Journal of Biological Sciences

Year: 2009 | Volume: 9 | Issue: 1 | Page No.: 12-20
DOI: 10.3923/jbs.2009.12.20

Molecular Analysis of Ganoderma species from Different Hosts in Peninsula Malaysia

L. Zakaria, N.S. Ali, B. Salleh and M. Zakaria

Abstract: Ganoderma isolates from different hosts namely, G. boninense from oil palm (Elaeis guineensis), G. philiplii from rubber (Hevea brasiliensis) and G. australe from forest trees were characterized using RAPD and PCR-RFLP of ITS+5.8S regions. RAPD primers of high G+C content of 80-100% [CRL-1 (5’CCAGCGCCCC), CRL-2 (5`CTGCCGCCGC), CRL-7 (5’GCCCGCCGCC3’), CRL-11 (5`CCACCGCGCC) and CRL-34 (5’GACCGCGCCC)] showed that the banding patterns from the same species generated similar patterns. Like RAPD, restriction analysis of ITS+5.8S regions using six restriction enzymes (Mspl, Bsu151, Hin61, Hindlll, Hinfl and Taql) also showed that restriction patterns from the same species generated similar patterns. From UPGMA cluster analysis of RAPD and PCR-RFLP of ITS+5.8S regions, Ganoderma species from the same host were clustered together. The results from the present study showed that RAPD and PCR-RFLP of ITS+5.8S regions could be used in characterization and taxonomic analysis of Ganoderma species from different hosts. Both techniques could also provide rapid procedure for differentiation of Ganoderma species in Peninsula Malaysia.

Keywords: PCR-RFLP, forest tree, tea, oil palm, rubber, Ganoderma and RAPD

INTRODUCTION

The genus Ganoderma was first described by Karsten (1881) based on a polyporoid species called Polyporus lucidus. Initially, the genus Ganoderma was characterised based on the presence or absence of laccate or shiny appearance of the upper surface of the fruiting bodies. Based on those characteristics, G. lucidum complex often comprised laccate species or specimens which show various degrees of laccate appearance and G. applanatum complex consist of all non-laccate species or specimens with dull appearance.

The genus Ganoderma comprises both economically and ecologically important species and widely distributed in tropical and temperate regions. Some of the species are sources of medicinal products, some are pathogens on perennial crops such as oil palm, tea, rubber, cocoa and as well as pathogen on forest trees. Ganoderma species is also well-known as decomposer of log and stump in tropical rain forest.

In Malaysia, a few Ganoderma species are associated with diseases of several economically important perennial crops. The most serious disease caused by Ganoderma is basal stem rot of oil palm (Elaies guineensis) which caused losses of more than 80% of oil palm planting (Turner, 1981). Ganoderma also infected rubber (Hevea brasiliensis) and tea (Camelia sinensis), causing red root rot disease. Ganoderma species are also found on trees in natural and plantation forest in Peninsula Malaysia (Lee, 1997).

The taxonomy of Ganoderma species is based mainly on morphological descriptions of the fruiting bodies, host specificity and geographical distribution (Seo and Kirk, 2000). However, the morphological concept for Ganoderma identification is still not well-established especially the tropical species. With the taxonomy of the genus poorly circumscribed and not universally accepted, Ryverden (1991) described the genus Ganoderma in a state of taxonomic chaos. Many researchers have also regarded that by using only morphological features are not sufficient for identification and characterization of Ganoderma species (Bazzalo and Wright, 1982; Gilbertson and Ryvarden, 1986).

For characterisation and identification of tropical Ganoderma species, descriptions by Steyaert (1967, 1972) were often used. However, Steyaerts species descriptions do not have an identification key which is essential to identify any species in a systematic manner. Because morphological characteristics have their limitation in allowing a reliable method for characterization of Ganoderma species, molecular techniques could be used to characterize and identify the species. With the development of PCR-based techniques, the used of molecular data for taxonomic purposes have been used widely to resolve conflicting data from morphological characteristics. Therefore, the objective of the study was to characterize Ganoderma species from different hosts in Peninsula Malaysia using molecular techniques of random amplified polymorphic DNA (RAPD) and Polymerase Chain Reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer and 5.8S gene (ITS+5.8S regions).

