An RT-PCR primer pair for the detection of Pospiviroid and its application in surveying ornamental plants for viroids

Journal of Virological Methods 116 (2004) 189–193

An RT-PCR primer pair for the detection of Pospiviroid and its application in surveying ornamental plants for viroids Hidayet Bostan a,1 , Xianzhou Nie b , Rudra P. Singh b,∗ b

a Department of Plant Protection, Faculty of Agriculture, Ataturk University, Erzurum 25240, Turkey Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, Fredericton, NB, Canada E3B 4Z7

Received 5 September 2003; received in revised form 24 November 2003; accepted 25 November 2003

Abstract A primer pair for reverse transcription-polymerase chain reaction (RT-PCR), based on the conserved sequences of the members of genus Pospiviroid was designed to yield a fragment of about 200 base pairs (bp). Since pospiviroids infect a large number of plants species and a few members of the genus Pospiviroid have been already detected in some ornamental plants, the primer pair was evaluated for its efficacy using ornamental plants. The method of return-polyacrylamide gel electrophoresis (R-PAGE) was used to determine the general presence of viroids in the test samples. Efficacy of the primer pair for members of genus Pospiviroid was demonstrated by the detection of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) in potato, Chrysanthemum stunt viroid and Iresine viroid in Verbena and Vinca species, and Citrus exocortis viroid in Impatiens species. Specificity of the primer pair became evident, where additional viroids were detected by R-PAGE in Coleus and Magilla species, but they were not amplified by the Pospiviroid primer. This primer pair would be of benefit in indexing ornamental plants in quarantine samples or in viroid-free certification schemes, irrespective of their actual identity. © 2003 Elsevier B.V. All rights reserved. Keywords: CEVd; CSVd; CLVd; IrVd; Impatiens; Verbena; Vinca; Viroids

1. Introduction Although viroid diseases could have existed (or perhaps pre-existed) virus infection of higher plants (Bar-Joseph, 2003), there is no direct established relationship of a natural host species and a viroid. Therefore, the origin of viroids is still an open question. Several recent outbreaks of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (closely related to PSTVd) have occurred in greenhouse tomato crops (Puchta et al., 1990; Singh et al., 1999; Elliott et al., 2001). Of these occurrences, only one was directly associated with an infected potato source, in that Solanum muricatum plants were present in adjacent greenhouses (Puchta et al., 1990). Absence of information about viroid introduction sources raises the possibility that PSTVd-like viroids may have other infection reservoirs besides the potato and that the latter could be only the secondary host for the viroid. It has been postulated that viroids presently assigned to natural hosts may have been acciden∗

Corresponding author. Tel.: +1-5064524843; fax: +1-5064523316. E-mail address: [email protected] (R.P. Singh). 1 Visiting scientist.

0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.11.014

tally introduced into them from the unrelated wild species (Diener, 1995). A potential source of such viroid introductions to crop plants may be through ornamental plants, which form a diverse group of plant collections and are often vegetatively propagated via en masse cuttings. Most of the 30 known (i.e., molecularly well-characterized) viroid species (Flores et al., 2003) affecting agricultural and horticultural plants have been recognized because they induce disease symptoms in their host plants. Approximately 20% of these well-defined viroids have been identified from ornamental plants. Many of which are carried symptomlessly in their host plants, but when introduced to cultivated plants like potato and tomato can cause severe disease symptoms (Hammond et al., 1989; Singh et al., 1992; Spieker, 1996a). Such severe visible symptoms in potato and tomato are difficult to differentiate from those symptoms expressed by PSTVd (Singh et al., 2003) and may lead to the imposition of plant quarantine despite being PSTVd-free. At present viroids isolated from ornamental plants include: Colummea latent viroid (CLVd) in Columnea erythrophae (Hammond et al., 1989), in Nematanthus wettsteinii (Singh et al., 1992) and (CLVd-B) in Brunfelsia undulata (Spieker, 1996b); Coleus blumei viroid (CbVd1) in

