Ann Microbiol (2012) 62:797–803 DOI 10.1007/s13213-011-0320-8

ORIGINAL ARTICLE

Sequence analysis of the ribosomal DNA internal transcribed spacer regions in heimii species

Shafiquzzaman Siddiquee & Weng Yan Yee & Khanam Taslima & Nur Hasan Nudin Fatihah & S. Vijay Kumar & Md Mainul Hasan

Received: 29 October 2010 /Accepted: 8 July 2011 /Published online: 29 July 2011 # Springer-Verlag and the University of Milan 2011

Abstract Termitomyces represents a very poorly known and belongs to the Tricholomataceae family. Based on the genus of fungi whose essential characteristic is that all morphological identification and the molecular analysis representatives of the genus are cultivated by conducted in this study, we have determined that the () in their nest. Many species of Termito- isolates represent the fungi named Termitomyces myces commonly form fruiting bodies which develop from heimii. combs within the nest. Identification based on morphological characteristics is problematic, tedious, and Keyword . Termites . Internal prone to error. Therefore, correct identification at the transcribed spacer (ITS) . Phylogenetic analysis species level is highly desirable. This is the first report on the identification of the Malaysian isolates of Termitomyces analyzed using the DNA sequence of their internal Introduction transcriber spacer regions (ITS1–5.8S–ITS2). The results clearly demonstrate that this group is clearly monophyletic The fungus-growing termites (Isoptera: Termitidae: Macro- termitinae) cultivate fungal symbionts of the monophyletic : genus Termitomyces (: Tricholomataceae: S. Siddiquee (*) S. V. Kumar Termitomyceteae) on specially constructed substrate combs Biotechnology Research Institute, located in single or multiple nest chambers (Wood and Universiti Malaysia Sabah JLN UMS, 88400 Kota Kinabalu, Sabah, Malaysia Thomas 1989; Aanen et al. 2002; Frøslev et al. 2003). e-mail: [email protected] These combs consist of finely fragmented dried plant and wood material with a high C/N ratio. Once fungal W. Y. Yee degradation occurs, the termites use both the fungal tissues Department of Biology, Faculty of Science, Universiti Putra Malaysia UPM, (mycelium and asexual fruiting bodies called nodules) and 43400 Serdang, Selangor, Malaysia the degraded plant materials. The relative proportions of fungal and plant material in the termite diet change between K. Taslima castes and between species (Leuthold et al. 1989; Traniello Department of Fisheries Biology and Genetics, Faculty of Fisheries, Bangladesh Agricultural University, and Leuthold 2000). Mymensingh, Bangladesh Termites belonging to the subfamily Macrotermitinae cultivate the mycelium of basidiomycetes on comb-like N. H. N. Fatihah structures made of small spheres of plant material which Department of Agricultural Science, Faculty of Agriculture & Biotechnology, Universiti Sultan Zainal Abidin (UNISZA), quickly passes through the termite gut. The fungi produce 20400 Kuala Terengganu, Malaysia small nodules which are consumed by the termites along with the degraded comb. In this way, the exosymbiotic fungus M. M. Hasan makes complex plant macromolecules for the termites, Department of Agricultural Botany, Patuakhali Science and Technology University, parallel to the function of the protozoic endosymbionts of Dumki, Patuakhali 8602, Bangladesh non-fungus growing termites (Rouland Lefevre 2000). During 798 Ann Microbiol (2012) 62:797–803 the rainy season, the fungal mycelium of the termite combs termites, such as T. heimii, have a small percentage of produce mushrooms which penetrate the termite nests and spores with two nuclei, thus predicting that a small soil to spread their spores (Heim 1977). percentage of spores are not required for out-crossing to The fungus Termitomyces heimii was first described by complete the sexual life cycle (Petersen 1995). Heim (1977) in Madras, Tamil Nadu, India in 1942 and is Species level identifications are usually difficult to be classified under the phylum Basidiomycota, class Hyme- resolved based on photo-microscopy. The unifying charac- nomycetes, order , and family Tricholomataceae. ters of the termitophilic mushrooms are the production of Termitomyces species are commonly found throughout the the pinkish spore, the termite association, and the subter- Equatorial region, Southeast Asia, and Southern Africa ranean elongation of the stipe called a pseudorhiza; most of (Pegler and Vanhaecke 1994; Tang et al. 2005) and survive the species in their termite