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) is the causal agentof the economically important sharka disease of stone fruit trees. To date, sixstrains of PPV have been recognized (PPV-M, -D, -Rec, -EA, -C, and -W),differing by their serological and molecular properties [1], the PPV-M, -D, and-Rec strains being the most common and widespread. The minor EA (El Amar)strain consists of divergent isolates, so far geographically limited to Egypt. TheoriginalElAmarisolatewaspartiallysequencedintheearly1990s[4]andwaslaterused to produce strain-specific monoclonal antibodies [3]. Recently, the completenucleotide sequence of an isolate belonging the EA strain and deriving from theoriginal isolate was reported [2]; (AM157175). We report here the completedfull-length sequence of the original ElAmar isolate kept in the virus collection ofINRA Bordeaux.This new sequence shows 3.8% divergence from theAM157175sequence, providing evidence for a higher variability within the PPV-EA strainthan in other PPV strains.

Serological reactivity to Plum pox virus (PPV) antisera has been described in several Prunus sources of Asian origin that are free of PPV infection. Using polyvalent or specific PCR assays, the presence of three closely related agents in two of these sources, Prunus mume cv. Bungo and P. persica cv. Ku Chu'a Hung, was demonstrated. Similarities in genome organization and sequence comparisons indicate that these agents should be regarded as members of the genus Foveavirus, their only singular trait being a very large (>800 nt) 3' non-coding region (NCR), as compared to the ca. 130-180 nt 3' NCR observed in other Foveaviruses. The three agents are very divergent from known Foveaviruses but are also significantly removed one from the others, with overall nucleotide sequence identity levels in the sequenced region of ca. 74-76% and of only 60.8-67.5% in their complete CP gene (61.9-71.3% amino acid sequence identity). Given the species discrimination criteria in the family Flexiviridae, these three agents should be regarded as three related yet distinct new viruses belonging to the Foveavirus genus, for which the names Asian prunus virus 1, 2 and 3 are proposed. Evidence is provided for the presence of variants of these new viruses in other Prunus germplasm of Asian origin.

International audience; Characterization of a seemingly new spherical virus isolated from severely affected plum trees in south-western France indicated that its divided genome is composed of two single-stranded, polyadenylated RNAs of approximately 7.4 and 3.7 kb. Its particles are composed of three coat protein subunits of approximately 23, 23.5, and 24.5 kDa. Partial sequencing of the genomic RNAs indicated that this new virus, tentatively named stocky prune virus (StPV), is distantly related to the two sequenced cheraviruses, cherry rasp leaf virus (CRLV) and apple latent spherical virus (ALSV). StPV should be regarded as a new member in the unassigned genus Cheravirus.

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Un nouveau virus, le virus X du bananier (Banana virus X, BVX), a été identifié chez le bananier en l'absence de symptômes associés, et une partie de son ARN génomique a été séquencée. L'analyse des 2917 résidus 3' terminaux de ce génome a permis de mettre en évidence 5 cadres ouverts de lecture. Sur la base d'homologies de séquence, des fonctions ont pu être attribuées à chacune des protéines correspondantes. Il s'agit respectivement d'une protéine associée à la réplication et comportant le motif S/TGx3Tx3NS/Tx22GDD caractéristique des ARN polymérases ARN dépendantes (RdRp) des virus Sindbis-associés, de trois protéines impliquées dans le mouvement et organisées en un "triple gene block" (TGBpl, TGBp2 et TGBp3) et d'une protéine de capside (CP). L'organisation du génome du BVX et des homologies de séquence le rapprochent de membres de la famille des Flexiviridae, dans laquelle il définit toutefois un nouveau genre, les Banexvirus. Le BVX produit plusieurs types d'ARN défectifs (D RNA) dérivés de son ARN génomique. Trois sites donneurs/accepteurs de recombinaison distincts comportant des répétitions directes et impliqués dans la synthèse des D RNA ont été mis en évidence. Tous trois sont situés dans les ORF codant les protéines TGBp1 et CP, et aboutissent à un ORF non fonctionnel, contrairement à la situation rencontrée chez d'autres membres de la famille des Flexiviridae. Une première étude épidémiologique a été conduite à petite échelle en Guadeloupe. Elle a révélé que le BVX était relativement répandu chez les bananiers sauvages ou cultivés. L'analyse moléculaire d'une partie de la séquence de l'ORF1 a également montré que contrairement à un autre Flexiviridae infectant le bananier, le Banana mild mosaic virus, les populations de BVX semblent montrer un faible niveau de diversité. (Texte intégral)

