Xanthomonas citri pv. citri is a clonal group of strains that causes citrus canker disease and appears to have originated in Asia. A phylogenetically distinct clonal group that causes identical disease symptoms on susceptible citrus, X. citri pv. aurantifolii, arose more recently in South America. Genomes of X. citri pv. aurantifolii strains carry two DNA fragments that hybridize to pthA, an X. citri pv. citri gene which encodes a major type III pathogenicity effector protein that is absolutely required to cause citrus canker. Marker interruption mutagenesis and complementation revealed that X. citri pv. aurantifolii strain B69 carried one functional pthA homolog, designated pthB, that was required to cause cankers on citrus. Gene pthB was found among 38 open reading frames on a 37,106-bp plasmid, designated pXcB, which was sequenced and annotated. No additional pathogenicity effectors were found on pXcB, but 11 out of 38 open reading frames appeared to encode a type IV transfer system. pXcB transferred horizontally in planta, without added selection, from B69 to a nonpathogenic X. citri pv. citri (pthA::Tn5) mutant strain, fully restoring canker. In planta transfer efficiencies were very high (>0.1%/recipient) and equivalent to those observed for agar medium with antibiotic selection, indicating that pthB conferred a strong selective advantage to the recipient strain. A single pathogenicity effector that can confer a distinct selective advantage in planta may both facilitate plasmid survival following horizontal gene transfer and account for the origination of phylogenetically distinct groups of strains causing identical disease symptoms.The genus Xanthomonas is comprised of strains that exhibit a high level of host specificity; over 125 different pathogenic variants (pathovars) of X. campestris that differ primarily in host range have been described (29). Host specificity in Xanthomonas can be due to gene-for-gene interactions involving avirulence genes that act in a negative fashion to limit host range (18,29,38) but also can be due to positive acting pathogenicity factors that condition host range in a host-specific manner, e.g., pthN and avrb6 of X. campestris pv. malvacearum (6, 70), opsX of X. campestris pv. citrumelo (32), and pthA of X. citri (60, 61).A highly clonal population structure is typical of many Xanthomonas pathovars that cause serious diseases (22). Surprisingly, some pathovars are comprised of clonal groups that are phylogenetically distinct but have similar or identical host ranges and cause identical disease symptoms. Examples of this phenomenon are observed for (i) common bean blight, caused by two major groups of strains within X. campestris pv. phaseoli that are only 20% related by DNA-DNA hybridization (28); (ii) bacterial spot of tomato and pepper, caused by two major groups of strains within X. campestris pv. vesicatoria (30) that are less than 50% related by DNA-DNA hybridization (53); and (iii) citrus canker disease, caused by two major groups of strains within X. citri (5) that a...