nests are connected to the comb in a state of obligate symbiosis with termites. through the basidiomes. The majority of the species have a In Malaysia, this edible wild fungus is ubiquitous, pronounced umbo or papilla, the ‘perforatorium’ (Heim especially during the months of July to August, probably 1977). Rouland Lefevre et al. (2002) demonstrated that triggered by the onset of heavy rains during those periods. identification solely based on morphological characteristics Among the Chinese in Malaysia, T. heimi is commonly is still inaccurate, inconclusive, inconsistent, and subject to known as “cendawan busut” or ‘termite mound mushroom’, human error. Due to the different morphological affinities, as “kulat tahun”, probably for its annual appearance, or as Termitomyces sp. was once placed under different genera, “kai ku”, which means chicken mushroom, due to its such as Lentinus, Entoloma, Pluteus,andArmillaria chicken-like taste and texture (Pegler and Vanhaecke 1994). (Frøslev et al. 2003). In Malaysia, it can be found in cocoa, oil palm, and rubber Molecular techniques have progressed very rapidly and estates, gardens, fruit orchards, such as the durian, mango, have been used extensively to discover and unravel the rambutan and ciku orchards, and pastures. T. heimii mysteries of unknown substances in various fields which normally appears at the same time with subterranean had previously been thought to be “impossible”, further termite’s nests located in the shade of trees, under unraveling the correct concepts and theories recognized conditions of low light intensity and high humidity (Lee long ago. Advanced molecular techniques have verified the and Wood 1971), with more than 300 basidiomata appear- correct identifications of microorganisms. With the advent ing from a single large termite’s nest, which last 3–5 days of molecular studies, such as gene sequencing, the correct before drying and withering (Pegler and Vanhaecke 1994). identification of various fungal species becomes more Termitomyces spp. are universally characterized morpho- conclusive and precise, and the phylogenic analysis logically based on the dome-looking shape of their between the different species and the co-evolutionary perforatorium on the pileus, which is well developed, and history of the organisms can be established and elucidated. the subterranean pseudorhiza, which are attached to the Molecular techniques have definitely proved to be useful fungal comb of the termite’s nest (Tang et al. 2005). In tools in distinguishing between organisms at the species contrast to other higher basidiomycetes, Termitomyces spp. level (Maxwell et al. 2005). do not form clamp connections between adjacent cells, thus The main aim of this study was to use gene sequencing making a heterokaryon difficult to differentiate from a data on T. heimii based on their internal transcribed spacer homokaryon (De Fine Licht et al. 2005). It has also been regions (ITS1 and ITS2), including the 5.8S gene of the reported that some species of Termitomyces have a rDNA. Thus, these studies are a combination of morpho- heterothallic mating system with multinucleate cells in both logical and molecular approaches, providing data that the homokaryon and heterokaryon phases. The heterothallic enable a correct identification of T. heimii species. mating system involves the germination of a single haploid basidiospore to produce a haploid mycelium, the homo- karyon, which then undergoes contact, fusion, and recipro- Materials and methods cal nuclear exchange through the mycelia between another compatible homokaryon to form a heterokaryon (usually Isolate of fungi heterokaryon has two nuclei), subsequently giving rise to fruiting bodies (De Fine Licht et al. 2005). Thus, in the Fungi fruiting bodies were collected from an oil palm heterothallic mating system adopted by some Termitomyces plantation at the Universiti Putra Malaysia campus during sp., such as the Termitomyces sp. associated with the termite the months of July to August, 2008 (Table 1). The natalensis, two compatible spores are ulti- outermost layer was peeled off and cut into 3 mm-length mately required to colonize a nest in order to complete the segments of fruiting bodies at the Mycology and Plant fungal life cycle (De Fine Licht et al. 2005). The Pathology Laboratory, Department of Biology, Faculty of Termitomyces sp. also supported by Odontotermes sp. Science, UPM. The segments were surface-sterilized in Ann Microbiol (2012) 62:797–803 799

