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Mycelial Cultivation of Phlebopus Spongiosus, an Edible Ectomycorrhizal Mushroom in Southern Vietnam

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Mycelial Cultivation of Phlebopus Spongiosus, an Edible Ectomycorrhizal Mushroom in Southern Vietnam 14 Le Thi Thuy Nhi et al. Ho Chi Minh City Open University Journal of Science, 7(1), 14-21 MYCELIAL CULTIVATION OF PHLEBOPUS SPONGIOSUS, AN EDIBLE ECTOMYCORRHIZAL MUSHROOM IN SOUTHERN VIETNAM LE THI THUY NHI, TRAN CHI HIEU Biotechnology Center of Ho Chi Minh City, Vietnam - [email protected] PHAN MY HANH, BUI VAN TAN, PHAM NGUYEN DUC HOANG Biotechnology Center of Ho Chi Minh City, Vietnam - [email protected] HO BAO THUY QUYEN Ho Chi Minh City Open University, Vietnam - [email protected] (Received: July 06, 2017; Revised: July 23, 2017; Accepted: August 08, 2017) ABSTRACT Phlebopus spongiosus is an endemic edible ectomycorrhizal mushroom (EEMM) in Southern Vietnam. Strains of Phlebopus spongiosus – collected from Pomelo orchards, Ben Tre Province, Vietnam – were cultured on different media, temperatures, pH and carbon/nitrogen sources to define mycelial growth conditions for spawn mushroom production. Phlebopus spongiosus BC-F0075 grew the fastest on the modified MS medium. The optimum temperature for mycelial growth was 30°C and the optimum pH was 4 – 5. BC-F0075 grew well on the MS media containing saccharose, glucose, fructose and maltose, but did not with lactose. Additionally, NH4H2PO4 was reported to be the best nitrogen source to the growth of BC-F0075 mycelia, whereas urea ((NH2)2CO) was not utilized as a nitrogen source. Keywords: Phlebopus spongiosus; mycelial cultivation; edible ectomycorrhizal mushroom (EEMM); chlamydospore. 1. Introduction Davoli 2012). In recent years, interest was Phlebopus spongiosus Pham & Har. shown in many EEMMs for cultivation, such Takah is a new tropical bolete in family as Tuber magnatum, Tricholoma matsutake, Boletinellaceae that was recently discovered Boletus edulis (Giomaro et al. 2005) and by Pham et al. (2012a). This mushroom was Phlebopus portentosus (Thongklang et al. described to be an ectomycorrhizal species 2010, Sanmee et al. 2010). P. spongiosus has with pomelo roots (Pham et al. 2012b). Most a close relationship with P. portentosus in the fruit bodies have been found in pomelo genus Phlebopus. P. portentosus is also one of orchards, thereby P. spongious is also named the most popular edible ectomycorrhizal as “nấm bưởi” in Vietnamese. P. spongiosus mushrooms in northern Thailand and China, is an endemic edible ectomycorrhizal because it has a large fruiting body and prized mushroom (EEMM) in Southern Vietnam. flavor. P. portentosus has been successfully EEMMs are considered delicacies, beneficial cultivated without a host plant (Ji et al. 2011, nutraceutical and medicinal value, therefore, Kumla et al. 2014), and thus has the potential they are sold at a relatively high price to produce fruiting bodies of EEMM in the compared to other foods. These mushroom absence of a host plant. could potentially be commercially measured This study aims to define mycelial growth in US$ billions (Hall et al. 2003, Sitta and conditions and suitable solid media for spawn Le Thi Thuy Nhi et al. Ho Chi Minh City Open University Journal of Science, 7(1), 14-21 15 mushroom production of Phlebopus spongious. Effect of temperature 2. Materials and Methods Phlebopus spongiosus BC-F0075 was Isolation grown on MS agar medium with different Mushroom samples were collected in temperatures at 20, 25, 30 or 35°C in the dark. pomelo orchards, Ben Tre Province, Vietnam, MS medium and strain BC-F0075 was chosen based on Phlebopus spongiosus described by because the good mycelial growth was obtained Pham et al. (2012a). Two samples were in the previous experiments. The experiment obtained named BC-F0075 and BC-F0076. was done in triplicate. Colony diameters at 15 Mycelia were isolated from the fruiting bodies days after inoculation were measured. and cultured on potato dextrose agar (PDA) Effect of pH medium (Himedia, India). Phlebopus spongiosus was grown in Ohta DNA analysis and phylogeny medium as a broth at pH range of 2 - 9. Two DNA was extracted from colonies on mycelium plugs (5 mm diameter) were cut media or from fruiting bodies for PCR from the growing edge of a 15 days-old amplification (Izumitsu, 2012). The set primer culture in MS medium and inoculated into of LR0R and LR5 was used to amplify the Ohta broth, and incubated for 15 days under LSU sequence (Vilgalys and Hester 1990). laboratory conditions (30 ± 2°C) on a PCR products were purified and sequenced by reciprocal shaker at 150 rpm. There were Macrogen, Inc. (Korea). The sequences were triplicate flasks for each treatment. At the 15th analyzed using ATGC ver 7.0.1 (Genetyx, day, the cultures were filtered with Whatman Japan). Phylogenetic relationships based on no. 1 filter papers, oven dried (60°C), and the LSU sequence (Pham et al. 2012a) were weighed to determine the