Antimicrobial Potential, Identification and Phylogenetic Affiliation of Wild Mushrooms from Two Sub-Tropical Semi-Evergreen Indian Forest Ecosystems

The diversity of wild mushrooms was investigated from two protected forest areas in India and 231 mushroom specimens were morphologically identified. Among them, 76 isolates were screened for their antimicrobial potential against seven bacterial and fungal pathogens. Out of 76 isolates, 45 isolates which displayed significant antimicrobial activities were identified using ITS rRNA gene amplification and subsequently phylogenetically characterized using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Sequencing of the ITS rRNA region classified the isolates into 16 genera belonging to 11 families. In total, 11 RAPD and 10 ISSR primers were selected to evaluate genetic diversity based on their banding profile produced. In total 337 RAPD and 312 ISSR bands were detected, among which percentage of polymorphism ranges from 34.2% to 78.8% and 38.6% to 92.4% by using RAPD and ISSR primers respectively. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of selected two methods were structured similarly, grouping the 46 isolates into two clusters which clearly showed a significant genetic distance among the different strains of wild mushroom, with an similarity coefficient ranges from 0.58 to 1.00 and 0.59 to 1.00 with RAPD and ISSR analysis respectively. This reporthas highlighted both DTR and MNP forests provide a habitat for diverse macrofungal species, therefore having the potential to be used for the discovery of antimicrobials. The report has also demonstrated that both RAPD and ISSR could efficiently differentiate wild mushrooms and could thus be considered as efficient markers for surveying genetic diversity. Additionally, selected six wild edible mushroom strains (Schizophyllum commune BPSM01, Panusgiganteus BPSM27, Pleurotussp. BPSM34, Lentinussp. BPSM37, Pleurotusdjamor BPSM41 and Lentinula sp. BPSM45) were analysed for their nutritional (proteins, carbohydrates, fat and ash content), antioxidant potential. The present findings also suggested that the wild edible mushroom strains do not have only nutritional values but also can be used as an accessible source of natural antioxidants.


Introduction
1. Dampa Tiger Reserve (DTR) (23˚23'N-23˚42'N; 92˚16'E-92˚25'E), which is one of the largest wildlife sanctuaries in Mizoram. It covers an area of % 500 km 2 (Fig 1) and the altitude of the reserve is from 800 to 1100 m above sea level with annual rainfall during the years of sampling ranges from 2000 mm to 2500. Maximum rainfall was observed mostly during June to August every year. The winter (October-January) is a cool dry season with few rainy days. Summer (February to May) is largely hot and dry, with occasional thundershowers and pre-monsoon rains in April-May.It has fine loam type of soil which covers over 48% of the total area of the state. The soil pH is %5. 34 and is rich in Nitrogen (N), Phosphorus (P) and Potassium (K). Temperature ranges from 12˚C to 25˚C during winter and between 22˚C to 35˚C during summer. DTR is recognised as sub-tropical semievergreen.
2. Murlen Nation Park (MNP) (23˚32'-23˚41'N; 92˚13'-92˚27'E) is located close to China Hills and covers an area of % 100 km 2 (Fig 1). Altitude ranges from 1,000-1,600 m and the average annual rainfall is about 2500 mm. MNP is classified as sub-tropical semi-evergreen and sub-montane forest. Winter is usually from October to mid-February and summer is from February/March to May. The pH of the soil is %5.92 and the soil type is mostly clayey fine soils which are usually deep and well drained.The temperature during winter ranges from 4˚C to 20˚C and 22˚C to 32˚C during summer.

Collection of wild mushrooms
Selected forests were visited for three consecutive years (2013-15) during rainy and winter sessions (June to December). Permission to collect wild mushrooms from the protected forests was obtained from the Chief Wildlife Warden, Environment and Forest Department, Government of Mizoram, India issued by Mr. LiankimaLailung, Conservator of Forest (WL), Mizoram, India. The wild mushrooms were identified in situ if possible or else the specimens were retained for later identification. The samples were collected in sterile paper bags and brought into the laboratory. Morphological parameters (pileus, lamellae, stipe, spore print and color) were assessed from fresh samples as per Largent and Stuntz [26]. Digital pictures were taken at natural habitat as well as under laboratory conditions and characterized morphologically up to genus level.
Tissue culture of wild mushrooms The collected fruiting bodies were used to raise mycelial culture as per the modified method of Bolla et al. [27]. Briefly, the collected fruiting bodies were cleaned thoroughly by using fabric paint brush. A small piece of tissue from the fruiting body was dipped in 0.05% sodium hypochloride (NaOCl) solution and washed three times with sterile distilled water. The samples were dried on sterilized tissue paper and transferred aseptically on to potato dextrose agar (Hi-Media, Cat No: MH096) media and incubated at 28˚C for 5-7 days.

