and Random Amplified Microsatellite (RAMS) - Semantic Scholar

Asia Pacific AsPac J. Mol.Journal Biol. Biotechnol., of Molecular Vol.Biology 13 (1),and 2005 Biotechnology, 2005 Vol. 13 (1) : 23-34

RAPD and RAMS of Ganoderma in Malaysia

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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 School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang. Institute of Bioscience, 3Department of Biology, Universiti Putra Malaysia, Serdang, Selangor, Malaysia 1

2

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

INTRODUCTION The basal stem rot of oil palm caused by Ganoderma is the most serious disease of field palms in Southeast Asia, particularly Malaysia and Indonesia. The disease was first reported by Thompson in 1931. Initially, the basal stem rot was thought to be a disease of older palms as it was found to infect mostly older palms, but in the 1960s the disease appeared in younger palms of 10 – 15 years old (Turner, 1981). In the last decade or so, palms as young as 1 year old were found to be infected by the disease (Arifin et al., 1989). The basal stem rot shortens the productive life of the oil palms and incurs considerable economic losses to the oil palm industry. A number of Ganoderma species have been reported to be associated with the basal stem rot of oil palm (Turner, 1981;

Abdullah, 1996; Idris, 1999). However, the most common species is G. boninense which was first reported by Ho and Nawawi (1985) and later Khairudin (1990) confirmed by pathogenicity tests that G. boninense was the causal agent of the disease. In several estates with a high disease incidence, G. boninense was also found to be the most common and virulent species (Idris, 1999). High disease incidence occurs in oil palm plantings that were previously planted with coconut, especially where old coconut trees were felled and the stumps buried or left on the ground to rot. Disease incidence is also high when old coconut trees were poisoned and left standing to rot. Although Ganoderma on coconut stumps exists as saprophyte, Turner *Author for Correspondence. Mailing address: School of Biological Sciences, Universiti Sains Malaysia,11800 Minden, Penang,Malaysia.Tel:604 – 6533506; Fax : 604– 6565125; Email : [email protected]

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AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005

(1981) suggested that old coconut stumps and felled coconut trunks which were colonised by Ganoderma could serve as inoculum to healthy oil palms. Pathogenicity tests have also shown that cross infection could occur between Ganoderma from oil palm and coconut (Abdullah, 1998; Idris, 1999). These studies suggest that Ganoderma on coconut stumps could assume an important role in the dissemination and development of the disease. However, it is not known whether the Ganoderma on coconut stumps is the same species as that on oil palm. As morphological characteristics of Ganoderma may vary with environmental conditions, they are unreliable as the sole criterion for distinguishing different species. Other characteristics, such as molecular characters, should be studied. Molecular characteristics of fungi are increasingly being used as additional taxonomic criteria in classification or to resolve controversies in taxonomic position of taxa. A wide range of molecular methods has been introduced, especially with the rapid development of polymerase chain reaction (PCR)-based techniques. One of the PCR-based techniques that has been widely used in characterisation of plant pathogenic fungi is random amplified polymorphic DNA (RAPD) which is based on PCR amplification of DNA fragment using single primer with an arbitrary nucleotide sequence (Williams et al., 1990). RAPD has been used in the characterisation of Aphanomyces euteiches strains that cause root rot disease (Malvick et al., 1998); in the identification of Colletotrichum fragariae from diseased strawberry (Martinez-Culebras et al., 2002); and in the characterisation of Fusarium moniliforme isolated from different hosts (Kini et al., 2002). Another PCR-based technique is random amplified microsatellite (RAMS), which combines several characteristics of RAPD and microsatellite analysis. In this technique, the DNA between distal ends of two closely related microsatellite is amplified. Hantula et al. (1996) has demonstrated that RAMS is applicable in studying genetic variation in fungi. In the present study, RAPD and RAMS analyses were used to determine the genetic relationship within and between G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps.

MATERIALS AND METHODS

Ganoderma isolates and DNA extraction. A total of 53 G. boninense isolates from infected oil palm and 15 isolates of Ganoderma from coconut stumps from different estates and areas in Malaysia were used in this study (Table 1). The isolates were cultured in yeast extract sucrose broth for 14 days after which their mycelia were harvested, lyophilized and used for DNA extraction following the phenol-chloroform method described by Raeder and Broda (1985). The DNA was dissolved in TE buffer and stored at –20oC until used. DNA was quantified using a spectrophometer (DM 65 Beckman).


