The 11th EAPR VIROLOGY SECTION MEETING Proceedings

Havlíèkùv Brod - Tøeš• Czech Republic 7 - 13th October, 2001 ............................................................. EUROPEAN ASSOCIATION FOR POTATO RESEARCH Virology section (2001 Trest) Proceedings of the 11th EAPR Virology Section Meeting: Trest, Czech Republic Compiler: Petr Dedic Organizing Committee: P. Dedic, Potato Research Institute Havlickuv Brod, Czech Republic J. Ptacek, Potato Research Institute Havlickuv Brod, Czech Republic N. Cerovska, Institute of Experimental Botany, CAS, Prague, Czech Republic Scientific Programme Committee: P. Dedic, Potato Research Institute Havlickuv Brod, Czech Republic C. Kerlan, INRA-ENSA Rennes, UMR BiO3P, F-35650 Le Rheu , France J. Matousek, Intitute of Plant Molecular Biology, CAS Ceske Budejovice, Czech Republic Place of the Meeting: Trest, Czech Republic, Zamecky hotel The individual contributions in this publication and any liabilities arising from them remain the responsibility of the authors. First Edition: September 2001 Editor: PRI, Potato Research Institute Havlickuv Brod, Dobrovskeho 2366, 58001 Havlickuv Brod, Czech Republic Printed by: Tisk Hermann & spolecnik, 2001 ..........................................................

CONTENT ORAL PRESENTATIONS Session 1:

"Variability of Potato virus Y"

Camille KERLAN, Miroslawa CHRZANOWSKA, Laurent GLAIS, Guillaume FREMONDIERE, Michel TRIBODET BIOLOGICAL AND MOLECULAR CHARACTERISATION OF VARIOUS PVYN W ISOLATES 1-3 NIE, X. SINGH, R.P. POSSIBLE EVOLUTION OF TUBER RINGSPOT NECROSIS STRAINS (PVYNTN ) FROM THE REGIONAL TOBACCO VEINAL NECROSIS (PVYN) STRAINS 4-6 Arie ROSNER, Ludmila MASLENIN NTN N DIFFERENTIATING PVY FROM PVY BY RESTRICTION CLEAVAGE PATTERNS, TRANSCRIPT CONFIRMATION POLYMORPHISIM AND BY ANNEALING TO REFERENCE RNA TRANSCRIPTS 7 Jiøí PTÁÈEK, Petr DÌDIÈ, Jaroslav MATOUŠEK THE DIVERSIFICATION OF POTATO VIRUS Y ( PVY)

8 - 11

Miros³awa CHRZANOWSKA IMPORTANCE OF DIFFERENT STRAINS OF PVY IN POTATO PRODUCTION AND BREEDING PROGRAM IN POLAND 12 - 14 Peter DOLNIÈAR MONITORING OF PVY STRAINS IN SLOVENIA

15 - 17

Urszula KACZMAREK, Ewa MOSAKOWSKA INTERACTION BETWEEN STRAINS OF THE POTATO VIRUS Y (PVY0 , PVYN - type Wilga, PVYNTN ) AND THE POTATO PLANTS OF TWO CULTIVARS. 18 - 20 Ewa TURSKA, Urszula KACZMAREK OCCURENCE OF THE POTATO VIRUS Y STRAINS (PVY) DURING LONG-TERM MULTIPLICATION OF SOME POTATO CULTIVARS IN POLAND. 21 - 24 HANDIZI, A., SERRA, J. A. & LEGORBURU, F. J. ESCAPING FROM PVY INFECTION IN THE FIELD: PLANTING DATES, APHID REPELLENTS AND RESISTANCE INDUCERS. 25

Jörg SCHUBERT, Jaroslav MATOUSEK, Dirk MATTERN, Frank RABENSTEIN, Petr DEDIC, Elena SUKHACHEVA NIB-MEDIATED RESISTANCE OF POTATOES TO PVY INFECTION 26 - 29 Ian BARKER, Jane MORRIS, David FIRMAN, Phil NORTHING, Richard LEACH, Sharon ELCOCK, Judith TURNER, Miles THOMAS , Keith WALTERS CONTROL OF POTATO VIRUS Y IN UK POTATO SEED CROPS 30

Session 2: "New diagnostic methods" Neil BOONHAM, Kathy WALSH, Hazel SWAN, Rick MUMFORD and Ian BARKER DIAGNOSIS OR DETECTION - DEVELOPING APPROPRIATE METHODS 31 - 33 Jaroslav MATOUŠEK ANALYSIS OF VIRUS AND VIROID VARIABILITY BY TGGE AND BY THE METHOD OF cDNA HETERODUPLEXES. 34 - 35 Rick MUMFORD, Anna BLOCKLEY, Kathy WALSH, Ian BARKER and Neil BOONHAM ADVANCES IN THE DIRECT DETECTION OF SPRAING FROM TUBERS 36 - 37 Session 3: " Soil-borne viruses" LEGORBURU, F. J. RATTLE AND MOP-TOP: PUZZLING VIRUSES FOR THE POTATO SEED PRODUCER. 38 J ROBINSON & M F B DALE PERSISTENT SYSTEMIC INFECTION OF POTATO CULTIVARS BY TOBACCO RATTLE VIRUS 39 - 40 H. XU and J. NIE DETECTION AND CHARACTERIZATION OF TOBACCO RATTLE VIRUS (TRV) ISOLATES USING RT-PCR AND RESTRICTION MAPPING 41 - 43 BROWNING I., CRAIGIE J., DARLING M., DARLING D. & HOLMES R. STUDIES ON DETECTION, TRANSMISSION TO PROGENY AND SYMPTOM EXPRESSION OF POTATO MOP TOP VIRUS IN POTATO 44 - 46 Maria SANDGREN, Eugene SAVENKOV and Anna GERMUNDSSON. CHARACTERISATION OF SCANDINAVIAN ISOLATES OF POTATO MOP-TOP VIRUS RNA 2 47 - 48

Session 4: "Quarantine considerations" JEFFRIES C, CHARD J INTERNATIONAL INFLUENCES ON POTATO QUARANTINE 49 - 52 J.Th.J. VERHOEVEN & J.W. ROENHORST HERBACEOUS TEST PLANTS FOR THE DETECTION OF QUARANTINE VIRUSES OF POTATO NOT TRANSMITTED VIA TRUE SEEDS 53 - 55 JEFFRIES C, JAMES C DEVELOPMENT OF AN EU DIAGNOSTIC PROTOCOL FOR THE DETECTION OF POTATO SPINDLE TUBER VIROID 56 - 57

Session 5: " Miscellaneous" A. NACHMIAS¹ and G. LOEBENSTEIN² TRENDS IN PRODUCTION OF VIRUS-TESTED TUBER SEEDS IN HOT CLIMATES BY RAPID PROPAGATION 58 Bogdan FLIS, Jerzy SYLLER, Iwona WASILEWICZ-FLIS INTERACTIONS BETWEEN POTATO PLANTS LEAFROLL VIRUS

AND

POTATO 59 - 61

Krzysztof TREDER, Jerrzy LEWOSZ PRODUCTION OF ANTIBODIES AGAINST POTATO LEAFROLL VIRUS USING COAT PROTEIN EXPRESSED IN ESCHERICHIA COLI AS AN ANTIGEN 62 - 64

POSTERS Group 1. I.A. RODKINA, G.A. YAKOVLEVA FIELD POTATO VIRUS Y RESISTANCE IN TRANSGENIC BELORUSKY 3 POTATO WITH PVY COAT PROTEIN GENE IN BELARUS. 65 - 66 Neil BOONHAM, Kathy WALSH and Ian BARKER POTATO VIRUS Y - DISCRIMINATING PVYN, PVYO , PVYC and PVYNTN ISOLATES 67 Karine CHARLET, Camille KERLAN CONTROL METHODS AGAINST POTATO TUBER NECROSIS RINGSPOT DISEASE : STRATEGIES IMPLEMENTED IN FRANCE 68 - 69 GLAIS L., TRIBODET M., KERLAN C MOLECULAR DETECTION OF PARTICULAR PVY ISOLATES: PVYNTN AND PVYN W 70 - 71 P. GUGERLI and M.-E. RAMEL STANDARDIZED LONG-TERM MONITORING OF POTATO VIRUS Y BY USING MONOCLONAL ANTIBODIES 72 - 73 KOSTIW M., ROBAK B. THE PRESSURE OF POTENTIAL APHID VECTORS OF POTATO VIRUS Y AT POTATO CROPS IN POLAND 74 - 75 O. LENZ, D. BYSTØICKÁ , I. MRÁZ, K. PETRZIK, P. DÌDIÈ and M. ŠÍP DNA - CHIP FOR DIAGNOSIS OF POTATO VIRUS ES 76 ŠKOPEK, J., PTÁÈEK, J., DÌDIÈ, P. AND MATOUŠEK, J. FRAGMENT CDNA-TGGE PROFILES THROUGHOUT PVY GENOME AS BASIS FOR VIRUS VARIABILITY ANALYSIS BY DNA HETERODUPLEXES. 77 Steen Lykke NIELSEN & Mogens NICOLAISEN VARIATION IN DANISH ISOLATES OF POTATO MOP-TOP VIRUS 78-79 T. MORAVEC, M.FILIGAROVÁ, P.DÌDIÈ, and N.ÈEØOVSKÁ POSSIBILITY OF USE OF RECOMBINANT COAT PROTEIN OF POTATO MOP-TOP VIRUS (PMTV) FOR VIRUS CHARACTERIZATION 80 - 82

Group 2. BURNS,R., GEORGE,E., JEFFRIES,C DEVELOPMENT OF A QUALITY ASSURANCE SYSTÉM FOR PLANT VIRUS TESTING 83 - 84 NISBET C, JEFFRIES C THE WORK OF THE UK POTATO QUARANTINE UNIT

85 - 86

MANADILOVA A.M., KUNAEVA R.M., TULEEVA G.T. EFFECT OF VIRAL INFECTION ON THE ACTIVITY OF ATPase IN POTATO LEAVES. 87 Miros³awa CHRZANOWSKA, Bo¿enna ZIELIÑSKA, Maria KAMIÑSKA REACTION OF POTATO CULTIVARS TO CUCUMBER MOSAIC VIRUS INFECTION. 88 - 89 Artur KRYSZCZUK, Miros³awa CHRZANOWSKA TRANSPORT OF POTATO VIRUSES THROUGH RESISTANT POTATO PLANTS 90 - 91 Jane MORRIS, Penny SMITH, Ian BARKER and Rick MUMFORD THE EFFECT OF PEPINO MOSAIC VIRUS ON UK POTATO CULTIVARS 92 Milan NAVRÁTIL, Pavla VÁLOVÁ , Tatiana KORMANOVÁ SPORADIC OCCURRENCE OF PHYTOPLASMAS IN POTATO PLANTS AND THEIR IDENTIFICATION IN THE CZECH REPUBLIC AND SLOVAKIA 93 - 94 Petr DÌDIÈ, Jitka SOUKUPOVÁ, Lubomír ŠANTRÙÈEK SEED POTATO CERTIFICATION SCHEME WITH POST-HARVEST VIRUS CONTROL IN CZECH REPUBLIC 95 - 97 DANKS,C., SHEPHERD,V. AND BARKER,I.: VALIDATION OF LATERAL FLOW DEVICES FOR USE IN THE ‘ONSITE’ DETECTION OF 5 MAJOR POTATO VIRUSES 98 Joëlle ROUZÉ-JOUAN, Sylvie TANGUY, Christine KERVARREC and Danièle GIBLOT DUCRAY. IDENTIFICATION OF APHID MEMBRANE(S) RESPONSIBLE OF TRANSMISSION SPECIFICITY OF POTATO LEAFROLL VIRUS (PLRV). 99 - 100

Biological and molecular characterisation of various PVYnW isolates Camille KERLAN 1 , Miroslawa CHRZANOWSKA2 , Laurent GLAIS1 , Guillaume FREMONDIERE1 , Michel TRIBODET1 1 2

INRA-ENSA Rennes, UMR BiO3P, F-35650 Le Rheu ([email protected]) Plant Breeding and Acclimatization Institute, Mlochow Research Center, 05-832 Rozalin, Poland

INTRODUCTION In the last two decades, a subgroup of particular PVYN isolates was reported in Poland, the first of such isolates being found in the potato cultivar Wilga and called PVYN Wi. These isolates were found to be more infectious than the older PVYN isolates, infecting a larger range of potato cultivars and not reacting with anti-PVYN specific antibodies (Chrzanowska, 1987, 1991, 1994). They have since spread up and are now prevalent in Poland. Similar isolates, sharing both PVYO and PVYN properties and also similar genomic traits, have been reported in Canada (Mc Donald and Singh, 1996), Spain (Blanco-Urgoiti et al, 1998) and France (Kerlan et al, 1999).

The objectives of the present study were to better characterise this PVYN subgroup in order to a) ascertain that PVYNW isolates belong to the PVYN strain group according to their behaviour in potato cultivars carrying HR resistance genes, b) check that PVYNW isolates do not induce superficial tuber necrosis; c) confirm the recombinant structure of the PVYNW genome and its heterogeneity in the 5’NTr-P1 region (Glais, 2000). MATERIAL AND METHODS Viral isolates. The range of viral isolates included four PVY reference isolates (PVYO Irl, PVYCNl503, PVYN Irl, PVYNTN Frorl); one reference Polish PVYN W isolate (PVYN Wi-P) and three other PVYN W isolates (PVYN N242, PVYN B11, PVYN Sp17) previously characterised at the genomic level (Glais et al, 1998; Glais, 2000); ten French field isolates were thought to be PVYN W isolates because of their particular behaviour in crops.

Biological typing. Three groups of potato cultivars (mechanically inoculated) were used : a) Polish cultivars, Drop, Lena, Perkoz and Jagoda, used for differentiating ”old” and ”new” PVYN isolates (Chrzanowska, 1994); b) Desiree and Eersteling which carry HR resistance gene to PVYO and PVYC, respectively, Maris Bard which carries HR resistance genes to PVYO, PVYC and PVYZ; c) Nicola and Bea which are highly susceptible to Potato tuber necrotic ringspot disease (PTNRD). Indicator plants also comprised Nicotiana tabacum cv Xanthi, N. tabacum cv Samsum and Chenopodium amaranticolor. Serological typing. Each isolate was tested in DAS-ELISA by using antiserum and monoclonal antibodies specific for PVY (Inra-Fnpppt), PVYN (Bioreba) and PVYO-C (Adgen). Immunocapture, nucleic acid amplification and restriction. IC-RT-PCR was performed as described by Glais et al (1998). Four genomic regions (5’NTr-P1, HC-Pro, P3 and CP) were separately amplified by specific primer pairs. Each amplified region was digested by seven to eleven restriction enzymes.

Phenetic analysis. RFLP data were analysed with the PHYLIP package (version 3,5) as described by Glais et al (1998) leading to a dendrogram for each amplified region.

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RESULTS I. Preliminary serological and biological typing

All of the 18 isolates reacted with anti-PVY antiserum and broad spectrum PVY specific Mabs. PVYO, PVYNW and field isolates reacted with anti-PVYO-C specific antibodies. Only PVYN and PVYNTN isolates, but not PVYNW and field isolates, were recognised by anti-PVYN specific antibodies. All four PVYNW isolates and the ten field isolates induced vein necrosis on N. tabacum Xanthi and Samsum and local lesions on C. amaranticolor. However, these local lesions were heterogeneous in size, number and time of occurrence. As expected, PVYN and PVYNTN did not induce any local lesion in this host. We can conclude that our ten field isolates are PVYN W isolates, since they have displayed the specific traits defined by Chrzanowska (1994). II.

Further biological characterisation in potato

1. Indexing on Polish cultivars differentiating old and new PVYN isolates (Table 1) This experiment was carried out with six French PVYN W isolates (referred to as 98…) which were compared to the standard Polish isolates, PVYN Wi, PVYN Ny and PVYO Lw isolates. In the four cultivars, Drop, Lena, Perkoz, and Jagoda, French isolates induced secondary infection (mosaic symptoms) as did PVYN Wi. They differed from PVYO LW and PVYN Ny in so far they moved to daughter tubers in the cultivars Drop, Lena and Perkoz. They also differed in the cultivar Jagoda from PVYN Ny in that they induced secondary infection, but without vein necroses. Differentiation according to primary symptoms was less clear, except in the cultivar Drop in which French isolates reacted as did PVYN Wi. In summary, in most cases, all French isolates behaved similarly to the standard PVYN Wi.

2. Indexing on cultivars carrying hypersensitivity genes On the cultivars Desiree, Eersteling and Maris Bard, all PVYNW isolates induced a more or less severe systemic mottle, as did the PVYN isolate. Three of them induced also a dwarfing of the plants. The PVYO and PVYC isolates induced hypersensitive reactions as expected with these pathotypes. The PVYNTN isolate induced a severe mottle on the top of these three cultivars and necrotic reactions on tubers of the cultivar Maris Bard. On cultivars Nicola and Bea, only the PVYNTN and PVYN isolates induced typical PTNRD symptoms (at harvest with PVYNTN and mostly during storage with PVYN). All PVYNW isolates induced a systemic mottle on the leaves, sometimes associated with vein necrosis, but no necrosis was observed on tubers, even after three months of storage. In summary, all PVYNW isolates behaved as the PVYN isolate and were close to the PVYNTN isolate on the three cultivars carrying HR genes, but unlike both PVYN and PVYNTN isolates, they did not induce any tuber necrosis in the cultivars Nicola and Bea. III.

Characterisation of the genome Polymorphism was studied by using the RFLP technique in four genomic regions: 5’NTr-P1, HC-Pro, P3, CP. Reference PVYO , PVYN , PVYNTN and PVYN W isolates showed profiles as expected from previous studies. Field isolates were not a homogeneous group; especially the 98N15 isolate, which differed in the 5’NTr-P1 and HCPro regions.

Phenetic analyses showed that all PVYNW isolates were clustered with PVYN and PVY in the 5’NTr-P1 region and in the HC-Pro gene, with PVYO and PVYNTN in the P3 gene, with PVYO and PVYC isolates in the CP gene. In the 5’NTr-P1 region, three subgroups were distinguished according to the profiles that were PVYN-type, PVYNTN-type and specifictype, respectively. NTN

2

DISCUSSION

The present study provides additional evidence of the spreading of PVYNW isolates and confirms that such isolates belong to the PVYN strain group and make up a distinct subgroup within this PVYN strain group, at the biological level as well at the genomic level. All of our 14 PVYNW isolates induced a systemic mottle without any necrotic reaction and consequently, none of them was associated with a hypersensitive reaction in one of the three potato cultivars. Hence, they belong to the PVYN strain group according to this criterion. However, these PVYNW isolates, unlike our reference PVYNTN and PVYN isolates, did not induce superficial necrosis in potato tubers in conditions highly favourable to the expression of tuber symptoms. Consequently, they are distinct from the other two PVYN subgroups, as already mentioned by Chrzanowska (1994). Molecular characterisation also confirms that PVYNW isolates are quite distinct from the other two PVYN subgroups. Indeed, their CP gene is PVYO–like at both the sequence and serological level and unlike PVYNTN, they have no recombination point within the CP region. Moreover, they shift from a PVYO cluster to a PVYN cluster according to the targeted region of the genome and hence are probably originated from recombination between PVYO and PVYN. Lastly, PVYNW isolates are heterogeneous in the 5’part of their genome and this polymorphism have led us to separate them into three divisions (Glais, 2000). The present study allows us to distinguish an additional division characterised by a PVYN-type in the 5’NTr-P1HC-Pro region and where the 98N15 isolate is the first member. REFERENCES

Blanco-Urgoiti, B. et al (1998). Eur. J. Plant Pathol. 104, 811-819 Chrzanowska, M. (1987). Hodowla Roslin i Nasiennictwo 5-6, 8-11 Chrzanowska, M. (1991). Potato Res. 34, 179-182 Chrzanowska, M. (1994). Phytopath. Polonica 8 (XX), 15-20 Glais, L. et al (1998). Archives Virol. 143, 2077-2091 Glais, L. (2000). Doctorat Université Rennes I, 139 p

Kerlan C., M. Tribodet, L. Glais, M. Guillet (1999). J. Phytopathology 147, 643-651 McDonald, J.G., R.P. Singh (1996). American Potato J. 73, 309-315 Table 1. Reactions observed after mechanical inoculation on four Polish potato cultivars differentiating ”old” PVYN (PVYNnonTN) and ”new” PVYN (PVYNW) isolates PVY isolate 98N2 98N5 98N8 98N10 98N12 98N15 PVYN Wi PVYN Ny PVYO LW

Cv Drop 1* 2* Mo, sn Mo Mo, sn Mo Mo, sn Mo Mo, sn Mo Mo, sn Mo Mo, sn Mo Mo, sn mo Ll, sn Ll, n -

* 1 : primary infection

Ll : local lesions

Cv Lena 1* 2* Ll, sn Mo Ll, sn Mo Ll, sn Mo Ll, sn Mo Ll, sn Mo Ll, sn Mo Ll, sn, mo Mo Ll, sn Ll -

Cv Perkoz 1* 2* Ll,sn Mo Mo Mo Mo, sn Mo sn Mo unclear Mo unclear Mo Mo Mo Ll, sn, mo Ll, sn, mo -

* 2 : secondary infection

Sn : systemic necrosis

Cv Jagoda 1* 2* unclear Mo Mo Mo Mo Mo Mo Mo Mo Mo Mo Mo Mo Mo Mo Mo, sn Mo -

- : not infected

Mo : mosaic

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POSSIBLE EVOLUTION OF TUBER RINGSPOT NECROSIS STRAINS (PVYNTN) FROM THE REGIONAL TOBACCO VEINAL NECROSIS (PVYN) STRAINS NIE, X. SINGH, R.P. Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, Fredericton, New Brunswick, Canada, E3B 4Z7

SUMMARY A segment from the 5' end, including part of the non-translated region, P1 coding region and part of the P2 gene were cloned and sequenced from eleven isolates of PVYN and PVYNTN, originating in Europe and North America. Phylogenetic analysis of the P1 coding region sequence indicated that PVYNTN isolates from Europe and North America were clustered with their respective PVYN strains. This indicates that the PVYNTN strains may be evolving from the prevailing PVYN strains in a given region. Using this information methods for the specific detection of North American PVYNTN strains were developed.