Evaluation for antifungal potential
All the isolates were screened for their in vitro antagonistic activity by dual culture technique [28,29]. Briefly, one 5 mm agar block of fully grown fungal pathogen was placed at the centre of the petridish containing PDA, and mushroom isolates were kept at the periphery of the plates. Petri dishes were incubated at 28˚C for 7 days and diameter of the fungus growth was measured as compared to control. All the experiments were carried out in triplicate and the percentage of inhibition was calculated using the formula: C-T/C×100; Where, C is the fungal pathogen control and T is the distance of the tested pathogen with the sample.

Evaluation for antibacterial potential
Antibacterial activity was determined by modified agar well diffusion method [30]. Briefly, an aliquot of the tested bacterial pathogens (50 μl) were spread on the surface of the modified LB agar plates using sterile L spreader and well of 6 mm diameter was prepared by using sterile cork borer. The wells were filled with 100 μl (1×10 −4 CFU/ml) of cell free extractof mushroom isolate and the plates were incubated at 37˚C for 24 h. All experiments were carried out in triplicates and the zone of inhibition was measured in mm.

Molecular characterization of Mushrooms
DNA extraction and ITS-rRNA gene amplification. Total genomic DNA was extracted from the freshly grown mycelial mat as described by Cenis [31] with minor modification. The internal transcribed spacer (ITS) region of the rDNA was amplified using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') [32]. PCR reaction mixture was carried out in a total volume of 25 μL containing, 1X PCR assay buffer with 1.5 mM MgCl 2 , 2.5mm dNTPs, 10 pmols of each primer, 50 ng of template DNA and 1 unit of Taq DNA polymerase. The PCR was carried out in the Applied Biosystems thermal cycler with an initial denaturation at 95˚C for 5 min, followed by 30 cycles of denaturation at 95˚C for 1 min, annealing at 57˚C for 1 min, extension at 72˚C for 2 min and a final extension at 72˚C for 7 min. The amplified PCR product was separated on 1.5% agarose gel and visualized under Bio-Rad Gel Documentation System XR+ (Hercules, CA, USA). The products were purified by using the Pure-link PCR Purification Kit (In-vitrogen) and sequenced commercially at Sci-Genom Labs Pvt. Ltd, Cochin, India.
Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) analysis. For RAPD profiling, eleven random decamer primers (Imperial Life Sciences (P) Ltd, (Table 1) were used to produce fingerprinting profiles. The reactions were assembled in a Veriti Thermal Cycler (Applied Biosystems, Singapore) in a total volume of 50 μl containing 5.0 μl of 10X reaction buffer, 1.0 mM MgCl 2 , 0.5 mMdNTPs, 5 pmols of each primer, 1 unit of Taq DNA polymerase and 50 ng template DNA. Negative control reaction without DNA was included in each set. The PCR amplification was carried out with the following conditions: initial denaturation at 95˚C for 5 min, followed by 35 cycles of denaturation at 94˚C for 35s, annealing at 32˚C for 45 s, extension at 72˚C for 2 min and a final extension of 10 min at 72˚C. The amplified PCR product was separated on 1.5% agarose gel using TAE buffer and examined under gel documentation system as described earlier. Ten ISSR primers were selected (Table 1) and amplification was done as per Du et al. [33].
The PCR reaction mixture was performed in 25 μl total volume, containing 50 ng of genomic DNA, 2.5 μl of 10X Taq Buffer with 15mM MgCl 2 , 2.0 μl of 2.5 mM dNTPs, 0.5 μl of 10 pmol ISSR primer,1 μl of DMSO (100%), 0.5 μl of BSA (10 mg/ml) and 2U Taq DNA Polymerase.The amplification conditions were as follows: initial denaturation at 94˚C for 4 min followed by 35 cycles at 94˚C for 50 s, at 45˚C for 50 s, and at 72˚C for 2 min with a final extension step at 72˚C for 7 min. The amplified fragments were separated on 1.2% agarose gel using 1X TAE buffer and the banding patterns were examined under gel documentation system asmentioned above. Phylogenetic analysis. The obtained ITS sequences were compared with type strains retrieved from GenBank/NCBI database using BlastN search program and were aligned using Clustal W [34]. Evolutionary models were selected according to BIC scores (Bayesian Information Criterion) and AICc values (Akaike Information Criterion, corrected) [35] using Mega 5.05 version [34]. The phylogenetic tree was constructed by neighbor-joining method using the model Kimura 3-parameter K2 + I (I = 0.67, R = 1.67) [36] taking Solanumkurzii (KC535794) as an out group. The robustness of the phylogenetic tree was tested by bootstrap analysis using 1,000 replicates using p-distance model [37]. Trees were viewed and edited by using program FigTree 1.3.1.
Amplified fragments of RAPD and ISSR were scored as present (1), or absent (0), the fragments produced were numbered sequentially and entered into a binomial matrix [38]. Only fragments bigger than 100 bases were considered for analysis. The binary matrix was used to calculate the Simple Matching (SM) coefficient, and a cluster analysis was performed using Unweighted Pair Group with Arithmetic Mean (UPGMA) algorithm supported by Numerical Taxonomy SYStem (NT SYS version 2.2).

