{'fraction_non_alphanumeric': 0.034557140341609435, 'fraction_numerical': 0.02764571227328755, 'mean_word_length': 5.101799623959173, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 4, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
A Study on the Diversity and Habitat Specificity of Macrofungi of Assam, India

Fungi play a significant role in industry, agriculture, medicine, food industry, textiles, and bioremediation (Danielson et al., 1989; Manzi et al., 1999; Krzywinski et al., 2009; Changet al., 2004). Fungi can be broadly classified into three different forms viz., yeast, moulds and macrofungi or mushrooms. Macrofungi are large, conspicuous sporebearing structures and have fleshy, tough umbrella like sporophores that bear holobasidia on the surface of gills or lamellae that hang down from the cap belonging to basidiomycetes and ascomycetes (few under Zygomycota) (Alexopoulus et al., 2000). Macrofungi are found in varied types of habitats, depending on the composition of tree species and other substrates. The distribution of macrofungal species is low in hot and dry seasons while they are abundant in spring and autumn due to favourable climate and abundance of flora during that time (Pilz et al., 2001). Similar to yeast and moulds, macrofungi also possess immense biological and economic impact since they serve as food, medicine, biocontrol agents and produce of bioactive compounds such as anti-oxidant, International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 12 (2017) pp. 275-297 Journal homepage: http://www.ijcmas.com


Introduction
Fungi play a significant role in industry, agriculture, medicine, food industry, textiles, and bioremediation (Danielson et al., 1989;Manzi et al., 1999;Krzywinski et al., 2009;Changet al., 2004). Fungi can be broadly classified into three different forms viz., yeast, moulds and macrofungi or mushrooms. Macrofungi are large, conspicuous sporebearing structures and have fleshy, tough umbrella like sporophores that bear holobasidia on the surface of gills or lamellae that hang down from the cap belonging to basidiomycetes and ascomycetes (few under Zygomycota) (Alexopoulus et al., 2000). Macrofungi are found in varied types of habitats, depending on the composition of tree species and other substrates. The distribution of macrofungal species is low in hot and dry seasons while they are abundant in spring and autumn due to favourable climate and abundance of flora during that time (Pilz et al., 2001). Similar to yeast and moulds, macrofungi also possess immense biological and economic impact since they serve as food, medicine, biocontrol agents and produce of bioactive compounds such as anti-oxidant,

