Abstract: Morphological and molecular diversity of Ganoderma species causing basal stem rot of coconut in Southern dry tracts of Karnataka, India was carried out during 2016-17. A total of 20 isolates were isolated from Chitradurga, Chikamagalore, Hassan and Tumkur districts of Karnataka and were identified based on morphological and molecular characteristics. Sporocarps and diseased root bits were found as good source for isolation of Ganoderma. In all the isolates there were high variability in cultural, morphological and molecular characteristics. The dendrogram generated from the cultural and morphological characteristics showed clear variations among Ganoderma isolates and formed two main clusters, one cluster consisted of 13 isolates and another cluster consisted of 7 isolates. Several isolates showed 100 per cent similarity in the morphological characters regardless of their geographical origin. All the Ganoderma isolates amplified a fragment of 650 bp with fungal universal primers (ITS1 and ITS4). The ITS gene sequences of five isolates viz., CG1 (MK 681870), CG7 (MK681871), CG11 (MK681872), CG14 (MK681873) and CG20 (MK681874) were deposited in NCBI gene bank. Taxonomic comparison of the isolates with NCBI database proved that the isolates were genetically related to Ganoderma spp. with 80-100 per cent identity. However, all the tested isolates could not amplify G. lucidum species specific markers which indicate its absence in the region. The phylogenetic analysis of the Ganoderma isolates (ITS1 and ITS4) of coconut with other known species of Ganoderma from GenBank emphasized the close relationship with India, China and Sri Lanka isolates. The isolate CG1 grouped with Ganoderma carnosum (KR 733545.1) with 98.97 per cent identity which is isolated from Sri Lanka and CG14 and CG20 grouped with G. applanatum (MF 072395.1) and G. gibbosum (OM 350473.1) with 98 to 99 per cent identity and CG7 and CG11 isolates of coconut grouped into distinct sub cluster and clearly indicated the species diversity in Ganoderma infecting coconut in Southern Karnataka.
Keywords: Coconut, DNA sequence, ganoderma wilt, ITS, phylogeny , variability.
Original Research Papers
Morphological and molecular diversity of Ganoderma spp. causal agent of basalstem rot of coconut in Southern dry tracts of Karnataka
Society for Promotion of Horticulture
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Coconut (Cocos nucifera L.) belonging to family Arecaceae is an important plantation crops of India providing livelihood to a substantial number of farm families. The versatile palm popularly known as ‘King of Palms’, ‘Tree of Heaven’, ‘Tree of life’, ‘Tree of Abundance’, as well as ‘God’s gift to mankind’, is grown in more than 93 countries within an area of 12.8 million hectares and production of 10.9 m MT (copra equivalent) in 2001. The total area and the production in Asian Pacific Coconut Committee (APCC) countries are estimated at 11.4 mha and 9.2 m MT respectively, which is 90 and 84 per cent of world area and production (Rethinam and Taufikkurahman 2002). In India, coconut palms are grown in an area of 2.17 million hectares with a production of 20,308.70 million nuts and a productivity of 9345 nuts/ha annually (CDB, 2019-20). Kerala ranks first in terms of area and production followed by Tamil Nadu, Karnataka and Andhra Pradesh, while, Tamil Nadu ranks first in the productivity followed by Andhra Pradesh and Kerala.
Coconut palms are normally affected by various biotic and abiotic stresses resulting in drastic reduction in yields. Among the various biotic stresses that affect coconut production in India, Basal Stem Rot (BSR) or Ganoderma wilt caused by Ganoderma applanatum Pers and G. lucidum (Leys). Karst. is a major constraint in coconut production, especially in dry tracts of Southern Karnataka. The disease is reported from various places all over the tropical world viz., India, Sri Lanka, West Indies, Seycheles, Guam etc., Though the disease was first recorded by Dr. Butler in the beginning of 20 th century and later by Venkatanarayan (1936) from Karnataka, a severe outbreak occurred in 1652 in Thanjavur district of Tamil Nadu and hence named as Thanjavur wilt. The disease is also reported from Andhra Pradesh, Kerala, Maharashtra, Gujarat and Orissa (Bhaskaran, 1994; Wilson et al, 1987). Ganoderma species are important wood decaying fungi occurring throughout the world. They are diverse in the tropics affecting plantation crops such as coconut, arecanut and oil palm by causing basal stem rot (Flood et al., 2000 and Pilotti, 2005) and they also affect ornamental and forest trees in tropical and temperate areas causing disease and wood rots of timber (Lee, 2000).
