Abstract
This work embodies the development of a real time loop mediated isothermal amplification (RealAmp) assay for the rapid detection of the cryptic tea phytopathogen, Exobasidium vexans, the causal organism of blister blight disease. Due to the widespread popularity of tea as a beverage and the associated agro-economy, the rapid detection and management of the fast-spreading blister blight disease have been a longstanding necessity. Loop-mediated isothermal amplification (LAMP) primers were designed targeting the E. vexans ITS rDNA region and the reaction temperature was optimized at 62 °C with a 60 min reaction time. Amplification of the E. vexans isolates in the initial LAMP reactions was confirmed by both agarose gel electrophoresis and SYBR Green I dye based colour change visualization. The specificity of the LAMP primers for E. vexans was validated by negative testing of seven different phytopathogenic test fungi using LAMP and RealAmp assay. The positive findings in RealAmp assay for E. vexans strain were corroborated via detecting fluorescence signals in real-time. Further, the LAMP assays performed with gDNA isolated from infected tea leaves revealed positive amplification for the presence of E. vexans. The results demonstrate that this rapid and precise RealAmp assay has the potential to be applied for field-based detection of E. vexans in real-time.
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Acknowledgements
The authors sincerely acknowledge Dr. Subhadeep Chatterjee, Head, Laboratory of Plant-Microbe Interactions, Centre for DNA Fingerprinting and Diagnostics, Hyderabad for his invaluable input in the preparation of the manuscript. The authors thank Ananda Tea Estate and Pathalipam Grant, Lakhimpur District, Assam, India, and Happy Valley Tea Estate, Darjeeling, West Bengal, India for providing the blister blight infected tea leaf samples used in the study. This work was supported by DBT, Govt. of India, Twinning Research Grant (Grant No. BT/427/NE/TBP/2013) to Tezpur University, Assam and NIPGR, New Delhi. Chayanika Chaliha would like to acknowledge DST, Govt. of India for her DST INSPIRE Fellowship (IF-150964).
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Eeshan Kalita: Conceptualization of the work, design of methodology, Validation of results, Writing, Review & Editing of the manuscript, Supervision of research, Funding acquisition. Chayanika Chaliha: design and execution of the methodology, experimentation, investigation and data analysis, writing the original draft, preparation of figures and manuscript formatting. Richa Srivastava: Experimentation and data acquisition. Lingaraj Sahoo: Resources for experimentation and Supervision of research. Praveen Kumar Verma: Supervision of research and Funding acquisition.
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Chaliha, C., Srivastava, R., Kalita, E. et al. Rapid and precise detection of cryptic tea pathogen Exobasidium vexans: RealAmp validation of LAMP approach. World J Microbiol Biotechnol 39, 52 (2023). https://doi.org/10.1007/s11274-022-03506-y
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DOI: https://doi.org/10.1007/s11274-022-03506-y