Efficacy of Pseudomonas chlororaphis subsp. aureofaciens SH2 and Pseudomonas fluorescens RH43 isolates against root-knot nematodes (Meloidogyne spp.) in kiwifruit

The Root-knot nematodes, Meloidogyne spp., are parasites of many crops and orchards, including kiwifruit trees. The Islamic Republic of Iran is among the leading kiwifruit producers in the world and M. incognita has been found as the dominant species responsible for severe loss of this crop. In order to evaluate the eff ectiveness of antagonistic bacteria on larval mortality, number of galls per plant and egg masses of nematode reduction, fifty local bacterial strains were isolated from root surrounding soils of kiwifruit plants in the northern production areas in Iran. Bacterial antagonists were characterized by morphological, physiological, biochemical and molecular methods. Two representative strains, showing the best nematicidal activity, were identif ed as Pseudomonas chlororaphis subsp. aureofaciens (isolate Sh2) and Pseudomonas fluorescens (isolate Rh43). They increased the percentage of larval mortality to 56:38% and 54:28% respectively in assays in vitro and showed excellent performance also in vivo with consistent reduction of number of galls (67:31% and 55:63%, respectively) and egg mass (86:46% and 84:29%, respectively) in plants. This study indicates that Pseudomonas chlororaphis subsp. aureofaciens isolate Sh2 and Pseudomonas fluorescens isolate Rh43 are good potential biocontrol agents for containing root-knot nematodes in kiwifruit trees.peer-reviewe

Simultaneous detection of the seven main tomato-infecting RNA viruses by two multiplex reverse transcription polymerase chain reactions

Cucumber mosaic virus, Tomato spotted wilt virus, Tomato mosaic virus, Tomato chlorosis virus, Pepino mosaic virus, Torrado tomato virus and Tomato infectious chlorosis virus cause serious damage and significant economic losses in tomato crops worldwide. The early detection of these pathogens is essential for preventing the viruses from spreading and improving their control. In this study, a procedure based on two multiplex RT-PCRs was developed for the sensitive and reliable detection of these seven viruses. Serial dilutions of positive controls were analysed by this methodology, and the results were compared with those obtained by ELISA and singleplex versions of RT-PCR. The multiplex and singleplex RT-PCR assays were able to detect specific targets at the same dilution and were 100 times more sensitive than ELISA. The multiplex versions were able to detect composite samples containing different concentrations of specific targets at ratios from 1:1 to 1:1000. In addition, 45 symptomatic tomato samples collected in different tomato-growing areas of Sicily (Italy) were analysed by multiplex RT-PCR, singleplex RT-PCR and commercially available ELISA tests. Similar results were obtained using the RT-PCR techniques, with a higher sensitivity than ELISA, revealing a common occurrence of mixed infections and confirming the presence of these seven virus species in ItalyPanno, S.; Davino, S.; Rubio, L.; Rangel, E.; Davino, M.; García Hernández, J.; Olmos Castelló, A. (2012). Simultaneous detection of the seven main tomato-infecting RNA viruses by two multiplex reverse transcription polymerase chain reactions. Journal of Virological Methods. 186(1-2):152-156. doi:10.1016/j.jviromet.2012.08.003S1521561861-