Table 1 Isolates of Termitomy- ces heimii analyzed in this Isolates no. Species name Isolates code Sampling date Host genotype study 1 Termitomyces heimii TH2 15 July, 2008 Oil palm 1A 2 T. heimii TH4 28 July, 2008 Oil palm 4B 3 T. heimii TH3 4 August, 2008 Oil palm 7D 4 T. heimii TH5 21 August, 2008 Oil palm 6 C

70% ethanol followed by 0.05% sodium hypochlorite (or et al. (2010a). Approximately, 50 mg of ground mycelium 5% Clorox bleaching agent) for 1–2 min, then rinsed with was added to 500 μl of extraction buffer [1 M Tris HCl, pH sterile distilled water, and blotted dry on sterilized What- 8.5, 1 M NaCl, pH 8.5, 1 M EDTA, pH 8.0, and 10% man no. 17 filter papers (Whatman, New York, NY). One or sodium dodecyl sulfate (SDS)]. The reaction tubes were three segments were placed on a potato dextrose agar then placed in a water bath for 8 h at 38°C. After (PDA) (Difco, Detroit, MI) plate and incubated for 3– incubation, 350 μl of buffered phenol and 150 μlof 6 days under ambient laboratory conditions (28±2°C, 12/ chloroform were added and homogenously mixed for 12-h light/dark photoperiod). After the incubation period, 10 min. The resulting suspension was subsequently the fungi colonies could be seen as small whitish growth centrifuged at 13,000 g at 4°C for 10 min. The upper spots. All subculturing of the isolates was done on a PDA aqueous layer was collected and transferred to a sterile plate and prepared as follows: 19 g of PDA was added centrifuge tube to which 3 μl of RNAse solution was 500 ml sterilized distilled water. Stock cultures were stored added. This solution was then incubated at 38°C in a water on agar slopes in 20-ml universal bottles maintained at 10– bath for 15 min. After incubation, an equal volume of 15°C until required for further analysis. chloroform was gently mixed and added to the sample for 10 min. The mixture was then centrifuged once more Liquid culture of isolates (13,000 g for 10 min at 4°C). The upper aqueous phase was again collected and transferred into a new tube. The DNA Isolates were cultured in potato dextrose broth (PDB) was precipitated with 250 μl of iso-propan-2-ol and kept (Difco) and prepared according to the manufacturer’s overnight at −20°C. The tube was centrifuged the next day specifications. Aliquots of 100 ml media were decanted (13,000 g for 10 min at 4°C). The resultant pellets were into individual 250-ml Erlenmeyer flasks, sealed with thoroughly washed twice with 500 μl of 70% ethanol, cotton wool stoppers in the flask mouths, and then vacuum-dried, and diluted in ddH2O. Finally, the DNA autoclaved. When cooled, each flask was aseptically pellets were suspended in 50 μlofddH2O and kept at −20°C inoculated with five agar discs of pure cultures of fungi if not used immediately. The quality of the DNA samples cut out from actively growing hyphal tips using a 5-mm- was checked by performing gel electrophoresis on 1.5% diameter cork borer. Once inoculated, the cotton wool agarose gel using a 50-bp ladder as a size standard (Promega, stoppers were replaced with aluminum foil and sealed with Madison, Wi). The electrophoresis was carried out in a 1× parafilm during the growth phase as static cultures under TBE (0.045 M Tris-borate and 1 mM EDTA, pH 8.2) room temperature conditions (28±2°C; 12/12-h dark/light running buffer at 70 V for 1–2 h. The gels were then stained photoperiod). The flasks were maintained as still cultures with ethidium bromide (0.5 μl/ml) and visualized under UV for 10–13 days under ambient laboratory conditions. The light. The appearance of bands indicated the presence of a mycelial mats were harvested by filtration through a DNA template which thereby allowed their use in a PCR. double-layered muslin cloth and washed several times with sterile water. The mycelial mats from each of the isolates PCR amplification of the ITS regions of the rDNA were then transferred into individual plastic bags (with excess liquid squeezed out), labeled, and frozen overnight Two universal primers were used in this study. The forward at −20°C. The frozen mycelia mats were immediately primer ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ground in a mortar and pestle that had been swabbed with the reverse primer ITS-4 (5′-TCC TCC GCT TAT TGA TAT ethanol prior to use. The slurry obtained was stored at −20°C GC-3′) were used for the amplification of ITS-1 and ITS-2 or used immediately for DNA extraction. regions of the rDNA, previously described by Siddiquee et al. (2010b). The PCR amplifications were performed in a DNA extraction methods total reaction volume of 50 μl made by mixing 50 ng

