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Detection of Xanthomonas albilineans in sugarcane stalks using lamp PCR and selective isolation assays

Duarte Dias V., Fernandez E., Da Cunha M.G., Roumagnac P., Comstock J.C., Rott P.. 2015. In : Book of abstracts. XI Pathology and IX Entomology ISSCT joint Workshop. Guayaquil : FIADE, p. 26-26. ISSCT Pathology Workshop. 11, 2015-09-14/2015-09-18, Guayaquil (Equateur).

Leaf scald, caused by Xanthomonas albilineans, is a major disease of sugarcane that can cause severe yield losses in susceptible cultivars. The disease is mainly controlled by use of resistant cultivars that need to be screened in breeding programs. Several diagnostic techniques are currently available to identify or to detect X. albilineans in infected stalks, including isolation on selective medium, serological and molecular methods. Each of these methods has advantages and disadvantage in terms of time and equipment/facilities needed, or sensitivity. In this study, we developed a colorimetric loop-mediated isothermal amplification (LAMP) assay, an easy, rapid and simple molecular method for identification of X. albilineans. This method requires only four specific primers, a DNA polymerase, and a regular laboratory water bath or heat block for DNA amplification. A positive reaction is indicated by a color change of the hydroxynaphthol blue (HNB) in the reaction solution from violet to sky blue. X. albilineans was detected by LAMP, PCR (polymerase chain reaction) and isolation on selective medium (XAS) in respectively 26, 26 and 27 out of 27 sugarcane stalks exhibiting symptoms of leaf scald in the field. The pathogen was also isolated from 33 out of 46 asymptomatic stalks using the selective agar medium. Only two of these asymptomatic stalks were found infected by X. albilineans using LAMP and PCR. Consequently, occurrence of pathogen stalk populations that are below the detection threshold of both LAMP and PCR limits the use of these two methods for detection of X. albilineans in plants showing no symptoms. However, LAMP can be used for easy and rapid identification of the pathogen in symptomatic plants or after isolation of the bacteria on agar medium. (Texte integral)

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