The movement proteins (NSm) of distinct tospoviruses peripherally associate with cellular membranes and interact with homologous and heterologous NSm and nucleocapsid proteins.

Tospovirus is the only genus containing virus species which infect plants in the Bunyaviridae family. The aims of this study were to understand the in vivo membrane association of the movement protein (NSm) of the tospovirus species Bean necrotic mosaic virus, Chrysanthemum stem necrosis virus, Tomato chlorotic spot virus and Tomato spotted wilt virus and the homologous and heterologous interactions among NSm and nucleocapsid protein (N). The results obtained by bimolecular fluorescence complementation (BiFC) assay and chemical treatments after membrane fractionation revealed that the four NSm proteins are associated with the biological membranes with the N- and C-termini oriented to the cytoplasm. BiFC analysis for protein-protein interactions showed: i) dimer formation for all NSm and N proteins; ii) interaction between NSm and the cognate N and iii) heterologous interactions between the NSm and N proteins. The implications of these interactions in the life cycle of tospoviruses are discussed.

Current status of viroid taxonomy.

Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of next-generation sequencing and bioinformatics for known and previously unrecognized viroids.

Ilarviruses of Prunus spp.: a continued concern for fruit trees.

Prunus spp. are affected by a large number of viruses, causing significant economic losses through either direct or indirect damage, which results in reduced yield and fruit quality. Among these viruses, members of the genus Ilarvirus (isometric labile ringspot viruses) occupy a significant position due to their distribution worldwide. Although symptoms caused by these types of viruses were reported early in the last century, their molecular characterization was not achieved until the 1990s, much later than for other agronomically relevant viruses. This was mainly due to the characteristic liability of virus particles in tissue extracts. In addition, ilarviruses, together with Alfalfa mosaic virus, are unique among plant viruses in that they require a few molecules of the coat protein in the inoculum in order to be infectious, a phenomenon known as genome activation. Another factor that has made the study of this group of viruses difficult is that infectious clones have been obtained only for the type member of the genus, Tobacco streak virus. Four ilarviruses, Prunus necrotic ringspot virus, Prune dwarf virus, Apple mosaic virus, and American plum line pattern virus, are pathogens of the main cultivated fruit trees. As stated in the 9th Report of the International Committee on Taxonomy of Viruses, virions of this genus are "unpromising subjects for the raising of good antisera." With the advent of molecular approaches for their detection and characterization, it has been possible to get a more precise view of their prevalence and genome organization. This review updates our knowledge on the incidence, genome organization and expression, genetic diversity, modes of transmission, and diagnosis, as well as control of this peculiar group of viruses affecting fruit trees.

Contribution of topology determinants of a viral movement protein to its membrane association, intracellular traffic, and viral cell-to-cell movement.

The p7B movement protein (MP) of Melon necrotic spot virus (MNSV) is a single-pass membrane protein associated with the endoplasmic reticulum (ER), the Golgi apparatus (GA), and plasmodesmata (Pd). Experimental data presented here revealed that the p7B transmembrane domain (TMD) was sufficient to target the green fluorescent protein (GFP) to ER membranes. In addition, the short extramembrane regions of p7B were essential for subsequent ER export and transport to the GA and Pd. Microsomal partitioning and bimolecular fluorescence assays supported a type II topology of p7B in planta. Mutations affecting conventional determinants of p7B membrane topology, such as the TMD secondary structure, the overall hydrophobicity profile, the so-called "aromatic belt," and the net charge distribution on either side of the TMD, were engineered into infectious RNAs to investigate the relationship between the MP structure and MNSV cell-to-cell movement. The results revealed that (i) the overall hydrophobic profile and the α-helix integrity of the TMD were relevant for virus movement, (ii) modification of the net charge balance of the regions flanking both TMD sides drastically reduced cell-to-cell movement, (iii) localization of p7B to the GA was necessary but not sufficient for virus movement, and (iv) membrane insertion was essential for p7B function in virus movement. Our results therefore indicate that MNSV cell-to-cell movement requires sequential transport of p7B from the ER via the GA to Pd, which is modulated by a combination of several signals with different strengths in the extramembrane regions and TMD of the MP.

First Report of Apricot latent virus and Plum bark necrosis stem pitting-associated virus in Apricot from Spain.