RAPD analysis. In a preliminary test, 20 random primers from Kit I (Operon Technologies, Inc.) were screened. The primers were ten bases long with 50% - 70% G+C content. From the test, 10 primers produced reproducible fragments. Each primer was further tested twice to verify the reproducibility and consistency of RAPD banding patterns. Four primers, which produced consistent patterns, were selected for RAPD analysis. They were OPI 01 (5’ACCTGGACAC3'), OPI 07 (5' CAGCGACAAG3'), OPI 12 (5' AGAGGGCACA 3') and OPI 14 (5' TGACGGCGGT 3'). RAPD analysis was repeated twice to ensure reproducibility and consistency of the banding patterns for each isolate. All reagents and standard molecular markers for PCR amplifications, namely, MgCl2, dNTPs, Taq polymerase as well as DNA template were optimised to obtain standardised reaction conditions for RAPD analysis. The reagents were obtained from Life Technologies GIBCO BRL. In the preliminary experiments, the concentrations of all the reagents and the PCR amplification protocol were the same as those reported by Williams et al. (1993) and Hseu et al. (1996). After optimisation of all the reagents, RAPD amplification was performed in a total volume of 25 µl reaction mixture which contained 2.5 mM MgCl2, 1X PCR buffer [20 mM Tris-HCl (pH 8.4) and 50 mM KCl ], 50 µM dNTP mix, 2.5 units Taq polymerase, 0.6 µM of primer and 3 - 5 ng template DNA. The RAPD amplification conditions used were based on those described by Williams et al. (1990). Amplifications were performed in a Perkin Elmer Thermalcycler (PE Applied Biosystem Model 2400) with the following protocols: 4 min of initial denaturation at 98oC, 45 cycles of denaturation at 95oC for 1 min, annealing at 36oC for 1 min and extension at 72oC for 2 min, and final extension at 72oC for 10 min to ensure complete extension of the amplification products. RAMS analysis. The primers used for RAMS analysis were adapted from Hantula et al. (1996). The primers were 5’BDB(ACA) 5, 5’DD(CCA) 5, 5’DHB(CGA) 5 and 5’YHY(GT)5G in which H, B, Y and D were used as 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 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 polymerase and 50 ng template DNA. The reagents were obtained from Life Technologies GIBCO BRL. The protocol for RAMS amplification was based on that of Hantula et al. (1996). Amplifications were performed in a Perkin Elmer Thermalcycler (PE Applied Biosystems model 2400) in a profile of 35 cycles. An initial denaturation of 10 min at 95 oC was carried out before the cycle began. Amplifications were done as follows: 30s denaturation at 95oC, 45s annealing at a temperature depending on the primer and 2 min extension at 72oC. Annealing temperature for



Table 1. List of locations of G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps

Host Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm

Code

Name and Location of Estate

EG 01 OP114 OP123 OP153 OP170 OP187 OP230 OP64 OP65 OP83 OP88 EGSE2 EGSE5 EGSE4 EGSE6 EGSE12 EGBD2 EGBD5 EGSK3 EGSK4 EGSK6 EGSK7 EGSK10 EGCN2 EGCN7 EGCN8 EGJS1 EGJS8 EGJS9 EGJS10 EGS2 EGS3 EGR1 EGR2 EGR4 EGR5 EGR6 EGR7 EGT EGL1 EGL2 EGL3 EGL5