genomic DNA, 10 mM dNTPs, 100 mM MgCl2, and 5,000 The total fungal DNA was extracted using the phenol– units/ml of Taq DNA polymerase (Bioron, Ludwigshafen, chloroform method, as previously described by Siddiquee Germany) placed in a reaction buffer concentration of 10× 800 Ann Microbiol (2012) 62:797–803

PCR buffer (200 mM Tris HCl, pH 8.4, and 500 mM KCl) and 1 μM primers of ITS1 and ITS4. The reactions were subjected to thermal cycle conditions performed in a Peltier Thermal Cycler-200 as follows: an initial denaturation of 5 min at 95°C followed by 36 cycles of 1 min at 94°C, an annealing temperature at 55°C for 1 min, extension at 72°C for 3 min, and a final extension at 72°C for 10 min.

Gel electrophoresis

The PCR products were run on a 1.5% agarose gel with a 100-bp ladder (Promega) as the size standard. The electrophoresis was performed in a 1× TBE running buffer (0.045 M Tris-borate and 1 mM EDTA, pH 8.2) at 70 V for 1–2 h. The gels were then stained with ethidium bromide Fig. 1 Sample of Termitomyces heimii (0.5 μl/ml) for 15–30 min and then visualized under UV light and photographed using an Alphamager 2200 ver. 5.5 the fungus on unique structures within their nests that are gel documentation systems (Alphamager, New Tippasandra, called fungus combs. These fungus combs are continuously Bangalore). provided with externally derived plant material (e.g., wood, dry grass, leaf litter), while the older parts, consisting of DNA sequencing and alignment partially degraded plant material and fungal mycelium and nodules (asexual fruiting bodies covered with conidia), are The PCR products (50 μl) were purified using a commer- consumed (Batra and Batra 1979; Rouland Lefevre 2000; cial kit (QIAquick PCR Purification kit; QIAGEN, Hilden, Aanen et al. 2002 ). All species in the genus Termitomyces Germany). The purified PCR products were sequenced are absolutely dependent on their termites since they have using Big Dye Teminator v3.1 kit on an ABI3130xl never been found free-living and are rapidly overgrown by sequencer (ABI, Foster City, CA). All of the sequence data other fungi when removed from their termite nest (Darlington were subjected to multiple alignments using the Bio-Edit 1994). In turn, the termites are completely dependent on Sequence Alignment Editor software. Multiple sequence Termitomyces fungi, since they have never been found alignments were performed in NEXUS format using the without Termitomyces, and experiments have shown that default option in CLUSTAL-X software ver. 1.83 (Thompson they are unable to survive without the fungus (Grassé 1959; et al. 1997) based on the algorithm of Waterman (1986). The Rouland Lefevre 2000). alignment was then optimized manually. A single gap was Termitomyces heimii is completely reliant on the termites; treated as a fifth nucleotide (A, C, G, T, and gaps). specifically, T. heimii cannot exist without the existence of Nucleotide sequences were then subjected to GenBank the Odontotermes sp. termites (Grassé 1959). In studies searching for comparison.

