Phylogenetic diversity and antioxidant activity of selected fungi from ethno-medicinal plants and soil

Endophytic fungi are prolific generators of bioactive metabolites with natural antioxidant properties that have a range of potential uses. In the present work, the antioxidant activity and phylogenetic diversity of endophytic fungi obtained from semi-arid terrestrial plants and the fungi obtained from the mangrove soil samples are investigated. The fungal isolates were identified by employing molecular characterisation and phylogenetic analysis by internal transcribed spacer (ITS) sequences. Fungi belonging to six taxonomic orders of Ascomycota associated with nine genera were identified, these being Diaporthales, Eurotiales, Hypocreales, Onygenales, Pleosporales and Xylariales. The antioxidant activity of the methanolic extracts of the fungal isolates was determined using modified 2,2’-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) methods. Amongst the fungal isolates, Aspergillus sp. AREF023 and Neocosmospora sp. AREF014, obtained from different parts of Datura metel, displayed the highest level of radical inhibition (80.76% and 70.62% respectively). GC/MS analysis of the active isolates confirmed the presence of known antioxidant phenolic compounds, including 2-tert-Butyl-5-hydroxymethyl-5-methyl-[1,3]dioxolan-4-one and 2,5-ditert-butylphenol, in the their crude extracts. Our findings suggest that fungal isolates from D. metel can be a sustainable resource for natural antioxidants.


Introduction
Endophytic fungi are diverse filamentous fungi that grow within plant tissues without causing any immediate harmful effects (Hyde and Soytong 2008;Debbab et al. 2013). These endophytic fungi are prolific producers of secondary metabolites and can serve as a valuable reservoir of lead molecules in the hunt for drug candidates against infectious diseases, cancer and many other disorders (Rana et al. 2020;Mishra et al. 2021a, b). Curvularia sp. G6-32, an endophytic isolate of medicinal plant Sapindus saponaria, produces asperpentyn, an epoxyquinone, which is a potential antioxidant (Polli et al. 2020). In addition, Diaporthe prunorum, an endophytic fungus obtained from Hypericum ascyron, produces terpenes and isocoumarins that have potential antimicrobial activity (Qu et al. 2020). The unique phenomenon of cross-talk existing between the colonizing endophytic fungi and the plant host results in the production of related and often the same bio-actives as originally produced by the host plant (Caruso et al. 2020). Endophytic fungi are novel source of fungal metabolites that sustainably help in the discovery of bioactive natural products.
The present study screened endophytic fungi isolated from medicinal plants and mangrove soil from two diverse regions for antioxidant activity. Firstly, the semi-arid region characterised by its intermediate climatic conditions between the desert and humid regions, secondly the semi-arid land forms, comprising of succulent, non-succulent and xerophytic perennials . Secondly, mangrove sediments are complex as compared to plant ecosystems in terms of a characteristic marine biome. The vegetation is well equipped to survive saline stress (Guillén-Navarro et al. 2015). There is no up to date published study that describes the phylogenetic study of endophytic fungi and soil fungi obtained from these two sources in India. With this rationale, the ethno-medicinal plants, D. metel, B. monnirei, C. citratus and O. tenuiflorum, and soil sediments from marine ecosystem were selected for the isolation of fungi. In the current study, we have identified, established the genetic relationship and screened the antioxidant activity of endophytic fungi isolated from some semi-arid terrestrial plants and the fungi obtained from the soil of the mangrove region of a marine area. Several of these endophytic and soil fungal isolates were found to be significantly active by inhibiting the DPPH and ABTS radicals and showed active spots on the DPPH bioautography assay. These bioactive endophytic and soil fungi isolates were further subjected to molecular characterisation and their chemical constituents were investigated using GC-MS.

Materials and method
Sampling sites and collection of samples

Terrestrial plants
One each healthy terrestrial plant parts, i.e. stem, leaves and roots of four species, Bacopa monnieri, Cymbopogon citratus, Datura metel and Ocimum tenuiflorum, was collected from Aravalli Bio-diversity Park (28°28′59.9″N 77°06′34.7″E) Gurgaon, Haryana, India. The identification and validation of plant species were done as previously described (Gamble 1925), and samples were preserved in our laboratory. Plant parts such as leaf, stem and roots were randomly cut with an ethanol-disinfected sickle and placed independently in sterile polythene bags. Endophytic fungi were isolated within 48 h of collecting the samples.

Soil samples
Nine mangrove soil samples were collected randomly in July 2018 from the marine area of Alibaugh (18°39′ 23.9544″N, 72°52′47.5248″E) Maharashtra, India. Soil samples, each with 1-km distance from the sea bed, were taken from the surface to a depth of 5-10 cm and placed in sterile zip a lock bag in an ice box and taken to the laboratory. The samples were then stored in the refrigerator (4°C) for fungal isolation.