Determination of Nutritional values of selected wild edible mushrooms
Ash content. Samples (1 g each) were dried for overnight in the oven maintained at 105˚C and weighed, then placed at 575˚C for 5 h. Then, the samples were weighed for final mass and difference between the mass at 105˚C and 575˚C was taken as ash content [39].
Carbohydrate content. Carbohydrate content was estimated by phenol sulphuric acid method with glucose as standard [40].
Protein content. Protein content was estimated by Bradford method using BSA as standard [41].
Fat content. The fat was extracted by Chloroform-methanol extraction and estimation method [42].
Determination of antioxidant potential of selected wild edible mushrooms Extract preparation. The lyophilized powder (1.5 g) was extracted by stirring with 40 mL of methanol (25˚C at 150 rpm) for 2 h and subsequently filtered through Whatman No. 4 paper. The residue was then extracted with 20 mL of methanol (25˚C at 150 rpm) for 2 h. The combined methanolic extracts were evaporated at 40˚C (rotary evaporator BUCHI R-210, Switzerland) to dryness and stored at 4˚C until analysis [43].
Determination of total phenolic content (TPC). Total phenolic content was measured according to the method given by Singleton et al. [44]. Briefly, 0.5 mL of sample was added to 4.5 mL of distilled water and was mixed with 0.2 mL saturated solution of Na 2 CO 3; finally 4.3 mL of distilled water was added to the solution. The reaction mixture was incubated for 60 min in the dark at room temperature and then the absorbance was measured at 725 nm using a microplate spectrophotometer UV-vis (Multiscan™ GO, Thermo Scientific, MA, USA). Total phenolic content was expressed as mg of Gallic acid equivalents (GAE) per gram of dry sample (mg GAE/g). The measurements were done in triplicate.
Determination of total flavonoids content (TFC). The flavonoids content was determined by the calorimetric method of aluminium chloride according to methodology previously described by Chang et al [45]. In this, 0.5 mL was mixed with 1.5 mL of 95% ethanol, 0.1 mL of 10% aluminium chloride, 0.1 mL of 1M potassium acetate and 2.8 mL of distilled water.
Finally, absorbance was measure at 415 nm using a microplate spectrophotometer UV-vis (Multiscan™ GO, Thermo Scientific, MA, USA) after incubation for 30 min in darkness at room temperature. The flavonoid content was calculated in mg Quercetin equivalents per g of dried sample (mg QAE/g).
Scavenging Activity of ABTS Radical. ABTS radical scavenging activity was determined according to Re et al [46] with some modifications. 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was dis-solved in water to a 7 mM concentration. ABTS radical cation (ABTS O+ ) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. The ABTS O+ solution was diluted with water to an absorbance of 0.70 (±0.02) at 734 nm. The reaction mixture consisted of 0.07 mL of extract and 3 mL of the ABTS radical. After incubation for 6 min, absorbance was determined in spectrophotometer at 734 nm. The antioxidant activity was calculated by using the following equation.
A control = Absorbance of negative control at the moment of solution preparation A sample = Absorbance of sample after 45 min The IC 50 values were calculated as the concentration of the sample (mg) required to scavenge 50% of the ABTS free radical. The IC 50 is often used to express the amount of concentration of extracts need to scavenge 50%of the free radicals.
Fourier Transform infrared Spectroscopy (FT-IR) analysis of selected wild edible mushrooms. The selected wild mushroom samples were dried and mixed with FT-IR grade potassium bromide (1:20; 0.02 of sample with KBr at a final weight of 0.4 g). The samples were grounded in agate pestle and mortar for obtaining pellets by hydraulic press. The absorbance Fourier transform infrared (FT-IR) spectra of the samples were recorded using JASCO FT-IR 6800 within the scanning range of 400-4000 cm -1 and 64 scans per second were recorded [47].

