Loop-mediated isothermal amplification (LAMP) based method for rapid and sensitive detection of ‘Candidatus Liberibacter asiaticus’ in citrus and the psyllid vector, Diaphorina citri Kuwayama

Citrus greening is a destructive disease of citrus in India and many citrus-growing regions around the world. The disease is associated with three Gram negative, fastidious and phloem-limited bacteria in the genus ‘Candidatus Liberibacter’. ‘Ca. L. asiaticus’ is the most wide spread and destructive species. Currently, there is no effective control method available to manage this disease, thus rapid detection, control of its psyllid vector population and removal of affected trees are commonly recommended to manage citrus greening. The present study was conducted to standardize a rapid and sensitive loop-mediated isothermal amplification (LAMP) protocol to detect ‘Ca. L. asiaticus’ in citrus and the psyllid vector Diaphorina citri Kuwayama. A set of six primers were identified from 16S rDNA region of Indian ‘Ca. L. asiaticus’ and the amplification reaction was optimized to 65 °C for 60 min. The amplified DNA produced a ladder-like band pattern on agarose gels, and visually produced an intense green color upon staining with SYBR green. The results were subsequently validated by PCR (polymerase chain reaction) and sequencing of the amplicon. The optimized LAMP protocol is rapid, highly sensitive and cost-effective method for the early detection of citrus greening in citrus groves and nurseries, and could be performed even in small laboratories located in remote places with limited resources.