Vol. 511996): 57-62. Resistance to potato virus A and potato virus Y in potato cultivars grown in Finland Jari RT. Valkonen 1 Department ofPlant Production, PO. Box 27, FIN-00014 University ofHelsinki, Finland Jukka P. Palohuhta The Seed Potato Center, Leppiojantie 25, FIN-91800 Tyrnävä, Finland Phenotypic expression of resistance to potato Y potyvirus (PVY) and potato A potyvirus (PVA) was tested in 24 potato cultivars and an advanced breeding line using graft-inoculation under controlled conditions in the glasshouse. Resistance phenotypes were determined based on symptom expression and systemic infection detected with DAS-ELISA. Tubers were harvested from the PVA-inoculated plants and tested for PVA with ELISA. Sixteen potato cultivars expressed hypersensitive resistance (HR) to the strain group Y° ofPVY. Ute expressed extreme resistance (ER) toPVY (strain groups Y° and Y N) and PVA, and eight cultivars (Amazone, Bintje, Fambo, Posmo, Record, Rosamunda, Satur- na and Van Gogh) expressed ER to PVA. These cultivars produced no PVA-infected tubers (tubers of Record were not tested). Matilda and Nicola expressed HR to PVA. The tubers of graft-inoculated Matilda produced no PVA-infected shoots, whereas shoots from Nicola tubers developed necrosis and severe mosaic symptoms and were PVA-infected based on results from ELISA. Comparison with purified PVA antigen (using ELISA) indicated that the secondarily infected shoots of the 14 PVA- susceptible cultivars contained 206-804 ng of PVA antigen per gram of leaf tissue. Key words: hypersensitive response, extreme resistance, potyvirus. Solarium tuberosum 'Current address: Institute of Biotechnology, P.O. Box 56, FIN-00014 University ofHelsinki, Finland Introduction Potato A potyvirus (PVA) and potato Y potyvirus (PVY) belong to the large and agriculturally im- portant family Potyviridae (Shukla et al. 1994). They can be transmitted mechanically, but are usually transmitted by aphids in the field in a non-persistent manner. PVA and PVY occur in potato worldwide and can cause yield losses up to 40% and 80%, respectively (Hooker 1981). Some potato cultivars (Solarium tuberosumL.) show necrotic lesions or top necrosis, i.e. a hy- persensitive resistance response (HR), follow- ing infection withPVY and/or PVA (Cockerham 1943, Ross 1952, MacLachlan etal. 1953, Jones 1990). Expression of HR can reduce the spread of PVY and PVA in the potato crop in the field, © Agricultural and Food Science in Finland Manuscript received February 1996 57 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=Pgi-tiweQMEghk8s.TKwLGkbBZPC1VKnk95-4Yg.m3xjbh7tyJT5bdxUqkbJ4MqC-boq8HY_iIa8Ayq2-ynxMj9HUtaBfxK873mMT3NsuFJADEWmIUZsC2uKazIsWEx_9uckDncdbWzZQw2Q-4x4Hi1bT12OAbjNEKrKlARQv5YmQ0u6Z8Gf-vVrR1YyqmcmoL1fANA_h47OeCRNNKSKfQZfgi-8UOnJOl4vd_5j_aq76-M2ormOkqmOB5g9soARfZnhtjvZc5tazBZp66F1vvp25uLiGla_utpMQOaeoxOLE8vCPCDgef9H9fo7egktTWjVPZA6551IfoA Valkonen, J. & Palohuhta, JResistance to PVA and PVY in potato cultivars in Finland (Research Note) but it seldom completely prevents it (Ross 1952). HR is usually virus strain-specific. Thus, virus isolates can be grouped according to their abil- ity to elicit a particular hypersensitivity gene or genes in potato, and this forms the basis of the strain groupconcepts for PVY (Jones 1990) and PVA (Valkonen et al. 1995). Extreme resistance (ER) to PVY and/or PVA, defined as a very low incidence of infection in intact plants with ex- tremely low virus titer in infected plants, is also known to exist in potato species. For reliable identification ofER in potato plants, graft-inocu- lation must be carried out (Benson & Hooker 1960, Valkonen et al. 1994). ER is functional against all strains of the potyvirus (Ross 1986, Valkonen 1994). Furthermore, ER to PVA and, in some potato genotypes, HR to PVA are also functional against PVY. HR and ER to PVY and/ or PVA in potato are dominantand inherited mono- genically (Cockerham 1943, 1970, Jones 1990, Ross 1986). PVY is economically the most important virus infecting potatoes in Finland (Kurppa 1983). Strains of the ordinary strain group (Y°) and the tobacco veinal necrosis strain group (YN ) ofPVY exist in Finland, of which YN seems to be the more prevalent (Kurppa 1983). Many