Data analysis
The data were calculated in mean ± SD of the values found in triplicates from antimicrobial screening and analysed by one way of analysis of variance (ANOVA) to compare statistical significant differences (P<0.05) between antimicrobial activities of all isolates using Duncan's multiple range test of SPSS software version 6.0. All the experiments were done in triplicates and the mean was taken. Relative abundance of mushroom isolates was compared between the selected natural forests by using Sigma Plot 12.0 version. Venn diagram was prepared online by using Bioinformatics and evolutionary genomics site (http://bioinformatics.psb.ugent.be/ webtools/Venn/).

Assessing Mushroom diversity
The present study documented 231 wild mushrooms, distributed across 64 genera belonging to 36 families, 14 orders and 06 classes. Morphologically, all isolates were classified into two Phylum Basidiomycota (n = 221, 95.67%) and Ascomycota (n = 10, 4.32%). Anatomical characteristics were also recorded of all collected fruiting bodies (Fig 2). Some of the economically important genus found are Lactarius, Trametes, Calocybe, Agaricus, Pleurotus, Schizophyllum, Cantharellus, Russula, Boletus, Termitomyces, Pholiota and Lentinus. Relative abundance of the wild mushrooms was compared between the two selected forests at the genus level (Fig 3) and the result showed that Boletus was the most abundant genus at DTR and MNP with 20.16 and 12.1% respectively. Genus Russula(10.48%) and Amanita (6.45%) were the most dominant genus in DTR as compared to MNP with 9.34% and 3.73% respectively. At the same time, genus Mycena and Trametes were dominant in MNP with 2.8% and 5.6% respectively. However, 19 genera were found in common among both the studied locations (Fig 4). Moreover, some rare genus like Favolaschia and Cookeina were found only in DTR. These results indicated that diversity of wild macrofungi varies between these two places due to different climatic conditions. MNP is known for its dense forest where only 1% sunlight can directly penetrate the forest on a sunny day. It has clayey fine soil which is usually with severe erosion. On the other hand, DTR being on a lower elevation with fine loamy soil, which is richer in humus than the soil type found in MNP. It also has an ideal climatic condition like higher humidity and warmer temperature to support mycelial growth of fungi. All these factorscould be the cause that led the higher diversity of wild mushrooms in DTR than in MNP.

Antimicrobial activity
Out of 231, 76 isolates could be grown on artificial media and were screened for their antimicrobial potential. All isolates were tested for their in vitro antagonistic activity against the selected fungal plant pathogens by dual culture plate assay and showed diverse antagonistic effects. Out of 76, 45 isolates showed positive growth inhibitory activity against atleast three fungal pathogens, with the percentage of inhibition ranging from 25% to 85% (Table 2). All isolates showed positive activity against Fusarium oxy f. sp. ciceri and F. oxysporum whereas C. albicans was the most susceptible pathogen against all wild macrofungi with the exception of 15 isolates BPSM (01, 05, 14, 22, 29, 30, 31, 33, 37, 38, 43, 45, 46, 47 and 48). Five isolates BPSM (31, 33, 37, 38 and 45) exhibited positive antagonistic activity against all the tested pathogens. Among them, isolate BPSM30 indicated highest antifungal activity (78%) against F. proliferatum whereas isolate BPSM41 showed maximum antifungal activity (80%) against F. oxy f. sp. ciceri. The strongest antagonistic activity was found in BPSM33 isolate against F. oxysporum with 85.71% inhibition as compared to control.
All isolates were tested for their antibacterial activity against three bacterial pathogens viz. P. aeruginosa, S. aureus and E. coli. Out of 76 isolates, 37 isolates showed positive effects against two out of three pathogens and all isolates showed positive effect against P. aeruginosa except isolates BPSM17 and BPSM32. E.coliis the strongest pathogen against all the tested isolates except BPSM (03, 13, 31, 33, 37, 38, 45 and 55). The result showed that isolate BPSM01 displayed greatest inhibitory effect (9.63 mm) followed by BPSM48 (9.6 mm) and BPSM47 (9.56 mm) against P. aeruginosa. The result indicated that isolates BPSM01 and BPSM47 had the most significant effect against S. aureusgrowth with 9.6 mm and 9.46 mm respectively. Isolate BPSM03 exhibited broad spectrum antibacterial activity against E. coli(5.36 mm) and S. aureus (4.36 mm), therefore it can be considered as the most potent isolate for further studies ( Table 2).