MATERIALS AND METHODS

Ganoderma isolates: Forty-five Ganoderma isolates were used in this study (Table 1). Based on morphological characteristics of basidiomata, Ganoderma from oil palm was identified as G. boninense, G. philippii from rubber and G. australe from forest trees. Eight Ganoderma isolates namely, G. philippii FP589, G. lucidum FP152 and G. australe FP104 from Forest Research Institute of Malaysia (FRIM); two G. boninense isolates of oil palm (SEL 28 and PER21) from Malaysian Palm Oil Board (MPOB) and three G. philippii isolates (CTEA1, CTEA2 and CTEA3) of tea from Rubber Research Institute of Malaysia (LGM) were used as reference isolates.

Isolation of Ganoderma mycelium: Isolation of mycelium was made from the basidiomata. Small pieces of basidiomata about 1x1 cm were cut and surface sterilized with 5% sodium hypochloride for 3 min, blotted dry and plate onto malt extract agar (MEA). Visible mycelia grown from the pieces of basidiomata were sub-cultured onto fresh MEA medium in Petri dishes.

DNA extraction: The Ganoderma isolates were cultured for 12 days on PDA broth, harvested and lyophilized. The DNA was extracted using DNeasy Plant Mini Kit (Qiagen) according to the manufacturers` instructions.

RAPD analysis: After primer screening, five RAPD primers with G+C content of 80-100% were used. The primers were CRL-1 (5`CCAGCGCCCC)`, CRL-2 (5`CTGCCGCCGC), CRL-7 (5`GCCCGCCGCC3`), CRL-11 (5`CCACCGCGCC) and CRL-34 (5`GACCGCGCCC) adapted from Kubelik and Szabo (1995). Amplification reactions were carried out in a 25μL reaction mixture containing 25 mM 10x PCR buffer (Promega, Madison, WI), 2.8 mM MgCl₂, 1 unit Taq polymerase, 0.7 mM dNTP mix, 0.4 mM each primer and 25 ng DNA template. Each reaction was overlaid with 25μL of mineral oil. PCR amplification was performed in a programmable thermal cycler (PCR DNA Engine Peltier Thermal Cycler Model PTC-100).The amplification starts with initial denaturation at 94°C followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 36°C for 1 min and elongation at 72°C for 2 min with a final elongation at 72°C for 10 min. After PCR, 5μL of the RAPD products was electrophoresed in 1.7% agarose gel in TBE buffer for 160 min at 500 mM and 80 V. After electrophoresis the gel was visualized by ethidium bromide (0.5μg mL^-1) staining. 1 kb marker (GeneRuler, Fermentas, Lithuania) and 100 bp marker (GeneRuler, Fermentas, Lithuania) were used as molecular marker to estimate the size of the RAPD bands.

PCR-RFLP analysis of ITS+5.8S regions: The ITS1 (5` TCC GTA GGT GAA CCT GCG G 3`) and ITS4 (5` TCC TCC GCT TAT TGA TAT GC 3`) primers were used to amplify the ITS+5.8S regions (White et al., 1990). Amplification reactions were conducted in a 25μL reaction mixture containing 2.5μL 10X PCR buffer (Promega), 3.0 mM MgCl₂, 1 unit of Taq polymerase (Promega), 0.3 mM dNTP mix, 0.4μM each of the primers and 25 ng DNA template. Each reaction was overlaid with 25μL of mineral oil. PCR amplification was amplified in a programmable thermal cycler (PCR DNA Engine Peltier Thermal Cycler Model PTC-100) using an initial denaturation step of 95°C for 2 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 63°C for 30 sec and elongation at 72°C for 2 min. The sizes of the amplified ITS+5.8S regions were estimated using 100 bp marker (Fermentas, Lithuania). The PCR product was separated in a 1.7% agarose gel after 160 min run at 80 V and 50 mA. Sizes of the restriction fragments were estimated using 1 kb DNA marker (Fermentas, Lithuania). The gels were stained with 0.5μg mL^-1 ethidium bromide.

