Rapid colourimetric detection and molecular surveillance of the invasive Bondar's nesting whitefly, Paraleyrodes...

Globalisation has accelerated the spread of Paraleyrodes bondari Peracchi, an important invasive pest of horticultural and plantation crops, necessitating rapid diagnostic tools for early detection and prevention of further spread into new geographical areas. In the present study, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene for the rapid detection of P. bondari were developed and validated. Molecular characterisation confirmed the identity of all specimens, with mtCOI sequences showing 100% identity to reference sequences. No amplification was detected in the nine non-target whiteflies, confirming the high analytical specificity of LAMP and RPA assays. Both assays achieved a detection limit of 10 pg µL^-1, comparable to that of quantitative polymerase chain reaction (qPCR). Validation using P. bondari (n = 102) collected from diverse agro-climatic regions of India and non-target species (n = 27) demonstrated high diagnostic performance, with colourimetric LAMP exhibiting a sensitivity, specificity and accuracy of 97.06%, 92.59%, and 96.12%, respectively, whereas the colourimetric RPA assay achieved a sensitivity, specificity, and accuracy of 99.02%, 96.30%, and 98.45% relative to qPCR. Both assays showed almost perfect agreement with qPCR (κ = 0.884 and 0.95, respectively). In addition, colourimetric detection using hydroxynaphthol blue (HNB) and SYBR Safe dyes enabled visual interpretation of results without specialised equipment. The differing operating temperature requirements of LAMP and RPA offer flexibility for implementation across different diagnostic settings.