1Department of Citriculture, National Institute of Fruit Tree Science, Nagasaki 859-2501, Japan; 2Faculty of Agriculture, Shizuoka University, Shizuoka 422-8017, Japan

Background and objectives
Citrus canker caused by Xanthomonas campestris pv. citri is one of the most important problems for production of citrus worldwide. Although the bacterium has a broad host range among Rutaceae, there have been no reports on differentiation in the virulence of the pathogen [1]. Nevertheless, lesions often appear to be variable in size, even on identical cultivars in the field. Variation has been associated with age of foliage or with the cultural condition of the hosts [2]; differences in pathogenicity of bacterial strains have npt been thought to be involved in the phenomenon. For breeding resistant cultivars and for future epidemiological studies, it is important to investigate the possibility of differentiation into pathovars.

Material and methods
Japanese isolates of X. c. pv. citri identified by serological and phage tests, and eight cultivars of citrus were used. The isolates were inoculated into the attached leaves of the cultivars by pricking. 40 days after inoculation, the diameters of lesions produced were measured to evaluate the aggressiveness of pathogenicity for each isolate. The rep-PCR method reported by Louws et al. [3], and RFLP analysis using hrp cluster of the bacteria as a probe, were applied to investigate the genetic background of the isolates.

Results and conclusions
At first, 10 isolates were inoculated into the citrus species Otachibana (Citrus otachibana), Navel orange (C. sinensis), Tachibana (C. tachibana), Yuzu (C. junos), Unshiu (C. unshiu), Mexican lime (C. aurantifolia), Grapefruit (C. paradisi) and Shikikitsu (C. madurensis). Despite their distinct field resistance [2], the inoculation test showed similar aggressiveness among the isolates on each plant except for Otachibana. An additional 30 isolates were further inoculated into Otachibana or Navel orange to investigate variability in detail. Although differentiation in pathogenicity was not observed on Navel orange, distinguishable differentiation into two groups (weak and aggressive) was clearly observed on Otachibana. When the rep-PCR amplification was performed using the ERIC sequences (ERIC1R and ERIC2) as primers, two groups could be separated by the presence of a 1.7-kb DNA fragment among otherwise identical fragments. All the isolates belonging to the aggressively pathogenic group on Otachibana showed this 1.7-kb DNA fragment, while none of the isolates belonging to the weakly pathogenic group showed this fragment. In contrast, the RFLP pattern of both groups was identical and distinguishable from that of X. c. pv. aurantifolii. These results indicate that differentiation of X. c. pv. citri can be observed on Otachibana, and this differentiation may be determined genetically.

1. Stall RE, Civerolo EL, 1991. Annual Review of Phytopathology 29, 399-420.
2. Koizumi M, Kuhara S, 1982. Bulletin of the Fruit Tree Research Station D-4, 73-92.
3. Louws FJ, Fulbright DW, Stephens CT, Bruijn FJ, 1994. Applied and Environmental Microbiology 60, 2286-2295.