INTRODUCTION In recent years, an increasing number of countries have reported occurrences of potato tuber necrotic ringspot disease (PTNRD) (Weidemann & Maiss, 1996; Singh et al.1998) caused by the tobacco veinal necrosis strain of potato virus Y (PVYN), belonging to the tuber necrosis (PVYNTN) sub-group (LeRomancer et al. 1994). Methods based on nucleotide sequences of primers from the P1 region of the virus genome, failed to differentiate PVYN from both the North American and European-type of PVYNTN. Often PVYNTN isolates homologous to European-type PVYNTN strains have been differentiated from PVYN isolates but not the North American-type (Weilguny & Singh, 1998). Most recently, PVYNTN strains from Japan have been reported (Ohshima et al. 2000) where the phylogenetic analysis using coat protein gene sequence clustered the Japanese PVYNTN distantly from the European isolates. These observations point out the possibility of PVYNTN strains existing as distinct regional sequence variants. Existence of such regional variants raises many questions about their diagnosis and origin. The objectives of this study were to determine whether or not PVYNTN and PVYN strains from different regions are related to each other; depending on the outcome, to design specific primers to differentiate the North American PVYNTN from the other PVYNTN and the local PVYN isolates .

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MATERIALS AND METHODS Viruses: PVYN isolates (N-27, N-Jg, N-5Yt, N-266 and N-394) and PVYNTN (Tu 619, Tu 648 and Tu 660) from North America have been used in previous studies and are well characterized for their biological and serological properties. Similarly PVYNTN isolates from Slovenia (Sl-44, Sl-50, Sl-64 ) were those used in previous studies (Weilguny& Singh, 1998;Singh et al.1998 ). Cloning and sequencing: RT-PCR amplified fragments of the 1056 nucleotides were cloned and sequenced. Phylogenetic analysis, restriction enzyme selection and primer designs were carried out using the GCG (University of Wisconsin) program 10.1.

RESULTS AND DISCUSSION To determine if various PVYNTN isolates have different or common origins and to effectively differentiate various PVYNTN from PVYN isolates, we determined the nucleotide sequence of the P1 coding region of eleven PVYN and PVYNTN isolates from North America and Europe. The deduced amino acid sequence comparison of the P1 gene indicated that the PVYNTN isolates of both the European and North Americantype had 97 to 99% sequence similarities to their regional PVYN isolates, compared to only 89 to 91% with their counterparts. In contrast, the sequence similarities of PVYNTN isolates were 70 to 73% with PVYO and 82-86% with PVYN-Wi isolate irrespective of the geographical region. The anomaly of the sequence data with regards to isolate PVYN-Uk , PVYN-Ru, and N-Fr, which clearly clustered with PVYO will be discussed. European-type PVYNTN isolates can be differentiated from PVYN isolates on the basis of a 3-primer RTPCR (Weilguny & Singh, 1998), but not the North American isolates. In this study we developed two approaches based on amplified fragment length polymorphism (AFLP) and competitive RT-PCR. The AFLP method utilized two restriction enzymes together, which yielded a unique pattern for the North American-isolates Tu 619 and Tu 660. In RT-PCR, use of a 2-primer combination amplified strongly both PVYN and PVYNTN isolates. Another 2-primer combination, amplified PVYNTN isolates Tu 619 and Tu 660 strongly, all PVYN isolates weakly, and the other PVYNTN isolates were not amplified . However, a combination of all the three primers in a competitive PCR format, amplified all PVYN and PVYNTN isolates, and yielded an additional band specific for the Tu 619 and Tu 660 isolates. This work demonstrated that the PVYNTN isolates may be evolving from the prevailing local PVYN isolates. It also shows that specific detection of the regional PVYNTN strains can be accomplished by designing primers differing only in the single base at their 3' end.

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REFERENCES LeRomancer, M., Kerlan, C. and Nedellec, M. 1994. Biological characterization of various geographical isolates of potato virus Y inducing superficial necrosis on potato tubers. Plant Pathol. 43:138-144.

Ohshima, K., Sako, K., Hiraishi, C., Nakagawa, A., Matsuo, K., Ogawa, T., Shikata, E. and Sako, N. 2000. Potato tuber necrotic ringspot disease occurring in Japan: Its association with Potato Virus Y necrotic strain. Plant Dis.: 84:1109-1115.

Singh, R.P. , Singh, M. and McDonald, J.G. 1998. Screening by a 3-primer PCR of North American PVYN isolates for European-type members of the tuber necrosis-inducing PVYNTN subgroup. Can. J. Plant Pathol. 20:227-233.

Weidemann, H.L. and Maiss, E. 1996. Detection of the potato tuber necrotic ringspot strain of potato virus Y (PVYNTN) by reverse transcription and immunocapture polymerase chain reaction. J. Plant Dis. Protect. 103:337-345. Weilguny, H. and Singh, R.P. 1998. Separation of Slovenian isolates of PVYNTN from the North American isolates of PVYN by a 3-primer PCR. J. Virol. Methods 71:57-68.

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Differentiating PVYNTN from PVYN by restriction cleavage patterns, transcript confirmation polymorphisim and by annealing to reference RNA transcripts Arie Rosner* and Ludmila Maslenin Department of Virology. The Volcani Center, Bet Dagan 50250, Israel Fax: +972-3-9604180, e-mail: [email protected] A procedure for the differentiation of PVYNTN from PVYN is described. It is based on the unique cleavage of their respective PCR products with strain specific restriction endonucleases. The PCR products corresponding to the 5' end of the N and NTN strains of PVY were cloned and sequenced, and a restriction map was constructed, which included common enzymes that were used for the differentiation of PVYNTN. Enzymes which uniquely cleave this strain were searched for. Unique, single cleavage of PCR products derived from the 5' end of the PVYNTN genome by Nco I, and that of the N-strain of PVY by Bgl II restriction endonuclease were demonstrated. The specific digestion patterns in polyacrylamide gel were used for the unequivocal differentiation between the N and NTN strains of the virus. Both single, and mixed infections with PVYNTN and PVYN were observed in field samples of potatoes by means of this procedure. Alternatively, PVYNTN was distinguished from PVYN on the basis of differences in electrophoretic mobility of their corresponding RNA transcripts. Transcripts were copied from PCR products of two PVY strains to which the T7 promoter was added. Single-strand and double-strand transcript heteroduplexes of the NTN and N strains exhibited different rate of migration in gel, which enabled strain differentiation. This procedure obviates the need for prior information on sequence divergence (e.g., nucleotide sequence, restriction sites etc.) among the virus isolates. An improved procedure for the resolution of RNA transcripts by electrophoretic gel retardation, mediated by annealing to specific homologous oligonucleotiedes was developed. Non-polymorphic but sequence-diverse RNA transcripts were copied from PCR products of the N and NTN strains of PVY. The transcripts were resolved by gel electrophoresis, because of the differential retardation effect caused by the binding of strain-specific homologous oligonucleotides. The two PVY strains were thus differentiated. A method for the differentiation of virus strains based on the shift in electrophoretic mobility of partially annealed RNA transcripts was tested. Oppositely oriented RNA transcripts of the NTN- and N-strains of PVY, complementary at their 3'-end variable (strain-specific) region, were annealed to form a partial duplex which moved more slowly in gel than heterologous (NTN + N) unpaired transcripts. Thus, the two virus strains could be identified by annealing to a known reference transcript. The rate of duplex migration was correlated with transcript lengths and could be tightly controlled thereby. Thus, a higher degree of resolution was obtained than with transcript conformation polymorphism, which is empirical and unpredictable in nature.

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THE DIVERSIFICATION OF POTATO VIRUS Y ( PVY) Jiøí Ptáèek1 , , Petr Dìdiè1 , Jaroslav Matoušek2 1 2

Potato Research Institute, Dobrovskeho 2366,Havlíèkùv Brod, CR, [email protected] Institute of Plant Molecular Biology, CAS, Èeské Budìjovice, Czech Republic

Introduction Isolates of PVY from potato have been classically sub-divided into three main strain groups on the basis of the symptoms on tobacco, Physalis floridana and potato cultivars (de Box and Huttinga, 1981). In addition to these basic criteria, PVY strains may also be differentiated by the reaction of other indicator plants and some potato cultivars carrying hypersensitive genes targeted against different PVY strain groups (Jones, 1990, Valkonen et al.,1994). Strain specific or better strain group specific monoclonal antibodies (MAbs) are also utilised (e.g. Gugerli and Fries, 1985, Rose and Hubbard, 1986, Singh et al., 1993). At the beginning of the eighties the potato tuber necrotic ringspot disease (PTNRD) appeared in potato crops (Beczner et al., 1984) and these isolates called PVY-NTN constitute a new sub-group of PVY-N (Le Romancer et al., 1994, van den Heuvel et al., 1994). Another particular group , PVY-Z, which overcome the hypersensitive resistance to PVY-C and PVY-O was found in Great Britain (Jones, 1990) and further different sub-group of PVYN isolates (Wilga-type) was reported in Poland (Chrzanowska, 1991). The causal agent of the PTNRD - the PVY-NTN isolates became wide-spread in most European countries during last few years. Currently, identification of PVY-NTN isolates is based primarily on the induction of necrotic symptoms on tubers of selected potato varieties which is unacceptably time -consuming. Neither a range of indicator plants, nor ELISA utilizing strain specific monoclonal antibodies were successfully used for this purpose and PVYNTN isolates could not be reliably distinquished from other members of PVY-N strain group. Only method based on variability of genome sequence seems to have the potential to discriminate these minor differences on the substrain level. Blanco-Urgoiti et al. (1996) developed a molecular typing method for classifying of the PVY isolates using coat protein region. Weidemann and Maiss (1996), Glais et al., (1996), Weilguny and Singh (1998), tried to utilise for strain specific differentiation RT-PCR directed to 5´NTR and P1 protein. Positive results with differentiation of PVY-NTN isolates obtained also Boonham et al. (1998) using primers derived from the CP region. The objective of the investigation presented was to compare a collection of PVY isolates by means of biological, serological and molecular genetic methods. Preliminary results were also published in Proceedings of Virological Section of EAPR, Baden/Austria (Dìdiè and Ptáèek, 1998)

8

Material and Methods Virus isolates The virus isolates originating from Czech Republic, Canada, France, Poland and Slovenia were collected and maintained in vitro usually on original potato plantlets or on tobacco in advance infected in the greenhouse.

Bioassay of PVY isolates on tobacco and potato plants Firstly Samsun tobacco plants at the 3-4 leaf stage were mechanically inoculated with selected PVY isolates and plants were maintained in a greenhouse with natural photoperiod and temperature regime. Presence of PVY was indicated (10-21 days later) by the development of characteristic symptoms and by a positive reactions in ELISA. The isolates were then transmitted from tobacco to potato cultivars Eersteling (hypersensitive to PVY-C), Desirée (hypersensitive to PVY-O), Maris Bard (hypersensitive to both PVY-O and PVY-C, as well as to PVY-Z (Jones, 1990). Cultivar Lukava was included as sensitive host pla nt for PTNRD. The plants and after harvest also daughter tubers and plants were repeatedly scored for visual symptoms. ELISA testing Broad-spectrum PVY antibodies (Bioreba or IPO-DLO Wageningen) and PVY-N, PVY-O/C MAbs (Bioreba, Adgen) in DAS-ELISA format (Clark and Adams, 1977) were used. Oligonucleotide primers All selected sets of primers were derived from PVY-NTN genome sequence, in the 5´ NTR and P1 protein region. (Weidemann and Maiss, 1996, Glais et al., 1996, Weilguny and Singh, 1998). RNA extraction Two various RNA isolation methods were compared: a-RNeasy Plant Total RNA Kit (Qiagen) and bimmunocapture on ELISA plates. Reverse transcription and Polymerase chain reaction First strand cDNAs were synthesized with Enhanced Avian RT-PCR Kit (Sigma) using reverse primers or random hexanucleotides in 0,2 ml PCR tubes or ELISA microplate. For the PCR, 5 µl of the reverse transcriptase reaction mixture (cDNA) were amplified in a total volume of 50 µl using Enhanced Avian RT -PCR Kit (Sigma) and selected primers according to: a) Weidemann and Maiss (1996): b) Glais et al. (1996): c)Weilguny and Singh (1998): The amplification products were analyzed in a 1 % agarose gel stained with ethidium bromide. The RT -PCR was performed in PTC-100 thermocycler (MJ Research), the gels were visualized by documentation system KS-3000 (Ultralum). Restriction digest 10 µl aliquots of PCR products amplified with procedure Glais et al. (1996) were restricted with 4 units of Taq I endonuclease at 65 o C for 2 h.

9

Results and Discussion The list of isolates selected for mutual comparison contains forty-four isolates of PVY. (See table 1.)

Serology and bioassay on tobacco and potato Two serotypes could be defined according to the reactions to the three monoclonals. All the PVY isolates serologically classified as PVY-N serotype, by means at least one of the PVY-N specific antibodies, also induced vein necrosis in tobacco. Five isolates did not react similarly with the both PVY-N antibodies. Seven isolates being serologically PVY-O induced mosaic symptoms on tobacco plants and it is possible to put them without any doubt among PVY-O strain. Specific hypersensitive reaction of potato cultivars – indicators clearly support this conclusion at five isolates, three isolates displayed a little different symptom pattern. However, eight isolates (and three others containing both serotypes) induced vein necrosis on tobacco, in spite of being the PVY-O serotyp. These isolates are possible to order into PVY-N Wi subgroup (Chrzanowska, 1991). The isolates of similar type found also in France and Canada (included in our experiments) and Spain (Blanco-Urgoiti et al., 1998) support the idea of their more frequent occurrence in potato fields. The further differentiation of the PVY-N isolates, especially of PVYNTN was in this part of experiments possible only according to development of necrotic ring symptoms on tubers (PTNR) of sensitive potato cultivars after artificial inoculation. It is important to mention here that no one isolate of PVY-O serotype was able to induce this necrotic symproms on tubers and contrary some isolates originally classified as PVY-N may after artificial inoculation of sensitive potato cultivars evoke PTNRD symptoms. Example of this are e.g. isolate Ny (Poland) and Tenor (Czech Republic). Some North American-type PVY-NTN isolates (TU 619, TU 660) failed to induce PTNRD after inoculation of sensitive cultivars we used. RT-PCR Both methods, extraction of total RNA by QIAGEN kit, and immunocapture technique, respectively yielded reliable and reproducable results and were also suitable for large-scale application. The immunocapture method seems to be easier and faster way to obtain suitable viral RNA. Utilizing specific primer combinations devised by Glais et al. (1996), Weidemann and Maiss (1996) and Weilguny and Singh (1998) it was possible to detect either all the isolates tested without further differentiation, or with selection of appropriate primer combinations to discriminate between common (PVY-O) and necrotic (PVY-N) strain group of PVY. Using specific pair of primers for PVY-O detection, there was observed the amplification product at sixteen PVY isolates. However five isolates of this strain group, parallely with other pair of primers, displayed also the specific product of PVY-N strain group. By means of protocol and primers 1-2 according to Weidemann and Maiss (1996) seventeen PVY isolates gave specific product for PVY-NTN. The same results were obtained by protocol and primers c-d (Glais et al., 1996) followed by digestion with Taq I, and also by the protocol and primers 3-4 (Weilguny and Singh, 1998) using annealing temperature 63 o C. Symptoms of PTNRD on sensitive potato cultivar Lukava were found only at fiveteen isolates and all were in agreement with RT -PCR differentiation. But twelve isolates with specific PVY-NTN products, in our experiments did not produce any necrotic symptoms on tubers of sensitive cultivar (isolates of Wilga type and double serotypes). Contrary, three Canadian isolates (TU 619-1, TU 619-4, TU 660 Cal) inducing PTNRD symptoms on the sensitive potato cultivar Jemseg (McDonald and Singh, 1995), did not produce any PVY-NTN specific RT-PCR product with any of method tested. But these three isolates did not produce any PTNRD symptoms on the sensitive potato cultivar Lukava. This indicates that these isolates differ from those collected in Europe. This work was supported by Grant Agency of Czech Republic, project number 522/00/0227 and by National Agency for Agricultural Research, project number 1167. The authors wish to thank Dr. J. McDonald (Canada), Dr. C. Kerlan (France) and Dr. Chrzanowska (Poland) who kindly supplied PVY isolates.

10

References BECZNER, L., HORVATH, J., , ROMHANYI, I., FORSTER, H. (1984): Studies on the etiology of tuber necrotic ringspot disease in potato. Potato Res., 27 : 339-352. BLANCO-URGOITI, B., TRIBODET, M., LECLERE, S., PONZ, F., PEREZ de SAN ROMAN, C., LEGORBURU, F. J., KERLAN, C. (1998): Characterization of potato potyvirus Y (PVY) isolates from seed potato batches. Situation of the NTN, Wilga and Z isolates. European J. Plant Pathol., 104, 811-819. BOONHAM, N., WALSH, K., BARKER, I. (1998):Towards he detection of PVY-NTN. Procc. Virol. Sect. EAPR, Baden/Austria, 69. CHRZANOWSKA, M. (1991): New isolates of the necrotic strain of potato virus Y (PVY-N) found recently in Poland. Potato Res., 34, 179-182. CLARK, M. F., ADAMS, A. N. (1997): Characteristic of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol., 34, 475-483. DE BOKX, J.,A., HUTTINGA, H. (1981): Potato virus Y. CMI/AAB, Descriptions of Plant Viruses No. 242. DEDIC, P., PTACEK, J. (1998):The possibility of specifis PVY-NTN detection. Procc. Virol. Sect. EAPR, Baden/Austria, 65-68. GLAIS, L., KERLAN, C., TRIBODET, M., MARIE-JEANNE TORDO, V., ROBAGLIA, C., ASTIERMANIFACIER, S. (1996): Molecular characterization of potato virus YN isolates by PCR - RFLP. Eur . J. Plant. Pathol., 102: 655-662. GUGERLI, P., FRIES, P.,(1983): Characterization of monoclonal antibodies to potato virus Y and their use for virus detection. J. Gen. Virol., 64, 2471-2477. JONES, R. A. C. (1990): Strain group specific and virus specific hypersensitive reactions to infection with potyviruses in potato cultivars. Ann Appl Biol., 117, 93-105. LE ROMANCER, M., KERLAN, C., NEDELLEC, M. (1994): Biological characterization of various geographical isolates of potato virus Y inducing superficial necrosis on potato tubers. Plant Pathol., 43, 138-144. MCDONALD, J.G., SINGH, R.P. (1995): Sensitivity of North American potato cultivars to PVY-NTN isolates. Abstr. Virol. Sect. EAPR, Bled/Slovenia, 35. ROSE, D. G., HUBBARD, A. L. (1986): Production of monoclonal antibodies for the detection of potato virus Y. Ann. Appl. Biol., 109, 317-321. SINGH, R. P., BOUCHER, A., SOMERVILLE, T. H., DHAR, A.K. (1993): Selection of a monoclonal antibody to detect PVY-N and its use in ELISA and DIBA assays. Canad. J. of Plant Pathol., 15, 293-300. VALKONEN, J, P. T., SLACK, S. A., PLAISTED, R. L. (1994): Use of the virus strain group concept to characterize the resistance to PVX and PVY-0 in the potato cv. Allegany. Am. Potato J., 71, 507-516. VAN DEN HEUVEL, J. F. J. M., VAN DER VLUGHT, R. A. A., VERBEEK, M., DE HAAN, P. T., HUTTINGA, H. (1994): Characteristics of a resistance-breaking isolate of potato virus Y causing potato tuber necrotic ringspot disease. Europ. J. Plant Pathol., 100, 347-356. WEIDEMANN, H.L., MAISS, E. (1996): Detection of the potato tuber necrotic ringspot strain of potato virus Y (PVYNTN ) by reverse transcription and immunocapture polymerase chain reaction (1996): J. Plant Dis. Protec., 103 (4): 337-345. WEILGUNY, H., SINGH, R.P. (1998): Separation of Slovenian isolates of PVY-NTN from the North American isolates of PVY-N by a 3-primer PCR. J. Virol. Methods, 71: 57-68.