Endophytic fungi
Isolation of the fungal isolates was done using a previous method of Qadri et al. (2013) with some modifications. Briefly, mature and excided small plant parts (leaves, stems and roots) were used for isolation of the fungal endophytes. The samples were washed for 1 min with 70% ethanol (v/v) and disinfected for 2 min using 2% sodium hypochlorite solution (v/v) followed by 20 s of brief rinsing with 70% (v/v) ethanol. Later they were rinsed with sterile distilled water. These plant parts were placed on potato dextrose agar (PDA) plate amended with 50 μg/ml chloramphenicol. PDA plates containing three pieces of plant material were incubated for 7-10 days at 28°C. Plating was done in triplicates.

Fungi from soil
Soil fungal isolates were isolated on water agar (WA) by employing soil plate method. Incubation was done at 28°C for 3-5 days, and checked for fungal colonies appearing in culture, and then a single colony was transferred on PDA plate and further incubated for growing pure culture. The cultures in petri dishes were incubated at 28°C for 7-10 days for observation. Plating of each soil sample was done in triplicate. All the isolates were deposited in the TERI Deakin Nanobiotechnology culture collection at 4°C.

Metabolite production
Potato Dextrose Broth (PDB, Himedia) was used for metabolite production. Three fungal plugs (8-9 mm in diameter) from 10-day-old culture were inoculated in 1000-ml Erlenmeyer flasks containing 250 ml of the medium. The flasks were incubated at 28°C for 8-10 days at 150 rpm. The growth conditions and time period had been optimised (Fig. S1) based on the assessment of growth kinetics using ergosterol estimation method (Axelsson et al. 1995). Ergosterol is the major sterol present in the cell membranes of filamentous fungi, and monitoring its level is a useful method for estimating growth kinetics.

Preparation of methanolic extract
For preparation of the extract, methanol was added slowly in the grown culture (1:1) (to avoid over heating), and kept at shaking for 6 h. The media was then filtered through sterile muslin cloth to separate mycelia. The filtrate obtained was then concentrated to 1/5 volume using rotary evaporator and diluted to 100 ml with distilled water. The diluted solution was then passed through Dianion HP20 column (Merck) and was eluted using 70% methanol. The obtained elute was concentrated in peer shape flasks, diluted with distilled water and lyophilised to obtain a powdered extract.

Thin layer chromatography (TLC) bio-autography
Each fungal extract was spotted and eluted with CHCl 3 :MeOH (85:15 v/v) on silica 60 F 254 TLC plate (Merck), followed by spraying with 0.2% DPPH solution in methanol and incubated for 30 min in dark. Active compounds appeared as yellow spots against a purple background.
Total antioxidant capacity assay DPPH assay DPPH radical scavenging assay was conducted according to the previous method of Blois (Blois 1958) with modifications. DPPH (Sigma) was dissolved in 100 ml of MeOH. The investigated samples were prepared by dissolving 1ml of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) in methanol and 0.5 ml of test sample at different concentrations (5 to 100 μg/ml) at room temperature. The absorbance at 517 nm was measured after 30 min versus the blank (0.5 ml of methanol instead of test sample in 1-ml DPPH solution). Positive controls ascorbic acid, tert-butylhydroquinone (TBHQ) and Trolox were also subjected to the same procedure for comparison. The analysis was carried out in triplicate, and the percentage (%) of inhibition was calculated by the following formula: The DPPH radical scavenging activity of each sample was expressed as Trolox equivalent antioxidant capacity (TEAC) (Shimamura et al. 2007). TEAC was calculated as follows:

ABTS assay
The ABTS radical scavenging assay of the fungal crude extracts performed in accordance with the method reported by Re et al (Re et al. 1999). Briefly, 2.45 mM potassium persulfate and 7 mM ABTS aqueous solution were mixed to generate ABTS + radical. The initial absorbance mixture solution was adjusted to 0.70 ± 0.02 at 745 nm. Different sample concentrations (5 to 100 μg/ml) and standard Trolox (1 to 50 μM) with ABTS + working solution were adjusted to a final volume of 1 ml and reacted at room temperature (25°C) for 6 min. The decrease in absorbance at 745 nm was recorded and calculated as half maximal inhibitory concentration (IC 50 ) for test samples and Trolox was calculated by plotting the scavenging capacity against the concentration. Trolox standard solution (final concentration 0-50 μM) was used. Results were expressed in terms of TEAC (Shimamura et al. 2007). TEAC was calculated as follows:

Molecular identification of the fungal isolates
Genomic DNA extraction Only the isolates which displayed optimum antioxidant activity were further subjected to sequencing and identification. For that, the pure culture obtained after isolation of the fungal isolates was grown in 250-ml Erlenmeyer flasks containing 50 ml of the medium, incubated at 28°C for 8-10 days at 150 rpm. The fungal mycelia obtained were freeze-dried and the genomic DNA was extracted by the CTAB (cetyl trimethylammonium bromide) method (Brent et al. 1995).

