Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses1

Journal of Virological Methods 74 (1998) 167 – 178

Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses1 V. Jacobi a, G.D. Bachand b, R.C. Hamelin a, J.D. Castello c,* a

Natural Resources Canada, Canadian Forest Ser6ice, Laurentian Forestry Centre, 1055 Rue du PEPS, P.O. Box 3800, Sainte-Foy, Que´bec G1V 4C7, Canada b Riley Robb Hall, Department of Agricultural and Biological Engineering, Cornell Uni6ersity, Ithaca, NY 14853, USA c State Uni6ersity of New York, College of En6ironmental Science and Forestry, Faculty of En6ironmental and Forest Biology, 1 Forestry Dri6e, Syracuse, NY 13210, USA Received 27 February 1998; received in revised form 18 May 1998; accepted 18 May 1998

Abstract Immunocapture (IC) RT-PCR assays were developed for detection of tomato (ToMV) and tobacco mosaic (TMV) tobamoviruses in spruce and pine extracts. When purified viruses were diluted in root or needle extracts of virus-free conifer seedlings, both IC-RT-PCR assays detected their respective target viruses at concentrations of 10 – 100 fg ml − 1. This compared to ELISA detection sensitivities of 1 ng ml − 1. Primers were designed from regions of high sequence diversity. Specificity of all primer pairs was confirmed by sequencing of PCR products. PCR distinguished more reliably between the two viruses than ELISA. Moreover, a multiplex IC-RT-PCR assay for the simultaneous detection and differentiation of TMV and ToMV was developed. When root extracts were seeded with both viruses simultaneously, the multiplex assay detected each virus at concentrations of 1 – 10 pg ml − 1. Six TMV and 18 ToMV isolates from various hosts, water samples and a soil sample were amplified and differentiated by multiplex IC-RT-PCR. No amplifications were observed against pepper mild mottle and ribgrass mosaic tobamoviruses and against six viruses belonging to other taxonomic groups. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Tobamoviruses; Detection and differentiation; Multiplex immunocapture RT-PCR

1. Introduction * Corresponding author. Tel.: + 1 315 4706789; fax: +1 315 4706934; e-mail: [email protected] 1 Sequence data from this article have been deposited with the GenBank Data Library under accession numbers AF042031, AF042032 and AF042033.

Tomato mosaic (ToMV) tobamovirus has been isolated from red spruce trees (Jacobi et al., 1992) and water, cloud, and soil samples in the north-

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eastern United States (Jacobi and Castello, 1991; Castello et al., 1995; Fillhart et al., 1998). A water isolate of the virus was transmitted subsequently to red spruce, black spruce, and balsam fir seedlings. A soil- and waterborne mechanism for spreading of ToMV from the roots of infected to noninfected conifer seedlings has been suggested (Jacobi and Castello, 1992). The same isolate of ToMV was later shown to reduce the growth of red spruce seedlings by more than 50% (Bachand et al., 1996). These findings raise questions concerning the importance of ToMV in coniferous trees. Further investigations into virus host range, distribution, and spread via water-, soil-, and airborne mechanisms (Fillhart et al., 1997) are needed to address these questions in more detail. Low virus concentration in conifer tissues (Jacobi and Castello, 1992; Jacobi et al., 1992; Bachand et al., 1996) and soil and water samples (Jacobi and Castello, 1991; Fillhart et al., 1998) has hampered progress. Moreover, the closely related tobacco mosaic tobamovirus (TMV) has been detected in forest tree species and in soil and water samples collected under forest stands (Nienhaus and Castello, 1989; Bu¨ttner and Nienhaus, 1989a,b; Fillhart et al., 1998). Finally, the presence of ToMV in field trees and greenhouse seedlings is not associated with obvious disease symptoms (Jacobi et al., 1992; Jacobi and Castello, 1992; Bachand et al., 1996). Consequently, ToMV detection in conifers has to rely solely on laboratory tests. In previous studies, a direct enzyme-linked immunosorbent assay (ELISA) has been employed for ToMV detection in tissue and soil samples, sometimes involving lengthy purification and concentration protocols (Jacobi et al., 1992; Jacobi and Castello, 1992; Fillhart et al., 1998). Moreover, cross reaction between ToMV and TMV antisera (Jacobi and Castello, 1991) limits serological differentiation of these two viruses by ELISA. The technique is therefore unsuitable for detecting mixed infections in tissues and monitoring both viruses separately in soil and water samples. A recent report of the presence of both TMV and ToMV in a soil sample collected in a forest stand from Tully,

