Bioprospection of Basidiomycetes and molecular phylogenetic analysis using internal transcribed spacer (ITS) and 5.8S rRNA gene sequence

Macrofungi belonging to the phylum Basidiomycota are mostly used as medicinal mushrooms in many countries. In the present study, hundred basidiocarp of macrofungi were collected from Tamilnadu during rainy season. The basidiocarp was found in association with root/trunk of living trees, wood log and decayed matter. Among the hundred basidiocarp, 49 were grown into axenic cultures. Notable variations in the macroscopic characteristics of the basidiome and culture morphology were observed. To study the genetic diversity, the molecular taxonomy of the isolates was carried out using internal transcribed spacer (ITS) and 5.8S rRNA gene sequence marker. Thirty-two strains belonging to the order Polyporales, Hymenochataeles and Russuales under the division Basidiomycota were classified based on phylogeny analysis. This study provides first evidence for the occurrence of species Fulvifomes fastuosus (LDCMY39 and LDCMY43) and Ganoderma wiiroense (LDCMY02, LDCMY08, LDCMY11, LDCMY17 and LDCMY19) from southern India. Molecular evidence for the existence of Phellinus badius was given for the first time as well. These data enhance our understanding on the diversity of macrofungi in India, which could be further exploited for biomedical applications.

The kingdom fungi are a distinct group of eukaryotic organisms encompassing about 1.5 M species 1,2 , where 77,000 fungal species are identified by ITS sequence and been reported in GenBank repository 3 . They are identified by filamentous mycelium, absence of motile cells and chlorophyll, presence of chitin-rich cell walls and secretion of external digestive enzymes to degrade the food. Their mode of reproduction is via asexual and sexual spores 4 . These are considered to be the key decomposers of terrestrial ecosystems and known to play crucial ecological role [5][6][7] . Wild mushrooms from the natural habitat have profound biological and economic impact due to their major role in ecosystem maintenance [8][9][10] . Destruction of environment is the major threat for fungal diversity; exploration of diversity of macrofungi and their taxonomy are acquired importance for reforestation programmes 11 .
The phylum Basidiomycota includes largely of fleshy fungi (e.g., mushrooms, toadstools, rusts) and ranked second with approximately 23,000 species 4 . Abundant growth of Basidiomycetes are prevalent in the rainy seasons where the environmental conditions such as temperature, relative humidity and sunshine are favourable, which aids them in the breakdown of dead organic tissue 12 . These are the potential indicators of environmental quality 13 . Many fleshy fungi are edible and harmless, but few are poisonous 14 . However, approximately 700 species of Basidiomycetes were reported to exhibit notable pharmacological activities 15,16 . These mainly aids in immune system enhancement, regulation of biorhythm, maintenance of homeostasis and are considered to be the biofactor of effective compounds to cure various diseases as anti-fungal, anti-inflammatory, anti-tumor, anti-viral, anti-bacterial, hepatoprotective, anti-diabetic, hypolipedemic, anti-thrombotic and hypotensive activities 17,18 .
Among the hundred basidiome collected only forty-nine isolates (49%) could be grown in axenic cultures. The mycelial growth significantly varied from 7 days to 30 days. The colour of the mycelia varies for each strain: white, orange white, yellowish white, pale yellow, greyish orange, light yellow, pale orange and brownish orange (Fig. 6, Table 2). The pure cultures of all isolates were stored in mineral oil till further use.
