RESUMO
In March 2009, in a sweet orange orchard (Citrus sinensis) cv. Valencia grafted on Swingle citrumelo (C. paradisi Macf. × Poncirus trifoliata L. Raf.) rootstock in Severínia County, São Paulo State, Brazil, approximately 40 trees were detected with small, necrotic, dark brown leaf spots. These lesions occurred whether or not citrus leafminer (Phyllocnistis citrella) was present and they were only found on leaves from branches arising from the rootstock. Sweet orange foliage was not affected even when in contact with infected rootstock branches. Symptoms were unusual and distinct from typical citrus canker lesions because the lesions were smaller and did not have erumpent margins. Typical yellow Xanthomonas colonies were isolated from the lesions on nutrient agar. The isolates were aerobic, gram negative, rod shaped, and they produced a dark pigment, which is characteristic of some Xanthomonas fuscans subsp. aurantifolii strains. Two reference strains were tested for pathogenicity on not fully expanded leaves of sweet orange, Swingle citrumelo, and key/Mexican lime (C. aurantifolia) plants by wound inoculation with a sterile needle previously dipped in a bacterial suspension (approximately 106 ml-1). Two plants of each species were used for inoculations in greenhouse conditions and six leaves were inoculated per plant. Each inoculated leaf received six point inoculations. These tests confirmed that the host range of this pathogen was restricted to Swingle citrumelo. Symptoms similar to those in the orchard were observed 3 weeks after inoculation and Koch's postulates were completed by reisolation of the bacterium and comparing it with the original isolates. Molecular fingerprinting with PCR-restriction fragment length polymorphism (RFLP) of the 16S-23S spacer region polymorphism (1) and ERIC- and BOX-PCR (2) was used to compare the new strain with 26 reference strains of Xanthomonas citri subsp. citri types A, A* and Aw, X. fuscans subsp. aurantifolii types B and C, and X. alfalfae subsp. citrumelonis. PCR-RFLP and ERIC-PCR showed that this new pathogen had the same profile as X. fuscans subsp. aurantifolii (B and C types). In BOX-PCR, this new strain had a unique profile, but it was still most similar to X. fuscans subsp. aurantifolii and very distinct from X. citri subsp. citri (A, A*, and Aw) and X. alfalfae subsp. citrumelonis strains. During the rainy season in Brazil, this new Xanthomonas strain is less aggressive than X. citri subsp. citri on Swingle citrumelo, inducing fewer lesions without erumpent margins even in young leaves severely infested by the citrus leafminer. The disease only occurred on trees that were separated from each other by 3 to 20 m, suggesting that the bacterium is spread by windblown rain and/or cultural practices. Xanthomonads pathogenic to citrus are of great importance for regulatory purposes worldwide. X. fuscans subsp. aurantifolii is only known to be pathogenic on lemons and limes in the field, and until now, has only been reported to infect lemons and limes in Argentina and key/Mexican lime in São Paulo (Brazil) (3). To our knowledge, this is the first report of a strain of this subspecies that infects Swingle citrumelo but not key/Mexican lime. References: (1) S. A. L. Destéfano and J. Rodrigues Neto. Summa Phytopathol. 28:167, 2002. (2) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994. (3) N. W. Schaad et al. Syst. Appl. Microbiol. 28:494, 2005.
RESUMO
AIMS: To have a PCR-based detection method for Xanthomonas axonopodis pv. citri (Xac) 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 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. 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.
