Hed sa med (Boletus griseipurpureus) is a popular edible ectomycorrhizal mushroom associated with stands of Melaleuca leucadendron and Acacia mangium in Thailand. Genetic variation of B. griseipurpureus basidiomes, obtained from the wild, was determined by internal transcribed spacer (ITS) sequence analysis. Sequences of B. griseipurpureus collections showed high similarity and constituted a monophyletic clade. The basidiomes were rich in protein and very low in fat confirming their value for human consumption

Aung-aud-chariya, A.

Bangrak, P.

Dell, B.

Kamlangdee, N.

Lumyong, S.

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Journal of Agricultural Technology 2012, Vol. 8(6): 1991-1998 1991  Preliminary molecular identification of Boletus griseipurpureus Corner from Thailand and its nutritional value    Amornrat Aung-aud-chariya1,2,3*, Phuwadol Bangrak1, Bernard Dell4, Saisamorn Lumyong5 and Niyom Kamlangdee1,2  1Ecology and Biodiversity Program, School of Science, Walailak University, Nakhon Si Thammarat, 80161, Thailand, 2Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok 10900, Thailand, 3Rajamangala University of Technology Srivijaya, Trang Campus, 92150, Thailand, 4Sustainable Ecosystems Research Institute, Murdoch University, Perth 6150, Australia, 5Microbiology Division, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand  Amornrat Aung-aud-chariya, Phuwadol Bangrak, Bernard Dell, Saisamorn Lumyong and Niyom Kamlangdee (2012) Preliminary molecular identification of Boletus griseipurpureus Corner from Thailand and its nutritional value. Journal of Agricultural Technology 8(6):1991-1998.  Hed sa med (Boletus griseipurpureus) is a popular edible ectomycorrhizal mushroom associated with stands of Melaleuca leucadendron and Acacia mangium in Thailand. Genetic variation of B. griseipurpureus basidiomes, obtained from the wild, was determined by internal transcribed spacer (ITS) sequence analysis. Sequences of B. griseipurpureus collections showed high similarity and constituted a monophyletic clade. The basidiomes were rich in protein and very low in fat confirming their value for human consumption.  Key words: Boletus griseipurpureus, Internal transcriped spacer (ITS), Ectomycorrhizal mushroom, Proximate composition  Introduction  In parts of Thailand (Dell et al., 2005) and nearby countries (e.g. Malaysia, Lee et al., 2009) wild edible mushrooms are important sources of food for local people (Rachabunditayasathan, 1996; Chansrikul, 1998; Dell et al., 2005; Sanmee et al., 2003; Phosri et al., 2004; Seehanan and Petcharat, 2008). Wild and cultivated mushrooms are considered to be good for human consumption because they are generally low in energy and fat but high in protein, fibre and carbohydrate (Cheung, 2010). They may also have strong antioxidant activity (Wong and Chye, 2009; Kumari et al., 2011). Many of these are ectomycorrhizal fungi, often associated with specific host trees.                                                           *Corresponding  author: Niyom Kamlangdee; Amornrat Aung-aud-chariya; e-mail: kniyom@gmail.com; tamornrat1975@gmail.com Journal of Agricultural Technology 2012 Vol. 8(6): 1991-1998 Available online http://www.ijat-aatsea.com ISSN 1686-9141  1992 Examples include Phlebopus portentosus (Sanmee et al., 2010), Amanita hemibapha (Sanmee et al., 2008), Astreus hygrometricus (Sanmee et al., 2003; Phosri et al., 2004) and Boletus griseipurpureus (Seehanan and Petcharat, 2008). Many wild edible ectomycorrhizal fungi fetch a higher price than cultivated mushrooms (Dell et al., 2005) because wild mushrooms fruit in a specific season and demand exceeds supply. Like Astreus in the north of Thailand, B. griseipurpureus can command a high price (US$3-4/kg). The latter species is a popular edible mushroom in the south of Thailand (Aungaudchariya et al., 2010). It has a bitter taste but is quite tasty in local cuisine. The proximate composition of some Thai edible ectomycorrhizal fungi has been determined (Sanmee et al., 2003) but B. griseipurpureus has yet to be quantified.  Boletus griseipurpureus is most frequently associated with Melaleuca leucadendron (Sa Med Khao or Cajeput tree) (Chandrasrikul et al., 2008; Aungaudchariya et al., 2010) and Acacia mangium. This mushroom has been reported as being ectomycorrhizal with M. leucadendron, A. auriculiformis, Gustavia gracellima, Eucalyptus camaldulensis and Casuarina equisetifolia (Seehanan and Petcharat, 2008) but this needs confirmation. Presently, it is unclear whether the B. griseipurpureus being collected for eating in Thailand is a single species or a species complex. In this first study, the internal transcribed spacer, or ITS region, of the nuclear ribosomal repeat DNA is used to probe intraspecific variation in B. griseipupureus in Thailand. Molecular genetic markers are extensively used for rapid identification of fungi (Moreau et al., 2006; Froslev et al., 2007). The ITS region has been widely used to identify inter- and intra- specific relationships within and among various fungal species. For example, ITS1 and ITS4 primers were used to differentiate between some edible Chinese and European boletes with similar morphology (Moor et al., 2002). In another study, ITS4 and ITS5 were used to discriminate Russula in the northeast of Thailand (Manassila et al., 2005).   