The taxonomy of basidiomycetes has traditionally been based on the morphological features of the basidiocarps. Identification based on the basidiocarp features, however, is prone to problems such as absence of basidiocarps during certain times of the year, their morphological plasticity and presence of cryptic species (Moncalvo and Ryvarden, 1997; Gottlieb and Wright, 1999). However, studies had shown that Ganoderma species were genetically heterogeneous since wide range of genetic variation were reported and caused by out crossing over generations and different geographical origins (Miller et al., 1999; Pilotti et al. , 2003). This leads to variation in their morphological characteristics even within same species (Hong et al., 2001). For these reasons, contemporary taxonomists employ morphological studies, mating tests, analyses of biochemical and DNA sequence information or combinations of these for identification of the pathogen. Recently, molecular approach has been adapted to identify Ganoderma species such a s thr ough multiplex polymerase chain reaction (PCR) which is a more rapid and precise approach (Idris et al., 2010; Wong et al., 2012). Disease management is an important aspect to sustain the palm industry. Accurate identification of the pathogen is pre requisite for designing management strategies. Hence, the present study was undertaken to investigate the diversity of Ganoderma species isolated from BSR infected coconut palms in terms of their molecular and morphological characteristics.
Different parts of the coconut palms such as diseased root bits/stem bits affected by Ganoderma wilt showing typical symptoms and sporocarps were collected from infected palms from various places of Southern Karnataka (Table 1). The samples were labeled and packed in polythene bags for the purpose of isolation of the causal organism.
Infected roots/ stem bits collected from infected palms were washed thoroughly with sterile water and cut into small bits/pieces and were surface sterilized in 1 per cent sodium hypochlorite solution for 30 seconds and rinsed with sterile distilled water thrice serially to remove the traces of sodium hypochlorite. After surface sterilization, diseased specimens were kept in sterilized bags along with wet cotton under room temperature for about 8 to 10 days. After 8 to 10 days of incubation period, slight mycelial growth was observed and that was transferred into potato dextrose agar (PDA) medium. The inoculated plates were incubated at room temperature (28 °C ± 2 °C) for 3-5 days to facilitate growth of the fungus. Later, the bit of fungal growth was transferred to PDA slants. The pure culture of the fungus was obtained by following hyphal tip culture technique under aseptic conditions. The isolated Ganoderma isolates of coconut were designated as CG1, CG2, CG3, CG4, CG5, CG6, CG7, CG8 , CG9, CG10, CG11, CG12 CG13, CG14, CG15,CG16, CG17, CG18, CG19 and CG20
Identity and designation of Ganoderma isolates of coconut and their source of collection
Note: CG-Coconut Ganoderma
The isolated fungus was sub-cultured on PDA slants and allowed to grow at 28 °C ± 2°C temperature for 8-10 days. The cultures so obtained were stored in refrigerator at 4°C for further studies and they were cultured periodically once in 2 to 3 months.
Twenty Ganoderma isolates of coconut isolated during course of investigation were used in variability study.
Twenty Ganoderma isolates [CG1, CG2, CG3, CG4, CG5, CG6, CG7, CG8, CG9, CG10, CG11, CG12, CG13, CG14, CG15, CG16, CG17, CG18, CG19 and CG20] of coconut collected from different geographic locations were cultured on PDA. The morphological characters like colony diameter/growth, biomass production, colony colour, colony margin, mycelial density, appearance of zones, reverse pigmentation etc were studied.
Mycelia plug (6 mm) from seven days old active culture was transferred onto the centre of a standard 9 cm PDA plate and incubated for 7 days at an ambient temperature (Idris et al., 2000). The test for all isolates with three replications was run simultaneously to avoid bias due to external factors. The diameter was measured daily and the number of days required for maximum growth of mycelium was also recorded. The colony texture, appearance of zone, reverse pigmentation colour, type of colony margin and mycelial density were recorded after seventh day of incubation.
