Genetic diversity among ‘Candidatus Liberibacter asiaticus’ isolates based on single nucleotide polymorphisms in 16S rRNA and ribosomal protein genes

Citrus Huanglongbing (HLB) is one of the most destructive and widespread diseases of citrus, caused by ‘Candidatus Liberibacter’, a non-cultured, phloem-restricted alpha-proteobacterium. In the present study, ‘Ca. Liberibacter’ was detected in symptomatic citrus plants growing in the Karnataka state (South India) by amplification of 16S rRNA and β-operon ribosomal protein (β-rp) genes, using PCR primers specific for African and Asian ‘Ca. Liberibacter’. Sequencing of the 16S rRNA amplified fragment revealed that ‘Ca. Liberibacter’, here identified belongs to the species ‘Ca. Liberibacter asiaticus’. The alignments of 16S rRNA and ribosomal protein gene sequences of all known ‘Ca. Liberibacter asiaticus’- related, allowed to recognize Single Nucleotide Polymorphisms (SNPs). Basing on SNP analysis, the strains were grouped in fourteen 16SrRNA SNP genetic lineages (16Sr-I to 16Sr-XIV) and three β-rp SNP genetic lineages (rp-I to rp-III). Only the strains of ‘Ca. Liberibacter asiaticus’ from the Karnataka state belonged to the genetic lineages 16Sr-I and rp-I. RFLP diagnostic tests on the discriminative 16S rRNA SNPs were set up to identify this lineage. These results revealed the presence of a new ‘Ca. Liberibacter asiaticus’ genetic lineage in the Indian sub-continent, where at least four genetically diverse SNP lineages were found. These findings could open new opportunities for in-depth studies on biological niches and traits of ‘Ca. Liberibacter asiaticus’.