Acta agriculturae slovenica, 83 - 1, junij 2004 str. 31 - 36 Agrovoc descriptors: solanum tuberosum, potyviruses, potato Y potyvirus, monitoring, identification, geographical distribution, strains, infection, necrosis, ELISA, Slovenia Agris category codes: H20 Agricultural Institute of Slovenia Crop and Seed Production Department COBISS Code 1.01 The incidence of potato virus Y strains in Slovenia Peter DOLNIČAR1 Received: March 24, 2004; accepted: May 24, 2004 Delo je prispelo 24. marca 2004; sprejeto 24. maja 2004 ABSTRACT The incidence of potato virus Y (PVY) strains and its infection pressure across Slovenia were monitored. Disease free tubers of the variety Igor were planted on different locations across Slovenia in 1997, 1998 and 2000. Tubers were harvested and visually evaluated for the presence of necrosis. They were tested by means of ELISA using polyclonal antibodies of PVY and monoclonal antibodies PVYN of Bioreba, and PVYN, PVYO and PVY O/C of Adgen. PVY NTN was the predominant strain on all locations in 1997 and 1998. The highest infection with PVYO was found in Pivka and Rakičan in the year 2000. PVYC strain was not found during the study. The infection with PVY was smaller in isolated parts of Slovenia like Brezula, Podčetrtek, Trbonje, Libeliče, Vrtojba and Tolmin. It also decreased from 1997 to 2000. Key words: potato, Solanum tuberosum L., PVY, strains, necrosis, infection IZVLEČEK PRISOTNOST RAZLIČKOV KROMPIRJEVEGA VIRUSA Y V SLOVENIJI V raziskavi smo preučevali prisotnost različkov krompirjevega virusa Y (PVY) in njegov infekcijski pritisk po Sloveniji. V letih 1997, 1998 in 2000 smo na različnih lokacijah po Sloveniji posadili gomolje sorte Igor, proste vseh bolezni in škodljivcev. Po izkopu pridelka smo vizualno ocenili prisotnost nekroz na gomoljih. Pri vseh gomoljih smo s serološko metodo ELISA določili prisotnost PVY. Uporabili smo poliklonska protitelesa PVY in monoklonska protitelesa PVYN proizvajalca Biorebe in monoklonska protitelesa PVYN, PVYO in PVY O/C proizvajalca Adgen. V letih 1997 in 1998 je bil na vseh lokacijah večinoma ugotovljen različek PVY NTN. Najmočnejšo okužbo z različkom PVYO smo ugotovili v Pivki in Rakičanu v letu 2000. Različka PVYC v raziskavi nismo odkrili. Okužba s PVY je bila manjša na območjih z dobro izolacijo v Brezuli, Podčetrtku, Trbonjah, Libeličah, Vrtojbi and Tolminu. Od leta 1997 do 2000 se je okužba s PVY zmanjšala. Ključne besede: krompir, Solanum tuberosum L., PVY, različki, nekroze, okužba M.Sc., SI-1000 Ljubljana, Hacquetova 17 1 32 Acta agriculturae slovenica, 83 - 1, junij 2004 1 INTRODUCTION Potato virus Y (PVY) has a world-wide distribution and is one of the most important pathogens which affect potato. Three main groups have been distinguished until now: PVYN, PVYO and PVYC. PVYO strain is found world-wide, whereas PVYN strain has been reported in Europe, North and South America and part of Africa (Stevenson et al., 2001). It spread in Europe in the seventies. A new PVYNTN strain was first found in Europe in 1984 (Beczner et al., 1984) and it was reported later on by numerous authors. The PVYNTN strains constitute a new sub-group of PVYN isolates (Boonham et al., 1998). According to Boonham and Nie with co-workers, PVYNTN could evolve from PVYN strain (Boonham et al., 1998, Nie et al., 2001). Further on, a different sub-group of PVYN isolates (Wilga type) was reported in Poland (Chrzanowska, 1991). PVYNTN isolates were confirmed in most European countries during the last decade. PVYNTN has also been the most serious potato disease in Slovenia in the last 13 years. It almost stopped seed potato production since it was first observed in 1986. Because of its effects on tuber quality it also changed the assortment of the varieties in production completely. The old Slovene varieties were not replaced only by resistant ones but also by some susceptible ones (Kus, 1995). Susceptible varieties are prone to the infection on foliage, but they do not show symptoms on tubers. Planting the infected tubers of susceptible varieties makes the virus survive from year to year. The infection of potato fields increased rapidly after the first appearance of the virus in Slovenia. Until 1994, a high infection pressure was observed. High aphid populations were also found during that period (Bitenc-Korinšek et al., 1995). After that, the infection grew lower along with lower aphid population. In the meantime, several resistant varieties were introduced into production. Lesser infection was observed on the fields and lesser necrosis found on tubers. Therefore, the following questions could be discussed: - Has the infection pressure of PVY NTN dropped during the past few years? - Is the infection rate similar across Slovenia? - Is the PVYNTN still the main strain in Slovenia? 