Research Article |
Corresponding author: Peter E. Mortimer ( petermortimer@mac.com ) Corresponding author: Jianchu Xu ( j.c.xu@cgiar.org ) Academic editor: María P. Martín
© 2019 Thatsanee Luangharn, Samantha C. Karunarathna, Peter E. Mortimer, Kevin D. Hyde, Naritsada Thongklang, Jianchu Xu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Luangharn T, Karunarathna SC, Mortimer PE, Hyde KD, Thongklang N, Xu J (2019) A new record of Ganoderma tropicum (Basidiomycota, Polyporales) for Thailand and first assessment of optimum conditions for mycelia production. MycoKeys 51: 65-83. https://doi.org/10.3897/mycokeys.51.33513
|
In this study a new record of Ganoderma tropicum is described as from Chiang Rai Province, Thailand. The fruiting body was collected on the base of a living Dipterocarpus tree. The sample is described on the basis of morphological characteristics and phylogenetic analyses, and compared with closely related taxa. Multigene phylogenetic analyses of LSU, ITS, and RPB2 highly support the placement of the G. tropicum group with isolates from China and Taiwan (Maximum likelihood 100%, Maximum parsimony 100%, and Bayesian posterior probabilities 1.00). The optimal media, pH, and temperature for mycelial growth of the G. tropicum strain KUMCC18-0046 was also investigated and is reported as: PDA, MEA, and YPD, at pH 7–8 and 25–28 °C, respectively. This is the first report on the successful growing conditions for mycelial production, but unfortunately fruiting could not be achieved.
Cultivation, medicinal mushroom, morphological characteristics, phylogeny, taxonomy
Ganoderma P. Karst. was established as a white rot fungus (
Ganoderma produces a high number of natural bioactive compounds, such as polysaccharides, triterpenoids, sterols, and secondary metabolites (i.e. ganoderic acid, ganodermanondiol, ganodermanontriol, and ganodermadiol), which can be used to remedy a wide range of diseases (
The taxonomy of Ganoderma has been a constant topic of debate due to the high levels of phenotypic plasticity in species such as G. lingzhi, G. lucidum, and G. sichuanense (
In October 2017, a single fresh basidiocarp of Ganoderma tropicum was collected on a living Dipterocarpus tree in a deciduous mixed rainforest dominated by Castanopsis and Dendrocalamus strictus during the dry season. The coordinates of the described area in Chiang Rai Province, Thailand are 19°49'23"N; 100°01'41"E, 41 m. The sample was then photographed and transported back to the laboratory where its fresh macroscopic details were described. The culture was aseptically isolated by using heat sterilized forceps, transferring sections of internal tissue from fruiting bodies onto potato dextrose agar (PDA) and incubated at 25 °C, for 21 days, under dark conditions (
Macro-morphological characteristics were described following the method by
Dried internal tissues of the basidiocarp were ground and total DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux). The ITS, LSU, and RPB2 genes were amplified by Polymerase Chain Reaction (PCR). The PCR amplifications were performed in a total volume of 25 μL of PCR mixtures containing 9.5 μL ddH2O, 12.5 μL of PCR master mix, 1 μL of DNA template, and 1 μL of each primer (10 μM). PCR amplification was carried out using primer pairs LROR/LR5 for the nuclear ribosomal large subunit 28S rDNA gene (LSU), ITS5/ITS4 for internal transcribed spacer rDNA region (ITS1, 5.8S rDNA and ITS2), and fRPB2-5F/fRPB2-7cR for the partial RNA polymerase second largest subunit region (RPB2) (
The sequence of the new record was subjected to standard BLAST searches of GenBank to determine the primary identity of the fungal isolate. All the other sequences of this study were retrieved from GenBank. All the sequences used to construct the phylogenetic tree are listed in Table
Fungal species and GenBank accession number of sequences used in this study.
