Visual flocculation-based detection of the oil palm pathogen Ganoderma boninense Pat. using recombinase polymerase amplification

Ganoderma boninense Pat., the causal agent of basal stem rot (BSR), is one of the most destructive threats to oil palm cultivation. Effective disease management requires rapid and accurate diagnostic tools to support timely intervention. In this study, a recombinase polymerase amplification (RPA) assay was developed and evaluated using two detection platforms, gel electrophoresis and flocculation, for the detection of G. boninense isolates collected from oil palm plantations in Thailand. Both platforms demonstrated high specificity, with no cross-reactivity against 12 other Ganoderma species, 20 additional mushroom species, or host plant DNA, and all samples were confirmed by internal transcribed spacer (ITS) sequence analysis. Using purified RPA products, the detection limits for genomic DNA were 50 pg/μL by gel electrophoresis and 25 pg/μL by flocculation. A simplified heat-lysis protocol enabled rapid DNA extraction from pure mycelial cultures of G. boninense and was fully compatible with the RPA assay. Field validation using 35 fungal samples identified 12 positives for G. boninense . The RPA-flocculation workflow could be completed within 50 min using minimal equipment, highlighting its potential utility for preliminary disease assessment in remote plantation settings with limited access to centralized laboratory facilities.