Representing 2% of world production, 20,000 ha of apricot (Prunus armeniaca L.), are cultivated in Spain, primarily in the southeast. A survey was conducted during the spring of 2008 in orchards in the region of Murcia to assess the incidence of several stone fruit viruses. Leaf and fruit samples from 160 trees from 40 orchards were collected randomly for reverse transcription (RT)-PCR analysis. Total RNA extracted (3) from leaves and fruits was tested by a multiplex one-step RT-PCR protocol with a mix of primers that detect eight distinct viruses (4). Amplicons of 250 bp expected for Plum bark necrosis stem pitting-associated virus (PBNSPaV), corresponding to part of the heat shock 70 protein gene, were obtained from four trees and amplicons of 700 bp expected for Apricot latent virus (ApLV), corresponding to part of the coat protein (CP) gene, were obtained from two trees. In all cases, amplicons were obtained using RNA extracted from leaf and fruit tissues. RT-PCR results were confirmed by uniplex RT-PCR with primers specific for each virus and dot-blot hybridization with virus-specific digoxygenin-labeled RNA probes (2). To further characterize the new viruses, we designed primers to amplify specifically the CP gene of ApLV (5'-CCCGACCATGGCTACAAGC-3' and 5'-TTGCCGTCCCGATTAGGTTG-3') and the minor CP gene of PBNSPaV (5'-GAACAAACTACAGCAGCACC-3' and 5'-CAAGGGTAGGACGGGTAACGC-3'). Amplicons of 1,500 and 950 bp corresponding to the ApLV and PBNSPaV CP genes, respectively, were purified from agarose gels and cloned in the pTZ57R plasmid (Fermentas, Burlington, Ontario, Canada). Blastp analysis of the full-length ApLV CP sequence from one infected tree (GenBank Accession No. GQ919051) revealed 86% amino acid (aa) similarity to the single full-length ApLV CP sequence available (No. AAC16234) and 79 and 66.9% similarity to Peach sooty ringspot virus (No. AAG48314) and Apple stem pitting virus (No. NP604468), respectively. Identity/similarity analysis of the full-length PBNSPaV minor CP genes using the Matrix Global Alignment Tool software, version 2.02 (1), revealed 98.8 to 99.6% aa similarity between the Spanish PBNSPaV isolates (Nos. GQ919047, GQ919048, GQ919049, and GQ919050) and 97.1 to 97.4% with the PBNSPaV isolate from the United States (No. EF546442). None of the six infected trees were associated with any particular field symptoms. Five infected trees were cv. Búlida and one was native cv. Murciana, which was infected with ApLV. All infected trees were located in geographically separated orchards. The incidence of ApLV and PBNSPaV was 1.25 and 2.5%, respectively. The low incidence of both viruses together with the scattered geographic distribution could be due to the recent introduction of virus-contaminated plants, although we cannot exclude that it is a consequence of a low dissemination rate. Even though no symptoms were observed, we cannot discard that the infection could affect fruit production or flowering or even cause a synergistic effect in mixed infection with other stone fruit viruses, a risk especially relevant considering the total area of cultivated apricot. To our knowledge, this is the first report of ApLV and PBNSPaV in Spain. References: (1) J. J. Campanella et al. BMC Bioinformatics 4:29, 2003. (2) M. C. Herranz et al. J. Virol. Methods 124:49, 2005. (3) D. J. Mackenzie et al. Plant Dis. 81:222, 1997. (4) J. A. Sánchez-Navarro et al. Eur. J. Plant Pathol. 111:77, 2005.

Multiplex polymerase chain reaction (PCR) and real-time multiplex PCR for the simultaneous detection of plant viruses.

Multiplex Polymerase Chain Reaction (PCR) can be used for the simultaneous detection of plant viruses. Multiple primer pairs or polyvalent primer pairs can be used to detect and identify several viruses in a single PCR.

First Report of Avocado sunblotch viroid in Avocado from Michoacán, México.

