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El control eficiente de la grasa de la judía causada por Pseudomonas syringae pv. phaseolicola se basa principalmente en la utilización de semilla libre del patógeno. La detección del patógeno en semilla se efectúa mediante métodos altamente sensibles basados en la detección por PCR de los genes responsables de la biosíntesis de la faseolotoxina, la cual, hasta ahora, se consideraba que era sintetizada por todas las cepas del patógeno con importancia epidemiológica. Sin embargo, en la Comunidad de Castilla y León, España, las epidemias de grasa de la judía en campo se asocian frecuentemente con cepas no toxigénicas de P. syringae pv. phaseolicola, que no pueden ser detectadas con los métodos moleculares y serológicos actuales. Los resultados presentados en este trabajo demuestran la existencia de aislados no toxigénicos de P. syringae pv. phaseolicola en zonas distintas de Castilla y León, lo que implica la necesidad de establecer una metodología fiable para la certificación de semillas de judía. Con este propósito, se presenta un sencillo protocolo en dos fases que permite la identificación de los dos tipos de aislados, y que se basa en una PCR multiplex con enriquecimiento a partir de extractos de semilla y en ensayos de patogenicidad.The efficient control of halo blight,  caused by Pseudomonas syringae pv. phaseolicola,  is primarily based on the use of pathogen-free seed. Detection of the pathogen in seeds is currently carried out with high-sensitive methods based on the detection by PCR of genes involved in the biosynthesis of phaseolotoxin,  which was believed to be produced by all strains of the pathogen with epidemiological importance. However,  field epidemics of halo blight in the county of Castilla y Leon,  Spain,  are often associated to nontoxigenic isolates of P syringae pv. phaseolicola,  which cannot be detected using current molecular and serological methods. The results presented in this work show the existence of nontoxigenic isolates of P syringae pv. phaseolicola in areas other than Castilla y Leon,  indicating the need to establish a reliable methodology for seed certification. A simple two-step methodology is presented with the aim to identify both types of isolates that is based on a multiplex enrichment PCR of seed soakates and on pathogenicity assays.This work was supported with grant AGL2004- 03143, from the Spanish Ministerio de Educación y Ciencia (MEC). A. Rico was funded by a fellowshipfrom the INIA. J. Murillo thanks the MEC for his support (grant PR2005-0299) during a sabbatical leave while this article was written

spanish journal of agricultural research

Short communication. Detection by multiplex PCR and characterization of nontoxigenic strains of Pseudomonas syringae pv. phaseolicola from different places in Spain