Potato virus Y (PVY) is the type member of the genus Potyvirus, family Potyviridae [1]. The virus has flexuous filamentous particles with a ca. 9.7-kb, single-stranded, positive RNA genome, excluding poly(A), and is transmitted by multiple species of aphids in a non-persistent manner [15]. PVY is an important pathogen affecting multiple crops of the family Solanaceae, e.g., potato (Solanum tuberosum L.), pepper (Capsicum annuum L.), tomato (Solanum lycopersicum L.), and others [11, 15, 22, 24]. PVY strains are distinguished biologically according to their host range or based on molecular properties [15, 22, 24]. Non-recombinant strains of PVY have been grouped into four main evolutionary lineages named PVYC, PVYO, PVYN, and Chile3 [19], corresponding to the biological properties of PVY isolates within these clades. The PVYC clade includes mainly pepper isolates that are poorly adapted to potato, while the PVYO and PVYN clades comprise potato isolates that are poorly adapted to pepper [5, 19, 22, 26]. The Chile3 clade comprises a single divergent isolate of PVY found in pepper that is unable to infect potato [19]. Phylogenetic analysis has revealed that the PVYC clade is quite diverse, having more than 8 % difference in the nucleotide sequence for the whole genome between individual PVYC isolates [6].

In the past, research on PVY diversity was mainly focused on potato [6, 12, 14, 17, 24] or pepper [2, 20, 22, 26] isolates, with tobacco isolates being a distant third [7, 10, 16]. Very little research has been conducted on PVY isolates found in tomato, in particular on PVYC isolates from tomato, which are largely under-studied both biologically and molecularly. In 2014, an isolate of PVY was collected from tomato on the island of Oahu, Hawaii. To address the knowledge gap regarding the biological and molecular properties of PVY isolates from tomato, we subjected this tomato isolate of PVY to complete characterization side-by-side with control isolates of PVY. Here, we studied this novel PVYC isolate, subjecting it to a host range study, serological and molecular typing, and whole-genome sequencing.

The original PVY-H14 isolate was collected in August 2014 from an infected tomato plant growing on a commercial farm near the village of Kahuku, Hawaii, on the island of Oahu. The infected plant was a field-grown cherry tomato that displayed symptoms typical of PVY infection, which included mosaic and necrotic lesions on leaves, and overall stunting of the plant. The original PVY-H14 sample was inoculated into tobacco (Nicotiana tabacum L., cv. Burley) and was maintained in tobacco through periodic re-inoculations to young healthy plants in an insect-free growth room as part of the PVY isolate collection maintained at the University of Idaho. This collection was periodically subjected to serological [21] and RT-PCR [3, 18] typing as part of the maintenance protocol to avoid contamination or strain mixtures and to ensure stable characteristics of the reference isolates. The reference PVY isolates Oz (PVYO) and Mont (PVYN) were used for comparisons.

The following indicator plants were used to determine the pathotype of the PVY-H14 isolate: C. annuum cv. Cal Wonder, Nicotiana tabacum cv. Burley, and Nicotiana benthamiana. All plants were mechanically inoculated with sap from PVY-infected tobacco at the 4- to 6-leaf stage as described previously [9]. Three potato cultivars with known genetic background were used to type the PVY isolates – Desiree, Maris Bard and King Edward. In addition, a North American cultivar, Russet Norkotah, which is known to be susceptible to a wide range of PVY strains [23], was used for inoculations. These potato indicators were maintained as virus-free tissue culture lines and inoculated mechanically at the 6- to 10-leaf stage [23]. All plants were grown in an insect-proof greenhouse before and after inoculations, in growth chambers under artificial light provided by fluorescent and incandescent lamps with an 18h day/6h night cycle and maintained at 23-26 °C. The observation of symptoms started 4-5 days after inoculation and was continued for 6-8 weeks. Three plants per species/cultivar were tested in a single experiment that always included the PVY-H14, PVY-Oz, and PVY-Mont isolates, plus a healthy control, and the experiment was repeated three times. Each infected plant was tested at 2 wpi (tobacco, pepper, N. benthamiana) or 3 wpi (potato) for systemic infection and to confirm the identity of the inoculated PVY strain, using TAS-ELISA and RT-PCR as described below.