Actas de Horticultura

El cultivo de los cítricos comenzó en Extremo Oriente hace unos 4.000 años, en las regiones que ocupan actualmente China y Japón. Los grandes movimientos migratorios que ocasionaron las conquistas de Alejandro Magno, la expansión del Islam y el descubrimiento de América favorecieron la expansión de este cultivo por todo el mundo. Sin embargo, fue a partir del siglo XVIII cuando la citricultura adquirió una relevancia económica, tanto desde el punto de vista industrial como ornamental. El movimiento de plantas fue acompañado por la difusión de diversos patógenos, aunque afortunadamente, sólo una parte de los presentes en las regiones de origen han llegado en las nuevas áreas de cultivo. En Italia, la superficie cultivada con cítricos es de aproximadamente 160.000 Ha y de éstas, alrededor del 60% se encuentran en Sicilia. En los últimos años ha cobrado relevancia la producción de cítricos destinados a fines ornamentales, con una producción media anual en Sicilia de unos 4,5 millones de plantas, lo que la convierte en el máximo productor de cítricos ornamentales de Europa. Entre estos se encuentran los limones ornamentales, distintos kumquats, calamondín, naranjo amargo, cidro, naranjo dulce, mandarinos y pomelos. Desde el punto de vista sanitario, hay que tener en cuenta que las plantas ornamentales que se venden por todo el territorio Europeo pueden actuar como reservorios y facilitar el tráfico y emergencia de nuevas enfermedades. Entre las enfermedades más comunes en los cítricos ornamentales se encuentra la exocortis, las protuberancias nerviales (vein enation), las manchas anulares (ringspot), la psoriasis, la tristeza, la variegación, las concavidades gomosas (concave gum), la impietratura, el stubborn y el Huanglongbing. En este artículo se describen las principales enfermedades que afectan a los cítricos ornamentales y que representan un riesgo en la Comunidad Europea

Actas de Horticultura

Los virus son organismos muy sencillos que se multiplican dentro de las células vegetales y aunque no suelen provocar la muerte de la planta, inducen síntomas que disminuyen el rendimiento y la calidad del cultivo reduciendo su valor comercial. Sin embargo, algunos síntomas de origen vírico tienen cierto valor ornamental y son explotados con fines comerciales, como ocurre con el virus del variegado del tulipán (Tulip breaking virus, TBV). Los síntomas más característicos que inducen los virus son enanismo, clorosis, necrosis y deformación foliar, variaciones de color de la hoja y en la flor en forma de mosaicos, moteados, rayas o manchas anulares. La mayoría de los virus se transmiten mediante organismos vectores que se alimentan de la planta: insectos, ácaros, hongos y nematodos; otros se dispersan por contacto físico y por las herramientas agrícolas (transmisión mecánica) y unos pocos se propagan por semilla. Dentro de los virus con más impacto en los cultivos ornamentales están el virus del mosaico del pepino (Cucumber mosaic virus, CMV), el virus del bronceado del tomate (Tomato spotted wilt virus, TSWV), el virus del mosaico del tabaco (Tobacco mosaic virus, TMV) y el virus del rayado del tabaco (Tobacco streak virus, TSV), que también causan graves daños en otros cultivos. El control de las enfermedades producidas por los virus se basa en la aplicación de medidas preventivas como el uso de material propagativo libre de virus mediante certificación de semillas o plántulas en vivero, eliminación de plantas infectadas en el cultivo y durante el proceso de comercialización, control de los vectores (insectos, hongos, nemátodos), desinfección de las herramientas, limpieza de malas hierbas (reservorios de virus) dentro y fuera del invernadero, etc. Para ello es necesario un monitoreo rutinario mediante la observación visual de síntomas y el uso de técnicas para el diagnóstico rápido, sensible y fiable tanto de los virus más importantes como de los de nueva implantación o virus emergentes

Efficacy of Pseudomonas chlororaphis subsp. aureofaciens SH2 and Pseudomonas fluorescens RH43 isolates against root-knot nematodes (Meloidogyne spp.) in kiwifruit

The Root-knot nematodes, Meloidogyne spp., are parasites of many crops and orchards, including kiwifruit trees. The Islamic Republic of Iran is among the leading kiwifruit producers in the world and M. in- cognita has been found as the dominant species responsible for severe loss of this crop. In order to evaluate the effectiveness of antagonistic bacteria on larval mortality, number of galls per plant and egg masses of nematode reduction, fifty local bacterial strains were isolated from root surrounding soils of kiwifruit plants in the northern production areas in Iran. Bacterial antagonists were characterized by morphological, physiological, biochemical and molecular methods. Two representative strains, showing the best nematicidal activity, were identifed as Pseudomonas chlororaphis subsp. aureofaciens (isolate Sh2) and Pseudomonas fluorescens (isolate Rh43). They increased the percentage of larval mortality to 56.38% and 54.28% respectively in assays in vitro and showed excellent performance also in vivo with consistent reduction of number of galls (67.31% and 55.63%, respectively) and egg mass (86.46% and 84.29%, respectively) in plants. This study indicates that Pseudomonas chlororaphis subsp. aureofaciens isolate Sh2 and Pseudomonas fluorescens isolate Rh43 are good potential biocontrol agents for containing root-knot nematodes in kiwifruit trees

Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy

[EN] Citrus tristeza virus (CTV) outbreaks were detected in Sicily island, Italy for the first time in 2002. To gain insight into the evolutionary forces driving the emergence and phylogeography of these CTV populations, we determined and analyzed the nucleotide sequences of the p20 gene from 108 CTV isolates collected from 2002 to 2009. Bayesian phylogenetic analysis revealed that mild and severe CTV isolates belonging to five different clades (lineages) were introduced in Sicily in 2002. Phylogeographic analysis showed that four lineages co-circulated in the main citrus growing area located in Eastern Sicily. However, only one lineage (composed of mild isolates) spread to distant areas of Sicily and was detected after 2007. No correlation was found between genetic variation and citrus host, indicating that citrus cultivars did not exert differential selective pressures on the virus. The genetic variation of CTV was not structured according to geographical location or sampling time, likely due to the multiple introduction events and a complex migration pattern with intense co- and recirculation of different lineages in the same area. The phylogenetic structure, statistical tests of neutrality and comparison of synonymous and nonsynonymous substitution rates suggest that weak negative selection and genetic drift following a rapid expansion may be the main causes of the CTV variability observed today in Sicily. Nonetheless, three adjacent amino acids at the p20 N-terminal region were found to be under positive selection, likely resulting from adaptation events.A.W. and S.F.E. were supported by grant BFU2012-30805 from the Spanish Secretaria de Estado de Investigacion, Desarrollo e Innovacion and by a grant 22371 from the John Templeton Foundation. The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the John Templeton Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Davino, S.; Willemsen, A.; Panno. Stefano; Davino, M.; Catara, A.; Elena Fito, SF.; Rubio, L. (2013). Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy. PLoS ONE. 8:66700-66700. doi:10.1371/journal.pone.0066700S66700667008Domingo, E., & Holland, J. J. (1997). RNA VIRUS MUTATIONS AND FITNESS FOR SURVIVAL. Annual Review of Microbiology, 51(1), 151-178. doi:10.1146/annurev.micro.51.1.151Grenfell, B. T. (2004). Unifying the Epidemiological and Evolutionary Dynamics of Pathogens. Science, 303(5656), 327-332. doi:10.1126/science.1090727Moya, A., Holmes, E. C., & González-Candelas, F. (2004). The population genetics and evolutionary epidemiology of RNA viruses. 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PLoS Pathogens, 6(10), e1001166. doi:10.1371/journal.ppat.1001166Vijaykrishna, D., Bahl, J., Riley, S., Duan, L., Zhang, J. X., Chen, H., … Guan, Y. (2008). Evolutionary Dynamics and Emergence of Panzootic H5N1 Influenza Viruses. PLoS Pathogens, 4(9), e1000161. doi:10.1371/journal.ppat.1000161Gómez, P., Sempere, R. N., Aranda, M. A., & Elena, S. F. (2012). Phylodynamics of Pepino mosaic virus in Spain. European Journal of Plant Pathology, 134(3), 445-449. doi:10.1007/s10658-012-0019-0Lefeuvre, P., Martin, D. P., Harkins, G., Lemey, P., Gray, A. J. A., Meredith, S., … Heydarnejad, J. (2010). The Spread of Tomato Yellow Leaf Curl Virus from the Middle East to the World. PLoS Pathogens, 6(10), e1001164. doi:10.1371/journal.ppat.1001164TOMITAKA, Y., & OHSHIMA, K. (2006). A phylogeographical study of the Turnip mosaic virus population in East Asia reveals an ‘emergent’ lineage in Japan. Molecular Ecology, 15(14), 4437-4457. doi:10.1111/j.1365-294x.2006.03094.xWu, B., Blanchard-Letort, A., Liu, Y., Zhou, G., Wang, X., & Elena, S. F. (2011). Dynamics of Molecular Evolution and Phylogeography of Barley yellow dwarf virus-PAV. PLoS ONE, 6(2), e16896. doi:10.1371/journal.pone.0016896MORENO, P., AMBRÓS, S., ALBIACH-MARTÍ, M. R., GUERRI, J., & PEÑA, L. (2008). Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Molecular Plant Pathology, 9(2), 251-268. doi:10.1111/j.1364-3703.2007.00455.xTatineni, S., Robertson, C. J., Garnsey, S. M., & Dawson, W. O. (2011). A plant virus evolved by acquiring multiple nonconserved genes to extend its host range. Proceedings of the National Academy of Sciences, 108(42), 17366-17371. doi:10.1073/pnas.1113227108Folimonova, S. Y. (2012). Superinfection Exclusion Is an Active Virus-Controlled Function That Requires a Specific Viral Protein. Journal of Virology, 86(10), 5554-5561. doi:10.1128/jvi.00310-12Bar-Joseph, M., Marcus, R., & Lee, R. F. (1989). The Continuous Challenge of Citrus Tristeza Virus Control. Annual Review of Phytopathology, 27(1), 291-316. doi:10.1146/annurev.py.27.090189.001451Davino, S., Rubio, L., & Davino, M. (2005). Molecular analysis suggests that recent Citrus tristeza virus outbreaks in Italy were originated by at least two independent introductions. European Journal of Plant Pathology, 111(3), 289-293. doi:10.1007/s10658-003-2815-zAlbiach-Marti, M. R., Mawassi, M., Gowda, S., Satyanarayana, T., Hilf, M. E., Shanker, S., … Dawson, W. O. (2000). Sequences of Citrus