Vietnamese Ganoderma: growth, peculiarities, and low-molecular composition compared to European and Siberian strains

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Turkish Journal of Botany Turk J Bot (2016) 40: 269-286 © TÜBİTAK doi:10.3906/bot-1410-15 http://journals.tubitak.gov.tr/botany/ Research Article Vietnamese Ganoderma: growth, peculiarities, and low-molecular composition compared to European and Siberian strains 1,* 2 2 3 1 Olga TSIVILEVA , Thao NGUYEN , Long VU , Nikolay YURASOV , Marina CHERNYSHOVA , 4 5 5 1 Alexander PETROV , Viktor GALUSHKA , Alexei MARKIN , Oleg KOFTIN 1 Institute of Biochemistry and Physiology of Plants and Microorganisms of the Russian Academy of Sciences (IBPPM RAS), Saratov, Russia 2 Southern Institute of Ecology, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam 3 Institute of Chemistry, N.G. Chernyshevskii Saratov State University, Saratov, Russia 4 Department of Botany and Genetics, Faculty of Biology and Soil Biology, Irkutsk State University, Irkutsk, Russia 5 Nanotechnology Department, Educational Research Institute of Nanostructures and Biosystems, N.G. Chernyshevskii Saratov State University, Saratov, Russia Received: 13.10.2014 Accepted/Published Online: 16.10.2015 Final Version: 08.04.2016 Abstract: The species from the genus Ganoderma are known to be extremely important macrofungi in fundamental biological, medicinal, and socioeconomic aspects. The present communication describes the brief morphological description, macro- and microscopic details, and chemical constituents of the representatives of five species of Ganoderma mushroom collected from different sites in Vietnamese National Parks. These species were Ganoderma colossus, G. neojaponicum, G. cattienensis, G. lucidum, and G. applanatum. Three additional herbarium strains of Ganoderma from European and Siberian regions have been implemented in the present framework for the purposes of comparison. Cultural characterization on solid and liquid fermentation, and scanning electron microscopy of morphology along with chemical analysis served as the supporting identification and comparison factors. Low-molecular-weight chemical constituents were evaluated by gas chromatography–mass spectrometry and gas-liquid chromatography techniques. Valuable substances (2-monolinolein, 2,3-dihydroxypropyl elaidate, fatty alcohol, fatty acid alkyl esters, and free fatty acids) detected in pigmented mycelia and submerged cultures have promising biotechnological applications including in food supplements, lipid-based drug delivery systems, and biodieselrelated items. The representative voucher specimens were deposited at the Herbarium of the Southern Institute of Ecology (Ho Chi Minh City, Vietnam) and were assigned accession numbers. Key words: Mushroom, Ganoderma, culture characteristics, chemical composition, gas chromatographic analysis, scanning electron microscopy 1. Introduction Many macrofungi observed in Vietnamese National Parks are wood rotting higher fungi, either edible or medicinal mushrooms. The greatest threat for many mushroom species is that of habitat loss and overharvesting of wild stocks; thus, by creating awareness of these issues, one enables a more sustainable use of these natural products (Mortimer et al., 2012). A part of contemporary research in mycology is encouraged by strategies for drug discovery as well as for monitoring and managing diseases caused by Ganoderma in woody crops and forest ecosystems (Moncalvo, 2005). The genus Ganoderma was established by Finnish mycologist Peter Adolf Karsten in 1881 for Polyporus lucidus W. Curt. In nonedible medicinal species, the xylotrophic * Correspondence: tsivileva@ibppm.sgu.ru mushroom Ganoderma (polypores) is the leader in terms of production. Extensive research over the last 10 years has provided evidence of the anticancer activities of both the triterpenoids isolated from Ganoderma (Wu et al., 2013) and the carbohydrate-enriched crude extracts from these fungi (Osińska-Jaroszuk et al., 2014). Ganoderma species exhibit a broad spectrum of antibacterial, antiviral (Gao et al., 2003), immunostimulatory (Osińska-Jaroszuk et al., 2014), cytotoxic (De S. Pereira et al., 2013), and antifungal (Sivaprakasam et al., 2011) activities. The studies of antibacterial action of Ganoderma extracts are not restricted to G. lucidum (Prasad and Wesely, 2008); other species are also active players (Ofodile et al., 2012; De S. Pereira et al., 2013; Osińska-Jaroszuk et al., 2014). 