Molecular analyses of nonribosomal peptide synthetase in Ganoderma boninense
Oil palm plays a significant role in Malaysia’s economy as Malaysia is the secondlargest palm oil producer after Indonesia. However, the basal stem rot disease (BSR) caused by G. boninense, a basidiomycete fungus has affected oil palm yields in most production areas. Many methods to control the d...
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| Format: | Thesis |
| Language: | English |
| Published: |
2015
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/117049/ http://psasir.upm.edu.my/id/eprint/117049/1/117049.pdf |
| Summary: | Oil palm plays a significant role in Malaysia’s economy as Malaysia is the secondlargest
palm oil producer after Indonesia. However, the basal stem rot disease
(BSR) caused by G. boninense, a basidiomycete fungus has affected oil palm
yields in most production areas. Many methods to control the disease have been
developed but to date, there is no control of BSR disease in the field. Fungi were
reported to produce variety of secondary metabolites. Some secondary
metabolites are toxic to host plant and may act as virulence factors. Secondary
metabolite especially produced by nonribosomal peptide synthetase (NRPS) has
recently gained interest due to its wide array of biological activities and virulence
factors. Hence, the understanding of fungal secondary metabolism of NRPS at
molecular level is essential. In this work, detection of NRPS in G. boninense was
achieved using PCR-based and biochemical methods. Primers targeted to NRPS
conserved region of adenylation (A) domain were used to molecular characterize
NRPS. To correlate NRPS and disease incidence during Ganoderma-oil palm
interaction, RT-PCR was done conducted using samples obtained from
glasshouse trial. Chrome Azurol S (CAS) agar plate assay with incorporation of
CAS-blue dye was used for siderophore detection. PCR products were
sequenced, translated and nucleotide sequences were searched against NCBI
database using BLAST tool. The PCR fragments showed similarity to the
conserved region of adenylation domain: A2 (LKAGxAYL(VL)P(LI)D, A3
(TSG(TS)TGxPKxV) and A5 (NxYGPxE). A-domain is important as it is the core
element of NRPS modules. A-domain acts as the selector and the activator of the
cognate substrate. Colour-change reaction in the CAS-blue agar showed the
production of siderophore by G. boninense. The colour change occurred as a
result of ferric iron transfer from the reagent complex to siderophore present in the
fungus. In other reports, NRPS genes are found to be involved in the biosynthesis
of siderophores, toxins involved in pathogenesis. RT-PCR of the oil palm samples
showed the same fragment size of NRPS as detected in Ganoderma fungus. Both
progenies of susceptible and tolerant oil palm seedlings showed visible disease
symptoms at 4 months after infection. However, RT-PCR of NRPS expressed as
early as 1 month after infection and consistent expression at 4 months after
infection onwards for both susceptible and tolerant progeny. Information related to
NRPS of G. boninense obtained in this study will facilitate the understanding of
the potential evolution of Ganoderma NRPS. Further studies and analysis of these
genes and their peptide products may identify important roles of secondary
metabolites produced by NRPS in Ganoderma physiology, ecology or fungal
pathogenicity. This study is the first evidence for the present of NRPS in the
genome of G. boninense and also its involvement in BSR disease of oil palm. |
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