The flasks containing 100 ml of sterilized potato dextrose broth (PDB) were inoculated with the 0.6 cm mycelial discs of Ganoderma isolates of coconut. Three replications were maintained for each treatment. The inoculated flasks were incubated at room temperature (28±2 °C) for 10 days, and then mycelial mat was harvested on a previously weighed Whatman No.4 filter paper and dried at 60 °C in a hot air oven till constant weight was obtained. The dry mycelial weight was recorded and expressed in mg 100 ml-1 broth and results were analysed statistically.
Qualitative data of cultural characteristics on solid media and bio mass were transformed into code and a numerical data matrix was generated (Table 2). The data was subjected to cluster analysis using multivariate statistical package (MVSP version 3.13). Similarity matrices were calculated using the simple matching coefficient and a dendrogram was generated using the unweighted pair group method of arithmetic averages (UPGMA) (Pilotti et al., 2004).
Cultural morphological characters and their corresponding codes used to describe Ganoderma isolates for assessment of cultural morphological characteristics
The isolates of Ganoderma species were identified through ITS (Internal Transcribed Spacer) region using universal primers ITS1 and ITS4 amplification.
All the chemicals were of analytical grade (M/s Sigma Ltd. and M/s Merck Ltd.). The following buffers and solutions were prepared : Extraction buffer (100 mmM Tris-HCl (pH 8); 20 mM EDTA (pH 8); 2 M NaCl; 3 % CTAB (w/v); 1 % PVP; 2 % β-mercaptoethanol (v/v); phenol : chloroform (24:1); potassium acetate 7.5 M; proteinase K, 0.05 mg ml-1 ; wash solution [15 mM ammonium acetate in 75 % (v/v) ethanol ]; TE buffer [10 mM Tris-HCl (pH 8), 1mM EDTA (pH 8)].
Fungal mycelia (100 mg) were ground to fine powder using liquid nitrogen. Pre-warmed extraction buffer (1 ml) was added to the samples and it was ground once more in the buffer. All the samples were transferred to 2.0 ml Eppendorf tubes and 5 µL proteinase K (10 mg ml-1) was added. The tube was incubated in 37 °C for 30 min and then at 65°C for another 30 min with frequent swirling. Samples were centrifuged at 10,000 x g for 10 min at RT and the supernatant was transferred to fresh Eppendorf tube. To the supernatant, 100 µL of 7.5 M potassium acetate was added and incubated at 4°C for 30 min. The samples were centrifuged at 13,000 x g for 10 min at RT; the supernatant was transferred to fresh tube, an equal volume of chloroform: isoamyl alcohol was added and mixed by gentle in version 30-40 times. The samples were centrifuged at 10,000 x g for 10 min at RT. The supernatant was transferred to a fresh tube and precipitated with 2/3 volume of isopropanol. The precipitated nucleic acids were collected and washed twice with the wash solution. The nucleic acid pellet so obtained was air dried until the traces of ethanol was removed and dissolved in an appropriate amount of TE buffer (50-70 µL). The nucleic acid dissolved in TE buffer were treated with ribonuclease (RNase 10 mgml-1), incubated at 37 °C for 30 min and stored at -20°C until further use. The experiment was repeated thrice and the results described as the mean of three independent experiments (Sambrook and Russel, 2001).
The quality and quantity of DNA was analyzed by running 2 µL of each sample mixed with 2 µL of 10x loading dye in one per cent agarose gel. The DNA from all the isolates produced clear sharp bands in one per cent agarose gel indicating good quality of the DNA. The DNA has been quantified by comparing with the 1 kb size marker (Genei, Bangalore) and by spectrophotometer (Nanodrop ND 1000).