269 TSIVILEVA et al. / Turk J Bot Ganoderma species are among those fungi that can thrive under hot and humid conditions and are usually found in subtropical and tropical regions (Moncalvo, 2000). There are numerous Ganoderma species that are native to Vietnam. Most likely Vietnamese Ganoderma contains not only similar substances to this genus mushrooms isolated elsewhere, but also several unusual or even unique secondary metabolites. There is a strong consensus among contemporary European mycologists that G. lucidum is probably restricted to western parts of Europe (Moncalvo, 2005). The distribution range of G. valesiacum Boud. includes areas of Siberia along with Europe, China, and Japan (Hong and Jung, 2004). Three herbarium strains of Ganoderma, of which two species are from Europe (lucidum and applanatum) and one is from Siberia (valesiacum), have been implemented in the present framework for the purposes of comparison with Vietnamese-origin data. Based on Index Fungorum and a literature survey (e.g., Douanla-Meli and Langer, 2009), the name Ganoderma colossus (Fr.) C.F. Baker instead of Tomophagus colossus (Fr.) Murrill was used throughout this work. The primary tasks posed for this work were to determine suitable storage and growth conditions of the nine strains of the mushrooms, and to research their low-molecular composition. Optimal growth of mycelia was tested by variation of the environmental parameters, i.e. the nutrient medium composition, carbon and nitrogen sources’ kinds and proportion, and thermal conditions of culture. Contemporary physicochemical methods (scanning electron microscopy (SEM), gas chromatography coupled with mass spectrometric detection (GC-MS), gas–liquid chromatography) were applied for further characterization of the cultures. The key objective of the present study was to generate baseline information on Ganoderma species of the largest protection areas in Vietnam, and to compare those with the species or strains of distinct geographical sampling. 2. Materials and methods 2.1. Fungal strains Collections were acquired at different localities of Cat Tien National Park, Bu Gia Map National Park, and Bidoup-Nui Ba National Park. Macromorphological characters were recorded from fresh specimens as usually done (Kour et al., 2013). Macroscopic characteristics such as shape, size, color and its change with age, odor, spore deposition of fresh samples, and its natural habitat were recorded. The photography was accomplished using a digital camera. The fungal specimens were brought to the laboratory for macro investigations. Each of the mushroom’s mycelia was introduced into the microbiologically individual noncontaminated 270 monoculture. For each isolation procedure, the surface of a massive freshly collected fruit body was disinfected, cut with a sterile scalpel, and the mycelium taken from the bulk of the fruit body was immediately placed in a petri dish with wort agar. Tissue isolation from a fruit body was followed by a series of passage procedures using the commonly accepted methods of experimental mycology. Specimens were identified on the basis of critical observations and perusal of relevant literature under the guidance of experts (Cao et al., 2012 and references therein). The mushroom cultures have been deposited in the Herbarium of Southern Institute of Ecology, Ho Chi Minh City, Vietnam. The herbarium cultures of G. lucidum 1315, G. applanatum 0154 (obtained from the collection of the Chair of Mycology and Algology, Moscow State University, Moscow, Russia), and G. valesiacum 120702 were implemented for comparison. The last of these fungi was collected a few years ago from the Siberian region by one of the authors (A.N.P.), and obtained for the purposes of this work from the collection of the Biology Department, Irkutsk State University, Irkutsk, Russia. 