The State of Michoacán, México cultivates approximately 100,000 ha of avocados (Persea americana M.) (4). During a survey from 2006 to 2007 in cv. Hass avocado groves in Tingambato County, in the State of Michoacán, deep yellow spots and streaks, which sometimes became necrotic or reddish, were observed on the skin of fruits and the pulp of the fruit also showed big yellow spots. Some young shoots developed fine, yellow streaks, and leaves of symptomatic trees sometimes showed irregular, white-to-yellow spots. These symptoms were similar to those recorded for Avocado sunblotch viroid (ASBVd) (3). To determine if ABSVd was associated with these symptoms, total RNA extracted (1) from the skin and pulp of symptomatic and asymptomatic fruits and also from leaves and bark of shoots from five trees collected in a commercial plot in Tingambato County was tested by a one-step reverse transcription (RT)-PCR protocol using one primer pair to amplify specifically the complete ASBVd genome sequence (3). All 30 samples of skin and pulp of fruits, leaves, and cortex of shoots from symptomatic trees yielded two PCR fragments with estimated sizes of 250 and 500 base pairs (bp) corresponding to the putative monomeric and dimeric forms of ASBVd, respectively. The 500-bp RT-PCR fragments obtained from the different samples were purified from an agarose gel and cloned. The 249-bp nucleotide sequence of the ASBVd genomic monomer was determined using the clones from the fruit skin from sample Arb No. 3 (GenBank Accession No. EU888588), pulp from sample Arb No. 5 (GenBank Accession No. EU888590), leaves from samples Arb No. 15 (GenBank Accession No. EU888589) and Arb No. 8 (GenBank Accession Nos. EU888591 and EU888592), and cortex of shoots from sample Arb No. 16 (GenBank Accession Nos. EU888593, EU888594, EU888595, EU888596, and EU888597). BLAST analysis of the ASBVd sequences showed a range of 98 to 100% nucleotide identity to ASBVd sequences (GenBank Accession Nos. AF404064, AF404051, or AF229821). A clone of the Michoacán ASBVd (GenBank Accession No. EU888593) was used to synthesize a Dig-High Prime-UTP-T7 (Roche, Mannheim, Germany) fluorescent riboprobe complementary to the ASBVd plus strand to perform a dot-blot analysis as described previously (2). All ASBVd samples positive by RT-PCR gave a strong signal in the dot-blot analysis. This riboprobe will be used to index the ASBVd in other commercial avocado groves in Michoacán. To our knowledge, this is the first report of ASBVd in Michoacán, México. References: (1) D. J. Mackenzie et al. Plant Dis. 81:222, 1997. (2) J. A. Sánchez-Navarro et al. Plant Pathol. 47:780, 1998. (3) R. J. Schnell et al. Plant Dis. 81:1023, 1997. (4) D. Téliz and A. Mora. El aguacate y su Manejo Integrado. Mundiprensa, Mexico City, 2007.

Low genetic variability in the coat and movement proteins of American plum line pattern virus isolates from different geographic origins.

Until very recently isolates of American plum line pattern virus (APLPV) had not been reported from outside North America. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of eight APLPV isolates from five Mediterranean countries were determined. Sequence analysis showed that both MP and CP genes are highly conserved irrespective of geographic origin. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that these proteins are under the effect of negative purifying selection. The MP and CP of APLPV possess most of the functional motifs described for other members of the genus Ilarvirus.

Mature monomeric forms of Hop stunt viroid resist RNA silencing in transgenic plants.

Viroids, small non-coding pathogenic RNAs, are able to induce RNA silencing, a phenomenon that has been associated with the pathogenesis and evolution of these small RNAs. It has been recently suggested that viroids may resist this plant defense mechanism. However, the simultaneous degradation of non-replicating full-length viroid RNA, and the resistance of mature forms of viroids to RNA silencing, have not been experimentally demonstrated. Transgenic Nicotiana benthamiana plants expressing a dimeric form of Hop stunt viroid (HSVd) that have the capability to cleave and circularize this viroid RNA were used to address this question. A reporter construct, consisting of a full-length HSVd RNA fused to GFP-mRNA, was agroinfiltrated in these plants and its expression was suppressed. Interestingly, both circular and linear HSVd molecules were stable and able to traffic through grafts in these restrictive conditions, indicating that the mature forms of HSVd are able, in some way, to resist the RNA-silencing mechanism. The observation that a full-length HSVd RNA fused to GFP-mRNA, but not circular and/or linear viroid forms, was fully susceptible to RNA degradation strongly suggests that structures adopted by the free mature monomer protect the pathogenesis-associated forms of the viroid from RNA silencing.

A peptide derived from a single-modified viroid-RNA can be used as an "in vivo" nucleolar marker.

Viroids are small, single-stranded, circular, non-coding pathogenic RNAs. Hop stunt viroid (HSVd) is characterized by possesses rod-like structure and replicate in the host nuclei. Green fluorescent protein (GFP) fusions with transit sequences or entire proteins can be used for deliberate labelling of particular cell compartments. Different GFP-fusions have been obtained to selectively illuminate different organelles and membranes in many cell types. However, as far as we know, examples for established efficient markers for nucleoli are scarce. In this work, a viroid-RNA was made translatable by inserting an ATG at position 1 and fused to the GFP. The results showed that the resultant fusion can be used as an efficient "in vivo" nucleolar marker in "real time" cellular observations. Thus, this construct can be a very useful tool to study processes related with nucleolus functions.

RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins.

Advances in structural and biochemical properties of carmovirus movement proteins (MPs) have only been obtained in p7 and p9 from Carnation mottle virus (CarMV). Alignment of carmovirus MPs revealed a low conservation of amino acid identity but interestingly, similarity was elevated in regions associated with the functional secondary structure elements reported for CarMV which were conserved in all studied proteins. Nevertheless, some differential features in relation with CarMV MPs were identified in those from Melon necrotic virus (MNSV) (p7A and p7B). p7A was a soluble non-sequence specific RNA-binding protein, but unlike CarMV p7, its central region alone could not account for the RNA-binding properties of the entire protein. In fact, a 22-amino acid synthetic peptide whose sequence corresponds to this central region rendered an apparent dissociation constant (K(d)) significantly higher than that of the corresponding entire protein (9 mM vs. 0.83-25.7 microM). This p7A-derived peptide could be induced to fold into an alpha-helical structure as demonstrated for other carmovirus p7-like proteins. Additionally, in vitro fractionation of p7B transcription/translation mixtures in the presence of ER-derived microsomal membranes strongly suggested that p7B is an integral membrane protein. Both characteristics of these two small MPs forming the double gene block (DGB) of MNSV are discussed in the context of the intra- and intercellular movement of carmovirus.

Functional analysis of the five melon necrotic spot virus genome-encoded proteins.

Function of the melon necrotic spot virus (MNSV) genome-encoded proteins (p29, p89, p7A, p7B and p42) has been studied. Protein-expression mutants of an infectious, full-length cDNA clone of a Spanish MNSV-Al isolate and a recombinant green fluorescent protein (GFP)-expressing virus were used in infection bioassays on melon plants. Results revealed that p29 and p89 are both essential for virus replication, whereas small proteins p7A and p7B are sufficient to support viral movement between adjacent cells operating in trans. It is also demonstrated that, in addition to its structural role as coat protein, p42 is an important factor controlling symptoms and is required for systemic transport. Moreover, both p42 and p7B, among all of the MNSV-encoded proteins, were able to delay RNA silencing in transient-expression assays on GFP-transgenic Nicotiana benthamiana plants. Finally, the presence of p42 also produced an enhancing effect on local spread similar to that of potyviral helper component proteinase (HC-Pro), probably due to its RNA silencing-suppression ability.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Identification of translocatable RNA-binding phloem proteins from melon, potential components of the long-distance RNA transport system.

Phloem proteins (P-proteins) are an enigmatic group of proteins present in most angiosperm species. The best characterized P-proteins (PP1 and PP2) are synthesized in companion cells, transported into sieve elements via pore plasmodesmata and translocated through the plant. Characteristics such as long-distance translocation, RNA-binding activity and capacity of increasing plasmodesmata exclusion size suggest that certain phloem proteins could be involved in RNA transport within the plant, forming translocatable ribonucleoprotein complexes with endogenous or pathogenic RNAs. Long-distance movement of RNA through the phloem is a process known to occur, but both the mechanisms involved and the components constituting this potential information network remain unclear. Here, we demonstrate that several melon phloem proteins have a wide RNA-binding activity. Serological assays strongly suggest that one of these proteins is the melon phloem protein 2 (CmmPP2). Mass spectrometry analysis undoubtedly identifies another one as the recently characterized melon phloem lectin (CmmLec17). Grafting experiments demonstrate that the CmmLec17 is a translocatable phloem protein, able to move through intergeneric grafts from melon to pumpkin. Translocatability and RNA-binding activity was also demonstrated for an uncharacterized protein of approximately 14 kDa. In light of these results the possible involvement of these phloem proteins in the long-distance transport of melon RNAs is discussed.

Genetic variability in the coat protein genes of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus.

