Development of PCR-based detection method for fruit bunch rot (FBR) disease in oil palm / Suhanah Jaabi

Fruit bunch rot (FBR) disease is considered as a new potential emerging disease of oil
palm in Malaysia. The occurrence of this disease has been reported in Perak and
Selangor states since 2012. The causal pathogen was identified as Marasmius
palmivorus Sharples (1928) is known to be as saprophytic apparently become parasitic
after it has attained a certain inoculum level of infection. The current study aimed to
develop molecular polymerase chain reaction (PCR) detection method for FBR disease
in oil palm. The use of molecular diagnostic assay offers specificity and rapid diagnostic
for an early disease detection in plantations. Therefore, a total of 39 samples were
collected from Perak and Selangor states by random sampling in the oil palm
plantations. Basidiocarps and rhizomorphs on diseased oil palm were collected prior
surface sterile and 8 pure isolates of M. palmivorus were successfully isolated. The
identity was confirmed by morphological and molecular identification. An artificial
inoculation of M. palmivorus on detached oil palm fruit using in vitro assay showed
positive infection. Ten species-specific primers were designed to amplify the Internal
Transcribed Spacer (ITS) of ribosomal gene cluster (rDNA) of M. palmivorus. Primers
designed in this study were screened for efficiency and specificity before optimized
using Gradient PCR for optimum annealing temperature (59 – 64ºC) for PCR
amplification. Validation assays performed by cross reaction with 34 different fungi
species includes 66 samples from different sources. The results obtained suggests that
the new designed primer could be employed to directly analyse sample from the field
with the aim of detecting the presence of the pathogen in diseased fruits. Molecular
analyses suggested that primer pair MP02 was able to amplify M. palmivorus at 64 ºC
annealing temperature in 30 cycles of PCR programme. This report for the first time
that species specific primer MP02 has revealed to be useful primer in PCR amplification
for the detection of M. palmivorus rDNA.