Multilocus, Multiplex Detection of Ganoderma zonatum from Environmental Samples

Ganoderma butt rot of palms is caused by a white rot basidiomycete fungus, Ganoderma zonatum. Typical symptoms include wilting of fronds that starts in the lower canopy and moves to the top. As wilting symptoms are also associated with other diseases and disorders, appearance of basidiomata on the trunks is necessary to confirm this disease. Basidiomata develop late in the disease cycle, making early diagnostics challenging. Here, we describe a DNA-based molecular diagnostic assay that could be used to confirm the presence of G. zonatum in palm trunks before conks are observed. Primers tailored to end on single-nucleotide polymorphisms (SNPs), that differentiate G. zonatum from 14 other Ganoderma taxa, were designed from multiple regions in four genes: internal transcribed spacer (ITS), RNA polymerase 1 (rpb1), rpb2, and translation elongation factor 1-α (tef1-α). A set of three primer pairs could successfully determine the incidence of G. zonatum with high specificity and sensitivity in different environmental samples such as sawdust collected from naturally infected palm trunks and soil samples containing G. zonatum basidiospores. This rapid PCR-based assay could potentially be used to detect inoculum sources of the fungus and track its movement and survival in different palm tissues and environments. Early detection of G. zonatum is a crucial step toward building and implementing better disease management strategies and mitigating potential risks from palm failures due to decay.