Arch Virol (2003) 148: 381–388 DOI 10.1007/s00705-002-0926-z
Identification of two nucleotide sequence sub-groups within Potato mop-top virus∗ Brief Report S. L. Nielsen and M. Nicolaisen Department of Plant Protection, Danish Institute of Agricultural Sciences (DIAS), Research Centre Flakkebjerg, Slagelse, Denmark Received May 27, 2002; accepted September 17, 2002 c Springer-Verlag 2002 Published online November 29, 2002
Summary. To evaluate the variation of Potato mop-top pomovirus from potato fields, 21 isolates were collected from different Danish locations. Reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) of regions of RNA2 was performed for all 21 isolates resulting in the establishment of two sub-groups of isolates. The nucleotide sequence of a region encoding part of the ‘readthrough protein’ of RNA2 was compared for 9 of these isolates. This sequence analysis confirmed the RT-PCR-RFLP grouping. The isolates were tested for symptom expression in indicator plants and grouped according to symptom development. No correlation between grouping based on symptom development and genotype was observed. ∗ Rust coloured rings, arcs and flecks in potato tubers known as spraing, are caused by infection with Potato mop-top virus (PMTV) or Tobacco rattle virus (TRV). PMTV, the type member of the genus Pomovirus, has tubular particles encapsidating three RNA molecules. RNA1 (6.1 kb.) encodes the viral RNA-dependent RNA polymerase, RNA2 (3.1 kb.) encodes the coat protein (CP) and a readthrough protein presumably involved in vector transmission, and RNA3 (3.0 kb.) encodes the triple gene block and a predicted protein of unknown function [15]. PMTV is transmitted by Spongospora subterranea f. sp. subterranea [1, 5]. The virus can survive in the soil for several years inside resting spores of S. subterranea ∗ Nucleotide
sequence data reported are available in the GenBank database under the accession numbers: AF508250–AF508257.
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f. sp. subterranea [2, 6]. An earlier survey of PMTV in Denmark showed that both the virus and its vector are widespread in potato production areas [10]. However, no comprehensive comparison of Danish isolates of PMTV has been carried out, although a few Danish isolates have been included in some studies: 1) a single Danish isolate was included in a test of monoclonal antibodies specific to PMTV [17] in which the Danish isolate was indistinguishable from other isolates collected from six countries; 2) Sandgren [14] did not find differences between four Danish isolates in their reaction to 11 anti-PMTV monoclonal antibodies, however some differences were detected between Danish and Swedish isolates. Sandgren et al. [15] compared RNA2 of two Swedish isolates (PMTV-S and -Sw), and found several nucleotide variations in the readthrough region, whereas the CP regions were almost identical. No differences in biological properties of these two isolates were reported. Mayo et al. [9] compared coat protein sequences of Scottish and Peruvian isolates and also found very limited variation. However, it is well documented that the same potato variety can show differential development of mop-top spraing symptoms when grown in Sweden, Finland or Denmark [18, 10, 12, 13]. This may be caused by different genotypes of PMTV, as suggested by Sandgren [13], although the effect also could be caused by factors such as climate or soil type as suggested by Nielsen and Mølgaard [10]. Harrison and Jones [4] compared 11 isolates of PMTV from Scotland and Northern Ireland and found differences in symptom development in three indicator plant species. The present study was carried out to investigate variation between a number of PMTV isolates from soils recovered from different potato production areas in Denmark. Parts of the nucleotide sequence of the RNA2 of 21 isolates of PMTV were investigated by sequencing and by RT-PCR-RFLP, using restriction enzymes selected to distinguish between the previously reported nucleotide sequences of PMTV-S and -Sw [15]. Furthermore, the response of 3 indicator plant species to infection by each of the 21 isolates was recorded. Forty-five soil samples were collected from fields in Jutland, where PMTV had been recorded previously. Soil samples were dried at room temperature and filtered to exclude particles greater than 80 µm. Plastic pots (4 ¾ ) were filled with peat moss and 2 ml of the 80 µm fraction were placed in the hole, where Nicotiana benthamiana seedlings were transplanted. Six weeks later root samples were tested by enzyme linked immunosorbent assay (ELISA) using polyclonal antibodies produced at Danish Institute of Agricultural Sciences (DIAS). PMTV positive roots were homogenised in phosphate buffer (pH 7.0) with polyethylene glycol 6000 (40 g/l) and inoculated to carborundum dusted leaves of N. benthamiana. Symptomatic leaves of these plants were used to inoculate all isolates to plants at the 4 leaf stage of each indicator plant species, N. debneyi, N. benthamiana and Chenopodium amaranticolor. This was replicated 3 times in the period September to January. Symptoms were recorded after 2 weeks and further 2–3 times, ending 4 weeks after inoculation. Plants were kept in a greenhouse at 20 ◦ C with supplementary light to 16 h with 50 W/m2 . Total RNA was isolated from infected N. benthamiana plants as described [16]. To minimize mutations, virus isolates had only been transmitted once to