Dusun Durian Estate, Banting, Selangor South Estate, Carey Island, Selangor South Estate, Carey Island, Selangor South Estate, Carey Island, Selangor South Estate, Carey Island, Selangor South Estate, Carey Island, Selangor South Estate, Carey Island, Selangor North Estate, Carey Island, Selangor North Estate, Carey Island, Selangor North Estate, Carey Island, Selangor North Estate, Carey Island, Selangor Selaba Estate, Teluk Intan, Perak Selaba Estate, Teluk Intan, Perak Selaba Estate, Teluk Intan, Perak Selaba Estate, Teluk Intan, Perak Selaba Estate, Teluk Intan, Perak Bagan Datoh Estate, Bagan Datoh, Perak Bagan Datoh Estate, Bagan Datoh, Perak Sungai Krian Estate, Bagan Serai, Perak Sungai Krian Estate, Bagan Serai, Perak Sungai Krian Estate, Bagan Serai, Perak Sungai Krian Estate, Bagan Serai, Perak Sungai Krian Estate, Bagan Serai, Perak Chersonese Estate, Kuala Kurau, Perak Chersonese Estate, Kuala Kurau, Perak Chersonese Estate, Kuala Kurau, Perak Jin Seng Estate, Bagan Serai, Perak Jin Seng Estate, Bagan Serai, Perak Jin Seng Estate, Bagan Serai, Perak Jin Seng Estate, Bagan Serai, Perak Serkam Estate, Serkam, Melaka Serkam Estate, Serkam, Melaka Regent Estate, Melaka Regent Estate, Melaka Regent Estate, Melaka Regent Estate, Melaka Regent Estate, Melaka Regent Estate, Melaka Tangkah Estate, Tangkak, Johor Lanadron Estate, Panchor, Muar, Johor Lanadron Estate, Panchor, Muar, Johor Lanadron Estate, Panchor, Muar, Johor Lanadron Estate, Panchor, Muar, Johor

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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 Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Oil palm Coconut Coconut Coconut Coconut Coconut Coconut Coconut Coconut Coconut Coconut Coconut Coconut Coconut Coconut

EGL8 EGN3 EGN4 EGN7 EGN8 EGN9 EGPK5 EGPK6 EGPK7 EGPK8 CN04 CN05 CN06 CNRT310 CNUB01 CNKK1 CNKK2 CNKK3 CNKK4 CNKK6 CNDDE2 CNBt61 CNBt62 CNBt63

Lanadron Estate, Panchor, Muar, Johor Nordanal Estate, Panchor, Muar, Johor Nordanal Estate, Panchor, Muar, Johor Nordanal Estate, Panchor, Muar, Johor Nordanal Estate, Panchor, Muar, Johor Nordanal Estate, Panchor, Muar, Johor Pitas Estate, Kudat, Sabah Pitas Estate, Kudat, Sabah Pitas Estate, Kudat, Sabah Pitas Estate, Kudat, Sabah Renggam, Johor Renggam, Johor Renggam, Johor Rengit, Batu Pahat, Johor Ulu Bernam, Teluk Intan, Perak Kuala Klanang, Banting, Selangor Kuala Klanang, Banting, Selangor Kuala Klanang, Banting, Selangor Kuala Klanang, Banting, Selangor Kuala Klanang, Banting, Selangor Dusun Durian Estate, Banting, Selangor Batu Enam, Banting, Selangor Batu Enam, Banting, Selangor Batu Enam, Banting, Selangor

(CCA)5 was 64oC, for (CGA)5 61oC, for (GT)5 58oC, and for (ACA)5 72oC. Final extension of 7 min at 72oC was performed after the cycles ended. Gel electrophoresis. The amplification products were separated by electrophoresis in a 1.75% agarose gel (SeaKem LE) using Tris Borate EDTA (TBE) pH 8.2 as running buffer. The electrophoresis was carried out at 5V/cm. After electrophoresis, the gels were stained with ethidium bromide and the patterns were visualized under UV light and photographed using a Kodak Polaroid camera. Comparisons of the banding patterns were made using the 100 bp marker as a molecular size standard.

Data analysis. The banding patterns generated by all the different primers were scored as presence (1) and absence (0) 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 reproducible in separate runs. The relationships between all the isolates could be more clearly represented by a similarity matrix. The similarity matrix

was constructed using simple matching coefficient according to the formula shown below: SMC = a + d / a + b + c + d where: a = number of shared bands b = number of bands present only in isolate 1 c = number of bands present only in isolate 2 d = number of bands absent in isolates 1 and 2. The Numerical Taxonomy System of Multivariate Program (NT-SYS) software package Version 1.8 (Rohlf, 1994) was used to analyse the data. A dendrogram was constructed using UPGMA (unweighted pair-group method with arithmetic averages) cluster analysis to infer the relationship between the 68 isolates from oil palm and coconut. RESULTS The four primers, OPI 01, OPI 07, OPI 12 and OPI 14 used in RAPD analysis produced reproducible and consistent banding patterns of the Ganoderma isolates. Primer OPI 01


