Haplotypes of "Candidatus Liberibacter solanacearum" identified in Umbeliferous crops in Spain

[EN] 'Candidatus Liberibacter solanacearum' is a phloem-limited Gram-negative bacterium that causes serious damage to different crops of the botanical families Solanaceae and Apiaceae. Five haplotypes have been described: LsoA and LsoB are present in solanaceous crops in America and vectored by the tomato/potato psyllid Bactericera cockerelli; LsoC affects carrots from Northern and Central Europe, and is transmitted by the carrot psyllid Trioza apicalis; haplotypes LsoD and LsoE are present in Southern Europe and Morocco in carrot and celery, and are associated with the psyllid Bactericera trigonica. Thirty-four 'Ca. L. solanacearum' isolates were collected in six different regions of Spain from distinct Apiaceae hosts (carrot, celery, parsley and parsnip) in eight consecutive years and were analysed. Their haplotypes were determined by a sequence analysis of 16S ribosomal RNA, the 16S-26S ribosomal RNA intergenic spacer, and the 23S ribosomal RNA and rplJ and rplL genes. Both haplotypes LsoD and LsoE were found across Spain, and no host specificity appeared between these two haplotypes. This is the first report of 'Ca. L. solanacearum' associated with parsley and parsnip.This work has been supported by grant INIA (RTA2011-00142). This paper is dedicated to the memory of F.J. Villaescusa (1981-2011). The technical support of S. Sanjuan and J.C. Ferrandiz from Agricola Villena Coop. V. is also acknowledged.Alfaro Fernández, AO.; Hernández-Llopis, D.; Font San Ambrosio, MI. (2017). Haplotypes of "Candidatus Liberibacter solanacearum" identified in Umbeliferous crops in Spain. European Journal of Plant Pathology. 149(1):127-131. https://doi.org/10.1007/s10658-017-1172-21271311491Alfaro-Fernández A., Cebrián M.C., Villaescusa F.J., Hermoso de Mendoza A., Ferrándiz J.C., Sanjuán S., Font M.I. (2012). ‘Candidatus Liberibacter solanacearum’ associated with Bactericera trigonica affected carrots in the Canary Islands. Plant Disease 96, 581.Bertolini, E., Teresani, G. R., Loiseau, M., Tanaka, F. A. O., Barbé, S., Martínez, C., Gentit, P., López, M. M., & Cambra, M. (2014). Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds. Plant Pathology, 64, 276–285.EPPO. (2013). Data sheets on pests recommended for regulation. Candidatus Liberibacter solanacearum. EPPO Bulletin, 43, 197–201.Green, M. J., Thompson, D. A., & Mackenzie, D. J. (1999). Easy and efficient DNA extraction from woody plants for the detection of phytoplasmas by polymerase chain reaction. Plant Disease, 83, 482–485.Hansen, A. K., Trumble, J. T., Stouthamer, R., & Paine, T. D. (2008). A new huanglongbing species, “Candidatus Liberibacter psyllaurous,” found to infect tomato and potato, is vectored by the psyllid Bactericera cockerelli (Sulc). Applied Environmental Microbiology, 74, 5862–5865.Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., & Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947–2948.Loiseau, M., Garnier, S., Boirin, V., Merieau, M., Leguay, A., Renaudin, I., Renvoisé, J.-P., & Gentit, P. (2014). First report of ‘Candidatus Liberibacter solanacearum’ in carrot in France. Plant Disease, 98, 839.Munyaneza, J. E. (2012). Zebra chip disease of potato: biology, epidemiology and management. American Journal of Potato Research, 89, 329–350.Munyaneza, J., Buchman, J., Upton, J., Goolsby, J., Crosslin, J., & Bester, G. (2008). Impact of different potato psyllid populations on zebra chip disease incidence, severity, and potato yield. Subtropical Plant Science, 60, 27–37.Munyaneza, J., Sengoda, V., Crosslin, J., de la Rosa-Lorenzo, G., & Sanchez, A. (2009). First report of ‘Candidatus Liberibacter psyllaurous’ in potato tubers with zebra Chip disease in Mexico. Plant Disease, 93, 552.Munyaneza, J. E., Fisher, T. W., Sengoda, V. G., & Garczynski, S. F. (2010a). First report of ‘Candidatus Liberibacter solanacearum’ associated with psyllid-affected carrots in Europe. Plant Disease, 94, 639.Munyaneza, J. E., Fisher, T. W., Sengoda, V. G., Garczynski, S. F., Nissinen, A., & Lemmetty, A. (2010b). Association of ‘Candidatus Liberibacter solanacearum’ with the psyllid Trioza apicalis (hemiptera: Triozidae). Journal of Economic Entomology, 103, 1060–1070.Munyaneza, J. E., Swisher, K. D., Hommes, M., Willhauck, A., Buck, H., & Meadow, R. (2015). First report of ‘Candidatus Liberibacter solanacearum’ associated with psyllid-infested carrots in Germany. Plant Disease, 99, 1269.Nelson, W. R., Fisher, T. W., & Munyaneza, J. E. (2011). Haplotypes of “Candidatus Liberibacter solanacearum” suggest long-standing separation. European Journal of Plant Patholology, 130, 5–12.Nelson, W. R., Sengoda, V. G., Alfaro-Fernández, A., Font, M. I., Crosslin, J. M., & Munyaneza, J. E. (2013). A new haplotype of ‘Candidatus Liberibacter solanacearum’ identified in the Mediterranean region. European Journal of Plant Pathology, 135, 633–639.Tahzima, R., Maes, M., Achbani, E. H., Swisher, K. D., Munyaneza, J. E., & De Jonghe, K. (2014). First report of ‘Candidatus Liberibacter solanacearum’ on carrot in Africa. Plant Disease, 98, 1426.Teresani, G. R., Bertolini, E., Alfaro-Fernández, A., Martínez, C., Tanaka, F. A. O., Kitajima, E. W., Roselló, M., Sanjuán, S., Ferrándiz, J. C., López, M. M., Cambra, M., & Font, M. I. (2014). Association of ‘Candidatus Liberibacter solanacearum’ with a vegetative disorder of celery in Spain and development of a real-time PCR method for its detection. Phytopathology, 104, 804–811.Teresani, G., Hernández, E., Bertolini, E., Siverio, F., Marroquín, C., Molina, J., de Hermoso Mendoza, A., & Cambra, M. (2015). Search for potential vectors of ‘Candidatus Liberibacter solanacearum’: population dynamics in host crops. Spanish Journal of Agricultural Research, 13, e10–002