potato cul- tivars express specific HR to Y°, whereas no HR to YN is known in cultivatedpotato. Thus, ER is required for the efficient control of all strains of PVY (Jones 1990). PVA is less prevalent than PVY in potatoes in Finland (Kurppa 1983). How- ever, recent observations suggest that in certain cultivars, e.g., Sabina, Puikula and Pito, and also Hertha and Hankkijan Timo, PVA is prevalent and more important than previously recognized. The strains of PVA infecting potatoes in Finland have not been systematically grouped according to the strain group concept for PVA (Valkonen et al. 1995). Moreover, theresistance to PVA has not been systematically examined in most of the potato cultivars currently grown in Finland. In this study, the phenotypic expression of resistance to PVA and Y° was examined in pota- to cultivars and an advanced potato breeding line, all of which are commonly grown, or are bred to be grown, in Finland and are included in the healthy seed potato production scheme at the Seed Potato Center, Tyrnävä. Experiments were carried out under controlled conditions in the glasshouse to permit reliable detection of the resistance phenotypes. Graft-inoculation was used to identify cultivars expressing ER, which provides efficient control of PVA and PVY in the field. Material and methods Pathogen-free minitubers of 25 potato cultivars (Table 1) were obtained from the Seed Potato Center, Tyrnävä. Tubers were planted in soil and plants were grown for virus inoculation under natural daylight in a glasshouse during March- May in 1995. The mean daily minimum and maximum temperatures were 18°C and 24°C. An isolate of PVA (A-9506) was obtained from potato cv. Hankkijan Tanu infected in the field in Tyrnävä. Tobacco ( Nicotiana tabacum L. cv. Samsun NN) was mechanically inoculated with the virus but developed no symptoms. The virus was back-inoculated mechanically to healthy plants of Hankkijan Tanu. It reacted se- rologically with polyclonal antibodies (Pab) to PVA (Boehringer Mannheim, Germany) and a monoclonal antibody to PVA (Mab no. 58/0; Browning et al. 1995) obtained from the Scot- tish Agricultural Science Agency (SASA), Scot- land. It did not react with Pabs to PVY, potato X potexvirus, potato M and S carlaviruses (Boeh- ringer Mannheim) and the Mab no. 53/8 to po- tato V potyvirus obtained from SASA. The iso- late of PVY (PVY°-UK) used in this study was described by Valkonen and Mäkäräinen (1993). A Finnish isolate of YN was kindly provided by A. Kuusela, Institute of Plant Protection, Agri- cultural Research Centre, Jokioinen, Finland. PVA, Y° and YN were maintained in potato cvs. Hankkijan Tanu, Pito and Valtti, respectively, in the glasshouse. Side-graft inoculation, using apical shoots of the virus-infected potato cultivars, was carried 58 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 57-62. Table 1. Phenotypic responses in potato cultivars following graft-inoculation with PVY° and PVA. Symptoms were ob- served and the virus litres were determined in the uppermost fully-expanded leaves using DAS-ELISA four weeks after graft-inoculation (PVY° and PVA) and four weeks after the emergence of the sprouts (tuber-borne infection with PVA). Potato virus Y° Potato virus A Tuber-borne infection Symptoms in Symptoms in Virus graft-inoculated Resistance graft-inoculated concentration (ng/g)' Resistance Cultivar plants' phenotypeb plants' Symptoms" Mean s.d. phenotypeb Amazone M S ns ns 0 ER Asterix NL H ns ns 475 130 S Bintje M S Npp ns 0 ER Fambo TN H ChS ns 0 ER Gloria MS ns Ru 277 65 S Hankkijan Tanu NL, SM H ns ns 689 171 S Hankkijan Timo NL, M H Mo Ru, M 362 137 S Hertha MS Y Y 259 130 S Kardal NL H M ns 251 38 S LH- 152-80 NL H ns ns 278 101 S Matilda TN H NL ns 0 H Nicola NL H TN NL, SM 78 31 H Posmo NL H Npp, ChS ns 0 ER Puikula M S M SM 300 59 S Record NL H Npp, ChS * * ER(?) Rosamunda TN H ns ns 0 ER Sabina M S M ns 445 84 S Satu NL, LD H ns ns 206 89 S Saturna NL H ns ns 0 ER Sieglinde (Siikli) NL H ns M 804 219 S Sini (Jo-0912) TN H ns ns 329 123 S Ute nsi ER ns ns 0 ER VanGogh SSI S ChS ns 0 ER Vento MM S MM ns 439 224 S Vital NL H ns Ru, M 262 57 S ”ChS, chlorotic spots; LD, leaf-drop; M, mosaic; MM, mild mosaic; Mo, mottle; NL, necrotic lesions