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Coleus blumei (Spieker et al., 1990); Chrysanthemum stunt viroid (CSVd) (Gross et al., 1982) and Chrysanthemum chlorotic mottle viroid (ChCMVd) (Navarro and Flores, 1997) in Chrysanthemum morifolium; Iresine viroid (IrVd) in Iresine herbstii (Spieker, 1996b); and Tomato apical stunt viroid (TASVd) in Solanum psuedocapsicum (Spieker et al., 1996). With exception of ChCMVd and CbVd1, all are members of the genus Pospiviroid, suggesting a greater likelihood that other members of Pospiviroid may be wide-spread in ornamental plants. However, because of the diverse group of plants and lack of symptoms in ornamental plants a systematic approach to determine the extent of viroid infection has not been possible and is not probable. Return-polyacrylamide gel electrophoresis (R-PAGE) (Singh and Boucher, 1987; Singh, 1991) a general method of viroid identification, based on the detection of denatured circular RNA structure has been used to a limited scale in the surveys of viroids of ornamental plants (Fonseca et al., 1989; Ramachandran et al., 1992; Singh et al., 1991). The reverse-transcription-polymerase chain reaction (RT-PCR) is a nucleotide sequence-specific method, which amplifies DNA based on the sequence identity. RT-PCR has been used for the identification of all groups of viroids except Coleviroid (Hadidi and Candresse, 2003) and in surveys of fruit trees (Faggioli et al., 2001; Ito et al., 2002; Shamloul et al., 2002). In the latter studies, specific primer pairs were used to detect specific viroid species either individually or using a multiplex RT-PCR protocol. Standard RT-PCR has been successful in detecting members of the genus Pospiviroid (Hadidi and Candresse, 2003) and high sequence homology between genus members limits the efficacy of a multiplex approach. In view of the difficulty in differentiating viroids from ornamental plants, a universal primer pair capable of amplification of all members of the genus Pospiviroid would be a desirable goal. The objective of this study, therefore, was to design a primer pair, which would detect all or most members of the genus Pospiviroid using standard RT-PCR. This article reports the evaluation of such a primer pair using potato and ornamental plants. The primer pair may be used in the potato certification programs, where total freedom from viroid-infection is practiced (Slack and Singh, 1998).

2. Materials and methods 2.1. Plant samples Potato tuber and leaf samples infected with PSTVd and TCDVd were obtained from an experimental field-plot at the Potato Research Centre, Fredericton, NB. Leaves and vines of ornamental plants were collected from a local nursery, which propagates annual and perennial flowers, bedding plants, hanging baskets and foliage plants, vegetables, shrubs and ornamental fruit trees. Samples were collected on the basis of visual inspection for leaf or flower variega-

tion, stunted leaves or plants, or those plants characterized by creeping or hanging vines or annual and perennial plants multiplied by cuttings. About 350 samples (250 ornamentals and 100 potato and indicator plants) were tested. 2.2. Nucleic acid isolation Nucleic acids were isolated from potato and ornamental plant leaves, and potato tubers as described previously (Singh and Boucher, 1987) with some modifications. In brief, 300 ␮l extraction buffer [0.53 M NH4 OH, 0.013 M disodium ethylenediaminetetraacetate (EDTA) adjusted to pH 7.0 with Tris, 4 M LiCl, 1% purified bentonite)] and 400 ␮l of Tris-saturated phenol (containing 0.1 g of 8hydroxyquinoline per 100 ml) was combined with five drops (200 ␮l) of sap. The sap was obtained from leaves or tuber tissues by a Tuber Slicer (Electrowerk, Behcke and Co., Hannover, Germany). The suspension was centrifuged (15 min, 12,000 × g) at 4 ◦ C, and the nucleic acids were precipitated from the aqueous layer with 2.5 vol of ethanol in the presence of 0.1 vol of 4 M sodium acetate (−20 ◦ C, overnight). The precipitate was collected by centrifugation (12, 000×g, 15 min, 4 ◦ C), washed with 70% ethanol, vacuum-dried, and dissolved in 200 ␮l (leaf sample) or 100 ␮l (tuber sample) of 1 × TBE (89 mM Tris–HCl, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) for R-PAGE and RT-PCR. 2.3. Return-polyacrlamide gel electrophoresis ( R-PAGE) R-PAGE was performed as described (Singh and Boucher, 1987). Briefly, 40% of glycerol and 10 ␮l of a solution consisting of 1% of xylene cyanol FF and 1% bromophenol blue was added to the nucleic acids dissolved in the ‘high salt’ TBE buffer (as above). First, electrophoresis separation of the nucleic acids was at constant current 46 mA for 2.5 h using an SE 600 series apparatus (Hoefer Scientific Instruments, San Francisco, CA) on 5% nondenaturing slab gels (5% polyacrylamide, 0.125% bis-acrylamide, 14 cm × 16 cm × 0.15 cm) in the high salt buffer, using 10 ␮l of sample in each well. For the second run, the buffer in both the upper and lower reservoirs was replaced with a ‘low salt’ buffer (1:8 dilution of the high salt buffer). About 2 l of the low salt buffer heated to boiling was poured into the lower electrophoresis chamber in which the first gel was immersed. These conditions denature the viroid. The remainder of the lower electrophoresis chamber was filled with 3 l of buffer heated to 73–75 ◦ C. The polarity was reversed and the electrophoresis was performed at 75 ◦ C (46 mA constant current, 2 h). Gels were stained using silver nitrate and photographed with an imaging system (Alpha Innoteck IS 1000, San Leandro, CA). 2.4. Primer design and reverse transcription-polymerase chain reaction (RT-PCR) Nucleotide (nt) sequences of nine Pospiviroid species (Steger and Riesner, 2003) from public databases (EMBL-

