Doi: 10.15446/agron.colomb.v34n1.53161

¹Laboratory of Industrial Microbiology, Faculty of Sciences, Universidad Nacional de Colombia. Medellin (Colombia)

² Laboratory of Cellular and Molecular Biology, Faculty of Sciences, Universidad Nacional de Colombia. Medellin (Colombia). mamarinm@unal.edu.co

ABSTRACT

Potato virus S (PVS) is a prevalent virus in potato fields in Colombia and the rest of the world. PVS has been classified into two separate lineages, PVSO (Ordinary) and PVSA (An- dean), which are genetically distinct. In this study, the com- plete genome sequence of a new PVS isolate (PVS_Antioquia) was obtained using High-throughput sequencing (Illumina HiSeq-2000) from tuber sprouts of Solanum phureja (var. Criolla Colombia). The PVS_Antioquia genome comprises 8,483 nt that code for six ORFs: RdRp (223 kDa), TGBp1-3 (25kDa, 12kDa, 7kDa) CP (32.3 kDa) and NABP (11 kDa) and share a high sequence identity with respect to the PVS_RVC (>95%) from Colombia, in contrast to 81 to 82% identity with respect to the PVSA and PVSO isolates from around the world. This genome information was used to design RT-qPCR primers that are specific for the Colombian PVS strains (RVC and Antioquia) which were validated in S. phureja leaf and tuber samples. These primers detected PVS in 80 and 60% of a set of fifteen leaf and tuber samples, respectively, suggesting a high incidence of this virus in the potato crops of Antioquia.

Key words: plant viruses, carlaviruses, diagnostic techniques, ELISA, PCR, potatoes.

RESUMEN

El Potato virus S (PVS) es un virus prevalente en los cultivos de papa de Colombia y otros lugares del mundo. Ha sido dividido en dos razas principales denominadas PVSO (Ordinaria) y PVSA (Andina), que representan a su vez dos linajes genéticos divergentes. En este trabajo se obtuvo la secuencia del genoma completo de un aislamiento de PVS denominado PVS_Antio- quia, por secuenciación masiva de nueva generación (Illumina HiSeq-2000) realizada sobre extractos de transcriptoma de tubérculos de Solanum phureja (var. Criolla Colombia). A partir de este genoma se diseñaron primers específicos para la detección por RT-qPCR de variantes colombianas de PVS (RVC y Antioquia), validándose su utilidad en pruebas de detección en tejido foliar y de tubérculos de S. phureja. El genoma de PVS_Antioquia tiene un tamaño de 8.483 nt que codifica para seis ORFs: RdRp (223 kDa), TGBp1-3 (25kDa, 12kDa, 7kDa) CP (32,3 kDa) y NABP (11 kDa), y comparte altos niveles de identidad con el aislamiento PVS_RVC (>95%) de Colombia y tan solo de 81 a 82% con representantes de PVSA y PVSO de diferentes países del mundo. Los primeros diseñados permitieron detectar el virus en 80 y 60% de 15 muestras foliares y 15 de tubérculos, respectivamente, lo que puede indicar la ocurrencia de altos niveles de incidencia de PVS y sus variantes en los cultivos de papa de Antioquia.

Palabras clave: virus de plantas, carlavirus, técnicas de diagnóstico, ELISA, PCR, papas.