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Les maladies virales constituent au niveau mondial d'importantes contraintes pour la multiplication et la diffusion de germplasm Musa et de nouvelles variétés de bananiers. Cette situation a motivé la mise en place d'un programme de recherche interdisciplinaire visant à contrôler le virus de la mosaïque en tirets du bananier (BSV) et le virus de la mosaïque atténuée du bananier (BanMMV) et à développer un itinéraire technique global pour la production de germplasm indemne de virus. Des outils immuno-moléculaires sensibles et spécifiques ont été développés pour la détection du BSV et du BanMMV. Leur utilisation, associée à celle d'outils existant, qui permettent la détection des autres virus infectant les espèces Musa, rendent possible la sélection de clones parentaux sains pour la production de masse de vitroplants certifiés indemnes de virus. Par ailleurs, les outils de détection du BanMMV ont permis d'étudier l'épidémiologie de ce virus en Guadeloupe et de montrer l'existence d'une transmission par voie non végétative. Ils ont également permis la mise en évidence et la caractérisation d'une nouvelle espèce virale qui infecte les espèces Musa sans provoquer de symptômes, pour laquelle le nom Banana virus X est proposé. La multiplication in vitro est l'une des causes principales d'activation des séquences BSV intégrées (EPRV BSV) au génome de Musa balbisiana. Elle demeure pourtant la méthode la plus communément utilisée pour multiplier et diffuser le germplasm Musa ainsi que les nouvelles variétés hybrides de bananier. Il est donc nécessaire d'évaluer et de quantifier les effets de la culture in vitro sur l'activation des EPRV BSV. Ce travail a été entrepris chez deux cultivars de plantains naturels triploïdes (AAB) ainsi que chez un plantain hybride tétraploïde (AAAB). Les résultats de cette étude confirment l'effet activateur de la culture in vitro mais ils montrent également que, pour les cultivars et l'hybride étudiés, le pourcentage de vitroplants infectés via une activation des EPRV BSV ne dépasse pas 20%, puis qu'il décroît lorsque le nombre de cycles de culture in vitro augmente. Des travaux sont en cours afin d'étudier le comportement au champ des vitroplants indexés négatifs au terme des étapes de culture in vitro. Enfin, des techniques immuno-cytologiques ont permis d'élucider la structure des EPRV BSV. Ces travaux ouvrent la voie à la caractérisation des mécanismes moléculaires d'activation des EPRV BSV et, à plus long terme, à la mise au point de stratégies de lutte.

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International audience; A novel virus was identified in banana (Musa spp). Analysis of the last 2917 nucleotides of its positive strand genomic RNA showed five open reading frames corresponding, from 5′ to 3′, to a truncated ORF coding for a replication-associated protein, three ORFs coding for a movement-associated triple gene block (TGB) and a capsid protein (CP) gene. This genome organization is similar to that of some members of the Flexiviridae family such as potexviruses and foveaviruses. This virus was named Banana virus X (BVX). Comparative sequence analysis showed that BVX is only distantly related to other members of the Flexiviridae family, in which it appears to define a new genus. BVX produces defective RNAs derived from its genomic RNA by non-homologous recombination. Three distinct pairs of donor/acceptor recombination sites involving short direct nucleotide repeats were characterized, accounting for deletions of 1268, 1358 and 1503 nucleotides. Contrary to the situation encountered for Potexviruses, these recombination sites are located within the TGB1 and CP genes and result in a truncated TGB1 protein.

