First report of Gymnopilus ochraceus Høil. 1998 (Agaricomycetes: Agaricales: Hymenogastraceae) from India and determination of bioactive components

A rare Gymnopilus species G. ochraceus Høil. collected from live Ficus platyphylla Del. tree is described and illustrated for the first time from India. Morphological and microscopic characters with mo - lecular and biochemical analysis has been discussed. To indicate that, 25 species of Gymnopilus have previously been recorded from India and the species Gymnopilus ochraceus is hereby reported for the first time, thus making a total of 26 species from India.

ISSN 0974-7907 (Online); ISSN 0974-7893 (Print) The genus Gymnopilus (family Hymenogastraceae) is represented by 200 species in the world (Kirk et al. 2008). Members of the genus are characterized by yellow to brown, ferruginous or purple fruiting bodies, saprotrophic nature, cortinoid to membranous veil and a rusty brown spore print (Kaur et al. 2015). The genus was confirmed by roughened basidiospores that range from verrucose to rugulose; capitate to sub-capitate, ventricose cheilocystidia and clamp connections present on almost all kinds of hyphae (Khan et al. 2017). Shape and size of the spores and cystidia are considered important characters for distinction among the species (Rees et al. 2004). Based on the pigments and the nonmycorrhizal habit, Kühner (1984) classified Gymnopilus together with Galerina Earle in the Strophariaceae family. Later, Singer (1986) placed it in family Cortinariaceae due to the ornamentation and lack of germinal pore of the basidiospores. Currently, according to the results of phylogenetic analysis, Gymnopilus forms an independent clade called "Gymnopilae" which is not related to any of the two families mentioned above by Matheny et al. (2006) and is currently placed in the family Hymenogastraceae (Kirk et al. 2008). 25 species of Gymnopilus have been reported from India (Kulkarni 1990;Thomas et al. 2003;Ministry of Environment and Forests 2011;Farook et al. 2013;Kumar et al. 2014;Kaur et al. 2015). Two species of Gymnopilus have been During the present study, mature fruiting body of G. ochraceus were collected on the living tree of Ficus platyphylla Del. from Kate Bhogaon, Panhala, Kolhapur district (M.S.), India. Morphological and microscopic analysis have been described in this paper. Analysis of ITS rDNA sequence was done to evaluate phylogenetic relationships and Gas Chromatography-Mass Spectrometry (GC-MS) was done for determination of bioactive components.
Abstract: A rare Gymnopilus species G. ochraceus Høil. collected from live Ficus platyphylla Del. tree is described and illustrated for the first time from India. Morphological and microscopic characters with molecular and biochemical analysis has been discussed. To indicate that, 25 species of Gymnopilus have previously been recorded from India and the species Gymnopilus ochraceus is hereby reported for the first time, thus making a total of 26 species from India.

Material and Methods a) Morphological and microscopic analysis
Morphological and ecological characters were noted in the field. Microscopic observations of fresh fruiting body was done with the help of 1.5% phloxine B staining and Lawrence & Mayo N-300M research microscope.

b) Identification of fungal strains
The identification of isolates was carried out at the sequencing facility of National Centre for Microbial Resource (NCMR), National Centre for Cell Science, Pune. At the facility, genomic DNA was isolated by the standard phenol/chloroform extraction method (Sambrook et al. 1989), followed by PCR amplification of the ITS regions using universal primers ITS1 [5'-TCC GTA GGT GAA CCT GCG G-3'] and ITS4 [5'-TCC TCC GCT TAT TGA TAT GC -3']. The amplified ITS PCR product was purified by PEG-NaCl precipitation and directly sequenced on an ABI® 3730XL automated DNA sequencer (Applied Biosystems, Inc., Foster City, CA) as per manufacturer's instructions. Essentially, sequencing was carried out from both ends so that each position was read at least twice. Assembly was carried out using Lasergene package followed by NCBI BLAST against sequences from type material for tentative identification (Boratyn et al. 2013).

c) GC-MS analysis
10% methanolic extract of dried fruiting bodies was sonicated for 60 minutes at 35°C followed by centrifugation at 10,000 rpm for five minutes. Supernatant was used for GC-MS analysis using Shimadzu, Japan TQ 8050 plus HS 20. Helium was used as the carrier gas at a flow rate of 1ml/min and an injection volume of 1.0 µL. Injector temperature was 250˚C; ion source temperature 250˚C. The oven temperature was 60˚C isothermal for 2.0 min, with an increase of 10˚C/ min to 250˚C, then 5˚C/min to 275˚C, ending with 10 min. isothermal at 275˚C. Detector voltage was 0.7ev.

Conclusion
The above described species G. ochraceus is reported for the first time from India. It clearly indicates that G. ochraceus is extremely rare species. Morphological and microscopic study along with ITS rDNA analysis confirms the species authentication. Many species of Gymnopilus are bitter or foul to taste, some are hallucinogenic and edibility of majority is unknown. There are many species and little consensus on identifying them. Microscopically they can be confused with Pholiota species and with the   deadly Galerina marginata complex. Microscopically, spores of Gymnopilus species are finely roughened (warty) and lack an apical pore.

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