Virosis en tomate transmitidas por semilla y su control

[ES] Las virosis transmitidas por semilla en el cultivo del tomate crean gran preocupación entre los productores, y son de especial atención en aquellos que se dedican al cultivo de variedades locales donde las semillas se extraen durante la campaña y son empleadas para cultivos posteriores con lo que la infección y dispersión de estos virus es mucho más frecuente. Entre los virus transmitidos por semilla en tomate destacan el virus del mosaico del tomate (ToMV) y el virus del mosaico del pepino dulce (PepMV). Ambos virus se caracterizan por transmitirse, además de por semilla, de manera mecánica fácilmente y son muy estables manteniéndose en los restos del cultivo anterior y en las infraestructuras empleadas durante el manejo del cultivo. Sin embargo, la localización de estos virus en las semillas contaminadas difiere, mientras que PepMV se localiza únicamente de manera superficial, ToMV puede encontrarse además en zonas más internas como en el endospermo. Esto hace que los tratamientos empleados para la desinfección de semillas infectadas con cada uno de estos virus sea distinto: mientras que PepMV puede ser inactivado con tratamientos químicos superficiales, el tratamiento para descontaminar semillas con ToMV debe ser térmico a elevadas temperaturas.[EN] Viral diseases transmitted through seed create a great concern among the tomato producers, especially those who use local varieties that harvest their own seeds from the previous growing season fruits. In this case the infection and spread of seed-transmitted viruses is more usual. ToMV and PepMV are the two main seed-transmitted viruses which affect tomato crops. Both viruses are easily mechanically and seed transmitted, and remain infective in the plant debris of the previous crop and in the crop structures. However, the location of the virus in the contaminated seed is different. PepMV is present only externally in the seed coat, but ToMV could be also found in the endosperm. Therefore seed treatments to inactivate these two viruses are different; while PepMV could be inactivated by external chemical treatments, ToMV infected seeds should be thermal treated in order to eliminate further seedling infections.Alfaro Fernández, AO.; Font San Ambrosio, MI. (2020). Virosis en tomate transmitidas por semilla y su control. En I Congrés de la Tomaca Valenciana: La Tomaca Valenciana d'El Perelló. Editorial Universitat Politècnica de València. 97-114. https://doi.org/10.4995/TOMAVAL2017.2017.6524OCS9711

