Asia J. Mol. Biol. of Molecular Biology and Biotechnology, 2005 AsPacPacific Journal Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia 23 Vol. 13 (1) : 23-34 Random Amplified Polymorphic DNA (RAPD) and Random Amplified Microsatellite (RAMS) of Ganoderma from Infected Oil Palm and Coconut Stumps in Malaysia Latiffah Zakaria1*, Harikrishna Kulaveraasingham2, Tan Soon Guan3, Faridah Abdullah3 and Ho Yin Wan2 1 School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang. 2 Institute of Bioscience, 3Department of Biology, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Received 20 September 2004 / Accepted 14 Jan 2005 Abstract. Random amplified polymorphic DNA (RAPD) and random amplified microsatellite (RAMS) analyses were used to determine the genetic relatedness within and between Ganoderma boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps from different locations in Malaysia. RAPD analysis using four random primers (5’ACCTGGACAC3', 5’CAGCGACAAG3', 5’AGAGGGCACA3' and 5’TGACGGCGGT3') showed variations of banding patterns within and between the isolates from oil palm and coconut stumps, indicating that they were genetically heterogeneous. There was no specific banding pattern that could differentiate between G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. RAMS analysis using four microsatellite primers, 5’BDB(ACA)5, 5’DD(CCA)5, 5’DHB(CGA)5 and 5’YHY(GT)5G, also showed variable banding patterns among the isolates from infected oil palm and coconut stumps. However, five common bands i.e. two bands (900 bp and 1200 bp) produced by primer (CGA)5, one band (1400 bp) by primer (ACA)5 and two bands (350 bp and 380 bp) by primer (CCA)5 were shown by all the G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. Dendrograms from cluster analysis based on UPGMA of RAPD and RAMS data showed that G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps did not cluster separately into two distinct clusters, but were clustered together, which indicated that both groups of Ganoderma are closely related. The finding that the Ganoderma isolates from coconut stumps are closely related to G. boninense isolates from infected oil palm would have an important bearing in the formulation of disease control measures and replanting procedures, especially in areas where the previous crop was coconut. Keywords. Ganoderma, oil palm, coconut, RAPD, RAMS Abdullah, 1996; Idris, 1999). However, the most common INTRODUCTION species is G. boninense which was first reported by Ho and Nawawi (1985) and later Khairudin (1990) confirmed by The basal stem rot of oil palm caused by Ganoderma is the pathogenicity tests that G. boninense was the causal agent of most serious disease of field palms in Southeast Asia, the disease. In several estates with a high disease incidence, G. particularly Malaysia and Indonesia. The disease was first boninense was also found to be the most common and virulent reported by Thompson in 1931. Initially, the basal stem rot species (Idris, 1999). was thought to be a disease of older palms as it was found to High disease incidence occurs in oil palm plantings that infect mostly older palms, but in the 1960s the disease were previously planted with coconut, especially where old appeared in younger palms of 10 – 15 years old (Turner, coconut trees were felled and the stumps buried or left on the 1981). In the last decade or so, palms as young as 1 year old ground to rot. Disease incidence is also high when old coconut were found to be infected by the disease (Arifin et al., 1989). trees were poisoned and left standing to rot. Although The basal stem rot shortens the productive life of the oil Ganoderma on coconut stumps exists as saprophyte, Turner palms and incurs considerable economic losses to the oil palm industry. *Author for Correspondence. Mailing address: School of Biological Sciences, Universiti Sains A number of Ganoderma species have been reported to be Malaysia,11800 Minden, Penang,Malaysia.Tel:604 – 6533506; Fax : 604– associated with the basal stem rot of oil palm (Turner, 1981; 6565125; Email : Lfah@usm.my 24 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia (1981) suggested that old coconut stumps and felled coconut RAPD analysis. In a preliminary test, 20 random primers trunks which were colonised by Ganoderma could serve as from Kit I (Operon Technologies, Inc.) were screened. The inoculum to healthy oil palms. Pathogenicity