f The Phloem-Limited Bacterium of Greening Disease of Citrus Is a Member of the α Subdivision of the Proteobacteria

Using the PCR, we amplified the 16S ribosomal DNAs (rDNAs) of an Asian strain and an African strain of the uncultured, gram-negative, walled, phloem-limited bacterium-like organism (BLO) associated with citrus greening disease. We evaded coamplification of chloroplast 16S rDNA by using restriction enzymes; the chloroplast 16S rDNA was sensitive to Bcl I digestion and resistant to Eco RI digestion, while the 16S rDNA of the BLO was resistant to Bcl I digestion and sensitive to Eco RI digestion. The 16S rDNA of the African BLO strain was amplified from Bcl I-digested DNA extracted from infected periwinkle leaf midribs. The Asian strain was isolated from plant extract by using a specific monoclonal antibody coated onto the surface of a PCR tube. The 16S rDNAs of the two BLO strains were cloned and sequenced. Comparisons with sequences of 16S rDNAs obtained from the GenBank data base revealed that the two citrus greening disease BLOs belong to the alpha subdivision of the class Proteobacteria. Even though their closest relatives are members of the alpha-2 subgroup, these BLOs are distinct from this subgroup as we observed only 87.5% homology between the 16S rDNAs examined. Therefore, the two BLOs which we studied probably are members of a new lineage in the α subdivision of the Proteobacteria. We propose the trivial name “liberobacter” for this new group of bacteria and will wait until additional characteristics have been determined before we propose a formal name.