2 MATERIALS AND METHODS Disease free tubers of the variety Igor were planted on 17 locations (Table 1) across Slovenia in 1997, 1998 and 2000. Igor is a very susceptible variety which shows symptoms after the infection with PVYNTN. 50 tubers were planted on each location in spring. Plants were exposed to the natural infection with PVY during growing period and harvested in September. After harvest they were transferred to the storage at the temperature of 10oC. Since it is known that most of the necrosis appears during storage, the tubers were visually evaluated three times: after harvest in September, at the end of October and in December. Tubers with and without necrosis were separated on each location. DAS-ELISA (Clark et al., 1977) was performed according to the supplier protocols using polyclonal antibodies PVY and monoclonal PVYN of Bioreba, and PVYN, PVYO and PVY O/C of Adgen. In 2000, the tubers were separated in two groups, with and without necrosis, and replanted next year. Plants were visually estimated and re-tested by means of ELISA. DOLNIČAR, P.: The incidence of potato virus Y strains in Slovenia 33 3 RESULTS AND DISCUSSION 3.1 The appearance of necrosis The appearance of necrosis on all harvested tubers is presented in Table 1. The rate of tubers with necrosis dropped from 1997 to 2000. The data in Komenda show 68.6% tubers with necrosis in 1997, comparing to 49.4% in 1998 and 15.1% in 2000. In Rakičan, 80.2% tubers with necrosis were observed in 1997, comparing to 14.0% in 1998 and 4.3% in 2000. We believe that the reason for this can be found in the lower infection during the growing season (Tables 2 to 4). Lower infection is the result of lower infection pressure which was achieved by planting resistant varieties and by higher proportion of good potato seed, and in the case of Rakičan, also by large drop in potato production in those years. The highest drop due to the same reasons was observed in Vrtojba, ranging from 80.6% tubers with necrosis in 1997 to 1.6% tubers with necrosis in 1998. Differences between locations within the same year were also observed (Table 1). There were 6.6% tubers with necrosis in Brezula and 80,2% in Rakičan in 1997, and 1.6% in Vrtojba comparing to 68.2% in Samotorica in 1998. A greater number of infected tubers without necrosis was found in 2000 than in the previous years. That also confirms the theory saying that some other external (probably environmental) factors influence the appearance of the necrosis. 3.2 The infection rate Table 1: The rates of tuber necrosis caused by PVY NTN in Slovenia in 1997, 1998 and 2000 Year Location Brezula Podčetrtek Trbonje Libeliče Idrija Novo mesto Miklavž Kranj Ljubljana Samotorica Komenda Zgornje Jablane Celje Rakičan Vrtojba Pivka Tolmin The highest infection rate was No. of tubers 144 433 371 236 206 797 72 661 482 331 440 739 598 409 422 1997 Necrosis (%) Yes 6.3 9.9 10.5 14.8 20.9 25.1 41.7 50.4 63.7 64.7 68.6 68.9 74.4 80.2 80.8 No 93.8 90.1 89.5 85.2 79.1 74.9 58.3 49.6 36.3 35.3 31.4 31.1 25.6 19.8 19.2 No. of tubers 419 283 243 361 302 356 125 268 250 1998 Necrosis (%) Yes 48.4 68.2 49.4 68.1 44.0 14.0 1.6 47.0 2.4 No 51.6 31.8 50.6 31.9 56.0 86.0 98.4 53.0 97.6 No. of tubers 46 73 92 92 92 2000 Necrosis (%) Yes 21.7 15.1 19.6 4.3 10.9 No 78.3 84.9 80.4 95.7 89.1 found in 1997 in some major potato growing areas (Ljubljana, Kranj, Komenda, Celje, Rakičan) and in Primorska region where early 34 Acta agriculturae slovenica, 83 - 1, junij 2004 potato is grown (Vrtojba). Remote areas in which potato is not grown were less infected. Similar picture was seen in 1998, except in Rakičan and Vrtojba, where most of the farmers gave up the potato production. It can be observed from the percentage of infected tubers of both groups (tubers with necrosis and without it) that the infection has been smaller in the last years (the first two columns of Tables 2 to 4). The reasons for that are the same as mentioned before. It could be concluded that the infection is lower because of lesser amount of highly infected fields, because higher rate of seed replacement in Slovenia and smaller populations of vectors in the last few years (Južnik et al., 1996). This trend can also be seen from the data of seed potato production (Južnik et al., 1997, 1998, 2000). 