Phylogenetic tree analysis

The interleaved NEXUS file was formatted using PAUP* (ver. 4.0b10; Swofford 2002) software. The phylogenetic tree was generated using parsimony in PAUP. Bootstrap tests with 1,000 replications were conducted to examine the reliability of the interior branches and the validity of the trees obtained.

Results and discussion

Mushrooms (basidiomes) of Termitomyces species are commonly found on termite mounds and collected by humans as food (Darlington 1994). The termites cultivate Fig. 2 Spore print of T. heimii showed brownish yellow spores Ann Microbiol (2012) 62:797–803 801

Fig. 3 T. heimii taken from Pegler and Vanhaeke (1994) (Fig. 2). The stipes are solid and white in color and have a cylindrical shape with a length measuring from 5 to 9 cm while the pseudorhiza of T. heimii (Fig. 3) which is attached to the Odontotermes sp. termite’s nest. The colony growth rate of T. heimii was 6.29 mm per day on PDA plates cultured at room temperature. The colony was initially watery-white (Fig. 4a), then turned a bright green color in the intact Petri dish after 7 days of culture (Fig. 4b). By the 7th to 8th day, 50–60% of the conducted on Termitomyces spp., it has been found the colony had a dark-green appearance, and the remaining 40– African and Southeast Asian region form a monophyletic 50% colonies were dull green or dirty green with compact group, while the symbiosis association of the fungi with the conidiophores throughout the whole Petri dish. The reverse termites has a single origin and no species of the fungi has side of all colonies of all isolates cultured on PDA agar had reverted to a free living state (Frøslev et al. 2003). a black coloration (Fig. 4c). Some studies have reported that the fungus combs of the Classical plays a key role in the given putative Termitomyces species associated with the Odontotermes sp. species names, but steps leading to the accurate identifica- termites (which include the T. heimii combs)trulyservedasa tion of species are very complicated, laborious, and direct food source (Rouland Lefevre 2000; Aanen et al. 2002) subjective and lead to incorrect identification. Davet and while the fungus combs of other Termitomyces sp. associated Rouxel (1997) mentioned that the traditional methods with the Macrotermes sp. termites only helped in the overestimate species, while those in the mycelial state or degradation of lignin due to more efficient utilization of those that have slow growth in culture are largely over- cellulose (Hyodo et al. 2003). It has also been reported that looked. These culturable isolates are those that can utilize the carbohydrate:lignin ratio of the Odontotermes sp. termites’ the energy source under the physical and chemical combs decreases with comb age, unlike Macrotermes sp. limitations of the growth medium. In our opinion, the combs, which increase with age. This difference suggests and taxonomic classification of Termitomyces fungi still has its supports the idea that T. heimii fungus combs are a direct food limitations. There are many species that appear to be similar source for the Odontotermes sp. colonies (Hyodo et al. 2003). under cultural conditions and exhibit a similar morphology Termitomyces heimii is characterized by a persistent but which in fact are completely different species. Process- annulus and a well-developed, umbonate-shaped perforato- ing of these cultures can be time-consuming and laborious rium which functions in soil penetration (Fig. 1). It when a large number of isolates have to be handled. During possesses a smooth, silky white, large pileus (measuring these processes, the risk of culture contamination is always about 8–12.5 cm in diameter) with a gray or brown center high, especially for the fast-growing fungi that overgrow

Fig. 4 a, b Top view of T. heimii cultured on potato dex- trose agar (PDA) after 4 days (a) and 7 days of culture (b). c Bottom view after 7 days of culture (mycelia colonies changed from white to black) 802 Ann Microbiol (2012) 62:797–803

