Rapid detection of the oil palm bunch rot pathogen Paramarasmius palmivorus by recombinase polymerase amplification

Paramarasmius palmivorus (Sharples) Antonín & Kolařík, a marasmioid fungus causing bunch rot in oil palm ( Elaeis guineensis Jacq.), poses a significant threat to oil palm production because infections often remain undetected until symptoms become advanced. Rapid and reliable diagnostic tools are therefore essential for effective disease management. In this study, a recombinase polymerase amplification (RPA)-based assay targeting the internal transcribed spacer (ITS) region was developed for the rapid detection of P. palmivorus . A total of 63 samples representing 18 marasmioid and 19 non-marasmioid species were collected from oil palm plantations in Thailand, and their identities were confirmed using ITS sequences. Species-specific primers designed from the ITS region enabled selective RPA amplification of P. palmivorus within 15 min at 42 °C, without cross-reactivity with 36 non-target fungal species or host plant DNA. Amplification products were detected by agarose gel electrophoresis and bridging flocculation. The assay achieved a detection limit of 10 pg/μL genomic DNA. In a preliminary evaluation of 32 basidiocarp and mycelium-infected plant samples, the assay was compatible with a rapid heat-lysis DNA extraction method and detected eight P. palmivorus -positive samples from crude DNA extracts. The entire workflow, from DNA extraction to bridging flocculation, was completed within 70 min using minimal equipment, demonstrating its potential for preliminary screening in plantations with limited laboratory infrastructure. These findings indicate that the RPA assay provides a rapid, sensitive, and specific approach for the detection of P. palmivorus , although further validation across multiple plantations and infection stages will be required before field deployment.