RAPD analysis
RAPD analysis was employed to detect genotypic variation among 45 selected strains using eleven random primers, which yielded 337 fragments. Amplified fragments in each strain were ranging from four to twelve bands which varied in size from 200 bp to 2500 bp. Of the 337 amplified bands, 272 were polymorphic with an average of 8.4 polymorphic fragments per primer. The percentage of minimum polymorphic ranging from 34.2% (R1) to maximum polymorphic of 78.8% (R5) was observed with an average of 56.8% polymorphism. Two  Table 2. Antimicrobial activity of wild mushrooms against selected bacterial and fungal pathogens.   (Fig 6A).

ISSR analysis
All the potential isolates were further screened to generate ISSR polymorphic DNA banding pattern using ten primers which varied in size from 200 bp to 2500 bp. Cluster B showed all the isolates belongs to Trametes in the RAPD analysis, while cluster B showed highly similar result in ISSR analysis (Fig 6B).

Antioxidant potential
Bioactive properties of the selected six edible mushrooms were assessed by three different assays measuring total phenolic contents, total flavonoids content and scavenging activity of ABTS radical (   In this study for scavenging activity of ABTS radical, the IC 50 values range from 3.25 to 11.43 mg/ml. The lower the IC 50 value of mushroom extract, the higher was their free radical scavenging activity. Among the mushroom extracts we have studied, S. commune BPSM01 with an IC 50 of 3.25 mg/ml and Pleurotusdjamor BPSM41 with an IC 50 of 3.73 mg/ml indicates a significant antioxidant activity in the methanolic extract of these mushrooms.

FTIR Analysis
Mushroom samples were analysed in ATR-FT-IR (Jasco, Japan) and different wave numbers, functional groups and their corresponding polymer of mushroom sample were presented in Table 7. From the results it was evident that, mushroom samples showed perfect peaks for cellulose, hemicelluloses and lignin at the corresponding wave numbers. Hemicellulose peak intensity of wave number 1740 cm -1 with functional group of ketone/aldehyde C = O stretch was greatly reduced in P. giganteus BPSM27 when compared to S. commune BPSM01, it shows that solubilization of the hemicellulose has taken place in P. giganteus BPSM27. The hemicellulose peak is totally absent in Pleurotus sp. BPSM34, Lentinussp. BPSM37 and Lentinula sp. BPSM45; it might be due to the complete solubilization of the hemicellulose part present in the samples. Reduction in lignin peak intensity of S. commune BPSM01 of wave number 2937 cm -1 with a functional group of C-H stretching was observed when compared to sample the rest of the studied mushrooms. Similarly reduction in peak intensity for lignin of wave number 1639 cm -1 and 1556 cm -1 was noticed in S. commune BPSM01 and Lentinussp. BPSM37 when compared to P. giganteus BPSM27, Pleurotussp. BPSM34, P. djamor BPSM41 and Lentinula sp.