Materials and methods  Sample collection  Basidiomes were collected from under M. leucadendron in Trang province, and from under A. mangium in Nakhon Si Thammarat and Songkhla provinces. Additional basidiomes were opportunistically purchased from city and roadside markets. In the laboratory, basidiomes were assigned to the B. griseipurpureus Corner taxon on the basis of macroscopic and microscopic features given in the type description (Corner, 1972).  Journal of Agricultural Technology 2012, Vol. 8(6): 1991-1998 1993  Molecular identification  Genomic DNA was extracted from five basidiomes per province and purified using the modified method of Lian et al. (2008). The ITS amplification was performed in a thermocycler with a 50 µl reaction mixture containing 1X PCR buffer, 25 mM MgCl2, 20 µM for each primer ITS5 (5´-GGAAGTAAAGTCGTAACAAGG-3´ and ITS4 (5´-TCCTCCGCTTA TTGATATGC-3´), 200-500 µg of genomic DNA and 5 units of Taq DNA polymerase (Invitrogen). The reaction mixture was initial denatured at 94 °C for 5 minutes and then subjected to 30 cycles of 1 minute at 94°C, 1 minute at 56°C, 1 minute at 72°C and to the final extension step of 10 minutes at 72°C. The PCR products were purified by purification kit (QIAGEN) according to the manufacturer’s instructions and then purified PCR products were ligated with pGEM® T-Easy vectors (Promega Corporation, USA). The ligation mixture was transformed into competent cells (Escherichia coli TOP10F) and the recombinant plasmids were extracted from E. coli cells by the alkaline lysis method. Then several clones were screened to find plasmids containing a specific DNA fragment. The nucleotide sequence was determined with an automatic DNA sequencer ABI 377 (Biodesign company, Thailand). After that BioEdit and Clustal W program were used to edit several sequences. Phylogenetic analysis of the aligned sequences was carried out by maximum likelihood analysis using MEGA (version 5.05). Bootstrapping was performed with maximum likelihood (ML) analysis using 1000 replicates.  Proximal composition  Basidiomes were manually cleaned to free them from extraneous material and dried at 60 oC. The proximate composition as protein, fat, ash, and fibre were determined on 100 g subsamples as described by Sanmee et al. (2003) and AOAC (1990) by the Science and Technology Service Center, Faculty of Science, Chiang Mai University.  Results   Basidiomes sourced from the two sides of Southern Thailand, namely Trang (West), Songkhla and Nakhon Si Thammarat provinces (East) were indistinguishable in colour (purple to gray-velvet) and size (pileus 1-7 cm diameter, stipe 3-8.5 cm). Overall, basidiome dimensions, and spore size and shape were similar to (data not presented) the type material from Malaysia described by Corner (1972) and Thai collections described by Seehanan and Petcharat (2008).   1994 The PCR products from the amplified rDNA were 700 - 800 bp in size on 1.5% agarose gels. The rDNA ITS regions were cloned and the nucleotide sequences were used to build a phylogenetic tree. The phylogenetic analysis was constructed using 14 sequences obtained from GenBank (http://www.ncbi. nlm.nih.gov) and 5 sequences obtained from B. griseipurpureus and submitted to GenBank as accession no. JQ726594, JQ726595, JQ726596, JQ726597 and JQ726598. Sequences of B. griseipurpureus collections showed high similarity when compared with other clades of the bolete lineage and constituted a monophyletic clade. In this first analysis, the B. griseipurpureus clade was more closely related to Tylopilus than to Boletus.  The proximal analysis of B. griseipurpureus basidiomes (Table 1) showed that basidiomes were rich in protein and very low in fat. The moisture content of the basidiomes was 10.8%.  