RESUMO
Exclusion and eradication or management based on an integrated approach with less susceptible varieties, copper-based bactericides, and windbreaks are the two main strategies used to prevent or control citrus canker. Field tolerance or resistance to citrus canker is not found in the most important commercial sweet orange cultivars, and pathogen-derived resistance has been developed and applied in different crops to obtain resistant genotypes to plant pathogens. We describe the development of DNA primers and probes based on the type III effector genes avrXacE1, avrXacE2, avrXacE3, avrBs2, pthA4, hpaF, and XAC3090 (leucine rich protein), and their application in the evaluation of the genetic diversity of the pathogen. A total of 49 haplotypes were identified in 157 strains by Southern blot analysis. No genetic polymorphism was detected by BOX elements - and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) analysis, nor with the genes avrBs2, XAC3090, and hpaF. Nei's genetic diversity indexes varied from 0.65 to 0.96 for subcollections of the pathogen. One or few haplotypes were most frequent in the strain collection, but several haplotypes were represented by solely one or few strains. The PthA4 probe resulted in the higher number of haplotypes identified in the Brazilian subcollections. Greater variation in the frequency of haplotypes occurred within subcollections (93.7%) than among subcollections. Only some haplotypes were genetically distant from all others, especially those originated from Rio Grande do Sul and Santa Catarina states. These bacterial effectors are widely spread in the collections and are useful for a better understanding of the host-pathogen interaction and the search for resistance genes in host and nonhost plants.
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.