tests have also primers were ten bases long with 50% - 70% G+C content. shown that cross infection could occur between Ganoderma From the test, 10 primers produced reproducible fragments. from oil palm and coconut (Abdullah, 1998; Idris, 1999). Each primer was further tested twice to verify the These studies suggest that Ganoderma on coconut stumps reproducibility and consistency of RAPD banding patterns. could assume an important role in the dissemination and Four primers, which produced consistent patterns, were development of the disease. However, it is not known selected for RAPD analysis. They were OPI 01 whether the Ganoderma on coconut stumps is the same species (5’ACCTGGACAC3'), OPI 07 (5' CAGCGACAAG3'), OPI as that on oil palm. 12 (5' AGAGGGCACA 3') and OPI 14 (5' TGACGGCGGT As morphological characteristics of Ganoderma may vary 3'). RAPD analysis was repeated twice to ensure reproducibility with environmental conditions, they are unreliable as the sole and consistency of the banding patterns for each isolate. All criterion for distinguishing different species. Other reagents and standard molecular markers for PCR characteristics, such as molecular characters, should be studied. amplifications, namely, MgCl2, dNTPs, Taq polymerase as Molecular characteristics of fungi are increasingly being used well as DNA template were optimised to obtain standardised as additional taxonomic criteria in classification or to resolve reaction conditions for RAPD analysis. The reagents were controversies in taxonomic position of taxa. A wide range of obtained from Life Technologies GIBCO BRL. In the molecular methods has been introduced, especially with the preliminary experiments, the concentrations of all the reagents rapid development of polymerase chain reaction (PCR)-based and the PCR amplification protocol were the same as those techniques. One of the PCR-based techniques that has been reported by Williams et al. (1993) and Hseu et al. (1996). widely used in characterisation of plant pathogenic fungi is After optimisation of all the reagents, RAPD amplification random amplified polymorphic DNA (RAPD) which is based was performed in a total volume of 25 µl reaction mixture on PCR amplification of DNA fragment using single primer which contained 2.5 mM MgCl2, 1X PCR buffer [20 mM with an arbitrary nucleotide sequence (Williams et al., 1990). Tris-HCl (pH 8.4) and 50 mM KCl ], 50 µM dNTP mix, 2.5 RAPD has been used in the characterisation of Aphanomyces units Taq polymerase, 0.6 µM of primer and 3 - 5 ng template euteiches strains that cause root rot disease (Malvick et al., DNA. 1998); in the identification of Colletotrichum fragariae from The RAPD amplification conditions used were based on diseased strawberry (Martinez-Culebras et al., 2002); and in those described by Williams et al. (1990). Amplifications were the characterisation of Fusarium moniliforme isolated from performed in a Perkin Elmer Thermalcycler (PE Applied different hosts (Kini et al., 2002). Biosystem Model 2400) with the following protocols: 4 min Another PCR-based technique is random amplified of initial denaturation at 98oC, 45 cycles of denaturation at microsatellite (RAMS), which combines several characteristics 95oC for 1 min, annealing at 36oC for 1 min and extension at of RAPD and microsatellite analysis. In this technique, the 72oC for 2 min, and final extension at 72oC for 10 min to DNA between distal ends of two closely related microsatellite ensure complete extension of the amplification products. is amplified. Hantula et al. (1996) has demonstrated that RAMS is applicable in studying genetic variation in fungi. In the RAMS analysis. The primers used for RAMS analysis were present study, RAPD and RAMS analyses were used to adapted from Hantula et al. (1996). The primers were determine the genetic relationship within and between G. 5’BDB(ACA) 5, 5’DD(CCA) 5, 5’DHB(CGA) 5 and boninense isolates from infected oil palm and Ganoderma sp. 5’YHY(GT)5G in which H, B, Y and D were used as from coconut stumps. degenerate sites and H = (A,T or C); B = (G or C); Y = (G, A or C); D = (G, A or T). PCR amplification was performed using a total volume of 25 µl in which the reaction mixture contained 1X PCR MATERIALS AND METHODS buffer [20 mM Tris-HCl (pH 8.4) and 50 mM KCl], 1.5 mM MgCl2, 0.2 mM of dNTP, 0.5 µM of primer, 2.5 units Taq Ganoderma isolates and DNA extraction. A total of 53 