2.2. Inoculum preparation All stock cultures were maintained on wort (4 Brix) agar slants, subcultured a few times a year, and stored at 4 °C. For inoculum preparation, all strains were initially grown on wort agar medium in a petri dish, and then transferred into the seed medium by punching out 5 mm of the agar plate culture with a self-designed cutter. Submerged culture was conducted in a 250-mL flask containing 70 mL of the medium. In the experiments aimed at biomass accumulation estimation, 1-L flasks containing 300 mL of nutrient liquid were used. These autoclaved nutrient liquids were seeded with 5% (v/v) of intermediate seeding submerged culture grown preliminarily on a synthetic medium containing D-glucose and L-asparagine, commonly used in our lab (Tsivileva et al., 2010). 2.3. Nutrient media The principal formulations of nutrient media used in the present work can be seen in Table 1. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) apart from the food components used for the lab-made liquid media. Potato broth was laboratory made by boiling potato in water for 20–30 min, filtering through a dense cloth to obtain a ratio of 800 g of potato to 1 L of cool final extract. The latter was 4-fold diluted before being used as a nutrient medium. Banana extracts were prepared from banana skin treated with 90 °C-water for 20 min at a proportion of 600 g of wet banana skin per 1 L of hot water, and was 4-fold diluted before being used as a nutrient medium. For preparing the solid media, 2% (m/v) of agar was added to the corresponding nutrient solutions. TSIVILEVA et al. / Turk J Bot Table 1. Media composition used in this study (concentration, g L–1). Formulation for solid media Component PMP MCM YMA CzGlc CzDex CzSuc CzLac CzMal CzY CzPe CzAm NaNO3 0 0 0 3 3 3 3 3 0 0 0 KH2PO4 0 0.46 0 1 1 1 1 1 1 1 1 K2HPO4 0 1 0 0 0 0 0 0 0 0 0 MgSO4∙7H2O 0 0.5 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 KCl 0 0 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 FeSO4∙7H2O 0 0 0 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 (NH4)2SO4 0 0 0 0 0 0 0 0 0 0 1.4 Glucose 0 20 10 30 0 0 0 0 20 20 20 Dextrose 0 0 0 0 30 0 0 0 0 0 0 Sucrose 0 0 0 0 0 30 0 0 0 0 0 Lactose 0 0 0 0 0 0 30 0 0 0 0 Maltose 0 0 0 0 0 0 0 30 0 0 0 Malt extract 10 0 3 0 0 0 0 0 0 0 0 Yeast extract 0 2 3 0 0 0 0 0 2 0 0 Peptone 1 2 5 0 0 0 0 0 0 2 0 Potato dextrose broth 24 0 0 0 0 0 0 0 0 0 0 Formulation for liquid media Component P PGGly PGY B BP BPY Glucose 0 20 20 0 0 0 Glycine 0 1.5 0 0 0 0 Potato broth 250 250 250 0 250 250 Banana extract 0 0 0 250 250 250 Yeast extract 0 0 2 0 0 2 2.4. Optimal temperature studies Optimal thermal condition searches for isolates of species under question in this work were carried out as presenting a useful, even if not decisive, characteristic in respect to identification, examination, and comparison. Cultivation of mycelia was performed in the dark at different temperatures in the range 21 °C up to 37 °С over 1 to 8 weeks depending on the given task to be solved in this work. 2.5. SEM technique For SEM studies, the fungal samples were placed on aluminum studs, sputter-coated with carbon (doublesided carbon tape), and then metalized with gold using an Emitech K450X+K350 (Emitech Limited, Kent, UK) confocal magnetron chamber sputtering from a Au target to a thickness of approximately 5 nm for 1 min at 20 mA of gun current. The gas pressure of argon was 1 × 10−1 mbar (10 Pa). The samples prepared were analyzed right after the gold deposition using a Mira II LMU scanning electron microscope (Tescan, Brno, Czech Republic) operating at 20 kV accelerating voltage. Chemical elemental analysis was performed with an energy-dispersive X-ray analysis system EDX INCA Energy 350 (Oxford Instruments, United Kingdom). Scanning electron micrographs were developed electronically with analySIS version 3.1 (Tescan, Czech Republic). Specimens were studied at a magnification from 300× to 50,000×. 