The ribosomal DNA (rDNA) unit contains genetic andnon-genetic or spacer r egion. Each repeat unitconsisted of a copy of 18S, 5.8S and 28S like rDNAand its spacer like internal transcribed spacers (ITS)and Inter-Genic Spacers (IGS). The rDNA has beenemployed to analyze evolutionary events because it ishighly conserved whereas ITS rDNA is more variable.Hence, it has been used for investigating the specieslevel relationships. The primers for amplification werecustom synthesized at Bangalore Genie Pvt. Ltd.,Bangalore and supplied as lyophilized products ofdesalted oligos. PCR was carried out in poly propylenetubes using univer sa l pr imer s IT S 1 (5' -AACGTTACCAAACTGTTA-3') and ITS 4 (5' -AAGTTCAGCGGGTATTCCT-3') and G. lucidumspecific primers GSF (5' -CCCTAAACCTCTCAAAGTCA-3') and GSR (5' -TATCGTACAGGTTCTCGTG -3). PCR amplification was performed in 25μl reaction mixture containing 10× reaction buffersupplied by the manufacturer, 100 ng of fungal DNA,each dNTP at a concentration of 0.5 mM, 20 Picomoles of each primer and 1 U of Taq DNA polymerase(NEB, USA). Thermo cycling conditions were 94o Cfor 5 min, followed by 30 cycles of 94o C for 30 sec,56o C for 1min and 72o C for 1min and a finalelongation step of 72o C for 5min.Separation of amplified products by Agarose gelelectrophoresisAgarose gel electrophoresis was performed to resolvethe amplified product using 1.4 per cent agarose in1X TBE (Tris borate EDTA) buffer, 0.5 mg ml-1 ofethidium br omide a nd loa ding buffer (0. 25 %bromophenol blue in 40 % sucrose). Four μL of theloading dye was added to 5 μL of PCR product andloaded to the agarose gel. Electrophoresis was carriedat 65 V for 1.5 hrs. The gel was observed under UVlight and documented using gel documentation unit.Morphological and molecular diversity of Ganoderma spp. causal agent
The ribosomal DNA (rDNA) unit contains genetic and non-genetic or spacer region. Each repeat unit consisted of a copy of 18S, 5.8S and 28S like rDNA and its spacer like internal transcribed spacers (ITS) and Inter-Genic Spacers (IGS). The rDNA has been employed to analyze evolutionary events because it is highly conserved whereas ITS rDNA is more variable. Hence, it has been used for investigating the species level relationships. The primers for amplification were custom synthesized at Bangalore Genie Pvt. Ltd., Bangalore and supplied as lyophilized products of desalted oligos. PCR was carried out in poly propylene tubes using universal primers ITS 1 (5' - AACGTTACCAAACTGTTA-3') and ITS 4 (5' - AAGTTCAGCGGGTATTCCT-3') and G. lucidum specific primers GSF (5- CCCTAAACCTCTCAAA GTCA-3) and GSR (5´- TATCGTACAGGTTCT CGTG -3).
PCR amplification was performed in 25 µl reaction mixture containing 10× reaction buffer supplied by the manufacturer, 100 ng of fungal DNA, each dNTP at a concentration of 0.5 mM, 20 Pico moles of each primer and 1 U of Taq DNA polymerase (NEB, USA). Thermo cycling conditions were 94o C for 5 min, followed by 30 cycles of 94o C for 30 sec, 56o C for 1min and 72o C for 1min and a final elongation step of 72o C for 5min.
Agarose gel electrophoresis was performed to resolve the amplified product using 1.4 per cent agarose in 1X TBE (Tris borate EDTA) buffer, 0.5 mg ml-1 of ethidium bromide and loading buffer (0.25 % bromophenol blue in 40 % sucrose). Four µL of the loading dye was added to 5 µL of PCR product and loaded to the agarose gel. Electrophoresis was carried at 65 V for 1.5 hrs. The gel was observed under UV light and documented using gel documentation unit.
The ITS region was sequenced from isolates of Ganoderma species to confirm the organism and to know the variability present in them. Homology search was done using BLAST algorithm (Basic Local Alignment Search Tool).
