Occurrence of Potato virus Y strain PVYNTN in foundation seed potatoes in Japan, and screening for symptoms in Japanese potato cultivars

Authors


E-mail: mchikhali@uidaho.edu

Abstract

In 2008 and 2009 seasons, a sudden increase in Potato virus Y (PVY) incidence was recorded in foundation seed potatoes in Hokkaido, northern Japan. This increase was obvious during the field inspection and the postharvest indexing. Molecular typing revealed that besides the previously reported strains of PVYO and PVYNA-N, the most common strain identified was the recombinant PVYNTN, with three characteristic recombinant junctions at the HC-Pro, VPg and CP regions. No potato tuber necrotic ringspot disease (PTNRD) was observed in foundation seed potatoes in correlation with the presence of PVYNTN. Moreover, an isolate with a typical PVYNTN recombinant genome, namely Eu-12Jp, did not induce PTNRD in 62 Japanese potato cultivars tested in both primarily and secondarily infected plants. Two cultivars carrying the extreme resistance gene Rychc were resistant to the infection with Eu-12Jp, which presents potential sources of resistance to PVYNTN. Eu-12Jp induced systemic mottle in potato cultivars Desiree and King Edward carrying resistance genes Ny and Nc, respectively, but induced a hypersensitive reaction in potato cultivar Maris Bard, with the Nz hypothetical resistance gene typical of the PVYZ strain group. Therefore, based on the genome structure and the reaction of the potato N resistance genes, Eu-12Jp should be classified as PVYZ-NTN, as described for isolates from Idaho, USA recently. This is the first report of PVYZ-NTN in Japan and the sudden and increased occurrence of PVYNTN/PVYZ-NTN represents a potential risk of PTNRD developing and increases the significance of PVY in Japan.

Introduction

Potato virus Y (PVY) is one of the most common and destructive viruses found in potato worldwide (Singh et al., 2008; Gray et al., 2010). It is the type species of the genus Potyvirus, family Potyviridae, with a single-stranded positive-sense genomic RNA of c. 9·7 kb (Berger et al., 2005). Based on genetic reactions in tobacco and potato indicators carrying the resistance genes Ny, Nc and Nz, potato isolates of PVY have been classified into five strain groups, PVYO, PVYC, PVYN, PVYZ and PVYE (Jones, 1990; Kerlan et al., 1999, 2011; Singh et al., 2008; Galvino-Costa et al., 2012). In addition, based on molecular genome structure, several strains of PVY have been defined including PVYNTN, PVYNA-N, PVYN-Wi, PVYN:O and PVYNTN-NW (Beczner et al., 1984; Chrzanowska, 1991; Le Romancer et al., 1994; Piche et al., 2004; Crosslin et al., 2006; Chikh Ali et al., 2007, 2010a; Singh et al., 2008; Hu et al., 2009a,b; Kerlan et al., 2011). The name PVYNTN was originally used to describe PVY isolates able to induce potato tuber necrotic ringspot disease (PTNRD) in susceptible cultivars (Le Romancer et al., 1994; Singh et al., 2008). The PVYNTN genome was found to be recombinant with spliced segments of PVYO and PVYN sequences with variable recombination patterns (Glais et al., 2002; Schubert et al., 2007; Ogawa et al., 2008). Later, PTNRD was demonstrated to be associated not only with PVYNTN but with other PVY strains, including PVYNA-N, PVYO, PVYN-Wi, PVYN:O and PVYNTN-NW; some of these strains were able to cause PTNRD at various, sometime low frequencies in susceptible potato cultivars (Boonham et al., 2002; Nie & Singh, 2003; Piche et al., 2004; Schubert et al., 2007; Ogawa et al., 2008; Chikh Ali et al., 2010a; Gray et al., 2010). This would suggest a general lack of connection between the PTNRD syndrome and a characteristic recombinant genomic structure of PVYNTN. Isolates of PVYNTN have been reported from most potato production areas of the world (Beczner et al., 1984; Le Romancer et al., 1994; Kerlan et al., 1999; Boonham et al., 2002; Glais et al., 2002; Boukhris Bouhachem et al., 2007; Chikh Ali et al., 2007; Karasev et al., 2008; Hu et al., 2009a; Visser & Bellstedt, 2009).