NY, underlines the need for an assay with greater specificity than ELISA (Fillhart et al., 1998). Recently, immunocapture reverse transcriptase-polymerase chain reaction (IC-RT-PCR) assays (Jansen et al., 1990) have been developed for the detection of several economically important fruit tree viruses (Wetzel et al., 1992; Candresse et al., 1995a,b; Rowhani et al., 1995; Jacobi et al., 1996). In IC-RT-PCR, the immunocapture of virions from crude plant extracts is carried out directly in PCR tubes in a fashion reminiscent of the ELISA assay. This step concentrates and pre-purifies the virus particles. RT-PCR is then undertaken on the trapped virus particles omitting the need for nucleic acid extraction. When compared with ELISA, IC-RT-PCR improved detection sensitivity by several orders of magnitude (Wetzel et al., 1992; Candresse et al., 1995a,b; Rowhani et al., 1995). Based on these promising results, we decided to develop a multiplex IC-RT-PCR test for the detection of TMV and ToMV in conifer tissue extracts. It was hypothesized that IC-RT-PCR would improve both detection sensitivity and detection specificity for TMV and ToMV when compared with ELISA. The objectives of this study were to (i) develop separate IC-RT-PCR tests for each virus and to optimize assay conditions; (ii) to develop a multiplex IC-RT-PCR test for the simultaneous detection of both viruses; (iii) to evaluate primer specificity against a range of TMV and ToMV isolates, other tobamoviruses, and plant viruses which are members of other taxonomic groups; and (iv) to compare sensitivity and specificity of the IC-RT-PCR approach with that of ELISA systems currently in use. To our knowledge, this is the first report of IC-RT-PCR tests for both TMV and ToMV and a multiplex IC-RT-PCR assay that can reliably detect and discriminate between two related plant viruses. This approach should also be amenable to the detection of other plant viruses (Nemchinov et al., 1995). A preliminary report has been published (Jacobi et al., 1997). Potential applications are discussed.

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2. Materials and methods

2.1. Plant material and inoculations Tobacco (Nicotiana tabacum L. cv. Turkish) and Chenopodium quinoa Willd. virus indicator plants were grown in Topgrow Mix (Tourbie`re Lafaille, Coaticook, Que., Canada), fertilized biweekly with all-purpose fertilizer (Lemoine Tropica Canada, Montreal, Que.), and maintained in controlled environment growth chambers (25°C day and at 20°C during night temperatures; 16 h light period at 175– 275 mmol s − 1 m − 2, and 8 h dark period). Carborundum-dusted leaves of indicator plants were inoculated either directly with infected leaf tissue or with purified virus preparations diluted in 10 mM phosphate buffer (pH 7.0). Virus-free white pine (Pinus strobus L.), red spruce (Picea rubens Sarg.) and black spruce (Picea mariana (Mill.) B.S.P.) seedlings were grown in Promix potting mixture (Premier Horticulture, Rivie`re-du-Loup, Que., Canada) from seed provided by the seed bank of the Laurentian Forestry Centre, Sainte-Foy, Que. Seedlings were maintained in a greenhouse at 18 – 23°C, 16 h daylength, and fertilized biweekly with quarter strength all-purpose fertilizer.

2.2. Tissue extracts Test samples consisted of three types: (i) crude extracts prepared from fresh tissues of herbaceous indicator plants and conifer seedlings; (ii) crude extracts prepared from dried leaf tissues of herbaceous indicator plants; and (iii) dilutions of purified viruses. The corresponding extracts were prepared as follows: (i) for ELISA, 0.1 g of tobacco or conifer tissues were macerated in ELISA extraction buffer (PBS-TP; Jacobi and Castello, 1992) to give a final dilution of 1:5. For IC-RTPCR, 0.05–0.1 g of tobacco or conifer tissues were extracted in ELISA extraction buffer containing 0.2% ovalbumin (PBS-TPO) to give a final dilution of 1:10. Acid-washed sand (Produits Chimiques ACP Chemicals, Saint-Le´onard, Que.) was added to aid tissue disruption. These crude stock extracts were incubated for at least 30 min at 4°C prior to diluting the supernatant a further

