Sequence and evolutionary analysis of ribosomal DNA from Huanglongbing (HLB) isolates of Western India

Citrus greening (Huanglongbing, HLB) is a widespread and economically important citrus disease all over the world. The disease is caused by a phloem-limited fastidious gram negative bacterium, Candidatus Liberibacter spp. which belongs to the alpha-proteobacteria group classified on the basis of its 16SrDNA sequence. Although the pathogen has been classified under three distinct groups, viz. Asian, African and American isolates, nothing is known about the status and the molecular variabilities among the Indian HLB isolates collected from different citrus cultivars grown in India. Five different HLB isolates showing variable symptoms based on their severity of infection on different citrus, viz. Mosambi, Rangpur lime, Cleopatra mandarin, acid lime and rough lemon, were studied by PCR amplification, sequence and evolutionary analysis of their 16S and 16S/23S rDNA intergenic regions. Analysis of the 16S/23S rDNA intergenic region separated all five Indian isolates from existing African isolates but failed to differentiate among Asian, American and Indian isolates. However, further analysis of complete 16S rDNA clearly indicated that Indian isolates fall within the Asian HLB group. Overall, our results suggest that all the five Indian HLB isolates taken for the current analysis belong to the Candidatus Liberibacter asiaticus strain, which showed distinct sequence variabilities and produced noticeable symptoms on the citrus trees. These results provide a robust framework for understanding how differences in pathogenicity among various HLB isolates may be related to evolutionary history.