11

IMPORTANCE OF DIFFERENT STRAINS OF PVY IN POTATO PRODUCTION AND BREEDING PROGRAM IN POLAND Miros³awa Chrzanowska Plant Breeding and Acclimatization Institute, 05-831 M³ochów, PL

Introduction Between 1983-1986 Potato virus Y (PVY) population changed considerably. PVYo (normal) strain was reduced and PVYN (necrotic) strain became dominant in Europe. Weidemann (1988) point out importance of control of PVYN in seed production. The mild strain PVYN Wi was characterized by Chrzanowska (1991). This strain is predominant in Poland. Severe strain PVYNTN is widespread in South of Europe (Becner et. al. 1984, Kus 1990). In Poland in 19871992, the ratio PVYN :PVYo was 86,5:13,5 (Kaczmarek 1995). In 1996-1997 PVYo infected 5% of tested potato or tobacco plants infected with PVY, PVYN - 95% and PVYNTN was sporadically identified (Chrzanowska and Doroszewska, 1997). The great variability of PVY population has important consequences in potato and tobacco production, in potato seed production and in breeding programs. Material and methods During last several years PVY was intensively investigated at M³ochów Research Center (IHAR) in cooperation with Dr. C. Kerlan (INRA, Le Rheu) and Dr. T. Doroszewska (IUNG, Pu³awy). Spreading of PVY under field condition was monitored using Nicotiana tabacum cv. Samsun as a bait plant. Identification of strains was done by serological and biological methods. The reaction of potato and tobacco cultivars to the inoculation with chosen PVY strains was investigated in greenhouse. Resistant tobacco Virginia A Mutant (VAM) and cv. Wiœlica were obtained from Dr. Doroszewska. For the serological identification of strains were used two kinds of Abs from Bioreba Co. and policlonal Abs from IHAR, Laboratory in Gdañsk. Results Different kind of PVY isolates were identified in Poland (Table 1). Table 1. PVY strains/isolates identified in Poland since 1956. Year PVY isolates obtained from: of isolation Potato Tobacco 1956 PVYN o 1970 PVY LW 1971 PVYNZ N 1974 PVY Ny 1981 PVYo E 1984 PVYN Wi 1986 PVYo P 1994 PVYN III (YNTN) NTN PVY 12/94 1999 2000

12

PVYNTN Gr PVYNTN Pu

References Berbeæ 1960 Chrzanowska 1973 Gajos 1971 Chrzanowska 1973 Œniegowski et. all 1981 Chrzanowska 1991 Pietrak 1986 Doroszewska 1997 Chrzanowska, Doroszewska 1997 Doroszewska 1997 Doroszewska 2000

In the last 20 years infection preasure under field condition at M³ochów was high. Since 1994 PVYNTN has been identified in 5-12% of tested samples of plants infected with PVYN. In the pathogen collection at M³ochów many isolates are maintained (Table 2). PVY isolates belonging to subgroups PVYN Wi or PVYNTN could be divided according to ability of breaking down the resistance of tobacco cvs. The same group of potato cultivars is susceptible to PVYNWi and PVYNTN (Zagórska et al. 2000). These two strains cause different disease symptoms on potatoes. Table 2. PVY strains/isolates maintained in the collection at M³ochów and criteria of their identification. Strains of PVY

Bioreba PVY Bioreba PVYN Gdañsk N. tabacum Samsun

YC LR, NL VP, NL ZB, NL

Yo LW* Wy E

+

+

+

+

vcl

vcl

YN (Ny) Bo, NL Bi, NL Gi, NL Ny* 20/98

YN (Wi) Wi** 28, 31, 33, 46/96 10, 15, 28, 49, 51/97 22/99 69/99 He – 01 Sa – 01 Bry – 96 Ze – 01 Wal (t) L-6 (t) TN (t) VS (t) L5 (t) 242/96 F 19/98

Criteria Serological identification: + + + Biological identification nv

+

- Ù

nÙ nÙ nÙ nÙ nÙ

Y NTN 12/94*** LB, F Ha, H 47/96 Wa 115-Pu 2/98 44/97 5/98 Orl, F Mu, H Di, A Gr 99 Gr 00 43/97 B, F YN III t

+

+ + +

nv

nv

n n n n n n n -

Ù Ù Ù

Ù Ù Ù Ù Ù Ù Ù

* PVYo LW, PVYN Ny - strains used in the selection of breeding clones since 1974 ** PVYN Wi used in the selection of parental lines at M³ochów since 1984 *** PVYNTN used for evaluation of resistance and reaction of potatoes since 1994 n Isolate able to break down the resistance of resistant tobacco VAM Ù Isolate able to break down the resistance of resistant tobacco cv. Wiœlica - Isolate not able to break down the resistance of respective control tobacco cvs. Consequences for potato production 1. In Poland mild necrotic strain PVYN type Wi predominant as identified in >90% of potato infected with PVY. Most of Polish cvs. are susceptible to PVYN Wi. PVYN Wi is moved to the North (it was found in North Poland, St. Petersburg, in Finland, Canada also in France). 2. PVYN Wi causes mild symptoms on potatoes but the infection may reduced the yield of tubers in 30-50% (Hane and Hamm, 1999).

13

Consequence for seed production 1. Many potato cultivars are susceptible to PVYN Wi strain. 2. The seed production area is shrinked because PVYN Wi is expansive to new places. 3. The negative selection in growing seed crop of cultivars reacting to PVY with mild symptoms is very difficult. 4. ELISA is effective under condition that antibodies used possess wide spectrum of specificity. Consequence for breeding potato resistant to PVY 1. None of PVY strain tested is able to infect extreme resistant potatoes (gene Ry sto). Besides 30% of cvs. registered in Poland extreme resistant to PVY, potat cvs. are susceptible to PVYN Wi and PVYNTN. 2. In commercial breeding when selection for PVY resistance is considered the applying of inoculation with PVYN Wi is recommended. Consequence for tobacco production 1. Recently breaking down of the resistance in tobacco cultivars previously resistant to PVY was noted. PVYN causes significant yield losses in tobacco production. 2. There is evident that the same PVY strains are identified in tobacco and in potato. More than 90% of diseased plants are infected with Wi type isolates. Exception is the strain PVYNZ which is not infecting potatoes but is rarely identified nowadays on tobacco. References Becner L., J.Horwath, I. Romhanyi, H. Forster (1984). Potato Res. 27, 339-352. Berbeæ J. (1960). Wiadomoœci Tytoniowe 1, 1-4. Chrzanowska M. (2000). Biul. IHAR 214, 231-238. Chrzanowska M., T. Doroszewska (1997). Phytopath. Polonica 13, 63-71 Chrzanowska M., T. Doroszewska, A. Chachulska (1997). Progress in Plant Protection. Inst. Plant Protection Poznañ, Vol.37, 327-329. Gajos Z. (1971). Zesz. Probl. Post. Nauk Roln. 115, 87-98. Chrzanowska M. (1973). Doctoral disertation. Inst. Ziemn. pp. 75. Hane D. C., P.B. Hamm (1999). Plant Dis. 83, 43-45. Jones R. A. C. (1990). Ann. appl. Biol. 117, 93-105. Kaczmarek U.(1995). Phytopath. Polonica 9, 15-23. Kerlan C., M. Tribodet, L. Glais, M. Guillet (1999). J. Phytopathology 147, 643-651. Kus M.(1990). Proc. 11th Trienn. Conf. EAPR Edinburgh UK, 8-13 June, 196. Marte M., G. Bellezza (1988). Phytopath. medit. 27, 169-172. Ohshima K., K. Sako, C. Hiraishi , a. Nakagawa, K. Matsuo, T. Ogawa, E. Shikata, N. Sako (2000). Plant Dis. 84, 1109-1115. Œniegowski Cz., A. Kowalska, I. Cieœlewicz, M. Chrzanowska (1981). Zesz. Probl. Post. Nauk Roln. 244, 167-175. Valkonen J. P. T. (1994). Plant Breeding 112, 1-16. Valkonen J. P. T., R. A. C. Jones, S. A. Slack, K. N. Watanabe (1996). Plant Breeding 115, 433438. Zagórska H., M. Chrzanowska, J. Pietrak (2000). Biul. IHAR 215, 293-303.

14

MONITORING OF PVY STRAINS IN SLOVENIA Peter DOLNIÈAR Agricultural Institute of Slovenia, Hacquetova 17, 1000 Ljubljana, Slovenia, E-mail: [email protected]

Introduction Potato YNTN virus has been the most serious potato disease in Slovenia in the last 12 years. It almost stopped seed potato production in Slovenia. Because of its effects on tuber quality it also completely changed the varieties in production. Our old varieties were changed not only by resistant varieties but also by some susceptible ones (Kus, M. 1995). Susceptible varieties are prone to the infection on the foliage, but they do not show symptoms on the tubers. Therefore they still have their market value, despite they were infected by the virus. One of the strategies for fighting the disease was more rigorous evaluation of new varieties to susceptibility to PVYNTN (Pepelnjak, M. 1995). All very susceptible varieties and varieties which showed symptoms on the tubers have been rejected in Slovenian national testing programme since then. Only resistant and some high quality susceptible varieties have been accepted on Slovenian national list. The infection of seed potato fields increased rapidly after the first appearance of the virus. Until 1995 and 1996 high infection pressure was observed. High aphid populations were also found during that period. After that, the infection get lower together with lower aphid populations. Since resistant varieties have been in production, less and less infection has been observed on the fields, and less and less necroses observed on tubers on infected fields of susceptible varieties. Therefore there was a need for re-evaluation of rate of infection pressure and next questions can be asked: - Did the infection pressure of PVY NTN drop during last few years? - Is the infection similar across Slovenia? - Is the PVYNTN still the main strain in Slovenia? - If it is not, do we have to re-evaluate the Slovenian national testing programme? Materials and methods Disease free tubers of variety Igor were planted on different locations across Slovenia in 1997, 1998 and 2000. Igor is very susceptible variety which shows symptoms after infection with PVYNTN . 50 tubers were planted on each location in spring. Tubers were harvested in September and transferred to the Agricultural Institute of Slovenia. Since it is known that most of the necroses appear during storage, the tubers were evaluated three times. The last evaluations were performed in December. Tubers with necroses and tubers without necroses were separated for each location. DAS-ELISA was performed after that. Polyclonal antibodies PVY and monoclonal YN of Bioreba and YN, YO and Y O/C of Adgen were used. Finally, in 2000 tubers were separated in two groups and repla nted next year. Plants were visually estimated and retested by ELISA.

15

Results and discussion The appearance of necroses on the tubers differs among locations. The highest infection was found in some major potato growing areas (Ljubljana, Kranj, Kome nda, Celje, Rakièan) and in Primorska region where grow early potato (Vrtojba –Bilje) in 1997. Remote not potato growing areas were less infected. Similar picture had been seen in 1998, except in Rakièan and VrtojbaBilje, where most of the farmers stopped with potato production (Table 1). From table 1 we can see that the infection pressure in general is getting smaller in the last years. The data in Komenda shows 68,6 % tubers with necroses in 1997, comparing to 49,4 % in 1998 and 15,1 % in 2000. Since only a part of infected tubes show necroses, the real infection was even higher. There are two main reasons for lower rates of tubers with necroses: smaller infection and lower rate of tubers with necroses when tubers are infected. We can say that the infection is lower because of less highly infected fields and seed change in Slovenia and smaller populations of vectors in the last few years. This trend can be seen also from the data of seed potato production (Južnik, M. et al, 1997; 1998; 2000) Table 1: The rates of tuber necroses caused by PVY Year

1997 Nu. of Necroses (%) Location tubers Yes No Brezula 144 6,3 93,8 Podèetrtek 433 9,9 90,1 Trbonje 371 10,5 89,5 Libelièe 236 14,8 85,2 Idrija 206 20,9 79,1 Novo mesto 797 25,1 74,9 Miklavž 72 41,7 58,3 Kranj-Primskovo 661 50,4 49,6 Ljubljana 482 63,7 36,3 Samotorica 331 64,7 35,3 Komenda 440 68,6 31,4 Zgornje Jablane 739 68,9 31,1 Celje - Žalec 598 74,4 25,6 Rakièan 409 80,2 19,8 Vrtojba-Bilje 422 80,8 19,2 Pivka Tolmin

NTN

in Slovenia from 1997 to 2000

1998 Nu. of Necroses (%) tubers Yes No

419

48,4

51,6

283 243 361 302 356 125 268 250

68,2 49,4 68,1 44,0 14,0 1,6 47,0 2,4

31,8 50,6 31,9 56,0 86,0 98,4 53,0 97,6

2000 Nu. of Necroses (%) tubers Yes No

46

21,7

78,3

73

15,1

84,9

92 92

19,6 4,3

80,4 95,7

92

10,9

89,1

Tubers were tested by ELISA with polyclonal antibodies of PVY and monoclonal YN of Bioreba and YN, YO and Y O/C of Adgen. We did not find any difference between polyclonal antibodies of PVY and monoclonal YN of Bioreba and YN of Adgen. All tubers with necroses had been positive using these antibodies. Some tubers without necroses had been also positive. We found more infected tubers without necroses in 2000 than in previous years. Only some tubers from locations close to Italian border were infected with PVYO . There was no infection with PVY C. Tubers were replanted in 2000. All tubers with necroses and some without them showed symptoms on plants. They were positive at ELISA testing again. From visual symptoms on tubers and ELISA results we can say that variety Igor was infected with PVY NTN . If there was a mixed infection with PVY N , we can not say.

16

Conclusions We can conclude: 1. PVY NT N strain is still the prevalent strain in Slovenia. 2. The infection was lower in some parts of Slovenia (isolated areas or areas with small potato production). 3. The infection pressure is smaller in the last few years. 4. We will probably have to change our national variety testing programme. Another year of exposure to virus infection will be needed in the part where we test virus resistance of varieties under natural conditions. Literature: Južnik M., Obal A., Pajmon A. 1997.- Semenarske informacije za leto 2000. Agricultural Institute of Slovenia, Ljubljana. Južnik M., Obal A., Pajmon A. 1998.- Semenarske informacije za leto 2000. Agricultural Institute of Slovenia, Ljubljana. Južnik M., Obal A., Pajmon A. 2000.- Semenarske informacije za leto 2000. Agricultural Institute of Slovenia, Ljubljana. Kus M. 1995.- The epidemic of the tuber necrotic ringspot strain of potato virus Y (PVY NTN) and its effect on potato crops in Slovenia. Book of abstracts, The 9th virology section meeting, Bled, p. 45-46. Pepelnjak M. 1995.- Testing to susceptibility of potato cultivars to potato tuber necrotic ringspot disease in Slovenia. Book of abstracts, The 9th virology section meeting, Bled, p. 42-43.

17

INTERACTION BETWEEN STRAINS OF THE POTATO VIRUS Y (PVY0 , PVYN - type Wilga, PVYNTN ) AND THE POTATO PLANTS OF TWO CULTIVARS. URSZULA KACZMAREK, EWA MOSAKOWSKA Plant Breeding and Acclimatization Institute, Branch Division Bonin. 76-009 Bonin, Poland [email protected] SUMMARY In 1998-2000 three series of experiments were carried out on two potato cultivars: Kolia (tolerant to PVYNTN) and Vital (susceptible). The reaction of the protection was the main result of the interaction effect between PVY0 , PVYN Wilga (PVYNW ), PVYNTN and the potato plants infected with these strains. According to DAS-ELISA (mabs YN specific, pabs Y) the prior infection with mild strains caused the protection against the infection with severe and aggressive strains of PVYNTN. The strain PVY0 protected the potato plants against the infection with PVYNW and PVYNTN, the strain PVYNW protected against PVYNTN. The effect of protection by mild strains against more severe strains has been visible as less severe infection of the potato plants with PVY, lower amount of tubers infested with severe strains and lower amount of tubers with PTNRD symptoms. The result of double infection was an appearance of intermediate strains that were not detectable by mabs YN specific, however on the basis of biological tobacco assay, they could accounted to PVYN strains. The strongest protection reaction against PVYNTN has been found in the cross-protection test. Only 4,0 % of the infested tubers has been detected by mabs YN in case of cv. Vital and 5,9 % in case of cv. Kolia in comparison with about 100 % infestation detected by polyclonal antibodies Y. INTRODUCTION The occurrence of the recombinant PVY isolates is known from some time when the molecular methods of the search were applied. According to recent investigation (Kaczmarek et all. 1998) the spread of PVYN strains in Poland was several fold higher (84,8%) than PVY0 (15,2%). However we have noted only 7,0 % content of PVYNTN. The rest of strains were accounted to the group of intermediate PVYNW (92,9 %) and these strains were undetected with mabs YN specific. The results, of the other field experiment indicated the presence of some progeny tubers from one plant could be infested with PVY0 and others from the same plant, could be infested with PVYNW (Kaczmarek, Mosakowska, 2000). Mixed infection with various PVY strains has been confirmed by Rosner and Maslenin, (1999) by unique single-restriction cleavage of PCR products. The occurrence of various PVY isolates during primary infection of the potato plants gives the opportunity to interaction between virus and plant resulting the generation of new strains. The purpose of this investigation was the explanation of phenomenon of the interaction between PVY strains and the infected potato plants on the background of the protective action of mild strains against severe ones and appearance of the new isolates. MATERIALS AND METHODS The potato plants cvs. Kolia and Vital (obtained from in vitro), after mechanical inoculation in the different combinations with three strains: PVY0 , PVYN and PVYNTN were examined in DAS-ELISA by using monoclonal antibodies - mabs YN specific (Bioreba), polyclonal antibodies - pabs Y (IHAR) and bioassay on Nicotiana tabacum cv. Samsun. The susceptible for PTNRD cv. Vital used also as the test plant. The schedule of plant assays involved: a) inoculation with single strains: PVY0 , PVYNW and PVYNTN; b) cross-protection test with each strain in sequence: first inoculation with PVY0 followed by PVYNW , PVY0 / PVYNTN, PVYNW / PVYNTN; c) simultaneous doubly- inoculation with mixture of sap, prepared from tobacco plants infected with particular strains. On that way following mixtures of the strain were used: PVY0 +PVYNW , PVY0 +PVYNTN, PVYNW +PVYNTN. The second inoculation, in crossprotection test was done after 4 or 7 days. In other series of this experiment, the progeny tubers 18