The geographic isolation of Japan and strict quarantine regulations, along with a good seed potato inspection system, have enabled the efficient control of potato viruses and kept the incidence and impact of potato viruses low. However, an accidental introduction of a new virus/strain may have dramatic effects due to shifts in the host–pathogen balance. A new type of PVY causing tobacco necrosis was first reported in Japan in 1983 and designated as PVYT (T stands for tobacco; Tomaru, 1983) which presented a significant threat to the tobacco industry. About a decade later, the PTNRD disease occurred in potato fields in Nagasaki prefecture, Japan, causing significant economic losses (Ohshima et al., 2000). The causal agent of PTNRD was identified as PVYNA-N (Ohshima et al., 2000; Ogawa et al., 2008). The genetic analysis of PVYNA-N populations in Japan suggested a close relationship with PVYNA-N from the US and Canada and a recent introduction of this strain into Japan. In 2008 and 2009 seasons, a sudden increase of PVY incidence was recorded in the foundation seed potatoes in Hokkaido-Chuo Station (Hokkaido-Chuo St.), and Shiribeshi Substation (Shiribeshi Subst.) of Hokkaido-Chuo Station, of the National Center for Seeds and Seedlings (NCSS), Japan. The present study aimed to identify and characterize the strain(s) responsible for the increase of PVY infection in these stations.

Materials and methods

PVY incidence and sample collection

The incidence of PVY was recorded during 2007–2009 in foundation seed fields in Hokkaido-Chuo St., Nishinosato, Kitahiroshima, Hokkaido prefecture; Shiribeshi Subst. of Hokkaido-Chuo St., Mihara, Makkari, Abuta, Hokkaido prefecture; and Unzen Station (Unzen St.), Unzen, Nagasaki prefecture, Japan. Up to 1·59 million plants of about 57 cultivars are planted and inspected visually at these stations annually. During the three seasons 2007–2009, potato plants showing virus-like symptoms (mainly necrosis, sometimes accompanied by mosaic, yellowing and crinkle) were noticed and removed during the roguing process. When removed plants had tubers, these were also checked visually for necrotic symptoms. Leaf samples were collected from plants with symptoms and kept at −80°C until use. Samples collected were tested by ELISA for PVY, Potato leafroll virus (PLRV), Potato virus S (PVS) and Potato virus X (PVX) as described previously (Maoka et al., 2010). After harvesting, 0·3% of all harvested tubers were randomly selected, grown in a greenhouse and tested for PVY, PVX, PVS and PLRV by ELISA.

Identification of PVY strains by multiplex PCR and sequencing

To identify the strain type, the multiplex PCR assay reported previously (Chikh Ali et al., 2010b) was used to test randomly selected PVY-positive potato samples, collected during the field inspection or postharvest indexing. The full-length genome sequence was determined for Eu-12Jp excluding the primer site at the 5′ terminus, as reported previously (Chikh Ali et al., 2007). To determine the 3′ end of Eu-12Jp, the reverse primer Oligo-dT/SacI [TACATgagctc(T)18] was used along with FY-2 (5′-AAGAGCCTTCACTGAAATGATG-3′) to amplify the 3′ terminal segment.

Based on the sequence of Eu-12Jp, a primer pair Eu12CPF (5′-ACCAAATCAGGAGATTTTACTC-3′) and Eu12CPR (5′-GTCTCCTGATTGAAGTTTACAG-3′) was designed to amplify a segment of 1344 nt at the 3′ terminus, including the complete CP region, of seven other isolates.