and/or vein necrosis; Npp, necrotic pin-point lesions; Ru, rugosity; SM, severe mosaic; SSI, systemic symptomless infection; TN, top necrosis; Y, yellow blotches; ns, no symptoms; nsi, no symptoms and no infection based on ELISA; *, not determined. bER, extreme resistance, based on no virus detected with ELISA and no necrosis observed except limited pin-point lesions; H, hypersensitive resistance response, based on the development of necrosis and virus titers detected with ELISA in the graft-inoculatedplants; S, susceptible, based on no necrotic response and high virus titers detected with ELISA. c Mean concentration from six plants; s.d., standard deviation; *, not determined. out according to Valkonen and Mäkäräinen (1993). Five plants of each potato cultivar were graft-inoculated with PVA and Y° two weeks after shoot emergence. Additionally, five plants of cv. Ute were graft-inoculated with YN. One shoot per plant was graft-inoculated with one virus-infected potato scion and other shoots were removed. One non-inoculated plant of each po- tato cultivar was grown as a control. Symptom development was observed until 28 days after inoculation. Virus litres were tested in the up- permost fully-expanded leaves using DAS-ELI- SA (Valkonen and Mäkäräinen 1993). PVA was tested using the Mab no. 58/0 and the alkaline 59 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Valkonen, J. & Palohuhta, J.: Resistance to PVA and PVY in potato cultivars in Finland (Research Note) phosphatase(AP)-conjugated Mab no. 58/0 ob- tained from SASA (Browning et al. 1995). PVA particles were purified from PVA-infected Tanu essentially as described by Browning et al. (1995). Virus concentration in the purified PVA preparation was determined spectrophotometri- cally and by comparison with known concentra- tions of a protein marker (Sigma) in gel electro- phoresis (SDS-PAGE; Laemmli 1970). PVA con- centrations in the leaf samples were determined by comparison with known concentrations of the purified PVA particles using DAS-ELISA. PVY was tested using the Pabs and AP-conjugated Pabs to PVY obtained from Boehringer. Tubers of the PVA-inoculated potato plants were harvested eight weeks after inoculation and stored at 3°C for three months. Six tubers ofeach potato cultivar were planted in soil and grown under natural daylight in the glasshouse in September-October. Symptoms were observed and PVA litres were tested as described above four weeks after shoot emergence. Results and discussion Of the of 25 cultivars tested, only Ute expressed ER to PVY and PVA (Table 1): no symptoms were observed, no Y°, YN and PVA were found following graft-inoculation, and no PVA was detected in the shoots grown from the tubers of the PVA-inoculated plants [ELISA readings (A405 ) were similar (0.00 - 0.02) for the graft- inoculated and non-inoculated Ute regarding detection of PVY and PVA]. Ute carries genes from the wild potato species S. stoloniferum Schlechdt. et Bché. (Ross 1986).Thus, it is likely that the comprehensive ER to PVY (and PVA) in Ute is controlled by the gene Ry sio from S. stoloniferum (Ross 1986). Sixteen cultivars expressed HR to Y°. These were not tested with Y N because it is known that the HR to Y° in S. tuberosum is not elicited by YN and that S. tuberosum has no HR to YN (Ross 1986, Jones 1990). Seven cultivars were susceptible and ex- hibited mosaic symptoms following infection with Y°. Van Gogh showed no symptoms (Table 1). This is consistent with the observations that Van Gogh can be severely infected with PVY in the field but remains symptomless (L. Pietilä, personal communication). Nine cultivars (including Ute) were extreme- ly resistant to PVA (Table 1). The necrotic pin- point lesions observed in the plants of Bintje, Posmo and Record, following graft-inoculation with PVA, were similar to the lesions described in a few extremely resistant potato genotypes graft-inoculated with PVY (Ross 1986). As no tubers of the graft-inoculated Record were test- ed for PVA infection, ER to PVA in this cultivar could not be confirmed (Table 1). This caution in the determination of theresistance phenotype of Record is based on the observation that the graft-inoculatedplants of many cultivars during