Genomic DNA was obtained and 5.8S ribosomal RNA gene segment was amplified using sequence specific primers. Thirty-two isolates were successfully sequenced and the size of the amplicon ranged from 599 bp to 902 bp. The sequences were deposited in GenBank and accession numbers were obtained (Table 3). Variation in genetic makeup was observed among the isolates from the same environment. Molecular phylogentic analysis was carried out using 52 ITS sequences in which 20 reference sequences were retrieved from GenBank, NCBI to clarify the variation among the sequences. The phylogenetic tree constructed using maximum likelihood (ML) method (Fig. 7). The basidiomycete species were clustered into three clades: Clade 1 -Polyporales, Clade 2 -Hymenochaetales and Clade 3 -Russuales. The three clades are detailed below: Clade 1: Polyporales -Found in all study sites except Ayyanar falls. Eighteen strains were grouped under this clade and fifteen sequences were further categorised under the family Ganodermataceae, two under Polyporaceae and one in Fomitopsidaceae. The isolated strains belong to the Polyporales were Coriolopsis caperata, Fomitopsis ostreiformis, Ganoderma resinaceum, Ganoderma sp., Ganoderma wiiroense and Trametes elegans. Coriolopsis caperata LDCMY42 collected from Nagamalai showed 99% similarity with the strain Coriolopsis caperata DK01 (AM237457). Monophyletic origin of Fomitopsis ostreiformis was determined with 100% bootstrap support. Five strains were identified as Ganoderma wiiroense (LDCMY19, LDCMY08, LDCMY11, LDCMY17 and LDCMY02) and showed highest similarity with the strains reported from United States of America (KT952361 and KT952363). Variations in the genetic makeup as well in the morphology of the Ganoderma wiiroense strains were observed. Majority of the Ganoderma strains were found to be stipitate. Based on molecular analysis, this is the first evidence for the occurrence of Ganoderma wiiroense from India.

Discussion
Fungi are ubiquitous in nature and distributed in all ecosystem. It can survive in diversified habitats such as air, water, soil, litter etc. It contains 1.5 million species, of which 74,000 species are named 4 . The phylum basidiomycota consist of 37% of all described fungal species 33 . Threats to fungi due to habitat destruction are a global concern as they play an important role in human welfare 19 . To understand the distribution and diversity of macrofungi in South India, the basidiomata were collected from living trees, wood log and leaf litters during the rainy season (November to January).
The Basidiomycetes were usually classified based on phenotypic traits; however, classification based on morphological characteristic features alone will be flawed and misleading and the use of molecular classification was found to be more reliable 34,35 . So far, only 5% of fungal strains were isolated as pure cultures and several described species were acknowledged only as herbarium specimens 19 . In the present study, pure culture ( Fig. 6) was raised from 49% of the isolates and the molecular data were obtained for 65% of the isolates. These molecular data helped in identification of the isolates and was used for construction of genetic diversity among the macrofungal isolates. Molecular phylogeny of the macrofungal isolates. The molecular systematics of macrofungi has been studied by various methods using DNA-DNA hybridization, restriction enzyme analysis -RFLP, rDNA, mtDNA and sequencing analysis of ITS 27 . Pectinase isoenzyme 36 , manganese superoxide dismutase 37,38 , ITS and 25S ribosomal sequences 34,35,39 were used to construct molecular phylogeny in macrofungal species. Later, ITS was used as a DNA barcode for fungal identification 32,40,41 . In this study, amplification of nuclear ribosomal ITS was used to identify the isolates. The identified isolates belong to three families namely Polyporales, Hymenochaetales and Russuales. The representative strains of the Polyporales from this study were Coriolopsis caperata, Fomitopsis ostreiformis, Ganoderma resinaceum, Ganoderma sp., Ganoderma wiiroense and Trametes elegans. The isolated strains belonging to Hymenochaetales were Fulvifomes fastuosus, Inonotus rickii, Phellinus sp. and Phellinus badius. Amylosporous sp. was the only strain found in our study from the family Russuales. We are the first to report the occurrence of Ganoderma wiiroense and Fulvifomes fastuosus with morphological and molecular evidence; and also provided the molecular evidence for Phellinus badius from India.   42 reported that G. lucidum (TVK1, India; GenBank FJ982798) was closer to G. wiiroense. In our study, we also found that the G. lucidum FJ982798 was closer to G. wiiroense than any other Ganoderma strains reported in this study.
The genus Phellinus belonging to the Family Hymenochaetaceae were important owing to their medicinal values 18,43 . Three hundred and sixty-seven Phellinus has been reported in the CBS (http://www.punenvis.nic.in/ bd_list.htm). In India, eighteen Phellinus species have been reported from Kerala 44,45 , P. nilgheriensis (Mont.) Cunn., P. shaferi from Gujarat 46,47 and P. badius was described morphologically from Punjab 48 . This study provides the first report on molecular evidence for P. badius from India.   Table 2  The genus Fulvifomes Murrill was segregated from Phellinus Quél., Murrill 49 and typified with F. robiniae (Murrill). It was not accepted as a separate genus and treated as a subgenus of Phellinus till 1999 50 . Later, comprehensive evidences based on molecular phylogenetic analyses proved that it as an independent genus closely associated with Aurificaria Reid and Phylloporia Murrill 51,52 . The key characteristics of Fulvifomes are pileate basidiocarps, a dimitic hyphal system, coloured basidiospores and absence of setae 51 . Species with resupinate basidiocarps and/or hymenial setae were included into Fulvifomes based on morphological studies 43 . Recently, species with monomitic hyphal system were included in Fulvifomes by Zhou 53 .