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1:10 in buffer to give a final dilution of 1:100 for use in IC-RT-PCR analysis. Extracts were prepared directly in 1.5 ml Eppendorf tubes (Gordon Technologies, Mississauga, Ont.) with plastic pestles (VWR Canlab, Ville Mont-Royal, Que.) attached to a drill. Pestles were soaked for 16–20 h in 0.5 N sodium hydroxide, 1% sodium dodecyl sulfate, soaked in several changes of deionized water, autoclaved, and reused; (ii) dried leaf tissues were extracted in PBS-TPO as described above and diluted to 10 − 3 for testing by IC-RTPCR; (iii) purified viruses were diluted in PBS-TP or PBS-TPO for testing by ELISA or IC-RTPCR, respectively, to concentrations ranging from 1 mg ml − 1 to 10 fg ml − 1.

2.3. Virus isolates and antisera The following tobamoviruses were used to develop the single-virus and multiplex IC-RT-PCR systems: WF-38 (ToMV) which had been previously transmitted from a stream water concentrate from Whiteface Mt., NY (Jacobi and Castello, 1991) and two isolates of the U1 strain of tobacco mosaic virus (TMV-U1Z; M. Zaitlin, Cornell University, Ithaca, NY; and TMV-U1D; W.O. Dawson, University of Florida, Lake Alfred, FL). Viruses were purified (Gooding and Herbert, 1967), and subjected to equilibrium density gradient centrifugation in cesium chloride. Immunoglobulin G (IgG) fractions purified from rabbit polyclonal antisera to TMV-U1Z and to ToMV-38 were used for all ELISA and IC-RTPCR experiments. Additional isolates of (i) ToMV and TMV; (ii) other tobamoviruses and (iii) viruses belonging to other taxonomic groups were used to verify the specificity of the multiplex IC-RT-PCR test (Table 1). Isolates under (i) and (iii) were from the virus collection of J.D. Castello, those under (ii) from the virus collection of the Pacific Agri-Food Research Centre, Summerland, B.C.

2.4. Primer design Primer sequences were based on the published sequences of genomes of TMV-L (type strain of ToMV, Ohno et al., 1984) and TMV 6ulgare

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Table 1 Taxonomic groups, isolate designations, and host and geographical origins of viruses used for ELISA and IC-RT-PCR assays Group

Virus

Isolate

Reference

Designation

Origin

Bromo6iruses

Brome mosaic virus

BMV

European beech, Germany

Cucumo6iruses

Cucumber mosaic virus Tobacco necrosis virus Cherry leaf roll virus Potato virus X Potato virus Y Tomato mosaic virus

CMV

—

TNV

Willow; Tully, NY

CLRV

European beech, Germany

PVX PVY CAT, HMF, WFb ToMV-C ToMV-DA ToMV-DW TMV-L ToMV-LC ToMV-M ToMV-PP ToMV-RST ToMV-RSW ToMV-ST TMV-U1Z

— — Streams and ponds, NY

Nienhaus and Castello, 1989 — — Jacobi and Castello, 1991

Cloud concentrate, Whiteface Mt., NY Dahlia, NY Dogwood, TN Tomato, Japan Lilac, Arnold Arboretum, MA Tomato, Malaysia Pitch pine, NY Red spruce, Tully, NY Red spruce, Whiteface Mt., NY Soil, Tully, NY —

Castello et al., 1995 — Reddick, 1989 Ohno et al., 1984 Castello et al., 1992 Abdul-Samad et al., 1996 — — Jacobi et al., 1992 Fillhart et al., 1998 —

TMV-U1D TMV-MA TMV-ST TMV-WA

— Moraine ash, NY Soil, Tully, NY White ash, Heiberg Memorial Forest, NY Willow, Tully, NY Pungent pepper; SC

— Castello et al., 1983 Fillhart et al., 1998 Castello et al., 1984

Sweet pepper; Richmond, B.C., Canada —

Beczner et al., 1997

Necro6iruses Nepo6iruses Potex6iruses Poty6iruses Tobamo6iruses

Tobacco mosaic virus

Pepper mild mottle virus

TMV-WI PMMV Ca10

Ribgrass mosaic virus

RiMV

Nienhaus and Castello, 1989 R. Provvidenti, Geneva, NY —

— Wetter, 1984

—

a

All virus isolates were provided by J.D. Castello except for PMMV, Ca10 and RiMV which were from the virus collection of the Pacific Agri-Food Research Centre, Summerland, B.C., Canada. b Comprise a total of eight water isolates.