3.3 PVY strains Tubers were tested by means of ELISA using polyclonal antibodies of PVY and monoclonal PVYN of Bioreba, and PVYN, PVYO and PVY O/C of Adgen. We did not find any difference between monoclonal antibodies PVYN of Bioreba and PVYN of Adgen. In 1997, most of the tubers with necrosis were positive when tested with both PVY polyclonal and monoclonal PVYN antibodies (Table 2). It appears that the reason for the necrosis on some tubers with necrosis was misdiagnosed and incorrectly annotated to the infection with PVY. That could be noticed in all three years. Only some tubers from the locations close to the Italian border were infected with PVYO. There was no infection with PVYC. Some tubers without necrosis were also found positive to monoclonal PVYN antibodies and, in Miklavž and Vrtojba, also to PVYO monoclonal antibodies. Table 2: The infection of tubers with different strains of PVY tested by means of ELISA in 1997 (in number of tested tubers) Location Brezula Podčetrtek Trbonje Libeliče Idrija Novo mesto Miklavž Kranj Ljubljana Samotorica Komenda Zgornje Jablane Celje Rakičan Vrtojba Tubers with necrosis No. of tubers 9 5 5003 10 20 10 22 41 29 75 56 54 60 86 40 20 40 PVY polyc. 9 10 10 17 35 28 75 55 47 56 69 40 20 40 PVYN 9 10 10 17 35 27 75 55 48 57 70 40 20 40 PVYO 0 0 0 0 0 1 0 0 0 0 0 0 0 0 PVYC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tubers without necrosis No. of tubers 38 40 40 20 21 21 40 30 10 20 PVY polyc. 6 6 1 10 1 7 15 - - 30 9 11 PVYN 8 00 6 1 11 1 7 14 30 9 7 PVYO 0 0 0 0 0 3 0 0 4 PVYC 0 0 0 0 0 0 0 0 0 From the visual symptoms on tubers and from the positive results with monoclonal PVYN antibodies it can be concluded that the variety Igor was infected mainly with PVYNTN. We could not decide if the infection with PVY was a mixed one. DOLNIČAR, P.: The incidence of potato virus Y strains in Slovenia 35 We did not find any infection with PVYO in 1998. Only the infection with PVYNTN was found in all locations (Table 3). Table 3: The infection of tubers with different strains of PVY tested by means of ELISA in 1998 (in the number of tested tubers) Tubers with necrosis Tubers without necrosis No. of PVY PVYO PVYC No. of PVY PVYO PVYC Location tubers 50 polyc. 43 PVYN 43 tubers 50 polyc. 4 PVYN 4 Pivka 0 0 0 0 Tolmin 6 4 4 0 0 50 0 0 0 0 Idrija 17 15 15 0 0 70 6 6 0 0 Kranj 40 35 35 0 0 - - - Ljubljana 60 53 53 0 0 70 24 24 0 0 Samotorica 50 50 50 0 0 50 42 42 0 0 Komenda 50 50 50 0 0 50 45 45 0 0 Zgornje Jablane 50 39 39 0 0 49 29 29 0 0 Celje 50 47 47 0 0 50 40 40 0 0 Rakičan 41 41 41 0 0 51 15 15 0 0 Vrtojba 2 2 2 0 0 50 3 3 0 0 PVYO was found in all locations in the year 2000 (Table 4). There was even more PVYO than PVYNTN in Pivka while in Rakičan PVYO infected tubers prevailed in the group with tubers without necrosis. We also found some mixed infections with both PVYNTN and PVYO strain. Table 4: The infection of tubers with different strains of PVY tested by means of ELISA in 2000 (in number of tested tubers) Tubers with necrosis Tubers without necrosis No. of PVY PVYO PVYC No. of PVY PVYO PVYC Location tubers 10 polyc. 10 PVYN 10 tubers 36 polyc. 30 PVYN 25 Idrija 0 0 12 0 Komenda 12 7 7 0 0 61 2 1 1 0 Celje 18 18 18 11 0 74 36 27 15 0 Rakičan 4 3 3 0 0 88 10 2 8 0 Pivka 10 5 4 1 0 82 16 2 14 0 Tubers were replanted in 2000. All tubers with necrosis and some without it showed symptoms on plants. They were found positive at ELISA testing. 36 Acta agriculturae slovenica, 83 - 1, junij 2004 4 CONCLUSIONS Based on the research carried out over several years we can conclude the following: 1. PVYNTN strain is still the prevalent strain in Slovenia, in spite of the fact that rather large infection with PVYO was found in the year 2000. 2. We could not decide if there was a mixed infection of PVY NTN with PVY N . 3. The infection was lower in some parts of Slovenia (isolated areas or areas with small amount of potato production). 4. The infection pressure has been lower in the last few years. 5 REFERENCES Beczner, L., Horvath, J., Romhanyi, I., Forster, H. 1984. Studies on the etiology of tuber necrotic ringspot disease in potato. Potato Research, 27, 1984, 3: 339-352. Bitenc-Korinšek, B., Južnik, M. 1995. The rate of infection with PVY and PLRV in seed potato production in Slovenia (1991-94). Bled, Book of abstracts, The 9th EAPR Virology Section Meeting: 55. Boonham, N., Walsh, K., Barker, I. 1998. Towards the detection of PVY-NTN. Baden, Book of abstracts, The 10th EAPR Virology Section Meeting: 69-70. Clark M.F., Adams A.N. 1977. 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