BIODIVERSITAS ISSN: 1412-033X Volume 21, Number 1, January 2020 E-ISSN: 2085-4722 Pages: 231-238 DOI: 10.13057/biodiv/d210130 First ectomycorrhizal syntheses between Astraeus sirindhorniae and Dipterocarpus alatus (Dipterocarpaceae), pure culture characteristics, and molecular detection NUTTIKA SUWANNASAI1, PREEYAPORN DOKMAI1, AKIYOSHI YAMADA2, ROY WATLING3, CHERDCHAI PHOSRI4, 1Department of Microbiology, Faculty of Science, Srinakharinwirot University. Bangkok 10110, Thailand 2Department of Bioscience & Biotechnology, Faculty of Agriculture, Shinshu University. 8304 Minami-minowa, Nagano 399-4598, Japan 3Caledonian Mycological Enterprises. Crelah, 26 Blinkbonny Avenue, Edinburgh, EH4 3HU, Scotland 4Faculty of Science, Nakhon Phanom University, Nakhon Phanom 48000, Thailand. email: [email protected] Manuscript received: 6 October 2019. Revision accepted: 24 December 2019. Abstract. Suwannasai N, Dokmai P, Yamada A, Watling R, Phosri C. 2020. First ectomycorrhizal syntheses between Astraeus sirindhorniae and Dipterocarpus alatus (Dipterocarpaceae), pure culture characteristics, and molecular detection. Biodiversitas 21: 231-238. This study provides the first mycorrhization of Astraeus sirindhorniae and its cultural characteristics on nutrient media. An attempt has been made to introduce spore suspension and mycelial inocula of A. sirindhorniae onto seedlings of Dipterocarpus alatus. After 6 months seedlings were harvested, measured for growth and morphological descriptions of the ectomycorrhizas formed with D. alatus seedlings were made. The fungus has increased the growth of D. alatus seedlings. Further, it can be confirmed that the primers designed (GAPK126F/GAPK379R) have been successful when applied for the detection of ectomycorrhizal formation of A. sirindhorniae in vivo. Keywords: Boletales, Dipterocarpaceae, DNA barcode, Ectomycorrhiza, edible mushroom INTRODUCTION 2014). As far as field observations are concerned, this Astraeus appears rather restricted in its distribution and Astraeus is a star-shaped fungus belonging to the family although consumed the taste and odor of A. sirindhorniae Diplocystidiaceae (Boletales, Basidiomycota) (Binder and are both similar to A. odoratus (Phosri et al. 2014) but it Hibbett 2006). A study developed by Phosri et al. (2013) has not yet become as popular as A. odoratus in terms of a clearly demonstrates that A. hygrometricus as previously natural food resource. circumscribed, to be not a single species but made up of a Dipterocarps are one of the most important families of number of cryptic species. It is widely distributed in warm timber trees in Thailand and Southeast Asia. They are temperate to subtropical and tropical regions, particularly widely distributed in lower Myanmar, Laos, Thailand, in sandy soils and forms ectomycorrhizal association with Cambodia, South Vietnam, the Andaman Islands, various tree species (Petcharat 2004, Phosri et al. 2004, Indonesia, and Malaysia. They produce high-quality wood 2007, 2013, Yomyart 2008, Kaewgrajang et al. 2013). It is and provide a source of non-timber forest products such as one of the uncultured fungi producing commercially resins and oils (Boontawee 2001). Because of their valuable mushrooms which are considered delicacies by important role in ecosystems, a research priority should be local people in the north and north-eastern parts of given to the Dipterocarpaceae although many reforestation Thailand and neighborhood to Laos. (Phosri et al. 2004). programs for the provision of high-quality timber have Three species of Astraeus are present in Thailand; A. focused on fast-growing exotic trees such as eucalypts asiaticus, A. odoratus and the recently described, A. (Brearley 2011). Over the past 50 years, former King sirindhorniae (Phosri et al. 2004, 2007, 2014). Bhumibol has raised the issue of the loss of dipterocarps Fresh basidiomes of A. asiaticus and A. odoratus are esp. Dipterocarpus alatus from Thailand and through a regularly found on sale in domestic markets during the Royal initiative, a re-afforestation program draws attention beginning of the rainy season in Thailand from May-June. amongst several sectors for the conservation of endangered Presently the wholesale price of fresh basidiomes ranges dipterocarp populations. Since then reforestation programs from 300-500 Baht ($8-14 US) per kilogram (Phosri in Thailand with dipterocarps species have been attempted pers.com.). Excess collections are preserved in saline and and resulted in a total of approximately 2,080 ha exported to neighboring countries. So far, A. sirindhorniae (Boontawee 2001). Members of the Dipterocarpaceae form has been recorded from only two localities in Thailand, viz. symbiotic root-inhabiting fungal associations with Chiang Mai and Chiyaphum provinces. The two locations hundreds of ectomycorrhizal (ECM) fungal species in which A. sirindhorniae was found are considered as (Watling and Lee 1995, 1998, 2007, Lee et al. 2002, 2003 highland dipterocarp forests, about 640 m. (Phosri et al. Brearley et al. 2003, 2006, 2007, 2011, 2012). Such reports 232 BIODIVERSITAS 21 (1): 