A new record of Ganodermatropicum (Basidiomycota, Polyporales) for Thailand and first assessment of optimum conditions for mycelia production

Abstract In this study a new record of Ganodermatropicum 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.

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 (Richter et al. 2015;Hapuarachchi et al. 2018b). Many compounds have been found in different species of Ganoderma, and extracts derived from G. tropicum contain phenolic compounds such gano-dermatropins A (1) and B (2), as well as compounds with antimicrobial activity (Hu et al. 2013). Ganoderma tropicum is recognized as a medicinal mushroom and has been recorded in the Chinese Pharmacopeia (Wu et al. 2013). The fruiting bodies contain natural triterpenes, primarily lanostanoid-type triterpenes, with potential use in chemotaxonomy (Ríos et al. 2012;Da Silva et al. 2013;Zhang et al. 2015).
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 (Pilotti et al. 2004;Wang et al. 2009;Cao et al. 2012;Dai et al. 2017;Loyd et al. 2018;Hapuarachchi et al. 2019). In an attempt to further our knowledge of the taxonomy this genus, we describe a specimen of Ganoderma tropicum as a new record for Thailand based on morphological characteristics and phylogenetic analyses, and the optimal conditions for mycelial growth of G. tropicum are also reported.

Sample collection and isolation
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 (Luangharn et al. 2017). After incubation, the agar surface was fully covered with white mycelium. The pure stock culture was then covered with mineral oil and deposited in the voucher culture collection of the Kunming Institute of Botany culture collection under the accession number KUMCC18-0046. The cultures are being maintained at 4 °C for further studies. The sample was then air dried at 45 °C for 48 hours until it was completely dehydrated. Finally, the herbarium material was deposited in the Herbarium of Mae Fah Luang University, Chiang Rai, Thailand (voucher number MFLU Herb. 17-1934) with duplicates in the Herbarium of Kunming Institute of Botany, Academia Sinica (HKAS), Yunnan Province, China (voucher number HKAS 97486).

Morphological study
Macro-morphological characteristics were described following the method by Lodge et al. (2004), while colors were recorded following (Ridgeway 1912). Macroscopic characteristics were determined according to the methodology described by Largent (1986). To observe microscopic characteristics, free-hand sections were made under a dissecting microscope (OLYMPUS SZ61) and mounted on a glass slide in 3-5% KOH, 1-3% Congo red, and Melzer's reagent for highlighting all tissues (Kreisel and Schauer 1987). Microphotography was done with a Nikon ECLIPSE Ni (Nikon, Tokyo, Japan) compound microscope, with a Canon EOS 600D (Tokyo, Japan) digital camera fitted on the top of the microscope. Basidiospores and hyphal system sizes, colour, and shapes were recorded and photographed. Measurements were taken using the Tarosoft® Image Framework program v. 0.9.0.7. The size and shape of basidiospores were followed [Q = L/W] and calculated considering the mean value of the lengths and widths in side view. The calculation was done by using at least 50 basidiospores from each basidiomata (Miettinen and Larsson 2006). The photographs were edited in Adobe Illustrator CS v. 3.

DNA extraction, PCR amplification, and sequencing
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 ddH 2 O, 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) (Vilgalys and Hester 1990;White et al. 1990;Liu et al. 1999). The PCR cycling amplification conditions incorporated the following modifications: LSU initial denaturation was at 95 °C for 3 min, followed by 35 cycles at 95 °C for 30 s, 56 °C for 45 s, 72 °C for 1 min, and a final extension of 72 °C for 10 min. The PCR cycling for ITS was as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles at 95 °C for 30 s, 55 °C for 1 min and 72 °C for 1 min and a final extension of 72 °C for 10 min. The PCR cycling for RPB2 was as follows: initial denaturation at 96 °C for 3 min, followed by 35 cycles at 95 °C for 35 s, 54 °C for 10 s, 72 °C for 10 s, and a final extension of 72 °C for 5 min. The sequencing of PCR products was carried out by Sangon Biotech Co., Shanghai, China. The nuclear ribosomal Internal Transcribed Spacer region (nrITS) of the fungi was amplified and the sequence was deposited in GenBank to obtain the accession number.

Sequence alignment and phylogenetic analyses
The sequence of the new record was subjected to standard BLAST searches of Gen-Bank 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 1; Amauroderma calcitum D.H. Costa Rezende & E.R. Drechsler-Santos (FLOR:50931) (Costa-Rezende et al. 2017) was used as the outgroup taxon. Sequences were aligned with MAFFT online server (Katoh and Standley 2013), and manually adjusted using Bioedit v. 7.2.5 (Hall 1999). Alignments were checked and optimized manually when necessary. Maximum parsimony (MP) analysis was performed with PAUP v. 4.0b10 (Swofford 2002). Maximum likelihood analyses (ML) were executed on the CIPRES webportal (Miller et al. 2010), performed using RAxML-HPC2 on XSEDE v. 8.2.8 (Stamatakis 2014), and carried out using raxmlGUI v. 1.3.1 (Silvestro and Michalak 2011). The best fitting substitution model for each single gene partition and the concatenated data set were determined in Mr-Modeltest 2.3 (Nylander 2004). Bayesian inference posterior probabilities (PP) with GTR+I+G model was used for each partition. Bayesian Markov Chain Monte Carlo (MCMC) analyses were conducted in MrBayes v. 3.2.2 (Huelsenbeck and Ronquist 2001). The number of generations was set at 1,000,000, with trees being sampled every 100 generations (a total of 10,000 trees), resulting in an average standard deviation of split frequencies below 0.01. Based on the tracer analysis, the first sampled topologies of 2000 trees representing 20% of burn-in phase were discarded. The remaining 8000 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (Larget and Simon 1999).
Phylogenetic   Optimal conditions for mycelial growth 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 2. All media petri dishes were incubated at 25 °C under dark conditions. In order to discover the mycelial growth rate, colony diameter (mm) was measured; and colony averages calculated by averaging the vertical and horizontal lengths. Mycelial characteristics on the agar surfaces were recorded. Mycelial density was determined by following Kadiri (1998) as very scanty (+), scanty (2+), moderate (3+), somewhat abundant (4+), and abundant (5+). The optimal conditions, growth rate, and mycelial density were carried out in five replicates. 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 (ANO-VA) analysis using the SPSS program (Softonic International SA, Barcelona, Spain).

