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

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The Asian type "Candidatus Liberibacter asiaticus" (Las, citrus greening organism) is severely damaging citrus production in Asia including Japan. Our previous study suggested that the bacteriophage-type DNA polymerase region (DNA pol) would be useful for molecular differentiation in different Southeast Asian Las isolates. Moreover, Las isolates originated from most of the Southeast Asian regions harbor the DNA pol gene, whereas Japanese ones lack this region. These preliminary findings lead us to a hypothesis that all Japanese isolates lack DNA pol. To try this hypothesis, we collected citrus leaf samples infected with Las throughout the Ryukyu Islands, and examined them by a duplex PCR that could simultaneously amplify two DNA fragments of DNA pol and nusG-rplK operon of Las. The duplex PCR was applied to the collection of 65 Las isolates. Both DNA pol and nusG-rplK operon were successfully amplified from nine isolates, whereas only nusG-rplK operon was amplified with the other 56 isolates. These nine isolates with DNA pol originated from the Hateruma, Irabu, Kohama, Miyako, Tarama, and Yonaguni Islands, which are geographically close to Taiwan. The nucleotide sequence of DNA pol of these nine isolates was identical, and was also the same as four Taiwanese isolates reported previously. These results suggest that Japanese Las isolates comprise at least two distinct genotypes, and the genotype that had DNA pol is highly homogeneous.