A polyvalent nested reverse transcription-polymerase chain reaction (RT-PCR) test using degenerate primers containing inosine (polyvalent degenerate oligonucleotides [PDO]) was developed for filamentous fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus. The 362-bp product was amplified from nucleic acid extracts obtained from Prunus and Malus leaf samples. All the viruses targeted were detected, demonstrating the polyvalence of the test. The variability of a collection of Apple chlorotic leaf spot virus isolates was analyzed using the sequence of the PDO RT-PCR amplified cDNAs. The technique was also used to screen stone fruit materials infected with known agents or with virus-like graft-transmissible diseases of unknown etiology. The results obtained further validated the broad specificity of the assay, with positive amplification obtained for uncharacterized or partially characterized viruses associated with cherry and peach disorders. Sequencing the amplified PCR products either directly or after cloning allowed the identification of variants of known agents and the tentative identification of two new agents, a Trichovirus and a Foveavirus. In addition, sequence comparisons demonstrated that the sequence of the targeted region is phylogenetically informative and of predictive taxonomic value.

Background: Banana and plantain (Musa spp) are an essential staple food and account for one of the most widely exported fruits in the world. This crop is susceptible to numerous pests and pathogens, including five reported and characterized viruses which are severe constraints to exchanges of Musa germplasm. A study of the molecular variability of Banana mild mosaic virus (BanMMV) isolates infecting banana in Guadeloupe lead to the identification of a short nucleotide sequence belonging to a distinct viral species. Methods: The partial nucleotide sequence of the RNA genome of this agent was determined from double stranded RNA following 3' RACE amplification. Results: Five open reading frames were identified from the analysis of the last 2917 nucleotides of RACE-amplified sequence. They correspond, from 5' to 3', to a truncated ORF encoding replication-associated protein, three ORFs encoding a movement-associated triple gene block (TGB) and a capsid protein (CP) gene. This deduced genome organization is similar to that of some members of the Flexiviridae family such as Potexviruses and Foveaviruses. Sequence comparisons suggest that this virus is a new virus species in the Flexiviridae family, for which the name Banana virus X (BVX) is proposed. Sequence comparisons also showed that BVX is only distantly related to other members of the Flexiviridae family, in which it does not appear to belong to any existing genus. There is currently no evidence that infection by BVX can cause any visible symptoms in Musa sp. Furthermore, it was shown that BVX produces defective RNAs derived from its genomic RNA by non-homologous recombination. Three distinct pairs of donor/acceptor recombination sites involving short direct nucleotide repeats were characterized. Contrary to the situation encountered for the Potexviruses, these recombination sites are located within the TGB1 and CP genes and the recombination event results in a truncated TGB1 protein. A small scale study of the molecular variability of BVX was performed on Musa samples collected in different locations in Guadeloupe. It showed that BVX is reasonably distributed in Musa plants in Guadeloupe and displays a very low level of molecular variability, which is in marked contrast to the situation encountered for BanMMV, another member of the Flexiviridae family infecting Musa spp.

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Phylogenic studies undertaken on Banana mild mosaic virus (BanMMV) populations led to the characterisation of a 230 nt sequence originating from a yet unidentified virus in one of the 69 accessions analysed. No visible symptom was associated with the presence of this virus. 3' RACE experiments were performed on double stranded RNAs purified from this accession, enabling the cloning and sequencing of about 2.3 kb of the 3' end region of the genome of this virus. Based on sequence comparisons, the analysis of the cloned sequence showed the presence of 5 open reading frames (ORFs) encoding an RNA dependent RNA polymerase (RdRp), three proteins involved in virus movement and organised as a triple gene block (TGB1, TGB2 and TGB3) and a coat protein, respectively. The genomic RNA ends in a short 3' non-coding region followed by a polyA tail. Sequence comparisons and phylogenic analyses performed on both nucleotide and amino acid sequences led to the conclusion that this virus, tentatively named Banana X virus (BanXV) seems to represent a new agent since it shows only distant relationships with viral sequences present in databanks. It probably belongs to a new genus within the Flexiviridae family and is very distantly related to members of other genera in this family such as the genus Foveavirus. Results of further molecular work aimed at unravelling BanXV expression strategy will also be presented and discussed (Texte intégral).