polymerase and 50 ng template DNA. The reagents were G. boninense isolates from infected oil palm and 15 isolates of obtained from Life Technologies GIBCO BRL. Ganoderma from coconut stumps from different estates and The protocol for RAMS amplification was based on that areas in Malaysia were used in this study (Table 1). The isolates of Hantula et al. (1996). Amplifications were performed in a were cultured in yeast extract sucrose broth for 14 days after Perkin Elmer Thermalcycler (PE Applied Biosystems model which their mycelia were harvested, lyophilized and used for 2400) in a profile of 35 cycles. An initial denaturation of 10 DNA extraction following the phenol-chloroform method min at 95 oC was carried out before the cycle began. described by Raeder and Broda (1985). The DNA was Amplifications were done as follows: 30s denaturation at dissolved in TE buffer and stored at –20oC until used. DNA 95oC, 45s annealing at a temperature depending on the primer was quantified using a spectrophometer (DM 65 Beckman). and 2 min extension at 72oC. Annealing temperature for AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia 25 Table 1. List of locations of G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps Host Code Name and Location of Estate Oil palm EG 01 Dusun Durian Estate, Banting, Selangor Oil palm OP114 South Estate, Carey Island, Selangor Oil palm OP123 South Estate, Carey Island, Selangor Oil palm OP153 South Estate, Carey Island, Selangor Oil palm OP170 South Estate, Carey Island, Selangor Oil palm OP187 South Estate, Carey Island, Selangor Oil palm OP230 South Estate, Carey Island, Selangor Oil palm OP64 North Estate, Carey Island, Selangor Oil palm OP65 North Estate, Carey Island, Selangor Oil palm OP83 North Estate, Carey Island, Selangor Oil palm OP88 North Estate, Carey Island, Selangor Oil palm EGSE2 Selaba Estate, Teluk Intan, Perak Oil palm EGSE5 Selaba Estate, Teluk Intan, Perak Oil palm EGSE4 Selaba Estate, Teluk Intan, Perak Oil palm EGSE6 Selaba Estate, Teluk Intan, Perak Oil palm EGSE12 Selaba Estate, Teluk Intan, Perak Oil palm EGBD2 Bagan Datoh Estate, Bagan Datoh, Perak Oil palm EGBD5 Bagan Datoh Estate, Bagan Datoh, Perak Oil palm EGSK3 Sungai Krian Estate, Bagan Serai, Perak Oil palm EGSK4 Sungai Krian Estate, Bagan Serai, Perak Oil palm EGSK6 Sungai Krian Estate, Bagan Serai, Perak Oil palm EGSK7 Sungai Krian Estate, Bagan Serai, Perak Oil palm EGSK10 Sungai Krian Estate, Bagan Serai, Perak Oil palm EGCN2 Chersonese Estate, Kuala Kurau, Perak Oil palm EGCN7 Chersonese Estate, Kuala Kurau, Perak Oil palm EGCN8 Chersonese Estate, Kuala Kurau, Perak Oil palm EGJS1 Jin Seng Estate, Bagan Serai, Perak Oil palm EGJS8 Jin Seng Estate, Bagan Serai, Perak Oil palm EGJS9 Jin Seng Estate, Bagan Serai, Perak Oil palm EGJS10 Jin Seng Estate, Bagan Serai, Perak Oil palm EGS2 Serkam Estate, Serkam, Melaka Oil palm EGS3 Serkam Estate, Serkam, Melaka Oil palm EGR1 Regent Estate, Melaka Oil palm EGR2 Regent Estate, Melaka Oil palm EGR4 Regent Estate, Melaka Oil palm EGR5 Regent Estate, Melaka Oil palm EGR6 Regent Estate, Melaka Oil palm EGR7 Regent Estate, Melaka Oil palm EGT Tangkah Estate, Tangkak, Johor Oil palm EGL1 Lanadron Estate, Panchor, Muar, Johor Oil palm EGL2 Lanadron Estate, Panchor, Muar, Johor Oil palm EGL3 Lanadron Estate, Panchor, Muar, Johor Oil palm EGL5 Lanadron Estate, Panchor, Muar, Johor 26 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia Table 1. List of locations of G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps (continued) Host Code Name and location of estate Oil palm EGL8 Lanadron Estate, Panchor, Muar, Johor Oil palm EGN3 Nordanal Estate, Panchor, Muar, Johor Oil palm EGN4 Nordanal Estate, Panchor, Muar, Johor Oil palm EGN7 Nordanal Estate, Panchor, Muar, Johor Oil palm EGN8 Nordanal Estate, Panchor, Muar, Johor Oil palm EGN9 Nordanal Estate, Panchor, Muar, Johor Oil palm EGPK5 Pitas Estate, Kudat, Sabah Oil palm EGPK6 Pitas Estate, Kudat, Sabah Oil palm EGPK7 Pitas Estate, Kudat, Sabah Oil palm EGPK8 Pitas Estate, Kudat, Sabah Coconut CN04 Renggam, Johor Coconut CN05 Renggam, Johor Coconut CN06 Renggam, Johor Coconut CNRT310 Rengit, Batu Pahat, Johor Coconut CNUB01 Ulu Bernam, Teluk Intan, Perak Coconut CNKK1 Kuala Klanang, Banting, Selangor Coconut CNKK2 Kuala Klanang, Banting, Selangor Coconut CNKK3 Kuala Klanang, Banting, Selangor Coconut CNKK4 Kuala Klanang, Banting, Selangor Coconut CNKK6 