Introduction

Potato virus Y (PVY) has emerged in recent years as one of the most important pathogens in potato, seriously affecting yield (Hane and Hamm 1999; Nolte et al. 2004) and quality of tubers by inducing potato tuber necrotic ringspot disease (PTNRD) in susceptible cultivars (Beczner et al. 1984; Le Romancer et al. 1994; Gray et al. 2010). The virus can be transmitted from plant to plant mechanically, however, in nature it is transmitted via vegetative propagation by seed tubers, or non-persistently by aphids (Kerlan 2006). Recombinant strains of PVY had become established in North America in 1990s and spread in both Canada and United States (Baldauf et al. 2006; Crosslin et al. 2002, 2005, 2006; Gray et al. 2010; Karasev et al. 2008; McDonald and Singh 1996).

PVY is a positive-sense, single-stranded RNA virus which belongs to the large family Potyviridae, genus Potyvirus; it has ca. 9.7-kb genome with poly(A) tail at the 3′-terminus and a covalently-linked VPg protein attached to the 5′-terminus (Adams et al. 2011). PVY expresses its genome as a single large polyprotein which is subsequently cleaved by three virus-specific proteases into 10 mature proteins (Fig. 1). PVY has elongated, helical particles composed of genomic ssRNA and one major species of 29-kDa capsid protein (CP) (Adams et al. 2011). Minor amounts of another PVY protein, HC-Pro, were found present in virus particles, in addition to CP and VPg that is covalently attached to the PVY RNA genome (Torrance et al. 2006).

Fig. 1
figure 1

Schematic diagram of the main types of PVY recombinants found in Europe and North America—PVYNTN, PVYN:O, and PVYN-Wi. Different shading marks origin of genome fragments either from PVYN (black) or PVYO (white) parental sequences. Positions of recombinant junctions, RJ 1 to 4, often used to distinguish PVY recombinants are labeled below the diagram. The ten PVY genes are drawn above the diagram approximately to scale

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Genetic Classification of PVY Strain Groups

PVY exists as a complex of strains or strain groups which can be distinguished by reactions towards a series of resistance genes in potato (Cockerham 1970; de Bokx and Huttinga 1981; Kerlan 2006; Singh et al. 2008). A group of strains eliciting hypersensitive resistance (HR) response in the background of Ny gene was named PVYO, and those eliciting HR in the background of Nc gene were named PVYC strains (Table 1). Existence of an additional resistance gene, Nz, was postulated based on HR reactions of a PVY strain group PVYZ that did not elicit HR in either Ny or Nc genetic backgrounds but elicited HR in cultivars like Maris Bard and Pentland Ivory carrying this putative Nz gene (Jones 1990; Singh et al. 2008). Isolates that could overcome Ny, Nc, and Nz genes, and did not elicit HR towards any of these potato N genes could be further distinguished based on their reactions in tobacco (Table 1)—those inducing vein necrosis in tobacco were classified into PVYN strain (Jones 1990; Singh et al. 2008), and those inducing only mosaic were classified into PVYE strain (Kerlan et al. 1999; Singh et al. 2008). Of these five original, historic PVY strain groups, only PVYN induced vein necrosis in tobacco (Table 1), and hence was named “necrotic” PVY strain (Cockerham 1970; de Bokx and Huttinga 1981; Boonham et al. 2002). It is important to note that this genetic classification scheme (Table 1) includes two strain groups, PVYO and PVYN, that are well-characterized molecularly, through serology, RT-PCR, and whole genome sequencing, but also two other strain groups, PVYZ and PVYE, whose molecular properties have not been studied until very recently (Kerlan et al. 2011; Galvino-Costa et al. 2012).

Table 1 Genetic classification of the PVY strains, with serological and molecular properties of the main genetic strains of PVY. The data compiled according to Singh et al. (2008), Dullemans et al. (2011), Kerlan et al. (2011), and Galvino-Costa et al. (2012)
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Molecular Classification of PVY Recombinants

Complete genome sequences are known for non-recombinant isolates from PVYO, PVYC, and PVYN strain groups (Robaglia et al. 1989; Turpen 1989; Singh and Singh 1996; Lorenzen et al. 2006a; Fomicheva et al. 2009; Dullemans et al. 2011; Karasev et al. 2011). In addition to these main, parental genomes, multiple recombinant PVY genomes have been discovered, built of segments of PVYO and PVYN sequences spliced in various combinations (Thole et al. 1993; Jakab et al. 1997; Glais et al. 1998, 2002a, b; Boonham et al. 2002; Nie and Singh 2003b; Lorenzen et al. 2006a; Chikh Ali et al. 2007, 2008; Schubert et al. 2007; Hu et al. 2009b, 2011; Karasev et al. 2011; Galvino-Costa et al. 2012). At least nine recombination patterns were revealed with PVYO and PVYN identified as parents, based on sequence analysis of multiple PVY genomes known to date (Hu et al. 2009a). These recombinant patterns had between 1 to 4 recombinant junctions (RJs), most of which were relatively conserved in their position in the PVY genome, especially in the P1, HC-Pro, and VPg-NIa regions (Hu et al. 2009a).