produced between 1 – 15 bands ranging from 400 – 2072 bp. Primer OPI 07 also produced 1 – 15 bands but the bands ranged from 300 – 2072 bp. For primer OPI 12, between 1 10 bands were produced with molecular sizes ranging from about 400 – 2072 bp and for primer OPI 14, between 3 – 14 bands ranging from 300 – 2072 bp were produced. None of the primers produced specific patterns that could differentiate between G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps as variations in banding patterns occurred within and between isolates of both groups of Ganoderma. Generally, there was a distinct pattern of bands amplified from each isolate regardless of whether it was from infected oil palm or from coconut stumps, although several bands were shared among the isolates. Both intense and faint bands were produced. Figure 1 and 2 show some representative RAPD banding patterns for some of the G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. From the similarity matrix based on simple matching coefficient, the similarity values between all the G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps were from 46% - 91%. Pair-wise comparison between the oil palm and coconut isolates showed similarity values of between 0.508 – 0.831 (about 51% - 83% similarities). Generally, Ganoderma isolates from the same estate had higher similarity (82% - 90%) when compared to the isolates from different estates (46% - 78%). The dendrogram based on UPGMA cluster analysis of RAPD bands is shown in Figure 3. There are overlapping of clusters which group all the Ganoderma isolates. The oil palm and coconut isolates were not separated into two distinct clusters but were clustered together, indicating close relationship. The dendrogram can be divided into several clusters and subclusters. The coconut isolates were clustered together with the oil palm isolates in Sub-clusters A, B, C and D. Subclusters E, F, G and Major Cluster II comprised only oil palm isolates. Isolates from the same estate in both groups of Ganoderma tended to cluster in the same cluster or sub-cluster. This is not surprising as the similarity values shown by the isolates from the same estate were very high which indicate very close relationship. The four microsatellite primers, (GT)5, (CCA) 5, (CGA)5 and (ACA)5, were able to generate amplification products for all the Ganoderma isolates tested. Variations of banding patterns were observed within and between G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. The banding patterns produced by using (GT)5 and (ACA)5 were not as highly polymorphic as those produced by using (CCA)5 and (CGA)5. Both intense and faint bands were produced by the Ganoderma isolates using the four microsatellite primers. Primer (GT)5 produced between 1 – 14 bands with molecular sizes ranging from 300 bp – 2072 bp and primer (ACA)5 produced between 2 – 7 bands ranging from 400 bp – 2072 bp. For primer (CGA) 5 which showed highly


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polymorphic patterns, 5 – 20 bands ranging from 200 – 2072 bp were produced and for primer (CCA)5 which also showed highly polymorphic patterns, 12 – 20 bands of 200 – 2072 bp were produced. Although RAMS analysis using the four primers resulted in polymorphic banding patterns, which indicated a high degree of genetic variation among the isolates from both groups of Ganoderma, five common bands were produced by all the Ganoderma isolates. They were 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. Figure 4 and 5 show some representative RAMS banding patterns of some G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. The similarity values from RAMS analysis showed a broad range of values from 0.531 – 0.972 (about 53% - 97% similarities) for G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. Pair-wise comparisons between oil palm isolates showed similarity values ranging from 53% - 97% and for comparisons between coconut isolates, similarity values ranged from 55% - 87%. As for pair-wise comparisons between oil palm and coconut isolates, similarity values were between 53% - 82%. The dendrogram based on UPGMA cluster analysis of RAMS bands is shown in Figure 6. Although the dendrogram can be divided into two major clusters, I and II, at about 66% similarity level, G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps did not cluster separately to form these two major clusters. The coconut isolates were clustered together with the oil palm isolates, especially in Sub-clusters 1C, IE and Major Cluster II. Most of the isolates were grouped according to their locality.