(Goelet et al., 1982). Two primer pairs were chosen for specific amplification of ToMV WF-38: (a) ToMV-1 (5%-TGG GCC CCA ACC GGG GGT3%; reverse primer, corresponding to nucleotides 6367 –6384 of TMV-L) and ToMV-3 (5%-TTC AAC AGC AGT TCA GCG AG-3%; forward primer, corresponding to nucleotides 5836 – 5855

of TMV-L) amplifying a 549 bp PCR product representing parts of the 3%-untranslated region and the viral coat protein gene; or (b) ToMV-5 (5%-CTC CAT CGT TCA CAC TCG TTA CT-3%; reverse primer, corresponding to nucleotides 5436–5458 of TMV-L) and ToMV-6 (5%-GAT CTG TCA AAG TCT GAG AAA CTT C-3%;

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forward primer, corresponding to nucleotides 4951 –4976 of TMV-L) amplifying a 508 bp PCR product within the viral movement protein gene. Specific amplification of TMV-U1 was achieved with primers TMV-1 (5%-GAC CTG ACA AAA ATG GAG AAG ATC T-3%; forward primer, corresponding to nucleotides 4948 – 4972 of TMV 6ulgare) and TMV-2 (5%-GAA AGC GGA CAG AAA CCC GCT G-3%; reverse primer, corresponding to nucleotides 5348 – 5369 of TMV 6ulgare) producing a 422 bp PCR product within the viral movement protein gene. Primer pairs TMV1/-2 and ToMV-5/-6 had been specifically designed for use in multiplex IC-RT-PCR such that the locations of TMV-1 and ToMV-6 within the genome of their respective target virus match exactly. Therefore, during RT-PCR with both primer pairs, assay specificity would not only rely on the number of mismatches between the sequences of a given primer and that of the heterologous virus at that primer binding site but also on direct competition between the TMV-1 and ToMV-6 primers for the same binding site, thus using the higher affinity of the homologous primer to outcompete its heterologous counterpart.

2.5. De6elopment of single-6irus IC-RT-PCRs In preliminary experiments with ToMV-38 and primer pair ToMV-1/-3, the effect of tissue concentration on assay performance was determined: root and needle tissues of white pine, red spruce, and black spruce seedlings were extracted in PBSTPO at 1/20, 1/100 and 1/500, seeded with purified virus, and assayed as outlined below. In addition, the effect on assay sensitivity of a 5 min denaturation step at 95°C prior to RT-PCR was assessed. The following protocol was adopted for all tests: thin walled or standard 0.5 ml microtubes for PCR (Gordon Technologies, Mississauga, Ont.) were coated with 50 ml tube − 1 of 2 mg ml − 1 of virus-specific IgG in sodium carbonate coating buffer (Clark and Adams, 1977). Tubes were incubated for 2 – 3 h at 37°C and the coating solution was pipetted off. Excess antibody was removed by adding 80 ml tube − 1 of PBS-T (phosphate buffered saline, pH 7.4, containing

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0.05% Tween 20), spinning for 15 s at 5000 rpm in a microfuge, and pipetting off the wash solution. Sample extracts were added (50 ml tube − 1) and incubated overnight at 4°C or 2–3 h at 37°C. Extracts were removed by pipette, 80 ml tube − 1 of PBS-T added, spun as above and the wash solution pipetted off. This PBS-T washing step was repeated once, and followed by a final wash with deionized water. RT-PCR was performed as a one-tube protocol in a final volume of 25 ml as follows: 20 ml tube − 1 of a solution containing primers, PCR buffer, Triton X-100, and dNTPs (Pharmacia Biotech, Baie d’Urfe´, Que.) were added, vortexed, spun briefly and overlaid with 40 ml of mineral oil (Sigma, St. Louis, MO). Samples were incubated at 95°C for 5 min and chilled immediately on ice for 5 min. Five microlitres tube − 1 of a solution containing Taq DNA Polymerase (Boehringer Mannheim Canada, Laval, Que.) and SUPERSCRIPT™ II RNase H − Reverse Transcriptase (Canadian Life Technologies, Burlington, Ont.) were added. Final concentrations of reagents and enzymes were as follows: 10 mM Tris–HCl, pH 8.3 (at 20°C), 50 mM KCl, 1.5 mM MgCl2, 0.3% Triton X-100, 0.4 mM each dNTP, 10 pmole each primer, 1 unit Taq DNA Polymerase, and 0.5 units Reverse Transcriptase. Tubes were spun briefly, and incubated in a PTC100 thermal cycler (MJ Research, Watertown, MA). Initially, the same incubation regime was used for all primer pairs: 42°C for 45 min and 92°C for 2 min, followed by 35 cycles of 92°C for 30 s, 58°C for 30 s, and 72°C for 1 min with a final extension at 72°C for 10 min. During development of the multiplex assay the incubation regime for primer pairs ToMV-5/-6 and TMV-1/2 was modified (see below), and also applied when these primers were used for single-virus tests. PCR products were separated in 1.5% agarose gels in TAE buffer (1x: 0.04 M Tris–acetate, 0.001 M EDTA), stained with ethidium bromide, and visualized under UV light.