Kuala Klanang, Banting, Selangor Coconut CNDDE2 Dusun Durian Estate, Banting, Selangor Coconut CNBt61 Batu Enam, Banting, Selangor Coconut CNBt62 Batu Enam, Banting, Selangor Coconut CNBt63 Batu Enam, Banting, Selangor (CCA)5 was 64oC, for (CGA)5 61oC, for (GT)5 58oC, and for was constructed using simple matching coefficient according (ACA)5 72oC. Final extension of 7 min at 72oC was performed to the formula shown below: after the cycles ended. Gel electrophoresis. The amplification products were SMC = a + d / a + b + c + d where: separated by electrophoresis in a 1.75% agarose gel (SeaKem a = number of shared bands LE) using Tris Borate EDTA (TBE) pH 8.2 as running buffer. b = number of bands present only in isolate 1 The electrophoresis was carried out at 5V/cm. After c = number of bands present only in isolate 2 electrophoresis, the gels were stained with ethidium bromide d = number of bands absent in isolates 1 and 2. and the patterns were visualized under UV light and photographed using a Kodak Polaroid camera. Comparisons The Numerical Taxonomy System of Multivariate Program of the banding patterns were made using the 100 bp marker (NT-SYS) software package Version 1.8 (Rohlf, 1994) was as a molecular size standard. used to analyse the data. A dendrogram was constructed using UPGMA (unweighted pair-group method with arithmetic Data analysis. The banding patterns generated by all the averages) cluster analysis to infer the relationship between different primers were scored as presence (1) and absence (0) the 68 isolates from oil palm and coconut. of band of a particular molecular size to compile a binary matrix which was then subjected to cluster analysis. Both faint and intense bands were scored if shown to be RESULTS reproducible in separate runs. The four primers, OPI 01, OPI 07, OPI 12 and OPI 14 used The relationships between all the isolates could be more in RAPD analysis produced reproducible and consistent clearly represented by a similarity matrix. The similarity matrix banding patterns of the Ganoderma isolates. Primer OPI 01 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia 27 produced between 1 – 15 bands ranging from 400 – 2072 bp. polymorphic patterns, 5 – 20 bands ranging from 200 – 2072 Primer OPI 07 also produced 1 – 15 bands but the bands bp were produced and for primer (CCA)5 which also showed ranged from 300 – 2072 bp. For primer OPI 12, between 1 - highly polymorphic patterns, 12 – 20 bands of 200 – 2072 bp 10 bands were produced with molecular sizes ranging from were produced. Although RAMS analysis using the four about 400 – 2072 bp and for primer OPI 14, between 3 – 14 primers resulted in polymorphic banding patterns, which bands ranging from 300 – 2072 bp were produced. None of indicated a high degree of genetic variation among the isolates the primers produced specific patterns that could differentiate from both groups of Ganoderma, five common bands were between G. boninense isolates from infected oil palm and produced by all the Ganoderma isolates. They were two bands Ganoderma sp. from coconut stumps as variations in banding (900 bp and 1200 bp) produced by primer (CGA)5, one band patterns occurred within and between isolates of both groups (1400 bp) by primer (ACA)5 and two bands (350 bp and 380 of Ganoderma. Generally, there was a distinct pattern of bands bp) by primer (CCA) 5. Figure 4 and 5 show some amplified from each isolate regardless of whether it was from representative RAMS banding patterns of some G. boninense infected oil palm or from coconut stumps, although several isolates from infected oil palm and Ganoderma sp. from coconut bands were shared among the isolates. Both intense and faint stumps. bands were produced. Figure 1 and 2 show some The similarity values from RAMS analysis showed a broad representative RAPD banding patterns for some of the G. range of values from 0.531 – 0.972 (about 53% - 97% boninense isolates from infected oil palm and Ganoderma sp. similarities) for G. boninense isolates from infected oil palm from coconut stumps. and Ganoderma sp. from coconut stumps. Pair-wise From the similarity matrix based on simple matching comparisons between oil palm isolates showed similarity coefficient, the similarity values between all the G. boninense values ranging from 53% - 97% and for comparisons between isolates from infected oil palm and Ganoderma sp. from coconut isolates, similarity