ISSN: 2319-7706 Volume 6 Number 12 (2017) pp. 275-297
Journal homepage: http://www.ijcmas.com Studies on the taxonomy and diversity of macrofungi have gained significance during the recent years, as many macrofungi are facing the risk of extinction because of habitat destruction. We report, here a systematic and detailed study on the diversity and distribution of wild mushrooms in the state of Assam, India, based on phenotypic and molecular characteristics. A total 44 samples were collected from various locations of Assam in different seasons during 2013-2015. Out of the total 44 samples, about 18 macrofungi species occurred during summer season, 9 species were recorded during rainy, 13 species were frequently found during autumn, 5 species in winter and 8 species were prominent during spring. The soil was found as major habitat for 18 collected species, while 10 species were collected from the tree/hardwood tree. This study indicated that the diversity and distribution of macrofungi are dependent on the plant community and the environmental conditions. Molecular characterization based on rDNA -ITS-((Internal transcribed spacer) sequences revealed that the 44 samples belonged to 16 macrofungal families. Out of the 44 samples, 23 samples were reported to be edible and amongst the rest 21 non-edible strains, 5 strains have medicinal properties, 6 strains are poisonous, two has industrial application and the rest have not been fully explored.
anti-diabetic, hypocholesterolemic, antitumor, anti-cancer, immunomodulatory, antiallergic, nephroprotective, and anti-microbial agents (Patel et al., 2012). Macrofungi are used in the bioremediation of industrial waste and in the accumulation of heavy metals from the environment (Tuli et al., 2013). There are about 2166 worldwide recognized edible species that include about 470 species possessing medicinal properties. Furthermore, macrofungi are useful indicators of health or age of an ecosystem (Boa et al., 2004).
Assam, situated in the Northeast of India is a constituent unit of the Eastern Himalayan Biodiversity Region; one of the two biodiversity "Hot Spots" in the country. The climatic conditions along with varied physical features in Assam, makes it rich in diversity of ecological habitats such as forests, grasslands, wetlands, which harbour and sustain wide range of floral and faunal species. Assam has a sub-tropical monsoon climate with an average rainfall of around 1,500 mm per year. The day-time temperature in summer, the rainy season, rises to around 35ºC and in winter cools to 25ºC with a night time minimum of around 10ºC which is favorsthe growth of most of the macrofungal species (Das et al., 2005). Although, macrofungi are good resources for agroindustrial, medicinal (Veena et al., 2012) and commercial purposes, very few studies have reported the macrofungal diversity of Northeast India (Khaund and Joshi, 2014). Several varieties of macrofungi are being consumed by the local tribes of Assam which are harvested from wild habitats but no efforts have been made to cultivate these macrofungi for commercial production. Moreover, there are no scientific reports related to characteristics of the wild edible macrofungi which are slowly disappearing due to habitat loss. Because of these reasons, the farmers and entrepreneurs of Assam have not been able to exploit its agro-economic potential. Therefore, it is crucial to document the diversity, distribution and abundance of these macrofungi in Assam. Such study will not only pave the way towards domestication and commercialization of the wild species for economic benefits but will also help in establishing a strong basis for molecular taxonomy. In this study we aimed to collect macrofungi from different habitats of Assam followed by their taxonomic evaluation using phenotypic and molecular characteristics.

Collection of macrofungi
Systematic and periodical survey of different forests, tea gardens and other habitats were undertaken during November, 2013 to April, 2015. For this study, the opportunistic sampling of macrofungi protocol, was followed (Mueller et al., 2004). The sampling method involved walking throughout the entire study region and collecting the macrofungi covering up to 80% of the area. Sampling of the ground-dwelling macrofungi involved careful extraction of the fruit body from the soil and leaf litter, ensuring that the basal surface was intact. The tree-dwelling macrofungi were sampled by cutting off the basidiomata from the trees without disturbing the spore surfaces present below. Different species exhibit different fruiting phenologies, which vary from month to month and at different altitudes and regions.
The survey and collection of mushroom was done from different locations of Assam viz. Nalbari, Barpeta, Jorhat, Sibasagar, Karbi Anglong, Kamrup, Golaghat, Goalpara, Tezpur, Tinsukia, Rangia and Nagaon under different agro-climatic conditions, during the year 2013-2015. The macrofungal samples were collected in different seasons of the year; rainy season (July and August), winter (December and January), spring (March and April) and summer (May and June) during 2013-15. Soft macrofungi were collected carefully by using forceps/free hand, while the macrofungi growing on wood were collected along with small part of wood. All collected samples were carefully wrapped in aluminum foil, placed in an air-tight zip-lock bag and labeled with the collection number, location, date, and other data. The photograph of the mushroom was taken in their natural habitat.

Morphological characterization
Morphological characterization of the macrofungal strains were carried out based on the cap size, cap structure, colour of the fruiting body, dimensions of stipe, gill shape and gill colour as described earlier (Kumar et al., 2015). Further, the fresh sporocarps of mushroom species were cultured to obtain vegetative mycelium from the tissue (small pieces cut from section between the pileus and stipe) using Potato Dextrose Agar (PDA) media and incubated at 25 ± 2°C.