Fungal species | Voucher | GenBank accession no. | References | ||
---|---|---|---|---|---|
ITS | LSU | RPB2 | |||
Ganoderma applanatum | Wei 5787a | KF495001 | KF495011 | – | GenBank |
G. applanatum | SFC20141001–24 | KY364255 | – | KY393273 |
|
G. australe | HUEFS: DHCR 417 | MF436676 | MF436673 | – |
|
G. austroafricanum | CMW 41454 | KM507324 | KM507325 | – |
|
G. chalceum | URM 80457 | JX310812 | JX310826 | – | GenBank |
G. destructans | CBS 139793 | NR_132919 | NG_058157 | – |
|
G. destructans | CMW 43670 | KR183856 | KR183860 | – |
|
G. enigmaticum | CBS 139792 | NR_132918 | NG_058156 | – |
|
G. enigmaticum | Ghana2/938397 | KR014265 | KR014266 | – | GenBank |
G. gibbosum | UB1 | KU569556 | KU570954 | – |
|
G. gibbosum | SPC2 | KU569547 | KU570946 | – |
|
G. lingzhi | Dai12441 | JQ781869 | – | – |
|
G. lingzhi | Li245 | JQ781863 | – | – |
|
G. lingzhi | Wu 1006–3 | JQ781858 | – | – |
|
G. lucidum | K175217 | KJ143911 | – | – |
|
G. lucidum | Dai11593 | JQ781852 | – | – |
|
G. lucidum | Dai2272 | JQ781851 | – | – |
|
G. lucidum | Rivoire 4195 | KJ143909 | – | – |
|
G. multiplicatum | CWN 04670 | KJ143913 | KJ143972 | KJ143972 |
|
G. multiplicatum | HMAS 242384 | JF915409 | – | JF915432 |
|
G. multiplicatum | Dai 9447 | KJ143914 | – | KJ143973 |
|
G. orbiforme | URM 83332 | JX310813 | JX310827 | – |
|
G. orbiforme | URM 83334 | JX310814 | JX310828 | – |
|
G. orbiforme | URM 83335 | JX310815 | JX310829 | – |
|
G. parvulum | URM 83339 | JX310817 | JX310831 | – |
|
G. parvulum | URM 83340 | JX310818 | JX310832 | – |
|
G. pfeifferi | 120818 | AY884185 | – | – | GenBank |
G. pfeifferi | JV 0511/11 | KF605660 | – | – | GenBank |
G. resinaceum | URM 83400 | JX310824 | JX310838 | – |
|
G. resinaceum | HSBU 200830 | KT343303 | – | – | GenBank |
G. resinaceum | HMAS 86599 | AY884177 | – | JF915435 | GenBank |
G. sessile | UMNMN8 | MG654281 | – | – | GenBank |
G. sichuanense | MFU16–2670 | KY404119 | – | – |
|
G. sichuanense | HMAS 251145 | JF915400 | – | – |
|
G. sichuanense | MFU16–2667 | KY244061 | – | – |
|
G. sichuanense | MFU16–2668 | KY244062 | – | – |
|
G. tropicum | HKAS: 76644 | KC222317 | – | – |
|
G. tropicum | Dai9724 | JQ781879 | – | – |
|
G. tropicum | HMAS 263143 | JF915410 | – | – |
|
G. tropicum | Wu 0407–2 | EU021458 | – | – |
|
G. tropicum | BCRC 37122 | EU021457 | – | – |
|
G. tropicum | KUMCC 18–0046 | MH823539 | MH823540 | MH883621 | This study |
G. valesiacum | CBS 428.84 | JQ520218 | – | – |
|
Amauroderma calcitum | FLOR: 50931 | KR816528 | KU315207 | – |
|
Phylogenetic trees and data files were figured in FigTree v. 1.4.0 (
Phylogenetic tree (RAxML) obtained from the DNA sequence data of LSU, ITS, and RPB2 datasets. Bootstrap values (BS) from maximum likelihood (ML, left), Maximum parsimony (MP, middle) greater than 70% and Bayesian posterior probabilities (PP) greater than 0.95 are indicated above the nodes as MLBS/MPBS/PP. The tree is rooted with Amauroderma calcitum FLOR:50931. Newly recorded species are indicated in black bold.
Seven different solid culture media were evaluated to determine the optimal media for the mycelial growth of the G. tropicum strain KUMCC18-0046, namely Czapek’s agar (CZA), malt extract agar (MEA), potato dextrose agar (PDA), rose Bengal agar (RBA), yeast extract agar (YEA), yeast malt extract agar (YMA), and yeast extract peptone dextrose agar (YPD). The media formulae used are shown in Table
Agar media reagents | Agar media composition (g/L) | ||||||
---|---|---|---|---|---|---|---|
CZA | MEA | PDA | RBA | YEA | YMA | YPD | |
Potato infusion | 4 | ||||||
Malt extract | 20 | 3 | |||||
Yeast extract | 3 | 3 | 10 | ||||
Peptone | 6 | 5 | 5 | 20 | |||
Dextrose | 20 | 20 | 20 | ||||
Glucose | 10 | ||||||
Saccharose | 30 | ||||||
Sodium nitrate | 33 | ||||||
Di-potassium phosphate | 1 | ||||||
Magnesium sulfate | 0.5 | 0.5 | |||||
Potassium chloride | 0.5 | ||||||
Ferrous sulfate | 0.01 | ||||||
Potassium dihydrogen phosphate | 1 | ||||||
Rose bengal | 0.033 | ||||||
Chloramphenicol | 0.1 | ||||||
Agar | 15 | 15 | 15 | 15 | 20 | 15 |
The optimal media shown for mycelial growth was then used to determine the optimal pH for mycelial growth. pH was adjusted to 4, 5, 6, 7, 8, and 9 with 1N HCl and 1N NaOH. The optimal temperature for mycelial growth was determined by using the highest growth rates of media and pH conditions under different dark conditions; including 15 °C, 20 °C, 25 °C, 28 °C, 30 °C, and 35 °C. After 10 days of incubation, five replicates of colony diameter were measured and calculated. The colony diameter was measured as described above.
