Agriculture and Agri-Food Canada, Potato Research Centre, PO Box 20280, Fredericton, New Brunswick, Canada E3B 4Z71
Agricultural Certification Services, NB Potato Agency, 245 Hilton Road, Unit 25, Fredericton, New Brunswick, Canada E3B 5N6 2
Author for correspondence: Rudra Singh.Fax +1 506 452 3316. e-mail singhr{at}em.agr.ca
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Abstract |
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Introduction |
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Potato spindle tuber viroid (PSTVd), with its numerous, well- characterized strains and isolates, was the first viroid to be discovered (Diener, 1971 ; Singh & Clark, 1971
), structurally characterized (Sänger et al., 1976
) and sequenced (Gross et al ., 1978
). It is the type species of the genus Pospiviroid (Flores et al., 1998
). About 40 PSTVd variant sequences are found in the GenBank and EMBL nucleotide databases. The potato (Solanum tuberosum) isolates represent the natural source of infection in the field (Schnö lzer et al., 1985
; Owens et al., 1992
; Herold et al., 1992
; Lakshman & Tavantzis, 1993
; Singh et al., 1993
; Gora et al., 1994
; Gora-Sochacka et al., 1997
). Other isolates are in vitro-generated PSTVd mutants (Hammond, 1992
, 1994
; Lakshman & Tavantzis, 1992
; Qu et al., 1993
; Wassenegger et al., 1994
, 1996
; Owens et al., 1995
; Hu et al., 1996
). Except for one isolate of 341 nt (Wassenegger et al., 1994
), such isolates are very similar in size and vary by 17 nt from the prototype strain (Gross et al., 1978
). Naturally occurring PSTVd isolates from greenhouse tomato ( Lycopersicon esculentum) and Solanum species (including S. muricatum) have also been reported (Puchta et al., 1990
; Owens et al., 1992
; Behjatnia et al., 1996
; Shamloul et al., 1997
). These isolates have sequence similarity of 9598% with PSTVd. Greenhouse tomato and pepino (S. muricatum) isolates are 35 nt shorter than the 359 nt PSTVd. It has been assumed that accidental transfers of PSTVd from symptomless pepino could have caused symptoms in greenhouse tomatoes, since pepino seed is generally infected with PSTVd (Puchta et al., 1990
).
In Canada, PSTVd has been eradicated (Singh, 1988 ) and has not been detected in potato fields since 1980. Thus, when approximately 2000 greenhouse tomato seedlings of cv. Trust, grown from seed imported from The Netherlands, suddenly developed chlorotic leaves and grew to become severely dwarfed and bunchy plants, i.e. symptomology typical of PSTVd in tomato (Singh & O'Brien, 1970
), a viroid aetiology was suspected. We report here identification of a viroid from these plants, designated tomato chlorotic dwarf viroid (TCDVd), which has sequence similarities of 8688% with PSTVd potato isolates and 8589% with other (greenhouse tomato, pepino, Solanum species) isolates. Considering that Mexican papita viroid (MPVd) (Martinez-Soriano et al., 1996
), which has 7880% sequence identity to PSTVd, has been proposed as an ancestor of crop viroids, TCDVd may represent an evolutionary link between MPVd and PSTVd.
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Methods |
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Viroid isolation, RTPCR, cloning and sequencing.
Total leaf nucleic acids were extracted and return-polyacrylamide gel electrophoresis (R-PAGE) was carried out (Singh, 1991 ). About 1·5 µg (6 µl) of nucleic acid was loaded on a 5% gel and the first cycle carried out under non-denaturing conditions in TBE buffer (89 mM Tris, 89 mM boric acid, 2·5 mM EDTA; pH 8·3) at 25 °C. The reverse cycle was carried out under denaturing conditions (1:8 diluted buffer, at 71 °C). RNA bands were visualized by silver staining.
An RTPCR for preliminary characterization of viroids was carried out using three primer pairs, based on the sequences of PSTVd isolates. Primers 2A (5' TGTTTCCACCGGTAGTAGC 3'; complementary to nt 254273) and 1S (5' ACTCGTGGTTCCTGTGGTTC 3'; identical to nt 1029) were designed for use with isolate FM (Herold et al., 1992 ). P1 (complementary to nt 256273) and P2 (identical to nt 274295) were from PSTVd (Gross et al., 1978
); P3 (complementary to nt 6893) and P4 (identical to nt 87110), for use with the Darwin isolate of PSTVd, were the primers used by Behjatnia et al. (1996)
.
For purification of viroid, total leaf nucleic acids from TCDVd- infected Nicandra, potato, Scopolia and tomato were separated in R-PAGE and unstained viroid bands (Singh et al., 1988 ) were cut out and incubated overnight at 37 °C in eluting buffer (0·5 M ammonium acetate, 0·1% SDS, 10 mM EDTA; pH 8·0). Eluted viroid RNA was precipitated with ethanol and suspended in TE (10 mM TrisHCl, pH 8·0, 1 mM EDTA). cDNA was synthesized and amplified with uracil-containing primers for subsequent cloning as in Rashtchian et al. (1992)
. Briefly, CUACUACUACUA was added onto primer P3 and CAUCAUCAUCAU onto P4 at the 5' end (Singh & Singh, 1995
). Amplification was carried out for 35 cycles (1 min each of denaturation at 94 °C, annealing at 55 °C and extension at 72 °C, with a final extension for 7 min). PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. Bands of approximately 400 bp were excised, extracted with TE, purified with Glass MAX spin cartridges (Gibco BRL) and then cloned into competent Escherichia coli DH
using the clone Amp 1 system (Gibco BRL). Viroid inserts were confirmed by PCR and agarose gel electrophoresis.