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ing to the manufacturer’s guidelines. Two cDNA clones of each fragment were sequenced in each direction, using a PE/ABI-ABI PRISM Big Dye Terminator cycle sequencing Ready Reaction Kit (PE Applied Biosystems). Sequences were subjected to BLAST program in pair wise comparison and sequence identity to other viroids was determined. When the sequence homology of the sequenced viroid sample was of 90–95% and above with a known viroid species, it was considered to be an isolate of that viroid and was assigned that name. Indicator herbaceous plants of known viroids were inoculated with sap containing viroids from the original host to determine plant responses. Irrespective of the plant reaction, they were retested by RT-PCR to ascertain the infection. Fig. 1. The accession numbers of viroid species exhibiting sequence identity with both forward (For) and reverse (Rev) Pospiviroid primer. Chrysanthemum stunt (CSVd): M19506; Citrus exocortis-tomato (CEVd-t): X53716; Columnea latent (CLVd): X15663; Iresine (IrVd): X95734; Mexican papita (MPVd): L78454; Potato spindle tuber (PSTVd) M88677; Tomato chlorotic dwarf (TCDVd): AF162131; Tomato planta macho (TPMVd): K00817; and Tomato apical stunt (TASVd): X06390.

EBI) were aligned using Clustal W (Multiple Sequence Alignment) program. A conserved nt stretch area shared by most species was selected for primer design. To balance the G:C content of forward primer additional nt (ATTA) were added at the 5 end of the primer (Fig. 1). Nucleic acid extract (2.5 ␮l) was incubated at 65 ◦ C for 5 min and chilled on ice for 3 min to denature the RNA and added to the reaction mixture. The final concentration of reagents was 20 ng/␮l of reverse primer (Fig. 1), 50 mM Tris–HCl, pH 8.3, 75 mM KCl, 10 mM DTT, 2.5 mM MgCl2 , 1.5 mM of each dNTPs, 20 U RNasin (Promega, Madison, WI) and 200 U Moloney murine leukemia virus-reverse transcriptase (MMLV-RT) (Gibco BRL). Samples were incubated for 1 h at 42 ◦ C for RT and subsequently incubated at 95 ◦ C for 3 min to terminate the reaction. PCR was performed using 2 ␮l cDNA mixture in 25 ␮l containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 200 ␮M each of dNTPs [dATP, dCTP, dGTP, and dTTP (Promega)], 0.1 ␮g each of the primers (Fig. 1) and 0.625 U of DNA polymerase (PE Applied Biosystems, Foster City, CA). Samples were amplified for 30 cycles using a Peltier Thermal Cycler (MJ Research, Watertown, MA). Annealing temperature was 62 ◦ C (30 s) denaturation (92 ◦ C, 30 s), and primer extension (72 ◦ C, 90 s), and a final extension (72 ◦ C, 10 min). Ten microliters of amplified products were separated by electrophoresis in a 2.05% agarose gel containing 0.5 ␮g/ml ethidium bromide and photographed under UV illumination with an imaging system. 2.5. Cloning, sequencing and viroid identification The PCR products of each viroid sample were cloned into pAMP1 vector (Gibco-BRL, Gaithersburg, MD) accord-