Data analysis: RAPD bands and restriction bands were recorded as presence (1) and absence (0) of particular bands, based on 100 bp and 1 kb markers to compile a binary matrix which was then subjected to cluster analysis. The relationships between all the Ganoderma isolates from different hosts and species are represented by a similarity matrix. The similarity matrix was obtained using simple matching coefficient which was based on total number of bands present in both isolates plus the number of bands absent in both isolates divided by the total number of different bands (Romesburg, 1984). All the data were analysed using the Numerical Taxonomy and Multivariate Analysis System (NTSYS-pc) software package version 2 (Rohlf, 2000). A dendrogram based on simple matching coefficient was constructed using unweighted pair group method with arithmetic averages (UPGMA).

RESULTS

RAPD analysis: The five primers (CRL-1, CRL-2, CRL-7, CRL-11 and CRL-34) produced reproducible and consistent banding patterns. CRL-1 produced bands with molecular sizes between 300-1750 bp bands; CRL-2 and CRL-34, from 250-1500 bp bands. For CRL-7 and CRL-11 yielded bands ranging from 375-2000 bp. Generally, for each primer, the RAPD banding patterns generated could differentiate the Ganoderma isolates from different hosts. Within the same species small variations of banding patterns were observed. Figure 1 shows RAPD banding patterns for some of the G. boninense isolates from oil palm.

The dendrogram based on UPGMA cluster analysis of RAPD bands is shown in Fig. 2, which clearly grouped the four Ganoderma species into different clusters. Ganoderma boninense from oil palm including the reference isolates PER71 and SEL28 were clustered in sub-cluster A, with similarity values ranging from 86-100%, and G. lucidum FP152 in sub-cluster B. G. philippii from tea, rubber and G. philippii FP589 were clustered in sub-cluster C. G. philippii isolates from rubber were grouped in the same sub-cluster as G. philippii FP589 in sub-cluster C1 and G. philippii tea isolates in sub-cluster C2. Within sub-cluster C1 and C2 of G. philippii isolates, the similarity value was 100% and similarity value between G. philippii rubber isolates and G. philippii tea isolates was approximately 70%. Sub-cluster D consist of all G. australe isolates from forest trees and G. australe FP104. The similarity values within the G. australe isolates were approximately 90-100%.

PCR-RFLP of ITS+5.8S regions: Primer pair of ITS1 and ITS4 amplified the ITS+5.8S regions of the Ganoderma isolates from different hosts. The size of the PCR products was not identical and ranged from 500 to 750 bp. The size of ITS+5.8S regions for G. boninense was 700 bp; 500 and 625 bp for G. philippii from rubber and tea, respectively and 700 bp for G. australe from forest trees. The size of PCR product for G. lucidum was 700 bp.

Estimated sizes of the restriction bands digested using the six restriction enzymes are shown in Table 2. From the six restriction enzymes used, PCR products of G. boninense isolates were not digested by MspI and HindIII.

Digestion of the ITS+5.8S regions with Hin61, HinfI, Taq1, Bsu151 and HindIII (Fig. 3) produced identical patterns for all G. boninense isolates including the reference isolates from MPOB, SEL28 and PER21.

All G. australe isolates from forest trees and G. australe FP104 also showed identical restriction patterns when digested using Hin61, HinfI, Taq1, Bsu151 and Msp1.

Restriction patterns of G. philippii isolates from tea was slightly different from G. philippii isolates from rubber and G. philippii FP589. G. lucidum FP152 showed restriction patterns which were different from the other Ganoderma species.

Cluster analysis of the restriction bands of ITS+5.8S regions also showed that the four Ganoderma species were clustered into different clusters (Fig. 4). The dendrogram is divided into two main clusters, I and II. Main cluster 1 comprised sub-cluster A and B. G. boninense isolates were grouped in sub-cluster A with similarity value of 100%.Sub-cluster B consisted of G. lucidum FP152 in sub-cluster B1 and G. australe isolates in sub-cluster B2. G. australe isolates exhibited 100% similarity value (Fig. 4). Main cluster II comprised two sub-clusters, C1 and C2 which clustered G. philippii isolates from rubber and tea, respectively. Like RAPD cluster analysis, G. philippii from rubber were grouped with G. philippii FP589 and G. philippii from tea formed separate sub-cluster. Within each sub-cluster, the isolates showed similarity value of 100%. Similarity value between G. philippii isolates from rubber and tea was approximately 70%.