Detección y cuantificación del virus del mosaico de la sandía por RT-PCR a tiempo real

El virus del mosaico de la sandía (watermelon mosaic virus, WMV) produce graves daños en varios cultivos de cucurbitáceas en todo el mundo. El control de enfermedades se basa en restringir la propagación del virus y en la obtención de variedades resistentes por mejora genética. Para poder aplicar estas estrategias de control es necesario disponer de herramientas para la detección sensible y la cuantificación precisa de WMV en plantas infectadas. En este trabajo, se desarrolló un procedimiento basado en la retrotranscripción seguida de una PCR a tiempo real con un par de iniciadores y una sonda TaqMan® específicos de WMV

Search for reservoirs of `Candidatus Liberibacter solanacearum¿ and mollicutes in weeds associated with carrot and celery crops

[EN] Currently, the main arthropod vectored pathogens associated with carrot and celery crop diseases are EiCandidatus Liberibacter solanacearumA ', Spiroplasma citri and different phytoplasma species. Mitigation strategies require elucidating whether these pathogens survive in the weeds of these Apiaceae crops, which can act as reservoirs. Weed surveys were conducted in a vegetative cycle (April to October 2012) in the spontaneous vegetation that surrounded crops affected by EiCa. L. solanacearumA ', S. citri and/or phytoplasmas. Sixty-three species of 53 genera that belong to 23 botanical families were collected in the main carrot and celery Spanish production area. Species were identified, estimating coverage and abundance, and conserved in herbarium. Samples were analysed by nested-PCR with universal primers for phytoplasmas detection, and were sequenced for identification purposes; by conventional PCR for S. citri and real-time PCR for EiCa. L. solanacearumA '. The only detected pathogens were EiCa. Phytoplasma trifoliiA ' (clover proliferation group 16Sr VI-A) in Amaranthus blitoides and Setaria adhaerens and EiCa. P. solaniA ' (stolbur group 16Sr XII-A) in Convolvulus arvensis. These pathogens were also sporadically detected in celery or carrot crops. Unexpectedly, neither EiCa. L. solanacearumA ' nor S. citri was detected in the weed samples, despite the relatively high prevalence of these pathogens (less than 66 % and 25 %, respectively) in the surveyed plots. This suggests that weeds do not play an epidemiological role as reservoirs in the spread of such organisms in the studied region. The use of pathogen-free seed lots and the control of vectors are crucial for preventing the introduction and spread of these economical important pathogens to new areas.This work has been supported by grant INIA (RTA2011-00142). G.R. Teresani was the recipient of a PhD grant from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Ministerio de Educacao, Brazil. This paper is dedicated to the memory of F.J. Villaescusa (1981-2011). The technical support of S. Sanjuan and J.C. Ferrandiz from Agricola Villena Coop. V. is acknowledged.Alfaro Fernández, AO.; Verdeguer Sancho, MM.; Rodríguez-León, F.; Ibañez, I.; Hernández, D.; Teresani, GR.; Bertolini, E.... (2017). Search for reservoirs of `Candidatus Liberibacter solanacearum¿ and mollicutes in weeds associated with carrot and celery crops. European Journal of Plant Pathology. 147(1):15-20. https://doi.org/10.1007/s10658-016-0984-915201471Alfaro-Fernández, A., Cebrián, M. C., Villaescusa, F. J., Hermoso de Mendoza, A., Ferrándiz, J. C., Sanjuán, S., & Font, M. I. (2012). First report of ˋCandidatus Liberibacter solanacearum´ in carrots in mainland Spain. Plant Disease, 96, 582.Bertaccini, A., & Duduk, B. (2009). Phytoplasma and phytoplasma disease: a review of recent research. Phytopathologia Mediterranea, 48, 355–378.Bertolini, E., Teresani, G. R., Loiseau, M., Tanaka, F. A. O., Barbé, S., Martínez, C., Gentit, P., López, M. M., & Cambrá, M. (2015). Transmission of Candidatus Liberibacter solanacearum in carrot seeds. Plant Pathology, 64, 276–285.Bové, J. M. (1986). Stubborn and its natural transmission in the Mediterranean area and the near east. FAO Plant Protection Bulletin, 34, 15–23.Bové J. M., Fos A., Lallemand J., Raie A., Ali Y., Ahmed N. (1988). Epidemiology of Spiroplasma citri in the old world. In: L. W. Timmer, S.M. Garnsey, L. Navarro, eds. Proceedings of the 10th International Organization of Citrus Virologist Conference, (295–299). Riverside, USA. (www.iocv.org/proceedings).Braun-Blanquet, J. (1932). Plant sociology: the study of plant communities (439 pp). New York: McGraw-Hill.Carretero, J. L. (2004). Flora arvense española (754 pp). Valencia: Las malas hierbas de los cultivos españoles. Phytoma