3. Results 3.1. The pospiviroid primer pair Nucleotides 89–108 in PSTVd, comprising the upper central conserved region (CCR), and nt 259–280 from the lower CCR formed the forward and reverse primers, respectively (Fig. 1). The primer pair was fully matched for pospiviroids, CEV-tomato isolate (CEVd-t), TASVd, CSVd, Mexican papita (MPVd), Tomato planta macho (TPMVd), PSTVd, TCDVd and two nt mis-match with IrVd (Fig. 1). However, there were six nt mis-matches with CLVd, including three mis-matches within the six nt from the 3 end of the forward primer and considerably higher in the reverse primer (Fig. 1). Therefore, all pospiviroids except CLVd were expected to be detected if present in the plant samples. 3.2. Detection of viroids by RT-PCR in potato Potato leaves and tubers (100 samples), known to be infected with PSTVd and TCDVd were evaluated with the Pospiviroid primer in an RT-PCR at a range of 55–62 ◦ C annealing temperatures. Both viroids were amplified at each temperature (data not shown) irrespective of the plant parts used, and therefore, were used as positive controls in subsequent RT-PCR with suspected ornamental samples. 3.3. Detection and identification of viroids by R-PAGE and RT-PCR in ornamental plants A total of 250 ornamental plant samples were tested. Most plants were free from viroids. In few cases, very faint amplification took place and no viroid bands were observed in R-PAGE from such samples. These samples were not pursued further. Only those plants in which viroid bands were obtained repeatedly and in different batches of samples by both R-PAGE and RT-PCR are presented (Table 1). Because of the varying sizes of the viroid species (from 354 to 372 nt), it was expected that fragment sizes in RT-PCR may vary (Fig. 2, lanes 5–6 versus lanes 7–8).

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Table 1 Detection of members of Pospiviroid in new host ornamental plants sampled at different times of the year Common name

Vinca major Double Impatiens Trailing Verbena Coleus species a

Family

Apocynaceae Balsamineaceae Verbenaceae Lamiaceae

Sampling months March

May

July

16/16a

27/31 5/9 4/5 0/4

10/15 9/13 8/9 0/3

6/9 2/3 0/4

Number of samples positive/number tested.

Fig. 2. Reverse transcription-polymerase chain reaction (RT-PCR) detection of five viroids from various host plants. Lanes 1–2, PSTVd (potato); lanes 3–4, TCDVd (potato); lanes 5–6, CSVd (Vinca); lanes 7–8, IrVd (Vinca); lanes 9–11, single and double infection of Verbena spp. by CSVd, CSVd + IrVd and CSVd, respectively; lanes 12–13, CEVd (Impatiens); lanes14–15, CbVd (Coleus); lane 16, PSTVd control; lanes 17–19, healthy and PCR mixture without template or primer as controls. M, Low DNA mass ladder.

The smallest fragment of 196 bp was expected for MPVd and the largest of 228 bp for IrVd (Table 2). The viroid fragments from RT-PCR were cloned and a minimum of two clones were sequenced. Sequences were compared with available databases and they were identified belonging to a viroid species, when sequence of the fragment matched to a known viroid. Thus, three viroid species were detected in three previously unrecorded viroid host plant species. In Vinca major and Verbena species both CSVd and IrVd were detected singly- or doubly-infected (Figs. 2 and 3, lanes 9–11). In Impatiens species only CEVd was detected. The frequency of infected ornamental plants within these three host species remained high through out the testing period (Table 1).When R-PAGE was used on plant samples, strong Table 2 Expected fragment sizes for the members of Pospiviroid with the primer pair used Viroids

Fragment length (base pairs)

