1 Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456-0462, USA
2 Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Ontario, Canada N1G 2W1
3 Agricultural Research Council, Plant Protection Research Institute, Private Bag X 134, Pretoria 0001, South Africa
Correspondence
Baozhong Meng
bmeng{at}uoguelph.ca
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences determined in this work are AY881626 and AY881627.
Present address: USDA-Pacific Basin Agricultural Research Center, 99 Apuni Street, Suite 204, Hilo, HI 96720, USA.
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
Vitis rupestris St George is the standard biological indicator used worldwide to diagnose RSP. In 1995, when we started to develop RT-PCR assays, St George plants were used as negative controls. Unexpectedly, these St George plants tested positive for GRSPaV. This finding was confirmed by consistent detection of GRSPaV by using RT-PCR and Western blotting in St George plants obtained from two sources; among the 29 St George plants tested, 23 were positive for GRSPaV (Meng et al., 2000, 2003
). Likewise, Minafra et al. (2000)
detected GRSPaV in the St George selection maintained at the University of Bari, Italy.
This finding triggered several questions. Were these St George plants also infected with a mixture of GRSPaV variants? Did these variants differ in genome sequence from the previously sequenced ones? Would these variants elicit RSP symptoms on the indicator St George plants? Most importantly, would infection of St George plants with these GRSPaV variants invalidate results of past indicator indexing experiments conducted in many countries?
To answer these questions, we first analysed the genetic diversity of GRSPaV in St George plants. dsRNAs were isolated from dormant cambium scrapings of ten St George indicator plants: five from Sidney, British Columbia, Canada, and the other five from Geneva, New York, USA. As a positive control, dsRNAs isolated from FrenchAmerican hybrid Bertille Seyve (BS) 5563 plants were also assayed. All of these plants repeatedly tested positive for GRSPaV (Meng et al., 1998, 1999a
, 2003
). Isolated dsRNAs were reverse-transcribed by using Moloney murine leukemia virus reverse transcriptase Superscript II (Invitrogen). Resulting cDNAs were PCR-amplified with primers 13 and 14 (Meng et al., 1999b
) by using AccuTaq LA DNA Polymerase (Sigma), cloned into pCR2.1 (Invitrogen), and recombinant plasmids transformed into JM109 cells (Promega). The cDNA products obtained by using primers 13 and 14 corresponded to nt 43734711 of the viral genome. We chose primers 13 and 14 for this analysis because they were designed based on the consensus sequence of multiple cDNA clones and because they could detect a wide spectrum of GRSPaV variants (Meng et al., 1999b
). After a quick screening by using PCR, recombinant plasmids were isolated by using a Qiagen Miniprep kit and sequenced on an ABI 373 sequencer using M13 forward and reverse primers.
In total, 52 clones from these ten St George plants and six from BS5563 plants were sequenced and their sequences were analysed by using MegAlign (DNAStar). The results showed that the nucleotide sequences of all six clones derived from BS5563 plants were identical to one another and differed from that of GRSPaV-1 by 12 %. In contrast, the cDNA clones derived from St George plants grouped into three clusters, SG1, SG2 and SG3, which differed from one another by 3·59·8 %. Among the ten plants assayed, seven were infected with SG1 alone, one with SG2 alone, one with SG1 and SG2, and the last (C1-2-3) with all three variants. Thus, SG1 was the predominant variant infecting the indicator St George plants, at least among those that we assayed. Interestingly, all 23 cDNA clones from the St George plants collected from British Columbia were identical and belonged to SG1 (data not shown).
In contrast, SG2, differing from SG1 by 9·8 %, was a minor variant, as it was detected in only two of the ten St George plants assayed. SG3 was detected in St George C1-2-3, the only plant in which all three variants were detected. GRSPaV-SG3 differed from SG1 by 5·3 % and from SG2 by 3·5 %. The frequency of GRSPaV-SG3 in this St George plant was so low that it was represented by only one of the 11 clones sequenced.
These data suggested the following: (i) three new variants of GRSPaV were identified among the St George plants assayed, with GRSPaV-SG1 being the predominant variant; and (ii) the hybrid BS5563 was probably infected with a single GRSPaV variant (which we designated here GRSPaV-BS) that is distinct from GRSPaV-1 and from those derived from St George plants. To unravel the relationship among GRSPaV-SG1, GRSPaV-BS and GRSPaV-1 at the genome level, we set out to sequence the entire genomes of GRSPaV-SG1 and GRSPaV-BS by using an RT-PCR-based stepwise approach (Fig. 1a). Again, the templates were dsRNAs isolated from St George plants infected with GRSPaV-SG1 only or from BS5563 plants. Initial clones were obtained by using RT-PCR and primers derived from GRSPaV variants for which sequences were available. Gaps between the initial clones were then bridged by using variant-specific primers. Furthermore, two independent approaches were used to obtain the 5'-end genomic sequences. In the first approach, dsRNAs were polyadenylated, reverse-transcribed and cDNAs were PCR-amplified by using primers d(T)17 and 28F3 (5'-CATCACGACTTGTCACAAACC-3'). Resulting RT-PCR products were cloned into pCR2.1 and sequenced. In the second approach, 5' rapid amplification of cDNA ends (RACE) was performed for GRSPaV-BS. Primer Race-3 (5'-GTGCTACCAAGCTGAGATC-3') was used in first-strand cDNA synthesis. cDNAs were purified by using a QIAQuick PCR Purification kit (Qiagen) and C-tailed by using terminal deoxynucleotidyl transferase (Fermentas). They were then PCR-amplified by using d(G)14 and virus-specific primers. Gel-purified PCR products were cloned and sequenced as described above. Clones obtained through both methods revealed identical sequences at the 5' terminus of the virus genome.