Two viroid clones, each of which infected Nicandra, potato, Scopolia and tomato, were initially sequenced in both directions by the dideoxy chain-termination method (Sanger et al ., 1977 ) using modified T7 DNA polymerase (Sequenase version 2.0, US Biochemical). Later, the sequencing was done using a PE/ABI 377 DNA sequencer and the PE/ABI-ABI PRISM BigDye Terminator cycle sequencing Ready Reaction Kit (PE Applied Biosystems). In order to confirm the sequence in the P3+P4 priming region, a second pair of primers (P5, P6) was designed. P5 is complementary to nt 342360 and P6 is identical to nt 120 of TCDVd (this study); these primers were synthesized with uracil NTPs, and amplification products were cloned and sequenced as before. Again, eight clones (two from each host) were sequenced as above. Sequence analysis by pair-wise comparison and multiple alignment, as well as calculation of secondary structures, were carried out using the Genetics Computer Group suite of programs, version 8.1 (University of Wisconsin, Madison, WI, USA).
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Results and Discussion |
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PSTVd is seed-transmitted and can persist in true potato seed for over 20 years (Singh et al., 1988 , 1991
). The possibility that TCDVd is seed-borne was tested by R-PAGE using 100 seeds of each indicator plant and two potato cultivars: a total of 700 seeds and 400 seedlings of N. physaloides, N . debneyi, P. angulata, P. floridana, tomato and potato cvs Atlantic and Superior were tested for seed-borne infection of TCDVd. No infection was detected by R-PAGE either in the seeds or plants generated from seeds. In parallel experiments, PSTVd infection of 7-year-old true potato seed was routinely detected by R-PAGE.
Because the differential migration pattern of TCDVd and PSTVd in R- PAGE allows the two species to be differentiated, cross-protection for viroid replication could be tested (Khoury et al., 1988 ). Ten tomato plants pre-infected with PSTVd FM (Herold et al., 1992
) and challenged with TCDVd supported replication of both viroids (Fig. 1
, lanes 69) similar to the singly infected plants (Fig. 1
, lanes 25 and 1013). The mild symptoms of PSTVd FM were replaced with the severe symptoms characteristic of TCDVd in doubly infected plants. We concluded that there was no interference by PSTVd in the multiplication and symptom expression of TCDVd in doubly infected plants.
RTPCR, cloning and sequence determination
Primer pair 2A+1S amplified two bands of different sizes from TCDVd and PSTVd. The main PSTVd band had a counterpart of similar mobility from the TCDVd-infected sources (Fig. 2 , upper panel). P1+P2 amplified PSTVd but not TCDVd (Fig. 2
, middle panel) and P3+P4 amplified a single band in both viroid infections (Fig. 2
, lower panel). These observations indicated that TCDVd might differ in sequence in the lower strand of the central conserved region, a difference which the primers P1+P2 were designed to detect. Since the P3+P4 primer pair, based upon the upper strand of the central conserved region (CCR), gave a full-length PCR product for both PSTVd and TCDVd, we suspected that there would be similarities of sequences in the upper strand of the CCR.
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When the TR sequences of TCDVd are compared with members of the genus Pospiviroid, various PSTVd isolates exhibit less than 90% identities, and TASVd, MPVd and Columnea latent viroid exhibit 93% identities. This indicates that the TR in TCDVd could have been derived from viroids other than PSTVd.
In addition to the extensive changes in the V and TR domains when compared to PSTVd, TCDVd has a nucleotide transition of A 45U45 in the Terminal Left (TL) domain, which is also found in PSTVd-N (Puchta et al., 1990
). The addition of U63 in the upper strand of the Pathogenicity (P) domain, the substitution of G311 in the lower strand of P domain and the transition U258
A 258 in the lower strand of the CCR are other changes in TCDVd. Nucleotide 258 is part of an ultraviolet light-sensitive structural element that is present in RNA molecules of hepatitis delta virus RNA, host 5S ribosomal RNA and 7SL signal recognition particle RNAs and PSTVd (Branch et al., 1990
). It is noteworthy that the transition of nt 258 was responsible for the failure of RTPCR amplification by primer pair P1+P2, based on the PSTVd sequence (Fig. 2
, middle panel). The single nucleotide mismatch at the 3' end of primer P1 prevented reverse transcription.
From this study it is concluded that TCDVd is a new viroid species, which has similarity to various members of the genus Pospiviroid but differs drastically in its Variable domain from other members. Infection of potato with this viroid may cause symptoms indistinguishable from those caused by PSTVd and could create plant quarantine problems. The origin of TCDVd in greenhouse tomato crops has been hard to determine. The tomato seeds were produced in The Netherlands and were exported to the USA, from where the seeds were imported into Canada. Extensive efforts to demonstrate seed transmission or presence of viroid in the seed have failed. Subsequent batches of tomato seeds from the same sources have produced healthy crops.
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Acknowledgments |
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Footnotes |
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References |
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Received 31 March 1999;
accepted 1 July 1999.
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