پاسخ ارقام سیب زمینی به پنج ایزوله متعلق به چهار سویه ویروس سیب زمینی y Response of Potato Cultivars to Five Isolates Belonging to Four Strains of Potato virus Y

Potato virus Y (PVY) is the type member of the genus Potyvirus in the family Potyviridae. It infects several important crops in the Solanaceae family, including potato, tobacco, tomato, and pepper (4,26). PVY is ubiquitous in distribution, causing significant yield loss and quality degradations on the potato crop worldwide (7,19,21,26,31). A great diversity in strains of PVY has been noted and several main strain groups have been classified (9,11,12, 21,32). In general, these strain groups can be categorized into nonrecombinant and recombinant categories. The former includes the common (ordinary) strain (PVYO), the potato stipple streak strain (PVYC), the tobacco veinal necrosis strain (PVYN), and its derivative, the nonrecombinant potato tuber necrotic strain (North American [NA]-PVYNTN) (15–۱۷). The latter includes the N:O group (PVYN:O or PVYN-Wilga) (6,18) and the recombinant PVYNTN, which can be further divided into European (Eu)-PVYNTN (15–۱۷) and PVYNTN-NW (2) or PVYNTN-HN2 (9,10). The two recombinant PVYNTN groups differ from each other at the third recombination junction (recombinant joint [RJ]3). In Eu-PVYNTN, the RJ3 occurs at the 3′ proximal end of the coat protein (CP) gene at approximately nucleotide 9,100, thus leading to a PVYN-type of CP and a PVYN serotype; whereas, in PVYNTN-NW/PVYNTN-HN2, the RJ3 is located prior to the CP gene at nucleotide 8,700, thus leading to a PVYO-type of CP and a PVYO serotype (2,9,10). It is noteworthy that Eu-PVYNTN has been reported in many potatogrowing areas in the world, whereas the PVYNTN-NW/PVYNTN-HN2 has only been reported in Syria and China to date (2,9,10). An increase in PVY incidence in potato crops has been observed in North America over the last decade (3,7,19,21,29). This increase is at least partially due to the emergence of novel PVY strains or isolates that may cause only mild symptoms in most commonly grown cultivars and by the increased production of susceptible cultivars that do not develop clear-cut symptoms, thus evading symptom-based rogueing and field inspections (7). Considering the significance of symptom recognition in PVY management, characterization of different potato cultivars in response to infection by different PVY strain or isolate groups is of great interest to potato growers and the field inspectors. Symptom expression in host plants upon PVY infection is determined by the virus strain, isolate, or variant type and the host plant species and its genotype (24,25). It is further affected by environmental factors (e.g., temperature and light intensity), plant physiological conditions (e.g., plant age), and, more importantly, whether the infection is primary (current season infection) or secondary (tuber-borne) (5,24,33). The symptoms induced by PVYO includes mild to severe mosaic, leaf and stem necrosis and leaf drop in many potato cultivars, and mosaic on tobacco (7,22,29). Symptoms elicited by PVYN are generally milder than PVYO in most potato cultivars, ranging from symptomless to mild to severe mosaic, with severe veinal, petiole and stem necrosis, and premature leaf death occurring on infected tobacco (4,17,21,22,28). PVYN:O and PVYNTN induce PVYN-like symptoms on tobacco plants and generally cause more severe symptoms, including distinct mosaic or chlorotic mottling on potato foliage, than PVYN. Moreover, PVYNTN can cause necrotic ringspots on tubers of susceptible potato cultivars (1,7). Despite the general knowledge of symptomatology of PVY strains on potato, no systematic studies have been carried out to elucidate symptom expression in potato cultivars after being infected with different PVY isolates or strains. This article reports a study of responses of 14 potato cultivars to five distinct PVY isolates belonging to four strains (PVYO, PVYN:O, Eu-PVYNTN, and NA-PVYN) in both primary and secondary infections under greenhouse and field conditions. Differential symptom expression and disease development for each cultivar in response to infection with different PVY isolates were investigated and presented. Materials and Methods Virus isolates and potato cultivars. Five PVY isolates— PVYO-FL (a severe PVYO isolate), PVYO-RB (a mild PVYO isolate), PVYN:O-Mb58, PVYN-Jg, and PVYNTN-Sl (14–۱۸)—were used in this study. Viruses were maintained in tobacco hosts in the greenhouse at the Potato Research Centre, Agriculture and AgriFood Canada (PRC-AAFC). Prior to the inoculation, the strain or isolate identity and purity was verified by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) as described previously (14–۱۸,۲۹). Virus-free tissue culture plantlets of 14 potato cultivars (namely, ‘AC Chaleur’, ‘CalWhite’, ‘Cherokee’, ‘Eramosa’, ‘Goldrush’, ‘Jemseg’, ‘Katahdin’, ‘La Rouge’, ‘Ranger Russet’, ‘Red LaSoda’, ‘Russet Burbank’, ‘Russet Norkotah’, ‘Superior’, and ‘Yukon Gold’) were obtained from the Plant Propagation Centre (New Brunswick Department of Agriculture, Aquaculture and Fisheries). The plantlets were transplanted to 6-in. (15.2 cm) pots containing premixed soil in the greenhouse with a cycle of 16 and 8 h (light and darkness, respectively). The ambient light was supplemented with artificial light or shading to give a light intensity of 90 µm2 /s. The temperature was 18 to 22°C and the humidity was 75%. Virus inoculation and symptom observation. For the primary infection experiments, four potato tissue culture plantlets of each cultivar were mechanically inoculated with PVY isolates on each of the three uppermost leaves at the six-leaf stage as described previously (29), and grown in a greenhouse. Plantlets inoculated with the inoculation buffer (mock) (10 mM phosphate buffer, pH 7.5, with 32 mM sodium sulfite) served as control treatments. Foliage symptoms were monitored daily after inoculation until harvest. For each pot, tubers were harvested and observed. Tuber symptoms, mainly necrotic ringspots or potato tuber necrotic ringspot disease (PTNRD), were checked at harvest and monthly post harvest for up to 4 months. The experiments were repeated two times. In the first repeat, the differences of tuber yield between virusinoculated plants and mock-inoculated (control) plants were visually observed and estimated. In the second repeat, the yield of each plant was measured and statistically analyzed using a t test. Tubers resulting from the above plants were used for the secondary infection experiments in both the greenhouse and field. The experiments were repeated two times, each using tubers resulting from one of the two primary infection experiments. For the secondary infection experiments in the greenhouse, one progeny tuber from each of the above primarily infected plants were planted in 6- in. (15.2 cm) pots and grown in a greenhouse under the conditions described above. Foliage symptoms, including the symptom type and symptom development, were recorded every other day after plant emergence until harvest. Upon harvest, tubers from each plant were observed for PTNRD. The tubers were reexamined for PTNRD monthly post harvest for up to 4 months. The impact of PVY infection on yield was measured as described above in the primary infection experiments. For the secondary infection experiments in the field, 10 progeny tubers of the primarily infected plants from each virus isolate– potato cultivar combination were planted in one row, 3 feet (91.4 cm) apart in the field plots at PRC-AAFC in the 2010 and 2011 growing seasons (late May to early October). All treatments of the same cultivar were planted side by side with the sequence of PVYO-FL, PVYO-RB, PVYN:O, PVYNTN, PVYN, and mock. The plants were tested for infection with the intended PVY isolates by RT-PCR and ELISA, as described below. The plants were managed with the regular management practices by the farm services staff at PRC-AAFC. Foliage symptoms, including the emergence rate, symptom type, and symptom development, were recorded twice a week until late August. Tuber symptoms were recorded at harvest and at 2 months post harvest. ELISA and RT-PCR. ELISA with the PVYO and PVYN serotype-specific antibodies MAb2 (PVYO) and 1F5 (PVYN) (Phytodiagnostics) was used to verify the serotypes of the inoculum prior to the mechanical inoculation, whereas ELISA with the serotypenonspecific antibody PVY-Poly (Neogen Europe) was used for the infection experiments. The ELISA was carried out as described previously (27,29) at the Agricultural Certification Services. For the primary infection experiments, leaves located above the inocu lated leaves were sampled at 21 days post inoculation (dpi) and used for ELISA; whereas, for the secondary infection experiments, leaves were sampled at 21 days post plant emergence (dpe) for the ELISA. Four sets of RT-PCR assays—including the P1-gene-based RTPCR (15), the RJ-based RT-PCR (17), the CP-gene-based PVYO variant differentiation RT-PCR (14), and the multiplex RT-PCR described by Lorenzen et al. (13)—were used to confirm the PVY isolate or strain purity and identity. Total RNA from leaves located above the inoculated leaves at 28 dpi in the primary infection experiments or from the upper leaves at 30 dpe in the secondary infection experiments was extracted using the sodium sulfite method (30). RT-PCR assays were performed as described in the above-mentioned articles.