271 TSIVILEVA et al. / Turk J Bot 2.6. GC-MS technique Dry samples were prepared from the mycelial mat, which developed yellow or brown pigmented areas. Solvent extraction of the mycelial samples was performed using a mixed solvent of ethanol:methanol:water (19:5:1, v/v). In GC-MS determinations, a Thermo Finnigan gas chromatograph–mass spectrometer, Trace GC-DSQ model (USA), was used. The principal conditions for separating and identifying the substances in mycelial extracts are listed in Table 2. The mass spectra reference source was “The National Institute of Standards and Technology (NIST) Library, 2011” (USA). The detected compounds were identified by comparing the mass spectra recorded in the experiment with the mass spectra from the library just mentioned. Within the framework of this study, a number of the organic substances were detected and identified. All these substances with more than 80% coincidence in their chromatographic patterns were repetitions of the corresponding standards from the reference library. Such level of identification probability is commonly referred to as “excellent or good correspondence”. 2.7. Fatty acids’ analysis Mycelia were air-dried at 35 °C and powdered before analysis. The dried samples were weighed with a standard analytical balance device. The samples were taken to be equal in dry-mass values, and then extracted with n-hexane (2 mL per 0.1 g) for 15 h using tightly stopped vessels at room temperature. The extracts were filtered through Whatman No. 4 paper. The residue was then extracted with two additional 2-mL portions of hexane. The combined extracts were evaporated to dryness in preliminarily weighed dark-glass vessels, weighed once again with the dry residues, and further used for methylation. This procedure was performed directly in the above vessels by means of redissolving in methylating agent (1 M HCl in methanol) at a ratio of about 1 mL of agent per 1 mg of dry residue, and stored at 84 °C for 16– 18 h. The methylating procedure was followed by careful evaporation in nitrogen flow, then washing thoroughly with chloroform:water (1:1) mixtures, collecting the combined chloroform fraction, and passing this fraction through sodium a sulfate filter to desiccate it. Thus, methyl esters of fatty acids as the constituents of the mushroom samples under question were prepared for being analyzed by means of gas-liquid chromatography. Fatty acid methyl esters from the extracts were analyzed using the Shimadzu GC-2010 gas-liquid chromatograph (Shimadzu, Japan) with an Equity-1 capillary column (Supelco, United States) and flame ionization detector. Fatty acids were identified by the retention time values of their methyl esters. The Bacterial Acid Methyl Esters CP Mix (Supelco) kit served as a set of standard substances including 11:0, 2-OH 10:0, 12:0, 13:0, 2-OH 12:0, 3-OH 12:0, 14:0, i-15:0, a-15:0, 15:0, 2-OH 14:0, 3-OH 14:0, i-16:0, 16:19, 16:0, i-17:0, 17:0, 2-OH 16:0, 18:29,12, 18:19, 18:0, 19:0, 20:0, as well as additional other methyl esters (Sigma) including 7:0, 8:0, 9:0, 10:0, 18:1, 20:2, 21:0, 22:1, 22:0, 23:0, 24:1, 24:0, where notations are given in accordance with the conventional practice for fatty acids. 2.8. Statistical analysis Quantitative data in this study were obtained in triplicate and are reported as average value ± standard deviation at a confidence level of 0.95. Table 2. Principal conditions of GC-MS determinations in this study. Parameter Mode description Mobile phase Helium (99.995% purity grade) with a 1.0 mL/min flow rate Column A 30-m-long TR-5MS, 0.32 mm internal diameter with a 0.25-µm-thick phase layer Thermal conditions in device, °C Injector (evaporator) 220, ionic source 220, MS Transfer Line 250 Electron energy 70 eV Total time for single sample analysis 56.50 min Programmed temperature Starting with heating at 80 °C for 5 min, then increasing the temperature at a rate of 3 °C/min to 220 °C, where it was maintained for 5 min Scanning interval 50–400 atomic units of mass (full scan) Gas flow mode Splitless Period before filament (electron source, cathode) switch-on In 3.50 min after injection Analyzed sample volume 2 µL 272 TSIVILEVA et al. / Turk J Bot 3. Results 3.1. Isolation, maintenance, and microscopic studies Morphological identification of the mushrooms was performed on the basis of macro- and microscopic characteristics of individual taxa and allowed us to present the following description (Table 3). The mushroom specimens’ morphology was visualized by SEM. Scanning electron micrographs can be seen in Figures 1–3 and Supplementary Material 1. Anatomy