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

First author: Kuchinotsu Citrus Research Station, National Institute of Fruit Tree Science, Kuchinotsu, Minami-shimabara, Nagasaki 859-2501, Japan; second, third, and ninth authors: National Institute of Fruit Tree Science, Fujimoto 2-1, Tsukuba, Ibaraki 305-8605, Japan; fourth and fifth authors: National Agricultural Research Center for Kyushu Okinawa Region, Suya 2421, Koshi, Kumamoto 861-1192, Japan; sixth author: Department of Entomology and Plant Pathology, Gadjah Mada University, Yogyakarta 55281, Indonesia; seventh and eighth authors: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, Taiwan.

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.

Additional keywords:citrus greening.