this study had similar ELISA readings (i.e., neg- ative) to the non-inoculated plants (data not shown). However, all six tubers of each of these cultivars produced shoots with high PVA titers, indicating that they were susceptible to PVA (Table 1). In Matilda and Nicola, necrotic lesions developed on the leaves and PVA was detected with ELISA following graft-inoculation, which indicated expression of HR. Fourteen potato cul- tivars were susceptible to PVA, but in many of them the shoots with secondary PVA infection (i.e., grownfrom the infected tubers) showed no symptoms (Table 1). Stegemann and Schnick (1985) listed the sus- ceptibility to PVY in many European potato va- rieties, 14 of which were included in this study. Susceptibilities were indicated by the numbers 1 (very low susceptibility) to 9 (very strong sus- ceptibility) and by 0 (resistant). Additionally, HR and ER were specifically indicated for a few cultivars. However, the strains and strain groups of the PVY isolates used, and the method of test- ing, were not reported, but the evaluations were likely to have been based on observations in the field. Thus, the previously reported resistances (Stegemann and Schnick 1985) and the pheno- typic resistance responses observed in this study cannot be readily compared. For example, no 60 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 57-62. cultivar which expressed characteristic HR to Y° in this study was listed as hypersensitive to PVY by Stegemann and Schnick (1985). Susceptibil- ity to PVA in Amazone, Bintje, Gloria, Nicola, Saturna, Sieglinde and Ute was low (2) or very low (1), and the susceptibility to PVA in Hertha was intermediate (5) according to Stegemann and Schnick (1985). Results from our study showed that Amazone, Bintje, Saturna and Ute express ER to PVA, Nicola expresses characteristic HR to PVA, and Gloria, Hertha and Sieglinde are susceptible to PVA. Record was hypersensitive to PVA according to Stegemann and Schnick (1985), whereas our results suggested that Record expresses ER to PVA. The differences between the results from this study and the list of Stegemann and Schnick (1985) are probably attributable to 1) the different virus strains used; 2) different methods used for testing the resist- ance expression; 3) the difficulty of observing HR reaction in the cultivars infected in the field in the previous studies, and 4) some cultivars which are susceptible to PVY or PVA following graft-inoculation can have quantitative resistance to PVY and/or PVA which is expressed as a low incidence of infection in the field if the infec- tion pressure, resulting from aphid infestation, is low (Davidson 1980). In conclusion, this study reports the pheno- typic expression of resistance to PVA and PVY in selected potato cultivars currently grown in Finland as determined under controlled condi- tions. The data are useful to agriculturalists and breeders as the utilizationand deployment strat- egies for resistance to PVA and PVY will be partially determined by the locally prevalent virus strain groups and the comprehensive or strain-group-specific resistance of the potato cultivars and breeding lines. Acknowledgements. We are grateful to Ulla Malkamäki for assistance in virus purification. Financial support from the Academy of Finland is gratefully acknowledged. References Benson, A.P. & Hooker, W.J. 1960. Isolation of virus X from “immune” varieties of potato, Sotanum tuberosum. Phytopathology 50: 231-234. Browning, 1.A., Burns, R., George, E.L. & Darling. M. 1995, Development and evaluation of ELISA assays in- corporating monoclonal antibodies for the detection of potato A potyvirus. EPPO Bulletin 25: 259-268. Cockerham, G. 1943. The reactions of potato varieties to viruses X, A, B and C. Annals of Applied Biology 30: 338-344. - 1970. Genetical studies on resistance to potato virus- es X and Y. Heredity 25: 309-348. Davidson,T.M.W. 1980. Breeding for resistance tovirus disease of the potato (Solarium tuberosum) at the Scot- tish Plant Breeding Station. 