of mycelia from agar (2 to 8 weeks old) was characterized by generative hyphae bearing or not clamp connections (Supplementary Material 1: Figure S1a), Figure 1. Ganoderma neojaponicum SIEbidoup swallowed fragments of skeletal hypha (scale bar is 10 µm). nonbranched or moderately branched skeletal hyphae, and relatively thin binding hyphae (Supplementary Material 1: Figures S2a and S3). The development of deeply wrinkled hyphae and different types of hyphae cell-wall overgrowth to form film-like structures was characteristic of G. applanatum SIE1304 (Supplementary Material 1: Figures S3a and S3b) and G. applanatum 0154 (Supplementary Material 1: Figures S3c and S3d)), and contributed greatly to the interstrain morphological similarity within species of G. applanatum under study. Another mycelial microstructure was visualized as capitate outgrowth on the mycelial cells (Supplementary Material 1: Figure S1b). Cuticular cells organized from generative hyphae were spherical and relatively large, about 14–20 µm in diameter (Supplementary Material 1: Figure S4b). Abundance of cuticular cells was characteristic for G. lucidum SIE1303 (Supplementary Material 1: Figure S6), in contrast to G. valesiacum with its low proportion of cuticular cells accompanied by the abundance of fibrous hyphae. Pileus crust was mainly composed of skeletal hyphae and swollen hyphal ends (Supplementary Material 1: Figure S7a). Surface tissue of the pileus outer layer was composed of closely packed, clavate-shaped palisade of pilocystidia intermixed with branching hypha-like cells with rare apical projections. Elongated pilocystidia of G. colossus had smooth heads without protrusions (Supplementary Material 1: Figure S4c). Chlamydospores were terminal or aroused as intercalary swellings on short side branches of generative hyphae of G. cattienensis and G. applanatum SIE1304 (Supplementary Material 1: Figures S2b, S7b, and S8). Chlamydospore formation was not observed in mycelial cultures of G. applanatum 0154. Other strains formed chlamydospores within 8 weeks. Isolates of G. colossus produced thick-walled globular chlamydospores (Figure 2a), bigger than those of other species (up to 20 µm in diameter), with cylindrical spines projecting from surface reticulations (Figure 2b). Even larger and more coarsely reticulate with few-µm Table 3. The representative voucher specimens of Ganoderma deposited at the Herbarium of the Southern Institute of Ecology. Number Systemic position Species Accession numbers Collected from 1 Kingdom: Fungi colossus SIE1301 Cat Tien National Park 2 Division: Basidiomycota neojaponicum SIEbgm Bu Gia Map National Park 3 Class: Agaricomycetes neojaponicum SIEbidoup Bidoup-Nui Ba National Park 4 Order: Polyporales cattienensis SIE1302 Cat Tien National Park 5 Family: Ganodermataceae lucidum SIE1303 Bidoup-Nui Ba National Park 6 Genus: Ganoderma applanatum SIE1304 Bidoup-Nui Ba National Park 273 TSIVILEVA et al. / Turk J Bot a b Figure 2. Ganoderma colossus SIE1301 chlamidospores formed on wort agar in 3 weeks: a - scale bar unit is 20 µm; b - full-length scale bar is 20 µm. a b Figure 3. Ganoderma colossus SIE1301 calcium salt crystals in: a - pileus tissue; b - mycelium on wort agar (full-length scale bars are 50 µm). projections were the G. cattienensis chlamydospores (Supplementary Material 1: Figure S2b). Contrastingly different were rather smooth surface and smaller size of G. applanatum SIE1304 chlamydospores abundant in tube (Supplementary Material 1: Figure S7c). 274 In the samples of G. colossus pileus and mycelium on wort agar, calcium salt microparticles were observed (Figure 3). The presence of calcium was proved by EDX analysis. TSIVILEVA et al. / Turk J Bot 3.2. Cultural characteristics on solid and liquid media The influence of nutrient media composition upon the mushroom culture growth on agar media was examined using different types of formulations and varying thermal conditions (Supplementary Material 2). Complex organic agar media enriched with nutrient components, including potato malt peptone medium (PMP), mushroom complete medium (MCM), and yeast malt extract (YMA), were tested (Table 1). In addition, Czapek-Dox (CzDox) broth-based agar media (Waksman et al., 1943) fortified with various carbon sources: glucose (CzGlc), dextrose (CzDex), sucrose (CzSuc), lactose (CzLac), and maltose (CzMal), as well as with various nitrogen sources: yeast extract (CzY), peptone (CzPe), and ammonium sulfate (CzAm), were examined for their effect on mycelial growth. The results are shown in Table 4. Table 4. Maximal growth rate (v, mm/day)* and mycelial layer thickness (l)** values of Vietnamese Ganoderma colonies on agar media.