The serological reactivity of PVY-H14 was tested in a TAS-ELISA format as described by Nikolaeva et al. [21]. All tests included control PVY isolates from the laboratory collection, with distinct serological patterns characteristic of PVYO, PVYN, and PVYO-O5 strains. In addition to a polyclonal antiserum [8, 13], four strain-specific monoclonal antibodies were used, Neogen-O/C (Neogen Europe, Ayr, Scotland) and MAb2 (Agdia, Elkhart, IN) recognizing PVYO, PVYO-O5, PVYN-Wi/N:O and PVYC; 1F5 (Agdia, Elkhart, IN) recognizing PVYN, PVYO-O5, and PVYNTN; and SASA-N (Scottish Agriculture Science Agency, Edinburgh, Scotland) recognizing PVYN and PVYNTN.

Two different multiplex RT-PCR assays were performed on PVY-H14, following the methods of Lorenzen et al. [18] and Chikh Ali et al. [4]. Control strains of PVY were obtained from the laboratory collection, and assays were conducted using the immunocapture (IC) RT-PCR format [3] on infected tobacco and pepper samples.

The nearly complete genome sequence of PVY-H14 was obtained by sequencing overlapping RT-PCR products as described by Karasev et al. [12]. The contigs obtained were assembled using SeqMan Pro of the Lasergene 9 Suite (DNASTAR). For the multiple sequence alignment, CLUSTALW, implemented in the program MEGA version 4 [28], was used with the default parameters. Sequence identity was checked using the BLAST program provided by the National Center for Biotechnology Information (NCBI). The aligned sequences were examined for evidence of recombination, using RDP4 Beta3 software. Phylogeny inference was conducted based on the whole genomes using the minimum-evolution method implemented in MEGA version 4, with 1,000 bootstrap replicates.

Four species of Solanaceae were tested for susceptibility to PVY-H14 infection: tobacco, pepper, N. benthamiana, and four cultivars of potato (Table 1). Tobacco, pepper, and N. benthamiana were susceptible to PVY-H14, developing a systemic infection within 2 weeks post-inoculation (wpi) that was easily detectable by ELISA and RT-PCR. In tobacco and pepper, PVY-H14 induced relatively mild symptoms, which were visible as mosaic and vein clearing at 2 wpi, similar to the symptoms induced by the control isolate Oz (PVYO). Another control isolate, Mont (PVYN), induced vein necrosis in tobacco but was unable to infect pepper (Table 1). PVY-H14 induced severe systemic necrosis in N. benthamiana, leading to plant death by 3 wpi (Table 1), in contrast to both Oz (PVYO) and Mont (PVYN), which induced mosaic and stunting in infected plants but no necrosis or death (Table 1). In potato, PVY-H14 induced necrotic lesions on inoculated leaves only in King Edward and Maris Bard, which were visible at 2 wpi (Supplementary Fig. 1), but no systemic infection in any of the four tested cultivars (Table 1). This contrasted with the PVY isolate Oz (PVYO), which induced HR in Desiree and Maris Bard and systemically infected King Edward and Russet Norkotah, and with Mont (PVYN), which systemically infected all four cultivars (Table 1).

Table 1 Symptoms induced by PVY-H14 in different indicator plants 1
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Serological typing of the PVY-H14 isolate is summarized in Table 2. PVY-H14 reacted positively with the strain-non-specific antibody Asc6 and with two O-specific antibodies, Neogen-O/C and Mab2, provided by two different commercial suppliers (Neogen and Agdia, respectively). It did not bind, however, to two N-specific antibodies, 1F5 and SASA-N, provided by Agdia and SASA, respectively (Table 2). Based on these ELISA data, PVY-H14 appeared to have an O serotype. When subjected to RT-PCR typing using the multiplex assay developed by Lorenzen et al. [18], PVY-H14 produced only one 267-bp band indicative of the presence of the PVYO sequence (Supplementary Table 1). In this respect, PVY-H14 was clearly different from the PVYN isolate Mont, producing characteristic bands of 328 and 398 bp (Supplementary Table 1). Nevertheless, a true PVYO sequence also produces a second, 689-bp band, like the PVY-Oz isolate (Supplementary Table 1). The RT-PCR typing utilizing the multiplex RT-PCR assay developed by Chikh-Ali et al. [3] produced no bands (Supplementary Table 1). The control isolate Oz (PVYO) produced two characteristic bands in this assay, 532 and 853-bp, while isolate Mont (PVYN) produced three characteristic bands, 398, 633, and 1,307-bp (Supplementary Table 1). The combination of a unique biological profile, O serology, and unusual molecular typing results suggested a new sequence, distinct from the currently known PVY genotypes.