The results revealed that there were cultural morphological variations between isolates of Ganoderma isolated from infected palms of coconut in Southern dry tracts of Karnataka. The colony diameter on 5th, 7th and 9th day after inoculation was significantly varied, where radial growth ranged from 1.87 to 8.53 cm on 5th day after inoculation. Similarly on 7th and 9th day after inoculation it ranged from 2.63 to 9.00 cm and 4.75 to 9.00 cm respectively. The number of days taken to cover full plate ranged from 7 to 18 days and most of the isolates covered entire plate in 7 days as noted in CG4, CG7, CG10, CG11, CG12, CG13, CG14 and CG20. However, some of isolates taken <10 days to cover entire plate. The bio mass production also varied significantly between different isolates and it ranged from 0.56 to 1.46 g/ 100ml. There were lot of variations observed with respect to colony/ mycelial characteristics viz., concentric rings. reverse pigmentation, density of mycelium and colony margin. However, there was not much variations were observed with respect to colour and texture of the colony (Fig.1 & 2 and Table 3)
Fig. 1:
Dendrogram showing relationships of Ganoderma isolates of coconut based on similarity matrix of cultural/morphological characteristics
Fig. 2:
Cultural morphological variability Ganoderma isolates. A1-A4, ) Ganoderma isolates on PDA and B) Ganoderma isolates on PDB
Cultural and morphological characteristics/variability of Ganoderma isolates of coconut
Note: + Present; - Absent; DAI-Days after Inoculation *Mean of three replications
The dendrogram generated from the cultural morphological characteristics showed clearly the variations among Ganoderma isolates and dendrogram formed two main groups (Fig.1). The isolate CG14 and CG5 are distinct. The complete similarity (100%) was found in several isolates of Ganoderma regardless of their geographical origin. All the isolates used under study showed high variability in cultural and morphological characteristics. Rakib et al. (2014) who had studied the genetic morphological variability of forty six isolates of Ganoderma causing basal stem rot and upper stem rot in oil palm stated that, there were significant variations within and between Ganoderma species in terms of their cultural morphology and basidiospore characteristics and they also reported that, cluster analysis of the cultural morphology and scattered plot of basidiospore features indicated that there was no distinct relationship within and between species, disease types or geographical origins of Ganoderma species.
The wide range of variation in morphological characteristic can be related to the heterogeneity of Ganoderma species. The cultural characteristic that appeared to distinguish G. zonatum from G. boninense and G. miniatocinctum was the strongly wavy characteristic of the colony in G. zonatum. However, this characteristic also varied and was not present in all of the G. zonatum isolates. Furthermore, the cultural appearances of fungi are also highly dependent on several factors such as type of media, pH and temperature (Adaskaveg and Gilbertson, 1989). Although similar (100 % similarity) cultural morphological features were observed between G3 and G4, G15 and G33, G19 and G27, and G30 and G31 based on the dendrogram generated, they were still genetically different based on the somatic incompatibility between the isolates. This showed that different genotype in Ganoderma species may express similar morphological features (phenotype). The dendrogram also showed same species of Ganoderma may be separated by up to 40 per cent dissimilarity, while different species of Ganoderma may have up to 92 per cent similarity. This indicates that Ganoderma species in an oil palm plantation could not be separated according to their species, disease type or geographical origins based on their cultural morphological features. More precise tool such molecular techniques/tools should be used to identify the Ganoderma species accurately.
Genomic DNA of different isolates of Ganoderma was isolated by CTAB method and the size was determined by resolving on one per cent Agarose gel. The concentration of DNA was determined using nanodrop equipment which was 75µg/µl.
The full length ITS rDNA region was amplified with ITS region with fungal universal primers (ITS1 and ITS4) and G. lucidum specific primers from the total genomic DNA of all the five isolates of Ganoderma.DNA amplicon was 600-650 bp in length in universal primers (Fig.3) and DNA was not amplified with G. lucidum specific primers and results revealed that, the G. lucidum species was absent in coconut isolates tested. Further, the species identity was confirmed with DNA sequencing.
The ITS rDNA fragments of Ganoderma isolates sequences were sequenced and DNA amplification from Ganoderma was observed at good specificity for the genus Ganoderma and approximately 600-650 bp product was exclusively amplified in all the isolates tested with fungal universal primers. DNA sequences of selected isolates of coconut was compared using bioinformatics tool like NCBI (National Centre for Bioinformatics) BLAST programme. Based on the sequence comparison, the identification of Ganoderma isolates was confirmed and all the ITS rDNA sequences of the isolates were confirmed as Ganoderma sp. with 80-100 per cent identity. The GenBank accession number for the ITS sequences for the isolates CG1, CG7, CG11, CG14 and CG20 were MK681870, MK681871, MK681872, MK681873, MK681874. and Phylogenetic tree of Ganoderma constructed with ITS region sequences is shown in Fig. 4.
The phylogeny of the Ganoderma isolates of coconut revealed that, the isolate CG1 grouped with Ganoderma carnosum (KR 733545.1) which is originated from Sri Lanka and CG14 and CG20 grouped with Ganoderma sp. (KR154930) and Ganoderma sp. (KM229652). These species were originated from India and CG7 and CG11 isolates of coconut grouped into distinct sub cluster and indicated the species
Fig. 3:
Gel picture showing PCR amplification of rDNA of Ganoderma isolates of coconut with ITS1, ITS4 and G lucidum primers
Legend: Lane M = 100bp Ladder; Lane 1-5 = Ganoderma isolates of coconut; Lane N = Negative control
diversity and dissimilarity of Ganoderma in Southern Karnataka.