Ed.Cebrián, M. C., Villaescusa, F. J., Alfaro-Fernández, A., Hermoso De Mendoza, A., Córdoba- Sellés, M. C., Jordá, C., Ferrándiz, J. C., Sanjuán, S., & Font, M. I. (2010). First report of Spiroplasma citri in carrot in Europe. Plant Disease, 94, 1264.Davis, R. M., & Raid, R. N. (2002). Compendium of Umbelliferous crop disease (110 pp).American Phytopathological SocietyEmber, I., Acs, Z., Munyaneza, J. E., Crosslin, J. M., & Kolber, M. (2011). Survey and molecular detection of phytoplasmas associated with potato in Romania and southern Rusia. European Journal of Plant Pathology, 130, 367–377.Fialová, R., Valová, P., Balakishiyevá, G., Danet, J. L., Safarová, D., Foissac, X., & Navratil, M. (2009). Genetic variability of stolbur phytoplasma in anual crop and wild plant species in South Moravia. Journal of Plant Pathology, 91, 411–416.Fujiwara, K. (1987). Aims and methods of phytosociology or "vegetation science", Papers on plant ecology and taxonomy to the memory of Dr. Satoshi Nakanishi. pp. 607–628.Green, M. J., Thompson, D. A., & MacKenzie, D. J. (1999). Easy and efficient DNA extraction from Woody plants for the detection of Phytoplasmas by polymerase chain reaction. Plant Disease, 83, 482–485.Gundersen, D. E., & Lee, I. M. (1996). Ultrasensitive detection of phytoplasmas by nested- PCR assays using two universal primer pairs. Phytopathologia Mediterranea, 35, 144–151.Haapalanien, M. (2014). Biology and epidemics of Candidatus Liberibacter species, psyllid-transmitted plant-pathogenic bacteria. Annals of Applied Biology, 165, 172–198.Herbario de la Universidad Pública de Navarra. (2012). http://www.unavarra.es/herbario. Accessed 2012.Herbario Virtual del Mediterráneo Occidental. (2012). http://herbarivirtual.uib.es/. Accessed 2012.Flora Ibérica. (2012). http://www.floraiberica.org/. Accessed 2012.Jomantiene, R., Maas, J. L., Dally, E. L., Davis, R. E., & Postman, J. D. (1999). First report of clover proliferation Phytoplasma in strawberry. Plant Disease, 83, 967.Jomantiene, R., Postman, J. D., Montano, H. G., Maas, J. L., Davis, R. E., & Johnson, K. B. (2000). First report of clover yellow edge Phytoplasma in Corylus (hazelnut). Plant Disease, 84, 102.Lee, I. M., Gundersen-Rindal, D. E., Davis, R. E., & Bartoszyk, I. M. (1998). Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic and Evolutionary Microbiology, 48, 1153–1169.Lee, I. M., Dane, R. A., & Black, M. C. (2001). First report of a member of Aster yellows Phytoplasma group and of clover proliferation Phytoplasma group associated with onion in Texas. Plant Disease, 85, 448.Lee, I. M., Bottner, K. D., Miklas, P. N., & Pastor-Corrales, M. A. (2004). Clover proliferation group (16SrVI) subgroup a (16SrVI-A) Phytoplasma is a probable causal agent of dry bean Phyllody disease in Washington. Plant Disease, 88, 429–429.Mateo, G., & Crespo, M. (2009). Manual Para la determinación de la flora valenciana (4ª ed.507 pp). Alicante: Librería Compás.Ed.Murphy, A. F., Cating, R. A., Goyer, A., Hamm, P. B., & Rondon, S. I. (2014). First report of natural infection by ‘Candidatus Liberibacter solanacearum’ in bittersweet nightshade (Solanum dulcamara) in the Columbia Basin of eastern Oregon. Plant Disease, 94, 1425.Nejat, N., Vadamalai, G., & Dickinson, M. (2011). Spiroplasma citri: a wide host range phytopathogen. Plant Pathology Journal, 10, 46–56.Schneider, B., Seemüller, E., Smart, C. D., & Kirkpatrick, B. C. (1995). Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In S. Razin & J. G. Tully (Eds.), Molecular and diagnostic procedures in mycoplasmology (Vol. Vol.I, pp. 369–380). San Diego: Academic Press.Teresani, G., Bertolini, E., Alfaro-Fernandez, A., Martínez, C., Tanaka, F. A., Kitajima, E., Rosello, M., Sanjuan, S., Ferrandiz, J. C., López, M. M., Cambra, M., & Font-San-Ambrosio, M. I. (2014). Association of ‘Candidatus Liberibacter solanacearum’ with a vegetative disorder of celery in Spain and development of a real-time PCR method for its detection. Phytopahology, 104, 804–811.Teresani, G., Hernández, E., Bertolini, E., Siverio, F., Marroquín, C., Molina, J., Hermoso de Mendoza, A., & Cambra, M. (2015). Search for potencial vectors of ‘Candidatus Liberibacter solanacearum’: population dynamics in host crops. Spanish Journal of Agricultural Research, 13, e10–002.Flora Vascular. (2012). http://www.floravascular.com/. Accessed 2012.Weed Science Society of America. (2012). http://wssa.net/weed/weed-identification/. Accessed 2012.Yokomi, R. K., Mello, A. F. S., Saponari, M., & Fletcher, J. (2008). Polymerase chain reactionbased detection of Spiroplasma citri associated with citrus stubborn disease. Plant Disease, 92, 253–260