of the potato plants cv. Vital which were infected with mild strains in previous year, have been inoculated with severe strains in the next year. RESULTS AND DISCUSSION The distinct interaction between PVY strains and potato plants has been observed on secondary infected plants and tubers (Tab. 1). The highest protection against severe and aggressive PVY strains has been obtained by previous inoculation with the mild strains. The strain of PVY0 caused 100 % protection of cv. Kolia against PVYNW , however this protection was on the 38,5 % level in case of cv. Vital. Prior infection with PVY0 has been long–wearing in cv. Vital, and has been better expressed in the next year, as the suppression of secondary infection with severe strain PVYNW to 0 % level (here not showed). The cross-protection exerted by prior infection with PVYNW against aggressive strain PVYNTN in cv. Kolia was better visible in comparison with PVY0 , while in cv. Vital was on the similar level. Prior infection with PVY0 of cv. Vital caused almost total protection against PVYNTN infection in progeny plants – only 4 % of plants were successfully infected with PVYNTN as measured with mabs -YN in DASELISA, whereas the polyclonal assay indicated 96 % of PVY infestation (here not showed). The different protection of cultivars by prior infection with mild strains of PVY against severe strains maybe caused by other susceptibility to each of PVY strain (Kaczmarek, Mosakowska, in printing). The percentage of PTNRD symptoms observed on tubers of cv. Vital was almost agreed to the mabs YN DAS-ELISA results obtained from the potato plants, however with tendency to decrease (Tab 1). Independent of the cultivar effect, in PVY combinations observed the occurrence of new PVY isolates, which have intermediate properties of PVY0 and PVYN. Some biological properties of these isolates were specific for PVYN (vein necrosis), despite of these isolates were undetectable with serum specific for PVYN strains (Dìdiè, Ptácìk, 1998; Blanco-Urgoiti et all., 1998). Percentage share of necrotic isolates was generally higher than PVY0 isolates. CONCLUSION Reported results (Kaczmarek et all., 1998) of the spread of PVY strains under natural conditions of Poland might be explained as the result of interaction between the PVY strains and potato cultivars. REFERENCES Blanco-Urgoiti B., Tribodet M., Leclere S., Ponz F., Perez de San Roman C., Legorburu F.J.and Kerlan C., (1998): Characterization of potato potyvirus Y (PVY) isolates from seed potato batches. Situation of the NTN, Wilga and Z isolates. Eur.J. of Plant Path. 104:811-819. Dìdiè P., Ptácìk J. (1998): The possibility of specific PVY-NTN detection. The 10th EAPR Virology Sect. Meet. Proceedings. Baden, Austria 5-10.07.1998: 65-68. Kaczmarek U., Turska E., Hnat A. (1998): The spread of PVY strains in some potato cultivars after multiplication in the field conditions in Poland. The 10th APR Virol. Sct. Meet.Proceedings. Baden, Austria, 5-10.07.1998: 116-122. Kaczmarek U., Mosakowska E. (2000): Reakcja wybranych odmian ziemniaka na infekcjê izolatami szczepów PVY0 i PVYN w warunkach naturalnych. Nasiennictwo ziemniaka – konferencja naukowa IHAR. Koszalin, 26-27.01.2000: 34-35. Kaczmarek U., Mosakowska E. (in printing): Comparison of the reaction of some potato cultivars to the infection with the strains PVY0 PVYN and PVYNTN. Phytopath. Pol. Rosner A. and Maslenin L. (1999): Differentiating PVYNTN by unique single-restriction cleavage of PCR products. Potato Res. 42: 215-221.

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Table 1 Interaction effect between PVY strains and the potato plants, the second inoculation after 4 or 7 days in cross-protection test. Bonin, 1998 – 2000

The combinations of PVY strains PVYO (control) PVYNW4 (control) PVYO/PVYNW PVYO+PVYNW PVYNTN (control) PVYO/PVYNTN PVYNW /PVYNTN PVYO+PVYNTN PVYNW +PVYNTN Control (not inoculated)

Cultivar Kolia Vital Kolia Vital Kolia Vital Kolia Vital Kolia Vital Kolia Vital Kolia Vital Kolia Vital Kolia Vital Kolia Vital

Secondary infection of the potato plants (%) Nicotiana tabacum - Samsun DAS - ELISA PVYO PVYN N 1 2 pabs Y mabs Y Vc Vn/1 Vn/2 100,0 n.i.3 100,0 0,0 0,0 84,8 0,0 86,7 0,0 0,0 100,0 n.i. 0,0 100,0 0,0 100,0 0,0 0,0 86,5 10,8 100,0 n.i. 100,0 0,0 0,0 97,5 0,0 38,5 59,0 0,0 100,0 n.i. 0,0 100,0 0,0 100,0 0,0 16,2 78,4 5,4 96,4 96,4 0,0 34,4 46,2 100,0 100,0 0,0 0,0 9,1 100,0 52,2 45,5 40,9 13,6 96,2 52,8 41,5 26,4 18,9 100,0 5,9 17,6 82,4 0,0 100,0 51,1 0,0 50,0 43,7 100,0 100,0 0,0 5,3 42,1 100,0 96,2 3,8 30,8 53,8 100,0 8,0 0,0 100,0 0,0 100,0 66,7 0,0 13,8 65,5 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0

Vn/3 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 15,4 90,9 0,0 11,3 0,0 6,3 52,6 11,6 0,0 20,7 0,0 0,0

Vital PTNRD ni ni ni 0,0 ni 0,0 ni 0,0 87,6 75,0 45,5 30,2 12,2 34,6 85,1 77,5 2,1 42,6 0,0 0,0

Explanation: 1 /-the symptom disease: Vc- vein clearing, Vn- vein necrosis, PTNRD – potato tuber necrotic ringspot disease; 2 /-the intensity of the symptoms disease: 1- mild; 2-distinct; 3-severe; 3 /-not investigated; 4 /-PVYN-Wilga

20

OCCURENCE OF THE POTATO VIRUS Y STRAINS (PVY) MULTIPLICATION OF SOME POTATO CULTIVARS IN POLAND.

DURING

LONG-TERM

Ewa Turska, Urszula Kaczmarek Plant Breeding and Acclimatization Institute, Branch Division Bonin, 76-009 Bonin, Poland [email protected]

INTRODUCTION Potato virus Y is one of the major problems in seed potato production in Poland. The factor favorable for the virus spread is the presence of virus infection sources in commercial fields planted mostly with seeds produced on the own farm without renewal for several years of multiplication. A significant role in virus Y epidemiology is ascribed to the incidence of the new isolates of its main strains in recent years: common Y0 and necrotic YN (Blanco – Urgoiti et al. 1998). Since the mid-1980s, the virus Y isolates of the necrotic strain group, causing the characteristic necrotic symptoms in tubers, are the object of many studies (Beczner et al. 1984, Kus 1992). The strain is described as YNTN . It was determined that this type of symptoms is associated with the potato genotypes and some cultivars reacting to the infection in a particularly clear way were accepted as indicator plants (Romancer, Kerlan 1991). There are few research data in the literature on the assessment of virus Y strains differentiation directly in the seed potato production. In a study done in Great Britain in which Record potatoes of various certification degrees, commercially grown at several locations, were checked for virus Y strain frequency, the necrotic strain was predominant (more than 90%), 3% of tubers were infected with both strains and only 3% with the ordinary PVY0 strain. In a study done in Poland, the incidence of more infectious virus Y isolates was signalized (Chrzanowska 1991, Kaczmarek at all. 1999). Many Polish cultivars were assessed for resistance to the 3 virus Y strains (Chrzanowska 1994), but the variation during the seed material reproduction was not investigated. The purpose of this study was the obtaining of information on the importance of differences of cultivar resistance to PVY0 , LW, PVYN Ny, PVYN W i strains, used commonly for testing, in the multiple reproduction of superelite.

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MATERIAL AND METHODS The investigations were conducted on the highly field–resistant to virus Y potato cultivars, notes 7–8 in 1– 9 scale, 1 being the most susceptible, but of differentiated resistance to virus Y strains (Chrzanowska 1994). The detailed resistance characteristics are presented in tab.1. Field multiplication after 3- or 4-year reproduction of SE was done in the high virus infection pressure area in 1996-1999. Each year after each consecutive reproduction, 400 tubers of each cultivar were sampled for PVY infection tests in the greenhouse indexation. All potato plants were tested by precipitation on latex for PVX, PVS, PVM and PVY presence and by DAS ELISA test for PRLV and PVY presence. The domestic antisera were used. In addition, the tests with Bioreba monoclonal antisera for PVY (so called isolate coctail) and PVYN (so called Mab) were done. For identification of virus Y strains, the Samsun tobacco plants were used and for YNTN strain – Vital and Rosalie potato cultivars. The investigation was co-funded by the Committee of Scientific Studies within 5P06B05411/96 research project. Tab. 1 Characteristics of cultivars in respect to virus resistance

Cultivar

Degree of field resistance (in 1-9 scale)

Resistance to PVY strain isolates

PLRV

PVM

PVY

Y0 LW

YN Ny

YN Wi

Bekas

7,5

3,5

8

immune

immune

immune

Vistula

6

2

8

resistant

resistant

resistant

Arkadia

8

3,5

8

partly resistant

partly resistant

partly resistant

Sumak

6

5

7,5

resistant

resistant

resistant

Aster

7,5

5

7,5

partly resistant

medium resistant

medium resistant

Orlik

5

5

7,5

partly resistant

partly resistant

medium resistant

Ekra

7,5

3,5

7

partly resistant

susceptible

susceptible

Irga

8

5

7

partly resistant

partly resistant

medium resistant

Explanation: Y0 LW – from Lipiñski Wczesny potato cultivar YN Ny – from Nysa potato cultivar (so called old strain) YN Wi – from Wilga potato cultivar (so called new strain)

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RESULTS After 3- or 4-year reproduction of 8 cultivars, only Irga and Orlik were more infected with PVY – it was 80% and 30% respectively, in 1999. The infection of Aster cultivar, of high field resistance and intermediate resistance to the particular PVY strains, similar to Orlik cultivar, was low–below 10% (Fig.1). The remaining cultivars were little or only sporadically infected. After first year of reproduction, the 3 cultivars (Irga, Aster and Orlik) were predominantly infected with the PVYN necrotic strain (Fig.1). According to the diagnostic results on tobacco plants, the frequency of common PVY0 strain was on the rise at various rate in consecutive years, depending on cultivar. At the high PVY infection of Irga cultivar, the share of common strain after 3 years was about 1/3. Simultaneously, within the necrotic strain isolate group, about 8% reacted positively with the Mab antiserum. These isolates were identified, on the ground of disease symptoms in tubers of potato test cultivars Vital and Rosalie, as PVYNTN . In the remaining cultivars, there were found cases of isolates causing necroses in tubers of potato test cultivars. However, no disease symptoms in tubers of cultivars tested were observed, despite keeping the samples under provocative room temperature for about 3 months.

% 90 80

YN

70

Y Mab

Y0

60 50 40 30 20 10 0

1997

1998

Irga

1999

1996

1997

1998

Aster

1999

1996

1997

1998

1999

Orlik

Fig. 1 The mean infection of cultivars, reproduced in 1996 – 1999, with potato virus Y.

DISCUSSION On the ground of results obtained, one can say that the susceptibility of potato cultivars to the main virus Y strains, determined in laboratory investigations (Chrzanowska 1994), could affect the differentiation of virus spreading rate in consecutive reproductions of superelite seeds, but it was not closely correlated with the previous (to individually strains resistance) assessment. In cultivars partially susceptible or susceptible to the PVY strain isolates studied, in which PVY infection spread was relatively quick, the strains PVY0 or PVYNTN can appear in higher frequency (Irga cultivar). However, in Ekra cultivar, rated as susceptible to the virus Y strains studied, only sporadic infection cases were found in field conditions. Practically it means the difference in seed production difficulties of Ekra and Irga cultivars. The results obtained confirmed the sporadic or lack of PVY infections during 4 years of reproduction of Bekas cultivar, immune from PVY and Vistula and Sumak – resistant to the PVY strain isolates studied. These cultivars should not pose difficulties in seed production.

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LITERATURE Baker H. !994. Incidence of potato virus Y infection in seed and ware tubers of the potato cv.Record. Ann. Appl. Biol. 124, 179– 183. Beczner L.,Horvath J., Romhanyi I.,Forster H.,1984: Studies on etiology of tuber necrotic ringspot disease in potato. Potato Research. 27, 339-352. Blanco-Urgoiti B., Tribodet M., Leclere S., Ponz F., Pérez de San Román C.,Legorburu F.J.,Kerlan C.. 1998. European Journal of Plant Pathology 104 811-819. Chrzanowska M. 1991. New isolates of the necrotic strain of virus Y (PVYN ) found recently in Poland. Potato Research 34. 179-182. Chrzanowska M. 1994. Differentiation of potato virus Y (PVY) isolates. Phytopath. Polonica 8 (XX): 15-20. Kaczmarek U., Turska E., Hnat A.1999: The spread of PVY strains in some potato cultivars after multiplication in the field conditions in Poland. Proceedings of the 10th EAPR Virology Section Meeting Baden, 116-122. Kus M. 1995: Investigations of the sensitivity of potato cultivars to tuber necrotic ringspot strain of potato virus Y (PVYNTN ). Proceedings of the 9th EAPR Virology Section Meeting Bled,135-138. Le Romancer M., Kerlan C. 1991: La maladie des nécroses annulaires superficielles des tubercules: une affection de la pomme de terre, due au virus Y. Agronomie 11.889-900.

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ESCAPING FROM PVY INFECTION IN THE FIELD: PLANTING DATES, APHID REPELLENTS AND RESISTANCE INDUCERS. Handizi, A. 1 , Serra, J. A. 2 & Legorburu, F. J.1 1

NEIKER-Basque Institute for Agricultural Research and Development Apartado 46, E-01080 VITORIA/GASTEIZ, Basque Country, Spain. [email protected], [email protected] 2 SAGRAL-Alavese Agricultural Services Polígono Industrial Lurgorri, E-01240 ALEGRÍA/DULANTZI, Basque Country, Spain.

In our previous meeting in Baden in 1998 results were presented on the control of PVY in the field by means of agrotextile fleece and sprays of mineral oil + pyrethroid. Fleece is too expensive for the individual farmer, but those results suggested that the repellent effect of the pyrethroid could help. Now we have tested an aphid repellent (neem oil) and a resistance inducer (benzothiadiazole, Bion ® from Novartis, now Syngenta). In addition to that, and since these chemical treatments must be applied weekly, we tried determining the most critical period of protection by a combination of planting dates and protection by fleece. No chemical control was as effective as the fleece protection. Crude neem oil gave the best control, followed by benzothiadiazole. Other commercial preparations containing neem oil or its active ingredient, azadiractin, were less effective. Crude neem oil also gave good control in three out of four farmer’s fields. The usual planting season in the area runs from mid May to mid June. Harvesting is usually done in September. Aphid flights usually peak in late June and have a minimum from mid July to mid August. Four planting dates were tested in 1999: late April, late May, mid June and mid July. The approximately three months of the potato (cv. Kennebec) vegetative cyle were divided into three periods of four weeks. The protection treatment consisted in fleece, during either the fist or second third, and mechanical haulm killing, for the last third. Protecting the first third of vegetative growth was the most effective, irrespectively of the planting date. Killing the haulms for the last third of vegetative growth didn’t improve the PVY control in any instance. This agrees with the phenomenom known as mature plant resistance. In 2000 the protection strategy was different: fleece protection was applied during either 4, 6 or 8 weeks after emergence, in order to determine the appropriate length of the protection period. Planting dates were early April, early May, early June and early July. Longer protection periods resulted, in general, in a better control. This effect was more pronounced when the longer protection period covered the aphid flight peak in June and early July. In both years PVY infection increased with the planting date, in contradiction to the received wisdom. As a result, our current recomendation for PVY control is to spray neem oil every seven to ten days from emergence until the end of the aphid peak flight, as determined by yellow water trapping.

25

NIB -MEDIATED RESISTANCE OF POTATOES TO PVY INFECTION Jörg Schubert*, Jaroslav Matousek+$ , Dirk Mattern*$, Frank Rabenstein*, Petr Dedic# , Elena Sukhacheva§$ * - Federal Centre for Breeding Research on Cultivated Plants, Institute for Resistance Research and Pathogen Diagnostics, Aschersleben, Germany; + - Czech Academy of Sciences, Institute for Plant Molecular Biology, Ceske Budejovice; Czech Republic# - Institute for Potato Breeding, Havlicku v Brod, Czech Republic; §- Institute of Biooragnic Chemistry, Moscow, Russia

Keywords: potato virus Y, potyviruses, potato, gene transfer, resistance, blue fluorescing protein, field

Abstract Potato plants of three different cultivars/lines were transformed with a truncated NIb gene of PVYN-CH605 which has been C-terminal fused to the enhanced blue fluorescing protein (EBFP, Clontech). Regenerated plants have been tested for resistance by mechanical inoculation with a PVYNTN strain. From plants remaining virus free tubers were harvested and emerging sprouts tested for secondary infection. 3 highly resistant and one immune clone could be identified. No correlation was found for level of GUS-activity, fluorescence of fused NIb-EBFP, RNA expression, number of inserts, and resistance. Under field conditions immunity of the clone was overcome. The three resistant clones showed recovery resistance. To two of them isolates were identified which could overcome their resistance too. Introduction While it is well documented that complete genes of potyviruses can confer resistance to virus infection little is known about applicability of truncated genes as well as fusion of them with certain proteins. An answer for this question would be important in context of higher biosafety of transgenes and for research on the mechanism of pathogen derived resistance. As an object for investigation we chose potato and PVY. Material and Methods The NIb gene used originated from a N-type strain (CH605) and was lacking 400 nt of the Cterminal part. Its sequence was typical for N-strains. The gene was fused to the 35S-CaMVpromoter (pRT100) and integrated into the binary vector pGPTV-KAN between the NPTII and GUS- i genes. In addition, to the C-terminal end of the NIb gene that of the enhanced blue fluorescing protein (EBFP) was fused in frame and integrated via pRT100 into the mentioned binary Agrobacterium plasmid. Transformed plants from two varieties ('Linda', 'Kamyk') and a dihaploid line (DH59) were transferred in a green house into soil. Resistance testing was performed with a PVYNTN strain (Hessen) in a climate chamber at 22 o C, 16h/8h light/dark with 10 plants/clone. Systemically infected leaves were tested by DAS ELISA (Bioreba) 35 dpi after mechanical inoculation (15 dpi in the case of primary infected leaves). Sprout testing for secondary infection was performed if the plants grown in a green house had reached a height of approximately 15 cm. Field experiments were performed with 3 replicates. Each single plot was surrounded by PVY infected plants (CH605). Virus was transmitted by naturally occurring aphids. Results and Discussion Only those plants were scored as resistant which showed 35 dpi OD values which were not or only slightly higher than those of the healthy control plants (x+2s). From these clones tubers $ - supported by grants from German Ministry of Education, Research and TEchnology

were harvested and tested for secondary infection. Using such a strong resistance selection criterion we intended to obtain clones with a resistance level comparable to extreme resistance.

26

This was reason to use a highly aggressive strain of PVYNTN. It has a sequence slightly different from that of the transgene. The results are summarised in Tab. 1. Table 1: Summarised results of testing for resistance (primary infection) Transgene variety/line: 'Kamyk' 'Linda' DH59 NIb-EBFP 54/1 58/3 NIb 178/0* 39/0 EBFP 13/0 1/0 *- number of tested independent clones/among them resistant

Only among plants transformed with the NIb fused to EBFP resistant clones were identified though much more clones with only NIb gene have been tested. EBFP seems to have either a stabilising function or targets the NIb to cell compartments where it can induce resistance. The results for resistance to primary infection were confirmed by testing for secondary infection. Data are presented in Tab. 2. Tab. 2: Summarised results of testing for resistance (secondary infection) clone number of individual plants which proved to be healthy slightly infected infected (0,05) OD) Linda/DH59 (control) 0 0 35 DH59 Nb67 44 0 10 DH59 Nb33 53 0 6 DH59 Nb146 55 1 (0,03)* 2 (2,7) DH59 Nb156 70 3 (0,02) 0 DH59 Nb93 35 2 (0,05) 0 Linda Nb 58 103 0 0 *- ELISA values

Clones Linda Nb58, DH59 Nb 93 and 156 appeared to be highly resistant while in the case of DH59 Nb 146 two sprouts out of 58 tested showed a high level of infection. Two other clones, DH59 Nb 67 and 33, revealed the same characteristics - most of the plants remained free of virus but some of them showed a high level of virus contamination. Together with the gene of interest a GUS-i gene was transferred. It was not possible to reveal any correlation between the intensity of fluorescence and level of resistance. This means that intensity of fluorescence can not be used as a pre-selection marker for resistance. In another experiment the virus concentration of primary infected leaves of the resistant clones was tested to get information on resistance mechanism. Linda Nb58 remained free of virus while DH59 Nb146 was free for the most part. The primary leaves of both other resistant clones were infected. This indicated that the type of resistance of Linda Nb58 was immunity while the three other clones revealed a recovery type. Linda Nb 58 was not infected by potato viruses A and V while three other resistant clones were. This is an indication that for the type of resistance also the site of integration into the genome is essential, not only degree of sequence homology to challenging virus. The number of integrated copies could play an important role too. In Fig. 2 a Southern blot of several clones is presented. One can see that resistant transgenic plants contain from three to ten insertions of the foreign gene. Interestingly, despite of the high number of insertions clone DH59 Nb93 displays only a weak GUS activity. The immune Linda Nb58 and DH59 Nb146 have both three copies of the NIb gene though differ in level and mechanism of resistance. The results demonstrated that the level of resistance does not correlate to the copy number.