Table 1. Proximate composition of Boletus griseipurpureus compared to three Thai edible ectomycorrhizal fungi  Composition (% dry wt) Boletus griseipurpureus Phaeogyroporus portentosus* Astreus hygrometricus* Russula nigricans* Ash  8.6 17.8 27.6 6.7 Crude fibre 15.0 8.8 10.8 9.6 Crude protein 31.4 24.2 14.0 22.6 Fat 0.9 2.8 2.7 4.8 Carbohydrate 33.3 46.4 54.4 56.3 *Sanmee et al. (2003)  Journal of Agricultural Technology 2012, Vol. 8(6): 1991-1998 1995   Fig. 1. The best-scoring maximum likelihood (ML) tree derived from the data. Numerals beside internal branches indicate bootstrap probabilities ≥50% based on 1000 replicates. Scale indicates expected number of substitutions per site. GenBank accession numbers are shown. JQ726594 = B. griseipurpureus from Nakhon Si Thammarat JQ726595 = B. griseipurpureus from Nakhon Si Thammarat 1 JQ726596 = B. griseipurpureus from Trang 1 JQ726597 = B. griseipurpureus from Trang 2 JQ726598 = B. griseipurpureus from Songkhla  Discussion  The ITS sequences which contained ITS1, ITS2 and the 5.8S rDNA gene, suggest a monophylogenetic clade among basidiomes of B. griseipurpureus which were collected from three locations in the south of Thailand. Whether this degree of similarity applies to populations of B. griseipurpureus in the northeast of Thailand (Seehanan and Petcharat, 2008; Pukahuta et al., 2009) remains to be determined. Furthermore, the assembled phylogenetic tree, though based on a small number of ITS sequences from GenBank, suggests that B. griseipurpureus has affinity with Tylopilus. It would therefore be of interest to examine a larger collection of boletes in the region to clarify relationships. As pointed out by Watling (2001), the genus Boletus circumscribed by Corner (1972) was very large and, at that time, included Tylopilus. Corner (1972) remarked that B. griseipurpureus was “deceptively like B. longipes”, a species that is known in Malaysia and Singapore, but so far has not been collected in Thailand. Thus, it would be of interest in the future to use  1996 molecular and other tools to better define species boundaries for B. griseipurpureus. The primary purpose of the current work was to determine whether the B. griseipurpureus being consumed by humans in southern Thailand is a single taxon, hence the ITS region was used in the analysis. To understand the phylogeny of such a large and diverse group as the Boletaceae requires large numbers of sequences for diverse gene regions in geographically diverse regions of the world (see Binder and Hibbett, 2006). Examples of such studies are those of Dentinger et al. (2010) and Drehmel et al. (2008) where ITS, LSU, ATP6, RPB1 and mtSSU sequences have been used to produce phylograms. At a species level, a combination of methods can be useful in determining new genera and species, as in the case with Spongiforma thailandica, a gasteroid bolete, where ITS and nuc-lsu were used (Desjardin et al., 2009).  As B. griseipurpureus is sought after for food in southern Thailand, it was of interest to determine some aspects of its food value. Table 1 compares the proximate composition of B. griseipurpureus with three other Thai edible fungi, including P. portentosus, another sought after ectomycorrhizal bolete. The crude protein of edible mushrooms typically range from 15 to 35% (Cheung et al., 2010) and values for wild edible northern Thai mushrooms were 14-24% (Sanmee et al., 2003). The concentration of protein in B. griseipurpureus was higher than in many ectomycorrhizal fungi (e.g. Agrahar-Murugkar and Subbulakshmi, 2005) but similar to that in Termitomyces globules (34%) (Gbolagade et al., 2006). In general, edible mushrooms are low in fat. The fat concentration in B. griseipurpureus was lower than in P. portentosus (Sanmee et al., 2003) but similar to C. bibarius (0.7%) (Gbolagade et al., 2006). The ash content of B. griseipurpureus lay at the lower end of the range (6-11%) of edible mushrooms (Cheung et al., 2010) and was similar to R. nigricans (Table 1). Most edible mushrooms have high carbohydrate contents but B. griseipurpureus (33%) was lower than other wild edible Thai mushrooms which ranged from 42 to 65% (Sanmee et al., 2003). Thus, the food value of B. griseipurpureus was high in terms of the relatively high protein and low fat contents.   Acknowledgement  This research is partially supported by the Center of Excellence on Agricultural Biotechnology, Science and Technology Postgraduate Education and Research Development office, Commission on Higher Education, Ministry of Education (AG-BIO/PERDO-CHE), grant from Utilization of Natural Products Research Unit at Walailak Universtiy, Graduated School grant from Walailak Universtiy and grant from Rajamangkala University Technology of Srivijaya Trang campus as well.  Journal of Agricultural Technology 2012, Vol. 8(6): 1991-1998 1997  References  AOAC (1990). Official Methods of the Association of Analytical Chemists. 15th Ed., AOAC, Washington. D.C., USA.  Agrahar-Murugkar, D. and G. Subbulakshmi (2005). Nutritional value of edible wild mushrooms collected from the Khasi hills of Meghalaya. Food Chem. 89:599-603. Aungaudchariya, A., P. Bangrak, B. Dell, and N. 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Preliminary molecular identification of Boletus griseipurpureus Corner from Thailand and its nutritional value

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Preliminary molecular identification of Boletus griseipurpureus Corner from Thailand and its nutritional value

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