A reverse transcription loop-mediated isothermal amplification assay for the detection of citrus exocortis viroid in Australian citrus

Citrus exocortis viroid (CEVd), the causal agent of exocortis, is a pathogen that is thought to infect all citrus varieties, although it is asymptomatic in most. Symptoms of exocortis develop on susceptible rootstocks, resulting in stunting and yield reduction. To aid the detection and management of CEVd, a rapid near-field assay was developed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the viroids in nursery and field trees. Over 240 CEVd sequences, including sequence variants from representative Australian isolates that induce mild and severe symptoms, were used in the design of the primers. The RT-LAMP successfully detected CEVd in a 1:1000 dilution (236 pg) of plant total RNA indicating high sensitivity, and also detected the viroid in rapid, crude plant extractions. The assay was highly specific to CEVd, given there was no cross-reactivity with other citrus-infecting pathogens. This new assay provides a simple, robust, specific, and sensitive method to detect CEVd in Australian citrus and to our knowledge, is the first RT-LAMP assay to detect any citrus-infecting viroid.