Discussion
More than 200 different mushroom species have been identified from different parts of the world. Previous researchers have shown the importance of mushrooms for the production of novel secondary metabolites having antimicrobial potential [48,49]. In India, several mushrooms had been reported for their medicinal properties like high antioxidant, antimicrobial and antitumor activities [49,50]. Few of them are Agaricusbisporus [51], Volvariellabombycina [52], Pleurotussajor-caju [53], Pleurotuspulmonarius [54], Lycoperdonperlatum [55], Lentinus tuber-regium [56] and Among the studied locations, it was revealed that the diversity of wild macrofungi was higher in DTR than in MNP. It is likely that the variation of wild macrofungi in different ecosystems is governed by different climatic conditions such as temperature, pH of soil, physiognomy, succession and geography. This finding was similarly reported by Angelini et al. [60], where sampling methods, soil and microclimate conditions affects diversity of macrofungal species. In addition, level of effectiveness amongst biota and the level of human disturbances may have resulted in difference in species recorded in our study. In our study, we found that diversity of wild macrofungi was higher in the rainy season as compared to the dry season. This finding was similarly reported by Andrew et al. [61] who stated that species diversity was higher in the rainy seasons. Chang and Miles [62] suggested that temperature may be the major physical factor which may affect mycelial growth in mushroom production.
All the wild macrofungi tested were found to have various degrees of antimicrobial effects against gram positive and gram negative bacterial pathogens. Similarly, a previous study has also reported antimicrobial activity from the extract of mushrooms [7]. In our study, the maximum inhibition zone was shown by Pleurotus pulmonarius (BPSM36) showing antibacterial activities against P. aeruginosa (9.4 mm) and S. aureus (7.7 mm). These findings were similarly reported by Chowdhury et al. [7] who stated that P. ostreatus has broad spectrum antibacterial activity. Isolate BPSM45 identified as Lentinula sp. showed antibacterial activity against all tested bacterial pathogens which was in concordance with the findings of Ishikawa et al. [63] who showed that Lentinula edodes has antibacterial action against B. cereus, S. aureus and E. coli. We found that all isolates were highly resistant against E. coli and C. albicans. Previous researchers have similarly reported that both the pathogens were found to be strongly effective against mushrooms [7,55]. Four strains showed significant antimicrobial activity against all the tested bacterial pathogens. Similar antibacterial results were reported by Barros et al. [64], Ramesh and Pattar [55], Chowdhury et al. [7] and Smolskaite et al., [11] who proved that mushrooms has strong antimicrobial activity and are potential candidates for the discovery of antimicrobial products.
All the isolates were screened for their in vitro antagonistic activity against three fungal pathogens and 45 isolates (19.48%) showed strong antimicrobial activity against at least two out of three tested pathogens. Interestingly, five isolates (BPSM 31,33,37,38 and 45) found to inhibit all selected fungal pathogens, similar findings were reported by Jagadish et al. [51] and Udu-Ibiam et al. [65] who demonstrated that medicinal mushrooms had found to have antagonistic potential. Isolate BPSM45 identified as Lentinula sp.Exhibited the highest percentage of inhibition (55.3%) against F. oxysporum, which is significantly higher than the finding reported by Osman et al. [66] who showed 22% and 17% inhibition of F. oxysporumby Lentinula edodes LC202 and Lentinula edodes LC2141. Most of the selected isolates in the present study showed antagonistic potential against F. oxysporum f. sp. ciceri, a potent pathogen causing diseases in several crops. The potential isolates (BPSM 31, 33, 37, 38 and 45) have shown strong antifungal activity which has the potential to develop as a biocontrol agent. Park et al. [67] has used ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATAGC-3') primers to amplify the ITS region to identify isolates of medicinal mushroom Hericium erinaceum whereas Dentinger et al. [15] have used ITS sequences to compare with COI and they reported that the overall performances of ITS and COI as barcode markers are similar. However, the barcode locus that has been chosen for animals, COI, will not work for mushrooms, rusts [68], and probably most other fungi [69], primarily due to the variable and unpredictable presence of large introns in the barcode region. The widely used ITS regions works well for identifying species [70,71] and shows that there is no better single molecular for barcoding mushroom species till today, and its versatility makes it possible to survey and discover new fungi. Subsequently, the Internal Transcribed Spacer (ITS) region of the ribosomal DNA (rDNA) has been extensively used as the de facto standard 'barcoding' marker for many years [71]. In accordance with the previous reports, we have used ITS region for the identification of selected wild mushrooms.