dry-biomass. analyzed using the Neighbor joining method Effect of different carbon and nitrogen in MEGA ver 6.0 with a Bootstrap analysis sources involving 1000 replication rounds (Tamura et In the testing of different carbon sources, al. 2013). MS agar media (without saccharose) was Effect of culture media supplemented separately with carbon sources Seven different solid media were used: comprising one of the followings: saccharose, Melin Norkans medium (MMN; Marx 1969), glucose, fructose, maltose or lactose. modified Murashige and Skoog medium (MS; In the testing of different nitrogen Straatsma et al. 1986), Fungus-host medium sources, MS agar media (without NH4NO3 (FH; Vaario 2000), Ohta medium (Ohta and KNO3) was supplemented separately with 1990), malt yeast extract agar (MYA 2%; nitrogen sources comprising one of the Merck, Germany), potato dextrose agar (PDA; followings: (NH4)2SO4, NH4OH, NH4Cl, Himedia, India) and modified potato dextrose NH4NO3, NH4H2PO4, (NH4)2HPO4, KNO3 or agar (PDA*; Ji et al. 2011). (NH2)2CO (urea). For standardized tests, 20 ml of media All cultures were incubated at 30°C in the were dispensed into 90-mm-diameter petri dark. The experiment was done in triplicate. dish. Plates were inoculated with a 5-mm- Colony diameters at 15 days after inoculation diameter plug of mycelium from the periphery were measured. of the growing colony on PDA after 25-days. 3. Results and discussion Cultures were incubated in darkness at 30 ± Isolation 0.5°C. The experiment was done in triplicate. Mushroom samples were collected in Colony diameters at 30 days after inoculation pomelo orchards (Fig 1a). Mycelia were were measured. cultured on potato dextrose agar (PDA) 16 Le Thi Thuy Nhi et al. Ho Chi Minh City Open University Journal of Science, 7(1), 14-21 medium and the colony covered the whole chlamydospore is an important type of asexual Petri dish after 45 days (Fig 2A). Mushroom spore, each spore is capable of germinating samples grew on MS medium with abundant and forming mycelia. Formation of clamp connections and chlamydospores. chlamydospores usually indicates good spawn Chlamydospores were light yellowish to production of Volvariella, therefore selection orange yellow in colour, thick-walled and had of strains is normally done based on the variable forms (Fig 2B, 2C). Chlamydospores chlamydospores’ production (Nannapaneni formed in intercalary or terminal mycelia. The and Subbiah 2016). Figure 1. Mushroom samples were collected in pomelo orchards A B C Figure 2. Isolate BC-F0075. (A) mycelium growth on PDA after 45 days, (B) clamp connections and chlamydospore, bar 10 µm, (C) chlamydospore, bar 10 µm DNA analysis and phylogeny group is a clade which is separated with other Phylogram base on the LSU sequence Phlebopus species in the genus Phlebopus (Fig showed that BC-F0075 and BC-F0076 clustered 3). P. spongiosus is a new tropical bolete in with Phlebopus spongiosus (AB673396) into a family Boletinellaceae that was recently group with 100% of sequence similarity. This described by Pham et al. (2012a). Le Thi Thuy Nhi et al. Ho Chi Minh City Open University Journal of Science, 7(1), 14-21 17 Figure 3. Phylogram of Phlebopus spongiosus in suborder Sclerodermatineae based on the LSU sequence. The outgroup was Suillus cavipes Effect of culture media discolor. Sanmee et al. (2010) reported that Phlebopus spongiosus BC-F0075 and Phlebopus portentosus WPPH2 turned brown BC-F0076 grew in all tested culture media or dark brown in the PDA and MNM media, (Fig 4). Mycelial growth of BC-F0075 was but did not in the MS medium. Small faster than BC-F0076. Additionally, BC- primordium-like structures with brown F0075 grew the fastest on the modified MS exudates were formed on modified MS medium, with the colony reaching 85.343 mm medium (Fig 5). Phlebopus portentosus in diameter after 30 days (Table 1). The CMUHH 121-005 grew the fastest and cultured plates turned brown in the PDA, formed primordium-like structures on MS PDA*, MYA and Ohta media, in contrast to medium (Thongklang et al. 2010) that look the MNM, MS and FH media, which did not similar to P. spongiosus BC-F0075. 18 Le Thi Thuy Nhi et al. Ho Chi Minh City Open University Journal of Science, 7(1), 14-21 Table 1 Mycelial growth of isolates on various solid media when incubated for 30 days Medium PDA PDA* MYA MNM Ohta MS FH Mycelial growth of BC- 68.540c 74.663b 51.646i 56.330g 49.333j 85.343a 63.456e F0075 (mm) Mycelial growth of BC- 51.420i 55.566h 45.093l 47.183k 51.143i 66.636d 60.450f F0076 (mm) Values with the same letter are not significantly different (p=0.05). Figure 4. Mycelial growth of isolates on various solid media when incubated for 30 days. Row 1 is colony of BC-F0075, row 2 is colony of BC-F0076 Figure 5. Primordia-like structure on MS medium Effect of temperature reported that the optimal temperature for Mycelia of BC-F0075 grew within a mycelial growth of P. portentosus WPPH2 temperature range of 20 - 35°C (Fig 6), but was 30°C.
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