The three most common recombinant patterns characteristic of PVYNTN, PVYN:O, and PVYN-Wi are presented on Fig. 1, with positions of the corresponding RJs indicated. All four RJs indicated on Fig. 1 were tested for possible use as a differentiating tool for typing PVY recombinants, and several primer sets exploiting single nucleotide polymorphism around these RJs are now available to type PVY recombinants in a uniplex or multiplex RT-PCR formats (Nie and Singh 2002, 2003a; Boonham et al. 2002; Lorenzen et al. 2006b; Rigotti and Gugerli 2007; Chikh Ali et al. 2010). Other non-recombinant, PVYNA-N (Nie and Singh 2003b), and recombinant genomes, e.g. NE-11 (Lorenzen et al. 2008), significantly different from PVYO and PVYN had also been reported. Most of these recombinant PVY variants were only studied by laboratory methods, and the only biological characterization conducted on some of them was tobacco test (Table 2). Interestingly, almost all PVY recombinants induced vein necrosis in tobacco, and thus would have been typed as PVYN if only this tobacco test had been used for PVY strain typing (see Table 2). Because of their recombinant nature, some PVY strain groups, like PVYN:O and PVYN-Wi, possessed PVYO serotype and PVYN symptomatology on tobacco, creating difficulties in correct differentiation of a PVY isolate. The recombinant PVYNTN strain group (Table 2) attracted the most attention, since necrotic tuber reaction, i.e. the PTNRD syndrome, was associated with this recombinant type (Beczner et al. 1984; Le Romancer et al. 1994; Glais et al. 2002b). Thus, multiple recombinant isolates of PVY are now classified based largely on genome properties of the isolates—the number and position of their RJs, which may be determined by RT-PCR assays or through genome sequencing. Tobacco reaction is not very useful for differentiating PVY recombinants, since most of them induce vein necrosis in tobacco (Table 2). Serology alone, based on PVYO and PVYN serotypes identified by monoclonal antibodies, also has limited usefulness in differentiating recombinant isolates of PVY, since it cannot distinguish PVYN:O/N-Wi from non-recombinant PVYO, or PVYNTN from non-recombinant PVYN (Table 2). Notably, very few of the recombinant isolates were tested on potato indicators (see Table 1), to determine their genetic type (for exceptions see Barker et al. 2009; Kerlan et al. 2011; Galvino-Costa et al. 2012), and consequently these two classifications, genetic and molecular, remain separate, creating confusions and making it difficult to navigate through the complexities of the PVY strain biology.

Table 2 Characteristics of the PVY strain groups classified based on their genome properties. Data compiled according to Singh et al. (2008), Lorenzen et al. (2006a, 2008), Barker et al. (2009), Kerlan et al. (2011), and Galvino-Costa et al. (2012)
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Merging Genetic and Molecular Classifications

Two factors made a seemingly minor, technical problem of merging the two systems of PVY strain classification quite a difficult endeavor. The first factor was related to a somewhat narrow group of researchers using potato indicators for typing PVY strains—these indicators essentially represented old European cultivars that were not universally available in every country with a PVY problem. To mitigate the problem of indicators’ availability, tobacco became the biological indicator of choice where potato indicators were not available. The logic behind this biological indicator change was to focus PVY strain detection on the “necrotic”, PVYN strain group (Boonham et al. 2002) which could overcome all N resistance genes in potato (see Table 1). Unfortunately, almost all recombinants induced vein necrosis in tobacco, and were thus indistinguishable from PVYN (Table 2). The second factor was related to fast changes in the composition of PVY isolates circulating in potato in Western Europe during late 1980s and early 1990s; the profound effect of these changes became obvious only later, when reliable molecular tools for PVY strain identification were developed (Nie and Singh 2002, 2003a; Boonham et al. 2002; Lorenzen et al. 2006b; Rigotti and Gugerli 2007; Chikh Ali et al. 2010). Specifically, certain types of genetically characterized PVY strains, like PVYC and PVYN, were largely displaced by new, recombinant types, like PVYN-Wi, PVYN:O, and PVYNTN, which were not characterized genetically (Blanchard et al. 2008; Kerlan and Moury 2008). Some of the recombinants, like PVYNTN, were initially noticed only because of their association with a new disease in potato, PTNRD (Beczner et al. 1984; Le Romancer et al. 1994). Throughout 1990s and 2000s, the main focus of the PVY differentiation was on molecular tools to distinguish various types of PVY recombinants. Attempts were made to apply these new molecular tools to type the old, historical PVY strains, like PVYZ and PVYE (Kerlan et al. 1999; Singh 2009; Kehoe and Jones 2011), but these were not very successful because the old isolates lost their infectivity in long-term storage. Some data retrieved for the non-infectious material with original PVYZ pointed at N-type serology (Singh 2009) or to PVYO-type capsid protein sequence present (Kerlan et al. 1999; Kehoe and Jones 2011), and were, thus, contradictory. A search for PVYZ and PVYE isolates among field isolates of PVY was the best strategy to characterize these historical strains.