Mexican papita viroid (MPVd), acc. # L78454 Tomato planta macho viroid (TPMVd), acc. # K00817 Potato spindle tuber viroid (PSTVd), acc. # M88677 Tomato chlorotic dwarf viroid (TCDVd), acc. # AF162131 Citrus exocortis viroid-tomato (CEVd-t), acc. # X53716 Tomato apical stunt viroid (TASVd): acc. # X06390 Iresine viroid (IrVd), acc. # X95734 Chrysanthemum stunt viroid (CSVd), acc. #M19506;

196 196 199 210 205 205 228 199

Fig. 3. Return-polyacrylamide gel electrophoresis (R-PAGE) detection of six viroids. Lane 1, PSTVd mild; lane 2, PSTVd severe; lanes 3–4, TCDVd; lanes 5–6, CSVd (Vinca), lanes 7–8, IrVd (Vinca); lane 9, IrVd single infection (Trailing Verbena) (TV); lane 10, double infection CSVd + IrVd (TV); lane 11, single infection CSVd (TV); lanes 12–13, faint bands CEVd (Impatiens); lanes 14–15, CbVd (Coleus) and lane 16, healthy potato control. Arrow indicates the viroid bands.

viroid bands were detected in Coleus species (Fig. 3, lanes 14–15), in addition to the three hosts detected using RT-PCR (Fig. 2). These viroid bands were not amplified in repeated attempts by the Pospiviroid primer (Fig. 2, lanes 14–15), indicating that the viroid bands in R-PAGE represent other viroid species different from the Pospiviroid genus. The location of the viroid band in R-PAGE of Coleus samples were indicative of a smaller-sized Coleviroid species (Singh et al., 1991) compared to the PSTVd and TCDVd. Use of coleviroids specific primer pair confirmed the prsence of a Coleviroid in these samples.

4. Discussion The objective of this study was to develop an RT-PCR protocol, which would detect most members of the genus Pospiviroid from a variety of plant species using a single primer pair. Five species of viroids were detected: PSTVd, TCDVd from potato; and CEVd, CSVd and IrVd from ornamental plants (Table 1). Although, not all known viroid species were encountered in this study, it may be extrapolated from the range of viroid species detected, that all known pospiviroids except CLVd can be detected using this primer pair. For example, the smallest Pospiviroid, the CSVd (354 nt) and the largest known viroids, CEVd and IrVd (372 nt) were detected in many ornamental plant samples (Fig. 2). Most viroid species except CLVd, have perfect match in primer sequence in their genomic RNA, therefore, if other pospiviroids were present in the ornamental plants they would have been detected. Probably a larger sampling of ornamental plants would have detected other viroid species belonging to genus Pospiviroid. The primer pair described here is specific to closely related Pospiviroid species, because some plants, for example Coleus contain the Coleviroid species but they were not

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amplified in RT-PCR with pospiviroid primer pair (Fig. 2) and many samples tested (Table 2). Similarly, it may have been the high mis-matches at the 3 end of primer in CLVd RNA that may have prevented the amplification of the viroid in pouch flower (Nemantanthus wettstenii) which is invariably infected (symptomlessly) with CLVd (Singh et al., 1992). Finding of CSVd in Vinca and Verbena and CEVd in Impatiens species at a high frequency (Table 1) indicates widespread viroid contamination of these species. These ornamentals are very popular, particularly the Impatiens species. It is not known, whether the viroids are seed-borne or are being spread through vegetative multiplications by contaminated tools. Majority of viroids are known to spread in nurseries and greenhouses by the use of viroidcontaminated tools (Singh, 1983). Impatiens plants grown from seed were viroid-free, while double-colour Impatiens, multiplied by cuttings, were invarably infected. This suggests that viroids may be primarily spreading by contaminated tools during multiplication. The availability of an RTPCR procedure using a generic Pospiviroid primer pair may prove useful in certifying viroid-free plants for nurseries.

Acknowledgements The Award of scholarship to H.B. by the Scientific and Technical Research Council of Turkey (TUBITAK), the assistance and generosity of the staff of Scott’s Nursery (Fredericton) in allowing frequent sampling of the plants, the technical assistance of A. Dilworth, and the editorial assistance of Dr. A. Singh (AgraPoint International Inc.) is highly appreciated.

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