Abundance and uniform distribution of genetic markers in any pathogen is necessary for applications like diversity analysis at various levels. Almost unlimited in number, they are widely and evenly distributed in the genome. Unaffected by other genes and environment, the genotype of any individual of a population with respect to DNA based markers can be determined unequivocally at any stage of the development non-destructively. In addition, it is possible to generate markers to suite specific applications without altering the genotype of the individuals. It is difficult to distinguish these species using traditional morphological and physiological differences. To understand existence of variation among the isolates of pathogens, PCR based technique with G. lucidum specific markers and ITS sequence was used in the present investigation.
Variations in morphological characteristics of Ganoderma have led many taxonomists to introduce biochemical and molecular methods to differentiate Ganoderma species. DNA amplification from Ganoderma was observed at good specificity for the genus Ganoderma and approximately 600-650 bp product was exclusively amplified in all the isolates tested with fungal universal primers. However, DNA amplification was not amplified with G. lucidum
Fig. 4:
Phylogenetic relationships of Ganoderma isolates of coconut inferred from the sequences of the ITS region
specific primers in the isolates tested. The sequencing and phylogenetic analysis of selected isolates Ganoderma infecting coconut revealed the Ganoderma species diversity in dry tracts of Southern Karnataka.
Nuclear rDNA, including the small and large subunits, 5.8S, and the Internal Transcribed Spacer (ITS) region, proved an ideal target for specific PCR primers, as each sequence is variable at the family, genus, or species level. Internal Transcribed Spacer (ITS) regions have been successfully used to generate specific primers capable of differentiating closely related fungal species. Amplification of target DNA through PCR with taxon-specific primers is a potentially more sensitive and accurate approach than conventional microscopic techniques. Nucleotide sequences from certain regions of the DNA reflect phylogeny at various taxonomic levels. Such regions need to be evolving at an appropriate rate in order to supply enough consistent differences to separate the taxa into statistically supported monophyletic groups. These regions must be present as a single copy in the genome or evolve as a single copy region in order to avoid comparisons of different copies in different species (paralogous comparisons) if the region exists as multicopy. Also, the region should have the same function in all organisms (Mitchell et al., 1995). The ribosomal RNA (rRNA) genes, certain ribosomal elongation factors, and genes from the nuclear and the mitochondrial genomes have been useful for DNA sequence analysis in fungi (Tan and Niessen, 2003; Moreau et al., 2006). Consequently, nucleotide sequence information from relatively conserved genes/ DNA segments such as the ITS (Moncalvo et al., 1995a, b; Smith and Sivasithamparam, 2000a), the mitochondrial small subunit (mtSSU) (Hong and Jung, 2004), and nuclear large subunit (LSU) (Lee et al., 2006) rDNA have been widely used in the taxonomy and phylogeny of Ganoderma species. This is because the variability of these regions, which is harboured mainly in the introns, provides sufficient resolution at various taxonomic levels.
Gottlieb et al. (2000) adopted rDNA analysis (ITS I and II of 5.8S rDNA) to identify South American isolates of Ganoderma and Elfvingia and found molecular and morphological agreement at the sub generic level, however this relationship was difficult to visualize at the species level. Singh et al. (2003) characterized 61 accessions using DNA finger printing technique and RAPD/ AFLP analysis which revealed highly significant genetic variability among G. lucidum isolates collected from coconut gardens in Coimbatore. Phylogenetic analysis of the ITS sequence data was used to resolve Australian Ganoderma isolates into five terminal clades, and showed that a number of isolates had been misnamed (Smith and Sivasithamparam, 2000a). Based on the phylogenetic analysis of the ITS and 5.8S sequence, Latiffah et al. (2002) showed that Ganoderma isolates from infected oil palm and coconut stumps belong to the same group as classified by PCR-RFLP. Gottlieb et al. (2000) also used ITS-based phylogenetic analysis together with PCR-RFLPs to elucidate the taxonomy of Ganoderma species in South America. They reported that molecular and morphological data agree at the subgeneric level, but that it was difficult to determine relationships at the species level.