values ranged from 55% - 87%. coconut stumps were from 46% - 91%. Pair-wise comparison As for pair-wise comparisons between oil palm and coconut between the oil palm and coconut isolates showed similarity isolates, similarity values were between 53% - 82%. The values of between 0.508 – 0.831 (about 51% - 83% dendrogram based on UPGMA cluster analysis of RAMS similarities). Generally, Ganoderma isolates from the same bands is shown in Figure 6. Although the dendrogram can estate had higher similarity (82% - 90%) when compared to be divided into two major clusters, I and II, at about 66% the isolates from different estates (46% - 78%). The similarity level, G. boninense isolates from infected oil palm dendrogram based on UPGMA cluster analysis of RAPD and Ganoderma sp. from coconut stumps did not cluster bands is shown in Figure 3. There are overlapping of clusters separately to form these two major clusters. The coconut which group all the Ganoderma isolates. The oil palm and isolates were clustered together with the oil palm isolates, coconut isolates were not separated into two distinct clusters especially in Sub-clusters 1C, IE and Major Cluster II. Most but were clustered together, indicating close relationship. The of the isolates were grouped according to their locality. dendrogram can be divided into several clusters and sub- clusters. The coconut isolates were clustered together with the oil palm isolates in Sub-clusters A, B, C and D. Sub- clusters E, F, G and Major Cluster II comprised only oil palm DISCUSSION isolates. Isolates from the same estate in both groups of The four primers used in RAPD analysis showed Ganoderma tended to cluster in the same cluster or sub-cluster. polymorphism within and between all the Ganoderma isolates This is not surprising as the similarity values shown by the from oil palm and coconut. However, these primers did not isolates from the same estate were very high which indicate produce specific patterns that could differentiate between G. very close relationship. boninense isolates from infected oil palm and Ganoderma sp. The four microsatellite primers, (GT)5, (CCA) 5, (CGA)5 from coconut stumps. Banding patterns produced by each and (ACA)5, were able to generate amplification products for primer were highly variable and most amplified bands were all the Ganoderma isolates tested. Variations of banding polymorphic, indicating genetic variation among all the patterns were observed within and between G. boninense Ganoderma isolates from both infected oil palm and coconut isolates from infected oil palm and Ganoderma sp. from coconut stumps. stumps. The banding patterns produced by using (GT)5 and Idris et al. (1996) have also reported variations in RAPD (ACA)5 were not as highly polymorphic as those produced by banding patterns in various Ganoderma isolates from different using (CCA)5 and (CGA)5. Both intense and faint bands were hosts such as oil palm, coconut, rubber, ornamental palm produced by the Ganoderma isolates using the four and hardwoods which originated from different geographical microsatellite primers. origins (Malaysia, Singapore, Indonesia, Papua New Guinea, Primer (GT)5 produced between 1 – 14 bands with Solomon Island, United Kingdom, United States of America molecular sizes ranging from 300 bp – 2072 bp and primer and Poland). They found polymorphism to be much greater (ACA)5 produced between 2 – 7 bands ranging from 400 bp within the isolates of G. boninense from oil palm and coconut – 2072 bp. For primer (CGA) 5 which showed highly hosts compared to the other Ganoderma species from other 28 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia Figure 1. RAPD banding patterns obtained using primer OPI 01 of G. boninense isolates from infected oil palm from two different estates and one isolate of Ganoderma sp. from coconut stump. Lane 1. coconut isolate. (1.CNUB01). Lanes 2–10: oil palm isolates (2.EGPK5, 3.EGPK6, 4.EGPK7, 5.EGPK8, 6.EGR2, 7.EGR6, 8.EGR4 , 9.EGS2, 10. EGS3). M-marker. Figure 2. RAPD banding patterns obtained using primer OPI 07 of G. boninense isolates from infected oil palm from two different estates and isolates of Ganoderma sp. from coconut stumps from four different areas. Lanes 1–5: oil palm isolates (1.EGR1, 2.EGR7, 3.EGCN2, 4. EGCN7, 5.EGCN8). Lanes 6–10: coconut iso1ates (6.CNKK2, 7.CNDDE2, 8.CN05, 9.CNRT310, 10.CN04). M – marker. AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia 29 Figure 3. Dendrogram from UPGMA analysis using simple matching coefficient based on RAPD bands of G. boninense isolates from infected oil palm and isolates of Ganoderma sp. from coconut stumps. 