DISCUSSION The four primers used in RAPD analysis showed polymorphism within and between all the Ganoderma isolates from oil palm and coconut. However, these primers did not produce specific patterns that could differentiate between G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. Banding patterns produced by each primer were highly variable and most amplified bands were polymorphic, indicating genetic variation among all the Ganoderma isolates from both infected oil palm and coconut stumps. Idris et al. (1996) have also reported variations in RAPD banding patterns in various Ganoderma isolates from different hosts such as oil palm, coconut, rubber, ornamental palm and hardwoods which originated from different geographical origins (Malaysia, Singapore, Indonesia, Papua New Guinea, Solomon Island, United Kingdom, United States of America and Poland). They found polymorphism to be much greater within the isolates of G. boninense from oil palm and coconut hosts compared to the other Ganoderma species from other

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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.



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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.

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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



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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.

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hosts. A preliminary study on RAPD of Ganoderma isolates from oil palm in Papua New Guinea by Pilotti et al. (1998) also showed variations, which led to difficulties in interpreting the results for comparison of individual isolates within a population. Later, Pilotti et al. (2000) observed genetic variation among sibling monokaryons of G. boninense and they suggested that sexual reproduction is important in maintaining genetic variation in G. boninense. In another preliminary study using RAPD analysis of Ganoderma isolates from oil palm in Indonesia, Darmono (1998) also found that the isolates were genetically variable; variations were observed in isolates from the same or different oil palm plantations. Polymorphic amplification products were produced by all Ganoderma isolates when the four microsatellite primers, (GT)5, (CGA)5, (CCA)5 and (ACA)5, were used, which indicate that the four microsatellite motifs exist abundantly in the genome of G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. RAMS analysis using the four primers resulted in variation in banding patterns within and between the isolates from infected oil palm and coconut, indicating a high degree of genetic variation among the isolates from both groups of Ganoderma. However, five common bands were produced in all the Ganoderma isolates by three primers, namely, (CCA)5, (CGA)5 and (ACA)5. These common bands have the potential to be developed as a diagnostic tool for early detection of the basal stem rot disease of oil palm. Sequence characterised amplified region (SCAR) markers could be developed from the common bands by cloning, sequencing and designing specific primers. These primers would be useful for diagnostic purposes such as to detect the disease in the oil palm tissues, especially the roots, or in the soil debris in oil palm field. Currently, detection of an infected palm occurs when basidiomata are formed and the palm shows obvious symptoms of the basal stem rot disease. At this stage, it is usually too late to save the palm. Early detection of the disease is very important as control measures can be carried out before the disease can cause severe damage to the palm. Furthermore, such a diagnostic kit can provide information on the mechanism of disease spread from sources of inoculum as any source of inoculum can be identified. Sequence characterised amplified region markers have been developed for identification and detection of plant pathogenic fungi such as Phoma sclerotiodes in the root tissues of alfalfa (Larsen et al., 2002), and Verticillium albo-atrum pathotypes PG1 and PG2 in xylem tissues of hop plants (Radisek et al., 2004). Genetic similarity values between all the isolates ranged from 46% - 91% for RAPD and from 53% - 97% for RAMS. The results show that G. boninense from infected oil palm and Ganoderma sp. from coconut stumps are genetically heterogeneous as a high level of variability was observed. The high degree of genetic variation could be due to the different geographical locations from which the isolates were obtained or it could indicate that the isolates may have