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N. benthamiana at the time of RNA extraction. Two parts of the PMTV RNA2 were amplified using the following primers: PMTV 129 (5 CTT TTG CGG GTG GGC TAC AGG 3 , corresponding to nt 129–149 in PMTV RNA2 ([15], GenBank accession number AJ243719)), was used together with PMTV 1019 (5 TCC CAC CAA CAA CAC TCA ATG CTC 3 , nt 996–1019), and PMTV 2032 (5 TTT GGT GAG TGG GGT AAT GGT A 3 , nt 2032–2053) together with PMTV 3050 (5 CCC TAA GCC GGA TAA TGA GTT T 3 , nt 3029–3050). Synthesis of cDNA was performed with a duration of 1 h using 1.4 µl RNA at 37 ◦ C in a final volume of 10 µl containing: 50 mM Tris, pH 8.3; 75 mM KCl; 3 mM MgCl2 ; 10 mM dithiothreitol; 0.5 mM of each dNTP; 2 µM downstream primer; 4 units of Rnasin (Promega, Madison WI, U.S.A.); and 26 units of M-MLV reverse transcriptase (Life Technologies, Roskilde, Denmark). PCR amplification was performed with 2.5 µl cDNA in a final volume of 25 µl containing: 10 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl2 ; 0.15 mM of each dNTP; 1 µM each primer; and 0.5 units of Taq DNA polymerase (Life Technologies). The PCR product PMTV 129/1019 was restriction digested with NheI (New England Biolabs, Beverly, MA, USA) and PMTV 2032/3050 was digested with EcoRV or AluI before analysis by gel electrophoresis in a 2% agarose gel stained with GelStar (BMA, Rockland, ME, U.S.A.) as DNA stain. The PCR products generated with primers PMTV 1998 (5 AGG ACA ATA TAA CGA CAG CG 3 , nt 1998–2017) and PMTV 2479 (5 CGG CAT TTA TGA CGG TTA GA 3 , nt 2479–2498) were precipitated with 1 vol. 5 M NH4Ac and 2.5 vol. of 96% EtOH, then re-dissolved in 10 µl H2 O. For sequencing 10 ng of PCR product was used in an ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). The sequencing primers were PMTV 1998 and PMTV 2479 together with primers designed according to published sequence data. Sequencing reactions were analysed on an Applied BiosystemsTM ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Twenty-one isolates of PMTV were baited from soil samples collected from potato fields distributed in the potato growing areas of Jutland. Symptom development was recorded in C. amaranticolor, N. benthamiana and N. debneyi for these 21 isolates. Symptoms in C. amaranticolor developed from chlorotic local lesions into lesions with concentric stippled rings; in N. benthamiana the first symptoms that appeared was systemic mosaic followed by chlorotic and later necrotic lesions; in N. debneyi chlorotic local ring spots and oak leaf patterns were observed together with veinal clearing and stipples. The isolates could be grouped according to symptom development: group 1 (weak symptoms: chlorotic local lesions in C. amaranticolor, faint systemic mosaic in N. benthamiana and chlorotic local ring spots in N. debneyi) consisted of 54-10, 54-14, 54-20, 54-21, 54-22, 54-23; group 2 (medium symptoms: chlorotic local lesions with concentric stippled rings in C. amaranticolor, mosaic with chlorotic lesions in N. benthamiana, and chlorotic local ring spots, oak leaf patterns, veinal clearing and occasionally stipples in N. debneyi) consisted of 54-9, 54-11, 54-12, 54-17, 54-27; group 3 (strong symptoms: like medium symptoms but always with very clear and distinct symptoms ending with necrotic lesions and stipples) consisted of 54-6, 54-13, 54-15, 54-16,
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Fig. 1. Agarose gel electrophoresis analysis of RT-PCR products amplified with primers PMTV 2032 and PMTV 3050 and digested with EcoRV. 1: 50 bp molecular marker; 2–6: isolates 54-6, 54-16, 54-19, 54-13 and 54-21, respectively Table 1. RT-PCR-RFLP and nucleotide sequences covering parts of the coat protein and readthrough region specific to either the A or the B subgroup. Nucleotide sequences of the region nt 2018–2478 of RNA2 were compared and placed in subgroup A or B Isolate no.
910a NheI
2329 EcoRV
2748 AluI
Nucleotide sequence
Isolate no.
910a NheI
2329 EcoRV
2748 AluI
Nucleotide sequence
54-6 54-8 54-9 54-10 54-11 54-12 54-13 54-14 54-15 54-16 54-17
B A B B A B A B B B A
B A B B A B A B B B A
B A B B A B A B B B A
B n.d. n.d. n.d. n.d. n.d. A n.d. n.d. B n.d.