30 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia Figure 4 . RAMS banding patterns obtained using primer (GT)5 of G. boninense isolates from infected oil palm from four different estates and isolates of Ganoderma sp. from coconut stumps from two different areas. Lanes 1-5, 7: oil palm isolates (1.EGPK6, 2.EGPK5, 3.EGPK7, 4.OP114, 5.OP65, 7.EGSE6). Lanes 6, 8-11: coconut isolates (6.CNDDE2, 8.CNKK1, 9.CNKK2, 10.CNBt62, 11.CNBt64). M – marker. 10 9 8 7 6 M 5 4 3 2 1 M Figure 5 . RAMS banding patterns obtained using primer (ACA)5 of G. boninense isolates from oil palm and isolates of Ganoderma sp. from coconut stumps. Note the common band of 1400 bp. Lanes 1-3: coconut isolates. (1.CNKK4, 2.CNRT310, 3.CNDDE2). Lanes 4-10: oil palm isolates (4.EGBD5, 5.EGR7, 6.EGL3, 7.EGL1, 8.EGPK5, 9.EGJS9, 10.EGJS1). M – marker. 10 9 8 7 6 M 5 4 3 2 1 M M 1 2 3 4 5 6 7 8 9 10 11 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia 31 Figure 6. Dendrogram from UPGMA analysis using simple matching coefficient based on RAMS bands of G. boninense isolates from infected oil palm and isolates of Ganoderma sp.coconut stumps. 32 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia hosts. A preliminary study on RAPD of Ganoderma isolates originated from the same species with a wide genetic base or from oil palm in Papua New Guinea by Pilotti et al. (1998) from closely related species. Idris et al. (1996) using Jaccard’s also showed variations, which led to difficulties in interpreting similarity coefficient to analyse RAPD patterns of 40 isolates the results for comparison of individual isolates within a from different Ganoderma species from different geographical population. Later, Pilotti et al. (2000) observed genetic areas reported that G. boninense from Malaysian oil palm and variation among sibling monokaryons of G. boninense and coconut showed similarity of 4.8% to 69.2% which also they suggested that sexual reproduction is important in indicated heterogeniety of the Ganoderma isolates. maintaining genetic variation in G. boninense. In another High levels of genetic variability are common in many preliminary study using RAPD analysis of Ganoderma isolates populations of sexually reproducing fungi such as Ganoderma. from oil palm in Indonesia, Darmono (1998) also found However, the sources and extent of the high degree of genetic that the isolates were genetically variable; variations were variation among the Ganoderma isolates from infected oil palm observed in isolates from the same or different oil palm and coconut stumps are unknown. In most eukaryotic plantations. organisms, the primary source of genetic variation is sexual Polymorphic amplification products were produced by all reproduction in which meiotic recombination occurs. More Ganoderma isolates when the four microsatellite primers, information, especially about Ganoderma, is required to explain (GT)5, (CGA)5, (CCA)5 and (ACA)5, were used, which indicate how and when fungal variability arose as the resulting that the four microsatellite motifs exist abundantly in the variability can affect the pathogen relationship with its host genome of G. boninense isolates from infected oil palm and and allows the fungi to adapt readily to changing Ganoderma sp. from coconut stumps. RAMS analysis using environmental conditions. the four primers resulted in variation in banding patterns Variability has also been reported to be related to the capacity within and between the isolates from infected oil palm and of the organisms to adapt to using different substrates. In coconut, indicating a high degree of genetic variation among new planting areas, especially after clean clearing, the inoculum the isolates from both groups of Ganoderma. However, five source may have to be able to survive in the soil for a long common bands were produced in all the Ganoderma isolates time before the oil palm trees can grow and infection occurs. by three primers, namely, (CCA)5, (CGA)5 and (ACA)5. These The Ganoderma inoculum must have the ability to undergo common bands have the potential to be developed as a some adaptation to be able to survive in the soil and to colonise diagnostic tool for early detection of the basal stem