Molecular characterization and phylogenetic analysis of the macrofungal strains
For Molecular characterization of the 44 macrofungal isolates were performed by sequencing the rDNA internal transcribed spacer (ITS) region. For DNA extraction, individual strains were cultured on PDA at 25 ± 2°C for 7 days to obtain mycelia. The genomic DNA was extracted from the pure mycelia using the protocol of . Amplification of the ITS (Internal transcribed spacer) region of the nuclear ribosomal repeat unit was done using universal primer set ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (Thatoi et al., 2014). PCR reaction mixture was prepared as per TaKaRa Ex Taq® DNA polymerase protocol (Takara, Japan) with 20 pmols of each primer, 2U Taq DNA polymerase and 50 ng genomic DNA in a final volume of 50 μL and amplification was performed using a thermal cycler (Applied Biosystems, USA). The PCR program was as follows: initial denaturing step of 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 49°C for 1 min and elongation at 72°C for 1 min; a final extension step at 72°C for 7 min. The amplified products were sequenced as per the BigDye Terminator sequencing protocol in an ABI 377 automated DNA Sequencer (Applied Biosystems, USA). The ITS rDNA sequence reads obtained after sequencing were assembled into contig using CodonCode Aligner (CodonCode Corporation, USA). Sequence similarity tool BLAST was employed to find the similarity of the sequences with known 16S rDNA sequences in the GenBank database and the sequences were deposited in GenBank of NCBI to obtain the accession numbers. Phylogenetic analysis of the isolates was carried out based on ITS-rDNA sequences in MEGA6 software (Tamura et al., 2013). A phylogenetic tree was constructed using the ITS-rDNA sequences of the macrofungal strains along with other similar or related macrofungal ITS-rDNA sequences retrieved from the NCBI GenBank. The sequences were aligned with Clustal W using default parameters and a neighbor-joining tree was constructed using MEGA6 software (http://www.megasoftware. net/) with Kimura-2 parameter correction and 1000-step bootstrap.

Sample collection and morphological characterization
A total of 44 macrofungal samples were collected from different places of Assam, viz., Nalbari, Barpeta, Jorhat, Sibasagar, KarbiAnglong, Kamrup, Golaghat, Goalpara, Tezpur, Tinsukia, Rangia and Nagaon ( Fig. 1) during the study period. The details of the sample collection sites are given in Table 1 The morphological taxonomic system of classification given by Ainsworth (1963) and modified by Webster (2007) was used for characterization of the macrofungal samples collected from different locations (Table 2). Macroscopic features of the carpophores pertaining to pileus, lamellae, stipe, etc. which play an important role in the taxonomy of macrofungi have been discussed. The photographs of the collected macrofungal samples are shown in Figure 2(a) and 2(b). The fruiting bodies of macrofungi were found to be distinctly different from each other. Some species that grew on wood had caps that attach directly to the wood (e.g. Pycnoporus, Ganoderma). In some cases, the cap was semi-circular and attached by the straight edge while in others the stalk (also called the stipe, or stem) may be central and support the cap in the middle and in Pleurotus, the stalk was off-central or lateral. The morphological data of the collected samples are detailed in Table 2. Similar studies were carried out by Ram et al., (2010) where several edible fleshy fungi were collected from the wilds in Eastern Uttar Pradesh during the rainy season on dead and decaying plant or animal remains. Kalita et al., (2016) documented wild edible macrofungi from Shyrwat and Upper Shillong Reserve Forests of Meghalaya, Northeast India where a total of 22 macrofungi were collected during the rainy season (July to September) 2014, and identified on the basis of macroscopic and microscopic characteristics. The diversity of wild macrofungi in Nagaland, India was reported by Toshinungla et al., (2016) where a total of 87 species of wild macrofungi were collected and identified and out of the collected macrofungi, 37 species were identified as edible, 21 species medicinal, 5 poisonous and 37 inedible/unclassified. Surveys were conducted in Narpuh Reserve Forest, Meghalaya, India to unveil the macrofungal diversity and identified a variety of macrofungi on the basis of macro-and microscopic characteristics.