Earlier studies based on morphological identification asserted that North American G. lucidum and European G. resinaceum belong to the same biological species (Adaskaveg and Gilbertson, 1986). Based on phylogenetic relationships and nucleotide sequence variations of the ITS (Moncalvo et al., 1995a, b) as well as the mtSSU (Hong and Jung, 2004), these two species were shown to be different. The gene phylogeny by Moncalvo et al. (1995b) has indicated that isolates that were morphologically identified as G. lucidum did not cluster together, neither did those identified as G. tsugae or G. resinaceum. In the phylogenetic analysis of Ganoderma species using mtSSU sequence data by Hong and Jung (2004), Ganoderma species were divided into six monophyletic groups ( G. colossus group, G. applanatum group, G. tsugae group, Asian G. lucidum group, G. meredithiae group, and G. resinaceum group) that included different species that were identified based on morphological characters. Species that were identified as G. lucidum were scattered over three of the groups, the Asian G. lucidum group, the G. resinaceum group and the G. tsugae group. Also, isolates that were identified as G. oregonense and G. oerstedii did not group together. These two studies indicate that some isolates were misidentified based on morphological characters since isolates that were identified as one thing do not form a monophyletic group.
From the preceding discussion it is clear that DNA sequence analysis of the ribosomal DNA region has provided an alternative approach to elucidate the taxonomy of Ganoderma. These techniques have played an important role in the taxonomy of Ganoderma, and have proved to be more reliable than other techniques that have been used for the same purpose. Misidentification and species synonyms based on morphological identification have been reduced using the molecular techniques. Among 5 isolates sequenced, isolate CG14 and CG20 are grouped in same cluster both in morphological and molecular phylogeny. However, other isolates viz., CG7 and CG11 which are genetically 100 per cent similar and grouped in same cluster are morphologically different as evidenced by grouping in different clusters in morphological phylogeny. In this study, a combination of cultural/morphological characteristics and molecular techniques allowed identification of groups within Ganoderma isolates of coconut and results indicated existence of morphological and molecular variability of Ganoderma isolates of coconut causing BSR in dry tracts of Southern Karnataka. Further, molecular characterization with G. lucidum species specific markers and fungal universal primers also indicated species diversity in Ganoderma causing basal stem rot/ Ganoderma wilt in coconut. In the present study based on phylogenetic analysis isolate CG1 was identified as G. carnosum with 98.97 per cent identity and isolates CG14 and CG20 showed maximum (98.96 to 99.46 %) identity with G. gibbosum and G. applanatum species and indicating the different species associated with Ganoderma wilt of coconut in dry tracts of Southern Karnataka. However, the species identity has to be confirmed by systematic investigation with polyphasic taxonomic approach to unravel the species diversity of Ganoderma causing basal stem rot in coconut in Karnataka.
The authors gratefully acknowledge ICAR-AICRP on palms, Agriculture Research Station, Arsikere and Department of Horticulture, Government of Karnataka for providing base line information on coconut cultivation and Department of Plant Pathology, University of Agricultural Sciences, GKVK, Bengaluru for providing facilities to carry out this work.
Identity and designation of Ganoderma isolates of coconut and their source of collection
Note: CG-Coconut Ganoderma
Cultural morphological characters and their corresponding codes used to describe Ganoderma isolates for assessment of cultural morphological characteristics
Fig. 1:
Dendrogram showing relationships of Ganoderma isolates of coconut based on similarity matrix of cultural/morphological characteristics
Fig. 2:
Cultural morphological variability Ganoderma isolates. A1-A4, ) Ganoderma isolates on PDA and B) Ganoderma isolates on PDB
Cultural and morphological characteristics/variability of Ganoderma isolates of coconut
Note: + Present; - Absent; DAI-Days after Inoculation *Mean of three replications
Fig. 3:
Gel picture showing PCR amplification of rDNA of Ganoderma isolates of coconut with ITS1, ITS4 and G lucidum primers
Legend: Lane M = 100bp Ladder; Lane 1-5 = Ganoderma isolates of coconut; Lane N = Negative control
Fig. 4:
Phylogenetic relationships of Ganoderma isolates of coconut inferred from the sequences of the ITS region
