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Confirmation of the Sequence of ‘Candidatus Liberibacter asiaticus’ and Assessment of Microbial Diversity in Huanglongbing-Infected Citrus Phloem Using a Metagenomic Approach

December 2009 , Volume 22 , Number  12
Pages  1,624 - 1,634

Heather L. Tyler,1 Luiz F. W. Roesch,1 Siddarame Gowda,2 William O. Dawson,2 and Eric W. Triplett1

1Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville 32611-0700, U.S.A.; 2Department of Plant Pathology, University of Florida, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred 33850, U.S.A.

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Accepted 9 July 2009.

The citrus disease Huanglongbing (HLB) is highly destructive in many citrus-growing regions of the world. The putative causal agent of this disease, ‘Candidatus Liberibacter asiaticus’, is difficult to culture, and Koch's postulates have not yet been fulfilled. As a result, efforts have focused on obtaining the genome sequence of ‘Ca. L. asiaticus’ in order to give insight on the physiology of this organism. In this work, three next-generation high-throughput sequencing platforms, 454, Solexa, and SOLiD, were used to obtain metagenomic DNA sequences from phloem tissue of Florida citrus trees infected with HLB. A culture-independent, polymerase chain reaction (PCR)-independent analysis of 16S ribosomal RNA sequences showed that the only bacterium present within the phloem metagenome was ‘Ca L. asiaticus’. No viral or viroid sequences were identified within the metagenome. By reference assembly, the phloem metagenome contained sequences that provided 26-fold coverage of the ‘Ca. L. asiaticus’ contigs in GenBank. By the same approach, phloem metagenomic data yielded less than 0.2-fold coverage of five other alphaproteobacterial genomes. Thus, phloem metagenomic DNA provided a PCR-independent means of verifying the presence of ‘Ca L. asiaticus’ in infected tissue and strongly suggests that no other disease agent was present in phloem. Analysis of these metagenomic data suggest that this approach has a detection limit of one ‘Ca. Liberibacter’ cell for every 52 phloem cells. The phloem sample sequenced here is estimated to have contained 1.7 ‘Ca. Liberibacter’ cells per phloem cell.

© 2009 The American Phytopathological Society