27

Fig. 2: Southern blot of transgenic plants after digestion with XbaI, probed with a nonradioactive probe of NIb. A-D: serial dilution of NIb insert; E, F: nontransformed Linda and DH59, respectively; G: DH59 Nb93; H: DH59 Nb146; I: DH59 Nb156; J: Linda Nb58; K: DH59 Nb51 (susceptible); Linda Nb 60 (susceptible).

Another question was whether level of RNA expression reflects level of resistance. In the light of the theory of post-transcriptional gene silencing (PTGS) one can expect, that low level of RNA expression or a high turnover rate of RNA, resulting in low level of available RNA, is a prerequisite for PTGS. Among susceptible plants we found the whole range of RNA concentrations (results not shown). This means that even plants with low expression levels, thought to be important for PTGS, were susceptible. Field experiments in 2000 and following sprout testing for secondary infection revealed the following results. All plants of the three resistant DH59 clones were infected. The immune line Linda Nb58 revealed 5 plants which were infected with PVY. Analysis of the resistance breaking isolates of Linda (isolates Linda 1 to 5) revealed mixtures of N– and O-strains (Adgen ELISAtest) and in one case probably a Wilga type isolate (isolate Linda 5) whic h was also highly infective on tomato. Sprout testing confirmed the results for Linda Nb58 as several heavily infected plants were found though most remained healthy. Most surprisingly data were obtained when the three resistant lines were investigated. They originated from two trials grown at a distance of 100 m. From the first trial plants of all three plots were free of virus. This was confirmed by back inoculations on tobacco. From the other trial clone DH146 showed some non-recovered plants. The isolates found belonged to the O-type. Under greenhouse conditions the isolates from Linda (isolate 2 and 5) and DH59 Nb146 could overcome resistance of corresponding clones too. Extensive testing with different virus isolates (DSM Braunschweig, collection of BAZ) revealed, that several isolates exist which can overcome immunity of Linda Nb58 (for other isolates under progress) – despite the fact that this clone is resistant to tested isolates of PVA and PVV. This means that other criteria than sequence homology of the transgene and challenging virus are important for stability. Comparison of sequences is under progress. Investigation of changes in concentration of transgene mRNAs was performed by means of a Northern blot. It is shown in Fig. 3. As one can see easily the concentration of the mRNAs between different non-inoculated potato clones does not show striking differences (see above). Different intensity of PVY- full length RNA-signals indicates different progress in infection (first symptoms after 12-14 days). Strains PVYO-Ad and PVY- NAd reached detectable levels much faster than isolate 5. In the case of successful infection it seems that the transgene mRNA reaches higher concentrations. Further data of interest are in lane 6 and 8: in both cases a reduced level of PVY- full length RNA is noticeable, a result of preinoculation with either PVYNTN-Hessen or PVA. The resistance mechanism was induced by these isolates but could be overcome by isolate Linda 5. In lane 18 the NIb- mRNA band clearly disappeared after infection with isolate Linda 2 suggesting a PTGS mechanism. 28

Fig. 3: Northern blot of total RNA of transgenic plants (corresponding ethidium bromide stained gel not shown) on Nylon membranes probed with radioactive labelled antisense RNA (8µg RNA applied, at 17 and 18 only 4 µg). Lane 19 was RNA- marker (Gibco), 20 corresponded to 0,1 µg of purified viral RNA (both cut off to avoid overexposure of film, but traces still visible). 1- non transformed potato, healthy; 2 - Linda Nb58, PVYN-Ad, 14 dpi; 3not infected; 4 - PVYNTN-Hessen, following samples 11 dpi; 5 – PVY, isolate 5; 6 – PVYNTNHessen, 2 days later inoculated with isolate Linda5 (=9 dpi); 7 – PVYO-Ad; 8- PVA, 2 days later inoculated with isolate Linda 5 (=9 dpi); 9 – PVYN-Ad; 10 – DH59 Nb156, not inoculated; 11 – isolate Linda 5, all following samples 9 dpi; 12 – PVYN-CH605; 13 – DH 59 Nb146, not inoculated; 14 – isolate Linda 5; 15 – isolate Linda 2; 16 - PVYN-CH605; 17 – DH59 Nb 93, not inoculated; 18 – isolate Linda 2. The process of suppression of resistance response was investigated more in detail for Linda Nb58 by means of ELISA too. Isolate Linda 5 is able to suppress resistance to PVA, thus supporting data from Northern blot. On the other hand, if the resistance reaction once is induced by PVA or an incompatible PVY strain the concentration of PVY Linda 5 remains lower than in the plants infected only with isolate Linda 5. In the case of double infections (2 days interval) the plants reacted with strong necrotic reactions to the cha llenging virus. Conclusions The results indicated, that one construct can induce different mechanisms of resistance. There exist several mechanisms by which the virus can actively block these resistance mechanisms. Kind and level of resistance do not mainly depend on level of sequence homology.

29

CONTROL OF POTATO VIRUS Y IN UK POTATO SEED CROPS Ian Barker, Jane Morris, David Firman, Phil Northing, Richard Leach, Sharon Elcock, Judith Turner, Miles Thomas & Keith Walters Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK Will discuss various aspects of PVY control in the UK including the following. Changes in potato seed production areas, incidence of aphid borne viruses, trends in pesticide usage, use of aphid monitoring schemes, use of modern diagnostic methods, efficacy of insectic ides and mineral oils.

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DIAGNOSIS OR DETECTION - DEVELOPING APPROPRIATE METHODS Neil BOONHAM, Kathy WALSH, Hazel SWAN, Rick MUMFORD and Ian BARKER Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK. [email protected] A wide spectrum of methods are currently available and in use in diagnostic laboratories on a day to day basis. The methods used are diverse and for viruses alone require laboratories to have a wide range of expertise to cope with most if not all of the following methods: electron microscopy, ELISA, PCR, RT-PCR, TaqMan, reverse PAGE or nucleic acid hybridisation, and finally bioassays such as inoculation of test plants. These methods tend to be suited to either diagnosis of disease or detection of a known pathogen. The former requires methods which are able to identify many targets in a highly parallel manor, whilst the latter focuses on one or a limited number of known targets, but often in a large number of samples. Work at CSL has been focused in these two areas, and using potato viruses as a model system, developments in each area will be illustrated. Some "multi-target" generic assays have found use in virus detection but most are suitable for a limited range of organisms only. Most notably electron microscopy, test plant inoculation, and more recently MALDI TOF (Matrix Assisted Laser DIssorption Time Of Flight mass spectrometry), are all "multi-target" methods. However each of these methods tend to be most suited to certain groups of targets. For example electron microscopy is most suitable for rod shaped viruses, whilst detection of low titres of spherical viruses is more difficult. Inoculation to test plants relies on the availability of ‘universal’ indicators, but again no single host exists for all viruses, some viruses are not mechanically transmissible and results may require follow up testing for identification. Finally MALDI-TOF has been used to detect a few plant viruses such as Tobacco mosaic virus and selected potexviruses in infected leaf material, however it is unlikely to be able to offer detection of low titre viruses direct from infected tissue. Finally, neither electron microscopy or MALDI TOF are suitable if no pathogen expressed protein is present, as is the case with viroids and some viruses. If a single ‘multi-target’ method could be developed that would be able to cope with the full range of organisms in a totally generic format, it would streamline and standardise a significant portion of diagnostic testing currently carried out. In recent years it is estimated that the amount of DNA sequence data in databases is doubling on average every 14 months, with approximately 6 billion base pairs being submitted every 2 months and whole genomes of a number of organisms have now been completely sequenced. This data holds the key to many new and exciting areas of scientific and medical discovery. The sheer volume of data poses a complex technological challenge, namely, how can such data be mined in a high throughput and cost effective fashion. Currently micro array technology is leading the way in offering researchers the ability to simultaneously examine the expression levels (presence of) of hundreds or thousands of genes in a single experiment. Already “micro arrays” or “gene chips” are being used that have 10 000 genes arrayed on the surface with current protocols allowing reliable detection of target down to only several copies per cell. So far the technology has mainly been applied in research fields for gene discovery, with only a few examples apparent in medical diagnostics, primarily for bacteria. It is envisaged that microarray technology could be exploited for the detection of not just viruses but for bacteria, fungi, invertebrates, phytoplasmas and viroids in a single generic test procedure. Up to 30 000 DNA probes can be arrayed onto a single glass microscope slide, which forms the microarray or gene chip. The DNA probes arrayed are gene sequences from each of the organisms that need to detected in a single assay. The gene chip can then be exposed to fluorescently labelled DNA/RNA from the sample to be tested and then fed into an micro-array reader to reveal if any of the targets were present in the sample. Thus, the end-user would only need to purchase an inexpensive “chip”, on a glass microscope slide from a specialist arraying company, and have access to a reader. Sample extraction, labelling and hybridisation can be achieved with standard laboratory facilities and minimal training. The 31

implications for success of the technique would be far reaching, enabling laboratories to screen for ‘all’ pathogens using a single diagnostic test. The paper will describe the production of microarrays, the development of extraction methods and labelling techniques for a selection of a number of common potato viruses. In addition, the relative sensitivity of the method compared to other commonly used detection methods such as ELISA and PCR and the levels of specificity achieved thus far will be discussed. Unlike diagnosis of disease, detection involves testing material for a limited number of known targets, in addition, detection also often requires large numbers of sample to be tested. A number of methods in common use are well suited for detection work, most notably ELISA, being easily scaled to take into account large sample numbers, it has a simple extraction method and is a very robust assay format. Molecular methods, in particular PCR are often developed, and are well suited to the detection of plant viruses. However the adoption of PCR for the routine detection of plant pathogens has been slow. Three of the main reasons for this are: firstly, the problem of running gels and interpreting results (particularly for large sample numbers). Secondly the problem of post-PCR manipulations, which leave sensitive PCR assays open to the risk of contamination and false positive results. Finally and perhaps most importantly, the ability to test large sample numbers. Adoption of TaqMan chemistry with real time detection (i.e. detection of PCR products during amplification) can help to alleviate each of these problems. However, developments need be made in a number of areas to improve the reliability, sensitivity and utility of PCR based methods. For example, control of virus disease within potato crops is most effectively carried out by planting seed potatoes which are carrying no or only a very low percentage of viral infection. Current virus testing methods for seed potatoes rely on the ‘growing-on test’. In which tubers (normally 100200) representing a seed stock are germinated, and the plants subsequently grown, to allow replication of the virus. The plants are then tested using ELISA. This method allows a turnaround time of between 4-6 weeks. Testing the tubers directly would lead to much improved turnaround times, however the titre of virus, particularly Potato virus Y (PVY) in seed tubers is very low and requires an assay which is considerably more sensitive than ELISA. A testing method based around PCR is thought to be sensitive enough to detect virus directly in the tubers, however, a number of aspects of PCR would need to be taken into account before a method based on PCR could be used. Firstly, the assay would need to be high throughput. Secondly, contamination when dealing with large sample numbers would have to be eliminated, as the best stocks would be virus free. Finally, the method would have to be robust, with appropriate controls to allow trouble shooting of the method. By using TaqMan chemistry and real time PCR product detection many of these questions can be answered. The method requires no post PCR manipulations, so is geared towards high throughput testing. Since it is a closed tube system, post PCR contamination is reduced, and the multiplex capabilities of the system, using different reporter dyes mean the incorporation of internal positive control reactions is straightforward and can be used to control for the presence of RNA extracted from the sample. TaqMan assays for PVY, PLRV, PVX, PMTV and TRV were evaluated for the direct detection of these viruses in tuber extracts. The method was compared with conventional glasshouse growing on tests, for the same tuber sample following the harvest of the 1999 crop. The results show a high degree of correlation between the direct testing using TaqMan and the growing on test following ELISA.

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ANALYSIS OF VIRUS AND VIROID VARIABILITY BY TGGE AND BY THE METHOD OF cDNA HETERODUPLEXES. Matoušek, J. Intitute of Plant Molecular Biology, CAS Èeské Budìjovice, CR [email protected] There is a need to use a more complex approach to characterise plant viruses and viroids, because the propagation of a homogeneous sequence in planta is rather an exception than the rule for most of these pathogens. Temperature gradient-gel electrophoresis (TGGE) and DNA heteroduplexes method provide the powerful tools to study dynamics of virus populations, mutated variants, quasispecies and strain mixtures. In this survey we present several examples for potato virus S (PVS), potato virus Y (PVY), potato spindle tuber viroid (PSTVd) and molecular pathogens infecting hop, hop latent viroid (HLVd) and apple mosaic virus (ApMV). For Central European isolates of potato virus S, which according to their inability to develop systemic infection in Chenopodium quinoa, resembled to ordinary strain of this virus we detected wide nucleotide variability by sequencing and by the TGGE method (Matoušek et al. 2000). Nucleotide comparisons of eight experimental clones for each (C-terminal part of the replicase, coat protein and 11K protein) revealed 55, 47 and 13 variable sites. However, it was found for isolate Kobra using temperature-gradient gel electrophoresis of a coat protein-derived 260 bp cDNA fragments that the variability is broader than expected, as at least seven dominant heteroduplexes were detectable for this single isolate, suggesting accumulation of quasispecies or mixed infections. The cDNA heteroduplexes method we developed recently to detect variability of 394bp cDNA fragments amplified by RT PCR from P1 gene using primers A3S4 (Weilguny and Singh, 1998). The standard cloned fragment of dominant sequence, which we derived from PVY isolate Nicola showed simple transition on denaturing gel and two end-melting points approx. at 41o and 48o C. A more complicated TGGE pattern of cDNAs was found for the whole isolate Nicola. This suggested the accumulation of some minor sequence variants of PVY in this isolate, which were identified latter by sequencing. Based on the TGGE pattern, 46o C was selected as the standard temperature for electrophoretic analysis of pre-formed heteroduplexes. The analysis of more than 40 PVY isolates from PVYN group, performed using the heteroduplexes method, clearly showed that in the most cases minor virus quasispecies are present accompanying the dominant form. In the most isolates of PVYN-Wilga type we found the mixtures of the major sequence variants having mutations, which led to several amino acid changes in P1. The same methods we used successfully for analysis of variability of plant viroids, like HLVd and recently PSTVd. According to our recent results we found that viroid replication under thermal stress leads to accumulation of mutations, localised mainly in the left part of the viroid structure (Matoušek et al. 2001). For the heat-induced PSTVd variants, "hot spots" we detected at positions 46, 47, 60, 309 and 317 changing pathogenic domain. Thus, these mutations could lead to evolution of highly pathogenic viroid strains. Such lethal strain, designated RG1 appeared presumably under uncontrolled thermal stress in experiments performed by Gruner et al. (1995). We found by the analysis of cDNA heteroduplexes that viroid mutants form secondary populations and that specific molecular evolution is initiated in infected plants, depending on cultivation conditions. It is of particular interest to analyse distribution of low level sequence variants, recombinant viruses, or some particular quasispecies within the virus or viroid populations. Quasispecies could exhibit differential adaptability ni different plant tissue and genotypes, at different ontogenetic stages, or/and under different growth conditions. TGGE and DNA hetroduplexes methods enable specific in vitro pre-selection of mutants by extracting, re-PCR and re-cloning of deviating cDNAs from polyacrylamide 34

gels. Such experiments we performed to analyse P1 PVY variants of Wilga type, low replicating thermomutants of HLVd and populations of ApMV. In the last example, we analysed RNA 3 component of ApMV, the 379 bp genome region encoding C terminal part of the coat protein (CP). TGGE pre-selection and cDNA library screening enabled to select five deviating ApMV sequence variants. Using gradient PCR, specific diagnostic primers were developed to detect quasispecies designated ApMV45, having double mutation in an "hot spot" region, which we identified within CP. It was determined by this specific diagnosis that ApMV45 prevail in virus population from young tissue over the old one at the levels from 1 to 10 %, depending on the plant genotype. In conclusion, our results demonstrate some advantages of electrophoretic methods based on thermodynamic and structural properties of cDNAs. Especially the high resolution of mixtures of sequence variants and virus or viroid populations, as well as the possibility to pre-select mutated cDNA forms, make these approaches suitable for easy analysis of complex populations of plant molecular pathogens like viruses and viroids. The experiments described were supported by several grants: GA ÈR 521/96/1308, GA ÈR 522/00/0227; NAZV EP9111 and GA ASCR S5051014.

References: Matoušek, J., Schubert, J., Dìdiè, P. , Ptáèek, J.: A broad variability of potato virus S (PVS) revealed by analysis of virus sequences amplified by reverse transcriptase-polymerase chain reaction. Can. J. Plant Pathol., 22: 29-37, 2000. Matoušek, J., Ptáèek, J., Dìdiè, P., Schubert, J.: Analysis of variability of P1 gene region of N strain of potato virus Y using temperature-gradient gel electrophoresis and DNA heteroduplex analysis. Acta virologica, 44: 40-46, 2000. Matoušek, J., Patzak, J., Orctová, L., Schubert, J., Vrba, L., Steger, G., Riesner, D.:The Variability of Hop Latent Viroid as Induced upon Heat-treatment.- Virology 287, 2001 (in press). Weilguny, H, Singh, R.P.: Separation of Slovenian isoaltes of PVYNTN from the North American isolates of PVYN by a 3-primer PCR. J.Virol.Methods 71: 57 – 68, 1998. Gruner, R., Fels, A., Qu, F., Zimmat, R., Steger, G., Riesner, D. (1995). Interdependence of pathogenicity and replicability with potato spindle tuber viroid. Virology 209: 60-69, 1995.