Seasonal occurrence of the macrofungal species
Climatic and edaphic conditions prevailing in Assam favoured the occurrence of diverse macrofungi. Our study revealed that 18 species occurred during the summer season, 9 species were recorded during rainy, 13 species were frequently found during autumn, 5 species during winter and 8 species grew mostly in the spring. Comparison of the seasonal occurrence is depicted in Figure 3. Some species were observed to overlap two seasons for e.g. Pleurotus sapidus, Chlorophyllum molybdites were found both in rainy and autumn season; Physisporinus vitreus in both autumn and winter season; Bolbitius titubans and Auricularia sp. grew both during summer and rainy season. Moisture is one of the major factors influencing the fruiting of macrofungi. During the rainy seasons, the availability of adequate moisture favor the growth of the macrofungi. Species diversity was higher in the rainy seasons compared to the early dry seasons, as there is adequate moisture available during the rainy seasons. While the macrofungal species collected in the early dry season was predominated by the Polyporales, since there is a decrease in rainfall and relative humidity along with increase in temperature and sunshine, most of the fleshy macrofungi cannot withstand these conditions. Singh and Prasad (2003) recorded a number of mushroom spp. during 1997-98 and 1998-1999. The occurrence of species was found to be greater in September during 1997-98 and in August in 1998-99, while July and October exhibited a lesser number of macrofungi. However, in another investigation, July month of each year (2002-05) showed a greater number of macrofungal species. A varying number of macrofungi of different species in different months may be due to low relative humidity, low temperature range, very little and no rainfall.
It was observed that the diversity of the macrofungal species depend on the habitat (Fig. 4). The highest numbers of samples were from Golaghat followed by Nagaon and Goalpara districts of Assam. The soil was found to be the best source of diverse macrofungal species followed by tree and deadwood. A variety of habitats i.e. soil, tree, wood, litter, lawn, etc. supports growth for macrofungi and soil was found as major habitat for 18 collected species, while 10 species were collected from the tree/hardwood tree. The variation observed in occurrence of mushroom species in various habitats may be due to their particular mode of nutrition; the macrofungi growing on the soil are symbiotic, on rotting and dead wood are saprophytic and on trees are parasitic. Cosmopolitan distribution of macrofungi have also been reported (Suryanarayanan et al., 2004), certain species of mushroom are associated with particular kinds of trees and plants (Hawksworth, 2001).      . 1= M3, 2=Assam, 3 =T1, 4=O, 5= Sap, 6=  CN,7=H10, 8= K, 9=S1,10= AM,11= HN,12= N1,13= V, 14= N2,15= P2,16= T2,17= H1,18=  C1,19= G2, 20=H2, 21=H4, 22= H7, . 25= AP3, 26= AP5, 27= AP6, 28= AP7, 29=  AP8, 30= AP9, 31= AP11, 32= AP12, 33= AP13, 34=AP14, 35= AP15, 36= AP16, 37= AP17,  38= AP1, 39= AP19, 40= AP20, 41= AP21, 42= AP2 43= AP24,  The genomic DNA was subjected to PCRamplification for the ITS region of the rDNA using ITS1 and ITS4 primers. ITS regions of fungal ribosomal RNA are highly variable sequences, which can be useful markers for determining fungal species. The approximate size of the amplified PCR products (~750bp) was purified and sequenced using ABI 377 automated DNA Sequencer (Applied Biosystems, USA) and resulting contigs were analyzed with CodonCode Aligner version 4.1 (CodonCode Corporation, USA). The consensus sequence obtained for each strain was compared with the ITS rDNA sequences of fungi available in NCBI through a BLAST search and the sequence data has been submitted to GenBank, NCBI and the accession numbers were obtained (Table 3). The sequences were aligned with the help of Codoncode Aligner and analysed for homology using nBLAST (NCBI) to obtain possible identities (Altschul et al., 1997). The BLAST results displayed 97-99% homology with the 44 isolates under study (Table 3). The comparison of ITS gene from different strain and different species indicates that there exists high species-specific homogeneity.
Many molecular markers have been applied in phylogenetic studies in order to decipher the relationships among species of Basidiomycetes (Shtaer et al., 2005;Shnyreva and Shtaer, 2006). However, internal transcribed spacer (ITS) I and II the two noncoding polymorphic regions, located between highly conserved regions of 18S, 5.8S and 28S rRNA genes (Agarwal et al., 2015), have been widely employed to assess the phylogenetic relationships of mushroom species (Choi et al., 2007). In India, Singh et al., (2006) first reported molecular characterization of mushroom using ITS primers specific to macrofungi and identified 18 mushroom germplasms based on ITS sequence polymorphism. Seven of these isolates were identified as Podaxis pistillaris, six belonged to Phellorinia herculean, four were identified as belonging to Phellinus igniarius and only one was identified as Gymnopilus subearlei. A recent report by Rajaratnam et al., (2012) indicated successful identification of wild macrofungi based on ITS (ITS1 and ITS4) sequence. Their study revealed that the wild mushroom belonged to Agaricomycetes which was a new report of a mushroom in India. Some of the wild edible macrofungi have also been reported from Manipur and Arunachal Pradesh of North East India (Sing et al., 2002). Few strains of Basidiomycetes have also been reported from Assam (Baruah et al., 1971). Verma et al., (1987) Verma et al., (1995) carried out a macrofungal survey of the North East Hill region of India and confirmed ninety-five species of higher fungi, among these, 85 species were new records. Recently, diversity of wild edible macrofungi from Khasi hills of Meghalaya, India have been reported and a total of 11 different species were identified based on spore morphology (Khaund and Joshi, 2013). Three new species of Russula (Russulasharmae, R. dubdiana and R. sikkimensis) from Sikkim (India) have been reported by Das et al., (2013).