In 2008, a seminal paper was published (Singh et al. 2008) that drew attention to the lack of biological characterization of PVY recombinants in potato cultivars with defined genetic background. The problem became very practical—PTNRD was clearly not confined to only PVYNTN recombinants, and new, specific tools to identify PTNRD-inducing PVY isolates were needed (Singh et al. 2008; Gray et al. 2010). Molecular and serological tools were useful to distinguish between various recombinant types of PVY, but they could not identify whether a particular recombinant, or a particular isolate would induce PTNRD in a particular potato cultivar.

This discussion paper (Singh et al. 2008) renewed interest in genetic classification of PVY strains, and prompted a series of publications addressing genetic typing of PVY recombinants on potato indicators. Hence, significant progress was made on biological characterization of PVY recombinants since 2008 (Barker et al. 2009; Kerlan et al. 2011; Galvino-Costa et al. 2012). This progress was based on biological characterization of PVY isolates on potato indicators and on concomitant whole genome sequencing. Importantly, molecular make-up for two genetic strains of PVY was determined, for PVYZ (Kerlan et al. 2011) and PVYE (Galvino-Costa et al. 2012). Both PVYZ and PVYE genomes turned out to be recombinant (Figs. 1, 2), which is consistent with the timing of their initial collection and characterization in the 1980s and early 1990s (Jones 1990; Kerlan et al. 1999). PVYE represented a new recombinant type associated with PTNRD (Galvino-Costa et al. 2012), with parental genomes identified as PVYNTN and PVY-NE 11, recombinants themselves (Lorenzen et al. 2006a, 2008). PVYZ, on the other hand, was found to have a well-known PVYNTN genome long known to be associated with PTNRD (Hu et al. 2009b; Kerlan et al. 2011).

Fig. 2
figure 2

Schematic diagram of a rare recombinant type of PVY, PVYE, with genome composed of two parental genomes, PVY NE-11 and PVYZ-NTN (Galvino-Costa et al. 2012). Positions of the two recombinant junctions, RJ2 and RJ3, characteristic of PVYZ-NTN are labeled below the diagram. Note that if only RJ2 and RJ3 are probed using a multiplex RT-PCR assay (Lorenzen et al. 2006b), PVYE will be mistaken for PVYZ-NTN

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This identification of PVYZ genome as having a typical PVYNTN recombinant structure came as a surprise, and prompted a study on interactions of ordinary PVYNTN isolates with the Nz resistance gene, present in cultivar Maris Bard (Kerlan et al. 2011; see Table 1). Subsequent biological characterization of four additional PVYNTN isolates on cultivars Desiree, King Edward, and Maris Bard indicated that two of them induced typical HR, and two—severe local and systemic HR-like necrosis in Maris Bard, while producing no HR or systemic necrosis in both Desiree and King Edward (Kerlan et al. 2011; Galvino-Costa et al. 2012). A similar reaction was reported by Barker et al. (2009) for a PVYNTN isolate PVYNTN-Slo in Maris Bard, i.e. local and systemic HR, while no HR on Desiree, Pentland Crown, King Edward, and Duke of York. Thus, it appears that both PVYZ and PVYNTN strain groups elicit HR in cultivars harboring Nz gene (Kerlan et al. 2011), and the only difference between PVYZ and PVYNTN isolates is their symptoms in tobacco: PVYZ induces mosaic and vein clearing, while PVYNTN induces vein necrosis (Tables 1, 2). Consequently, Kerlan et al. (2011) proposed that PVYZ and PVYNTN are biologically very close or identical in potato and should be named PVYZ-NTN. In an amended genetic classification, tobacco symptoms were suggested to be subordinate to symptoms in potato indicators (Kerlan et al. 2011). Hence two of the five original, historic strains of PVY were found to have recombinant genomes (see Table 1).