59th Annual Report of the Scottish Plant Breeding Institute, p. 100-108. Hooker, W.J. 1981. Compendium of potato diseases. American Phytopathological Society and Michigan State University. St. Paul, MN, USA. 125 p. Jones, R.A.C. 1990. Strain group specific and virus spe- cific hypersensitive reactions to infection with potyvirus- es in potato cultivars. Annals of Applied Biology 117:93- 105. Kurppa, A. 1983. Potato viruses in Finland and their iden- tification. Journal ofThe Scientific Agricultural Society of Finland 55: 183-300. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of Bacteriophage T 4. Nature 227: 680-685. MacLachlan, D.S., Larson, R.H. & Walker, J.C. 1953. Strain relationships in potato virus A. Research Bulletin No. 180. University of Wisconsin, Madison, Wl, USA. 35 p. Ross, H. 1952. Studies on mosaic resistance in the po- tato. Proceeding of the Conference on Potato Virus Dis- eases. Wageningen-Lisse, The Netherlands, p. 40-47. - 1986. Potato breeding - problems and perspectives. Journal of Plant Breeding, Supplement 13. 132 p. Shukla, D.D., Ward, C.W. & Brunt, A.A. 1994. The Potyviridae. C.A.B. International, Wallingford, UK. 516 p. Stegemann, H. & Schnick, D. 1985. Index 1985 of Eu- ropean potato varieties. Mitteilungen aus der Bio- logischen Bundesanstalt fur Land- und Forstwirtschaft. Heft 227. Paul Parey, Berlin. 126 p. Valkonen, J.P.T. 1994. Resistance to viruses in cultivat- ed and wild potato species (Solarium spp,). Plant Breed- ing 112:1-16. - & Mäkäräinen, E. 1993. Symptom expression and ac- cumulation of potato virus Y (PVY°) and potato leaf roll virus in thirteen potato cultivars. Agricultural Science in Finland 2: 33-40, Slack, S.A. & Plaisted, R.L. 1994. Use of the virus strain group concept to characterize the resistance to PVX and PVY° in the potato cv. Allegany. American Potato 61 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Valkonen, J. & Palohuhta, J.: Resistance to PVA and PVY in potato cultivars in Finland (Research Note) Journal 71: 507-516. on hypersensitive responses in potato, serological prop- erties, and coat protein sequences. Plant Disease 79: 748-753. Puurand, U., Slack, S.A., Mäkinen, K. & Saarma, M. 1995. Three strain groups of potato A potyvirus based SELOSTUS Suomessa viljeltävien perunalajikkeiden kestävyys A- ja Y-virusta vastaan Jari Valkonen ja Jukka Pekka Palohuhta Helsingin yliopisto ja Siemenperunakeskus Tutkimuksessa testattiin 24 perunalajikkeen ja yhden kotimaisen perunan jalostuslinjan kestävyyttä peru- nan A- ja Y-virusta vastaan. Testattavat perunat saas- tutettiin viruksilla varttamalla ne kasvihuoneessa. A- viruksella saastutetuista kasveista otettiin talteen mukulat, jotka idätettiin ja versot testattiin. Viruskes- tävyyden tyyppi määritettiin oireiden perusteella ja mittaamalla viruspitoisuudet ELISAm avulla. Kuu- dessatoista lajikkeessa havaittiin hypersensitiivistä kestävyyttä (yliherkkyyttä) Y-viruksen roturyhmää Y° vastaan, mikä ilmeni kuoliolaikkujen tai latvakuolion kehittymisenä. Ute havaittiin äärimmäisen kestä- väksi Y- ja A-virusta vastaan. Amazone, Bintje, Fam- bo, Posmo, Record, Rosamunda, Saturna ja Van Gogh olivat äärimmäisen kestäviä vain A-virusta vastaan. Äärimmäinen kestävyys ilmeni siten, että kasveihin ei kertynyt ELISA:IIa havaittavia viruspitoisuuksia. Lisäksi kasvit pysyivät oireettomina tai lehtiin ilmes- tyi pistemäisiä kuoliolaikkuja tai pieniä kloroottisia laikkuja. A-viruksella saastutettujen äärimmäisen kestävien lajikkeiden mukuloista kasvaneissa versois- sa ei ollut A-virusta. Matildassa ja Nicolassa ilmeni hypersensitiivistä kestävyyttä A-virusta vastaan. Ma- tildan mukuloista kasvaneissa versoissa ei ollut A- virusta, mutta Nicolan mukuloista kasvaneet versot olivat viroottisia ja niissä ilmeni kuoliota sekä sel- keitä mosaiikkioireita. A-viruksen tartuttamat, viroot- tisista mukuloista kasvatetut versot olivat oireettomia useimmissa lajikkeissa. Tämän tutkimuksen perus- teella ainoastaan Ute on luonnollisen kestävyytensä ansiosta suojattu sekä Y- että A-virukselta. Hypersen- sitiivisen kestävyyden tehokkuus vaihtelee lajikkei- den kesken ja eri ympäristöoloissa, ja kohdistuu vain tiettyyn tai muutamaan virusrotuun. Joissakin tässä tutkimuksessa alttiiksi todetuissa lajikkeissa Y- tai A- virussaastunta voi olla alhainen pellolla, mikä perus- tuu näiden lajikkeiden kykyyn vastustaa kirvojen le- vittämää virusta. 62 AGRICULTURAL AND FOOD SCIENCE IN FINLAND