Tristeza Virus Separated in Time and Space Are Essentially Identical. Journal of Virology, 74(15), 6856-6865. doi:10.1128/jvi.74.15.6856-6865.2000Rubio, L., Ayllon, M. A., Kong, P., Fernandez, A., Polek, M., Guerri, J., … Falk, B. W. (2001). Genetic Variation of Citrus Tristeza Virus Isolates from California and Spain: Evidence for Mixed Infections and Recombination. Journal of Virology, 75(17), 8054-8062. doi:10.1128/jvi.75.17.8054-8062.2001Silva, G., Marques, N., & Nolasco, G. (2011). The evolutionary rate of citrus tristeza virus ranks among the rates of the slowest RNA viruses. Journal of General Virology, 93(2), 419-429. doi:10.1099/vir.0.036574-0Mawassi, M., Mietkiewska, E., Gofman, R., Yang, G., & Bar-Joseph, M. (1996). Unusual Sequence Relationships Between Two Isolates of Citrus Tristeza Virus. Journal of General Virology, 77(9), 2359-2364. doi:10.1099/0022-1317-77-9-2359Vives, M. C., Dawson, W. O., Flores, R., L√≥pez, C., Albiach-Mart√≠, M. R., Rubio, L., … Moreno, P. (1999). The complete genome sequence of the major component of a mild citrus tristeza virus isolate. Journal of General Virology, 80(3), 811-816. doi:10.1099/0022-1317-80-3-811Martín, S., Elena, S. F., Guerri, J., Moreno, P., Sambade, A., Rubio, L., … Vives, M. C. (2009). Contribution of recombination and selection to molecular evolution of Citrus tristeza virus. Journal of General Virology, 90(6), 1527-1538. doi:10.1099/vir.0.008193-0Vives, M. C., Rubio, L., Sambade, A., Mirkov, T. E., Moreno, P., & Guerri, J. (2005). Evidence of multiple recombination events between two RNA sequence variants within a Citrus tristeza virus isolate. Virology, 331(2), 232-237. doi:10.1016/j.virol.2004.10.037D’Urso, F., Sambade, A., Moya, A., Guerri, J., & Moreno, P. (2003). Variation of haplotype distributions of two genomic regions of Citrus tristeza virus populations from eastern Spain. Molecular Ecology, 12(2), 517-526. doi:10.1046/j.1365-294x.2000.01747.xSambade, A., Rubio, L., Garnsey, S. M., Costa, N., Muller, G. W., Peyrou, M., … Moreno, P. (2002). Comparison of viral RNA populations of pathogenically distinct isolates of Citrus tristeza virus : application to monitoring cross-protection. Plant Pathology, 51(3), 257-265. doi:10.1046/j.1365-3059.2002.00720.xReed, J. C., Kasschau, K. D., Prokhnevsky, A. I., Gopinath, K., Pogue, G. P., Carrington, J. C., & Dolja, V. V. (2003). Suppressor of RNA silencing encoded by Beet yellows virus. Virology, 306(2), 203-209. doi:10.1016/s0042-6822(02)00051-xFolimonova, S. Y., Robertson, C. J., Shilts, T., Folimonov, A. S., Hilf, M. E., Garnsey, S. M., & Dawson, W. O. (2009). Infection with Strains of Citrus Tristeza Virus Does Not Exclude Superinfection by Other Strains of the Virus. Journal of Virology, 84(3), 1314-1325. doi:10.1128/jvi.02075-09Kong, P., Rubio, L., Polek, M., & Falk, B. W. (2000). Virus Genes, 21(3), 139-145. doi:10.1023/a:1008198311398Powell, C. A., Pelosi, R. R., Rundell, P. A., & Cohen, M. (2003). Breakdown of Cross-Protection of Grapefruit from Decline-Inducing Isolates of Citrus tristeza virus Following Introduction of the Brown Citrus Aphid. Plant Disease, 87(9), 1116-1118. doi:10.1094/pdis.2003.87.9.1116Roistacher C, Dodds J. (1993) Failure of 100 mild Citrus tristeza virus isolates from california to cross protect against a challenge by severe sweet orange stem pitting isolates. Proc 12th Conf IOCV: 100–107.Ayllón, M. A., Rubio, L., Sentandreu, V., Moya, A., Guerri, J., & Moreno, P. (2006). Variations in Two Gene Sequences of Citrus Tristeza Virus after Host Passage. Virus Genes, 32(2), 119-128. doi:10.1007/s11262-005-6866-4Ayllón, M. A., Rubio, L., Moya, A., Guerri, J., & Moreno, P. (1999). The Haplotype Distribution of Two Genes of Citrus Tristeza Virus Is Altered after Host Change or Aphid Transmission. Virology, 255(1), 32-39. doi:10.1006/viro.1998.9566Sentandreu, V., Castro, J. A., Ayllón, M. A., Rubio, L., Guerri, J., González-Candelas, F., … Moya, A. (2005). Evolutionary analysis of genetic variation observed in citrus tristeza virus (CTV) after host passage. Archives of Virology, 151(5), 875-894. doi:10.1007/s00705-005-0683-xMatos, L. A., Hilf, M. E., Cayetano, X. A., Feliz, A. O., Harper, S. J., & Folimonova, S. Y. (2013). Dramatic Change in Citrus tristeza virus Populations in the Dominican Republic. Plant Disease, 97(3), 339-345. doi:10.1094/pdis-05-12-0421-reDavino, S., Davino, M., Sambade, A., Guardo, M., & Caruso, A. (2003). The First Citrus tristeza virus Outbreak Found in a Relevant Citrus Producing Area of Sicily, Italy. Plant Disease, 87(3), 314-314. doi:10.1094/pdis.2003.87.3.314aRUBIO, L., AYLLONl, M. A., GUERRI, J., PAPPU, H., NIBLETT, C., & MORENO, P. (1996). Differentiation of citrus tristeza closterovirus (CTV) isolates by single-strand conformation polymorphism analysis of the coat protein gene. Annals of Applied Biology, 129(3), 479-489. doi:10.1111/j.1744-7348.1996.tb05770.xLarkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., … Higgins, D. G. (2007). 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Molecular characterization of corsican isolates of Citrus tristeza virus

