Multilocus sequence analysis of ‘Candidatus Phytoplasma asteris’ associated with phyllody of cucumber in India and development of loop‐mediated isothermal amplification (LAMP) assay for its detection

A survey assessed cucumber phyllody disease incidence in Chikkaballapura, Bengaluru Rural, Bengaluru Urban, and Bagalkote districts of Karnataka, India. The average disease incidence ranged from 8 to 18 %. A total of 9 samples showing phyllody and little leaf symptoms were collected and association of phytoplasma was confirmed through 16S rRNA gene amplification, using universal primer pair P1/P7, and followed by nested PCR using primer R16F2n/R16R2. Further, characterization through multilocus sequence analysis targeting secY and rpl22 genes confirmed all nine strains as part of the 16SrI group ('Candidatus Phytoplasma asteris'). Additionally, four weed species (Lecusa aspera, Parthenium hysterophorus, Acaranthus sp., and Amaranthus viridis) found in cucumber fields tested positive for phytoplasma (16SrI) disease. In-silico RFLP analysis of the amplified F2n/R2 region of the 16S rRNA gene indicated that two phytoplasma strains (CuPP1 and CuPP3) from Chikkaballapura belonged to subgroup X (16SrI-X), one phytoplasma strain (CuPP2) belonged to subgroup B (16SrI–B). While the remaining strains from Bangalore Urban, Bengaluru Rural, and Bagalkote districts belonged to subgroup B (16SrI–B). A loop-mediated isothermal amplification (LAMP) detection protocol was also developed for 'Ca P. asteris', targeting the phytoplasma 16S ribosomal DNA. Comparison of assay sensitives demonstrated that LAMP technique is highly efficient and could detect the presence of phytoplasma up to 50 fg of template DNA. LAMP-based detection offers superior ease of use, cost-effectiveness, and rapid results. To the best of our knowledge, this is the first report of phytoplasma causing phyllody disease in cucumber crop in India. •Infected cucumber plants showed little leaf, phyllody, leaf proliferation, and shortened internodes.•‘Ca. Phytoplasma asteris’ strain (16SrI) detected in cucumber phyllody symptoms via multi-locus gene sequence analysis (MLST).•A Loop‐mediated isothermal amplification (LAMP) assay was developed for detecting phytoplasma.•The present study reveals genetic diversity and impact of phyllody phytoplasma on cucumbers.•This is the first report of ‘Ca. Phytoplasma asteris’ (16SrI) causing cucumber phyllody in India.