Malaysian Applied Biology Journal

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47_05_15

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Malays. Appl. Biol. (2018) 47(5): 119–127

 

COMPARISON OF DNA EXTRACTION AND DETECTION

OF Ganoderma, CAUSAL OF BASAL STEM ROT DISEASE

IN OIL PALM USING LOOP-MEDIATED ISOTHERMAL

AMPLIFICATION


MADIHAH, A.Z.1,2*, MAIZATUL-SURIZA, M.2, IDRIS, A.S.2, BAKAR, M.F.A.3, KAMARUDDIN, S.1,

BHARUDIN, I.1, ABU BAKAR, F.D.1 and MURAD, A.M.A.1


1School of Biosciences and Biotechnology, Faculty of Science and Technology,

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

2Ganoderma and Diseases Research for Oil Palm (GanoDROP) Unit, Biology Research Division,

Malaysian Palm Oil Board (MPOB), Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia

3Malaysia Genome Institute, Jalan Bangi Lama, 43000, Kajang, Selangor, Malaysia

*E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Accepted 19 October 2018, Published online 30 November 2018


ABSTRACT

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 ?L-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 ?L-1 in less than 40 min at 65ºC; whereas a minimum of 0.02 ng ?L-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.

Key words: Oil palm, basal stem rot, Ganoderma, loop-mediated isothermal amplification, LAMP

 

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