Phylogenetic analysis
Phylogenetic analyses of the endophytes were carried out by the acquisition of the ITS1-5.8s ITS2 ribosomal gene sequencing. ITS region of rDNA was amplified and sequenced with universal primers ITS4 (5′ -TCCT CCGCTTATTGATATGC-3′) and ITS1 (5′-CCGT AGGTGAACCTGCGG-3′). PCR amplification was performed according to the methods of Rajeendran et al (Rajeendran et al. 2017). The amplicons obtained were gel purified by amicon ultra columns (Millipore, USA) and 20-40 ng was used for sequencing at Eurofins Laboratory Pvt, Ltd. (Bengaluru, India). Finch TV software (http://www.geospiza.com/Products/finchtv.shtml) was used for assembling sequences and homology was determined using BLASTn against the NCBI Gen Bank database (Altschul et al. 1997). A phylogenetic tree was constructed using the sequences displaying maximum homology. CLUSTAL W was employed for multiple sequence alignment and MEGA X was used for phylogenetic and molecular evolutionary analyses .
Following the Tamura-Nei model and maximum likelihood algorithm, phylogenetic reconstruction was done with bootstrap values calculated from 1000 replicate runs (Tamura and Nei 1993).

GC/MS analysis
The volatiles and major chemical components of the fungal extracts were analysed using Agilent 6890 GC-MS coupled to Agilent 5873 (EI mode, 70 eV). The GC conditions were as follows: 1-min split less time, helium carrier at 1.0 ml/min, oven temperature from 70 to 135°C at 2°C/min, for 10 min; then to 220°C at 4°C/min, 10 min; and finally to 270°C at 3.5°C/min, for 20 min. For analysis HP-5MS capillary column (0.32 mm × 30 m × 0.25 μm), GC injector with temperature set at 280°C and MS transfer line temperature at 290°C were used. The detected compounds were compared with the mass spectra from the NIST library for their identification (Proestos et al. 2006).

Results
Isolation of endophytic fungi, fungal isolates from soil and screening for antioxidant activity In total, 43 fungal isolates were obtained from different parts of the plant and mangrove soil. From them, 17 antioxidantrelated fungal isolates (10 endophytic fungi and 7 soil fungi) obtained after initial screening of the pure isolates are illustrated in Table S1. Methanol extracts of these fungal isolates were screened for antioxidant potential using two assays. Extract antioxidant activity was tested at a concentration of 1000 μg/ml.

TLC bio-autography analysis
Amongst the various solvent elution tested, chloroform/ methanol/acetic acid (8.5:1.5 by volume) gave the best separation of compounds from the 70% methanol extracts with retention factor values (R f ) ranging from 0.15 to 0.87. The existence of strong adsorbing sites, as depicted on TLC plate (Fig. S2), confirms the suitability of this solvent system for separation and mobility of the fungal extracts. The baseline of all the methanolic fractions indicated a range of deep to light yellow colour, indicating the presence of antioxidant constituents in varying amounts.
The spots of R f 0.87-0.71 displayed bright yellow spot on the TLC plate confirming the presence of antioxidant compounds in these extracts. Radical scavenging activity of methanol extracts from all fungal isolates against DPPH and ABTS + radical is shown in Table 1. The IC 50 values were obtained in the range of 33-616 μg/ml for the DPPH assay and 14-167μg/ml for ABTS assay. The lower the IC 50 and the higher the TEAC value, the greater is the antioxidant activity. Amongst all the 17 fungal crude extracts, isolate AREFF023, identified as Aspergillus sp. obtained from the internal tissues of roots of D. metel, exhibited promising DPPH scavenging activity (IC 50 33.49 μg/ml, TEAC of 0.3 and 80.76% of DPPH radical inhibition) and also against ABTS + (IC 50 14.33 μg/ml and 75.18 % of ABTS + radical inhibition). The second most potent isolate was Neocosmospora sp. AREF014, which showed moderate antioxidant activity (IC 50 35.39 μg/ml against DPPH and TEAC of 0.28; and IC 50 33.50 μg/ml against ABTS + ).