Sequence analysis

Sequence analysis was carried out using the dnasis software (Hitachi Software Engineering Co., Japan). The program clustalW (Thompson et al., 1997) provided with mega v. 4 (Tamura et al., 2007) with the default parameters was used for multiple alignment of sequences determined in the present study and those of all PVY isolates with complete genomes available in GenBank (as of 25 November 2010). Alignments were built based on the whole genome and CP-3′-NCR sequences. The aligned sequences were checked for recombination according to Chikh Ali et al. (2010a) using rdp3 (Martin et al., 2005). The recombination points were confirmed and plotted with simplot v. 3.5.1 (Lole et al., 1999) and the recombinant sites numbered based on the visual observation. Sequence identity was performed using the blast program provided by the National Center for Biotechnology Information (NCBI). Phylogeny inference was conducted using the neighbour-joining (NJ) method implemented in mega v. 4 (Tamura et al., 2007) with 1000 bootstrap replicates.

Biological characterization

Eu-12Jp was collected from a potato cultivar, Toyoshiro, in Shiribeshi Subst. in 2009 and subjected to a single local lesion isolation using the potato cv. Konkei-59. Then it was propagated in Nicotiana tabacum cv. Samsun. To confirm the strain type, Eu-12Jp was inoculated on two plants each of N. tabacum cv. Samsun and differential indicator potato cultivars, Desiree (Ny:nc:nz), Maris Bard (Ny:Nc:Nz) and King Edward (ny:Nc:nz). Virus-free tubers of these potato cultivars were obtained from the Genebank Project of the National Institute of Agrobiological Sciences (NIAS), Tsukuba, Japan with the following accession numbers: Desiree, MAFF95008717; Maris Bard, MAFF30010615; and King Edward, MAFF30010501. This experiment was repeated on two occasions. Buffer-inoculated potato and tobacco plants of each cultivar were used as healthy controls. This experiment was carried out in a greenhouse at 22°C and natural light conditions of the summer season in Japan. Inoculated plants were tested for PVY infection by ELISA about 4 weeks after inoculation. After harvest, tubers were kept at room temperature and evaluated for necrosis weekly for 2 months.

Reaction of Japanese potato cultivars to Eu-12Jp

To study the symptoms of primary and secondary infections and the ability to induce PTNRD, 62 potato cultivars grown in Japan were inoculated with Eu-12Jp. Virus-free potato tubers were planted in pots and mechanically inoculated with Eu-12Jp at the 5–6 leaf-stage. Four leaves per plant and five plants per cultivar were inoculated. Plants were allowed to grow in a greenhouse at 20°C and natural light conditions. Temperatures above 20°C were recorded occasionally during the daytime. Symptom observation, starting 1 week after inoculation, was carried out daily for 4 weeks. Upper leaves were collected 1 month after inoculation and PVY infection was tested by ELISA. When all plants of a given cultivar were negative for PVY, the inoculation test was repeated on this cultivar in the second year. Evidence of tuber necrosis symptoms was evaluated on two occasions, after harvest and before planting in the next season. Harvested potato tubers were kept at 4°C for about 6 months and planted again to test the symptoms of the secondary infection in pots under greenhouse conditions. Because of limited space in the greenhouse, some tubers were planted and grown under a screen house. During the entire growing season, the secondary symptoms of foliage were recorded, and upper leaves were collected and tested by ELISA for PVY infection as described above. Tubers were harvested from the secondarily infected plants, and necrotic symptoms were checked just after harvesting and finally 6 months after storage at 4°C. All these experiments were performed in 2009 and 2010 at NCSS headquarters, Fujimoto, Tsukuba, Ibaraki prefecture, Japan.

Results

PVY incidence in foundation seed potatoes

In the field inspection of the 2007 season, numbers of PVY infected potato plants, identified visually and confirmed by ELISA, were very low; 4, 3 and 0 at Hokkaido-Chuo St., Shiribeshi Subst. and Unzen St., respectively (Table 1). Starting from 2008, a sudden increase in PVY infection was recorded during the field inspection in Hokkaido-Chuo St. and Shiribeshi Subst. (Table 1). In 2009, a total of 110 and 259 PVY-infected plants were recorded in Hokkaido-Chuo St. and Shiribeshi Subst., although the overall percentage of PVY-infected plants remained quite low (Table 1). Unlike the situation in northern Japan, no such increase in PVY incidence was observed at Unzen St. in Nagasaki, although a single PVY-infected plant was identified in 2008 (Table 1). No other viruses were detected in all samples collected during the growing seasons in the three stations. In the postharvest indexing, an increase of PVY infection was noticed in Hokkaido-Chuo St. but no changes were found in Shiribeshi Subst. and Unzen St. (Table 1). Single PLRV and PVS infections were found in Hokkaido-Chuo St. and Shiribeshi Subst., respectively, in the postharvest indexing of the 2008 season.