rot disease a new substrate. It has been reported that Ganoderma on felled of oil palm. Sequence characterised amplified region (SCAR) trunks and stumps of either coconut or oil palm exists as markers could be developed from the common bands by saprophyte but the fungus can become a pathogen when oil cloning, sequencing and designing specific primers. These palm trees are planted nearby (Turner, 1981). primers would be useful for diagnostic purposes such as to Genetically heterogeneous Ganoderma isolates from infected detect the disease in the oil palm tissues, especially the roots, oil palm and coconut stumps suggest that disease spread or in the soil debris in oil palm field. Currently, detection of through root-to-root contact of a diseased palm to a healthy an infected palm occurs when basidiomata are formed and palm may not be very prevalent. As clonal spread was not the palm shows obvious symptoms of the basal stem rot evident, disease infection and spread could have originated disease. At this stage, it is usually too late to save the palm. through basidiospore which colonise stumps or felled trunks Early detection of the disease is very important as control which could then act as infection foci, or from secondary measures can be carried out before the disease can cause severe inoculum sources such as infected residues from coconut damage to the palm. Furthermore, such a diagnostic kit can palms or oil palm left in the soil. Miller et al. (1999) reported provide information on the mechanism of disease spread that the analysis of RFLPs of mtDNA using the restriction from sources of inoculum as any source of inoculum can be endonucleases Hae III and Msp I revealed heterogeneity among identified. Sequence characterised amplified region markers isolates of Ganoderma of oil palms from different localities as have been developed for identification and detection of plant well as from neighbouring palms and from within individual pathogenic fungi such as Phoma sclerotiodes in the root tissues palms. A study by Abdullah (2000) on the incidence of basal of alfalfa (Larsen et al., 2002), and Verticillium albo-atrum stem rot of oil palm on a former coconut plantation also pathotypes PG1 and PG2 in xylem tissues of hop plants suggested that disease infection and spread was from (Radisek et al., 2004). secondary inoculum such as infected residues from coconut Genetic similarity values between all the isolates palms left in the soil. Somatic incompatibility study has also ranged from 46% - 91% for RAPD and from 53% - 97% for shown that G. boninense isolates from infected oil palm and RAMS. The results show that G. boninense from infected oil Ganoderma from coconut stumps are genotypically distinct palm and Ganoderma sp. from coconut stumps are genetically individuals and not clones of single genotypes (Latiffah et heterogeneous as a high level of variability was observed. al.., 2002). All these studies imply that the Ganoderma The high degree of genetic variation could be due to the population in oil palm is derived from diverse inoculum different geographical locations from which the isolates were sources. obtained or it could indicate that the isolates may have AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 RAPD and RAMS of Ganoderma in Malaysia 33 Although G. boninense isolates from infected oil palm and Hseu, R.S., Wang, H.H., Wang, H.F. and Moncalvo, J.M. 1996. Ganoderma sp. from coconut stumps are genetically Differentiation and grouping of isolates of Ganoderma heterogeneous, cluster analysis of RAPD and RAMS bands lucidum complex by Random Amplified Polymorphic showed that the isolates from the two groups did not cluster DNA-PCR compared with grouping on the basis of separately into two major distinct clusters, but instead were Internal Transcribed Spacer Sequences. Applied and clustered together showing close relationship. The finding Environmental Microbiology 62 (4): 1354 – 1363. that Ganoderma sp. from coconut stumps is closely related to G. boninense from infected oil palm lends support to the Idris, A.S., Thangavelu M. and Swinburne T.R. 1996. The suggestion that Ganoderma on felled coconut trunks or stumps use of RAPD for identification of species and detection can act as infection foci for dissemination of disease to oil of genetic variation in Ganoderma isolates from oil palm, palm. 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