Data analysis was carried out using statistical programs (SPSS) with five replicates (n = 5). All data were compared to obtain a mean separation using Tukey’s test (p < 0.05) followed by post-hoc tests. The results are expressed in a one-way analysis of variance (ANOVA) analysis using the SPSS program (Softonic International SA, Barcelona, Spain).
Phylogenetic analyses were inferred from the combined LSU, ITS, and RPB2 sequences, comprising 44 taxa, including 19 Ganoderma species with Amauroderma calcitum FLOR: 50931 as the outgroup taxon. The dataset comprised 2223 total characters, of which 1961 were constant, 176 variable characters were parsimony-informative, and 86 characters were parsimony-uninformative. The tree topologies were grouped into nine distinct clades, including five laccate clades of G. tropicum, G. sichuanense, G. lingzhi, G. orbiforme, G. lucidum, and two other laccate clades with one non-laccate clade, and an outgroup clade. The phylogenetic analyses showed considerably high support for the G. tropicum strain KUMCC18-0046 being closely related to the laccate G. tropicum isolates of China and Taiwan (MLBS = 100%/ MPBS = 100%/ PP = 1.00).
Basidiome. Sessile, dimidiate. Pileus shape. Semicircular to dimidiate or conks, up to 7–12 cm in length and 4–8 cm in width, up to 1.5 cm thick. Pileus surface. Dark brown (9F5) at the base, slightly brownish red (10C8) at center, reddish gray (10B8) extending to the margin, light yellow (1A5) to yellow (2A6) under basidiocarp with grayish yellow (4C7) to brown (6D7 to 6F6) close to tube layer on upper surface of pileus glabrous, weakly to strongly laccate, glossy and shiny, smooth, spathulate, shallow sulcate several layers thick, consistency furrows, thicker at the base than the margin, covered by a thin and hard crust, and light in weight when dried. Context trimitic, irregular cuticle cells, mostly light yellow (1A5) to yellow (2A6) close to crust, grayish yellow (4C7), brown (6D7 to 6F6) to dark brown (9F5), near the tubes, dense context layer, thick near the base, tough to break when dried. Hymenophore. Grayish yellow (4C7). Basidiospores. Mostly oblong ellipsoid and broadly ellipsoid with double wall (ganodermoid) at maturity, (7.3–)7.6–10.1(10.8) × (10.1)10.6–13.3(13.9) μm (x̄ = 9.1 × 12.2 μm, n = 50) (including myxosporium), (5.4–)5.6–8.3(8.6) × (8.3)8.4–12.5(12.9) μm (x̄ = 7.1 × 10.6 μm, n = 50) μm (excluding outer myxosporium), light brown (6D6–6D8), reddish brown (9F6) to dark brown (9F8), usually with one end tapering, and usually overlaid by a hyaline myxosporium. Tubes. 2–7 mm long, up to 80–170 µm wide, and sulcate at different levels. Stipe. Lateral, up to 1.5 cm thick, dark brown (9F5). Margin. reddish gray (10B8), up to 0.3–0.7 cm thick, round, tough and hard, thicker towards the margin. Pore. Angular, 4–7 per mm; pore diameter up to 65–120 µm. Pore surface Pale yellow (2A3) to light yellow (2A5) and brown (6D7) to dark brown (6F6) when touched. Hyphal system Generative hyphae up to 0.80–2.85 μm (x̄ = 1.45, n = 50) in diameter, colorless, thin-walled, some thick-walled, branched, with clamp connections; binding hyphae 1.00–3.10 µm (x̄ = 2.05, n = 50), colorless, thin-walled, much-branched, clamped; skeletal hyphae up to 1.45–4.25 μm (x̄ = 2.35, n = 50), colorless, thick-walled, unbranched or with a few branches in the distal end. Culture characteristics. Initially white (4A1) to yellowish white (4A2), pale yellow (4A3) when growing, become orange white (5A2), pale orange (5A3), light orange (5A4–6A5) and some reddish yellow (4A6) to dark brown (9F8) around the plugged circle of active mycelium after incubation for 3 weeks. Odor. Distinctive odor when fresh and dried.