International audienceTristeza disease, caused by the Citrus tristeza virus (CTV), is the most important viral disease for almost all the citrus species. Main syndromes are quick decline, stem pitting, vein clearing and seedlings yellows but expression and intensity of symptomatology depends on the interactions of the factors involved in the pathosystem such as host-strain-rootstock-climate-vector. Several isolates of CTV have been studied all over the world. Two Corsican isolates have been previously characterized, the K strain from Marumi kumquat, known to be symptomless on Mexican lime, and the Cal-1 from calamondin inducing stem pitting, but no sequences of p20 gene are published or available in GenBank. Two additional CTV strains were recently found in Corsica. The isolates LA5 and CO3 were detected in two different orchards of 40 year old Clementine on sour orange trees that didn’t show any specific symptoms ascribable to tristeza. Results of the molecular characterization of these isolates are reported. CTV presence was ascertained in young shoots collected in field by RT-PCR using specific primers for the gene p20. The amplicons obtained were analyzed by cloning and sequencing. Sequences analysis pointed out high similarity between the two isolates (98%). Multiple alignment analysis with all the p20 CTV gene sequences deposited in GenBank evidenced the identity of the virus detected in the two orchards. Respect to the already described mild, severe and atypical groups of CTV isolates, phylogenetic analysis revealed a new cluster including both LA5 and CO