Fig. 5 Banding patterns pro- these will continuously be associated with the wrong taxa. duced of T. heimii isolates using 1 2 3 4 5 M the primer ITS1 and ITS4 In our opinion, an essential link between data and taxa can provide a means to verify the taxonomic characters of the strains sequenced and some of the morphological character- 500bp istics. Otherwise, a species level identification study cannot be corrected and will remain uncorrected, and the user has to rely on the person making the misidentification. The ITS1 and -2 regions of the rDNA of the isolates (TH2, TH3, TH4 and TH5) were successfully amplified by PCR with expected sizes of about 530 bp in length (Fig. 5). After sequencing, the newly identified sequences were submitted to and deposited in a GenBank under accession number EU443836 for the ITS 1 and 2 regions of the rDNA. Following homology searching against the Gen- the entire medium. As such, it is recommended that a multi- Bank or the proprietary fungal DNA databases, the new disciplinary approach be used for the accurate identification sequences of isolates were found to share 100% similarity of Termitomyces species. in the ITS regions with those of T. heimii. A phylogenetic Molecular data have revolutionized systematic and relationship was established through the alignment and phylogenetic research and are now routinely used system- cladistic analysis of homologous nucleotide sequences atically in laboratories. Most journals publishing molecular among these fungal species (Fig. 6); isolates closest to the phylogenies require that the sequences be deposited in genus Termitomyces are shown. According to this phyloge- accessible repositories, such as a GenBank. However, as netic analysis, isolates could be classified to the genus more molecular data become available, there is a growing Termitomyces as a species variant of T. heimii. concern about the taxonomic origin of these data (Nilsson The spacer regions of the ITS 1 and 2 of the rDNA are et al. 2006; Peterson et al. 2007). Although some taxa widely and routinely used in the analysis of species appear to be well-outlined and easy to recognize, others can relationships using a phylogenetic reconstruction method only be identified by a handful of specialists. Nowadays, in various organisms. It has been successfully applied in the there are a large number of sequences deposited in analysis of the phylogenetic relationship between T. heimii GenBank that are incorrectly labeled and, unless remedied, species and the results are in agreement with those from

Fig. 6 Phylogenetic trees (Parsimony) showing the rela- tionship of isolates with other related fungal species retrieved from GenBank based on their sequence of the internal tran- scriber spacer regions ITS1 and -2 of the rDNA Ann Microbiol (2012) 62:797–803 803 phylogenetic analysis based on gene sequencing of the Heim R (1977) Termites et champignons. Les champignons termitophiles ’ ’ ITS1 and -2 regions of the rDNA. Our results show that the d Afrique Noire et d Asie méridionale. Éditions Boubée, Paris Hyodo F, Tayasu I, Inoue T, Azuma JI, Kudo T, Abe T (2003) ITS technique is an effective tool in the reconstruction of Differential role of symbiotic fungi in lignin degradation and evolutionary relationship among these species and allow food provision for fungus-growing termites (Macrotermitinae; organisms to be identified at the species level. To the best of Isoptera). Functional Ecol 17:186–193 our knowledge,, this is the first report of T. heimii Lee KE, Wood TG (1971) Termites and Soils, 1st edn. Academic, London identification using an approach which combines the Leuthold RH, Badertscher S, Imboden H (1989) The inoculation of DNA sequence of the ITS1 and -2 regions of the rDNA newly formed fungus comb with Termitomyces in Macrotermes and morphological characteristics. colonies (Isoptera, Macrotermitinae). Insectes Sociaux 36:328– Molecular data in phylogenetic analyses will definitely 338 Maxwell A, Jackson SL, Dell B, Hardy GESJ (2005) PCR- continue to proliferate in the future and lead to large identification of Mycosphaerella species associated with leaf amounts of information easily retrievable by researchers. disease of Eucalyptus. 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