Isolation of DNA from the uncultured "Candidatus Liberobacter" species associated with citrus Huanglongbing by RAPD

Candidatus Liberobacter, the uncultured bacterium associated with citrus Huanglongbing (HLB) disease, is an alpha-Proteobacteria, and two species, "Candidatus L. africanum" and "Candidatus L. asiaticum," have been characterized by sequence analysis of the 16S rDNA and beta operon (rplKAJL-rpoBC) genes. These genes were isolated by PCR and random cloning of DNA from infected plants. However, this strategy is laborious and allowed selection of only three Liberobacter DNA fragments. In this paper, we described isolation of additional genes using Random Amplified Polymorphic DNA (RAPD). In total, 102 random 10-mer primers were used in PCR reactions on healthy and Liberobacter-infected plant DNA. Eight DNA bands amplified from infected plant DNA were cloned and analyzed. Six of them were found to be part of the Liberobacter genome by sequence and hybridization experiments. On these DNA fragments, four genes were identified: nusG, pgm, omp, and a hypothetical protein gene. These results indicate that RAPD can be used to clone DNA of uncultured organisms.