Comparison of DNA extraction and detection of Ganoderma, causal of basal stem rot disease in oil palm using loop-mediated isothermal amplification.
Ganoderma boninense, a phytopathogenic fungus in the causal of the basal stem rot (BSR) disease of oil palm in Malaysia. Current detection method requires longer time and subjected to cross-reactivity with other fungi. The aim of this study is to develop fast and reliable method for detecting Ganoderma using loop-mediated isothermal amplification (LAMP). Several protocols of DNA extraction were compared to produce good quality of genomic DNA for LAMP detection. The highest concentration was achieved at 2197.8 ng µL-1 when using polyvinylpyrrolidone and sodium dodecyl sulphate method. Several genes from transcriptomic data were selected for LAMP detection such as mating genes (STE3 and STE12), regulatory genes (MnSOD and lac) and G. boninense novel genes (BUG1, BUG2, BUG3 and BUG4). Sensitivity test revealed the ability of LAMP detection at DNA concentration of 0.002 ng µL-1 in less than 40 min at 65°C; whereas a minimum of 0.02 ng µL-1 was required to detect Ganoderma using polymerase chain reaction (PCR). Out of eight set of LAMP primers designed, only bug1A primer combination was able to distinguish between Ganoderma pathogenic strains (G. boninense, G. zonatum and G. miniatocinctum) and Ganoderma non-pathogenic strain (G. tornatum) and other actinomycetes/basidiomycetes fungi. Result was confirmed by conventional PCR indicating that LAMP is more sensitive and better potential in detecting Ganoderma pathogenic to oil palm.