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ADVANCES IN THE DIRECT DETECTION OF SPRAING FROM TUBERS Rick MUMFORD, Anna BLOCKLEY, Kathy WALSH, Ian BARKER and Neil BOONHAM Central Science Laboratory, Sand Hutton, York, North Yorkshire, YO41 1LZ, UK. E-mail: [email protected]

Spraing is an economically-important disease of potatoes, which is common throughout Europe and North America. The disease itself is caused by either of two different viruses, namely Tobacco rattle virus (TRV; genus Tobravirus) and Potato mop-top virus (PMTV; genus Pomovirus). The symptoms of the disease are mainly expressed on the tubers, where they appear as internal, brown necrotic arcs, lines or spots. Symptoms can also be expressed externally, resulting in rings on the surface of the tuber. As a result of these symptoms, the quality of the affected tubers is reduced, often to the extent that an entire crop destined for processing or pre-packaging for supermarkets, may be rejected. Spraing is difficult to control as both of the causal agents are soil-borne. In the case of TRV, the virus is transmitted by free-living nematodes (Trichodorus and Paratrichodorus spp.), while PMTV is transmitted by the powdery scab fungus Spongospora subterranea. As a result of these differences and as the susceptibility of different potato cultivars to each virus varies greatly, an accurate diagnosis has to be made before control measures are attempted. However, until recently diagnosis has been very difficult as traditional methods (e.g. visual symptoms, electron microscopy, mechanical transmission and ELISA using polyclonal antisera) have proved extremely unreliable. More recently the situation has improved, with the production of PMTV-specific monoclonal antibodies and the development of a reliable ELISA assay. However, even this type of technology is inappropriate in the case of TRV, as in potato crops the NM-type isolates (that exist solely as infectious RNA 1, with no associated proteins) are prevalent, and these cannot be detected by serological means. As a result, the development of molecular methods, such as reverse transcription-polymerase chain reaction (RT-PCR), have proved necessary for TRV detection. While diagnostic methods are now available for both the spraing-causing viruses, the requirement for two different tests, combined with the problems associated with conventional PCR (e.g. contamination risks, gelrunning, use of toxic chemicals), has added considerable time and cost to the overall testing procedure. Further problems including lack of sensitivity also exist, which combined to make spraing testing both complicated and not totally reliable. In order to circumvent these problems, a multiplex assay based upon TaqMan chemistry was developed (Mumford et al., 2000). TaqMan is a molecular detection method, that combines polymerase chain reaction (PCR) with fluorescent detection. The system works by using a oligonucleotide detection probe, which is designed to bind to the target DNA between a pair of target-specific PCR primers and which is labelled at each end with two dyes: a reporter and a quencher. While the probe is intact, any fluorescence emitted by the reporter dye is absorbed by the quencher. However, if the two dyes become spatially separated (e.g. if the probe is degraded), then fluorescence is emitted. This general principle is called Fluorescence Resonance Energy Transfer or FRET. During a TaqMan assay, if target DNA is present, the primers will bind to it and DNA amplification will occur, as in PCR. As this occurs, the probe (which will also be bound to the target sequence) is digested by the Taq polymerase, which has an exo nuclease activity. This releases the reporter dye from the quencher, and an increase in fluorescence is detected, indicating that the sample is positive. In practice, simultaneous amplification and detection is achieved by running assays on the ABI Prism 7700 machine, which combines a thermal cycler (as used in PCR) with a fluorescent detection system. Overall, TaqMan has many advantages over conventional PCR, especially as it removes the need for gel running, reduces the overall risks of cross-contamination and has proved to be far more sensitive. The new multiplex TaqMan assay has been extensively tested, in order to prove it’s reliability. Over 50 different field isolates of TRV and PMTV were collected from all over the UK and elsewhere in Europe, and were tested using the new assay. All were correctly identified using TaqMan . The new assay was also shown to be far more sensitive than both the existing TRV PCR and PMTV ELISA tests. In fact the TaqMan assay was 10,000 times more sensitive than the ELISA assay, and 100-fold more sensitive than the already sensitive TRV RT-PCR test. In addition to being reliable and sensitive, the new assay is also very rapid, allowing samples to be tested in under 24 hours in some cases. In addition to the development of a reliable detection system for spraing, further studies have been carried out to develop RNA extraction methods that permit high-throughput testing, especially directly from tubers. While current extraction methods are reliable and relatively quick for small sample numbers, they are labour intensive and are not practicable once sample numbers start to increase. A variety of different approaches have been examined, including commercially available spin columns, probe capture and magnetic extraction. The preliminary results from these studies are presented. The third area of work discussed in this paper relates to preliminary studies that have been carried out to assess the efficiency of TRV transmission from infected seed tubers. Recently it has been suggested that the seed transmission of TRV through to progeny tubers is more common than previously thought. This could obviously 36

have major implications for seed certification schemes. In order to help address this issue, work has been carried out in order to ascertain the significance of seed transmission, including the timing, distribution and overall efficiency of systemic spread. Reference: Mumford R.A., Walsh K., Barker I. & Boonham N. (2000). The detection of potato mop-top and tobacco rattle viruses using a mu ltiplex real-time fluorescent RT-PCR assay (TaqMan ). Phytopathology 90: 488-493.

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RATTLE AND MOP-TOP: SEED PRODUCER.

PUZZLING VIRUSES FOR THE POTATO

Legorburu, F. J. NEIKER-Basque Institute for Agricultural Research and Development. Apartado 46, E-01080 VITORIA/GASTEIZ, Basque Country, Spain. [email protected]

Tobacco rattle tobravirus (TRV) and potato mop-top pomovirus (PMTV) induce sympoms in the tuber flesh (spraing), becoming a problem for the ware potato grower. However, in the current certification schemes, viruses are dealt with by the seed growers. The potato seed certification schemes are built upon the idea of succesive field multiplications of clean starting material until it reaches the maximum tolerable infection. TRV and PMTV differ greatly from the other potato viruses in being soil-borne, rather than tuber borne. The seed grower is used to fight aphids and rogue infected plants, but is also used to a fresh start every year, one of the most critical points of success being the health status of his/her starting plant material. These viruses, however, are transmitted only erratically to the daughter tubers but, more seriously, stay in the soil for years. The induction of necrotic arcs in the tuber flesh is a sign of the hypersensitive response of the host. Due to this incompatible reaction, the amount of virus available for diagnosis is low and erratic and, eventually, the virus can die out and give rise to a healthy plant in the next generation. Although PMTV particles are fragile, it can be reasonably detected in tubers by ELISA. For TRV, however, this is an almost impossible task, because over eight serotypes have been described. PCR diagnostic methods have greatly improved the possibility of properly diagnosing individual tubers. But, the main source of infection being the soil, it would be more appropriate to diagnose soils by means of bait plants. The ability of producing necrotic arcs in the tuber flesh is higly variety-dependent. The genetic of the potato resistance and tolerance to these viruses is poorly understood, due to the difficulty in uniformly testing potato genotypes. It has been long aknowledged that PMTV can be transmitted by the seed tuber in the spores of its vector, the powdery scab protist. However, no provisions against PMTV are made in the current sed certification schemes. For TRV, only recently the possibility of transmission from symptomless tubers of certain varieties has been described. In principle, the risk of introducing TRV into a given field is lower than for PMTV, since the nematode vectors of TRV are not carried on the seed tuber and the species present in the soil should match the virus serotype carried by the tuber. More research is needed to assess the risk of TRV transmission by the seed tuber. Once stablished in a field, both viruses will stay for years. In the case of PMTV, its vector’s spores are long lasting. For TRV, both the virus and its vectors are extremelly polyphagous and will survive in many crops and weeds. Erradication is extremely difficult, TRV- carrying nematodes will reappear one year after soil fumigation and only recently weedfree lucerne growing has been described as a means of control.

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PERSISTENT SYSTEMIC INFECTION OF POTATO CULTIVARS BY TOBACCO RATTLE VIRUS D J Robinson & M F B Dale Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK [email protected]

Tobacco rattle virus (TRV) has a bipartite RNA genome. RNA-2 includes the coat protein gene and is rather variable in sequence, giving rise to a large number of serotypes and strains. RNA-1 is less variable, and contains the genes for RNA replication and movement within the infected plant. RNA-1 can infect plants systemically on its own; isolates of this kind do not produce nucleoprotein particles and are known as NM-type. Particle-producing isolates contain both RNA-1 and RNA-2, and are called M-type. TRV is transmitted by trichodorid nematodes, and occurs mainly on lighter soils. Each virus strain is transmitted by only one or a few species of nematode (Ploeg et al., 1992). TRV is one of the causes of spraing disease of potatoes, but only certain potato cultivars develop spraing symptoms when exposed to viruliferous nematodes; others remain symptomless. However, even spraing-susceptible cultivars do not develop spraing symptoms after manual inoculation of virus to their leaves, and testing for susceptibility to spraing involves the exposure of growing plants to viruliferous nematodes. This is routinely done in the glasshouse, using pots of field soil that have been shown to contain viruliferous nematodes by prior bait tests with tobacco seedlings (Dale & Solomon, 1988). This procedure has proved more reliable than directly planting in the field, because of the patchy distribution of viruliferous nematodes within most fields. Work at SCRI (Xenophontos et al., 1998) showed that among the potato cultivars that do not develop spraing symptoms in these pot tests, some are truly resistant to infection by TRV, but others can become infected with TRV without developing spraing symptoms. This type of infection differs in several ways from that in spraingsensitive cultivars. Thus, infections associated with spraing symptoms are not fully systemic and are transmitted to only a proportion of daughter tubers, and virus recovered from such infections is usually of the NM type. In contrast, infections in cultivars that do not show spraing symptoms are transmitted to all daughter tubers and can persist through many vegetative generations, and M-type virus can be recovered from all parts of the plants. Indeed, such cultivars are truly susceptible to infection by TRV, whereas spraing symptoms seem to be a manifestation of a form of resistance to the virus. Furthermore, infected plants of TRV susceptible cultivars, such as Wilja, can serve as sources for virus acquisition by vector nematodes (Xenophontos et al., 1998). In field experiments, plants grown from a chronically-infected stock of cv. Wilja emerged and established more slowly than uninfected plants. Foliar symptoms comprising reddish or yellow discolorations on the edges or tips of leaflets appeared on only a few leaves on each infected plant (Xenophontos et al., 1998). Although these symptoms were indicative of infection, they were not sufficiently distinctive to be diagnostic. The progeny tubers on infected plants were significantly decreased in size, but greatly increased in number. Overall, there was a loss in yield of up to 50%. Moreover, most of the tubers from infected plants were misshapen, with a remarkable amount of secondary growth and growth cracking. Tuber quality traits were also affected, with notably a 3% decrease in dry matter content and a worsening of after-cooking blackening (Dale et al., 2000). Glasshouse tests on additional cultivars have extended the list of those that can become systemically infected with M-type TRV when exposed to viruliferous nematodes to include King Edward, Marfona, Nadine, Rocket, Romano, Santé, Saxon, Shepody, Wilja, and probably Home Guard. These cultivars are all listed as having moderate resistance to spraing disease, and between them occupy 20 – 25% of the UK potato acreage. Infected stocks of these cultivars have been produced and are currently being assessed in the field for effects of infection on yield and quality . Systemically-infected but spraing-free tubers are likely to pass unrecognized in seed potato stocks, especially because they occur in cultivars that have a moderately high rating for resistance to spraing by conventional criteria. However, if such seed tubers are planted at a site at which the appropriate vector nematode species is present, the nematodes could acquire the virus and transmit it to other plants in the same or subsequent crops. This provides a previously unrecognized mechanism for the dissemination of TRV to new sites. Only one isolate of TRV was used in the experiments reported above, and there is some evidence for variation among isolates in their effects on different potato cultivars. For example, isolates that cause spraing disease in cultivars normally considered resistant have been reported. Thus, international trade in seed tubers may lead to the dissemination of TRV isolates over long distances and their introduction into places far from their origin, with unwelcome results.

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References: Dale MFB & Solomon RM. (1988). A glasshouse test to assess the sensitivity of potato cultivars to tobacco rattle virus. Annals of Applied Biology 112, 225-229. Dale MFB, Robinson DJ, Griffiths DW, Todd D & Bain H. (2000). Effects of tuber-borne M-type strain of tobacco rattle virus on yield and quality attributes of potato tubers of the cultivar Wilja. European Journal of Plant Pathology 106, 275-282. Ploeg AT, Brown DJF & Robinson DJ. (1992). The association between species of Trichodorus and Paratrichodorus vector nematodes and serotypes of tobacco rattle tobravirus. Annals of Applied Biology 121, 619630. Xenophontos S, Robinson DJ, Dale MFB & Brown DJF. (1998). Evidence for persistent, symptomless infection of some potato cultivars with tobacco rattle virus. Potato Research 41, 255-265.

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Detection and Characterization of Tobacco Rattle Virus (TRV) Isolates Using RT-PCR and Restriction Mapping H. XU and J. NIE Canadian Food Inspection Agency, Centre for Animal and Plant Health, 93 Mount Edward Road, Charlottetown, PEI, Canada, C1A 5T1, E-mail: [email protected] Tobacco rattle virus (TRV) is the type member of Tobravirus. TRV consists of two types of interdependent particles, a long (180 to 200×22 nm) and a short (50 to 110×22 nm) particle. The TRV genome contains two RNAs, RNA1 (in the long particles) and RNA2 (in the short particles). Distinct strains have been confirmed: strain M (containing both RNA1 and RNA2), strain NM (containing only RNA1), California strain (isolated from Capsicum frutescens in California), Oregon strain (isolated from potato in Oregon), PRN strain (isolated from potato in Scotland), and strain PSG (isolated from potato in the Netherlands)(Harrison, 1970, Robinson & Harrison, 1989). TRV has a wide host range that includes many species of weeds and economically important crops (Robinson & Harrison, 1989). Potato stem mottle, necrotic rings and tuber internal necrosis (spraing) caused by TRV are of great concern to potato production (Jeffries, 1999). TRV is weakly immunogenic, which usually results in antisera of low tire. Serological detection is unreliable because of considerable diversity in amino acids of CP of different isolates (Jeffries, 1999). Isolates of the NM strain can not be detected by serological methods because of the absence of coat proteins. Nucleic acid hybridization (Robinson, 1989) and RTPCR (Robinson, 1992, Weidemann, 1995) have been developed to detect TRV. In this study, an Oregon strain including a severe isolate (OS)(ATCC PV-64) and a yellow isolate (OY)(ATCC PV-73), a PRN strain (ATCC PV526) and several unidentified TRV isolates from imported potato samples (cv. Florida Red) from the USA and the virus collection of the Centre for Animal and Plant Health (CAPH) were used in this study. These isolates were propagated in potato (Yukon Gold) and tobacco (Samsun) plants. Several other potato viruses including potato Y Potyvirus (PVY), potato S Carlavirus (PVS), potato leafroll Polerovirus (PLRV), potato mop-top Furovirus (PMTV), and potato spindle tuber viroid (PSTVd) from the CAPH virus collection were also included in this study. Total RNAs were extracted from leaves or tubers using TRI-REAGENTT M (Molecular Research Center, Inc.) following the recommendations of the supplier. Total RNAs extracted from 100 µl of tissue sap were suspended in 100 µl DEPC-water and used as undiluted stock solution of RNA extracts. Five- fold serial dilutions were then made from these solutions to evaluate the sensitivity of RT-PCR. Oligonucleotide primers specific to the conserved sequences of TRV RNA1 that correspond to ORF4 encoding a 16 KDa protein of unknown function (Hamilton et al., 1987, Robinson, 1992) were specific for TRV in PCR-based tests and detects a wide range of variants. In this study, primers ptrF2 and R2 were modified from primers A and B (Robinson, 1992) by removing a base from the 3' end of each primer. Primers ptrF3 (homologous to 6213-6232: 5'ATGGTTGGTGTCACACGTAG-3') and R3 (complementary to 6556-6577: 5'AACAGTCTATCACAGAAACAG-3') were newly designed based on sequence analysis of TRV isolates. Standard two-step RT-PCR was evaluated using various primer sets and TRV isolates. The first strand cDNAs were synthesized by MMLV-RT (Life Technologies) using 0.5 µM antisense primers and 1 µl of the RNA extract in a 20 µl reaction mixture. One µl of the first strand cDNA solution was mixed with 24 µl PCR reaction mixture containing 0.25% BLOTTO (De Boer et al., 1995), 1.5 mM MgCl2 , 50 µM of each dNTPs, 0.5 µM of each primer and 2.5 units of Taq Gold DNA polymerase (Biocan). PCR amplification was performed in a Thermolyne Amplitron II thermocycler for 35 cycles. PCR products were analyzed by 41

electrophoresis in 1.5% agarose gel and the ethdium bromide stained gel was visualized under UV light. Annealing temperature was evaluated in a temperature gradient thermocycler (Biometra) and the optimum temperature for all primers used in this study was 58°C. Primer pair A/B failed to amplify the RNAs of TRV-PRN and several unidentified isolates. However, the modified primer set ptrR2/F2 and the new primer set, ptrF3/R3, yielded amplified products in PCR from all isolates tested. PCR with these primers was highly sensitive and reliable even when the stock solutions of RNA extracts were diluted in DEPC water to 1/15,625. The primers were specific for the virus, as only TRV RNAs were amplified when RNAs of healthy tissues and several other potato viruses and PSTVd were also present. To evaluate the feasibility of screening potato tubers in composite samples by RT-PCR on a large scale as required in the seed certification program, the tissue sap from TRV infected and non- infected dormant tubers were mixed in a ratio of 1:24, 1:49, 1:99, and 1:499 (infected: healthy) prior to total RNA extraction. PCR products were obtained from the RNA extracts of all composite samples including the mixture at the ratio of 1:499 after amplification for 30 - 35 cycles. Amplification for 28 or 30 cycles yielded PCR products from the RNA extracts of composite samples in 1:24, 1:49 and 1:99 mixture, but not from the composite sample mixture at 1:499. PCR products from various isolates were purified with PEG 8000 followed by precipitation with 0.25 M KAc and 95% ethanol. The nucleotide sequence of the PCR products were then determined by automated sequencing using the dye terminator cycle sequencing method. The sequence analysis revealed some variation among TRV strains or isolates as compared to known ORF4 sequences of TRV RNA1. The sequences determined by Kawchuk et al. (1997) and by Pérez et al. (2000) shared only 91.6% (424/463) and 90.7% (420/463) identical bases, respectively, with that of TRV-OS. TRV-OS and several other strains/isolates shared 99.1% identical bases in the compared region. Aliquots of PCR products (50

20 6-19 0 >20 6-19 0

Name of potato cultivar Alicja, Barycz, Baszta, Kuba * Beata, Bekas, Dunajec, Omulew Klepa Anielka, Bzura, Hinga Danusia, Fregata, Jasia, Maryna, Meduza, Nimfy, Sante, Vistula

* all tubers with internal necroses REFERENCES Chrzanowska M. 2000. (Abstr.) 12th Congr. FESPP. Plant Physiol. Biochem, vol.38, 225 Delhey R. 1974. Phytopath. Z. 80: 97-119. Muchalski T. 1998. Metody wyró¿niania genotypów ziemniaka odpornych na wirus nekrotycznej kêdzierzawki tytoniu (tobacco rattle virus, TRV). Doctoral dissertation. IHAR. pp. 83. Project supported by the State Committee for Scientific Research 5PO6A-030-17p02

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REACTION OF POTATO CULTIVARS TO CUCUMBER MOSAIC VIRUS INFECTION. Miros³awa Chrzanowska, Bo¿enna Zieliñska, Maria Kamiñska* Plant Breeding and Acclimatization Institute, 05-831 M³ochów Center, PL *Research Institute of Pomology and Floriculture, ul. Pomologiczna 18, 96-100 Skierniewice, PL Several crops including cucumber, squash, tobacco, lettuce, tomato, leguminous and ornamental species are infect with Cucumber mosaic virus (CMV) under field conditions (Tobias et al. 1982, Kamiñska 1995, 1996). In France, CMV was found in about 40% of samples collected from tobacco plants (Blancard et al. 1994).The virus was also identified on tobacco grown in Poland (Doroszewska, personal comm.). In Europe, CMV was occasionally reported on potato grown in England, Germany and Estonia (MacArthur 1958, Bode 1975, Agur 1975). CMV is not significant disease factor on potatoes. In the last years the reaction of potatoes to CMV was characterized by Valkonen et al. (1995), Celebi et al. (1998), Valkonen and Rokka (1998), Valkonen and Watanabe (1999). For studies the reaction of Polish potato cultivars three virus isolates characterized previously by Korbin and Kamiñska (1998) were used for the inoculation. Materials and methods Experiments were done in IHAR Research Center at M³ochów in 1999-2000. Virus isolates were collected from cucumber (CMV-J and CMV-M) and Asiatic lily (CMV-26P). These three isolates infect potato cultivar Desiree causing systemic symptoms. Healthy tubers of 22 Polish and 5 foreign potato cultivars were obtained from breeding stations as a basic seed. From each cultivar 18 plants were grown in the greenhouse. Inoculum was prepared from tobacco plants (cv. Samsun) infected with CMV. Potato plants were inoculated mechanically. Observations of local and systemic symptoms were lead up to 5-6 weeks after inoculation, ELISA using antibodies from Bioreba Co. was done for potato plants inoculated with CMV-J. Tubers of inoculated plants and also tubers from the second vegetative progeny were collected and stored for six months. They were presprouted and planted into pots in greenhouse. Upper leaves of inoculated plants and samples of leaves from secondary infected plants were used for the dry inoculation of Chenopodium quinoa indicator plants. Results and discus sion From three CMV isolates used for inoculation of potato plants, isolates CMV-J (from cucumber ) and CMV-P26 (from lily) appeared the most infectious for potato (102 and 93 infected plants on 135 inoculated once, respectively). CMV-M, other isolate from cucumber, was very mild and less infectious for potatoes (25/135). All inoculated cultivars reacted with local chlorotic symptoms excepted cv. Gloria (Table 1). The isolate CMV-J was detected in 10% of inoculated plants by ELISA and in 22% by bioassay on C. quinoa. According to Valkonen & Watanabe (1999) potatoes are resistant to CMV. In their experiments potatoes remain non- infected following inoculation or develop detectable amounts of CMV only in inoculated leaves. The resistance of potato to CMV is function of slow virus cell-to-cell movement (Valkonen & Rocca, 1998). Results of presented experiments confirm this conclusion. In our potato cultivars necrotic reaction to CMV was not observed. From 27 tested cultivars: 26 showed local chlorotic symptoms, 12 showed primarily systemic symptoms (necroses or mosaic, malformation of leaves). In 5 cultivars the virus was present in second and third vegetative progeny.

