Early in planta detection of Xanthomonas axonopodis pv. punicae in pomegranate using enhanced loop-mediated isothermal amplification assay

Abstract

Bacterial blight in pomegranate caused by Xanthomonas axonopodis pv. punicae (Xap) is an increasing threat for pomegranate cultivation in India. To prevent the economic losses, it is pivotal to detect the infection in latent stages rather than in later stages. We have developed an enhanced method termed as loop-mediated isothermal amplification (LAMP) technique to evaluate for the latent detection of Xap in pomegranate using six set of specific primers. Three DNA intercalating dyes were used, such as Ethidium bromide, hydroxynaphthol blue (HNB) and SYBR Green resulted in visualising the positivity for LAMP assay. The reaction time and temperature were to be 65{degrees}C from 30 min onwards, for the dyes and its sensitivity was observed up to 10-7 ng in the LAMP assay. For field applicability, LAMP assay detected Xap on 7th day post infection while the PCR amplified Xap after 11th day post infection. Finally, the specificity of LAMP assay was validated to be positive with ten Xap isolates for its accuracy and 29 non-Xap bacterial isolates showed negative results. Moreover, this method could be used as a better alternative to PCR based methods, for early detection of the pathogens.