The analysis of genetic diversity is important to understand the genetic variation among the closely related organisms based on their genetic makeup. In RAPD analysis, both Pleurotus sp. (BPSM10 and BPSM34) has been closely clustered together. This finding was similarly reported by Yin et al. [20] and Ro et al. [72] who reported that phenotypic characteristics of fruiting bodies of. P. eryngii clustered together based on RAPD grouping. It proved that effectiveness of the molecular marker in the genetic diversity analysis of mushrooms at the genus level. The results obtained in the present study provided information regarding the genetic diversity of the strains by combined RAPD/ISSR analysis. In the present study, both RAPD/ ISSR fingerprinting analysis clearly showed closely related Schizophyllum sp. strains and formed a mono-subclade at the coefficient of 90% and 95% respectively. Similarly Auriculariapolytricha (Isolates BPSM14, 22 and 31) clustered together in both RAPD and ISSR technique, which further proved that the selected markers can be used to differentiate at the genus level. The RAPD technique has been successively used to differentiate among different strains of Agaricus bisporus [73], Ganoderma lucidum complex [74], Lentinula edodes [75]; Agaricusbisporus [76]. RAPD has the potential to survey the entire genomes, and often identifies multiple loci in a single reaction. RAPD can provide a practical tool for breeders and spawn companies to protect novel strains [77]. Trametes strains were clustered together and showed high levels of genetic diversity in ISSR markers, and been used in the identification and taxonomy for more than two decades [78]. It is a very systematic, perceptive and successful method to studygenetic diversity under species level which had been used in Lentinula edodes [79], Cordycepssinensis [80], Auriculariapolytricha [81] and Agaricus bisporus [82].
Among the nutritional contribution of the selected six mushrooms we have studied, Lentinula sp. BPSM45 showed the highest ash content 21.406 g/100g. On the other hand, our study revealed the highest energetic contribution of Panusgiganteus BPSM27 among the selected mushrooms, due to the highest content of carbohydrates and proteins with low fat content. The level of antioxidant activity in mushrooms is largely varied from one species to another and the solvent used for extraction. This could be due to their capability to extract bioactive substances [83]. Apart from these two factors, geographical locations may also affect the bioactive properties of wild mushrooms which could be due to their differences in relation with growth conditions, genetic factors, geographical variations and analytical procedures [84,85,86].
Phenolic group is the major antioxidant compound found in mushrooms, [87]. There are also reducing agents like ascorbic acid and flavonoids contributing in lower values [88,89]. Phenolic components are potential antioxidants which can donate hydrogen to free radicals to terminate the chain reaction of lipid oxidation at the initial stage. This is due to the phenolic hydroxyl groups which help the phenolic compounds to scavenge radicals [83]. Wong et al [88] reported in methanolic extract of S. commune 25.03 mg GAE/g where as the TPC of S. commune BPSM01 was 55.19 mg GAE/g which is the highest TPC in this study. Flavonoids were not detected in P. Ostreatusin the findings of Matilla et al [90] and Chowdhury et al [91]. It is reported by USDA that mushrooms are considered as non-sources of flavonoids and our finding agrees with the abovementioned reports as the highest TFC observed is 0.744 mg QAE/g.
The DPPH and ABTS assays are known for their reproducibility; ease of application and low cost [92] The ABTS radical scavenging activity of is a more sensitive radical for assessment of antioxidant activity. The discoloration of ABTS radical cation shows the ability of the antioxidants to deactivate these radical species by donating either electrons or hydrogen atoms [93]. As shown in Table 5, the average scavenging activity of ABTS radical is 6.805 mg/ml which is slightly higher than S. commune BPSM01 with an IC 50 of 3.25 mg/ml which is the most significant antioxidant activity in the methanolic extract of these mushrooms.

Conclusions
The macrofungal diversity from two protected forest areas in India was undertaken and their antimicrobial potential was investigated. For the first time 231 wild macrofungi were collected and documented from the selected locations. The significant antifungal and antibacterial activity was found in Microporus sp. and Schizophyllum commune respectively. Further testing of these strains for the discovery of novel and efficient antimicrobials should be considered. Microporus sp. strain BPSM 33 showed the highest antagonistic potential against the phytopathogen F. oxysporum with 85.71% of inhibition and could be used for the development of biocontrol agent against F. oxysporum. Furthermore, the genetic variation among the potential strains showing antimicrobial potential was considered by using RAPD and ISSR molecular markers. We reported that the selected markers can be used for assessing genetic diversity of wild mushrooms, considering both marker systems gave similar results in the present study. We also conclude that the wild edible mushroom strains does not have only nutritional values but also can be used as an accessible source of natural antioxidants.