Loop-mediated isothermal amplification assay for pre-symptomatic stage detection of Xanthomonas axonopodis pv. punicae infection in pomegranate

Bacterial blight in pomegranate caused by Xanthomonas axonopodis pv. punicae (Xap) is an increasing threat to pomegranate cultivation in India. To prevent economic losses, it is pivotal to detect the infection in latent stages rather than in symptomatic stages. We have developed an enhanced loop-mediated isothermal amplification (LAMP) technique for the detection of latent Xap infection in pomegranate using six sets of pathovar-specific primers. The DNA-intercalating dye ethidium bromide was standardized for visualizing positive amplification in the LAMP assay. The optimum reaction time was 30 min or more at 65 degrees C, and assay sensitivity was enhanced down to femtogram (fg) amounts of template DNA (0.153 fg) in the LAMP assay. In artificially inoculated plants, the enhanced LAMP assay detected Xap on the 7th day post inoculation (dpi), while PCR detected Xap only after the 11th dpi. Finally, the specificity of the LAMP assay for Xap was validated against 31 bacterial strains belonging to 22 species. The LAMP assay developed in this study is highly specific, sensitive and robust and can be used as an improved alternative for PCR-based methods for the early detection of Xap infection in pomegranate. Early detection of Xap in pomegranate ensures quality fruits with increased yield by adopting precautionary measures that ensure improved monetary benefit to farmers.