Diversity Within Strain Groups

In the course of the 2004–2006, U.S.-wide survey of seed potato crops, more than 4,000 PVY positive samples were typed using biological reaction on tobacco, serological profiling with PVY specific monoclonal antibodies, and RT-PCR profiling using multiplex assay identifying the two most prominent recombinant junctions in the PVY genome (Gray et al. 2010). An unusual sub-group of isolates was revealed during this survey, which was assigned to the PVYO strain based on the absence of recombinant junctions (RT-PCR assay), failure to induce vein necrosis in tobacco, and positive reaction to PVYO-specific monoclonal antibody MAb2 (Ellis et al. 1996; Karasev et al. 2010; Gray et al. 2010). Nevertheless, this sub-group of isolates was distinguished from other, ordinary PVYO isolates by a distinct serological marker, i.e. positive reaction towards one PVYN-specific monoclonal antibody, 1F5 (Karasev et al. 2010). This sub-group was named PVYO-O5 (Karasev et al. 2010), and subsequently clearly identified as belonging to the PVYO strain group through whole genome sequencing conducted on multiple, more than 30 isolates displaying this unusual serological marker (Karasev et al. 2011). Remarkably, almost all PVYO-O5 isolates grouped into a distinct, separate evolutionary lineage when whole genomes were subjected to phylogenetic analysis, suggesting that this serological marker reflected a more profound diversity within the PVYO strain (Karasev et al. 2011). Isolates belonging to this PVYO-O5 lineage were initially found in the U.S. (Gray et al. 2010; Karasev et al. 2010), but later identified in Canada (Nie et al. 2011), and in Australia (Kehoe and Jones 2011). Although biologically PVYO-O5 isolates were found similar to ordinary PVYO, they demonstrated substantial differences in certain biological features, notably in their interaction with the Ny gene in potato—reaction against PVYO-O5 was much more severe than against PVYO in cultivars bearing Ny gene (Karasev et al. 2011). It is important to point out that PVYO-O5 was found ecologically expanding during the 3-year PVY survey (Gray et al. 2010), and thus hypothesized to have certain evolutionary advantages over the ordinary PVYO isolates (Karasev et al. 2010).

Similarly the PVYN:O/N-Wi strain can be divided into the two groups PVYN:O and PVYN-Wi (sometimes referred to as the a and b subgroups) (Lorenzen et al. 2006a) by the presence of an extra recombination junction (Fig. 1). Additionally, these groups can be further subdivided on biological properties. The U.S./Canadian survey (Gray et al. 2010) identified significant numbers of variants within the PVYN:O/N-Wi strain that did not cause necrosis in tobacco. Furthermore, the isolates within all variant groups differ in their ability to cause PTNRD. The diversity of isolates within the PVYN:O/N-Wi appears to be significant, although the spatial and temporal characteristics of distribution are unknown since recent surveys have not generally used the biological tests that can separate the variant groups and molecular characterization has not been completed.

Concluding Remarks

The biological and genetic diversity of PVY is vast and it is clear the research community will continue to struggle with taxonomic schemes until the molecular determinants regulating the biological and genetic properties of the various strain groups and variant groups within strains can be better identified. This should then lead to improved diagnostic capabilities that can more efficiently categorize PVY isolates into a meaningful and understandable taxonomy. Nevertheless, this is likely to be a continually moving target given the changes is strain composition in recent times. The PVYN strain has all but disappeared from potato in the U.S. and Canada, with only a handful of isolates being identified in surveys of the national seed potato crop since 2004. The predominance of the PVYO strain also appears to be waning in many of the potato production regions of the U.S. and similar findings have been reported from other geographic regions. The recombinant PVYN:O/N-Wi and PVYNTN are becoming prevalent in potato production and the reasons are not fully understood. In general, the recombinant strains tend to induce foliar symptoms that are milder that the PVYO strain. This has compromised the efficiency of seed certification inspections and on-farm rogueing operations and perhaps has led to increased virus incidence in seed stocks. Also contributing is a practice of some seed certification schemes to allow a higher tolerance from mild mosaic than severe mosaic that can also lead to a selection of recombinant types of PVY. Regardless of the reasons, the recombinant strains of PVY are on the increase and they have the potential to significantly increase the consequences of PVY on the potato crop due to the ability of some of these strains/variants to cause PTNRD. Currently PVY is a major disease concern of seed growers, if the PTNRD strains of PVY continue to increase and become endemic, PVY could become a major disease concern for the entire potato industry, growers and processors, as well as consumers.