originated from the same species with a wide genetic base or from closely related species. Idris et al. (1996) using Jaccard’s similarity coefficient to analyse RAPD patterns of 40 isolates from different Ganoderma species from different geographical areas reported that G. boninense from Malaysian oil palm and coconut showed similarity of 4.8% to 69.2% which also indicated heterogeniety of the Ganoderma isolates. High levels of genetic variability are common in many populations of sexually reproducing fungi such as Ganoderma. However, the sources and extent of the high degree of genetic variation among the Ganoderma isolates from infected oil palm and coconut stumps are unknown. In most eukaryotic organisms, the primary source of genetic variation is sexual reproduction in which meiotic recombination occurs. More information, especially about Ganoderma, is required to explain how and when fungal variability arose as the resulting variability can affect the pathogen relationship with its host and allows the fungi to adapt readily to changing environmental conditions. Variability has also been reported to be related to the capacity of the organisms to adapt to using different substrates. In new planting areas, especially after clean clearing, the inoculum source may have to be able to survive in the soil for a long time before the oil palm trees can grow and infection occurs. The Ganoderma inoculum must have the ability to undergo some adaptation to be able to survive in the soil and to colonise a new substrate. It has been reported that Ganoderma on felled trunks and stumps of either coconut or oil palm exists as saprophyte but the fungus can become a pathogen when oil palm trees are planted nearby (Turner, 1981). Genetically heterogeneous Ganoderma isolates from infected oil palm and coconut stumps suggest that disease spread through root-to-root contact of a diseased palm to a healthy palm may not be very prevalent. As clonal spread was not evident, disease infection and spread could have originated through basidiospore which colonise stumps or felled trunks which could then act as infection foci, or from secondary inoculum sources such as infected residues from coconut palms or oil palm left in the soil. Miller et al. (1999) reported that the analysis of RFLPs of mtDNA using the restriction endonucleases Hae III and Msp I revealed heterogeneity among isolates of Ganoderma of oil palms from different localities as well as from neighbouring palms and from within individual palms. A study by Abdullah (2000) on the incidence of basal stem rot of oil palm on a former coconut plantation also suggested that disease infection and spread was from secondary inoculum such as infected residues from coconut palms left in the soil. Somatic incompatibility study has also shown that G. boninense isolates from infected oil palm and Ganoderma from coconut stumps are genotypically distinct individuals and not clones of single genotypes (Latiffah et al.., 2002). All these studies imply that the Ganoderma population in oil palm is derived from diverse inoculum sources.


Although G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps are genetically heterogeneous, cluster analysis of RAPD and RAMS bands showed that the isolates from the two groups did not cluster separately into two major distinct clusters, but instead were clustered together showing close relationship. The finding that Ganoderma sp. from coconut stumps is closely related to G. boninense from infected oil palm lends support to the suggestion that Ganoderma on felled coconut trunks or stumps can act as infection foci for dissemination of disease to oil palm. This would have an important bearing in the formulation of more effective disease control measures and replanting procedures, especially in areas that are previously planted with coconut.


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Hseu, R.S., Wang, H.H., Wang, H.F. and Moncalvo, J.M. 1996. Differentiation and grouping of isolates of Ganoderma lucidum complex by Random Amplified Polymorphic DNA-PCR compared with grouping on the basis of Internal Transcribed Spacer Sequences. Applied and Environmental Microbiology 62 (4): 1354 – 1363. Idris, A.S., Thangavelu M. and Swinburne T.R. 1996. The use of RAPD for identification of species and detection of genetic variation in Ganoderma isolates from oil palm, rubber and other hardwood trees. In: Proceedings of the 1996 PORIM International Palm Oil Congress (Agriculture) ed. D. Arifin. pp. 538 – 551.

REFERENCES

Idris, A.S. 1999. Basal stem rot (BSR) of oil palm (Elaeis guineensis Jacq.) in Malaysia : Factors associated with variation in disease severity. Ph.D Thesis. University of London, London, UK.

Abdullah, F. 1996. Taxonomic and electrophoretic studies of selected species of Ganoderma (Karst.). Ph.D Thesis. Universiti Pertanian Malaysia, Selangor, Malaysia

Khairudin, H. 1990. Basal stem rot of oil palm: Incidence, Etiology and Control. MSc Thesis. Universiti Pertanian Malaysia, Serdang, Selangor, Malaysia.

Abdullah, F. 1998. Characterization, pathogenicity and cross pathogenicity studies of Ganoderma causal pathogen of basal stem rot of oil palm. UPM Research Report 1998. Volume 2. Universiti Putra Malaysia.

Kini, K.R., Leth, V. and Mathur, S.B. 2002. Genetic variation in Fusarium moniliforme isolated from seeds of different host species from Burkina Faso based on random amplified polymorphic DNA analysis. Journal of Phytopathology 150 : 209-212.