Table 1. Potato virus Y (PVY) incidence in fields of foundation seed potatoes at three stations of the National Center for Seeds and Seedlings in Japan, as revealed by field inspection and postharvest indexing
Year Hokkaido-Chuo stationShiribeshi substationUnzen station
No. plants/tubers inspectedaNo. plants infectedb (%)No. plants/tubers inspectedNo. plants infected (%)No. plants/tubers inspectedNo. plants infected (%)
  1. a

    Total plants inspected visually during the growing season, of which those showing virus-like symptoms were subjected to ELISA.

  2. b

    Total number of PVY positive samples as confirmed by ELISA.

  3. c

    Inspection during the growing season in which plants showing virus-like symptoms were inspected.

  4. d

    After harvesting, 0·3% of all harvested tubers were randomly selected, grown in a greenhouse and tested.

2007Foliage inspectionc358722 4 (0·0011)906232 3 (0·0003)4717460 (0)
Postharvest indexingd4646 3 (0·065)3607 1 (0·028)24835 (0·2)
2008Foliage inspection273588 22 (0·008)909459 90 (0·0099)4122041 (0·0002)
Postharvest indexing6035 7 (0·12)5541 4 (0·072)22808 (0·35)
2009Foliage inspection217696110 (0·0505)942631259 (0·0275)2900590 (0)
Postharvest indexing4137 15 (0·36)10522 3 (0·029)22187 (0·32)

Identification of PVY strains by multiplex PCR

Thirty-eight (68%) PVY-infected potato samples, including the isolate Eu-12Jp, produced three PCR products of 1307, 633 and 441 bp, characteristic of PVYNTN of the A type, with three recombination points at HC-Pro/P3, VPg and CP regions (Table 2; Chikh Ali et al., 2010b). PVYNTN was detected in PVY-infected samples collected from Hokkaido-Chuo St. (15 samples), Shiribeshi Subst. (18 samples) and Unzen St. (five samples; Table 2). Besides PVYNTN, PVYNA-N (12 samples; 22%) and PVYO (three samples; 5%; Table 2) were detected. Three samples were mixed infections with PVYO and PVYNTN.

Table 2. Identification of Potato virus Y (PVY) strains using a multiplex PCR assaya in 56 randomly selected PVY positive samples collected during field inspection and postharvest tuber indexing
 Hokkaido-Chuo stationShiribeshi substationUnzen station
  1. a

    Chikh Ali et al., 2010b; NTN, PVYNTN; NA, PVYNA-N; O, PVYO; NTN+O, mixed infection of PVYNTN and PVYO.

YearNTNNAONTN+ONTNNAONTN+ONTNNAONTN+O
20070011
200810007001
200914421117005100
Total15432187015100

Sequence analysis

The fully determined sequence of Eu-12Jp excluding the primer site at the 5′ terminus was 9675 nt and encoded a polyprotein of 3061 amino acids. Sequence data of Eu-12Jp will appear in the DDBJ/EMBL/GenBank nt sequence databases with the accession number AB702945.

In the recombination analysis, three crossover points were detected at nucleotide positions 2419, 5844 and 9183 of the Eu-12Jp genome (Fig. 1a). In the recombination analysis of the partial segments at the 3′ terminus, a recombination point was detected in the C terminus of the CP region of the seven isolates sequenced (Fig. 1b).

Figure 1.

Plots of similarity (generated by simplot) showing the recombination points in the HC-Pro, VPg and CP of the Eu-12Jp genomes (a) and the CP region of Chuo-10 (b) with comparison to PVY-Oz (dashed line) and N 605 (solid line) as representative isolates of PVYO and PVYN, respectively. Arrows indicate the location of the recombination points. Each point plotted is the percentage identity within a sliding window 200 bp wide centred on the position plotted, with a step size between points of 20 bp.