|
|
Concerning the coding regions, sequence identities varied depending on individual ORFs. For example, when compared in their entirety, the ORF1s of the three variants were 85·086·5 % identical in nucleotide sequence and 91·692·7 % identical in amino acid sequence (Table 1). However, closer examination revealed that more than 50 % of the amino acid differences clustered in a region between aa 451 and 750 (Fig. 1c
), which was flanked by the methyltransferase and protease domains of the polypeptide deduced from ORF1 (Meng & Gonsalves, 2003
). Conceivably, this region may have been under less selective pressure during evolution than other regions. A similar trend was observed for nucleotide differences among these ORF1s.
ORF2 appeared to be less conserved. For example, the amino acid sequences of the ORF2 translation products of the three variants had identities of 93·2 % (between GRSPaV-SG1 and GRSPaV-1), 86·5 % (between GRSPaV-SG1 and GRSPaV-BS) and 86·9 % (between GRSPaV-1 and GRSPaV-BS). When GRSPaV-1 and GRSPaV-SG1 were compared with GRSPaV-BS, most of the differences were found in the C-terminal halves of the deduced polypeptides. On the other hand, the capsid proteins of the three variants were more conserved, having amino acid identities ranging from 90·8 to 96·2 % (Table 1). Contrary to ORF2, most of the differences (7580 %) were found in their N-terminal halves (data not shown). A similar trend held true when the nucleotide sequences of ORF5 were compared.
To determine whether GRSPaV-SG1 could induce RSP symptoms, an indicator indexing experiment was conducted. Graft inoculation was carried out in 1999 according to Martelli (1993), except that the inocula used were 46 cm long stem pieces instead of chip buds. Indicator St George plants were obtained by rooting dormant cuttings from mother plants C1-2-8 (GRSPaV-negative) and C1-2-10 (GRSPaV-positive). Resulting indicator plants were grafted with inocula from the following source plants: St George C1-2-8 (GRSPaV-negative), St George C1-2-10 (GRSPaV-positive) and Seyval (GRSPaV-positive). Five replicates were included for each of the rootstock/scion combinations. Additionally, 15 plants each of non-grafted St George C1-2-8 and St George C1-2-10 were included as negative controls (Table 2
). Grafted and non-grafted indicator plants were maintained in the greenhouse to ensure successful virus transmission through callus formation at the grafting union. These plants were subsequently transplanted to, and maintained in, the field plot until June 2004, when all plants were removed from the soil and wood symptoms were observed.
|
It remains a mystery how the indicator St George became infected with GRSPaV in the first place. As the indicator St George plants from British Columbia, New York and Bari all tested positive for GRSPaV, and all of these plants presumably originated from the Foundation Plant Services of the University of California at Davis, CA, USA, infection of St George by GRSPaV might have occurred in the original source plants. A possible scenario is that the mother plant(s) carried the virus to begin with and that, due to its asymptomatic nature, the virus has escaped detection ever since. Alternatively, the mother plant(s) may have been free of GRSPaV, but became infected later on as a result of transmission via an unknown insect vector or grafting.
GRSPaV has been detected in many countries where grapevines are grown (Zhang et al., 1998; Meng et al., 1999b
; Nolasco et al., 2000
; Stewart & Nassuth, 2001
; Tarnowski et al., 2002
; Dovas & Katis, 2003
; Espinha et al., 2003
; Petrovic et al., 2003
). Despite its ubiquitous occurrence, the aetiological role of GRSPaV in RSP remains unclear. An ultimate solution to this enigma may be provided through the use of infectious cDNA clones. We have recently created such full-length cDNA clones and are currently testing their infectivity.
In summary, through sequence analysis of cDNA clones corresponding to a representative genomic region of the virus, we detected three new variants of GRSPaV in a sample of the indicator St George plants, with GRSPaV-SG1 being the predominant variant. The genomes of GRSPaV-SG1 and another new variant from the grapevine hybrid BS5563 were sequenced, revealing a genome structure identical to that of GRSPaV-1. Lastly, we demonstrated experimentally that infection of St George plants with GRSPaV-SG1 is asymptomatic.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Dovas, C. I. & Katis, N. I. (2003). A spot nested RT-PCR method for the simultaneous detection of members of the Vitivirus and Foveavirus genera in grapevine. J Virol Methods 107, 99106.[CrossRef][Medline]
Espinha, L. M., Gaspar, J. O., Kuniyuki, H. & Camargo, L. E. A. (2003). Molecular detection of Rupestris stem pitting-associated virus in grapevines in Brazil. Fitopatol Bras 28, 206.