Ganoderma tropicum is diagnosed as having a distinctly dimidiate, smooth, spathulate pileus, with a laccate or glabrous dark brown slightly brownish red upper surface, usually tough when dried; margin usually has a reddish gray surface, round and hard; pore surface pale yellow when young, light yellow when mature, and becoming brown or dark brown when bruised; basidiospores are described as ellipsoid, with size range of (7.3–)7.6–10.1(10.8) × (10.1)10.6–13.3(13.9) μm (including myxosporium), (5.4–)5.6–8.3(8.6) × (8.3)8.4–12.5(12.9) μm (excluding outer myxosporium); context trimitic, abundant generative hyphae with branches; thin-walled, binding hyphae; and skeletal hyphae with clamp connections.
Morphology of Ganoderma tropicum strain KUMCC18-0046 A, B Mature basidiocarps C Margin D Pore characteristics E, F Culture after incubation at 25 °C for 21 days G, J Basidiospore in KOH K, L Generative hyphae of context in KOH M Skeletal hyphae N Skeletal hyphae and binding hyphae O Sparing branch hyphae. Scale bars: 1 cm (A–C); 500 µm (D); 1 cm (E, F); 5 µm (G–I); 15 µm (J); 20 µm (K, L, O); 10 µm (M, N).
Solitary on living Dipterocarpus species in deciduous forests.
Thailand, Chiang Rai Province, 19°48'24"N, 100°03'54"E, 836 m, October, 2017.
In our study of the seven different agar media, mycelial growth (mm), growth rates (mm/day), and mycelial density were screened as an indication of favorable growth of Thai G. tropicum (Table
Effect of various agar media on mycelial growth (mm) and mycelial growth rates (mm/day) of Ganoderma tropicum strain KUMCC18-0046, incubated at 25 °C for 10 days. Values with the same letter are not significantly different (p < 0.05).
Agar media | Colony diameter | Growth rate | Mycelial density |
CZA | 16.70 ± 0.13e | 3.50 | + |
MEA | 41.20 ± 0.12a | 8.40 | 5+ |
PDA | 42.20 ± 0.44a | 8.50 | 4+ |
RBA | 27.70 ± 0.08c | 4.20 | 3+ |
YEA | 21.00 ± 0.08d | 5.70 | 2+ |
YMA | 36.90 ± 0.13b | 8.10 | 5+ |
YPD | 40.40 ± 0.40a | 8.40 | 5+ |
Mycelial morphology and colony color characteristics differed on each agar media (Fig.
Characteristics of Ganoderma tropicum strain KUMCC18-0046 mycelial cultures were incubated at 25 °C for 10 days on different agar media A Czapek’s agar (CZA) B Malt extract agar (MEA) C Potato dextrose agar (PDA) D Rose Bengal agar (RBA) E Yeast extract agar (YEA) F Yeast malt extract agar (YMA) G Yeast extract peptone dextrose agar (YPD). Scale bars: 1 cm.
All pH values from 4–9 were suitable for promoting mycelium growth of G. tropicum, however, the most favorable pH range was shown to be pH 7–8, followed by pH 9 (Table
Effect of pH on mycelial growth (mm) and mycelial growth rates (mm/day) of Ganoderma tropicum strain KUMCC18-0046, incubated for 10 days. Values with the same letter are not significantly different (p < 0.05).
pH | Colony diameter | Growth rate | Mycelial density |
4 | 30.50 ± 0.14d | 5.50 | 3+ |
5 | 45.90 ± 0.10c | 7.30 | 4+ |
6 | 46.80 ± 0.10c | 8.10 | 4+ |
7 | 57.50 ± 0.12ab | 8.50 | 5+ |
8 | 58.90 ± 0.05a | 8.50 | 5+ |
9 | 56.10 ± 0.07b | 8.10 | 5+ |
Ganoderma tropicum mycelial growth increased when going up from 15–25 °C and 28 °C, after which it started to decline again, with the most suitable temperature for mycelial growth being between 25 °C and 28 °C. Although the mycelia could grow between 15–35 °C, growth appeared to be drastically suppressed at 15 °C and 35 °C (Table
Effect of temperature on mycelial growth (mm) and mycelial growth rates (mm/day) of Ganoderma tropicum strain KUMCC18-0046, incubated for 10 days. Values with the same letter are not significantly different (p < 0.05).