Table 2 Serological reactivity of PVY-H14 and two control isolates with different monoclonal antibodies tested in TAS-ELISA
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The nearly complete, 9,620-nt genome of PVY-H14 was determined by Sanger sequencing of overlapping RT-PCR fragments and deposited in the GenBank database under the accession number KX580384. A single, 3,061-aa polyprotein was encoded by an open reading frame starting at position 116 and ending at position 9,301. This polyprotein was found to encode 10 cistrons typical of PVY and other potyviruses, including capsid protein (CP) at its C-terminus.

When subjected to a BLASTN search of the GenBank database, the PVY-H14 sequence was found to be the most closely related to the sequence of the PRI-509 isolate (91 %) belonging to the PVYC clade of PVY [6]. In pairwise comparisons (Supplementary Table 2), the levels of sequence identity of PVY-H14 to clades of PVY ranged from 82 % for RRA-1 (PVYNA-N) to 91 % for PRI-509 (PVYC). Apparently, PVY-H14 had a genome quite divergent from the other known PVY sequences and, based on a sequence difference of 9-18 %, might represent a new strain. In phylogenetic reconstructions conducted with 31 non-recombinant whole genome sequences from all major clades of PVY, PVY-H14 was placed within a diverse PVYC clade that included PVY isolates found in potato, pepper, tomato, and tobacco (Fig. 1) and grouped phylogenetically most closely to an isolate MN from tobacco found in North Carolina [7] and another uncharacterized tobacco isolate NC57 (GenBank accession no. DQ309028). No evidence of recombination was found when the PVY-H14 sequence was analyzed using the programs from the RDP suite (not shown). Based on the very long branch lengths, which reflected the evolutionary distances (Fig. 2), sequences grouped into this PVYC clade appeared to be much more diverse than sequences of the PVYO and PVYN clades (Fig. 1), making it difficult to conclude if PVY-H14 represents a new strain of PVY.

Fig. 1
figure 1

Maximum-likelihood phylogenetic tree for the full genomes of non-recombinant potato virus Y isolates. Sequence alignment was done using ClustalW provided in MEGA 4, and the tree was drawn using the RDP 4 program for PVY isolates with no detected recombination. Bootstrap values higher than 70 % are shown at the corresponding nodes

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PVY isolates from potato have long been known to be poorly adapted to replication in pepper, and pepper isolates of PVY are known to replicate poorly in potato, while both tobacco and tomato serve as permissive hosts for both groups of PVY isolates [5, 19, 25]. This biological difference is supported by the phylogenetic separation between the PVYC lineage comprising pepper-infecting isolates, and PVYO and PVYN lineages comprising potato-infecting isolates [17, 19]. The host range determined here for PVY-H14 suggested it belonged to the PVYC strain group, since it was unable to systemically infect any of the four tested cultivars of S. tuberosum but it easily established a systemic infection in C. annuum and the two Nicotiana spp. tested (Table 1). N. benthamiana provided a clear distinction between systemic necrosis induced by PVY-H14 leading to plant death by 3 wpi, and mosaic and stunting without necrotic reaction for both Oz (PVYO) and Mont (PVYN) isolates (Table 1).

Two laboratory methods were used for PVY-H14 strain assignment: serotyping using a series of commercial monoclonal antibodies and multiplex RT-PCR. Although widely used for differentiation of potato-infecting strains of PVY [3, 18], RT-PCR typing proved to be not very useful for detection and characterization of PVY-H14. Only the older methodology [18] produced a single band, which could have been erroneously interpreted to indicate the presence of PVYO, while the more advanced technology [3] produced no bands and gave no clues as to what type of PVY strain was present in the sample. The potential misidentification of PVY-H14 as a PVYO isolate was made easier due to its serotype identified as PVYO using monoclonal antibodies available commercially.

The novel isolate PVY-H14 found in tomato in Hawaii exhibited only 91 % nucleotide sequence identity to the closest isolate, PRI-509 from the same PVYC clade (Supplementary Table 2). This low sequence identity and the long branches in phylogenetic reconstructions characteristic of PVYC clade (Fig. 1) indicate that the PVYC lineage is diverse, and its classification may be more complicated than currently accepted. The precise classification of PVYC isolates will have to wait for more pepper and tomato isolates from this lineage to be characterized biologically and through whole-genome sequencing.