54-18 54-19 54-20 54-21 54-22 54-23 54-24 54-25 54-26 54-27
A B B A A B B B A B
A B B A A B B B A B
A B B A A B B B A B
A B n.d. A A n.d. n.d. n.d. A B
n.d. = not determined. a Nucleotide position on the RNA2 of PMTV-Sw (GenBank AJ243719)
54-19, 54-26. The remaining isolates showed inconsistent variations between plant species and replicates. Symptom development in the 3 indicator plant species was generally as described previously [3, 4] and the existence of differences in symptom development between isolates were in accordance with Harrison and 䉴 Fig. 2. Multiple alignment of nucleotide sequences encoding part of the readthrough protein. Nucleotide positions are numbered according to the PMTV-Sw sequence (GenBank AJ243719). Only nucleotide positions that are different from the PMTV-S isolate are shown. Subgroup-specific nucleotides are shaded. The EcoRV site at position 2329 is underlined
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Jones [4] who found differences between PMTV isolates inoculated to N. tabacum cv. Xanthi-nc, N. debneyi and Datura stramonium. One of the isolates in this investigation (PMTV-T) developed much more severe symptoms than the others, although it should be noted that this isolate has later been found to carry deletions in its genome probably due to propagation by mechanical transmission for several years [15]. The PMTV isolates were then RT-PCR-RFLP typed. Amplicons obtained with PMTV 129/1019 were restriction digested with NheI and amplicons obtained with PMTV 2032/3050 were digested with EcoRV or AluI. One example of agarose gel electrophoresis of digestion products is given in Fig. 1. All 21 isolates were typed by this method and they could be placed into 2 sub-groups, PMTV-A or PMTV-B according to RT-PCR-RFLP pattern (Table 1). There was full correspondence in the grouping obtained with each set of primer pair and restriction enzyme. As the next step, the nucleotide sequence of the region 2018–2478, covering a part of the readthrough protein encoding RNA, was determined for 9 isolates comprising 5 of the RT-PCR RFLP sub-group A and 4 of sub-group B. This region was chosen because Sandgren et al. [15] found a relatively high degree of variation within this part of the genome among different isolates. The region includes the EcoRV site used for RT-PCR-RFLP in this study. Sequence comparison of the 9 isolates revealed 2 subgroups, with group-specific variation found at 8 nucleotide positions of the 461 nucleotides analysed (Fig. 2). In addition to the 8 nucleotides that differed consistently between the two groups, a few other nucleotides were found to differ in individual isolates. The nucleotide sequence subgrouping was in accordance with that found using RT-PCR RFLP. The sequences were compared to sequences deposited in the GenBank. The sequences of subgroup PMTV-A were found to be almost identical to the nucleotide sequence of PMTV-S, and the sequences of subgroup PMTV-B were found to be almost identical to PMTV-Sw, the two isolates collected in Sweden [15]. As only a few isolates from other countries have been sequenced, it is not known whether both the A- and Bsubgroup can also be found in other countries. However, the complete sequence of the CP and readthrough region of PMTV has been determined for a few isolates [7, 11, 15]. Ignoring deletions in some of these isolates (PMTV-T and PMTV-S) [15], the variation between these isolates has been found to be small, especially in the CP region. This observation is supported by the present study as only 8 of 461 nucleotides were found to differ (98% identity) between the two subgroups A and B. We identified two subgroups of Danish PMTV isolates on the basis of nucleotide sequences. The isolates also differed in symptom development in 3 indicator plants. However, no correlation between genotypic and phenotypic groupings could be established. Neither could a correlation between geographic origin and any of the two other characters be established. Whether these two subgroups of isolates show different characteristics with regard to virulence in potato remains to be determined. These studies indicate that in efforts to obtain resistance against PMTV, it will be prudent to include isolates of both subgroups.
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Acknowledgements The authors wish to thank Estrella A/S for providing infected soil samples. This work was supported by a grant from The Directorate for Food, Fisheries and Agri Business under the Danish Ministry of Food, Agriculture and Fisheries: Detection and control of diseases in potatoes, HØJ99-4, subproject B.
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16. Spiegel S, Martin RR (1993) Improved detection of potato leafroll virus in dormant potato-tubers and microtubers by the polymerase chain-reaction and ELISA. Ann Appl Biol 122: 493–500 17. Torrance L, Cowan GH, Pereira LG (1993) Monoclonal antibodies specific for potato mop-top virus, and some properties of the coat protein. Ann Appl Biol 122: 311–322 Author’s address: Steen Lykke Nielsen, Department of Plant Protection, Danish Institute of Agricultural Sciences (DIAS), Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark; e-mail:
[email protected]