Fulvifomes fastuosus was described by Bondartseva and Herrera 54 . There are 162 reports available in GenBank on the genus Fulvifomes based on molecular data and among them only 18 sequences were on F. fastuosus. The species F. fastuosus was described from China 43 , Thailand 55 and Sri Lanka 56 . In this study based on molecular phylogeny, two strains collected from Lady Doak College, Tamilnadu, India were identified as Fulvifomes fastuosus.

Macro and micromorphological characteristic features of G. wiiroense, P. badius and F. fastusosus.
The identification based on molecular means has been checked with the macro-and micro-morphological characteristic features and were found to be similar with the reported strains. However, the observation on basidiospores was different from the other reports for P. badius and F. fastuosus. The basidiospores of P. badius are ovoid to subglobose to globose and 4-6 × 4-5.5 μm 44 . Singh and colleagues 48 reported that basidiospores were broadly ellipsoid to subglobose. Our observation shows the P. badius basidiospores were ellipsoid and 4.21-5.54 × 2.83-4.13 μm. The basidiospores of F. fastuosus were subglobose, thick-walled, smooth 4.49 × 4.01 μm 56 . According to Dai 43 , the basidiospores are 5-6.1 × 4.2-5.6 μm. Our observations shows the basidiospores were 3.4-5.7 × 3.1-4.2 μm, which was smaller than Dai 43 , but similar to Ediriweera et al. 56 . However, the variation in the ratio (Q) was the same as previously reported of F. fastuosus strains. The variation in the size of basidiospores might be due to their geographical niche as well as depending on their nutrients from the host species.
Host preference by the macrofungal isolates. There are several factors that influence the distribution of fungi namely ecological niche, climatic conditions, host/substrate type, distribution of fauna and flora 19 . To study host preference, basidiomata were collected from the living trees, wood log, and leaf litters. Later, the basidiomata was identified by molecular classification.
In India, the information on Ganoderma was first published in the early 1900s 57 . Nearly 144 hosts were recorded in India 58 . Among them coconut, betelnut, Casuarina, Areca catechu, Dalbergia sissoo and Toona ciliata 59,60 was observed as obvious host of Ganoderma sp. In India and Sri Lanka, Cocus nucifera showed high incidence as a host for Ganoderma species 58,[61][62][63] . From this study, it was observed that Ganoderma sp. grown on the following host species: Albizzia sp., Tamarindus sp., Azadirachta sp. and Coccus nucifera. Fomitopsis ostreiformis belonging to Ganodermataceae has the host species Albizzia sp., and Coriolopsis caperata from wood log. The newly reported Ganoderma wiiroense has been collected from the trees of Albizzia sp., ( Table 1).
The species Fulvifomes fastuosus belongs to the family Hymenochaetaceae and reported to have medicinal properties 43 . The F. fastuosus has been reported in the trees of Xylocarpus granatum 55 . In this study, F. fastuosus were found in the host trees of Albizzia sp.
The genera Phellinus have wide host range. Globally Quercus sp. is the more susceptible host and, in India Mangifera sp. followed by Acacia, Artocarpus and Albizzia are the predominant host of Phellinus 64,65 . It was observed that Albizzia sp. is the host preferred by the genera Phellinus.
The genera Amylosporus was first reported in India among the Asian countries 66 with bamboo as their host 67 . In this study, the Amylosporus sp. was found in the host Nerium sp. and Albizzia sp. Interestingly from this study, Albizzia sp. is found to be the host preferred by most of the macrofungal isolates. This might be due to the abundance of this species in the vicinity of the collected macrofungi.