*** Complex media Formulation G. colossus SIE1301 G. neojaponicum SIEbgm V l l V l V l V l V PMP 15.9 ± 0.8 4 4 11.2 ± 0.6 5 18.7 ± 1.1 5 15.2 ± 0.9 4 9.92 ± 0.50 5 MCM 12.5 ± 0.6 4 4 10.7 ± 0.5 5 14.6 ± 0.7 4 14.7 ± 0.8 4 6.64 ± 0.35 5 YMA 12.5 ± 0.7 3 4 10.7 ± 0.6 5 14.6 ± 0.6 5 14.3 ± 0.8 4 7.11 ± 0.37 4 CzDox 10.8 ± 0.5 1 2 10.6 ± 0.3 3 12.5 ± 0.5 1 11.7 ± 0.6 1 5.93 ± 0.28 1 10.2 ± 0.4 G. neojaponicum SIEbidoup G. cattienensis SIE1302 G. lucidum SIE1303 G. applanatum SIE1304 l Mineral media fortified with carbohydrates Formulation G. colossus SIE1301 G. neojaponicum SIEbgm G. neojaponicum SIEbidoup G. cattienensis SIE1302 G. lucidum SIE1303 G. applanatum SIE1304 V l V l V l V l V l V l CzDex 11.5 ± 0.5 4 11.8 ± 0.6 4 12.0 ± 0.7 5 13.9 ± 0.7 5 16.2 ± 0.8 5 10.0 ± 0.4 5 CzSuc 13.3 ± 0.6 4 11.4 ± 0.7 4 11.6 ± 0.5 5 13.7 ± 0.6 4 16.2 ± 0.7 4 7.12 ± 0.36 5 CzLac 10.6 ± 0.4 4 9.90 ± 0.57 4 10.1 ± 0.7 5 11.9 ± 0.5 4 14.5 ± 0.5 4 2.14 ± 0.21 5 CzMal 15.4 ± 0.8 4 10.6 ± 0.5 11.0 ± 0.6 4 11.7 ± 0.6 4 16.0 ± 0.6 4 9.62 ± 0.48 5 4 Mineral media fortified with nitrogen sources Formulation G. colossus SIE1301 G. neojaponicum SIEbgm G. neojaponicum SIEbidoup G. cattienensis SIE1302 G. lucidum SIE1303 V V l V l V l l G. applanatum SIE1304 V l V l CzY 17.7 ± 0.9 4 7.64 ± 0.33 5 7.93 ± 0.40 5 18.7 ± 1.2 4 14.6 ± 0.7 5 9.22 ± 0.47 5 CzPe 16.6 ± 0.9 4 7.02 ± 0.38 5 7.20 ± 0.34 5 16.6 ± 0.9 2 15.5 ± 0.8 5 8.73 ± 0.44 5 CzGlc 14.9 ± 0.6 3 9.00 ± 0.43 4 9.41 ± 0.47 5 14.7 ± 0.7 3 13.3 ± 0.7 4 6.01 ± 0.33 5 CzAm 15.0 ± 0.8 3 6.54 ± 0.33 4 7.03 ± 0.38 4 17.5 ± 1.1 3 15.3 ± 0.8 5 5.81 ± 0.30 5 Different thermal conditions Temp., °C G. colossus SIE1301 G. neojaponicum SIEbgm G. neojaponicum SIEbidoup G. cattienensis SIE1302 G. lucidum SIE1303 G. applanatum SIE1304 21 6.81 ± 0.34 3 13.7 ± 0.7 5 7.52 ± 0.37 4 12.4 ± 0.5 3 8.91 ± 0.46 3 6.33 ± 0.32 4 27 12.9 ± 0.5 4 10.3 ± 0.6 4 11.2 ± 0.4 16.3 ± 0.7 5 15.2 ± 0.7 10.0 ± 0.6 37 15.0 ± 0.7 3 No growth 5 5 5 *Average value ± standard deviation **Estimated by an ascending 5-point scale ranging from “1 - very thin” to “5 - very thick” layer ***See abbreviations in Table 1 275 TSIVILEVA et al. / Turk J Bot The effect of complex media on the submerged mycelial growth of various Ganoderma strains was investigated in flask 21-day culture. We examined different organic compositions capable of inducing Ganoderma growth on liquid media at 24 and 28 °C. Biotechnologically available components and agricultural wastes were tested (Table 1), including potato broth (P), potato-glucoseglycine (PGGly), potato-glucose-yeast extract (PGY), banana extract (B), banana-potato extract (BP), and banana-potato-yeast extract (BPY). The relevant growth characteristics are presented in Table 5. Note that the biomass values in “g per 300 mL” instead of “g x∙L–1 “ appeared to be closer to the experiment. Both strains of G. lucidum occasionally produced fruiting bodies on agar plates (Supplementary Material 2). In vitro fruiting appeared as projections from the white (G. lucidum 1315) or yellowish (G. lucidum SIE1303) mycelium. Primordia formation was easily observed for almost all the other species in culture on colored mycelium (Supplementary Material 2). For characterizing the fungal development under the conditions of liquid phase cultivation, the absolutely dry biomass of the mushroom mycelium was estimated. Among the 9 fungal cultures examined, the relatively high yield in mycelial biomass production was achieved in PGY broth. In particular, Ganoderma valesiacum 120702 and G. colossus showed favorable growth parameters in PGY medium with dry biomass values of 2.56 and 2.37 g per 300 mL, respectively. 