The phloem-limited bacterium of greening disease of citrus is a member of the alpha 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 BclI digestion and resistant to EcoRI digestion, while the 16S rDNA of the BLO was resistant to BclI digestion and sensitive to EcoRI digestion. The 16S rDNA of the African BLO strain was amplified from BclI-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 alpha 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.

[1]  K. Schleifer,et al.  Taxonomic notes: a proposal for recording the properties of putative taxa of procaryotes. , 1994, International journal of systematic bacteriology.

[2]  W. Fitch,et al.  Construction of phylogenetic trees. , 1967, Science.

[3]  J. Nourrisseau,et al.  Marginal chlorosis, a new disease of strawberries associated with a bacteriumlike organism , 1993 .

[4]  D. Gowanlock,et al.  Rickettsia-like and mycoplasma-like organisms associated with two yellows-type diseases of strawberries in Queensland , 1979 .

[5]  P. Oberholzer,et al.  Citrus psylla, a vector of the greening disease of sweet orange. , 1965 .

[6]  J. Bové,et al.  The Greening Organism is a Gram Negative Bacterium , 1984 .

[7]  A. Dabek.  Wissadula Proliferation in Jamaica II. Psyllid transmission and antibiotic therapy , 1983 .

[8]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[9]  J. Bové,et al.  Transmission of the organism associated with citrus greening disease from sweet orange to periwinkle by dodder , 1983 .

[10]  J. Bové,et al.  Study of the Greening Organism (GO) with Monoclonal Antibodies: Serological Identification, Morphology, Serotypes, and Purification of the GO , 1991 .

[11]  J. Bové,et al.  Witches' broom, a lethal mycoplasmal disease of lime trees in the sultanate of Oman and the United Arab Emirates. , 1991 .

[12]  S. Goodison,et al.  16S ribosomal DNA amplification for phylogenetic study , 1991, Journal of bacteriology.

[13]  J. Bové,et al.  Production of Monoclonal Antibodies Recognizing Most Asian Strains of the Greening BLO by in Vitro Immunization With an Antigenic Protein Purified From the BLO , 1993 .

[14]  J. Bové,et al.  Monoclonal antibodies against the bacterial-like organism associated with citrus greening disease. , 1987, Annales de l'Institut Pasteur. Microbiology.

[15]  R. Mierendorf,et al.  Direct sequencing of denatured plasmid DNA. , 1987, Methods in enzymology.

[16]  C R Woese,et al.  The phylogeny of purple bacteria: the alpha subdivision. , 1984, Systematic and applied microbiology.

[17]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[18]  S. P. Capoor,et al.  Diaphorina citri Kuway., a vector of the greening disease of Citrus in India. , 1967 .

[19]  J. Bové,et al.  Influence of temperature on symptoms of California stubborn, South Africa greening, India citrus decline and Philippines leaf mottling diseases. , 1974 .

[20]  J. Bové,et al.  Transmission of Indian citrus decline by Trioza erytreae (Del Guercio), the vector of South African greening. , 1976 .

[21]  C. Woese,et al.  Bacterial evolution , 1987, Microbiological reviews.

[22]  R. Mierendorf,et al.  [58] Direct sequencing of denatured plasmid DNA , 1987 .

[23]  J. Bové,et al.  Use of a monoclonal antibody to detect the stolbur mycoplasmalike organism in plants and insects and to identify a vector in France. , 1992 .