Introduction

Potato virus S (PVS), a virus first described in the Netherlands in 1952 (de BruynOuboter, 1952), is a member of the Carlavirus genus (Betaflexiviridae family) with high incidence in potato fields around the world (Cox and Jones, 2010). PVS virions consist of flexuous rods that are 610-710 to 12-15 nm in size with a positive-sense RNA genome of approximately 8,500 nt in length and six open reading frames (ORFs) encoding an RNA-dependent RNA-polymerase (RdRp, 223 kDa), the triple gene-block proteins (TGBp1-3, 25kDa, 12kDa, 7kDa) involved in virus cell-to-cell movement, a viral coat protein (CP, 32.3 kDa) and a NABP (cysteine-rich nucleic-acid-binding protein) (11 kDa) (Martelli et al., 2007). PVS can be transmitted in a non-persistent manner by aphids, such as Aphis fabae, A. nasturtii, Myzus persicae and Rhopalosiphum padi; mechanical means and/or infected seed tubers (Wardrop et al., 1989; Lambert et al., 2012). Depending on the ability to cause systemic infection or not in the experimental host Chenopodium sp., PVS has been classified into two strains: PVSA (Andean) and PVSO (Ordinary), respectively (Hinostroza-Orihuela, 1973; Lin et al., 2009). More recently, Cox and Jones (2010) proposed the acronyms PVSO–CS for isolates that invade Chenopodium sp. systemically, but are not within clade PVSA and PVSA-CL to divergent PVSA isolates that cannot infect Chenopodium sp. systemically and suggested that the term PVSA should be applied strictly to members of the genetically distinct clade. The natural host range of PVS seems to be limited because it has only been reported to infect the sweet cucumber (Solanum muricatum Ait.) (Dolby and Jones, 1988) and different potato species, such as Solanum tuberosum L. (Cox and Jones, 2010) and S. phureja Juz. & Buk (Guzmánet al., 2010; Gutiérrez et al., 2013).

Tipically, PVS strains do not induce visible symptoms in the majority of potato varieties, but, when they do, consist of mild leaf symptoms such as mosaics, leaf rugosity, vein deepening and leaf bronzing (Salari et al., 2011). Although the disease caused by PVSO may be symptomless in the leaves and tubers of infected potatoes, the incidence may reach 100% with yield losses of 15% (Matoušeket al., 2005). Additionally, PVS co-infection enhances the severity of other viruses, such as Potato virus A (PVA), Potato virus Y (PVY) and Potato virus X (PVX) (Salari et al., 2011; Ny- alugwe et al., 2012).

Viral diseases are one of the most limiting factors in the production of S. phureja in Colombia, of which PVS has been shown to have a very high incidence (up to 40%) in the main potato producing provinces of Colombia: Antio- quia, Boyaca, Cundinamarca and Nariño (Gil et al., 2013, Gutiérrez et al., 2012, 2013; Gutiérrez-Sánchez, 2014). The coat sequence analysis suggested the existence of at least two PVS variants in Colombia, related to PVSO and PVSA (Gil et al., 2013). A third strain was found by high-trough put sequencing of the foliar transcriptome of S. phureja var. Criolla Colombia (Gutiérrez et al., 2013). The high incidence of PVS in potato crops in Colombia, as well as the presence of new variants never detected elsewhere, highlights the need to better study this genetically distinct clade of PVS that infect S. phureja. In this study, we performed a high-throughput transcriptome sequencing analysis of S. phureja tuber sprouts infected by PVS in the province of Antioquia (Colombia), in order to characterize its sequence properties and to design a set of primers that would be useful in Real-time reverse transcription-PCR (RT-qPCR) for PVS detection.

Materials and methods

Plant samples and DAS-ELISA tests

For this study, fifteen S. phureja tuber samples from different storage cellars in the municipalities of La Union (six samples) and Yarumal (nine samples) and an equal number of leaf samples from S. phureja plots at the flowering stage in the municipalities of Entrerrios (fourth samples from one plot) and Marinilla (11 samples from three plots) were used (Tab. 1). Each tuber and leaf sample consisted of three tubers and six to eight leaflets, respectively. The samples were initially tested for PVS infection by DAS-ELISA using the PSA 40000 kit from Agdia (Elkhart, IN), which uses polyclonal antibodies and alkaline phosphatase enzyme conjugates as capture and detection reagents, respectively.

