Confirmation of the sequence of "Candidatus Liberibacter asiaticus" and assessment of microbial diversity in Huanglongbing-infected citrus phloem using a metagenomic approach

The citrus disease, Huanglongbing (HLB), is a highly destructive disease in many citrus-growing regions of the world. As Koch's postulates have not been completed and the putative causal agent of this disease, Candidatus Liberibacter asiaticus, is difficult to culture, progress in studying this disease has been limited. 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 infected with a defined isolate of HLB. A culture independent analysis of 16S rRNA in these sequences showed the only bacterium present within the phloem metagenome was Ca L. asiaticus. No viral or viroid sequences were identified within the metagenomic DNA. 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 contained less than 0.2-fold coverage of five other alphaproteobacterial genomes. Thus, phloem metagenomic DNA provide a PCR-independent means of verifying the presence of Ca. L. asiaticus in infected tissue and strongly suggest no other disease agent is present in phloem.