Primers based on the rpf gene region provide improved detection of Xanthomonas axonopodis pv. citri in naturally and artificially infected citrus plants.
OBJECTIVE
To have a PCR-based detection method for Xanthomonas axonopodis pv. citri (Xac) using primers designed in a specific region of its genome.


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 28 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.


CONCLUSIONS
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.
Coletta-Filho
H D
HD
Centro APTA Citros Sylvio Moreira, Instituto Agronômico de Campinas, Cordeiropolis, SP, Brazil. helvecio@centrodecitricultura.br
Takita
M A
MA
Souza
A A
AA
Neto
J R
JR
Destéfano
S A L
SA
Hartung
J S
JS
Machado
M A
MA
D001424:Bacterial Infections / Q000382:microbiology*; D001426:Bacterial Proteins / Q000235:genetics*; D002957:Citrus / Q000382:microbiology*; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005784:Gene Amplification / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D010935:Plant Diseases / Q000382:microbiology*; D018515:Plant Leaves / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods; D014974:Xanthomonas / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D016207:Cytokines; D017931:DNA Primers; D004269:DNA, Bacterial; C113704:resuscitation-promoting factor, bacteria