2.6. Multiplex IC-RT-PCR The protocol for the multiplex assay was identical to the single-virus assays described above except for the following modifications: PCR tubes

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were coated with antibodies to both viruses at concentrations of 2 mg ml − 1 each; RT-PCR was carried out with primer pairs ToMV-5/-6 and TMV-1/-2 in the same mix and the incubation regime was changed as follows: 50°C for 45 min and 94 for 2 min, followed by 35 cycles of 94°C for 30 s, 62°C for 45 s, and 72°C for 1 min with a final extension at 72°C for 5 min. To verify specificity of the modified assay, a total of 18 ToMV and six TMV isolates, three tobamoviruses other than ToMV and TMV and six viruses belonging to other taxonomic groups were tested (Table 1). The assay also was used to confirm the presence of both ToMV and TMV in a forest soil sample. Soil was collected on February 3, 1997 from Heiberg Memorial Forest, Tully, NY, within 10 m of a small stream from which ToMV had been recovered previously (Jacobi and Castello, 1991). The soil concentrate (Fillhart et al., 1998) was rubbed onto carborundum-dusted leaves of C. quinoa plants. Eleven local lesions appeared on the inoculated leaves. The lesions were of four types: (1) six lesions were round and necrotic, (2) one lesion was necrotic with a chlorotic halo, (3) four lesions were necrotic rings, and (4) one chlorotic lesion was derived from the single chlorotic lesion with halo. The different lesion types were carefully separated and re-inoculated onto individual C. quinoa plants. Infected leaf tissues (samples 1–4) as well as a composite of the original preparations (sample 5) were assayed by multiplex IC-RT-PCR.

2.8. ELISA and IC-RT-PCR comparison Direct double antibody sandwich (DAS)ELISA (Clark and Adams, 1977) was conducted as described by Bachand et al. (1996). Samples were considered positive, if the mean absorbance at A405 of two sample wells was greater than the mean A405 plus three standard deviations of two wells containing either ELISA extraction buffer or virus-free root or needle extracts. Buffers and tissue extracts prepared from healthy seedlings of white pine, red spruce and black spruce were prepared for each assay as previously described. To compare assay sensitivities and specificities, buffers and extracts were seeded with either purified ToMV-38 or TMV-U1D at concentrations ranging from 1 mg ml − 1 to 10 fg ml − 1 and tested with each assay in the homologous and heterologous combination.

3. Results

3.1. IC-RT-PCR de6elopment A 10 − 2 dilution of root and needle extracts in PBS-TPO worked best for detection of ToMV in tissues of all three conifer species tested. The incorporation of a 5 min denaturation step at 95°C prior to addition of enzymes and RT-PCR improved assay sensitivity to 10–100 fg ml − 1 ToMV-38 as compared with 10–100 pg ml − 1 ToMV-38 without denaturation (data not shown).

2.7. PCR product sequencing 3.2. ELISA and IC-RT-PCR comparison Purified ToMV-38 was diluted to 0.5 mg ml − 1 in healthy conifer root extract and amplified either with primer pairs ToMV-1/-3 or ToMV-5/-6 using the initial RT-PCR conditions described above for single-virus detection. Purified TMVU1D was amplified in the same way using primer pair TMV-1/-2. IC-RT-PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Chatsworth, CA) and sequenced on an ABI PRISM™ 377 DNA Sequencer (Universite´ Laval, Sainte-Foy, Que.). Generunner Version 3.00 (Hastings Software) was used for sequence analyses.