Abdullah, F. 2000. Spatial and sequential mapping of the incidence of basal stem rot of oil palm (Elaeis guineensis) on a former coconut (Cocos nucifera) plantation. In Ganoderma Diseases of Perennial Crops, ed. J. Flood, P.D., Bridge and P. Holderness.. CABI Publishing, Wallingford, United Kingdom. pp. 183 – 194. Arifin, D., Singh, G., and Lim T.K. 1989. Ganoderma in Malaysia – Current Status and Research Strategy. In Proceedings of the 1989 PORIM International Palm Oil Development Conference-Module II: Agriculture. ed. S. Jalani and D. Arifin. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia. pp. 249 - 297

Larsen, R.C., Gritsenko, M.A., Hollingsworth, C.R. and Gray, F.A. 2002. A rapid method using PCR-based SCAR markers for the detection and identification of Phoma sclerotiodes: the cause of brown root rot disease of alfalfa. Plant Disease. D-2002-0627-01R (online publication). Latiffah, Z., Abdullah F., Tan S.G., Harikrishna, K. and Ho, Y.W. 2002. Morphological and growth characteristics, and somatic incompatibility of Ganoderma from infected oil palm and coconut stumps. Malaysian Applied Biology 31 : 37 – 48.

Darmono, T.W. 1998. Variation among isolates of Ganoderma sp. from oil palm in Indonesia. Second International Workshop on Ganoderma Diseases. MARDI, Serdang, Malaysia. 5 – 8 October, 1998.

Martinez-Culebrsa, P.V., Barrio, E., Suarez- Fernandez, M.B., Garcia-Lopez, M.D. and Querol, A. (2002). RAPD analysis of Colletotrichum species isolated from strawberry and the design of specific primers for the identification of C. fragariae. Journal of Phytopathology 150 : 680 – 686.

Hantula, J., Dusabenyagaasani M. and Hamelin R.C. 1996. Random Amplified Microsatellites (RAMS) – a novel method for characterization of genetic variation within fungi. European Journal of Forest Pathology 26 : 159 – 166.

Malvick, D.K., Grau, C.R. and Percich, J.A. 1998. Characterisation of Aphanomyces euteiches strains based on pathogenicity and random amplified polymorphic DNA analyses. Mycological Research 102 : 405 – 475.

Ho, Y.W. and Nawawi A. 1985. Ganoderma boninense Pat. from basal stem rot of oil palm (Elaies guineensis) in Peninsular Malaysia. Pertanika 8 : 425 – 428.

Miller, R.N.G., Holderness M., Bridge P.D., Chung G.F. and Zakaria M.H. 1999. Genetic diversity of Ganoderma in oil palm plantings. Plant Pathology 48: 595 – 603.

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Pilotti, C.A., Sanderson, F.R., Aitken, E.A.B. and Bridge P.D. 1998. Variability of Ganoderma sp. in Papua New Guinea. Second International Workshop on Ganoderma Diseases. MARDI, Serdang, Malaysia, 5 – 8 October, 1998. Pilotti, C.A, Sanderson, F.R., Aitken, E.A.B and Bridge, P.D. 2000. Genetic variation of Ganoderma sp. from Papua New Guinea as revealed by molecular (PCR) methods. In Ganoderma Diseases of Perennial Crops. ed. J. Flood, P.D. Bridge and P. Holderness, pp. 195–204. CABI Publishing, Wallingford, United Kingdom. Radisek, S., Zalskega, C., Jakse, J. and Javornik, B. 2004. Development of pathotype specific SCAR markers for detection of Verticillium albo-atrum isolates from hop. Plant Disease 88 : 1115 – 1122. Raeder, U. and Broda P. 1985. Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology 1 : 17 –20. Rohlf, F.J. 1994. NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Version 1.8. Exeter Publishing Ltd. Setauket, New York, USA. Thompson, A. 1931. Stem rot of the oil palm in Malaya. Bulletin Department of Agriculture, Straits Settlements and F.M.S., Science Series 6, 23. Turner, P.D. 1981. Oil Palm Diseases and Disorders. Oxford University Press. Williams, J.G.K., Hanafey, M.K., Rafalski, J.A. and Tingey, S. 1993. Genetic analysis using random amplified polymorphic DNA markers. Methods on Enzymology 218 : 704 – 40. Williams, J.G.K., Kubelik A.R., Lival, K.J., Rafalski J.A. and Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18 : 6531-6535.