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Prunus davidiana (Carr.), a wild species with poor fruit quality that is related to peach [Prunus persica (L.) Batsch], is used as a source of resistance to pests and diseases in peach breeding programs. Two genotypes of P. davidiana were studied for fruit biochemical composition and compared to three genotypes of P. persica (`Summergrand', `Bailey' and `Pamirskij'), and two P. persica × P. davidiana hybrids. Fruit of P. davidiana clones had higher malic acid, neochlorogenic and cryptochlorogenic acid and lower sucrose concentrations than fruit of all P. persica genotypes, even poor-quality Bailey. Differences in biochemical composition could be related to sensory evaluation. P. persica × P. davidiana hybrids had intermediate values between their parents for neochlorogenic acid concentration. They were similar to the P. persica parent for total soluble sugar, malic and citric acid, amino acid and catechin concentrations, indicating possible rapid progress for fruit quality in a breeding program.

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The identification of genes involved in variation of peach fruit quality would assist breeders in creating new cultivars with improved fruit quality. Major genes and quantitative trait loci (QTLs) for physical and chemical components of fruit quality have already been detected, based on the peach [ Prunus persica (L.) Batsch] cv. Ferjalou Jalousia((R)) (low-acid peach) x cv. Fantasia (normally-acid nectarine) F(2) intraspecific cross. Our aim was to associate these QTLs to structural genes using a candidate gene/QTL approach. Eighteen cDNAs encoding key proteins in soluble sugar and organic acid metabolic pathways as well as in cell expansion were isolated from peach fruit. A single-strand conformation polymorphism strategy based on specific cDNA-based primers was used to map the corresponding genes. Since no polymorphism could be detected in the Ferjalou Jalousia((R)) x Fantasia population, gene mapping was performed on the almond [ Prunus amygdalus ( P. dulcis)] cv. Texas x peach cv. Earlygold F(2) interspecific cross from which a saturated map was available. Twelve candidate genes were assigned to four linkage groups of the peach genome. In a second step, the previous QTL detection was enhanced by integrating anchor loci between the Ferjalou Jalousia((R)) x Fantasia and Texas x Earlygold maps and data from a third year of trait assessment on the Ferjalou Jalousia((R)) x Fantasia population. Comparative mapping allowed us to detect a candidate gene/QTL co-location. It involved a cDNA encoding a vacuolar H(+)-pyrophosphatase ( PRUpe;Vp2) that energises solute accumulation, and QTLs for sucrose and soluble solid content. This preliminary result may be the first step in the future development of marker-assisted selection for peach fruit sucrose and soluble solid content.

International audience; Analysis of the P1C124 source (associated with the cherry necrotic mottle leaf (CNML) disease) revealed the presence of two different viral agents. The complete nucleotide sequence of one of these agents, P1A, had an overall nucleotide sequence similarity of 83% with a previously sequenced North American isolate of Cherry green ring mottle virus (CGRMV) and should therefore be regarded as an European isolate of CGRMV. Approximately 2 kb of the 5′ end of the genome of the second agent, P1B, were also sequenced and were shown to be 82% homologous with Cherry necrotic rusty mottle virus (CNRMV), another member of the Foveavirus genus. The possible involvement of CGRMV-P1A and of CNRMV-P1B in the etiology of the CNML disease is discussed.

International audience; Peach asteroid spot (PAS) and peach sooty ringspot (PSRS) are two diseases of stone fruit trees of unknown aetiology. The use of a cRNA probe of the newly described Apricot latent virus (ApLV), a tentative member of the Foveavirus genus, indicated the presence of cross-hybridizing agents in PAS isolate LA2 and in PSRS isolates Caserta 12 and Clava J4. Analysis of dsRNA patterns revealed in each case the presence of a major dsRNA band of about 9.6 kbp. The purified dsRNAs were used to obtain cDNA clones for isolates LA2 and Caserta 12. Sequence analysis of a 1.1 kbp cDNA clone from isolate LA2 showed very high homology with the known ApLV sequence, indicating that this isolate represents a closely related variant of ApLV. Sequence analysis of a 3.06 kbp Caserta 12 cDNA clone representing the 3′ region of the genome revealed a genomic organization similar to that reported for other members of the Foveavirus genus, including the triple gene block and a large, 43.6 kDa coat protein. Sequence comparison with the CP gene of ApLV, the only sequenced region so far for this virus, showed an overall homology of 78%. These results indicate that the foveavirus represented by the Caserta 12 isolate of PSRS disease may be regarded as a distant variant of ApLV. The present results indicate that the viral agents associated with peach asteroid spot and peach sooty ringspot diseases might be variants of the recently described ApLV.