Phylogenetic diversity of cultivable fungi
Amongst the seventeen isolates, eleven (Fig. 1) displaying commendable antioxidant activities were subjected to sequencing for their molecular characterisation. A further categorisation of these isolates was carried out into nine genera, namely Alternaria, Aspergillus, Auxarthron, Curvularia, Diaporthe, Monascus, Neocosmospora, Talaromyces and Xylaria which belong to six orders Diaporthales, Eurotiales, Hypocreales, Onygenales Pleosporales and Xylariales, found in phylum Ascomycota. Amongst the isolated endophytic fungi Alternaria and Aspergillus was the predominant genus, with relative frequency of 18.18%, while the remaining genera Auxarthron, Neocosmospora, Monascus, Talaromyces, Xylaria, Curvularia and Diaporthe were found with relative frequency of 9.99% The phylogenetic tree (Fig. 2)   one newly identified endophytic isolate with three sequences of Xylaria, Neocosmospora and Diaporthe sp. obtained from NCBI. Third and the last clade had two subclades in which one endophyte sequence clustered with three sequences of Curvularia sp. and other sub cluster contained two endophyte isolates sequences clustered with Alternaria sp. Characterisation data from this study have been submitted in the NCBI GenBank, accession codes MT013399 to MT013409 (Tables 2 and 3).

Detection of bioactive metabolites of the methanol extracts by GC-MS analysis
Considering the highest antioxidant activity obtained from the methanol extracts of Aspergillus sp. AREF023 and Neocosmospora sp. AREF014, a tentative identification of the metabolites from these two isolates was carried out using GC-MS. Chemical diversity was observed in the constituents of both the extracts, belonging to diverse classes of alkenes, di-ethers, esters, fatty acids, phenols, polysaccharides, organic acids and hydrocarbons. A total of 20 compounds constituting 96.3% of the relative area in the methanol extract and 95% similarity with the standard mass spectra were revealed from the GC-MS analysis of the AREF023 extract ( Fig. 3a) ( Table 4). The methanol extract of AREF023 indicated the presence, amongst others, of phenolics, including 2-tert-butyl-5-hydroxymethyl-5-methyl-[1,3]dioxolan-4-one (8.97%), a common antioxidant molecule which is used in a range of applications. Similarly, analysis of AREF014 showed the presence of 7 compounds, having resemblance almost 97% with the standard masses in the library and representing 100% of the relative area in the extract (Fig. 3b) ( Table 5). The extract clearly showed the presence of the phenolic compound, 2,5-ditert-butylphenol (36.45 %), which is widely used as a dietary antioxidant compound.

Discussion
According to the antioxidant assays, amongst all the isolates found, Aspergillus sp. AREF023 displayed the highest antioxidant activity, followed by Neocosmospora sp. AREF014, comparable to that of the standards used, Trolox, TBHQ and ascorbic acid. Scavenging activity is directly proportional to the concentration of extracts and varies in their activity because of the varying metabolite composition. This is the first report of the antioxidant activities of endophytic fungi identified as Aspergillus sp. AREF023 obtained from D. metel.

Conclusion
The results in this study have established that the endophytic f u n g a l i s o l a t e s , A s p e r g i l l u s s p . A R E F 0 2 3 a n d Neocosmospora sp. AREF014 contain metabolites rich in phenolics and flavonoids which are the major contributors to antioxidant activities in extracts from these strains. The extract with the highest phenolic content displayed the highest  antioxidant capacity. The endophytic fungi Aspergillus sp. and Neocosmospora sp. are excellent sources of naturally derived antioxidants. However, the toxicity of these fungal extracts having antioxidant activity needs further investigation, particularly in regard to their applicability as neutraceutics and pharmaceutics. In addition, the mechanisms of antioxidant activity of these extracts required investigation in order to gain more insight into their scavenging behaviour in food and biological systems.
Acknowledgements The authors are grateful to Ms. Deep Rajni for support with GC-MS facility.
Author contribution MG conceptualised and designed the work. RCM performed the experiments, carried out data analysis, manuscript drafting and editing. SKD and ND helped in the identification and phylogenetic analysis of the fungal strains. RK contributed in the GC-MS analysis. MG and CJB contributed toward the critical revision and final approval of the manuscript. All authors contributed to the article and approved the submitted version.
Funding The research activities of the authors are supported by The Energy and Resources Institute, India, and Deakin University, Australia. Rahul Mishra (Candidate ID -217450416) is supported by a Deakin University HDR scholarship.
Data availability All data generated or analysed during this study are included in this article (and its supplementary information files). The names of the repository/repositories and accession number(s) can be found in the article/ supplementary material.

Declarations
Conflict of interest The authors declare no competing interests.