TMV and ToMV ELISA systems detected their target viruses at 1 ng ml − 1. However, they also reacted to the respective heterologous viruses if these were present at concentrations equal to or greater than 10–25 ng ml − 1 (Table 2). Therefore both ELISA assays failed to distinguish clearly between TMV and ToMV. In contrast, the singlevirus IC-RT-PCR tests detected as little as 0.1 pg ml − 1 of purified homologous virions corresponding to 1500 particles ml − 1. This is a 10000-fold increase in detection sensitivity when compared with ELISA. Cross reaction to the respective het-

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erologous viruses were not observed if these were present at concentrations equal to or below 1 mg ml − 1, the highest virus concentration tested (Table 2). The multiplex system was as sensitive as single-virus IC-RT-PCRs if only one virus at a time was captured (Fig. 1A and B). In addition, neither the TMV-1/-2 nor the ToMV-5/-6 primer pairs amplified their respective heterologous viruses (Fig. 1A and B). A 10- to 100-fold reduction in detection sensitivity was observed when equal amounts of both viruses were captured and amplified simultaneously (Table 2, Fig. 1C).

3.3. Specificity of multiplex IC-RT-PCR The multiplex assay amplified all 24 isolates as expected, i.e. in accordance with their previous designation as either TMV or ToMV (results for sixteen isolates are presented in Fig. 2). Neither the two PMMV isolates and RiMV nor the six viruses belonging to other taxonomic groups were amplified (data not shown). The assay could clearly correlate the different lesion types obtained after inoculation of C. quinoa with a soil eluate concentrate from Tully, NY, to either TMV or ToMV infection (Fig. 3). These results Table 2 Comparison of the sensitivity and specificity between ELISA and IC-RT-PCR assays for detection of tomato mosaic (ToMV) and/or tobacco mosaic (TMV) virusesa Test virus

TMV-U1D ToMV-38 Both a

ELISAb

IC-RT-PCRb

TMV

ToMV

TMV

ToMV

Both

1 10 – 25 nt

10 1 nt

0.1 nd 0.1

nd 0.1 0.1

0.1 0.1 1–10

Purified preparations of ToMV-38 and/or TMV-U1D were either diluted in the respective extraction buffers or in conifer root and needle extracts prepared as described in the text. Detection limits for ELISA are given in ng/ml, those for IC-RT-PCR in pg/ml. b TMV, ToMV, single-virus IC-RT-PCR; both, multiplex ICRT-PCR; nd, not detected; nt, not tested. Sensitivities of single-virus IC-RT-PCR assays in the presence of both viruses refer to detection of the respective homologous viruses (heterologous viruses were not amplified); sensitivity of the multiplex IC-RT-PCR assay refers to the simultaneous detection of both viruses.

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were later confirmed by bioassay onto black turtle beans (Phaseolus 6ulgaris L. cv. Black Turtle 1). Only sample 1 yielded red local lesions indicative of TMV, whereas the other extracts did not produce symptoms, indicating the presence of ToMV but not TMV (Fillhart et al., 1998).

3.4. Sequence analysis ToMV sequences were aligned with the sequence of TMV-L and the TMV sequence with that of TMV 6ulgare. Sequencing results confirmed the viral origin of the amplified products and revealed some differences between our TMV and ToMV isolates and the respective reference sequences (GenBank accession numbers AF042031, AF042032, and AF042033). Part of the ToMV-1/-3 PCR product overlapped a 347 bp PCR product of ToMV-38 previously sequenced (Castello et al., 1995). The two sequences in this region matched exactly.

4. Discussion This study was conducted to develop an ICRT-PCR assay for detection of TMV and ToMV in conifer tissues that would improve both detection sensitivity and specificity when compared with the ELISA systems currently in use. The IC-RT-PCR approach was chosen rather than hybridization or RT-PCR tests because of (i) high sensitivity due to the initial immunocapture enrichment step followed by PCR amplification; (ii) high specificity due to the combination of virus specific antibody capture and primer specificity; and (iii) virus detection directly in crude plant extracts rather than in partially or highly purified nucleic acid preparations. Studies on the detection of economically important fruit tree viruses have documented the potential of IC-RT-PCR to overcome limitations for virus detection in crude tissue extracts of woody plants (Wetzel et al., 1992; Candresse et al., 1995a,b; Jacobi et al., 1996). Initial experiments during development of the single-virus tests indicated that a denaturation step at 95°C on immobilized virions prior to addition of enzymes and RT-PCR increased assay sensitiv-

