Evaluation of Genetic Diversity Among ‘Candidatus Liberibacter asiaticus’ Isolates Collected in Southeast Asia

Abstract

The aim of this study was to investigate the genetic diversity and relationships among 'Candidatus Liberibacter asiaticus' isolates from different hosts and distinct geographical areas in Southeast Asia. Genetic diversity among 'Ca. Liberibacter asiaticus' was estimated by sequencing four well-characterized DNA fragments: the 16S ribosomal DNA (rDNA) and 16S/23S intergenic spacer regions; the outer membrane protein (omp) gene region; the trmU-tufB-secE-nusG-rplKAJL-rpoB region (gene cluster region); and the bacteriophage-type DNA polymerase region. The sequences of the 16S rDNA and 16S/23S intergenic spacer regions were identical among all 'Ca. Liberibacter asiaticus' isolates. In contrast, nucleotide substitutions were observed in both the omp gene and the gene cluster regions. However, extended bacteriophage-type DNA polymerase sequences acquired by thermal asymmetric interlaced polymerase chain reaction provided the most sequence diversity among isolates. Phylogenetic analysis of the bacteriophage-type DNA polymerase sequences revealed three clusters in the Southeast Asian 'Ca. Liberibacter asiaticus' population. All Indonesian 'Ca. Liberibacter asiaticus' isolates clustered in one group. The other clusters were not correlated with geographic distribution. The differences in genetic sequences did not reflect differences in the original citrus host (mandarin or pummelo). These results suggest that the bacteriophage-type DNA polymerase region would be useful for molecular differentiation between different Southeast Asian 'Ca. Liberibacter asiaticus' isolates.

Distribution of Two Distinct Genotypes of Citrus Greening Organism in the Ryukyu Islands of Japan

“…All the isolates sequenced in this study were 3,609 nucleotides long, and were identical among isolates. Moreover, sequence alignment was carried out using the sequences obtained in this study and the previously reported sequences of the Southeast Asian isolates 16 . Interestingly, the nine Japanese isolates sequenced in this study were identical with four Taiwanese isolates (TW2, TW3, TW5, and TW6, data not shown).…”

“…Our previous study suggests that the polymorphic DNA pol may be useful for molecular differentiation among the Southeast Asian Las isolates 16 .…”

“…DNA pol was chosen as a variable region to differentiate isolates, while nusG-rplK operon was selected as a conserved region among isolates to verify the presence of Las. One primer set of DNA pol was previously reported as GODNPFW1 (5'-TCCTGAGAATTACACACAAAC-3') and GODNPRV1 (5'-TCTAAGTCTATCCTGTAACCC-3') 16 . The other primer set of nusG-rplK operon was previously reported as MHO353 (5'-GTGTCTCTGAT GGTCCGTTTGCTTCTTTTA-3') and MHO354 (5'-GAACCTTCCACCATACGCATAGCCCCTTCA-3') 8 .…”

“…Among the analyzed isolates in this study, nine isolates that possessed the DNA pol were selected, and the sequence of the DNA pol and flanking regions were determined as described previously 16 and compared among isolates. Sequence data were assembled using BioEdit version 5.0.9 6 .…”

“…Methods to distinguish Las isolates are fundamental for ecologic and epidemiologic studies. We reported genetic diversity of Las in Southeast Asia previously 16 . The degree of genetic diversity was different in each gene region of Las, and the most diverse gene region was the bacteriophage-type DNA polymerase region (DNA pol).…”