Results and discussion

Genome features of PVS_Antioquia

The analysis of High-throughput sequencing results con- firmed the presence of a PVS strain, with a genome of 8,483 nt (excluding the 3' poly-A tail), closely related to strain PVS_RVC (95% nucleotide identity), that shares 81 to 82% nucleotide identity with respect to PVSA and PVSO isolates. The assembled sequence was deposited in GenBank under accession KR152654 with PVS_Antioquia as the strain name. No evidence of recombination was found with the program RDP3 (not shown). PVS_Antioquia has 5' and 3' untranslated regions (UTR) of 58 and 104 nt, respectively, and contains six ORFs, as expected. ORF1 (59-5986) codes for the RNA-dependent RNA polymerase (RdRp, 223 kDa) that contains the following functional domains: methyl- transferase (Pfam:PF01660, residues 43-352), Carlavirus endopeptidase (Pfam:PF05379, 1000-1087), RNA helicase (Pfam:PF01443, 1175-1432) and RdRp (Pfam:PF00978, 1553-1967). ORF2 (5973-6704), ORF3 (6682-7008) and ORF4 (6972-7172) comprise the triple gene block (TGB): TGBp1 is predicted to be a 243 residue protein (26.9 kDa) with viral RNA helicase domain (Pfam:PF01443, 40-235), TGBp2 is composed of 108 residues (Pfam:PF01307, 11.7 kDa) and contains a plant viral movement motif (4-104) while TGBp3 comprises 66 residues (7.2 kDa) and contains a 7kDa viral protein motif (Pfam:PF02495, 6-65). The viral coat protein is predicted to be a 294 residues protein (32.3 kDa) encoded by ORF5 at positions 7214-8098 and contain Pfam domains PF08358 (48-99) and PF00286 (108-247), typical of Carlavirus CPs. ORF6 (8095-8379) codes for

High-throughput sequencing of the S. phureja transcriptome was performed on a bulk of the tuber-seed sprouts. The bulk sample was ground using liquid nitrogen followed by RNA extraction with the GeneJET Plant RNA Purification mini kit (Thermo Fisher Scientific, Waltham, MA). The library was constructed with the TruSeq RNA Sample Preparation kit (Illumina, San Diego, CA) and rRNA was depleted with the TruSeq Stranded Total RNA with Ribo- Zero Plant kit (Illumina, San Diego, CA). Sequencing was performed with the Illumina HiSeq 2000 System Provided by Macrogen (Seoul, South Korea), which resulted in a pair- end library of 24,817,068 reads for a total of 4,963,413,600 bp. Adapter sequences and low quality bases were removed with SeqTK (https://github.com/lh3/seqtk). The PVS genome was assembled with de novo reconstruction of the S. phureja transcriptome with Trinity (Grabherr et al., 2011) and confirmed by mapping with Bowtie2 (Langmead and Salzberg, 2012). The final contig was assembled from a total of 40,451 paired-end reads with an average depth of 949X. ORFs codifying for viral proteins were identified with BLASTX (Gish and States, 1993). Sequence alignments were performed with MUSCLE (Edgar, 2004). Phylogenetic trees were calculated in MEGA6 (Tamura et al., 2013) using the Maximum Likelihood method based on the General Time Reversible model with a Gamma distribution (+G parameter = 0.4964). Nonsynonymous (Ka) and synonymous (Ks) substitution rates (denoted as Ka and Ks, respectively) were estimated using the software KaKsCalculator (Zhang et al., 2006) with the LPB method (Pamilo and Bianchi, 1993). Recombination analysis was done with the program RDP3 (Martin et al., 2010).