DISCUSSION

Genetic variations of the Ganoderma isolates from different hosts were detected in both RAPD and PCR-RFLP of ITS+5.8S regions. RAPD analysis generated more variable banding patterns than PCR-RFLP analysis. This could be due to different capacity of the two analyses to reveal variations. In RAPD, the random primers scan the entire genome for priming sites while PCR-RFLP of ITS+5.8S regions characterise restriction sites within a specific region. Thus, RAPD can produce more variable banding patterns compared to PCR-RFLP of ITS+5.8S regions. However, both analyses give similar results in which the analyses produce distinct cluster which clustered the Ganoderma species into separate clusters.

In general, in PCR-RFLP of ITS +5.8S regions, the sum of the restriction bands agreed with the estimated size of the PCR product or the undigested band, however, some discrepancies were observed. There were cases where the total size of the digested bands were either larger or smaller then the undigested bands. Cases where the digested bands were smaller occurred after digestion using HinfI in G. boninense isolates and G. australe isolates from forest trees; MspI for G. philippii isolates of rubber and TaqI for G. australe isolates from forest tree. This may be due to several factors such as smaller bands were not resolve on the gel or lost during gel running (Gottlieb et al., 2000), difficulties in visualizing, smaller bands or bands of equal sizes co-migrating together on the gel (Cooke and Duncan, 1997). Cases where total restriction bands were larger then the PCR product were observed in restriction patterns of Bsu151 for G. philippii isolates of tea and digestion using HindIII for G. boninense isolates from oil palm. The occurrence could be due to polymorphism within the recognition site (Cooke and Duncan, 1997) or the presence of mixed rDNA type which could cause by intragenomic differences within the ITS regions (Hibbett, 1992).

PCR products of the ITS+5.8S regions of G. boninense isolates from oil palm were not digested using MspI which indicate there`s no restriction site within the PCR product. In contrast, HindIII could only digested PCR products of G. boninense isolates from the oil palm.

In RAPD, although variations were observed within and between the Ganoderma species from different hosts, the similarity values within species were very high, which indicate high degree of similarity among the isolates. G. boninense showed the most variable banding patterns with similarity values ranged from 86-100%. Highly variable banding patterns of G. boninense isolates were also observed by Idris (2000), Pilotti et al. (2000) and Latiffah et al. (2005). Besides RAPD, Random Amplified Microsatellites (RAMS) using microsatellite primers also generated variable banding patterns of G. boninense from oil palm (Latiffah et al. 2005).

Ganoderma boninense is a laccate species and has been widely studied as the species is the main pathogen of oil palm, causing basal stem rot disease. However, the identity of the species remains controversial as phenotypic variations were reported by Ho and Nawawi (1985) and Latiffah et al. (2002). Ganoderma boninense was also found on coconut stumps and trunks. These coconut stumps and trunks could become the sources of inoculum of basal stem rot disease (Turner, 1981). Like G. boninense on oil palm, G. boninense on coconut stumps were also variable in their phenotypic features as well as molecular characteristics using RAPD and RAMS analysis (Latiffah et al., 2005). In Malaysia, Ganoderma sp. is not known to be a pathogen of coconut (Navarantnam, 1964; Turner, 1981) and only colonise the stumps and trunks of the coconut palm.

Although there were other Ganoderma species reported to be associated with basal stem rot of oil palm, in this study only G. boninense was identified. Ganoderma boninense has been reported to be the most common species infecting oil palm in Malaysia (Ho and Nawawi, 1985; Idris et al., 2000; Latiffah et al., 2002) as well as in Indonesia (Darmono, 1998) and Papua New Guinea (Pilotti et al., 2000).

