrogen Corp,
Carlsbad, CA). Two independent clones were sequenced from both orientations and a consensus
sequence was obtained for each isolate. Nucleotide and predicted amino acid sequences were aligned
and compared using Vector NTI Advance10 software (Invitrogen). Multiple sequence alignments were
performed using ClustalW (BioEdit version7.0.5.3 [Ibis Therapeutics, Carlsbad, CA]). Phylogenetic
analysis was carried out by the neighbor-joining method using PAUP Version4.0b10 software (Sinauer
Associates, Inc., Sunderland, MA). Corresponding sequences of GLRaV-2 isolates available in GenBank
(AF039204, USA; AY881628, South Africa; Y14131, Italy; Y15890, France) were included in these
analyses.
Results and Discussion
In pair-wise comparisons, sequences from all forty seven isolates showed 83 to 92% identity at the
nucleotide level and 91 to 96% identity at the amino acid level. Phylogenetic analyses revealed
segregation of these forty seven isolates into three clusters. Cluster I consisted of twenty three isolates
from three different cultivars (Cabernet sauvignon, Pinot noir and Merlot) in six different vineyards. Cluster
II consisted of eighteen isolates from one cultivar (Cabernet sauvignon) in two vineyards. Cluster III was
represented by six isolates from another cultivar (Sangiovese) in a different vineyard. The results indicate
wide spread distribution of cluster I isolates when compared to cluster II and III isolates. Pair-wise
nucleotide sequence identities between these clusters are shown in the table below. The amino acid
sequence identities between isolates in cluster I and II were 91 to 93%, between cluster I and III was 93%
and between cluster II and III was 94 to 96%. Isolates in cluster I are identical to isolates from USA
(AF039204) and Italy (Y14131). Isolates within cluster II and cluster III are as distantly related to each
other as either of the clusters to the isolates in cluster I. In addition, isolates in cluster II and III are
distantly related to isolates from Italy (Y14131), New York (AF039204), South Africa (AY881628) and
France (Y15890).
50
In DAS-ELISA, 4 out of 8, 3 out of 3, and none out of 2 isolates from cluster I, II and III, respectively,
tested positive for GLRaV-2. These results indicate serological variability among the GLRaV-2 isolates
tested in this study.
In summary, the present study clearly documents the occurrence of GLRaV-2 molecular variants in WA
vineyards. The results also highlight the need for RT-PCR testing to complement ELISA test results in
order to detect all possible variants of GLRaV-2. Further studies are in progress towards a
comprehensive understanding of virus variability which will enable the development of robust assays for
the detection and discrimination of a broad range of GLRaV-2 variants.
Acknowledgements
This work was funded by Washington Wine Commission, WSDA Nursery Assessment Funds,
Washington State Commission on Pesticide Registration, USDA Northwest Center for Small Fruits
Research, and Washington State University (New Faculty Seed Grant, ARC-CAHNRS).
References
Gugerli, P. (2003). Grapevine leafroll and related viruses. Pages 23-24 In: The 14th ICGV Conference,
Locorotondo, Italy.
Karasev, A.V. 2000. Genetic diversity and evolution of closteroviruses. Ann. Rev. Phytopathol.38: 293324.
Martin, R.R., Eastwell, K.C., Wagner, A., Lamprecht, S. and I. E. Tzanetakis, I.E. 2005. Survey for viruses
of grapevine in Oregon and Washington. Plant Disease 89: 763-766.
Naidu, R.A., Soule, M.J. and Jarugula, S. 2006. Single and mixed infections of Grapevine leafrollassociated viruses in Washington State vineyards. Phytopathology 96:S83.
Table: Percent nucleotide sequence identities between GLRaV-2 isolates from Washington State (WA)
and corresponding sequences in the GenBank.
WA
Cluster I
WA
Cluster II
WA
Cluster III
Y14131
AF039204
AY881628
Y15890
WA
Cluster I
isolates
WA
Cluster II
isolates
WA
Cluster III
isolates
Italy
(Y14131)
New York
(“PN” isolate,
AF039204)
100
85-86
88
100
100
100
92
85
85
86
92
100
90
88
88
90
100
100
100
89
89
100
86
86
87
100
South Africa
(“93/955”
isolate,
AY881628)
89
France
(“FR6”
isolate,
Y15890)
92
51