Primer design and RT-qPCR detection of PVS

Alignment of Colombian CP sequences, obtained in this and previous studies (Gil et al., 2013; Gutiérrez et al., 2012, 2013; Gutiérrez-Sánchez, 2014), allowed for the identification of sequences that are useful for designing RT-qPCR primers that are specific for the PVS variants found in Colombia with the aid of the program Primer3Plus (Un- tergasser et al., 2012). Validation of the RT-qPCR primers PVS_gen_F and qPVS_gen_R was performed on the fifteen tuber samples and fifteen leaf samples collected in the different municipalities of Antioquia. RNA was extracted from 100 mg of ground tissue using the GeneJET Plant RNA Purification kit (Thermo, Fisher Scientific, Waltham, MA) and eluted in 40 μL of DEPC treated water; the purity and concentration were determined by absorbance readings at 260 and 280 nm using a Nanodrop 2000C (Thermo Fisher Scientific, Waltham, MA). Retrotranscription was performed for 30 min at 50°C in 20 μL containing 200 U of Maxima Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA), 1X RT Buffer, 0.5 mM dNTP Mix, 100 pmol Oligo(dT)18, 20 U de RiboLock RNase Inhibitor and 100-500 ng of total RNA. For the qPCR, the Maxima SYBR Green/ROX qPCR Master Mix (2X) kit (Thermo Fisher Scientific, Waltham, MA) was used in 25 μL of reaction containing 12.5 μLmix, 10 μL DEPC water, specific primers PVS_gen_F (5'ATG CCG CCY AAA CCA GAT CC 3') and qPVS_gen_R (5'AGC ATK GCT TCY TCA TTT TGC CCT G 3') at 0.3 μM and 50-100 ngc DNA. The amplification cycles consisted of 10 min at 95°C to activate the polymerase, followed by 35 cycles at 95°C for 15 s and 53°C for 45 s, using a Rotor-Gene Q-5plex Platform (Qiagen, Hilden, Germany); fluorescense was measured after each amplification cycle. The PVS positive control was obtained from a PVS infected potato leaf-tissue and a virus-free sample was used as the negative. The samples were considered positive if they exhibited fluorescence values higher than the threshold before cycle 35 (Schena et al., 2004). Primer specificity was verified by High Resolution Melting in the 50 and 99°C range; the identity of the amplicons was con- firmed by Sanger sequencing of five amplification products (including the positive control), previously purified with the QIAquick Gel Extraction (Qiagen, Valencia, CA) kit. Sequences were edited manually with MEGA6 (Tamura et al., 2013) and compared against the NCBI database using BLASTN  (www.ncbi.nlm.nih.gov/BLAST).

Results and discussion

Genome features of PVS_Antioquia

The analysis of High-throughput sequencing results confirmed the presence of a PVS strain, with a genome of 8,483 nt (excluding the 3' poly-A tail), closely related to strain PVS_RVC (95% nucleotide identity), that shares 81 to 82% nucleotide identity with respect to PVSA and PVSO isolates. The assembled sequence was deposited in GenBank under accession KR152654 with PVS_Antioquia as the strain name. No evidence of recombination was found with the program RDP3 (not shown). PVS_Antioquia has 5' and 3' untranslated regions (UTR) of 58 and 104 nt, respectively, and contains six ORFs, as expected. ORF1 (59-5986) codes for the RNA-dependent RNA polymerase (RdRp, 223 kDa) that contains the following functional domains: methyl- transferase (Pfam:PF01660, residues 43-352), Carlavirus endopeptidase (Pfam:PF05379, 1000-1087), RNA helicase (Pfam:PF01443, 1175-1432) and RdRp (Pfam:PF00978, 1553-1967). ORF2 (5973-6704), ORF3 (6682-7008) and ORF4 (6972-7172) comprise the triple gene block (TGB): TGBp1 is predicted to be a 243 residue protein (26.9 kDa) with viral RNA helicase domain (Pfam:PF01443, 40-235), TGBp2 is composed of 108 residues (Pfam:PF01307, 11.7 kDa) and contains a plant viral movement motif (4-104) while TGBp3 comprises 66 residues (7.2 kDa) and contains a 7kDa viral protein motif (Pfam:PF02495, 6-65). The viral coat protein is predicted to be a 294 residues protein (32.3 kDa) encoded by ORF5 at positions 7214-8098 and contain Pfam domains PF08358 (48-99) and PF00286 (108-247), typical of Carlavirus CPs. ORF6 (8095-8379) codes for a 94 a.a. protein (10.6 kDa) and contains a cysteine-rich nucleic-acid-binding protein motif (Pfam:PF01623, 1-89). Ten polymorphic sites were detected in the PVS_Antioquia assembly: A1657G, A1933T, G3169A, T3817C, A3821G, A4828C, G4837A, A6050G, C6131T, C7600T; substitution A3821G results in amino acid change I1255V within RdRp (Fig. 1).

