Multilocus genotyping of ‘Candidatus Phytoplasma aurantifolia’ associated with witches’ broom disease in Citrus spp.

Witches’ broom disease of lime (WBDL), associated with ‘Candidatus Phytoplasma aurantifolia,’ is a serious disease of acid lime in Southern Iran. In order to characterize ‘Ca. P. aurantifolia’ strains, a multilocus sequence analysis protocol was developed based on different genomic loci, including 16S rRNA, 16S-23S rRNA intergenic spacer (IS), ribosomal protein genes rpl22 and rps3, cell division protein, and group II intron reverse transcriptase/mature gene, in 46 phytoplasma strains. Analyses of virtual RFLP, phylogenetics, and DNA sequence identity of 16S rRNA of Iranian phytoplasma strains associated with symptomatic citrus plants indicated that the Iranian strains were classified in ribosomal subgroup 16SrII-B. In total, three genotypes were recognized based on 16Sr virtual RFLP profiles. Phylogenetic analysis based on the rp and ftsH gene sequences showed clustering of all the strains as one group. Comparative sequence and phylogenetic analysis of group II intron genes indicated the highest differentiation of ‘Ca. P. aurantifolia’–related strains in four genotypes. The polymorphism of group II intron reverse transcriptase/mature genes fragmentation provides finer differentiation of closely related ‘Ca P. aurantifolia’ strains and reveals higher genetic diversity than previously reported.