Molecular characterization of corsican isolates of Citrus tristeza virus

International audienceTristeza disease, caused by the Citrus tristeza virus (CTV), is the most important viral disease for almost all the citrus species. Main syndromes are quick decline, stem pitting, vein clearing and seedlings yellows but expression and intensity of symptomatology depends on the interactions of the factors involved in the pathosystem such as host-strain-rootstock-climate-vector. Several isolates of CTV have been studied all over the world. Two Corsican isolates have been previously characterized, the K strain from Marumi kumquat, known to be symptomless on Mexican lime, and the Cal-1 from calamondin inducing stem pitting, but no sequences of p20 gene are published or available in GenBank. Two additional CTV strains were recently found in Corsica. The isolates LA5 and CO3 were detected in two different orchards of 40 year old Clementine on sour orange trees that didn’t show any specific symptoms ascribable to tristeza. Results of the molecular characterization of these isolates are reported. CTV presence was ascertained in young shoots collected in field by RT-PCR using specific primers for the gene p20. The amplicons obtained were analyzed by cloning and sequencing. Sequences analysis pointed out high similarity between the two isolates (98%). Multiple alignment analysis with all the p20 CTV gene sequences deposited in GenBank evidenced the identity of the virus detected in the two orchards. Respect to the already described mild, severe and atypical groups of CTV isolates, phylogenetic analysis revealed a new cluster including both LA5 and CO

Molecular characterization of corsican isolates of citrus tristeza virus

International audienceTristeza disease, caused by the Citrus tristeza virus (CTV), is the most important viral disease for almost all citrus species. Syndromes and symptoms expression depend on the interactions of factors involved in the pathosystem, host-strain-rootstock-climate-vector. Several strains of CTV have been studied all over the world. Two Corsican isolates have been previously characterized, K strain from Marumi kumquat, known to be symptomless on Mexican lime, and Cal-1 from calamondin inducing stem pitting. No sequence data are published or available in GenBan

Molecular characterization of corsican isolates of citrus tristeza virus

International audienceTristeza disease, caused by the Citrus tristeza virus (CTV), is the most important viral disease for almost all citrus species. Syndromes and symptoms expression depend on the interactions of factors involved in the pathosystem, host-strain-rootstock-climate-vector. Several strains of CTV have been studied all over the world. Two Corsican isolates have been previously characterized, K strain from Marumi kumquat, known to be symptomless on Mexican lime, and Cal-1 from calamondin inducing stem pitting. No sequence data are published or available in GenBan