The sequence comparison between the PVS_Antioquia and PVS_RVC revealed a total of 310 transitions and 68 transversions for a global transition/transversion ratio of 4.56. With 60 amino acid substitutions, RdRp is the most divergent protein (96.96% aa identity); the most variable region corresponded to the one connecting the peptidase and methyl transferase domains (Fig. 2). The overall Ka/ Ks ratio for the RdRp ORF was 0.12, suggesting a strong negative selection for the whole protein; however, a sliding window analysis revealed three regions undergoing neutral selection: 640-670, 739-778, 1196-1226 (Ka/Ks of 0.97, 1.00 and 0.84, respectively) and a segment within the helicase domain under positive selection (1263-1277, Ka/Ks of 3.22) (Fig. 2). The triple gene block proteins TGBp1-3 showed a high degree of conservation among the PVS lineages (Fig. 2), with measured Ka/Ks ratios of 0.08, 0.05 and 0.0 that suggested a strong negative selection. TGBp1 shares 98.3% identity at the amino acid level with respect to PVS_RVC and contains four aminoacid substitutions: Y123H, C138S, E140G and T228S. TGBp2 has only one aminoacid substitution E69G while TGBp3 is 100% identical to its homo- log in PVS_RVC. The CP had a total of four amino acid substitutions (V31A, Q35E, P37S and E54G) with respect to PVS_RVC and a sliding window analysis revealed a N-terminal region to be under neutral selection (Ka/Ks=1.08, 23-41). The 11K protein had the highest global Ka/ Ks ratio with a value of 0.8, suggesting a mutation rate close to neutrality; however, residues 7-32 had a local Ka/Ks ratio of 1.23, indicating a slighty positive selection; the following substitutions were observed for this protein: G7S, Y14N, I28V, H40R and P81S. Recombination analyses performed on the PVC_Antioquia genome discarded recombination with other PVS strains as the mechanism of emergence for this new lineage. Failure to detect this PVS variant in previous studies was probably due to the lack of sequence information on Andean PVS strains; future studies would probably confirm the presence of this PVS lineage in other regions of Colombia and South America.

The phylogenetic analysis of the complete PVS genomes showed that isolate PVS_Antioquia clustered with PVS_RVC in an independent clade with a bootstrap of 100%. This clade is a sister group to Andean strains composed of BB-AND from Brazil (Geraldino et al., 2012) and RL5 from Colombia (Gutiérrez et al., 2012) (Fig. 3A). Using a partial segment of the CP sequence, the cluster analysis suggested the existence of four distinct groups (Fig. 3B). The first group comprised PVS^O strains and included three Colombian strains isolated in Colombia (Quincha- Cundinamarca, Valle de María-Antioquia and Chasques- Cundinamarca). The second group corresponded to isolates Peruvian and Vltava, of which the latter has been shown to be a recombinant strain between PVS^O and PVS^A (Geraldino et al., 2012). Group three comprised PVS^A isolates BB-AND from Brazil, Q5 from Chile, Guizhou CP01 from China and four Colombian isolates (Suras-Nariño, and El Roble2, RL5, Aldana Bajo from Antioquia). The fourth clade seemed to be a lineage derived from the PVS^A group and comprised PVS strains infecting S. phureja such as PVS_Antioquia as well as nine other Colombian isolates from Nariño, Antioquia and Cundinamarca, reported by Gil et al. (2103) and including PVS_RVC (Gutiérrez et al., 2013). CP sequences isolated from S. tuberosum (Gil et al., 2013) clustered in the same clade as PVS_Antioquia, suggesting that this PVS lineage does not have S. phureja as its unique host; however, we propose to name this derived lineage as PVS^P in reference to the first known host (S. phureja). Future studies on local S. tuberosum and S. phureja varieties from the South-American Andes will demonstrate whether PVS^P has a generalized distribution in countries that are part of the center of origin of the Potato or not. Additionally, it will be of great interest to determine the pathogenic characteristics of members of the PVS^P lineage (i.e local or systemic lesions) on Chenopodium sp., which will allow a biological comparison with respect to PVS^O and PVS^A.