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Fig. 1. (A – C) Sensitivity and specificity of multiplex IC-RT-PCR for detection of tomato mosaic (ToMV-38) and tobacco mosaic (TMV-U1D) viruses. All tubes were coated with purified Immunoglobulin G to both viruses and all RT-PCR reactions contained primer pairs TMV-1/-2 and ToMV-5/-6. Healthy black spruce root extract diluted at 10 − 2 in PBS-TPO served as diluent for purified TMV-U1D (A), purified ToMV-38 (B), or both viruses (C). Lane numbers are identical for each section of the figure: 100 bp DNA ladder (1+13); TMV-U1D (2-10A), ToMV-38 (2-10B), or both viruses (2-10C) at final concentrations of 1 mg/ml, 100-, 10-, and 1 ng/ml, 100-, 10-, and 1 pg/ml, 100- and 10 fg/ml; healthy black spruce root extract (11A-C); blank (12A-C).

ity. Consequently, the denaturation step also was included in the final protocol for simultaneous detection of TMV and ToMV by multiplex ICRT-PCR. Others also employed manipulations such as heating, freeze – thaw cycles and addition of detergents to aid disruption of captured virus

particles and improve assay performance (Wetzel et al., 1992; Wyatt et al., 1993; Minafra and Hadidi, 1994; Barbara et al., 1995; Kokko et al., 1996; Lemmetty et al., 1997). Efficacies of these treatments appear to vary substantially between different viruses and, therefore, the best condi-

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Fig. 2. (A – B) Amplification and differentiation of twelve ToMV and four TMV isolates by multiplex IC-RT-PCR. All tubes were coated with purified Immunoglobulin G to both viruses and all RT-PCR reactions contained primer pairs TMV-1/-2 and ToMV-5/-6. (A) Purified preparations of nine ToMV isolates were diluted to 10 ng/ml in PBS-TPO, captured, and amplified: 100 bp DNA ladder (1 +13), HMF-32 (2), HMF-35 (3), HMF-36 (4), WF-38 (5), WF-39 (6), CAT46 (7), CAT-49 (8), WF-51 (9), ToMV-C (10), PBS-TPO used for immunocapture (11), blank (12). (B) Three ToMV and four TMV isolates were extracted from dried or fresh infected leaf tissues of herbaceous indicator plants in PBS-TPO at a dilution of 10 − 3, captured and amplified: 100 bp DNA ladder (1+ 13), TMV-WA (2); TMV-MA (3), ToMV-PP (4), TMV-WI (5), ToMV-RST (6), TMV-ST (7), ToMV-ST (8), PBS-TPO used for immunocapture (9), healthy tobacco leaf extract used for immunocapture (10), mix of TMV-WA and ToMV-RST (11), blank (12).

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tions for any particular virus are difficult to predict and need to be assessed individually. Detection of TMV and ToMV in conifer root and needle crude extracts by IC-RT-PCR is at least 104 times more sensitive than ELISA when only one virus is trapped at a time. In the presence of equal amounts of captured particles of both viruses, the IC-RT-PCR is still 102 –103 times more sensitive than ELISA for detection of individual viruses. A direct comparison with ELISA detection sensitivities in the presence of both viruses was not possible as the ELISA assay could not reliably distinguish between TMV and ToMV. The IC-RT-PCR detection limit of 100 fg/ml of either TMV or ToMV corresponds to 1500 particles/ml. In some cases, bands were visible even at 10 fg ml − 1 of virus. These results compare well with those reported for other plant viruses. The IC-RT-PCR detection limits for apple chlorotic leaf spot virus (ACLSV) and plum pox virus (PPV) were 0.1 pg ml − 1 corresponding to approximately 1000 particles of ACLSV or 2000 particles of PPV (Candresse et al., 1995a,b). Detection limits for prune dwarf (PDV), prunus necrotic ringspot (PNRSV), grapevine fanleaf (GFLV), and cherry leaf roll (CLRV) viruses ranged from 0.2 to 2 pg ml − 1 (Rowhani et al., 1995). Due to the lack of quantitative data on multiplex IC-RT-PCR detection of other plant viruses, we could not contrast our results with those of others. In contrast with ELISA systems, which could not discriminate between TMV and ToMV, our experiments confirmed the specificity of primer pairs TMV-1/-2 and ToMV-5/-6 for their respective target viruses in both single-virus and multiplex IC-RT-PCR assays. Based on this finding, the multiplex system was used for all further tests. Not only could the primers distinguish between TMV and ToMV but they also were able to amplify a wide range of isolates of each virus. Primer pairs specific for ACLSV or PPV have been used successfully with single-virus IC-RTPCR assays to amplify different isolates representing the host and geographic range of each virus (Candresse et al., 1995a,b). Our primers did not amplify three other tobamoviruses and one member of each of six other taxonomic groups. This is

