Comparison of resistance to potyviruses within Solanaceae: infection of potatoes with tobacco etch potyvirus and peppers with potato A and Y potyviruses

The reaction of several cultivated potato varieties (Solarium tuberosum L.) to three strains of tobacco etch potyvirus (TEV-F, TEV-Mex21 and TEV-ATCC) and the reaction of several pepper lines (Capsicum annuum L. and C. chinense L.) to two strains of potato Y potyvirus (PVY^O and PVY^N) and one strain of potato A potyvirus (PVA-M) was tested. The potato varieties included in this study carried resistance genes against PVY, PVA and potato V potyvirus, but all were susceptible to TEV and developed mottle and mosaic symptoms. TEV was readily transmitted by mechanical inoculation from tobacco and potato to potato, whereas transmission from pepper to potato occurred infrequently. TEV was transmitted through potato tubers, and from pepper to potato plants by aphids. Lack of detectable systemic infection following graft-inoculation indicated extreme resistance to PVY^O and PVA in several pepper lines. No pepper line was systemically infected with PVY^N following mechanical inoculation (graft-inoculation was not carried out with PVY^N). The development of necrotic lesions following mechanical and graft-inoculation indicated hypersensitive response to PVY^O in several pepper lines which resembled the resistance responses to these potyvirus strains in potato. Results of this study together with previous work indicate that C. annuum cv. Avelar is resistant to four potyviruses [PVY, PVA, pepper mottle potyvirus (PepMoV) and some isolates of TEV]; C. annuum cv. Criollo de Morelos and C. chinense PI 152225 and PI 159236 are resistant to three potyviruses (PVY, PepMoV and PVA; and PVY, PepMoV and TEV, respectively); C. annuum 9093–1 and 92016–1 are resistant to PVY and PepMoV; and C. annuum cv. Jupiter and C. annuum cv. RNaky are resistant to PVY^N and PVA.     

Extreme resistance to potato virus V in clones of Solanum tuberosum that are also resistant to potato viruses Y and A: evidence for a locus conferring broad-spectrum potyvirus resistance

 Extreme resistance to the potato V potyvirus (PVV) was found in four potato cultivars that contain Ry genes from Solanum stoloniferum. When plants of these cultivars, were inoculated by grafting in shoot tips from PVV-infected tomato plants, necrotic symptoms developed in some cultivars, although a full hypersensitive reaction was not elicited, while other cultivars were symptomless. PVV replication was not detected in any of the inoculated plants by ELISA, an infectivity assay of leaf extracts by manual inoculation to Nicotiana benthamiana indicator plants, or by ‘return grafting’ of shoot tips taken from newly developed shoots of the potato plants to virus-free indicator plants of tomato. These methods readily detected PVV infection in inoculated plants of cv ‘Flourball’, which does not contain an Ry gene and is susceptible, and in cvs ‘Maris Piper’ and ‘Dr Macintosh’, which contain gene Nv conditioning a hypersensitive reaction to inoculation. One of the Ry-containing cultivars, ‘Barbara’, has been previously shown to contain two genes that control extreme resistance, defined as no viral replication in intact plants, to the potyviruses potato viruses Y and A (PVY and PVA). These genes are: Ry _sto , which conditions resistance to PVY and PVA, and gene Ra, which conditions resistance to PVA only. It was found that in genotypes from a progeny of the cross ‘Barbara’ (Ry _sto /Ra)בFlourball’ (ry/ra), extreme resistance to PVV segregated with gene Ry _sto . It is proposed that either gene Ry _sto conditions broad-spectrum extreme resistance to the distinct potyviruses PVY, PVA, and PVV or that Ry _sto represents a family of genetically closely linked genes each controlling resistance to a specific virus. Received: 27 December 1996 / Accepted: 9 June 1997     

Strain group specific and virus specific hypersensitive reactions to infection with potyviruses in potato cultivars

When 12 potato cultivars were inoculated with isolates (one each) of potato virus Y (PVY) ordinary (Y^o), C (Y^c) and tobacco veinal necrosis (Yⁿ) strain groups, potato virus A (PVA) and potato virus V (PVV), none of them responded hypersensitively to Yⁿ. However, with Y^o, Y^c, PVA and PW specific hypersensitive reactions developed depending on isolate-cultivar combination which were all independent of each other. When field isolates of PVY thought to be Y^oor Y^cwere inoculated to the same 12 cultivars, two did not fit into either strain group giving hypersensitive reactions in only two cultivars instead of seven with Y^oor eight with Y^c. These two isolates may represent a previously unreported PVY strain group (Y^z). When Y^owas graft-inoculated to seedlings of the cross Desiree × Maris Piper (hypersensitive × non-hypersensitive for Y^o), the segregation ratio obtained for non-hypersensitive:hypersensitive reactions was close to 1:1 suggesting that a single dominant gene (Ny_tbr) determining Y^ospecific hypersensitivity may be present in cv. Desiree (simplex condition). In tests using PVV and Desiree × Maris Piper (non-hypersensitive × hypersensitive for PVV) seedlings, the segregation ratio obtained was close to 1:5 indicating that a single dominant gene (Nv) determining PVV specific hypersensitivity may be present in cv. Maris Piper (duplex condition). Cultivars Corine, Pirola and clone G5457(4) which each carry one of the extreme resistance genes (Ry) from Solanum stoloniferum were graft-inoculated with Yⁿ, Y^o, Y^c, PVV and PVA. G5457(4) gave a strong localised hypersensitive reaction in all instances, while cv. Pirola did so with all except PVA to which it was immune. In cv. Corine a severe localised hypersensitive reaction developed with PVA, generalised hypersensitivity with PVV but an immune response with the three PVY strain groups. Large-scale grafting of Yⁿto plants of cvs Corine and Pirola gave no evidence of selection of a strain which overcomes Ry genes.     

Expression of genes for resistance to potato virus Y in potato plants and protoplasts

Plants of several potato clones with major gene resistance to potato virus Y (PVY) developed necrotic local lesions and systemic necrosis after manual inoculation with common (PVY_o) or veinal necrosis (PVY_N) strains of the virus. The clones reacted similarly, although their resistance genes are thought to be derived from four different wild species of Solarium. Mesophyll protoplasts from each clone became infected when inoculated with RNA of PVY_o by the polyethylene glycol method. The proportion of protoplasts infected, assessed by staining with fluorescent antibody to virus particles, was similar to that of protoplasts of susceptible potato cultivars. In contrast, plants of potato cultivars Corine and Pirola, which possess gene Ry from S. stoloniferum, developed few or no symptoms when manually inoculated or grafted with PVY_o. Moreover, only very few protoplasts of these cultivars produced virus particle antigen after inoculation with PVY_o RNA. The extreme resistance to PVY of cvs Corine and Pirola was therefore expressed by inoculated protoplasts whereas the resistance of the necrotic-reacting potato clones was not.     

Transfer of resistance to potato leafroll virus, potato virus Y and potato virus X from Solarium brevidens to S. tuberosum through symmetric and designed asymmetric somatic hybridisation

Resistance to potato leafroll virus (PLRV), potato virus Y (PVY^o) and potato virus X (PVX) was studied in symmetric and asymmetric somatic hybrids produced by electrofusion between Solanum brevidens (2n=2×=24) and dihaploid S. tuberosum (2n=2×=24), and also in regenerants (B-hybrids) derived through protoplast culture from a single somatic hybrid (chromosome number 48). All of the somatic hybrids between 5. brevidens and the two dihaploid lines of potato cv. Pito were extremely resistant to PLRV and PVY^oand moderately resistant to PVX, irrespective of their chromosome number and ploidy level (tetraploid or hexaploid). Most (56%) of the asymmetric hybrids of irradiated S. brevidens and the dihaploid line of potato cv. Pentland Crown (PDH40) had high titres of PVY^osimilar to those of PDH40, whereas the rest of the hybrids had PVY^otitres less than a tenth of those in PDH40. Three B-hybrids had a highly reduced chromosome number (27, 30 and 34), but were however as resistant to PLRV, PVY^oand PVX as 5. brevidens. Two asymmetric hybrids and one B-hybrid were extremely resistant to PLRV but susceptible to both PVY and PVX. The results suggested that resistance to PLRV in 5. brevidens is controlled by a gene or genes different from those controlling resistance to PVY and PVX, and the gene(s) for resistance to PVY and PVX are linked in S. brevidens.     

The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance

Hypersensitive resistance (HR) is an efficient defense strategy in plants that restricts pathogen growth and can be activated during host as well as non-host interactions. HR involves programmed cell death and manifests itself in tissue collapse at the site of pathogen attack. A novel hypersensitivity gene, Ny-1, for resistance to Potato virus Y (PVY) was revealed in potato cultivar Rywal. This is the first gene that confers HR in potato plants both to common and necrotic strains of PVY. The locus Ny-1 mapped on the short arm of potato chromosome IX, where various resistance genes are clustered in Solanaceous genomes. Expression of HR was temperature-dependent in cv. Rywal. Strains PVY^O and PVY^N, including subgroups PVY^NW and PVY^NTN, were effectively localized when plants were grown at 20°C. At 28°C, plants were systemically infected but no symptoms were observed. In field trials, PVY was restricted to the inoculated leaves and PVY-free tubers were produced. Therefore, the gene Ny-1 can be useful for potato breeding as an alternative donor of PVY resistance, because it is efficacious in practice-like resistance conferred by Ry genes.     

Rescuing abortive inter-EBN potato hybrids through double pollination and embryo culture

Summary Regeneration of inter-EBN hybrids among potato species was achieved using embryo rescue techniques. Tetraploid hybrids between 4x(2EBN) Solanum stoloniferum x 4x(4EBN) S. tuberosum Gp. Andigena as well as diploid hybrids between 2x(1EBN) S. chancayense x 2x(2EBN) S. chacoense were obtained by culturing immature hybrid embryos in nutrient medium. Identification of appropriate embryo developmental stages was critical in developing a suitable protocol for rescuing viable hybrid embryos. The use of IVP clones as the second pollinator in 4x(2EBN) x 4x(4EBN) crosses reduced premature fruit drop and helped to identify triploid hybrids. Morphological and cytological examination confirmed true hybridity for a few of the regenerated plants. Male sterility and meiotic abnormalities were characteristic of the hybrids. Several S. stoloniferum-Andigena hybrids were successfully backcrossed to Gp. Andigena.Cooperative investigation of Vegetable Crops Research Unit, USDA, Agricultural Research Service, and the Wisconsin Agricultural Experiment Station Note: Reference to a specific brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of similar nature not mentioned     

Strong resistance to potato tuber necrotic ringspot disease in potato induced by transformation with coat protein gene sequences from an NTN isolate of Potato virus Y

The potato cv. Igor is susceptible to infection with Potato virus Y (PVY) and in Slovenia it has been so severely affected with NTN isolates of PVY causing potato tuber necrotic ringspot disease (PTNRD) that its cultivation has ceased. Plants of cv. Igor were transformed with two transgenes that contained coat protein gene sequence of PVY^NTN. Both transgenes used PVY sequence in a sense (+) orientation, one in native translational context (N‐CP), and one with a frame‐shift mutation (FS‐CP). Although most transgenic lines were susceptible to infection with PVY^NTN and PVY^O, several lines showed resistance that could be classified into two types. Following manual or graft inoculation, plants of partially resistant lines developed some symptoms in foliage and tubers, and virus titre in the foliage, estimated by ELISA, was low or undetectable. In highly resistant (R) lines, symptoms did not develop in foliage and on tubers, and virus could not be detected in foliage by ELISA or infectivity assay. Four lines from 34 tested (two N‐CP and two FS‐CP) were R to PVY^NTN and PVY^O and one additional line was R to PVY^O. When cv. Spey was transformed with the same constructs, they did not confer strong resistance to PVY^O.     

Expression of the PVYO coat protein (CP) under the control of the PVX CP gene leader sequence: protection under greenhouse and field conditions against PVYO and PVYN infection in three potato cultivars

Coat protein-mediated resistance (CPMR), resistance conferred as a result of the expression of viral coat proteins in transgenic plants, has been illustrated to be an effective way of protecting plants against several plant viruses. Nonetheless, consistent protection has not been achieved for transgenic plants expressing the coat protein of potato virus Y (PVY), the type member of the potyvirus family. In this report, three different potato cultivars were transformed with a chimeric construct consisting of the capsid protein (CP) coding sequences of PVY flanked by the AUG codon and the translational enhancer from the coat protein gene of potato virus X (PVX). These cultivars were shown to express high levels of PVY CP and confer a high degree of protection against PVY^o and PVY^N under both greenhouse and field conditions. In addition, transgenic plants infected with potato virus A (PVA), a related potyvirus, exhibited a delay in virus accumulation, which could be easily overcome with increasing virus concentrations. Received: 26 October 1995 / Accepted: 14 June 1996     

Behavior of potato gametoclonal plants against the necrotic strain of potato Y potyvirus

A total of 59 Solanum tuberosum androgenetic plants have been obtained through anther culture, 47 of which derived from a tetraploid clone, seven from a diploid hybrid, and five from an anther-derived clone. About two thirds of the anther-derived plants were dihaploids, a few were monohaploids (5.08%) or aneuploids (6.78%), whereas the tetraploid genotype generated about a third of tetraploids. Seven hundred twenty seven R₁ plants arisen from tubers of the androgenetic potatoes were mechanically inoculated with the necrotic strain of the potato Y potyvirus (PVY^N) and grown in a glasshouse. Fifty days after inoculation, the presence of PVY^N in R₁ plants was detected by DAS-ELISA (Double Autibody Sandwich). Only three plants (0.4%) of genotype H2-258 exhibited local necrotic symptoms (hypersensitivity reaction) suggesting the presence of the N _y gene, and this extreme resistance is epistatic to hypersensitive resistance. The immunity (R _y-gene) to PVY^N was retained through anther culturing and present at all levels of ploidy. The pattern of segregation for immunity was differentiated according to the ploidy level of the anther-derived plants. This changed segregation pattern may be due to a loss of resistance during the culturing, when an endoreduplication has taken place or to the possible regeneration from Second-division restituted unreduced microspores. Anyway, this segregation pattern must be taken into account when gametoclones are used in genetic studies. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 572–578. The text was submitted by the authors in English.     

Inheritance of resistance to potato viruses Y and A in progeny obtained from potato cultivars containing gene Ry:evidence for a new gene for extreme resistance to PVA

Extreme resistance in cultivated potato (Solanum tuberosum) to potato viruses Y and A (PVY and PVA) conditioned by the presence of Ry genes introduced from Solanum stoloniferum was described by Cockerham (1970). Cockerham detailed a number of genes which controlled a variety of reactions, including extreme resistance to both viruses (i.e. little or no visible reaction of plants and no viral replication following graft and manual inoculation) controlled by gene Ry _sto. In the present study, cvs Pirola and Barbara, which contain a Ry gene, were found to have extreme resistance to PVY isolates from the ordinary (PVY°), veinal necrosis (PVY^N) and potato tuber necrotic ringspot (PVY^NTN) subgroups, and PVA. The inheritance of this phenotype was examined in seedling progenies obtained by crossing Barbara and Pirola with susceptible cultivars. Segregation data for resistance to PVY and PVA in a progeny involving cv Pirola best fitted a genetical model of one gene controlling extreme resistance to both PVY and PVA, although the possibility that there are two genes, each controlling resistance to one virus but closely linked, cannot be excluded. Segregation data from progenies involving cv Barbara best fitted a genetical model in which there are two independent genes, one controlling extreme resistance to PVA and PVY and a second gene controlling extreme resistance to PVA but not to PVY. This previously unrecognised gene conferring extreme resistance to PVA only, should be given the notation Ra in keeping with nomenclature used for other resistance genes.     

Recombination of strain O segments to HCpro‐encoding sequence of strain N of Potato virus Y modulates necrosis induced in tobacco and in potatoes carrying resistance genes Ny or Nc

Hypersensitive resistance (HR) to strains O and C of Potato virus Y (PVY, genus Potyvirus) is conferred by potato genes Ny_tbr and Nc_tbr, respectively; however, PVY N strains overcome these resistance genes. The viral helper component proteinases (HCpro, 456 amino acids) from PVY^N and PVY^O are distinguished by an eight‐amino‐acid signature sequence, causing HCpro to fold into alternative conformations. Substitution of only two residues (K269R and R270K) of the eight‐amino‐acid signature in PVY^N HCpro was needed to convert the three‐dimensional (3D) model of PVY^N HCpro to a PVY^O‐like conformation and render PVY^N avirulent in the presence of Ny_tbr, whereas four amino acid substitutions were necessary to change PVY^O HCpro to a PVY^N‐like conformation. Hence, the HCpro conformation rather than other features ascribed to the sequence were essential for recognition by Ny_tbr. The 3D model of PVY^C HCpro closely resembled PVY^O, but differed from PVY^N HCpro. HCpro of all strains was structurally similar to β‐catenin. Sixteen PVY^N605‐based chimeras were inoculated to potato cv. Pentland Crown (Ny_tbr), King Edward (Nc_tbr) and Pentland Ivory (Ny_tbr/Nc_tbr). Eleven chimeras induced necrotic local lesions and caused no systemic infection, and thus differed from both parental viruses that infected King Edward systemically, and from PVY^N605 that infected Pentland Crown and Pentland Ivory systemically. These 11 chimeras triggered both Ny_tbr and Nc_tbr and, in addition, six induced veinal necrosis in tobacco. Further, specific amino acid residues were found to have an additive impact on necrosis. These results shed new light on the causes of PVY‐related necrotic symptoms in potato.     

Variations in resistance against Phthorimaea operculella in wild potato tubers

Tuber resistance can contribute to current management strategies against the potato tuber moth, Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae), in field and stored potatoes. Wild potatoes represent a potential source of novel resistance traits against the moth. We assessed resistance in three wild potato species, Solanum multiinterruptum Bitt., Solanum sparsipilum (Bitt.) Juz. & Buk., and Solanum wittmackii Bitt. against neonate and developing tuber moth larvae. All three species had high levels of resistance but accessions of S. sparsipilum and S. wittmackii were significantly more resistant. Resistance in S. multiinterruptum was generally concentrated in the tuber periderm, whereas in S. sparsipilum and S. wittmackii resistance was mainly cortex‐based. Unidentified cortex‐resistance factors in all three species reduced survival and increased larval and pupal development times, but had no apparent effects on the pupal weights of survivors. A high proportion of larvae abandoned or died within tubers of S. wittmackii, which has particularly high levels of unidentified cortex‐based defenses. Resistance decreased in S. multiinterruptum and S. sparsipilum as tubers sprouted but was more stable in S. wittmackii. Periderm‐based resistance was more stable than cortex‐based resistance in S. multiinterruptum during sprouting. In contrast, cortex‐based resistance was stable in tubers of S. wittmackii as these sprouted, and resistance may have increased on some older sprouting tubers. Solanum multiinterruptum and S. sparsipilum are proposed as potential sources of resistance against the potato tuber moth.     

Molecular examination of a chromosome region that controls resistance to potato Y and A potyviruses in potato

 The gene Ry _adg that confers resistance to potato Y potyvirus (PVY) in the cultivated potato [Solanum tuberosum subsp. andigena, line 2x(v-2)7] is located on chromosome XI in a segment that contains three other known resistance genes in other syntenic solanaceous species. One of them is the gene N that controls resistance to tobacco mosaic tobamovirus in tobacco and has previously been isolated and sequenced. Three sequence-related, resistance gene-like (RGL) DNA fragments (354–369 bp) highly homologous to the gene N were PCR-amplified from the potato line 2x(v-2)7. Two RGL fragments (79 and 81% homologous to the N gene) co-segregated with Ry _adg among the 77 F1 progeny tested. These RGLs may originate from a resistance gene family on chromosome XI. The potato line 2x(v-2)7 also expressed resistance to potato A potyvirus (PVA), which was controlled by another locus on chromosome XI mapped ca. 6.8 cM distal to Ry _adg . Received: 18 December 1997 / Accepted: 30 December 1997     

Molecular and Biological Characterization of Potato virus Y Isolates from Vietnam

Eight isolates from different potato growing regions in Vietnam were characterized. All were highly pathogenic in some potato cultivars, but did not overcome the extreme resistance of Solanum stoloniferum and Solanum demissum. RT‐PCR analysis revealed that all of these isolates are recombinants. Sequence data for 4 isolates were obtained, and their reaction in potato cultivars harbouring specific N genes was determined. Different phylogenetic analyses of viral sequences confirmed previous results that the recombinant isolates evolved from different parental sequences. One of the Vietnamese isolates investigated had a specific structure. The need for a clear classification of PVY^NWi isolates is discussed.     