Primer design and RT-qPCR detection of PVS

During the initial stages of this study, it was noticed that previously published primer sets designed to detect PVS failed to amplify some ELISA-positive samples even after exhaustive modifications of both RT-PCR and RT-qPCR protocols (data not shown). A similar situation was observed in a study by Gil et al. (2013) using RT-PCR where the authors where obliged to combine primers PVSCPF and PVSR reported in different papers (Nie and Singh, 2001; Ali et al., 2008) due to amplification problems using the original published sets. Sequence alignment of these primers with respect to the genomes of PVS_Antioquia, RVC and RL5 confirmed the presence of several mismatches at the annealing sites that could affect PCR efficiency (Fig. 4A, B). As these amplification problems could be detrimental to any PCR-based diagnostic tool aimed at detecting Colombian PVS isolates, a new primer set (PVS_gen_F and qPVS_gen_R) was designed for specific detection by RT-qPCR of PVS variants from Colombia. Primers PVS_gen_F and and qPVS_gen_R were tested by RT-qPCR using fifteen tuber and leaf samples obtained at different locations in Antioquia. This trial resulted in positive amplification curves for 80 and 60% of the leaf and tuber samples analysed. The Ct values suggested a higher viral titer in the leaf tissues (Ct in the 7.12-30.08 range) than in the tubers (Ct 28.76-33.94). The specifity of the reaction was confirmed by the melting curve analysis, which resulted in Tm values in good agreement with the positive control (86.96±1°C) (Fig. 4C, D). Sanger sequencing of four randomly chosen samples and for the positive control confirmed amplification of the PVS coat region. Two sequences showed higher identity with regions at positions 7218-7301 of the coat of PVS-RVC (98% identity) while the remaining two samples did so with positions 7215-7300 of the PVS-RL5 genome (100% identity).

In a previous study, Gil et al. (2013) detected the presence of PVS in 40% of the 320 S. tuberosum and S. phureja leaf samples from the four main potato producing provinces of Colombia: Antioquia, Boyaca, Cundinamarca and Nariño. A separate study on the accessions from the Coleccion Central Colombiana de papa using immunoprinting and DAS-ELISA with polyclonal antibodies revealed infections levels of 61.3 and 85%, respectively (Franco-Lara et al., 2009; Guzmánet al., 2010). These reports underscore the need to use stricter parameters in the tuber seed certification program of Colombia, which tolerates PVS levels of 1, 2 and 5% in basic, registered and certified tuber seeds, respectively (ICA, 2015). Similar studies in the rest of the world have revealed high levels of PVS incidence when tuber-seed certifications programs do not reach all farmers in a region or due to the use of diagnostic tests with low sensitivity. For example, a three year study in seven provinces of Iran using two hundred and forty potato samples with one or more symptoms of leaf mosaic, distortion, mottling and yellowing, detected PVS in 18.2% of the samples with a predominance of the PVSO lineage (Salari et al., 2011). In China, Wang et al. (2011) reported an average PVS infection level of 16.3%, reaching values of 22.6 and 26.7% in the provinces of Heilongjiang and Yunnan, respectively. In Costa Rica, Vásquezet al. (2006) evaluated the incidence of PVS and distribution at different altitudes, finding PVS to be present in 19% of the 600 tested samples. The highest PVS incidence was found at middle altitutes because the virus was detected in 60% of the plots of this region with an average infection of 75%.

Due to the increasing importance of S. phureja for internal use as well as a potential export crop, it is important to establish appropriate certification guidelines using highly sensitive and specific techniques designed to detect local PVS variants. Especially in international trade, tubers and in vitro plants can carry PVS. It would be interesting to further investigate the biological features of isolates belonging to PVSP, including the host range, transmission by aphids and overall agronomical effects as well as the ability of the members of new proposed lineage PVS^P to cause systemic and/or non-systemic infections in Chenopodium sp. and its synergistic interaction with other potato viruses.

Conclusions

Complete sequencing of a PVS strain obtained from S. phureja tuber sprouts in Antioquia (Colombia) confirmed the existence of a new lineage of this virus, for which we propose the acronym PVSP. Based on this genome sequence, primers PVS_gen_F and qPVS_gen_R, targeting the CP region, were designed for use in RT-qPCR detection of this virus. The validation of this molecular test on fifteen tuber samples and fifteen leaf samples revealed high levels of incidence of PVS in both tissues (60 and 80%, respectively), suggesting that urgent measures are required to strengthen current S. phureja seed certification programs in Colombia.

Acknowledgment

This work was funded by the Universidad Nacional de Colombia (Grants VRI: 19438 and 26737) and the International Foundation for Science (Sweden, Grant C/4634-2).

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