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The protein level and regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, involved in malic acid synthesis) was studied during the fruit development of two grape (Vitis vinifera L.) varieties, ‘Cabernet Sauvignon’ and ‘Gora Chirine’, with berries of normal and low organic acid content, respectively. The protein level and in vitro activity were higher in the low-acid variety than in the normal-acid variety for most stages. In vivo PEPC activity, measured using 14 CO2 labelling, was significantly higher in the low-acid variety than in the normal-acid variety about 1 week before and 1 week after veraison (the day which corresponds to the onset of ripening). However, partitioning into malate was the same for both varieties. Antibodies raised against the N-terminal part of SorghumPEPC recognised the grape berry PEPC, indicating the presence of the consensus phosphorylation site involved in PEPC regulation. PEPC phosphorylation status was estimated by studying sensitivity to pH and malate. Grape berry PEPC appeared more sensitive to low pH and malate during ripening (IC50 malate, 0.2–0.7 mM) compared to during the earlier stages of development (IC50 malate, 1.2–2 mM) for both varieties. Therefore, in the normal-acid variety, PEPC seems to participate in controlling malic acid accumulation but does not seem to control the differences in malic acid concentration observed between the two varieties.

International audience; The synthesis of organic acids was studied during fruit development of two peach (Prunus persica L, Batsch) cultivars, Fantasia and Jalousia, having fruits with high and low organic acid content, respectively. The malate content was higher in cv. Fantasia than in cv, Jalousia at the end of the first rapid growth stage (50 days after bloom [DAB]). Malate and citrate contents,were higher in Fantasia than in Jalousia during the second rapid growth stage (from 100 DAB to maturity). The expression of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), which is involved in organic acid synthesis, was studied during peach fruit development. PEPC mRNA levels, and protein levels on a total soluble protein basis, peaked at 23 and 108 DAB in Fantasia. In Jalousia, they were very low at 23 DAB and reached levels similar to Fantasia at 108 DAB, For both cultivars, in vitro PEPC activity expressed on a dry weight basis was maximal at 24 DAB, decreased from 24 to 60 DAB, and then remained constant. The activity of peach fruit PEPC appeared extremely sensitive to malate (I-0.5 of 100 mu M for Fantasia and 65 mu M for Jalousia at pH 7.3) and low pH, PEPC may participate in the control of organic acid accumulation during fruit development in the normal-acid fruit of Fantasia, However, mechanisms other than organic acid synthesis might account for the differences in acidity between normal-acid and non-acid peach fruit.

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Changes in metabolites were studied during the fruit development of two greenhouse grown peach [Prunus persica (L.) Batsch] cultivars with low acidity (`Jalousia') or normal acidity (`Fantasia'). Both cultivars had the same sucrose concentration in fruit mesocarp at maturity. In the fruit juice, pH was higher and titratable acidity was lower for `Jalousia' than for `Fantasia' from 80 days after bloom to maturity. At four different times during fruit development, in vivo 13C NMR spectroscopy was used to measure the vacuolar pH of fruit mesocarp. At 55 days after bloom, the vacuolar pH of fruit mesocarp was not significantly different between `Jalousia' and `Fantasia', whereas the juice pH was different between cultivars. The three major organic acids in fruit mesocarp were malic, citric, and quinic acids for both cultivars. Citric acid concentrations were similar in both cultivars until ≈85 days after bloom and then became significantly higher in `Fantasia'. A significantly higher concentration in malic acid in `Fantasia' than in `Jalousia' was observed from the end of the first growth phase to maturity. At maturity, `Fantasia' fruit had two and five times more malic and citric acid, respectively, than `Jalousia' fruit. The differences observed between `Jalousia' and `Fantasia' fruit for malic and citric acid concentrations accounted for the difference in titratable acidity. The differences in acid concentration appeared during the plateau between the two rapid growth phases of the fruit, i.e., far before the onset of maturation. The three major amino acids were asparagine, glutamic acid, and proline for both cultivars. Their concentration followed similar patterns in acid and low-acid fruit.