Available data suggests that lettuce big-vein disease is caused by the ophiovirus Mirafiori lettuce big-vein virus (MLBVV) but not by the varicosavirus Lettuce big-vein-associated virus (LBVaV), although the latter is frequently associated with the disease. Since the disease occurs worldwide, the putative coat protein (CP) open reading frames of geographically distinct isolates of MLBVV and LBVaV were sequenced. Comparison of both nucleotide and amino acid sequences showed a high level of sequence similarity among LBVaV isolates. Phylogenetic analysis of LBVaV CP nucleotide sequences showed that most of the Spanish isolates clustered in a phylogenetic group whereas English isolates were more similar to the USA isolate. An Australian isolate was closely related to the Dutch isolate. Genetic diversity among MLBVV CP nucleotide sequences was higher ranging from 0.2% to 12%. Phylogenetic analysis of MLBVV CP nucleotide sequences revealed two distinct subgroups. However, this grouping was not correlated with symptom development on lettuce or the geographic origin of the MLBVV isolates. Finally, a quick method based on RFLP analysis of RT-PCR amplicons was developed for assigning MLBVV isolates to the two subgroups.

First Record of Hop stunt viroid in Canada.

"Tissue-printing" hybridization (3) for Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) was used to assess the sanitary status of stone fruit accessions in the Canadian Clonal Genebank (CCG) located in Harrow (Ontario). The Prunus spp. accessions in the CCG are primarily of Canadian origin; other countries of origin include the United States, the United Kingdom, Hungary, the Czech Republic, the Former Soviet Union, Spain, New Zealand, and Italy. All Prunus spp. accessions were donated to the Genebank from Canadian or American sources. Leaves were harvested in November 2003 from 336 trees (116 peach and nectarine, 84 sweet and sour cherries, 54 plum, 44 apricot, and 38 of other cherries) representing 267 accessions. No visible symptoms were observed during the collection of the accessions to be evaluated. The petioles were excised at the base and imprinted on a nylon membrane in triplicate for each sample. The membranes were air dried and submitted by mail to the laboratory. The digoxigenin-labeled riboprobes used for hybridization were obtained by T7 RNA polymerase transcription of the linearized plasmids pHSVd (1) and pPLMVd (2). Thirty stone fruit samples were infected by viroids. PLMVd occurred in 28 peach and nectarine samples, representing the following cultivars and selections: Harblaze Hardired, Harko, Earlyvee, Harbelle, Harken, Harland, Harrow Beauty, Harrow Rubirose, HW264, Redhaven, Silver Gold, Suncling, V68101, Vanity, Veeglo, Velvet, Vesper, Villa Doria, and Vulcan. PLMVd-infected samples represented 24.1% of the tested peaches and nectarines. PLMVd finding confirms previous reports of the viroid in Canada from British Columbia and Ontario. Two CCG apricot accessions, 'Bulida' and 'Velkopavlovicka', were found to be infected with only HSVd, representing 4.5% of tested apricot samples. These samples, determined to be positive by tissue-printing hybridization, were also positive by reverse transcription-polymerase chain reaction (RT-PCR) (1). In addition, nucleotide sequences of the PCR products were obtained. The 'Bulida' isolate showed 100% homology to a Spanish isolate, apr9, while the 'Velkopavlovicka' isolate showed 99% homology to an Italian isolate. Since HSVd has not been previously reported in Canada (4), to our knowledge, this report documents its first detection in the country. This report may prompt the inclusion of regular testing for HSVd in existing Prunus spp. virus testing programs in Canada. References: (1) N. Astruc et al. Eur. J. Plant Pathol. 102:837, 1996. (2) M. Badenes et al. Acta Hortic. 472:565, 2001. (3) V. Pallás et al. Page 135 in: Virus and Virus-Like Diseases of Stone Fruits, with Particular Reference to the Mediterranean Region. A. Myrta et al., eds. CIHEAM-IAMB, 2003. (4) R. Singh et al. Page 255 in: Viroids. A. Hadidi et al., eds. CSIRO Publishing, Australia, 2003.

Detection of melon necrotic spot virus in water samples and melon plants by molecular methods.

Melon necrotic spot virus (MNSV) is a water and soil-borne pathogen affecting species of the Cucurbitaceae family both in hydroponic and soil crops. Molecular methods for detecting MNSV in water samples, nutrient solutions and melon plants were developed. For this purpose, water samples from a water source pool of a hydroponic culture or from the recirculating nutrient solution were concentrated by ultracentrifugation or PEG precipitation followed by RT-PCR analysis. Both concentration methods were suitable to allow the detection of MNSV and represent, as far as we know, the first time that this virus has been detected in water samples. A non-isotopic riboprobe specific for MNSV was obtained and used to detect the virus in plant tissue. Different parts of mechanically infected plants were examined including the roots, stems, inoculated cotyledons and young leaves. Excluding the inoculated cotyledons, the tissues showing the highest accumulation levels of the virus were the roots. The potential inclusion of such tools in management programs is discussed.

First Report of Peach latent mosaic viroid on Peach in Uruguay.