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Fig. 3. Detection of TMV and ToMV by multiplex IC-RTPCR in a soil concentrate from Tully, NY. After inoculation of the concentrate onto C. quinoa leaves, eleven local lesions, segregated into four types, appeared. The lesion types were carefully separated and re-inoculated onto individual C. quinoa plants. These leaf tissues (samples 1 to 4), as well as a composite of the original preparations (sample 5), were extracted at 10 − 3 in PBS-TPO and assayed by multiplex IC-RTPCR: 100 bp DNA ladder (1 + 9), samples 1–5 (2–6), PBS-TPO used for immunocapture (7), blank (8).

important because tobamoviruses other than TMV and ToMV may be present in forest ecosystems including soil and water samples. Similarly, the six members of other virus groups were isolated previously from either forest trees or from soil and water samples collected under forest stands (Nienhaus and Castello, 1989). The detection of both TMV and ToMV by multiplex IC-RT-PCR in leaf tissue extracts of C. quinoa plants inoculated with a concentrated soil eluate prepared from soil collected in a forest stand in Tully, NY, provided further evidence of the utility of this assay. To confirm the presence of both viruses by traditional methods would have involved agar-gel double-diffusion test anal-

ysis (Jacobi and Castello, 1991), or inoculation of two herbaceous indicator plant species each producing symptoms only in response to infection by either TMV or ToMV even in the presence of both viruses. Gel diffusion tests require substantial amounts of virus and antiserum, which may not always be available. Few indicator plants distinguish reliably between TMV and ToMV: Phaseolus 6ulgaris cv. Black Turtle 1 is susceptible to infection by TMV and some isolates of ToMV but only TMV infection will result in the production of red, necrotic local lesions; Cucumis sati6us L., on the other hand, produced local lesions if ToMV was present in the inoculum, but no symptoms were observed upon inoculation with TMV (Jacobi and Castello, 1991; Fillhart et al., 1998). One must also consider that it takes between 3 to 7 days for symptoms to develop whereas IC-RTPCR can be performed in 1 day, including gel analysis. The multiplex IC-RT-PCR assay for detection and differentiation of TMV and ToMV should be useful in the following applications: tests on field trees to monitor virus distribution and host range; tests on virus-inoculated greenhouse seedlings to study infection mechanisms and virus movement; and tests on environmental samples such as water, soil, cloud and fog. Its high sensitivity may help to eliminate the need for sample purification and concentration prior to virus detection. The assay should also be applicable to detect and differentiate TMV and ToMV in their major solanaceous hosts tobacco, tomato, and pepper. In addition, it is a quick screening tool to ‘type’ unknown isolates as either TMV, ToMV, or both and verify the authenticity of virus stock cultures, be it dried leaf material or purified virus preparations. Recently, IC-RT-PCR assays have been used to amplify isolates of ACLSV and PPV directly from their woody hosts and sequence cloned PCR products to study the molecular variability of these viruses without the need to transmit them to herbaceous indicator hosts (Candresse et al., 1995a,b). Beet necrotic yellow vein virus (BNYVV) was amplified by IC-RT-PCR and PCR products subjected to single-strand conformation polymorphism (SSCP) analysis to identify mixed infections and to assign BNYVV isolates to specific strain groups (Koenig et al., 1995).

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Further studies have been initiated in our laboratories on the use of multiplex IC-RT-PCR for detection of TMV or ToMV in virus inoculated conifer seedlings and in field trees. In addition, we have begun to sequence and SSCP-analyse ICRT-PCR amplified PCR products from a range of isolates of each virus to determine their molecular variability.

Acknowledgements The authors thank Dr D.M. Rochon, Pacific Agri-Food Research Centre, Summerland, BC, Canada, for kindly providing the two isolates of PMMV and an isolate of RiMV to V. Jacobi and Drs N. Abdul-Samad, W.O. Dawson, J. Hamacher, R. Provvidenti, B.B. Reddick and M. Zaitlin for providing various virus isolates to J.D. Castello. We also thank Pamela Cheers for editorial services.

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