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Sorbitol and sucrose are the two main soluble carbohydrates in mature peach leaves. Both are translocated in the phloem, in peach as in other rosaceous trees. The respective role of these two soluble carbohydrates in the leaf carbon budget, and their phloem loading pathway, remain poorly documented. Though many studies have been carried out on the compartmentation and export of sucrose in sucrose-transporting species, far less is known about sorbitol in species transporting both sucrose and sorbitol. Sorbitol and sucrose concentrations were measured in several tissues and in sap, in 2-month-old peach (Prunus persica L. Batsch) seedlings, i.e. leaf blade, leaf main vein, petiole, xylem sap collected using a pressure bomb, and phloem sap collected by aphid stylets. The sorbitol to sucrose molar ratio depended on the tissue or sap, the highest value (about 7) found in the leaf main vein. Sorbitol concentration in the phloem sap was about 560 mM, whereas that of sucrose was about 140 mM. The lowest sorbitol and sucrose concentrations were observed in xylem sap collected from the shoot. The volume of the leaf apoplast, estimated by infiltration with 3 H-inulin, represented about 17% of the leaf blade water content. This volume was used to calculate a global intracellular concentration for each carbohydrate in the leaf blade. Following these simplifying assumptions, the calculated concentration gradient between the leaf's intracellular compartment and phloem sap is nil for sorbitol and could thus allow for the symplastic loading of the phloem of this alditol. However, infiltration of 14 C-labelled source leaves with 2 mM p-chloromercuribenzenesulfonic acid (PC-MBS), a potent inhibitor of the sucrose carrier responsible for phloem loading in sucrose-transporting plants, had a significant effect on the exudation of both labelled sucrose and sorbitol from the phloem. Therefore, in peach, which is a putative symplastic loader according to minor vein anatomy and sorbitol concentration gradients, apoplastic loading may predominate.

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The winegrape cv. Cabernet Sauvignon and the tablegrape cv. Gora Chirine (both Vitis vinifera L.) differ in the acidity of their berry juice. The changes in pH, titratable acidity and the concentration of compounds of the berry juice were measured from 20–30 d before veraison to harvest on plants cultivated in the glasshouse. The difference in acidity between the juice of the two varieties appeared before veraison. At harvest, the berry juice of Gora Chirine had a pH of 5.2 and titratable acidity (TA) of 32 meq/L while that of Cabernet Sauvignon had a pH of 3.2 and TA of 150 meq/L. To evaluate cellular compartmentation in the berry pericarp, vacuolar pH was measured on intact berries at two growth stages, using 13C nuclear magnetic resonance (NMR). Vacuolar pH did not differ significantly between the two cvs. The lower TA of Gora Chirine juice could be attributed to lower concentrations of all three major acids, malic, tartaric and citric, and to a higher concentration of potassium. At veraison, the malic acid concentration in the berry juice of Cabernet Sauvignon was about seven times higher than that of Gora Chirine. The accumulation of hexoses, especially glucose, occurred earlier during the berry growth cycle in Gora Chirine than in Cabernet Sauvignon. The osmotic potential of the Gora Chirine juice, before veraison, was lower than could have been expected because of low acidity. This was due to the compensatory effect of early accumulation of glucose. Amino acid concentration was significantly higher in Gora Chirine than in Cabernet Sauvignon, especially just before veraison. The role of organic acids in berry acidity and osmotic potential, and various metabolic processes that may lead to the difference in organic acid concentration, are discussed.

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