Molecular typing of Coorg black pepper yellows phytoplasma by multiple gene analysis

In previous work, Coorg black pepper yellows phytoplasma (CBPYp), a 'Candidatus Phytoplasma asteris'-related strain, was identified in association with black pepper plants exhibiting yellows symptoms in southern India. In the present study, multiple gene (16S rRNA, tuf, rplV-rpsC, secY and secA) sequence analyses were carried out for finer characterisation of CBPYp isolates identified in seven plants. Nucleotide sequences of each gene studied were identical among all the CBPYp isolates here analysed. Comparison of virtual restriction fragment length polymorphism (RFLP) patterns, validated by actual digestion of polymerase chain reaction (PCR) products, revealed that CBPYp is a member of subgroups 16SrI-B, rpI-L, tufI-B, secYI-L and secA1-A. Interestingly, alignments of nucleotide sequences with other 'Candidatus Phytoplasma asteris'-related strains revealed the presence of CBPYp-specific single nucleotide polymorphisms (SNPs), located in restriction sites for endonucleases not used for conventional classification. CBPYp-specific SNPs in genes 16S rRNA, tuf and secA were detectable by virtual and actual RFLP assays, while SNPs present in rplV-rpsC and secY genes were not located in any restriction recognition site. CBPYp-specific SNPs can be used as molecular markers for the specific identification of CBPYp and for future research focused on investigating epidemiology and ecology of CBPYp in India.