To conclude, we have identified and report two new macrofungal species G. wiiroense and F. fulvifomes and molecular evidence for P. badius from India. It was observed that Albizzia sp., as the host preferred by most of the macrofungal isolates. Our data provide the existence of G. wiiroense in India; however, we were unable to trace of out the origin of how G. wiiroense might have cross boundaries. We can only speculate G. wiiroense already exists in India; because of the lack of intense mycological study prior, this is the first report on it. These data gains us insight on macrofugal diversity in India, which can be used for the prospection of macrofungi in biomedical and industrial applications.

Methodology
Sample Collection and culture of isolates. Fresh basidiomata of the wild mushrooms belonging to the division basidiomycota were collected from different locations in Dindigul (Ayyanar falls), Madurai (Lady Doak College Campus, Nagamalai, Pudhupatti), Coimbatore (Kovai Kutralam), Thenkasi and Tirunelveli, Tamilnadu (India) during 2013-2017 on rainy seasons i.e., November to January. The basidiomata were cleaned and aseptically transferred to the lab. After surface sterilization with 70% ethanol, small pieces from the contextual layer of basidiomata 68 were transferred to sterile potato dextrose agar (PDA) medium supplemented with streptomycin. The plates were incubated at 37 °C for 5-7 days. The pure culture was obtained by continuous sub culturing and used for further analysis. The isolates were stored in PDA plates and slants. The basidiomata were then dehydrated with naphthalene balls for future studies.
The radial growth of the mycelium of all the isolates on the PDA medium was measured using a ruler. Five-millimetre mycelial plugs were removed from the growing edge of the 7-day-old pure culture and inoculated on to the centre of the 80 mm petriplates containing PDA. According to Tomkin 69 and our observation, the growth is not constant in the early stage. The lag phase was shorter (1 day) in some strains and longer (5 days) in some strains. The radial/lateral expansion was measured after three days (i.e., 3 rd day for strains with shorter lag phase and 7 th day for strains with longer lag phase) in diameter (in mm), and the number of days taken to completely colonize 80 mm petridish was recorded. All the measurements were made in triplicates. The representative voucher specimens were deposited in the Department of Biotechnology, Lady Doak College, Madurai, Tamilnadu, India. Taxonomical identification of the isolates was carried out based on molecular identification methods.
After identification, the macromorphological characteristic features such as shape, color, hymenial surface of the basidiomata were studied according to published description 70 . Microscopical observations (hyphal system, presence/absence of setae and basidiospores) were carried out using brightfield microscope (Olympus system microscope model CX41). Slides were prepared using 5% KOH and cotton blue 71 .

Molecular characterization of the isolates. Genomic DNA Isolation, PCR amplification and sequencing.
Genomic DNA of all the isolates were extracted as described by Moncalvo et al. 35 . 10 mg of mycelial biomass was homogenized with 3% SDS extraction buffer (3 g SDS, 50 mM Tris, 150 mM NaCl and 80 mM Na 2 EDTA) and  incubated at 60 °C for 20-30 min. The 5.8S nuclear ribosomal RNA gene was amplified using ITS1 (CTTGGTCAT TTAGAGGAAGTAA) and ITS4 (CAGGAGACTTGTACACGGTCCAG) primers 30 . PCR amplification was carried out using the following condition: initial denaturation (95 °C, 2 min), denaturation (94 °C, 45 sec), annealing (50 °C, 45 sec), extension (72 °C, 1.30 min), final extension (72 °C, 5 min). The PCR products were purified and sequenced (Chromous Biotech Pvt. Ltd, Bangalore). The sequences were read bidirectionally for both strands of the entire ITS1, 5.8S rDNA and ITS2 region. The DNA sequence obtained from both the strands was edited and contig assembly was carried out using DNA Baser sequence assembly software (V.4.36.0). The assembled sequences were submitted to GenBank Database.
Phylogenetic analysis. Additional ITS sequences of Basidiomycetes were downloaded from GenBank to clarify the interspecies relationship. The phylogenetic tree was constructed by maximum likelihood (ML) analysis in MEGA 6 software 72 . The tree inference options were set as follows: Heuristic Method Nearest-Neighbor-Interchange (NNI) with the very strong branch swap filter with 1000 bootstrap replicates, gaps were treated as missing.