Analysis of nucleotide sequence variation of citrus greening organism and application for rapid and sensitive detection from infected citrus

Citrus greening is a serious disease of citrus which limits production in many parts of Asia and Africa. The Asian causal agent is designated Candidatus Liberibacter asiaticus (Las). Genetic difference among isolates of Las is little known. So far, no nucleotide substitution was found in the 16S/23S region. This study looked into in more detail on the genetic difference of Southeastern Las. Fragments of the tufB-secE-nusG-rplKAJL- rpoB region and the phage-type DNA polymerase region were sequenced after PCR amplification. A few nucleotide substitutions were observed in the tufB-secE-nusG- rplKAJL-rpoB region at a ratio of one out of about 2000 nucleotides. In contrast, there was a highly variable region in the upstream of the DNA polymerase gene. The results suggested that conserved sequence in the rpl-KAJL operon would be applicable for stable detection of Las utilizing gene amplification, whereas the variable part in the phage-type DNA polymerase region would be useful for differentiation of Southeastern Asian Las. Based on these findings, a simple and reliable detection method by using Loop-mediated Isothermal Amplification (LAMP) was developed.