Goheen, A. C. (1988). Rupestris stem pitting. In Compendium of Grape Diseases, p. 53. Edited by R. C. Pearson & A. C. Goheen. St Paul, MN: American Phytopathological Society Press.
Hull, R. (2002). Matthews' Plant Virology, 4th edn. San Diego, CA: Academic Press.
Martelli, G. P. (1993). Rugose wood complex. In Graft-Transmissible Diseases of Grapevines: Handbook for Detection and Diagnosis, pp. 4554. Edited by G. P. Martelli. Rome: Food and Agriculture Organization of the United Nations.
Martelli, G. P. & Jelkmann, W. (1998). Foveavirus, a new plant virus genus. Arch Virol 143, 12451249.[CrossRef][Medline]
Meng, B. & Gonsalves, D. (2003). Rupestris stem pitting-associated virus of grapevines: genome structure, genetic diversity, detection, and phylogenetic relationship to other plant viruses. Curr Top Virol 3, 125135.
Meng, B., Pang, S., Forsline, P. L., McFerson, J. R. & Gonsalves, D. (1998). Nucleotide sequence and genome structure of grapevine rupestris stem pitting associated virus-1 reveal similarities to apple stem pitting virus. J Gen Virol 79, 20592069.[Abstract]
Meng, B., Johnson, R., Peressini, S., Forsline, P. L. & Gonsalves, D. (1999a). Rupestris stem pitting associated virus-1 is consistently detected in grapevines that are infected with rupestris stem pitting. Eur J Plant Pathol 105, 191199.[CrossRef]
Meng, B., Zhu, H.-Y. & Gonsalves, D. (1999b). Rupestris stem pitting associated virus-1 consists of a family of sequence variants. Arch Virol 144, 20712085.[CrossRef][Medline]
Meng, B., Goszczynski, D. E. & Gonsalves, D. (2000). Detection of Rupestris stem pitting associated virus in the indicator Vitis rupestris "St George" and sequence analysis. In Extended Abstracts of the 13th Meeting of the ICVG, pp. 4344, 1217 March 2000, Adelaide, South Australia.
Meng, B., Credi, R., Petrovic, N., Tomazic, I. & Gonsalves, D. (2003). Antiserum to recombinant virus coat protein detects Rupestris stem pitting associated virus in grapevines. Plant Dis 87, 515522.
Minafra, A., Casati, P., Elicio, V., Rowhani, A., Saldarelli, P., Savino, V. & Martelli, G. P. (2000). Serological detection of grapevine rupestris stem pitting-associated virus (GRSPaV) by a polyclonal antiserum to recombinant virus coat protein. Vitis 39, 115118.
Nolasco, G., Mansinho, A., Teixeira Santos, M., Soares, C., Sequeira, Z., Sequeira, C., Correia, P. K. & Sequeira, O. A. (2000). Large scale evaluation of primers for diagnosis of rupestris stem pitting associated virus-1. Eur J Plant Pathol 106, 311318.[CrossRef]
Petrovic, N., Meng, B., Ravnikar, M., Mavric, I. & Gonsalves, D. (2003). First detection of Rupestris stem pitting associated virus particles by antibody to a recombinant coat protein. Plant Dis 87, 510514.
Rowhani, A., Zhang, Y. P., Chin, H., Minafra, A., Golino, D. A. & Uyemoto, J. K. (2000). Grapevine rupestris stem pitting associated virus: population diversity, titer in the host and possible transmission vector. In Proceedings of the 13th Meeting of the ICVG, p. 37, 1217 March 2000, Adelaide, Australia.
Santos, C., Santos, M. T., Cortêz, I., Boben, J., Petrovic, N., Pereira, A. N., Sequeira, O. A. & Nolasco, G. (2003). Analysis of the genomic variability and design of an asymmetric PCR ELISA assay for the broad detection of Grapevine rupestris stem pitting-associated virus. In Proceedings of the 14th Meeting of the ICVG, pp. 126127. 1217 September 2003, Locorotondo (Bari), Italy.
Stewart, S. & Nassuth, A. (2001). RT-PCR based detection of Rupestris stem pitting associated virus within field-grown grapevines throughout the year. Plant Dis 85, 617620.
Tarnowski, C. G., Worlock, P. A., Ulanovsky, S. & Gómez Talquenca, S. (2002). First report of Rupestris stem pitting associated virus in Argentina. Plant Dis 86, 921.
Zhang, Y.-P., Uyemoto, J. K., Golino, D. A. & Rowhani, A. (1998). Nucleotide sequence and RT-PCR detection of a virus associated with grapevine rupestris stem-pitting disease. Phytopathology 88, 12311237.
Received 9 December 2004;
accepted 13 January 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |