First report of saffron latent virus in Crocus sativus from Hungary.

In early April of 2018 we sampled asymptomatic autumn flowering Crocus plants (Fig. S1.) in a private collection in Hajdú-Bihar county, Hungary. From each species (Cr. kotschyanus subsp. kotschyanus, Cr. sativus, Cr. speciosus) 200 mg leaf sample was collected from 5 neighboring shoot, which were treated as one sample. ELISA tests were carried out in duplicates using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) on the samples (Agdia, Elkhart, IN, USA). A sample was considered positive if the absorbance was at least three times greater than that of the negative control. Only one sample tested positive; the absorbance values of Cr. sativus leaves were 0.013 and 0.014, while the negative controls were 0.002 and 0.003, respectively. The samples were further tested by RT-PCR for potyviruses (Salamon and Palkovics 2005), tomato spotted wilt virus (TSWV) (Nemes and Salánki 2020) and nepovirus subgroup A (Digiaro et al. 2007). Total nucleic acid was extracted with the phenol-chloroform method of White and Kaper (1989), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) using random hexamer primer. The samples were negative for TSWV and nepovirus subgroup A, but a single PCR product of ~ 1700 nucleotide (nt) was amplified with potyvirus specific primers and cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA). The 1726 nt long insert sequence, including the complete coat protein region was determined and deposited in the NCBI GenBank database (Accession No: OR425160). Digestion of the original PCR products with restriction enzyme SacI yielded only the predicted restriction fragments (364 / 1362 bp), indicating the presence of only a single potyvirus in the infected sample. BLASTn analysis of the CP cistron revealed the highest nt identities to saffron latent virus (SaLV) Iranian isolates (GenBank AccNo.: MN990394 - 85.44%, MN990415 - 85.39% and RefSeq: NC_036802 - 84.05%). For phylogenetic analyses MEGA11 (Tamura et al. 2021) was used. The resulting Maximum Likelihood tree (Fig. S2) showed that all Iranian SaLV isolates grouped together, while the Hungarian isolate is on an adjacent branch, separate from other virus species, and supported with 100% bootstrap values. From these results, it appears that the Hungarian isolate has been separated from the Iranian clade, and has evolved separately as a distinct lineage. We were unable to fulfill Koch's postulates as all available Crocus sativus plants were infected with SaLV. Latent potyvirus infection of Crocus species, by bean yellow mosaic virus (BYMV), iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV) and turnip mosaic virus (TuMV) has been reported by Grilli Caiola and Faoro (2011). SaLV was first reported from Iran (Parizad et al. 2017), but to our knowledge has never been reported from Europe or from any current EPPO member state. Since Crocus species can be asymptomatic virus reservoirs, it is important that any certification scheme for production should require laboratory tests to prove the health of the plants; or advise growers to keep possible high value susceptible crops such as breeding material and nuclear stocks at a distance from crocuses to mitigate virus transmission between stocks. It is also advisable to grow infected lots far from healthy stocks and protected wild hosts. To our knowledge, this is the first report of SaLV from Hungary and from Europe.

First report of meadow saffron breaking virus on wild Colchicum autumnale from a stricly protected Natura2000 site at a Hungarian National Park.

In mid-April of 2018 light green to greenish yellow linear stripes (Fig. S1.) were observed on the foliage of meadow saffron (Colchicum autumnale) plants - which are native to Hungary - at a strictly protected Natura2000 site maintained by the Duna-Ipoly National Park (DNPI). By autumn, during the flowering season, flower breaking symptoms (Fig. S2.) were noticed, which indicated possible viral infection. With the permit of the Government Office of Pest County and the DNPI, 200 mg leaf sample was collected from one symptomatic plant in spring 2021 and stored at -70 °C until further processing. At the time of the sampling about 2.5 % of the ~ 5000 meadow saffron were symptomatic. Multiplex RT-PCR testing of the sample and an asymptomatic C. autumnale plant for cucumber mosaic virus, tomato spotted wilt virus (Nemes and Salánki 2020) and Nepovirus subgroup-A (Digiaro et al. 2007) gave negative results. The asymptomatic plant also tested negative for potyviruses (Salamon and Palkovics 2005). The asymptomatic (healthy) C. autumnale plant was inoculated with leaf sap of the sample (0.02M Sörensen's phosphate buffer pH 7.2 + 2 % PVP-40 (m/v)) resulting in symptoms of flower breaking in autumn of 2021, and linear stripes on the foliage in spring 2022, identical to symptoms on the originally infected plant. ELISA tests were carried out on the source plants in duplicate using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) (Agdia, Elkhart, IN, USA). Absorbance values were 1.519 and 1.530, while the negative controls were 0.003 and 0.007, respectively indicating potyvirus infection of the sample. Molecular tests were carried out on the source and inoculated plant samples in 2022. Total nucleic acid was extracted with the modified CTAB protocol of Xu et al. (2004), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) with poly T2 (5'-CGGGGATCCTCGAGAAGCTTTTTTTTTTTTTTTTT-3') primer (Salamon and Palkovics 2005). PCR amplification was carried out with poty7941 (5'-GGAATTCCCGCGGNAAYAAYAGYGGNCARCC-3') and poly T2 primers as described earlier (Salamon and Palkovics 2005). A PCR product of ~ 1.6 kb was obtained in each case (Fig. S3.), cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA) and transformed into E. coli DH5α strain. The obtained 1642 nucleotide (nt) sequence encompassing the complete coat protein (CP) was determined (Accession No: OP057214). The virus sequence present in the source and inoculated plants shared 100% nt identity. EcoRV digestion of the PCR products yielded two restriction fragments (369/1273 bp), indicating the presence of a single potyvirus in the infected plant tissue (Fig. S3.). BLASTN analysis of the CP cistron revealed highest nt identity (93.91 %) to meadow saffron breaking virus (MSBV) isolate FR GenBank Acc. No.: AY388995. MSBV was first reported in the Alsace region of France at an INRA research station in cultivated meadow saffron plants showing similar symptoms and the disease reached 100% incidence within a year (Poutaraud et al. 2004). Potyviruses are transmitted mechanically and by aphids (Inoue-Nagata et al. 2022). The spread of MSBV could lead to the infection and decline of the population of Colchicum in protected ecosystems. To our knowledge, this is the first report of MSBV on wild meadow saffron plant from a strictly protected Natura2000 site at a Hungarian National Park.

Peanut stunt virus movement protein is the limiting factor in Capsicum annuum infection.

Cucumber mosaic virus (CMV) is one of the most devastating plant viruses, with more than 1,200 species of host plants. The host range and economic importance of peanut stunt virus (PSV) are mostly limited to legumes, despite the similar taxonomy and genome structure with CMV. Since no data are available on the background of the limited host range of PSV, RNA 3 recombinant and reassortant viruses were generated (C12P3, P12C3, C12CP3, C12PC3, C12PΔC3) to study their infection phenotype on a common host (Nicotiana benthamiana) and on a selective host (Capsicum annuum cv. Brody). The PSV movement protein (MP) was not able to function with the coat protein (CP) of CMV unless the C-terminal 42 amino acids were deleted from the PSV MP. As a result of the inoculation experiments, MP was considered the protein influencing symptom phenotypes on N. benthamiana and responsible for the host range difference on the pepper. Since plasmodesmata (PD) localization of viral MPs is essential for cell-to-cell movement, subcellular localization of GFP-tagged MPs (CMV-MP-eGFP, PSV-MP-eGFP) was observed. In the case of CMV-MP-eGFP, clear colocalization with PD was detected in both hosts, but PSV-MP-eGFP was not tightly connected to the PD in N. benthamiana and barely localized to the PD in C. annuum epidermal cells. Measuring Pearson correlation coefficients (PCCs) also supported the visual observation.

First report of Coniella granati causing leaf spot of pomegranate (Punica granatum L.) in Hungary.

Pomegranate (Punica granatum L.), the hystoric fruit and ornamental crop native to Iran and North India is widely planted in the Mediterranean and became popular in the house gardens of northest parts of Europe (Fernandez et al. 2014) including Hungary. In August 2020 necrotic black lesions and serious defoliation were observed on 60% of 1-3 year old pomegranate trees (cv. Wonderful) in a horticultural nursery near Gödöllo, Hungary (47°36'00.9"N 19°21'26.5"E). Symptoms started as small irregular dark brown spots on the leaves, which later increased in size (2.6 ± 0.9 mm). Ultimately, the entire leaf turned yellow, defoliation resulted in damage on (6) - 8 - (15)% of the leaves. Then, black pycnidia with unicelled, elliptical to fusiform, colourless conidia (Avg. 50 conidia: 2.4 - (3.6) - 3.9 × 10.2 - (13,1) - 17.9 µm) developed on the surface. These morphological features matched those described earlier by Van Niekerk et al. (2004) and Alvarez et al. (2016) for C. granati. Conidia from pycnidia were directly transferred to potato dextrose agar (PDA) by sterile needle. The plates were incubated at 24°C in the dark. Light yellow colonies with whitish aerial mycelia and later black globose pycnidia were observed. Mass of conidia oozed from pycnidia after 15 days of incubation. Pathogenicity tests were carried out on 1-year-old potted P. granatum trees (cv. Wonderful) with 5 replicates in the greenhouse. Ten, randomly selected leaves were inoculated per plant. 7-mm mycelial plugs from the edge of 10-day-old colonies were placed directly on disinfested (2% NaOCl solution, than sterile distilled water) leaves. The plants were covered with plastic film for 3 days after inoculation (26±3°C and 87±3% relative humidity). Pathogenicity was also tested on nonwounded, surface-disinfested fruits by mycelial plugs in 3 × 3 replicates. Inoculated fruits were placed in large grass vessels for 15 days (24±2°C and 80±5% relative humidity). Uncolonized, sterile PDA plugs were used as controls in both cases. Dark brown legions developed after 9-12 days on the plants in the greenhouse. On pomegranate fruits, the fungus colonized the fruit after 7-8 days, followed by fruit rot. In some cases, after 2 weeks pycnidia developed on the skin surface. No decay were present on control leaves or fruits. The pathogen was reisolated from all infected tissues and identified as C. granati, thus fulfilling Koch's postulates. For molecular identification, total genomic DNA of the isolate was extracted from the growing margins of colonies on PDA and partial sequence of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (tef1) were amplified by PCR using primers described by Alvarez et al. (2016). Sequence data of the Hungarian isolate of the ITS region (GenBank acc. no. MW581953) showed 99.8% identity (559 bp out of 560 bp) with C. granati sequences deposited in GeneBank (Acc. nos. MH860368, MH855389 and KX833582). Considering tef1 sequence of the Hungarian isolate (OM908764) obtained had complete identity with other published C. granati isolates (KX833676, KX833682). C. granati has been previously reported on pomegranate from Europe (Palou et al. 2010, Pollastro et al. 2016). Based on morphological and molecular studies, this is the first record of C. granati in Hungary. The economic importance of this disease in currently limited in Hungary due to pomegranate is rather an ornamental crop, however, the first cultivation trials have been already started. There is a risk that the spread of the pathogen began with the infected propagating material, as a result the disease may outbreak anywhere in the country.

The 28 Ser Amino Acid of Cucumber Mosaic Virus Movement Protein Has a Role in Symptom Formation and Plasmodesmata Localization.

Cucumber mosaic virus (CMV, Cucumovirus, Bromoviridae) is an economically significant virus infecting important horticultural and field crops. Current knowledge regarding the specific functions of its movement protein (MP) is still incomplete. In the present study, potential post-translational modification sites of its MP were assayed with mutant viruses: MP/S28A, MP/S28D, MP/S120A and MP/S120D. Ser28 was identified as an important factor in viral pathogenicity on Nicotiana tabacum cv. Xanthi, Cucumis sativus and Chenopodium murale. The subcellular localization of GFP-tagged movement proteins was determined with confocal laser-scanning microscopy. The wild type movement protein fused to green fluorescent protein (GFP) (MP-eGFP) greatly colocalized with callose at plasmodesmata, while MP/S28A-eGFP and MP/S28D-eGFP were detected as punctate spots along the cell membrane without callose colocalization. These results underline the importance of phosphorylatable amino acids in symptom formation and provide data regarding the essential factors for plasmodesmata localization of CMV MP.

Genetic Diversity of Potyviruses Associated with Tulip Breaking Syndrome.

Tulip breaking is economically the most important viral disease of modern-day tulip growing. It is characterized by irregular flame and feather-like patterns in the flowers and mosaic on the foliage. Thirty-two leaf samples were collected from cultivated tulip plants showing tulip breaking syndrome from Hungary in 2017 and 2018. Virus identification was performed by serological (ELISA) and molecular (RT-PCR) methods. All samples proved to be infected with a potyvirus and evidence was provided that three potyvirus species could be identified in the samples: Lily mottle virus (LMoV), Tulip breaking virus (TBV) and Rembrandt tulip-breaking virus (ReTBV). Recombination prediction accomplished with Recombination Detection Program (RDP) v4.98 revealed potential intraspecies recombination in the case of TBV and LMoV. Phylogenetic analyses of the coat protein (CP) regions proved the monophyletic origin of these viruses and verified them as three different species according to current International Committee on Taxonomy of Viruses (ICTV) species demarcation criteria. Based on these results, we analyzed taxonomic relations concerning potyviruses associated with tulip breaking syndrome. We propose the elevation of ReTBV to species level, and emergence of two new subgroups in ReTBV.

Contribution of cell wall peroxidase- and NADPH oxidase-derived reactive oxygen species to Alternaria brassicicola-induced oxidative burst in Arabidopsis.

Cell wall peroxidases and plasma membrane-localized NADPH oxidases are considered to be the main sources of the apoplastic oxidative burst in plants attacked by microbial pathogens. In spite of this established doctrine, approaches attempting a comparative, side-by-side analysis of the functions of extracellular reactive oxygen species (ROS) generated by the two enzymatic sources are scarce. Previously, we have reported the role of Arabidopsis NADPH oxidase RBOHD (respiratory burst oxidase homologue D) in plants challenged with the necrotrophic fungus Alternaria brassicicola. Here, we present results on the activity of apoplastic class III peroxidases PRX33 (At3g49110) and PRX34 (At3g49120) investigated in the same Arabidopsis-Alternaria pathosystem. ROS generated by Arabidopsis peroxidases PRX33 and PRX34 increase the necrotic symptoms and colonization success of A. brassicicola. In addition, the knockdown of PRX33 and PRX34 transcript levels leads to a reduced number of host cells showing an extracellular burst of ROS after inoculation with A. brassicicola. Our results also reveal an age-dependent transcript distribution of ROS-producing peroxidase and NADPH oxidase enzymes, and some potential new components of the RBOHD, PRX33 and PRX34 signalling networks.

A single point mutation in Tomato spotted wilt virus NSs protein is sufficient to overcome Tsw-gene-mediated resistance in pepper.

The nonstructural protein (NSs) of Tomato spotted wilt virus (TSWV) was previously identified as an avirulence determinant for Tsw-based resistance on pepper. The NSs of wild-type (WT) and resistance-breaking (RB) TSWV strains isolated in Hungary had only two amino acid substitutions (104, 461). We have analysed the ability of the NSs and their point mutant variants to trigger Tsw-mediated hypersensitive responses and RNA silencing suppressor (RSS) activity in patch assays. We identified a single amino acid change at position 104 (T-A) that was responsible for the necrosis induction or loss, while a significant difference was not detected in the RSS activity of the two parental strains. We have successfully complemented the infection of the WT strain on resistant pepper cultivar with the infectious S RNA transcript of the RB strain and the WT-T104A point mutant. Our work provides direct evidence that a single amino acid change can induce an RB phenotype.

Phylogenetic analysis of Tomato spotted wilt virus (TSWV) NSs protein demonstrates the isolated emergence of resistance-breaking strains in pepper.

Resurgence of Tomato spotted wilt virus (TSWV) worldwide as well as in Hungary causing heavy economic losses directed the attention to the factors contributing to the outbreak of this serious epidemics. The introgression of Tsw resistance gene into various pepper cultivars seemed to solve TSWV control, but widely used resistant pepper cultivars bearing the same, unique resistance locus evoked the rapid emergence of resistance-breaking (RB) TSWV strains. In Hungary, the sporadic appearance of RB strains in pepper-producing region was first observed in 2010-2011, but in 2012 it was detected frequently. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there is no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, we concluded that the RB isolates evolved separately in geographic point of view, and also according to the RB mechanism.

Comparison of the nucleotide sequences of wheat dwarf virus (WDV) isolates from Hungary and Ukraine.

Wheat dwarf virus (WDV) is the most ubiquitous virus in cereals causing huge losses in both Hungary and Ukraine. The presence of barley-and wheat-adapted strains has been confirmed, suggesting that the barley strain is restricted to barley, while the wheat strain is present in both wheat and barley plants. Five WDV isolates from wheat plants sampled in Hungary and Ukraine were sequenced and compared with known WDV isolates from GenBank. Four WDV isolates belonged to the wheat strain. Our results indicate that WDV-Odessa is an isolate of special interest since it has originated from wheat, but belongs to the barley-adapted strain, providing novel data on WDV biology and raising issues of pathogen epidemiology.

Characterization of a natural Plum pox virus isolate bearing a truncated coat protein.

Plum pox virus (PPV) isolates were collected in Hungary from plum varieties. PCR targeting the 3' genomic region resulted in a shorter PCR product in the case of the B1298 isolate bearing a 135-nucleotide deletion in frame in the N-terminal part of the coat protein (CP). The isolate was aphid-transmissible and the virion diameter was reduced compared to PPV-SK68. Detectability of this isolate by Western blot varied according to the antibody used. Integration of the deleted CP gene into an infectious PPV clone had no effect on infectivity and symptomatology. In competition experiments, B1298 had a considerable advantage in virus accumulation.

No recombination detected in artificial potyvirus mixed infections and between potyvirus derived transgenes and heterologous challenging potyviruses.

Risk-assessment studies of virus-resistant transgenic plants (VRTPs) focussing on recombination of a plant virus with a transgenic sequence of a different virus should include a comparison of recombination frequencies between viruses in double-infected non-transgenic plants with those observed in singly infected transgenic plants to estimate recombination incidence in VRTPs. In this study, the occurrence of recombination events was investigated in non-transgenic plants double-infected with two different potyviruses, as well as in potyviral genomes in singly infected transgenic plants expressing potyvirus sequences. Different potyviruses, namely Potato virus A (PVA), Tobacco vein mottling virus (TVMV), two strains of Potato virus Y (PVY-O, PVY-H) and two strains of Plum pox virus (PPV-NAT, PPV-SK68), were used in three combinations for double infection of a common host. Furthermore, transgenic plants expressing either potyviral coat protein (CP), helicase (CI) or polymerase (NIb) coding sequences (PPV-NAT-CP, PVY-CI, PVY-NIb) were singly-infected with a heterologous potyvirus, which was not targeted by the respective transgenic resistance. To identify recombinant potyviral sequences, a sensitive RT-PCR was developed to detect up to one recombinant molecule out of 10(6) parental molecules. In 304 mixed infected non-transgenic plants, 92 mixed and 164 single infected transgenic plants screened for recombinant sequences no recombinant potyviral sequence was found. These results indicate that recombination events between different potyviruses in mixed infections and between a potyvirus infecting a potyvirus-resistant transgenic plant are likely to be very infrequent.

Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the plum pox virus helper component proteinase suppressor of gene silencing.

Helper component proteinase (HC-Pro) of Plum pox virus is a multifunctional potyvirus protein that has been examined intensively. In addition to its involvement in aphid transmission, genome amplification and long-distance movement, it is also one of the better-studied plant virus suppressors of RNA silencing. The first systematic analysis using pentapeptide-insertion scanning mutagenesis of the silencing suppression function of a potyvirus HC-Pro is presented here. Sixty-three in-frame insertion mutants, each containing five extra amino acids inserted randomly within the HC-Pro protein, were analysed for their ability to suppress transgene-induced RNA silencing using Agrobacterium infiltration in transgenic Nicotiana benthamiana plants expressing green fluorescent protein. A functional map was obtained, consisting of clearly defined regions with different classes of silencing-suppression activity (wild-type, restricted and disabled). This map confirmed that the N-terminal part of the protein, which is indispensable for aphid transmission, is dispensable for silencing suppression and supports the involvement of the central region in silencing suppression, in addition to its role in maintenance of genome amplification and synergism with other viruses. Moreover, evidence is provided that the C-terminal part of the protein, previously known to be necessary mainly for proteolytic activity, also participates in silencing suppression. Pentapeptide-insertion scanning mutagenesis has been shown to be a fast and powerful tool to functionally characterize plant virus proteins.

Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a 'shooter mutant' A. tumefaciens.

In this work, Potato virus Y (PVY) resistant potatoes were generated using an environmentally safe construct. For this purpose, a 'shooter' mutant Agrobacterium-based transformation system was used. The isopentenyl transferase gene (ipt) present on the Ti plasmid of 'shooter' strains enhances shoot regeneration and can be used as a phenotypic selection marker. The introduced marker-free binary vector carried a hairpin construct derived from the coat protein gene of PVY-NTN strain in order to induce gene silencing. Transformation resulted in high regeneration rates (1.4-5.7 shoots per explant). With pre-selection for the ipt (+) phenotype the transformation frequency was 24-53%, while without selection 12-28% of the shoots were PCR positive. The presence of the transgene was verified by Southern hybridization. In 16 of 31 challenged transformant lines PVY could be detected neither by RT-PCR nor by back inoculation. A 62.5% of these resistant lines proved to be also ipt-free. This transformation system was reproducible in four potato cultivars, suggesting that it could easily be adapted for other species.

Geographically and temporally distant natural recombinant isolates of Plum pox virus (PPV) are genetically very similar and form a unique PPV subgroup.

Natural recombinant Plum pox virus (PPV) isolates were detected in Albania, Bulgaria, Czech Republic, Germany, Hungary and Slovakia. Despite different geographical origins and dates of isolation, all the recombinant isolates were closely related at the molecular level and shared the same recombination breakpoint as well as a typical signature in their N-terminal coat protein sequence, suggesting a common origin. Biological assays with four recombinant isolates demonstrated their capacity to be aphid-transmitted to various Prunus hosts. One of these isolates had a threonine-to-isoleucine mutation in the conserved PTK motif of its HC-Pro and showed a drastically decreased, although not abolished, aphid transmissibility. The complete genome sequence of one of the recombinant isolates, BOR-3, was determined, as well as some partial sequences in the HC-Pro and P3 genes for additional natural recombinant isolates. Analysis of the phylogenetic relationships between the recombinant isolates and other sequenced PPV isolates confirmed that the recombinant isolates form a phylogenetically homogeneous lineage. In addition, this analysis revealed an ancient recombination event between the PPV-D and M subgroups, with a recombination breakpoint located in the P3 gene. Taken together, these results indicate that recombinant isolates represent an evolutionarily successful, homogeneous group of isolates with a common history and unique founding recombination event. The name PPV-Rec is proposed for this coherent ensemble of isolates.

Characterization of Hungarian isolates of zucchini yellow mosaic virus (ZYMV, potyvirus) transmitted by seeds of Cucurbita pepo var Styriaca.

Zucchini yellow mosaic virus (ZYMV) has emerged as an important pathogen of cucurbits within the last few years in Hungary. The Hungarian isolates show a high biological variability, have specific nucleotide and amino acid sequences in the N-terminal region of coat protein and form a distinct branch in the phylogenetic tree. The virus is spread very efficiently in the field by several aphid species in a non-persistent manner. It can be transmitted by seed in holl-less seeded oil pumpkin (Cucurbita pepo (L) var Styriaca), although at a very low rate. Three isolates from seed transmission assay experiments were chosen and their nucleotide sequences of coat proteins have been compared with the available CP sequences of ZYMV. According to the sequence analysis, the Hungarian isolates belong to the Central European branch in the phylogenetic tree and, together with the ZYMV isolates from Austria and Slovenia, share specific amino acids at positions 16, 17, 27 and 37 which are characteristic only to these isolates. The phylogenetic tree suggests the common origin of distantly distributed isolates which can be attributed to widespread seed transmission.

The DNA form of a retroviroid-like element characterized in cultivated carnation species.

Carnation small viroid-like RNA (CarSV RNA) is a small (275 nt), circular molecule which is unique among plant viroid-like RNAs in having a tandemly repeated homologous DNA. This DNA form was found fused to DNA sequences of carnation etched ring caulimovirus (CERV) in certain Spanish carnation plants. The observation of a growth abnormality consisting of extensive shoot proliferation in cultivated carnations in Hungary prompted the molecular analysis of these plants, in which both CarSV RNA and DNA forms were detected. Several CarSV DNA sequences were characterized in various Dianthus caryophyllus cultivars which were symptomless or showed different symptoms. CarSV DNA forms showing minor sequence heterogeneities and deletions occurred in the same plant. Unit-length CarSV DNA sequences were proven to accumulate in the plant cell nucleus. The plants studied here were not infected by any of the viruses (including CERV) or other cellular pathogens described previously in carnation.