Enhanced early detection of Ganoderma orbiforme in oil palm: a nested PCR approach for improved basal stem rot disease management

Basal stem rot (BSR), caused by Ganoderma orbiforme (syn. G. boninense ), is among the most economically devastating diseases of oil palm, with yield losses reaching 80%. Early detection is challenging because of the pathogen’s prolonged asymptomatic infection period. This study developed a nested PCR protocol for detecting G. orbiforme in soil and artificially inoculated oil palm seedling tissues. Among the six primer pairs evaluated, Gan1/Gan2 demonstrated selectivity for G. orbiforme K4/2 at 50 °C. Conventional PCR detected the pathogen in soil at 0.02–1.50 g of inoculum per 500 g of soil but was unreliable at relatively low concentrations and produced nonspecific amplification in plant tissue controls. Nested PCR employing Nest1_F/Nest1_R primers in the second round achieved 4–tenfold greater sensitivity, detecting G. orbiforme at 0.005 g per 500 g of soil (1:100,000 fungus-to-soil ratio) where conventional PCR failed. Additionally, it eliminated nonspecific amplification. In inoculated seedlings assessed after 12 months, nested PCR yielded consistent positive signals at all stem sampling heights (0–30 cm) in all inoculated plants, with no amplification in uninoculated controls. Visual disease severity assessment revealed that inoculated seedlings remained asymptomatic, demonstrating that symptom-based observation could not reliably identify early infection and underscoring the necessity of molecular detection for BSR diagnosis. Biochemical analysis revealed significantly reduced lignocellulose, cellulose, and lignin contents in inoculated tissues ( p < 0.05), consistent with active white rot degradation. This nested PCR protocol shows promise as a sensitive and selective diagnostic tool for G. orbiforme under semicontrolled conditions. Field validation using naturally infected palms is needed before broader deployment.