Evidence for utility of the same PCR-based markers for selection of extreme resistance to Potato virus Y controlled by Rysto of Solanum stoloniferum derived from different sources

The tuber‐bearing wild potato species, Solanum stoloniferum, carries a dominant gene, Ry_sto, which confers extreme resistance (ER) to Potato virus Y (PVY). This gene was introgressed to cultivated potato germplasm (Solanum tuberosum) using accessions of S. stoloniferum maintained in European gene banks. It is mainly used in potato breeding programmes in Europe. Ry_sto was recently mapped to potato chromosome XII. However, in this study, a different accession of S. stoloniferum (PI275244; Haw1293) was used as a female parent in a cross to obtain a diploid (2n = 2x = 24) potato population of 112 F1 genotypes. From this accession, ER to PVY has been introgressed to the potato breeding programmes at the International Potato Center (Peru). As expected, ER to PVY was inherited in a dominant, monogenic fashion in the F1 population. Marker‐specific choices of DNA polymerase and adjustments of PCR conditions were made to optimise marker detection. The corresponding gene (Ry_sto) was mapped to the chromosome XII using the previously described and new cleaved amplified polymorphic sequence (CAPS) markers, which are based on the restriction fragment length polymorphism loci GP122 (six markers) and GP269 (one marker), and the simple sequence repeat marker STM0003. Four GP122‐based CAPS markers and STM0003 detected the same genotypes expressing ER to PVY. Because of a few recombinants, that is ER genotypes lacking the markers and the genotypes that react with necrosis but contain the markers, the marker distance from Ry_sto was estimated as 15.2 cM in this F1 population. However, the distance may be less if necrosis was considered an altered response also controlled by Ry_sto. The markers also specifically detected independent European potato cultivars that express ER to PVY derived from S. stoloniferum. Phylogenetic analysis of the sequences amplified from the GP122 locus of S. stoloniferum and potato cultivars further confirmed that the Ry_sto gene from independent accessions of S. stoloniferum can be selected using the same markers and the protocols described in this study.     

Factors underpinning the responsiveness and higher levels of virus resistance realised in potato genotypes carrying virus-specific R genes

Responses to Potato virus A (PVA, genus Potyvirus) segregate to three phenotypic groups in a diploid cross between Solanum tuberosum subsp. andigena and a highly interspecific potato hybrid. The aim of this study was to compare gene expression between the progeny genotypes which react with hypersensitive response (HR) to PVA, allow PVA accumulation in inoculated leaves but restrict PVA infection to the inoculated leaf by blocking systemic movement [non-necrotic resistance (nnr)], or are susceptible (S) and systemically infected with PVA. Expression levels of ca 10 000 genes were compared using probes arranged in a microarray format, and real-time RT-PCR was applied for quantitative comparison of the expression of selected defense-related genes (DRGs). Results showed that a few DRGs were autoactivated in HR genotypes at an early stage of plant growth in the absence of PVA infection, which was not observed in the two other phenotypic groups (nnr and S). More detailed studies on the DRGs encoding a beta-1,3-glucanase, a chitinase and a basic PR-1b protein showed that autoactivation of the genes was not evident in vitro and up to 2 weeks of growth in soil in a controlled growth cabinet but was apparent 2 weeks later. Hence, autoinduction of these DRGs in the HR genotypes could be associated with growth stage, environmental factors or both. Furthermore, a number of other DRGs were induced in the inoculated leaves of HR genotypes as a response to infection with PVA, which was not observed in nnr and S genotypes. These results provide some novel information about factors underpinning the higher levels of virus resistance realised in potato genotypes carrying virus-specific R genes and suggest that part of the resistance is attributable to additional ‘minor’ genes functioning simultaneously, hence adding to the overall responsiveness and level of resistance against infection. These results also imply that some genotypes might be more responsive to chemical induction of pathogen and pest resistance, which could be considered in screening of progenies in plant-breeding programs.     

Production of monoclonal antibodies for the detection of potato virus Y

Monoclonal antibodies (McAb) were obtained to potato virus Y (PVY) after immunisation of BALB/c mice with purified PVY, tobacco necrotic strain (PVY_n). Spleen cells from a mouse showing a high serum titre were used for fusion with X63NS1 myeloma cells. Hybridomas were selected in medium containing HAT. Culture supernatants were screened for antibody production against PVY, ordinary strain (PVY₀) and PVY_n using indirect ELISA. Clones of interest were further cross-reacted with 12 isolates each of PVY₀ and PVY_n and two isolates of potato virus A (PVA) and healthy sap. For further trials, two clones which reacted specifically with PVY_n isolates and one which detected all PVY isolates except two of potato virus C (PVC) were selected.