Peach latent mosaic viroid (PLMVd) (2) is widely distributed and causes yellow, chlorotic mosaics and delayed foliation, flowering, and ripening. Infected fruits display a cracked suture and are often dented, misshapen, frequently flattened, and discolored. In the greenhouse, PLMVd natural isolates are divided into severe or latent strains depending on whether they induce leaf symptoms on seedlings of the peach indicator GF-305. PLMVd was detected in 2001 during a survey in three locations in the Canelones Department, the main peach producing area in Uruguay. Fifty samples were tested for the presence of five viruses: Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern virus (APLPV), Plum pox virus (PPV) and Apple chlorotic leaf spot virus (ACLSV); samples were also tested for the viroids affecting stone fruits, Hop stunt viroid (HSVd) and PLMVd. The analyses were completed with molecular hybridization using specific nonisotopic riboprobes for each virus (4). PLMVd, undescribed in Uruguay, was detected in 9 of 50 samples in three peach cultivars, Scarlet Pearl, EarliGrande, and Barcelo. The PLMVd-positive sample for 'Scarlet Pearl' showed mild mosaic symptoms on leaves whereas the two PLMVd-positives of 'EarliGrande' showed clear calico type symptoms. The remaining PLMVd-positive samples belonged to 'Barcelo' and showed no symptoms or mild chlorosis. The first two cultivars were imported from the United States, a source with a high percentage of PLMVd infections in peach germ plasm (1). In five of nine PLMVd-positive samples, the viroid occurred with PNRSV and in one with PDV and PNRSV. PLMVd has previously been reported in Brazil (3), but to our knowledge, this is the first report of PLMVd in Uruguay. These results reveal the importance of following strict sanitary practices with plant material used for propagation. Molecular tools are available to prescreen scion and rootstock sources for PLMVd. References: (1) M. L. Badenes and G. Llácer. Acta Hortic. 309:565, 1998. (2) R. Flores et al. Res. Virol. 141:109, 1990. (3) A. Hadidi et al. Plant Dis. 81:154, 1997. (4) V. Pallás et al. Detection of plant RNA viruses by non-isotopic dot-blot hybridization. Pages 461-468 in: Plant Virus Protocols: From Virus Isolation to Transgenic Resistance. G. Foster and S. Taylor, eds. Humana Press, Totowa, NJ. 1998.

Molecular variability of twenty-one geographically distinct isolates of Carnation mottle virus (CarMV) and phylogenetic relationships within the Tombusviridae family.

The complete nucleotide sequence of a Spanish isolate of Carnation mottle carmovirus (CarMV) has been determined. Phylogenetic analyses were carried out with the replicase, coat protein (CP) and the putative movement proteins (p7 and p9) of CarMV with the homologous proteins of representative members of the different genera included within the family Tombusviridae. These analyses revealed that phylogenetic trees obtained depended on the protein analyzed, and that the best correlation with taxonomy grouping was observed with the replicase and, to a lesser extent, with CP phylogenies. This result indicates that speciation has evolved as a consequence of different selection pressures to different genomic regions. In addition, the CP, p7 and p9 coding sequences of twenty-one CarMV isolates from nine different countries have been determined. Comparative analyses revealed that CarMV isolates separated in time and space show a very high genetic stability. A division in three protein motifs is proposed for the p7 movement protein, based on the homology data presented here and on our previous identification of RNA binding sequences and structural characterization of the protein. Interestingly, a remarkable covariation in the amino acid sequence was found for the CP between Pro164 (located at the S domain) and Lys331 (within the P domain), by which a change Pro164 --> Ala correlated with a change Lys331 --> Asn, strongly suggesting the existence of tertiary interactions between these two regions of the protein. In addition, this perfect covariation allows to segregate the 23 CarMV isolates characterised so far into two main groups that we propose to name as group PK and group AN for further studies.

Identification of an in vitro ribonucleoprotein complex between a viroid RNA and a phloem protein from cucumber plants.

We used the interaction of Hop stunt viroid (HSVd) and cucumber plants to investigate the involvement of phloem proteins in the systemic transport of RNA molecules. A ribonucleoprotein complex, stable even at high salt and temperature conditions, was detected in vitro between HSVd-RNA and the phloem exudate obtained from sectioned internodes from cucumber plants. The phloem protein 2 was recovered from this ribonucleoprotein complex and its RNA-binding properties as demonstrated by gel retardation analysis. The involvement of this protein in the movement of RNAs in cucumber is discussed.