Phylogenetic analysis
Phylogenetic analysis was carried out by comparing the query sequences against a nonredundant database for comparative analysis of ITS partial sequences. The phylogenetic tree generated through the Neighbor-Joining (NJ) method was well supported by the bootstrap values within their nodes. The phylogenetic analysis using partial ITS rRNA gene sequences revealed that the isolate showed closeness with each other. However, further such studies involving more number of isolates (taxa) can better depict their phylogenetic position at a community level. Phylogenetic analysis using the Neighbour-Joining tree of the identified strains is shown in Figure 5. The evolutionary history was inferred using the Neighbor-Joining method and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Efronet al., 1996). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. All the 44 macrofungal isolates were grouped into ten clusters (Fig. 5)

Classification of the macrofungal species
Classification of the macrofungal species (Table 4) revealed that all the 44 strains belonged to the division Basidiomycota. The macrofungal species can be distributed into 16 families viz.
It was observed that, among the 44 samples, 23 strains were edible and among the 21 nonedible strains, 4 strains have medicinal properties, 6 strains are poisonous, one has industrial application and the rest are not fully explored. Singer (1986) recognized 230 genera and 5658 species spread over 17 families in order Agaricales. Previous reports worldwide indicated the edibility and medicinal properties of these species and a complete list of all macrofungal species and countries can be retrieved from www.wildusefulfungi.org (Schwarze et al., 2011).
Scientific classification of the identified strains based on their molecular data has been depicted in Figure 6. The Basidiomycota contains about 30,000 described species, which is 37% of the described species of true fungi (Kirk et al., 2001). Zhao et al., (2011) provide an overview of recent work on understanding phylogenetic relationships within the Agaricales, a major group within the Agaricomycotina that is the largest clade of mushroom-forming fungi (Matheny et al., 2006).
A timely research regarding isolation, identification and characterization of the existing mushroom flora is essential due to over exploitation and habitat destruction. Thus, our study was an attempt to survey; collect valuable wild macrofungi and their identification based on phenotypic and molecular characteristics, to know the mycotreasure available in this region that is yet to be fully explored. In this study we observed that 23 strains were edible and the rest 21 were non-edible. Among the nonedible strains, 4 strains have medicinal properties, 6 strains are poisonous, one has industrial application and the rest are not fully explored. Further studies on this less explored may provide valuable information as macrofungi have a great potential for the production of useful bioactive metabolites and drugs. The list of macrofungi in this study provides the baseline information needed for the identification based on their morphological and molecular characteristics.