3.3. Chemical composition of Ganoderma pigmented mycelia The Ganoderma species under study demonstrated the presence of yellow and brown pigmentation when growing on agar slants. The corresponding samples’ description is given in Table 6. At 20 °C on wort agar this culture occurred intensively yellow in 4 weeks, but it was not observed under the conditions of relatively high temperature (27 °C or higher). By applying the latter thermal conditions, which appeared favorable to G. cattienensis at solid-phase growth (see Table 4), we observed the G. cattienensis SIE1302 grayish-white mycelium remaining unchanged in color for a period as long as 3 weeks on wort agar. At the same temperature of 27 °C, but on PGGly agar media, brown mycelium of G. colossus was observed in 24 days of culture. Table 5. Growth of Ganoderma cultures on liquid media (dry biomass, g per 300 mL).* Fungus G. colossus SIE1301 G. neojaponicum SIEbgm G. cattienensis SIE1302 G. lucidum SIE1303 G. lucidum 1315 G. applanatum SIE1304 G. applanatum 0154 G. valesiacum 120702 Temp., °C Liquid media formulation P PGGly PGY B BGGly BPY 24 1.39 ± 0.15 1.61 ± 0.22 2.04 ± 0.26 1.43 ± 0.16 1.68 ± 0.13 1.96 ± 0.16 28 1.61 ± 0.18 1.87 ± 0.21 2.37 ± 0.26 1.57 ± 0.18 1.82 ± 0.21 2.31 ± 0.20 24 1.71 ± 0.17 1.88 ± 0.26 2.03 ± 0.23 1.68 ± 0.15 1.85 ± 0.22 2.00 ± 0.22 28 1.29 ± 0.16 1.31 ± 0.15 1.41 ± 0.13 1.38 ± 0.17 1.41 ± 0.19 1.50 ± 0.18 24 0.65 ± 0.09 0.68 ± 0.08 0.86 ± 0.11 0.77 ± 0.07 0.92 ± 0.08 1.08 ± 0.11 28 0.98 ± 0.12 1.15 ± 0.15 1.47 ± 0.18 0.95 ± 0.10 1.10 ± 0.14 1.42 ± 0.15 24 0.61 ± 0.08 0.75 ± 0.07 0.78 ± 0.08 0.64 ± 0.06 0.73 ± 0.08 0.77 ± 0.08 28 1.05 ± 0.16 1.28 ± 0.16 1.36 ± 0.16 1.02 ± 0.12 1.24 ± 0.12 1.32 ± 0.17 24 1.78 ± 0.26 2.18 ± 0.31 2.31 ± 0.27 1.74 ± 0.19 2.13 ± 0.19 2.26 ± 0.18 28 1.11 ± 0.14 1.36 ± 0.13 1.35 ± 0.17 1.18 ± 0.10 1.33 ± 0.14 1.41 ± 0.16 24 0.74 ± 0.11 0.90 ± 0.11 1.17 ± 0.11 0.73 ± 0.09 0.90 ± 0.11 1.16 ± 0.11 28 1.18 ± 0.16 1.43 ± 0.18 1.99 ± 0.25 1.11 ± 0.15 1.33 ± 0.12 1.85 ± 0.18 24 0.91 ± 0.12 1.10 ± 0.13 1.53 ± 0.16 0.92 ± 0.08 1.13 ± 0.12 1.46 ± 0.19 28 0.61 ± 0.08 0.74 ± 0.08 0.96 ± 0.11 0.62 ± 0.06 0.76 ± 0.09 0.98 ± 0.09 24 1.43 ± 0.23 1.78 ± 0.25 2.56 ± 0.25 1.49 ± 0.17 1.87 ± 0.25 2.51 ± 0.24 28 0.80 ± 0.11 0.99 ± 0.08 1.93 ± 0.17 0.83 ± 0.41 1.04 ± 0.11 1.70 ± 0.19 *Average value ± standard deviation 276 TSIVILEVA et al. / Turk J Bot Table 6. Pigmented mycelia samples studied by GC-MS analysis. No. Sample code Sample details Species Strain 1 G colosyel G. сolossus 2 G colosgr 3 SIE1301 Mycelium color yellow Agar medium wort Culture age, days 30 Culture temp., °C 20 G. сolossus SIE1301 grayish-white wort 30 27 G colosbr G. сolossus SIE1301 brown PGGly 24 27 4 G neojyel G. neojaponicum SIEbgm yellow PGGly 24 27 5 G appSIEyel G. applanatum SIE1304 yellow wort 30 27 6 G luc1315yel G. lucidum 1315 yellow wort 30 27 7 G lucSIEyel G. lucidum SIE1303 yellow wort 30 27 8 G valesbr G. valesiacum 120702 brown wort 30 27 9 G сattbr G. cattienensis SIE1302 brown wort 30 27 We studied extracts from the vegetative mycelia by means of a gas chromatography–mass spectrometry (GCMS) detection method. The substances detected are given in Table 7. Data are presented in the ascending order of retention time values. The results of analyses presented in Table 8 are for each sample separately. A relative level of each component (as a ratio of its chromatographic peak square absolute value to the total sum of square values in the given fungal sample), expressed as mass percent, is displayed. Table 8 also contains the values of the molar percentage of each chemical component calculated with respect to the total moles of the components detected in the sample. 3.4. Evaluation of fatty acids in submerged mycelia Free fatty acids were analyzed in the form of their methyl esters (Song et al., 1989) by a gas-liquid chromatography method. The samples were grown on submerged culture, and the description is given in Table 9. The fatty-acid composition of total lipids of submerged mycelium of different Ganoderma strains under study can be seen in Supplementary Material 3. Culture conditions were as in Table 9. Data were given as percentages of total fatty acids in the fungal sample. The total sum of fatty acids of each group (saturated, monounsaturated, polyunsaturated fatty acids, short-chain