Temperature (°C) | Colony diameter | Growth rate | Mycelial density |
---|---|---|---|
15 | 17.40 ± 0.07c | 4.00 | 1+ |
20 | 29.70 ± 0.04b | 6.30 | 3+ |
25 | 43.50 ± 0.06a | 8.50 | 5+ |
28 | 43.70 ± 0.04a | 8.50 | 5+ |
30 | 30.40 ± 0.13b | 6.40 | 4+ |
35 | 17.90 ± 0.11c | 4.30 | 2+ |
In this study, we introduce a new record of G. tropicum strain KUMCC18-0046, which was collected from Chiang Rai Province, Thailand. Ganoderma tropicum serves as the sister group to G. multipileum, G. parvulum, and G. destructans (ML = 73%, MP = 81%). This finding is consistent with those of
Ganoderma tropicum has been widely reported in tropical areas; however, no specimens have been recorded in Thailand prior to this study (
However, G. flexipesis can be differentiated from G. tropicum by its small basidiocarps and long stipe, while G. lingzhi has usually distinctive sessile basidiocarps, a dark brown context, and mostly irregular cuticle cells (
Our morphological analyses show that the Thai G. tropicum strain has a semicircular to dimidiate pileus, a pileus size between 4–8 cm in width, 7–12 cm in length, and up to 1.5 cm thick. The basidiospores are mostly oblong ellipsoid and broadly ellipsoid in shape, with double walls, (7.3–)7.6–8.2–10.1(–10.8) × (10.1–)10.6–11.7–13.3(–13.9) μm (x̄ = 9.1 × 12.2 μm, n = 50), and (5.4–)5.6–7.1–8.3(–8.6) × (8.3)8.4–10.8–12.5(–12.9) μm (x̄ = 7.1 × 10.6 μm, n = 50) mm (excluding outer myxosporium); the pore surface is pale yellow (2A3) with pore are 4–7 per mm, and the tubes are 2–7 mm long with a light yellow to dark brown context. The original description of G. tropicum has the basidiospores fasciculate, 7–9 × 10–12 µm with 4–5 µm of hymenia hyphae (Tai et al. 1979). These characteristics are in accordance with the basidiospore sizes we recorded for the Thai strain of G. tropicum. The strain of G. tropicum from South America shares much in common with the Thai strain; however, notable differences in the South American strain include light brown ovoid basidiospores, a pileus of dark to black coloring at the base, and a blunt to slightly round margin. Our results of G. tropicum are in accordance with the description of
The optimal conditions for mycelial growth were investigated based on medium, pH, and temperature. The best growth rates were obtained using PDA, MEA, and YPD media. These three media are composed of high concentrations of dextrose as a carbon source, while various forms of carbon sources have been reported as affecting fungal mycelial growth (
This study confirmed the new record of Ganoderma tropicum from Northern Thailand based on morphological characteristics together with phylogenetic analyses. The optimal conditions for promoting the mycelial growth of G. tropicum were investigated and the best media and pH for mycelia growth were found to be PDA, MEA, and YPD media at pH 7–8, respectively. The optimal temperature was found to be 25–30 °C.
We appreciate the kind support given by the University of Chinese Academy of Sciences, Beijing 100049, China; Key Laboratory for Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China; and Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China. This research was supported by the Key Research Program of Frontier Sciences, CAS, Grant No. QYZDY-SSW-SMC014. We thank the Germplasm Bank of Wild Species, Kunming Institute of Botany, Kunming 650201, Yunnan, China for enabling our molecular laboratory work. Dr Olivier Raspé is thanked for his help with phylogenetic analyses. Dr Saowaluck Tibpromma, Nimali I. de Silva and Subashini Chathumini are thanked for their help and valuable suggestions. Kasiphat Limsakul and Wilawan Punyaboon are acknowledged for their invaluable assistance. Samantha C. Karunarathna thanks CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (No. 2018PC0006) and the National Science Foundation of China (NSFC) for funding this work under the project code 31750110478. Peter E. Mortimer thanks the National Science Foundation of China (NSFC) project codes 41761144055 and 41771063. The authors also thank William A. Julian for his contributions to the English editing. This research is part of the CGIAR-FTA Program.