Optimal media conditions for mycelial growth and characteristics of mycelial cultures
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 3). After 10 days of incubation, the agar surface was fully colonized with a white (6A1) to pale orange (6A2-6A3) mycelium. The best mycelium colony diameter was observed on PDA, MEA, and YPD media, following YMA, RBA, YEA, and CZA, respectively.
Mycelial morphology and colony color characteristics differed on each agar media (Fig.  3). For instance, the morphological characteristics of G. tropicum growth on CZA medium were expressed as a very scanty, cotton colony (Fig. 3A). The colony on YEA medium was similar to that of CZA, although YEA exhibited greater density and biomass (Fig. 3E). Mycelial morphological characteristics on MEA and YPD were similar; both were expressed as an abundant (5+) massive cottony colony with orange white to pale orange (6A1-6A3) colony (Fig. 3B, G). The PDA medium, by contrast, showed a somewhat abundant (4+) white cotton colony (Fig. 3C), which was a slightly dark golden yellow (5A7) colony after 18 days of incubation (Fig. 2E, F). Moderate colony growth (3+) was observed on RBA (Fig. 3D). Abundant white massive cottony mycelia, with a radius from the center towards the edge of the petri dish, were observed on the YMA medium (Fig. 3F). Furthermore, filamentous colonies were observed in all media, except for CZA and YEA.

Optimal pH conditions for mycelial growth
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 4).  56.10 ± 0.07 b 8.10 5+ Table 5. 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).

Discussion
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 Wang et al. (2012), as well as Yang and Feng (2013), whose studies indicated that G. tropicum forms a sister clade with G. multipileum, exhibiting macro-morphological characteristics of strongly echinulate basidiospores (Wang et al. 2009). Ganoderma tropicum and G. multipileum were also shown to be the sister groups of G. lingzhi, which is in the same clade of species distributed in China (Wang et al. 2012). Ganoderma tropicum has been widely reported in tropical areas; however, no specimens have been recorded in Thailand prior to this study (Wang et al. 2009;Zhang et al. 2015). The morphological characteristics of the G. tropicum strain from our study are similar to other G. tropicum specimens described from other tropical areas, including in mainland China (Cao et al. 2012;Wang et al. 2012;Hapuarachchi et al. 2018a), South America (Gottlieb and Wright 1999), and Taiwan (Wang et al. 2009). Although there is a high degree of variability in the macro-morphological characteristics of Ganoderma tropicum specimens found around the world, certain common characteristics can be seen. These common characteristics include a distinct reddish-brown pileal surface, with sessile to dimidiate basidiocarps. Furthermore, there are some similarities between G. tropicum and other Ganoderma species. According to Cao et al. (2012), among the Chinese Ganoderma species, G. flexipes, G. multipileum, G. sichuanense, G. lingzhi, and G. tsugae are morphologically similar to G. tropicum, having a reddishbrown pileus surface, ellipsoid basidiospores, and cuticle cells.
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 (Cao et al. 2012;Wang et al. 2012). Ganoderma multipileum is distinguished from G. tropicum by having mostly concentric growth zones in context, and varying the homogeneous context when maturity (Wang et al. 2012). Ganoderma sichuanense is separated from G. tropicum by its usually formed flabellate to reniform, concave or convex basidiocarps, and also by its ovoid basidiospores which are truncate at the apexes (Yao et al. 2013). Ganoderma tsugae is separated from G. tropicum by absence of the melanoid bands, and also thin dissepiments when mature (Wang et al. 2012).
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 (Simonic et al. 2008;Dang et al. 2018). Although identical fungal species are able to grow on different agar media, the morphological characteristics of the mycelia can be expressed differently, and we therefore conclude that each ingredient in each agar medium affects the morphological characteristics of the resultant culture. The optimal pH was evaluated by using the PDA media, and pH 7-8 was found to be the optimal pH range. Here, we found that G. tropicum grows well in an alkali pH range, as its fruiting body was also collected on the substrate at pH 8 in nature (data not shown). The optimal PDA media at pH 7, incubated within the temperature range at 25-28 °C, were found to be the most suitable for G. tropicum mycelial growth, while temperatures lower than 15 °C or higher than 30 °C are not suitable for mycelial growth. Luangharn et al. (2017) reported that the non-laccate Thai strain of G. australe grew well on PDA media, at a pH of 7-8 and at temperature range of 25-30 °C. This study revealed similar mycelial growth conditions with other Ganoderma species that have been previously evaluated (Lee et al. 2008;Jo et al. 2009;Magday et al. 2014). In light of medicinal mushroom consumption trends, G. tropicum has a high potential for commercial production. Further studies will evaluate the best method to optimize the mushroom spawn and growing substrate for bringing Thai G. tropicum cultivation into high-yield production, and also establish whether other Ganoderma species remain to be discovered in Thailand.

Conclusion
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. nan, 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.