The genome of Eu-12Jp shared highest identity of 99·77% with isolate v942490 (EF016294; UK), 99·76% with isolate L26 (FJ204165; USA), 99·74% with isolate N4 (FJ204164; USA) and 99·73% with isolate HR1 (FJ204166; USA) and isolate 423-3 (AY884982; USA). The complete amino acid sequence of the polyprotein of Eu-12Jp had the highest identity with HR1 (99·9%). The phylogenetic analysis of the sequenced genome grouped Eu-12Jp tightly within the PVYNTN isolate group, particularly close to those from Idaho, USA (Fig. 2). The partial sequences of Japanese PVYNTN isolates sequenced shared high similarities regardless of their geographic origin and showed a similar identity profile to Eu-12Jp (Table 3). The name and sequences of isolates with partial genomes determined will appear in the DDBJ/EMBL/GenBank nt sequence databases as follows: Eu-Chou10Jp (AB702950), Eu-Chou11Jp (AB702951), Eu-Chou20Jp (AB702952), Eu-Chou21Jp (AB702953), Eu-Un3Jp (AB702954), Eu-Un4Jp (AB702955) and Eu-Un6Jp (AB702956).

Table 3. Identities (%) of a nucleotide sequence of 1344 nt consisting of the 3′ terminus including the partial NIb, complete CP region and 3′ NCR of seven Japanese PVYNTN isolates with PVY isolates available in GenBank
 Eu-Chou10JpaEu-Chou11JpEu-Chou20JpEu-Chou21JpEu-Un3JpbEu-Un4JpEu-Un6Jp
  1. a

    Chuo: isolate from Hokkaido Chuo station.

  2. b

    Un: isolate from Unzen station.

Eu-Chou10Jp
Eu-Chou11Jp99·8
Eu-Chou20Jp99·899·9
Eu-Chou21Jp99·899·999·9
Eu-Un3Jp99·399·599·499·4
Eu-Un4Jp99·599·799·699·699·2
Eu-Un6Jp100·099·999·899·899·399·6
N4 (FJ204165)99·999·999·899·899·499·699·9
HR1 (FJ204166)99·899·899·899·899·399·599·8
L26 (FJ204164)99·899·899·899·899·399·599·8
v942490 (EF016294n) 99·7699·999·899·899·499·699·8
Figure 2.

Neighbour-joining trees of all PVY isolates based on complete nucleotide sequences generated by the program mega v. 4 (40) with 1000 bootstrap replicates. The scale bar shows the number of substitutions per residue. Numbers at the nodes indicate the bootstrap values (%) of the cluster at the right.

Biological characterization and reaction of Japanese potato cultivars to Eu-12Jp

To confirm the molecular typing, Eu-12Jp was subjected to a more rigorous biological testing. In N. tabacum cv. Samsun, Eu-12Jp induced severe veinal necrosis, which was accompanied by stunting, mosaic and vein clearing. In the potato cv. Desiree, Eu-12Jp induced few small necrotic lesions 8 days after inoculation, but inoculated leaves remained vigorous and no wilting or HR reaction was observed. About 10 days after inoculation, systemic mottle appeared on upper non-inoculated leaves followed by a few necrotic lesions along the veins of upper leaves about 2 weeks after inoculation. In cv. King Edward, systemic mottle and crinkle started to appear about 10 days after inoculation without any visible signs of local or systemic necrosis. In cv. Maris Bard, yellowing and wilting of inoculated leaves appeared at 10 days after inoculation accompanied by systemic mottle. Two weeks after inoculation, inoculated leaves became fully necrotic and while some leaves dropped, others remained attached to the stems. Then stem necrosis started to appear followed by systemic top necrosis.