and long-chain acids, and hydroxy acids) was analyzed. In the total lipidic extracts from the submerged mycelia, 9,12-octadecadienoic, hexadecanoic, and 9-octadecenoic acids dominated. The ratio of the mass fractions of these three compounds to that of the total compounds ranged between 61% (G. applanatum 0154) and 94% (G. lucidum 1315). Therefore, the short-chained and very-longchained acids were not a major fraction of free fatty acids of Ganoderma mycelia total lipids. One can see from Supplementary Material 3 that the high degree of unsaturation of the mycelial total lipids was characteristic for G. valesiacum that contained the greatest (about 58%) amount of C18:2. Somewhat lower relative quantities of the linoleic acid percentage (both values about 47%) were found for G. neojaponicum SIEbgm and G. lucidum SIE1303. The maximal level of C18:1 (about 33% of the sum of total fatty acids) occurred in G. colossus. The pool of unsaturated lipids of this species comprised also the monounsaturated C14:1 (tetradecenoic) acid, which was detected in G. colossus (both cis and trans isomers) and G. applanatum SIE1304 (cis isomer only). 4. Discussion 4.1. Isolation and maintenance of fungal cultures We isolated the starter cultures from the fruiting bodies of mushrooms picked up in the National Parks of Vietnam, the rich and valuable biodiversity of which has been facing challenges. Bidoup-Nui Ba National Park is one of the largest protection areas in Vietnam. Cat Tien National Park is one of six biosphere reserves recognized by UNESCO in Vietnam. Bu Gia Map National Park is a conservation area of rare and precious species of fauna and flora rich in the southeastern region. The importance of selecting suitable short-term and long-term storage of the fungi was to ensure they remained viable throughout this investigation, as well as for future reference and use. It was also desirable to obtain maximum growth of the fungal mycelium in a liquid cultivation medium so that sufficient material was available to pursue further studies on its biological activity. Most fungal cultures can be maintained on agar by subculturing at 2- to 6-month intervals. Agar to agar is a relatively cheap method of storage, with little time 277 TSIVILEVA et al. / Turk J Bot Table 7. List of substances detected in mycelial extracts by GC-MS analysis. Chemical name and schematic structure 1-octen-3-ol Detected in sample codes (absolute values of GC peak squares, ×10–5) G valesbr (5.67) > G lucSIEyel (5.15) OH 3-octanol G valesbr (2.40) > G lucSIEyel (2.00) OH Ethyl tetradecanoate O G colosyel (8.20) O Ethyl pentadecanoate G colosyel (25.6) > G colosgr (8.78) > G appSIEyel (6.88) > G lucSIEyel (6.70) > G сattbr (4.78) O O Ethyl 9-hexadecenoate O G colosgr (59.0) > G colosyel (45.7) O Hexadecanoate (palmiate) G colosyel (121) > G colosgr (59.0 ) > G сattbr (25.6) > G valesbr (26.2) > G lucSIEyel (23.8) HO O Ethyl hexadecanoate (ethylpalmiate) O O 1-octadecanol (stearyl alcohol) OH G colosyel (245) > G colosgr (92.5) > G сattbr (84.5) > G lucSIEyel (31.1) > G appSIEyel (25.3) > G colosbr (12.2) > G luc1315yel (9.51) > G neojyel (6.84) G colosyel (123) > G valesbr (98.2) Ethyl heptadecanoate O G appSIEyel (5.38) O 2-monolinolein (glyceryl monolinoleate) HO O HO G valesbr (301) > G luc1315yel (232) > G colosbr (204) O 2,3-dihydroxypropyl elaidate (2-monoolein; glyceryl monooleate) HO HO O G colosbr (204) > G valesbr (48.1) > G neojyel (32.1) O 9,12-octadecadienoate (linoleate) HO O G colosyel (87.7) > G colosgr (81.7) > G valesbr (27.4) > G сattbr (22.0) > G lucSIEyel (21.1) > G appSIEyel (12.1) 9-octadecenoate (oleate) HO O Ethyl 9,12-octadecadienoate (ethyllinoleate) O O Ethyl 9-octadecenoate (ethyloleate) O O Ethyl octadecanoate (ethylstearate) O O 278 G colosyel (87.7) > G colosgr (81.7) G valesbr (340) > G colosyel (309) > G colosgr (132) > G сattbr (103) > G appSIEyel (101) > G lucSIEyel (82.0) > G luc1315yel (48.7) > G neojyel (20.5) G colosyel (271.43) > G valesbr (244.28) > G сattbr (126.35) > G colosgr (118) > G lucSIEyel (39.7) > G appSIEyel (23.5) > G colosbr (14.9) > G luc1315yel (9.06) G colosyel (67.3) > G colosgr (28.6) > G сattbr (17.3)
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