231-238, January 2020 fail to indicate that members of the genus Astraeus are (MS) (Marx 1969, Straatsma et al. 1986). The initial pH frequent, hypogeous associates. They form putative was adjusted to 6 for all types of media with 1N HCL or ectomycorrhizal associations with several host tree species 1N NaOH. Mycelium plugs with 6 mm diameter obtained including Shorea siamensis, S. roxburghii, S. farinosa, D. from 4 weeks old culture were placed in the middle of the alatus, D. intricatus, D. Obtusifolius and Hopea odorata agar plate. The cultures were then incubated at 30°C in (Petcharat 2004, Phosri et al. 2004, 2007, 2013, Yomyart darkness for 4 weeks. The optimum pH studies were 2008, Kaewgrajang et al. 2013, 2019). This strongly performed in 25 ml MNC broth by inoculation with a suggests that members of this genus are very important mycelium plug (6 mm diameter) of A. sirindhorniae. The ectomycorrhizal components. pH was adjusted from 4-8 (Stoll and Blanchard 1990, The present study was undertaken as A. sirindhorniae Sanmee et al. 2010). Cultures were then incubated at 30°C had not been maintained in culture nor its ecological for 4 weeks. The optimum temperature was determined parameters demonstrated. An attempt was made therefore from cultures on MNC agar medium adjusted to pH 6. to study the effects of the cultural conditions on the Fungal cultures were incubated for 4 weeks at different biomass production of A. sirindhorniae. Further to detail temperatures viz. 25, 30, 37°C and at room temperature in descriptions for the fungal characteristics and its Thailand (30±2°C). Mycelial growth in all experiments was mycorrhizal morphologies. An additional aim was to evaluated by measuring weight of mycelia (biomass) after produce primers designed for A. sirindhorniae as a means drying the mycelium at 60°C for 48 hours. The of molecular detection in nature after out-planting into the experiments were set in a completely randomized design field. with five replicate plates/flasks per treatment. Mycorrhizal synthesis MATERIALS AND METHODS Dipterocarpus alatus seeds collected from Yasothon province were rinsed several times with tap water, and Fungal isolation and cultures soaked in distilled water overnight. For surface The basidiomes of A. sirindhorniae collected from a sterilization, the seeds were treated with 5% hydrogen o o single population (N 16 24’ 13.194” and E 101 34' peroxide (H2O2) solution for 15 min and rinsed three times 47.089", elev. 640 m asl.) in dry deciduous forests, with sterile water. All seeds were then placed in plastic associated with Dipterocarpus tuberculatus Roxb., Shorea baskets (35 x 45 cm) covered with moist paper until obtusa Wall. and Shorea siamensis Miq. Phu Khieo germination. The germinated seeds with 3-4 cm root length Wildlife Sanctuary, Chiyaphum province, were isolated for were individually transplanted into polyethylene bags (5 pure culture on modified Norkrans’s “C” agar medium cm x 11 cm) containing 250 cm3 autoclaved sandy loam (MNC) (Yamada and Katsuya 1995). The cultures were soil (2.0 g kg-1 of N, 1.7 g kg-1 of P, 42 mg kg-1 of K, 387 incubated at 30°C for 4-5 days. Fungal mycelium was then mg kg-1 of Ca, 37 mg kg-1 of Mg, pH 7.3, Department of transferred to new medium and observed for clamp- Soil Science, Faculty of Agriculture, Kasetsart University) connections under the microscope (Olympus BX40). and peat moss in a 1:1 (v/v) ratio. They were routinely Mycelial cultures were maintained on MNC slants at 4°C watered with distilled water for a month. Mycelial inocula and were routinely sub-cultured every 2 months. Some and spore suspensions of A.sirindhorniae were transferred mature basidiomes of A. sirindhorniae were kept at 4°C for to 1 month old D. alatus seedlings. The mycelium the spore-inoculum experiments. inoculum was prepared in a 250 cm3 glass bottle, containing 200 cm3 vermiculite and peat moss in a 1:1 (v/v) Molecular identification of fungi ratio. Thirty milliliters of MNC broth with pH 6 was then Pure cultures of fungal isolates were confirmed by the added to a bottle prior to autoclaving at 121°C for 15 min. sequencing of internal transcribed spacer (ITS) region. Three mycelial plugs of A. sirindhorniae (isolate Genomic DNA was extracted from fresh fungal mycelium GACM13-6) were introduced into the mixture and using Plant Genomic DNA Extraction kit (Farvogen). The incubated at 30°C for 4 weeks. This mixture was used as ITS region was amplified using primers ITS1F and ITS4B the mycelial inoculum. A spore suspension was prepared (Gardes and Bruns 1993). The PCR reaction and cycles from dried mature basidiomes with a drop of Tween 80 in were followed by Phosri et al. (2014). Amplicons obtained sterile distilled water to ensure homogeneous dispersion. were purified using PCR/Gel Purification kit (Farvogen) Spore density was measured by using hematocytometry and sequenced at the 1st BASE (Malaysia). The DNA and adjusted to 108 spores/ml. Fifty cubic centimeter of sequences were manually checked and compared to mycelial inocula or 5 mL of spore-suspension was added to GenBank database using BLAST program.