In the Japanese potato cultivars, Eu-12Jp did not show symptoms on inoculated leaves and upper non-inoculated leaves in three cultivars, Fugenmaru, Konafubuki and Sakurafubuki (Table 4). PVY was detected in upper leaves of cv. Fugenmaru by ELISA, indicating a symptomless or latent infection, but not in those of cvs Konafubuki and Sakurafubuki, which remained virus-free. Thirty-five cultivars showed no symptoms on the inoculated leaves but showed symptoms on upper leaves. Twenty-four cultivars showed necrotic symptoms on inoculated leaves and mosaic or crinkle on upper leaves. The most severe symptoms of the primary infection were necrosis followed by leaf drop observed on the cultivars Inca-no-hitomi, Inca-no-mezame and Hokkai 98. Cultivars May Queen, Red Moon and Nishiyutaka showed the most severe secondary symptoms such as stunting or mosaic, but no necrosis was found (Table 4).

Table 4. Reaction of Japanese potato cultivars to sap inoculation by the Japanese PVYNTN, Eu-12Jp
No.CultivarPrimary symptomsaSecondary symptoms
Inoculated leavesUpper leaves
  1. Weak symptoms are shown in lower case letters.

  2. a

    nt: not tested; CHL: cholorosis; CR: crinkle; LD: leaf drop; M: mosaic; N: necrosis; NRS: necrotic ring spot; NS: necrotic spot; ST: stunt; VN: vein necrosis; Y: yellowing; –: no symptom.

1Konafubukint
2Sakurafubukint
3Fugenmarumcr
4YukitsuburaCHL, cr, ym
5Aiyutakamm
6Dejimamm, cr
7Hanashibetsumm, cr
8Tawara MagatamamM, cr
9SetoyutakamM, CR
10Andes-redmM, CR
11Hokkai 50mM, CR
12ChériemM, CR
13Tawara Yodelm, yM, CR
14Norking Russetm, crm, cr
15Oojirom, crm, CR
16Okhotsk Chipm, crM, cr
17Northern Rubym, crM, cr
18Touyam, crM, CR
19Atlanticm, crM, CR
20Hikarum, crM, CR
21Shadow Queenm, cr, yM, cr
22SayumimurasakiMcr
23AndoverMm, cr
24BenimaruMM
25RanranchipM, crm, cr
26HarukaM, crm, cr
27San-en-imo (Vermont Gold Coin)M, crm, CR
28Beni-akariM, crM, CR
29ShepodyM, crM, CR
30Star RubyM, crM, CR
31KitamurasakiM, crM, CR
32KitakamuiM, CRM, CR
33Early starchM, CRM, CR
34YukirashaM, CRM, CR
35Ground PechkaM, CR, yM, CR
36Tawara KobanM, CR, yM, CR
37Tawara Choemon UdaiM, CR, yM, CR
38May QueenM, CR, yM, CR, ST
39Dansyakuimo (Irish Cobbler)nm, crm, CR
40Pirukavnmcr, vn
41Tawara Altair Hikoboshivn, Ymm, cr
42MatildaNSvn, crcr
43HokkaikoganeNSm, crM, CR
44Kita-akariNS, Ym, crM, CR
45Tawara MurasakiVNm, yM, CR
46EniwaVN, LDm, nm
47Norin 1VN, N, nsmmCR
48Saya-akaneVN, Y, LDmm
49WheelerVN, NRS, Y, LDmM, cr
50ToyoshiroVN, NRS, Y, LDm, crM, CR
51SayakanM, CR, yM, CR
52Inca-no-hitomiN, Y, LDM, cr, yCR
53MusamaruNm, CR, ym, CR
54Inca-no-mezameN, Y, LDN, Y, crnt
55Red MoonNSM, crM, CR, ST
56WaseshiroNS, YM, CRM, CR
57Hokkai 98NS, Y, LDM, CR, NRSM, CR
58Tawara MagellanNRS, yM, crM, CR
59Tawara Polaris HokkyokuseiVNM, crm, cr
60CynthiaVNM, CRm
61NishiyutakaVN, YM, crM, cr, ST
62Snow MarchVN, Y, LDM, crM, CR