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Table 1. Reaction of 27 potato cultivars after inoculation with CMV Potato cultivars

Bzura, Desiree, Drop, Irys, Karlena, Lawina, Orlik, Vistula, Vital

Number of plants showed local lesions* 10 - 15

Potato cultivars which were infected with CMV systemically** Bzura, Desiree, Irys, Karlena, Lawina, Orlik, Sante Ania, Kos

Ania, Baszta, Kos, Lena, Meduza, 6 - 10 Muza, Nimfy, Omulew, Sante Aster, Bila, Glada***, Gloria, Hinga, 0-5 Aster, Bila, Tokaj Ibis, Irga, Tokaj, Triada * - on 15 plants inoculated ** - the virus was detected on C. quinoa *** - no symptoms bold - potato cvs infected with CMV in second and third vegetative progeny

References Celebi F., P. Russo. K. Watanabe, J.P.T. Valkonen, S.A. Slack (1998): Resistance of potato to cucumber mosaic virus appears related to localization in inoculatrd leaves. Amer. J. Potato Res. 75, 195-199 Kamiñska M. (1996): Virus infection of lilies in Poland. Phytopath. Polonica 11, 51-58 Korbin M., M. Kamiñska (1998): Characterization of cucumber mosaic cucumovirus isolates. Phytopath. Polonica 16, 71-84 MacArthur A.W. (1958): A note on the occurrence of cucumber mosaic virus in potato. Scott. Plant Breeding Stn. Rep. 75-76 Tobias I., D.Z. Maat, H. Huttinga (1982): Two Hungarian isolates of cucumber mosaic virus from sweet pepper (Capsicum annuum) and melon (Cucumis melo): identification and antiserum preparation. Neth.J.Pl.Path. 88, 171-183 Valkonen J.P.T., S.A. Slack, K.N. Watanabe (1995): Resistance to cucumber mosaic virus in potato. Ann. appl. Biol. 126, 143-151 Valkonen J.P.T., K.N. Watanabe (1999): Autonomous cell death, temperature sensitivity and the genetic control associated with resistance to cucumber mosaic virus (CMV) in diploid potatoes (Solanum spp.) Theor appl Genet 99, 996-1005

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EFFECT OF VIRAL INFECTION ON THE ACTIVITY OF ATP ase IN POTATO LEAVES. Manadilova A.M., Kunaeva R.M., Tuleeva G.T. M.A. Aytkhozhin Institute of Molecular Biology and Biochemistry, 480012 Dosmuchamedova 86, Almaty , Kazakstan. [email protected]

Potatoes are the second most important crop in Kazakstan. Studies of viruses in potato fields in Kazakstan by ELISA, showed a high infection by potato virus X ( PVX) and virus Y ( PVY ).

It is known, that infection of plants by fungus, bacteria is followed by the cha nge of the activity of such hydrolytic enzyme as ATPase. This enzyme plays an important role in many processes of vegetative organism. ATPase has a large effect in the increase of plant’s resistance to various diseases. This work has been aimed at studying of the post-infection changes in the activity of Na, K - and Ca, Mg - ATPase and their role at virus infection. The effect of X and Y viruses of potato on activity ATPase has been studied, using various sorts of potato ( Sante – resistant to PVX; Lady Rozetta - resistant to PVY ; Tamasha - sensitive to X and Y).

It was shown, that the character of post- infection changes in the activity of enzyme depends both on the sort of a potato and on subcellular enzyme localization. So, in the potato leaves resistant to Y, the activity of cytoplasm Na, K- , Ca, Mg - ATPase exceeds the control, reaching the max. in 7 days after the infection. As for the leaves, resistant to PVX, no difference was observed. In the leaves, sensitive to X virus , the increase in Ca, Mg- activity was observed in the following day after the infection. As for the leaves, sensitive to Y no difference was shown. The activation of Ca, Mg- ATPase in cell walls was observed in an hour after the infection. Taking Ca, Mg – ATPase the activity exceeds the control in 3-4 times.

During isoelectric focusing in the gradient pH 4 – 10 one acidic isoenzyme ( pI 3,5) and two alkaline ones ( pI 8,5 ; 9,0 ) were obtained, but there were no differences in isoforms between healthy and infected plants. The increase in activity plays an important role in getting resistant to virus stress . When ion of Ca and Mg are added the enzymes reach the highest degree of activity. It is supposed, that the flow of divalent cations into cells, takes part in the induction of protective reaction in plants.

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THE WORK OF THE UK POTATO QUARANTINE UNIT NISBET C, JEFFRIES C Scottish Agricultural Science Agency, East Craigs, Edinburgh, EH12 8NJ, UK [email protected]

[email protected]

All potato material entering the EU for planting is prohibited (Plant Health Directive 2000/29/EC, formerly 77/93/EEC). However, such material may enter under a derogation specified in Commission Directive 97/46/EC (“The Scientific Work Directive”). It establishes the conditions under which harmful organisms and certain plants can be introduced and moved within the community, for trial or scientific purposes and for work on varietal selections. Potato material must undergo official post-entry quarantine testing. In the UK this is done at the UK Potato Quarantine Unit (UKPQU), Scottish Agricultural Science Agency (SASA). Established in 1981, it was the first potato quarantine unit world-wide to use the technique of micropropagation as a means of containment within quarantine (Miller-Jones & Howell 1986). The work of the UKPQU is overseen by an Interdepartmental Committee comprising representatives of the UK Government’s Agricultural Departments (Department for Environment, Food and Rural Affairs, Department of Agriculture and Rural Development - Northern Ireland, and the Scottish Executive Environment and Rural Affairs Department). Also represented are private and public plant breeders and commercial and research interests. The testing that material receives exceeds the requirements specified in Directive 97/46/EC. Material is tested for PSTVd by RNA probe or return-PAGE. ELISA and bioassay are used to test for the viruses APLV, APMV, AVBO, PBRV, PLRV, PMTV, PVA, PVM, PVS, PVT, PVV, PVX, PVY, PYV, TSWV, potato latent virus and potato rough dwarf virus. The indicator plants used are Chenopodium amaranticolor, C. murale, C. quinoa, Nicotiana benthamiana, N. bigelovii, N. clevelandii, N. debneyi and N. tabacum “White Burley”. Immunofluorescence microscopy and bioassay to eggplant are used to test for Clavibacter michiganensis subsp. sepedonicus. A nutrient and selective medium respectively are used to test for Erwinia chrysanthemi and Ralstonia solanacearum. All tests other than bioassay are conducted at least twice. The material is also grown in the glasshouse for one complete vegetative cycle to observe symptoms of any harmful organism.

In vitro material, which has cleared quarantine, is issued with an EEC Plant Passport. It may then be moved and planted in other Member States without further quarantine testing. The plant passport issued by the UKPQU also acts as a Potato Germplasm Health Statement, with details of the testing done as recommended by FAO/IPGRI (Jeffries 1998). About 50 lines are tested annually in quarantine. The UKPQU also offers a range of other services to its customers. Normally only 4 in vitro plants of a line are released from quarantine. However, if required plants can be multiplied to produce thousands of plants of each line for release. Approximately 20,000 plants are produced annually for this service. In addition, the UKPQU offers virus elimination for those lines failing quarantine because of virus infection (3-5 lines annually).

The success of potato quarantine depends on knowledge of the organisms that affect potato and the availability of good diagnostic methods (Jeffries et al. 1993). SASA continues to make contributions to these areas (Brattey et al. 1998, Harris et al. 1986, Jeffries 1991, 1996, 1998). Moreover, it continuously revises and develops its procedures, often in collaboration with colleagues in the EC and internationally (Harju et al. 2001), to ensure that it stays at the forefront of testing technology.

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References Brattey C, George E, Burns R, Goodfellow HA, Jeffries CJ, McDonald JG, Badge JL, Foster GD (1998) A newly described carlavirus infecting potato. Abstract 1.11.33. Offered Papers. Abstracts Volume 2. International Congress of Plant Pathology 1998, Edinburgh, Scotland. Harju VA, Henry CM, Cambra M, Janse J, Jeffries C (2001) Diagnostic protocols for organisms harmful to plants – DIAGPRO. OEPP/EPPO Bulletin 30, 365-366. Harris PS, James CM, Kelly P (1986) Use of a cDNA probe for sensitive detection of potato spindle tuber viroid in potato quarantine. Pp 113-125 in Viroids of Plants and their Detection. Inernational Seminar, August 1220, 1986, Warsaw Agricultural University. Jeffries CJ (1991) Saving a potato gene bank. Pp15-16 in Agricultural Scientific Services Annual Report 1990-1991. Jeffries CJ (1996) Potato Quarantine Testing in the European Community. Pp 55-61 in Proceedings of the 9th EAPR Virology Section Meeting, Bled, June 18-22, 1995. Jeffries C (1998) FAO/IPGRI Technical Guidelines for the Safe Movement of Germplasm. No.19. Potato (ISBN 929043-390-6). Jeffries CJ, Chard JM, Brattey C (1993) Coping with plant health risks posed by gene bank collections of potato. Pp 145-156 in Plant Health and the European Single Market. British Crop Protection Council Monograph, No 54. Miller-Jones DN; Howell PJ (1986) Micropropagation as an aid to quarantine procedures for potato material. Pp 257-260 in Plant Tissue Culture and its Agricultural Applications (eds LA Withers and PG Alderson). Butterworths, London.

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DEVELOPMENT OF A QUALITY ASSURANCE SYSTEM FOR PLANT VIRUS TESTING REAGENTS BURNS R, GEORGE E, JEFFRIES C Scottish Agricultural Science Agency, East Craigs, Edinburgh, EH12 8NJ, UK [email protected]

[email protected]

[email protected]

The Scottish Agricultural Science Agency (SASA) carries out tests on a range of plant samples for the presence of plant pathogens. The majority of these tests are carried out in furtherance of the Agency’s responsibilities for the health of Scottish seed potatoes and for quarantine testing of imported potato material. In support of this work the SASA Antibody Unit was established in 1986 to produce reagents for the detection of plant pathogens by ELISA. Since then it has developed test systems for many indigenous and non-indigenous viruses PVA, PVM, PVS, PVV, PVX, PVY, PVYc, PVYo/c, PVYn , PLRV, PMTV, TBRV, AVB-O, potato latent virus, PVT, TRSV-Ca/PBRSV. Most of the reagents provided by the Unit are produced from monoclonal antibodies. With the rise in awareness of Quality Assurance in all industries, a programme of QA development was undertaken to incorporate and develop existing methods into a Quality System compliant to BS EN ISO 9002. The scope of the System is the “Manufacture and Supply of Antibody-Based Diagnostic Systems for Plant Virus Detection”. The main areas that are covered by the Quality System are: Control of raw materials All materials used for the manufacture of reagents are controlled to ensure their quality prior to their use in reagent production. Where possible raw materials are obtained from Quality Assured sources. Process control of reagent manufacture Monoclonal antibodies are derived from Quality Assured cell banks held by the Unit and all stages of antibody production and reagent manufacture are covered by the System. All critical equipment used for production is monitored and maintained according to detailed procedures. The use of polyclonal antibodies is also defined and controlled within the System. Quality control of intermediate and final products Quality control checks are made at various stages of manufacture and records of all tests performed are maintained within the System. All appropriate checks must have been made prior to reagents being accepted for issue to clients. Stock control The stock of manufactured reagents is controlled to ensure that the quality of products is maintained during storage and that adequate supplies are available at all times. Reagents are stored under environmentally controlled conditions to ensure optimum shelf life. Receipt of client orders and dispatch of reagents Client requests for products enter the System on receipt and are processed according to defined procedures. The dispatch of products is also controlled to ensure rapid response to client requests. The initial assessment visit for ISO 9002 accreditation was carried out by the British Standards Institute on 17 July 2001 and the auditor has recommended that registration should be granted.

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POSSIBILITY OF USE OF RECOMBINANT COAT PROTEIN OF POTATO MOP-TOP VIRUS (PMTV) FOR VIRUS CHARACTERIZATION 1

T. MORAVEC, 1 M.FILIGAROVÁ, 2 P.DÌDIÈ, AND 1 N.ÈEØOVSKÁ

1

INSTITUTE OF EXPERIMENTAL BOTANY CAS, PRAGUE, RESEARCH, HAVLÍÈKÙV BROD, CZECH REPUBLIC

2

INSTITUTE FOR POTATO

Introduction: Potato mop-top virus (PMTV) was first described in Scotland and Northern Ireland (Calvert and Harrison,1966) and is now known to occur in various part of Europe, South America, Canada, China and Japan. In 1983 rare occurrence of PMTV was recorded also in Czech territory (Novak et al., 1983). PMTV causes a wide range of symptoms in haulms and tubers which vary depending on the potato cultivar and environmental conditions (Kurppa 1989, Harrison and Jones 1971). This variation in symptom expression causes difficulties in the identification of the virus disease. The virus is in field conditions transmitted by the motile zoospores of the plasmodiophoromycete fungus Spongospora subterranea (Wallr.) Lagerh. (Jones and Harrison 1969, Arif et al., 1995), which causes powdery scab on tubers. Effective and environmentally acceptable chemical control of the fungal vector is not commercially available, and there are no sources of resistance or tolerance to PMTV that have been deliberately used in breeding programs (Barker et al., 1998). After reclassification of furoviruses PMTV has been placed in the proposed new genus Pomovirus (Torrance and Mayo 1997). The particles of PMTV are tubular and rigid, 18 - 22 nm in diameter and 100-150 nm or 250-300 nm in length. Discrepancies in reported length are probably due to the fragility of the particles, which readily disintegrate and uncoil from one end (Kassanis et al., 1972). Capsid that forms the shell of the virus consists of the single type of protein subunit encoded by a distinct virus gene. The entire genome of PMTV consists of three different positive strand RNA molecules, RNA 1, 2 and 3 (Kallender et al., 1990), of approximately 6.5, 3.2 and 2.4 kb, respectively. The complete nucleotide sequence (6043 nt ) of RNA 1 was determined only recently (Savenkov et al.,1999). There are many difficulties involved in the diagnosis of PMTV because this virus infects tubers and haulms erratically and often occurs in concentrations under the detection level. Both Mills (1987) and Kurppa (1990) reported difficulties in using ELISA for diagnosing PMTV and attributed them to the high background reaction of polyclonal antibodies and the uneven distribution of the virus in potato plants and tubers. Polyclonal antisera and a panel of MAbs have been produced against the Scottish isolate PMTV-T by Torrance et al., (1993). The data obtained by sequencing of the coat protein gene (Reavy et al., 1997, 1998), proved that a number of PMTV isolates seems to be highly conserved in this region. Nevertheless a considerable differences in virulence and significant variation among isolates in biological properties were already observed by Harrison and Jones (1970). For further characterization of this virus we produced the recombinant coat protein in E.coli which will be used for raising antisera for the detection and subcellular localization of viral proteins in plants. Methods: We used two isolates of PMTV from the Czech Republic – Pacov and Korneta which from symptomatic potato tubers were transmitted to N. benthamiana and subsequently propagated in N.debneyi. cDNA of PMTV RNA 3 was obtained by immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) carried out on infected leaves sap using commercially available PMTV antibodies (Adgen,UK), forward and reverse primers MT-CP5A and MT-CP3A corresponding, in sequence of RNA3 (AJ243719), to nt 313-333 and to nt 822-841, respectively. The reaction was carried in 30 cycles of 30 s denaturation at 94 °C, 30 s annealing at 55 °C and 1 min elongation at 72 °C. 80

The RT-PCR products were directly cloned to pUC57T/A (Fermentas) using 3'-A overhangs generated by Taq polymerase and were sequenced using an ALFexpressII Sequencer with the AutoRead Sequencing Kit (AP Life Science). Plasmid pUC57 was used for subcloning of insert into several different bacterial expression systems. As an alternative the Gateway cloning technology (Life Technologies) was used. Results and discussion: Cloning of PMTVCP gene into expression vectors: The PCR product containing CP coding region was cloned into plazmid pUC 57. Fidelity of the clones was confirmed by sequencing. Both isolates used (Korneta and Pacov) possessed coat protein of the same sequence, which was then submitted to GenBank (AF393507). Comparing this very conservative region with other known sequences we found almost 100% homology with the isolate Sw (Sandgren, 2001). This could be considered as an interesting result, comparing with other plant RNA viruses (e.g. potyviruses), commonly performing less than 98% homology by sequencing even the identical starting material. Clone containing coat protein of isolate Korneta was used for further experiments. NcoI (NEB) and XhoI (TaKaRa) fragment carrying the CP gene was ligated into expression plazmids and transformed into E.coli. After optimization of the cultivation (time, temperature, inductor concentration) the cells were harvested and the presence of desired protein was proved by immunoblot using commercial monoclonal antibodies against PMTV (Adgen).(Fig.1). Gateway cloning technology: This cloning technology eliminates traditional subcloning. This is simple two-step cloning process based on site- specific recombination. After cloning the gene of interest into the Entry Vector, it is possible to transfer the gene simultaneously into many destination vectors for expression in bacterial, yeast, insect and mammalian cells. At first the RT PCR reaction was done with our PMTVCP gene and specially designed primers containing the extra part of sequences compatible to two distinct att recombination sites in entry vector. After that our gene in entry vector was simultaneously transferred into two destination vectors, in the first our protein was expressed as a fusion protein with GST and in the second in fusion with 6xHis tag in E.coli BL21. The presence of PMTVCP protein was proved after expression in the same way as in previous experiment. Conclusions: We determined coat protein coding sequences of two field isolates of PMTV from the Czech Republic. From the comparison with other known sequences we can conclude, that it is a very conservative part of the PMTV genome. By both cloning methods (traditional cloning and Gateway) of CP gene of PMTV into expression vectors we obtained expressed PMTVCP protein, which was proved by immunological methods. From comparison of both types of cloning, we can say that by both methods we reached the goal. The new method is much simpler and not so time consuming, but rather expensive. After isolation PMTVCP will be used for preparation of polyclonal and monoclonal antibodies, which will be used not only for research of antigenic properties of this virus, but we hope also for purposes of routine diagnosis.

