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Title: PRIMERS BASED ON THE RPF GENE REGION PROVIDE IMPROVED DETECTION OF XANTHOMONAS AXONOPODIS PV CITRI IN NATURALLY AND ARTIFICIALLY INFECTED CITRUS PLANTS.

Author
item COLLETTA-FILHO, H.D. - CENTRO DE CITRICULTURA
item TAKITA, M.A. - CENTRO DE CITRICULTURA
item DE SOUZA, A.A. - CENTRO DE CITRICULTURA
item NETO, J.R. - INSTITUTO BIOLOGICO
item DESTEFANO, S.A.L. - INSTITUTO BIOLOGICO
item Hartung, John

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2005
Publication Date: 2/1/2006
Citation: Colletta-Filho, H., Takita, M., De Souza, A., Neto, J., Destefano, S., Hartung, J.S. 2006. Primers based on the rpf gene region provide improved detection of xanthomonas axonopodis pv citri in naturally and artificially infected citrus plants.. Journal of Applied Microbiology. 100(2):279-285.

Interpretive Summary: Citrus bacterial canker (CBC), is a serious disease and it occurs in both São Paulo, Brazil and Florida, USA. The disease is caused by the bacterium Xanthomonas axonopodis pv. citri. In both regions, infected trees must be destroyed, often along with surrounding trees that look healthy but which may be infected. Losses and control costs may exceed one hundred million dollars annually. Current diagnostic tests are very good, but must be improved to detect new strains of the pathogen that have appeared in recent years in Florida and in southwestern Asia. We identified a unique arrangement of genes in the citrus canker pathogen and used this knowledge to develop a new specific and sensitive diagnostic test for the citrus canker pathogen. We show that our new test is able to detect and identify all strains of the citrus canker bacterium, including the new strains from Florida and southwestern Asia. This represents a significant improvement in the technology used to detect this pathogen. The unique arrangement of genes that we found may contribute to the control of the disease process itself, and so will be of interest to plant pathologists studying the basic biology of plant diseases.

Technical Abstract: Aim: To have a PCR based detection method for all Xanthomonas axonopodis pv. citri (Xac) strains using primers designed in a specific region of its genome. Methods and Results: A Xac-specific region was identified inside the rpf gene cluster of strain IAPAR 306 in an analysis of its complete genomic sequence. Two primers were designed, Xac01 and Xac02, which, when used in a standard PCR assay, direct the amplification of a 581 bp fragment from DNA of strains belonging to Xac from different regions around the world including unusual American and Asian strains. This product was not observed when DNA from strains of the closely related X. a. aurantifolli and X. a. citrumelo were used as templates. Extracts prepared from twenty-eight Xanthomonads of other species, and epiphytic bacteria isolated from citrus also failed to produce products with these primers. Amplification was obtained from cells grown in vitro, from extracts of both fresh and dried citrus canker lesions, and from washes of inoculated but asymptomatic leaf surfaces. In sensitivity tests, this PCR technique detected as few as 100 cells. Conclusions: Primers Xac01 and Xac02 provide specific and sensitive detection of Xac in all citrus tissues where the pathogen is found. Significance and Impact of the study: This PCR-based diagnostic test is suitable for monitoring asymptomatic plants in areas where the bacteria is endemic, in plant quarantine and regulatory situations, and also for obtaining an accurate diagnosis in a very short time. These are important characteristics for any assay to be used for the management of citrus canker disease.