Discussion

In the 2008 and 2009 seasons, a sudden increase in PVY incidence was recorded at two stations of foundation seed potato production in Hokkaido Island, northern Japan. This increase was obvious during the field inspection and the postharvest indexing. Molecular identification of samples collected during this period revealed that isolates with recombinant genomes in the HC-Pro/P3, VPg and CP coding regions typical of the PVYNTN were the most common, comprising more than 65% of PVY-positive samples tested. The occurrence of PVYNTN was not restricted to Hokkaido Island, as it was also detected in a third station of foundation seed potato production in Unzen, Nagasaki, southern Japan, indicating a wide geographic distribution. Based on whole and partial genome sequence analysis, PVYNTN isolates from Japan were found to be closely related to PVYNTN and PVYZ-NTN isolates from Idaho, USA, described during a PVY outbreak in 2007–2008 (Karasev et al., 2008; Hu et al., 2009b; Kerlan et al., 2011) and a PVYNTN isolate reported from the UK (v942490, GenBank accession no. EF016294). In the confirmatory tests in potato differential cultivars, Eu-12Jp induced systemic mottle in potato cultivars Desiree and King Edward carrying resistance genes Ny and Nc, respectively, but induced a hypersensitive reaction in cv. Maris Bard which carries the Nz triggered by the PVYZ strain, including the recently described PVYZ-NTN (Kerlan et al., 2011). Therefore, considering the reaction of the potato N resistance genes and the genome identity, Eu-12Jp should be classified as an isolate of PVYZ-NTN. Unlike PVYZ-NTN, the infection with Eu-12Jp was not accompanied by PTNRD in all infected potato plants. However, this would not alter the classification of Eu-12Jp as PVYZ-NTN, because the tuber necrotic response has not yet been used for PVY strain classification. Moreover, it is well known that the field infection of PVYNTN is not always accompanied by the development of tuber necrosis even at infection rates of 50–70% (Beczner et al., 1984). It was also reported that the molecular identification of PVYNTN isolates does not always imply the ability to cause PTNRD (Xu et al., 2005). In addition, the ability of PVYNTN to cause PTNRD in Japanese potato cultivars has not been documented so far. To further investigate the ability of PVYNTN isolates from Japan to induce PTNRD, they should be tested on susceptible cultivars to PTNRD such as Yukon Gold, Nadine, Hermes and Nicola, as was recommended previously (Singh et al., 2008). Therefore, even if the occurrence of the PVYNTN in foundation seed potatoes has not been accompanied by PTNRD, one cannot exclude the potential risk of PTNRD in certain cultivars under favourable conditions. Recently, Ogawa et al. (2012) identified three isolates collected from Kyushu and Honshu islands as PVYNTN based on the genomic structure; however, this is the first report of PVYNTN from the Hokkaido region, northern Japan in correlation with the increased PVY infection in foundation seed potatoes in that region.

Foliage symptoms on inoculated cultivars with Eu-12Jp varied from latent to very severe. Primary symptoms generally displayed necrosis on inoculated leaves which make them more obvious than the symptoms of secondary infection. However, in many cultivars, the systemic symptoms were more apparent in the secondary infection.

The current study should be supported by a survey of PVYNTN infection in commercial seed potato fields in Hokkaido for a better understanding of the epidemiology of PVYNTN. Two cultivars, Konafubuki and Sakurafubuki, with the PVY extreme resistance gene Rychc (Sato et al., 2006), showed extreme resistance to PVYNTN when mechanical inoculation was used, but this resistance needs to be further verified using graft/aphid inoculation.

In conclusion, isolates identified molecularly as PVYNTN were responsible for the sudden increase of PVY infection in two foundation seed potato stations in Hokkaido Island, Japan. The occurrence of PVYNTN was not accompanied by PTNRD, which suggests that the value of this phenotype to identify PVYNTN is limited and more value should be given to the genomic structure. Based on the genome identity and reaction of potato resistance genes Ny, Nc and Nz, a Japanese PVYNTN isolate was classified as PVYZ-NTN that was recently described for isolates reported from Idaho, USA. This is the first report of PVYZ-NTN in Japan. The reaction of all potato cultivars in Japan to the PVYZ-NTN was reported for both primarily and secondarily infected potatoes.

Acknowledgements

This research was partially funded by the Japan Society for the Promotion of Science (JSPS) which granted a postdoctoral fellowship to the first author. The authors wish to thank Drs K. Asano, M. Mori and K. Hosaka for their useful suggestions.

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