Reverse transcription-duplex-polymerase chain reaction for simultaneous detection of Citrus tristeza virus and \u2018Candidatus Liberibacter\u2019 from citrus plants

Tristeza and Huanglongbing are the most important diseases of citrus caused by Citrus tristeza virus (CTV) and 'Candidatus Liberibacter' species, respectively. A reverse transcription duplex polymerase chain reaction (RT-d-PCR) was developed to detect these pathogens simultaneously from infected citrus plants. Consistence of the technique was validated and specificity of the primers was confirmed by sequence analysis. Concurrent detection of CTV and 'Ca. Liberibacter' spp. decreases the risk of contamination and saves time in comparison to standard techniques (i.e., nested PCR) and reduces the cost as compared to high-end techniques (i.e., quantitative PCR), especially for the selection and production of pathogen free citrus plants for certification programs

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

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 P-operon ribosomal protein (beta-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 beta-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'