Initially, the Ganoderma species associated with red root disease of rubber and tea was identified as G. pseudoferreum. However, Steyaert (1975) considered G. pseudoferreum to be synonymous with G. philippii. Unlike G. boninense, G. philippii is a secondary pathogen on rubber and therefore the species has not been studied extensively. For rubber, major losses from root diseases are caused by Rigidosporus lignosus and Phellinus noxious (Liyanage, 1997). In both analysis, G. philippii from rubber and the reference isolate G. philippii FP589 were clustered into different sub-clusters from G. philippii from tea. According to Varghese and Chew (1973), G. philippii on rubber and tea was closely related which could explain the clustering of G. philippii isolates into two different sub-clusters and the similarity based on RAPD and RFLP of ITS+5.8S regions was approximately 70%.

Ganoderma australe from forest trees were clustered with the reference isolate, G. australe FP104 in both RAPD and RFLP of ITS+5.8S regions analyses, which indicate very close relationship. Ganoderma australe is non-laccate species and are often found in tropical and sub-tropical regions. In Malaysia, G. australe have been found on mango (Mangifera indica) stumps (Abdullah et al. 1997) and as wood degrading fungi in forest reserves and plantation forests. However, there`s no proper record on the occurrence of G. australe on different types of forest trees in Malaysia. Similar to G. boninense, phenotypic variations of this non-laccate species have been reported by many researchers (Steyaert 1980; Gottlieb and Wright, 1999). Existence of biological species of G. australe was reported by Yeh et al. (1995) and Kaliyaperumal and Kalaichelvan (2007) in Taiwan and southern India, respectively.

Ganodrema lucidum FP152 was clustered separately from other Ganoderma species in both RAPD and PCR-RFLP of ITS+5.8S regions analyses. Ganoderma lucidum is well known for its medicinal value and as parasite causing root rot of perennial crops and forest trees in temperate regions. Initially, Navaratnam (1961, 1964) reported that G. lucidum was the causal agent of basal stem rot of oil palm in Malaysia, but later Steyaert (1972) found that G. lucidum was only limited to the temperate region. Moncalvo and Ryvarden (1997) suggested that based on molecular data G. lucidum might be restricted to European region.

RAPD and PCR-RFLP of ITS+5.8S regions have been used widely to characterize and to solve taxonomic problems of plant pathogenic fungi. Although the use of RAPD has been criticized for its undesirable characteristics as reported by Devos and Gale (1992), Blackeljau et al. (1995), Van De Zande and Bijlsma (1995) and Staub et al. (1996), the limitation can be overcome by vigorous optimization of PCR components. By using primers with high G+C content, it could increase efficiency of annealing and also increase the number of amplification products, which can detect variations within and between species (Kubelik and Szabo, 1995). RAPD allow a large number of samples to be analyse within short period of time and can be performed rapidly.

ITS+5.8S regions are located between 18S and 28S genes of ribosomal subunit. The regions have been used to determine variation at intraspecific and interspecific level. The ITS regions are highly conserved, the sequences are polymorphic and provide useful tools for taxonomic studies. The ITS+5.8S regions have been used to characterize Ganoderma species such as studies by Moncalvo et al. (1995a, b) on Ganoderma species from tropical and temperate regions; a study by Smith and Sivasithamparan (2000) on Ganoderma from Australia and Gottlieb et al. (2000) used the regions as an aid to the taxonomy of Ganoderma species from southern South America.

From RAPD and PCR-RFLP ITS+5.8S regions analyses, species-specific markers could be developed. Species-specific markers are useful to detect Ganoderma inoculum in the soil or in infected tissues especially on oil palm plantation. Early detection of any diseases is very important as control measures can be carried out before the disease can cause severe damage.

Characterization of Ganoderma species from different hosts especially from economically important agricultural crops is important to achieve effective disease control management. The present study demonstrate the applicability of RAPD using high G+C content and PCR-RFLP of the ITS+5.8S regions to characterize and analyze genetic variation of Ganoderma species from different hosts in Peninsula Malaysia. Both techniques could also provide rapid procedure for differentiation of different Ganoderma species.

REFERENCES