First report of cucurbit chlorotic yellows virus infecting watermelon and zucchini in the Canary Islands, Spain

This work was funded by grants from Spanish Ministerio de Econolnia, industria y competitividad (RTA2017-00061-C03-02) and from Instituto Valenciano de Investigaciones Agrarias (IVlA) (51912), both co-funded by the European Regional Development Fund (ERDF).Alfaro Fernández, AO.; Espino De Paz, AI.; Botella-Guillen, M.; Font San Ambrosio, MI.; Sanauja, E.; Galipienso, L.; Rubio, L. (2022). First report of cucurbit chlorotic yellows virus infecting watermelon and zucchini in the Canary Islands, Spain. Plant Disease. 106(7):1-1. https://doi.org/10.1094/PDIS-10-21-2296-PDN11106

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

Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Olea Europaea Geminivirus

A real-time loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of the Olea europaea geminivirus (OEGV), a virus recently reported in different olive cultivation areas worldwide. A preliminary screening by end-point PCR for OEGV detection was conducted to ascertain the presence of OEGV in Sicily. A set of six real-time LAMP primers, targeting a 209-nucleotide sequence elapsing the region encoding the coat protein (AV1) gene of OEGV, was designed for specific OEGV detection. The specificity, sensitivity, and accuracy of the diagnostic assay were determined. The LAMP assay showed no cross-reactivity with other geminiviruses and was allowed to detect OEGV with a 10-fold higher sensitivity than conventional end-point PCR. To enhance the potential of the LAMP assay for field diagnosis, a simplified sample preparation procedure was set up and used to monitor OEGV spread in different olive cultivars in Sicily. As a result of this survey, we observed that 30 out of 70 cultivars analyzed were positive to OEGV, demonstrating a relatively high OEGV incidence. The real-time LAMP assay developed in this study is suitable for phytopathological laboratories with limited facilities and resources, as well as for direct OEGV detection in the field, representing a reliable method for rapid screening of olive plant material

Estudio de la flora arvense como posible reservorio natural de agentes implicados en el nuevo problema fitosanitario que afecta a los cultivos de apio y zanahoria en Villena (Alicante)

Resumen: En primavera de 2008 apareció una nueva patología en cultivos de apio (Apium graveolens) y zanahoria (Daucus carota) de la empresa Agrícola Villena Coop. V de Villena (Alicante). que provocó importantes pérdidas en ambos cultivos. Con el fin de identificar posibles reservorios naturales de los agentes implicados en su desarrollo, se tomaron muestras de las especias arvenses presentes en campos de apio y zanahoria y sus alrededores y se realizaron análisis moleculares para la detección de fitoplasmas, Spiroplasma citri y “Candidatus Liberibacter solanacearum”. Se procesaron 135 muestras de flora arvense, algunas presentaban alteraciones en su desarrollo, pero la gran mayoría eran asintomáticas. Se identificaron 58 especies pertenecientes a 23 familias. De los tres agentes fitopatógenos analizados, únicamente se detectaron fitoplasmas en Setaria adhaerens y Amaranthus blitoides