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Figure 1: Expression of fusion proteins containing PMTV-CP in E.coli (GATEWAY) BL21 (DE3) cells containing relevant plasmid were grown in LB media in 37°C. After reaching OD 1, the culture was split and to one part IPTG to a final concentration 0,5 mM was added. After 3 hours incubation the cells were harvested, lysed in 2x Laemmli buffer and subjected to SDS-PAGE. A) Nitrocellulose membrane stained with PonceauS after blotting of SDS gel containing lysates of E.coli cells. Line M contains molecular size standard, line 1=PMTV purificate, line 2 = fusion protein PMTV/GST – after O/N induction , line 3 = the same- after 3 h induction; line 4 = fusion protein PMTV/6xHis –O/N- induction, 5 = the same after 3 h induction, 6 = the same expression system without induction; NK = BL21 cells + pET22 as a control. B) Immunoblot analysis of total SDS-soluble proteins. Presence of desired protein was proved with commercial monoclonal antibodies against PMTV (Adgen). References Arif, M., Torrance, L., and Reavy, B. (1994): Potato Research 37, 373-381. Arif,M. - Torrance,L. - Reavy,B.: Ann Appl Biol, 126, 493-503, 1995 Barker,H.- Reavy,B. - McGeachy,K.D. - Dawson,S.: Mol Plant Microbe Interact, 11, 626-633, 1998 Calvert,E.L.- Harrison,B.D.: Plant Pathol, 15, 134-139, 1966 Harrison,B.D. - Jones,R.A.C.: Ann Appl Biol, 65, 393-402, 1970 Harrison,B.D. - Jones,R.A.C.: Ann Appl Biol, 68, 281-289, 1971 Jones,R.A.C. - Harrison,B.D.: Ann Appl Biol, 63, 1-17, 1969 Kallender,H. - Buck,K.W. - Brunt,A.A.: Neth J Plant Pathology, 96, 47-50, 1990 Kashivazaki,S. - Scott,K.P. - Reavy,B. - Harrison,B.D.: Virology 206, 701-706, 1995 Kassanis,B. - Woods,R.D. - White,R.F.: J Gen Virol, 14, 123-132, 1972 Kurppa,A.: EPPO Bull.,19, 593-598, 1989 Novak,J.B. - Rasocha,V. - Lanzova,J.: Ochr rostl, 19, 161-167, 1983 Reavy,B. - Sandgren,M. - Barker,H. - Heino,P. - Oxelfelt,P.: Eur J Plant Pathology 103 : 829-834, 1997 Reavy, B. - Arif, M. - Cowan, G. H. - Torrance, L.:(1998): J Gen Virol, 79, 2343-2347, 1998. Sandgren, M., Savenkov, E. I., and Valkonen, J. P. (2001):. Arch Virol 146, 467-77. Savenkov,E.I. - Sandgren,M. - Valkonen,J.P.T.: J Gen Virol 80, 2779-2784, 1999 Scott,K.P., Kashivazaki,S. - Reavy,B. - Harrison,B.D.: J Gen Virol,75, 3561-3568, 1994 Torrance,L., Cowan,G.H. - Pereira,L.G.: Ann Appl Biol, 122, 311-322, 1993 Torrance,L. - Mayo, M.A.: Arch. Virol., 142, 435-439, 1997

This work is supported by a grant from the Grant Agency of the Czech Republic No. 522/01/1121

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VARIATION IN DANISH ISOLATES OF POTATO MOP-TOP VIRUS Steen Lykke Nielsen & Mogens Nicolaisen The Danish Institute of Agricultural Sciences, Department of Plant Protection, Research Centre Flakkebjerg, DK 4200 Slagelse. Phone +45 58 11 33 00, e-mail: [email protected]

Abstract Twenty isolates of PMTV were baited from Danish fields and symptom development in Nicotiana debneyi, N. benthamiana and Chenopodium amaranticolor after mechanical leaf inoculation were compared. Sixteen of the isolates could be grouped according to symptom development and differences in severity of symptom expression Two isolates developed very strong symptoms, 4 strong, 5 medium and 5 weak symptoms. Sequence analysis and/or restriction fragments length polymorphism (RFLP) analysis of PCR fragments derived from parts of the coat protein and read through region was carried out. The isolates could be grouped in two major groups according to this analysis. One group was similar to the PMTV-T and the other to PMTV-Sw according to Sandgren et al. (2001). There was no correlation between sequence type and symptom development in indicator plants. Introduction Spraing provoked by infection of potato mop-top virus is a serious and increasing problem in Danish potato production. Establishing of tests of resistance of potato cultivars to spraing and virus infection necessitates knowledge of the variation of PMTV. Only very limited information is available of possible biological and genetical variations in the Danish population of PMTV however, for which reason the present investigation was realised. Materials and methods Twenty isolates of PMTV were isolated from soil samples taken from PMTV infected fields using Nicotiana benthamiana as bait plant. Presence of PMTV in roots was confirmed by DAS-ELISA. The isolates were transferred by mechanical inoculation to leaves of new N. benthamiana where the isolates were maintained and propagated. Symptom development was followed in N. debneyi, N. benthamiana and Chenopodium amaranticolor. Two plants per species per plot and three replicates at different periods. Symptom reading first time after 2 weeks and further 23 times ending 4 weeks after inoculation. A ~3000 bp region of the coat and readthrough proteins was analysed by RT-PCR using 3 specific primer pairs using standard methods. PCR products were sequenced or digested with the specific restriction enzymes Alu I, Eco RV, Nhe I. Results Symptom development. Differences in symptom development between isolates appeared as 1) how fast symptoms developed, 2) how severe the same kind of symptoms developed (numbers and extend of spots and distinctness of symptoms), 3) in a progressive development of different kinds of symptoms how many kinds an isolate provoked and 4) consistence in differences between isolates in all replicates and in all plant species. An overview of weak and strong symptoms in the 3 indicator plant species are shown in Table 1.

Table 1. PMTV-symptoms development in three indicator plants in order of appearance. C. amaranticolor N. benthamiana Symptoms Weak/at first Chlorotic local lesions Weak systemic mosaic Strong/later

Chlorotic local lesions with concentric stippled rings

Strong systemic mosaic and chlorotic and necrotic systemic lesions

N. debneyi Chlorotic local ringspots and thistle leaf patterns Ditto and veinal clearing and stipples

On basis of differences seen in symptom development the 20 isolates could be grouped into 4 groups, where symptom development was consistent in all 3 indicator plant species and replicates, while in 4 isolates inconsistent variations were found between plant species and replicates. Two isolates developed very strong symptoms, 4 strong, 5 medium and 5 weak symptoms. Sequence analysis. Of the 20 isolates, 12 belonged to the PMTV-Sw group and 6 belonged to the PMTV-T group according to their sequence or RFLP analysis. Two isolates were not analysed.

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Discussion and conclusions In 20 isolates of PMTV sampled from different Danish fields it was possible to show existence of biological differences in symptom development in 3 indicator plant species primary based on differences in severity and how far a progressive development of symptoms reached. Harrison & Jones (1970) compared 11 isolates of PMTV from Scotland and Northern Ireland and also found differences in symptoms development in 3 indicator plants and one of the isolates developed much severe symptoms than the others. Sandgren (1996) found no difference between 4 Danish isolates in their reaction to 11 anti-PMTV monoclonal antibodies. The sequence analysis revealed 2 groups of sequence variants, PMTV–Sw and -T. It has not been possible to correlate these two groups to any biological or geographical differences. Literature Harrison, B.D. & Jones, R.A.C. 1970. Host range and some properties of potato mop-top virus. Ann. appl. Biol. 65, 393-402. Sandgren, M. On spraing in potato. A soil borne virus disease in potato, significance, detection and variability. Dissertation Uppsala 1996. Swedish University of Agricultural Sciences. Sandgren, M, Savenkov, E.I. & Valkonen, J.P.T. 2001. The readthrough region of Potato mop-top virus (PMTV) coat protein encoding RNA, the second largest RNA of PMTV genome, undergoes structural changes in naturally infected and experimentally inoculated plants. Arch. Virol. 146, 467-477.

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Fragment cDNA-TGGE profiles throughout PVY genome as basis for virus variability analysis by DNA hetero- duplexes. Škopek, J.1,3 , Ptáèek, J.2 , Dìdiè, P. 2 and Matoušek, J.1 1

IPMB AS CR, Branišovská 31, 370 05 Èeské Budìjovice; 2 Institute for Potato Research, Dobrovského 2366, 58001 Havlíèkùv Brod; 3 South Bohemian University, Branišovská 31, 370 05 Èeské Budìjovice; Seven regions within PVY genome were analyzed using temperature gradient-gel electrophoresis (TGGE). Temperature conditions suitable for gel electrophoresis of cDNA heteroduplexes described previously (Matoušek et al., 2000) were suggested for each region. For PCR products derived from PVY NTN (Nicola isolate) at positions 238-490, 1121-1370, 38074071, 5605-5872, 6350-6589, 8031-8290, 8783-9011 (positions are according to sequence AC M95491), 41.1, 40.6, 41.0, 39.1, 38.3, 38.0, 40.6 o C were determined as the melting points, respectively. Some regions we analyzed in detail. These new data completed our previous work (Matoušek et al., 2000), where significant variability was detected in P1 region of PVY. Probably less variable was the region covering HC-Pro ORF. However, also in this region 12 mutations were observed for isolate Nicola and eight of them led to aa changes. Clear discrimination between N-serotypes (including NTN) and O-serotypes of PVY isolates was achieved when RT-PCR was performed using primers derived from the region covering Nib-CP boundary. This region seems to be helpful for virus classification using molecular genetic methods. Specific primers were prepared and assayed using gradient RT-PCR to follow up incidence of selected PVY quasiforms in different potato genotypes and plant tissues. The approaches based on the thermodynamic parameters of cDNA fragments make possible to perform complex analysis of PVY throughout all genome. This complex approach is, in general, necessary for better understanding of specific sequence changes of plant viruses. References: Matoušek, J., Ptáèek, J. , Dìdiè, P., Schubert, J.: Analysis of variability of P1 gene region of N strain of potato virus Y using temperature-gradient gel electrophoresis and DNA heteroduplex analysis. - Acta virologica, 44: 40-46, 2000. Acknowledgements This work was supported by GA ÈR 522/00/0227; NAZV EP9111 and GA ASCR S5051014.

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DNA - CHIP FOR DIAGNOSIS OF POTATO VIRUSES O. Lenzb, D. Bystøická a,b , I. Mráza , K. Petrzika , P. Dìdièc and M. Šípa,b a

Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, b University of South Bohemia, Branišovská 31, 370 05 Èeské Budìjovice and cPotato Research Institute, Dobrovského 2366, 580 01 Havlíèkùv Brod, Czech Republic, e-mail: [email protected] Parallel detection of potato viruses is planned using the new microarray technology. This approach consists in anchoring an array of virus-specific sequences on a glass plate and subsequent hybridization with labeled samples of nucleic acids from the tested plants. The importance of potato as nutrition source makes a reliable screening of its viral pathogens an economical necessity. In our project, we focus on the seven most widely distributed potato viruses: PVY and PVA potyviruses, PVX potexvirus and PVS and PVM carlaviruses, potato leafroll luteovirus and the newly detected potato mop-top potamovirus. To achieve this goal, viral RNA was isolated from infected indicator plants. Primers for amplification of conservative as well as variable regions of the virus genomes were prepared and RT - PCR was performed. 400 600 bp DNA amplicons were cloned to pBSK(+) vector. The corresponding E. coli clones are kept in the LB medium containing 15% of glycerole. The clones will be used for design of a DNA-chip. Selected sequences will be immobilized on a glass plate and used for parallel detection of the above-mentioned viruses.

Acknowledgement: This project is supported by the grant No. 522/01/1105 of the Grant Agency of the Czech Republic.

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THE PRESSURE OF POTENTIAL APHID VECTORS OF POTATO VIRUS Y AT POTATO CROPS IN POLAND KOSTIW M., ROBAK B. Plant Breeding and Acclimatization Institute, 76-009 Bonin, Poland

SUMMARY The results show that the aphids not feeding on potatoes started the spring migration earlier or much earlier than potato aphids. Their frequency was also much higher during the early stage of plant vegetation. These two findings seem to confirm the meaningful role the non-potato aphids play in the transmission of non-persistent viruses, mainly during the early stage of plant vegetation. INTRODUCTION There are many aphid species which appear on potato plants, but do not feed on them. Many of them have also the ability to transmit the non-persistent viruses on the stylet. The transmission is effected during the trial pricks of an insect looking for the proper host plant. According to many authors (Kostiw 1987), PVY can be transmitted by more than 40 aphid species. Some of them can be the vectors of Potato virus M and Potato virus S. The purpose of this study was the comparison of spring migration date, relative share in population and frequency dynamics of several aphid species, not feeding on potatoes, and those aphids for which the potatoes are common host plants in Poland. MATERIAL AND METHODS The study was conducted at 5 locations in various parts of Poland in 1997-2000 (Fig.1). The occurrence of alate aphids was studied from May till August, using the yellow water traps (YWT). Two traps per field were placed on the ground. The traps (23 cm of diameter) were filled with water containing detergent and were emptied 5 times a week. The aphid species were identified. In calculation of the relative share of particular aphid species (altogether 10 species were studied), the total (100%) comprised the mean numbers of 4 years. The calculations were done on transformed numbers, using the logarithmic transformation lg (n+1), n meaning the number of aphids. RESULTS AND DISCUSSION The earliest migration of aphids was observed at Stare Olesno, on average on May 23 (mean of 10 species). As first at that location, Cavariella aegopodii (Scop.) was trapped, on May 8, and the latest was A. nasturtii – on June 4. Somewhat later the spring migration started at Jadwisin (on average on May 26). As the first Rhopalosiphum padi L. and C. aegopodii were trapped – on May 17 and 18, and the latest were Cryptomyzus galeopsidis (Kalt.) and Hyperomyzus lactucae (L.) – on June 5 and 9, respectively. At Bonin the migration started on average on May 31, and as first H. lactucae occurred – on May 18. A. frangulae appeared as late as July 1. At the remaining 2 locations, the beginning of spring migration was still later – at Zamarte on June 4 on average and at Szyldak as late as June 14. On the earliest date began the migration of C. aegopodii – it appeared on average on May 21. Somewhat later appeared R. padi and Cavariella theobaldi (Gill et Bragg) – on May 24 and 25, respectively, and then Brahycaudus helichrysi (Kalt.), Aphis fabae complex, H. lactucae and C. galeopsidis – on May 27, 28, 29, and 30, respectively. As the latest occurred the aphids feeding 74

on potatoes: A. nasturtii, M. persicae and A. frangulae – on average on June 8, 10, and 25, respectively. The predominant species during the first part of the growing season (May-June) were R. padi (16,6%), A. fabae complex (15,3%), C. aegopodii (12,6%), B. helichrysi (11,9%) and H. lactucae (11,5%). Much smaller share during this part of growing season had the potato aphids – M. persicae (8,1%), A. nasturtii (5,7%), and A. frangulae (only 2,1%). Potato aphids were more frequent during the second part of growing season (July-August). The share of M. persicae was 17,1% and A. nasturtii – 13,4%. Only A. frangulae had a small share (6,7%), however, it was 3 times larger than during the first period (2,1%). Besides, the frequency of C. galeopsidis was notable (13,3%) and above all the A. fabae complex (18,5%). It should be noted that also during the first period its share was large (15,3%), what means these species were frequent dur ing the whole growing season. The other species (C. aegopodii, C. theobaldi, R. padi, B. helichrysi and H. lactucae) had smaller or much smaller share during the first period. The results demonstrate much more frequent occurrence of the non-potato aphids during the earlier growing season (May till second decade of June) and much less frequent occurrence during the later growing season – in comparison to potato aphids, regardless of location. The obtained results indicate that during the early part of growing season, the non-potato aphids appear to play a significant role in epidemiology of non-persistent viruses, especially PVY. The arguments of this conclusion are the early start of spring migration and higher, in comparison to potato aphids, frequency during the early period of plant vegetation. Fig. 1. Locations of yellow water traps in different regions of Poland

Bonin Zamarte

Szyldak Jadwisin

Stare Olesno

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IDENTIFICATION OF APHID MEMBRANE(S) RESPONSIBLE OF TRANSMISSION SPECIFICITY OF POTATO LEAFROLL VIRUS (PLRV). Joëlle ROUZÉ -JOUAN, Sylvie TANGUY, Christine KERVARREC and Danièle GIBLOT DUCRAY. Institut National de la Recherche Agronomique, UMR Biologie des Organismes et des Populations appliquée à la Protection des Plantes, BP 35327, 35653 Le Rheu Cedex, France E-mail : [email protected]

Potato leafroll virus (PLRV, genus Polerovirus, family Luteoviridae) (Mayo & d'Arcy, 1999) is one of the most important viruses that cause damage in potato crops worldwide. It is transmitted by aphids in a persistent manner and is restricted to the phloem tissue. Aphid vectors ingest virus particles with plant sap when feeding in the phloem tissues of infected plants. In order to complete their route in the vector, virions must associate with cellular components in order to cross two epithelial membranes and to persist in aphid haemolymph. The first one is the gut membrane that virions cross by an endocytosis -exocytosis mechanism to move from the gut lumen into the haemolymph. Garret et al. (1993) have shown that in Myzus persicae (Sulz.) the site of passage of PLRV is the midgut, whereas for most Luteoviridae the site of passage is the hindgut (reviewed by Gildow, 1999). Once in the haemolymph, virus particles circulate throughout the aphid body cavity and must cross the accessory salivary gland (ASG) basal lamina and plasmalemma membrane before arriving in the salivary canal, so they can be inoculated to other plants (reviewed by Gildow, 1999). Moreover, van den Heuvel et al. (1994) have revealed an affinity between PLRV particles and symbionin, a chaperon protein produced by Buchnera endosymbionts. Such an association of PLRV and other luteoviridae with symbionin in the haemolymph suggests that it is essential for capsid integrity and for the success of transmission (Hogenhout et al., 1996). PLRV transmission depends on several parameters such as aphid species, clone, morph and instar (Kennedy et al., 1962; Björling & Ossiannilsson, 1958; Upreti & Nagaich, 1971; Hinz, 1966; Robert et Maury, 1970), and virus isolate (Tamada et al.,1984, Jolly & Mayo, 1994). However, little is known about the respective roles of the gut and ASG membranes in transmission efficiency and specificity. Most results, obtained with BYDVPAV or CYDV-RPV suggest that the ASG membranes play a crucial role (Peiffer et al., 1997). Concerning PLRV, few studies have been carried out, although Gildow (1982) has shown that PLRV particles attached to ASG membrane and has concluded that interactions occur at this site. Recently, Bourdin et al. (1998) have observed that PLRV strain 14 2 could not be transmitted after acquisition in infected plants by some clones belonging to the Myzus persicae complex. To determine where the transmission process was blocked inside the aphid's body, we have used three acquisition procedures : plant to plant transmission, membrane feeding on purified virus preparations and microinjection of purified virus directly into the haemolymph of aphids (Rouzé-Jouan et al., 2001). In each procedure, three aphids were transferred to each of heathly Physalis floridana seedlings. The transmission efficiency in the test plants was assessed by ELISA tests. Our results have allowed us to conclude that Myzus persicae gut membrane filters virus particles and, therefore regulates PLRV movement through the vector. Moreover, comparison of coat protein (CP) and readthrough protein (RTP) sequences between poorly and readily transmissible strains have shown that PLRV-14 2 differed from the others isolates by amino acid changes in both of these proteins. This suggests that changes in CP and /or RTP affect the transmission of PLRV-14 2 by reducing its recognition at the gut membrane. Recently, we have expanded these results by studying what happens in others potato aphid species such as Aulacorthum solani (KLTB.), Macrosiphum euphorbiae (THOS.) and Aphis nasturtii (KLTB.). A. solani and M. euphorbiae show considerable differences in the efficiency with which they transmit PLRV but little is known about these species. The role of A. nasturtii in the epidemiology is not well established although the occurrence of this species in potato crops is increasing. The results of our experiments will be presented and discussed as regards the role of the gut and ASG membranes. Also, a particular attention will be given to the role of aphid saliva in virus transmission.

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REFERENCES : Björling, K., & Ossiannilsson, F.(1958). Socker 14, 1-13. Bourdin, D., Rouzé, J., Tanguy, S., & Robert, Y. (1998). Plant Pathology, 47, 794-800. Garret , A., Kerlan, C.& Thomas, D.(1993). Archives of virology, 131, 377-392. Gildow , F.E. (1982). Phytopathology 72, 1289-1296. Gildow, F.E. (1999). In the Luteoviridae, pp. 88-111. Edited by H. G. Smith & H. Barker. Wallingford: CABI. Hinz, B. (1966). V. Wissenchaftliche Zeitschrift der Universsität Rostock. Mathematische-naturwissenschaftliche Reihe 15, 289-293. Hogenhout, S.A., Verbeek,M., Hans, F.Houterman, P.M., Fortass, M., vander Wilk, F., Huttinga, H., van den Heuvel, J.F. J.M. (1996). Agronomie 16, 167-173. Jolly, C. A. & Mayo, M.A. (1994). Virology 201, 182-185. Kennedy, J.S., Day, M.F.& Eastop, V.F. (1962) London : Commonwealth Institute of Entomology. Mayo, M.& d'Arcy, C.J. (1999). In the Luteoviridae, pp 15-22. Edited by H. G. Smith & H. Barker. Wallingford: CABI. Peiffer, M.L., Gildow, F.E. & Gray , S.M. (1997). Journal of General Virology 78, 495-503. Robert, Y.& Maury, Y. (1970). Potato Research 13, 199-209. Rouzé -Jouan, J., Terradot, L., Pasquer, F., Tanguy, S.& Giblot Ducray-Bourdin, D. (2001) Journal of General Virology 82, 17-23. Tamada, T., Harrison, B.D. & Roberts, I.M. (1984). Annals of applied Biology 104, 107-116. Upreti, G.C.& Nagaich, B.B. (1971). Phytopathologische Zeitschrift 71, 163-168. van den Heuvel, J.F.J.M., Verbeek, M. & van der Wilk, F.(1994). Journal of General Virology 75, 2559-2565.

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