GC-MS Analysis, Antioxidant and Antifungal Studies of Different Extracts of Chaetomium globosum Isolated from Urginea indica

To discover new natural resources with biological effects, the chemical investigation of antioxidant and antimicrobial activities of extract's Chaetomium globosum isolated from roots of Urginea indica. Gas chromatography-mass spectrometry (GC-MS) analysis demonstrated the presence of the major chemical constituents present in the methanol extract (1,3-oxathiolane, 1,3-cyclopentadiene, 5-(1-methylethylidene), 5,9-hexadecadienoic acid, methyl ester, decane), chloroform extract (acetic acid, diethoxy-, ethyl ester, 2,2-bis(ethylsulfonyl)propane, 3-methyl-2-(2-oxopropyl) furan), and hexane extract (3-hexanone, 4,4-dimethyl, decane,2,6-dimethyldecane, decane, 2,4,6-trimethyl, decane, 2,4,6-trimethyl, 1-butanesulfinamide, 1,1,2,2,3,3,4,4,4-nonafluoro-N-methyl, decane). The total compound identified (56.2%) in chloroform extract, (54.72%) in hexane extract, and (65%) in methanol extract. The antioxidant effects were performed using diphenylpicrylhydrazyl radical (DPPH). The results showed that the methanol extract showed significantly the highest anti-DPPH with an IC50 value of 37.61 ± 1.37 μg/mL, followed by chloroform and hexane extracts with IC50 values of 40.82 ± 3.60 and 45.20 ± 2.54 μg/mL, respectively. The antifungal activity of extracts was evaluated against pathogens fungi including Fusarium oxysporum, Rosellinia necatrix, Cladosporium xanthochromaticum, and Sclerotinia sclerotiorum. Methanolic and chloroform extracts showed maximum inhibition against all test pathogens, while hexane extract showed minimum inhibition.


Introduction
Endophytes are microorganisms, frequently bacteria or fungi, that live within a plant for at least part of its life cycle without causing discernable disease. Usually, endophytic fungi are extensively dispersed in all kingdoms of the plant kingdoms. They generate advantageous plant secondary metabolites and utilize them for medical, agricultural, and industrial usage.
Endophytic fungi are a new type of microbes isolated from plants that have received attention due to their ecological nature [1]. Endophytes are the repository of new and unique secondary metabolites; these metabolites can serve as a marvelous origin of drugs for anti-inflammatory, antioxidant, anticancer, antimicrobial, and antidiabetic activities. In current years, mushrooms known as higher fungi are a rich source of secondary metabolites as compared to metabolites derived from plants. There are three categories of mushrooms, saprotrophic, parasitic, and symbiotic. There are millions of species of mushrooms but only 4% of species are considered to be safe for edible. Nowadays, natural products from mushrooms are gaining the attention of researchers. Secondary metabolites of higher fungi (mushrooms) are an underexplored resource compared to plant-derived secondary metabolites. An increasing interest in mushroom natural products has been noted in recent years. Mushrooms are more nutritious, but other than nutrition mushrooms are also proficient in producing valuable bioactive secondary metabolites, these metabolites have great uses in the medicinal field. There are different categories of bioactive metabolites produced from mushrooms such as lectins, polysaccharides, phenolic, terpenoids, polyphenolics, and other organic compounds like ceramides. These metabolites possess immunomodulating properties. Anticancer compound 9methoxycamptothecin is isolated from endophytic fungi. Taxol, which is a potent anticancer compound, widely used for the treatment of different types of cancer, produces by different species of endophytic fungi isolated from different medicinal plants. Vinblastine is an anticancer compound produced by the endophytic fungi Alternaria sp. These are the major anticancer metabolites produced by endophytes. All over the world, cancer is the main killer disease. Every year, more than six billion people suffer from cancer, and the discovery of these fungal endophytes is a great achievement for human welfare. These metabolites have remarkable biological and industrial applications. Secondary metabolites produced via endophytic fungi bear in various fascinating activities useful in different fields.
C. globosum endophytic strain isolated from Ginkgo biloba inhibited the growth of pathogens and acted as a strong biocontrol agent [2]. In another study, nine new cytochalasan alkaloids were isolated from the C. globosum strain obtained from a common pillbug (Armadillidium vulgare). These cytochalasins showed the capability to inhibit cancer cells [3]. Chaetoviridin A, a strong antifungal compound isolated from Chaetomium globosum, inhibits the growth of Verticillium dahlia (a soil-born pathogen) causing disease in the cotton plant. The inhibition of this pathogen improves the yield of the cotton plant [4]. Medicinal plants generally possess endophytes with comparable secondary metabolites and therapeutic activities. Urginea indica is a distinguished Indian medicinal plant with various medicinal properties. With this motivation, the medicinal plant U. indica was chosen for the isolation of the endophytic fungus C. globosum from its roots. C. globosum is known as a biocontrol fungus because it can kill pathogens due to the production of secondary metabolites having strong antifungal and insecticidal potential [5]. This work is the first report of the isolation of endophytic fungi C. globosum from U. Indica, a potent medicinal plant, and checking the antifungal activity. The purpose of the study is to isolate the endophytic fungus Chaetomium globosum pure culture from the medicinal plant U. indica, and the preparation of extract was done. Testing of extract should be done to check the presence of essential compounds present in the fungus responsible for antimicrobial and antifungal activities. The best extract among the three extracts should be taken further for fungicide studies. The present study shows the isolation and characterization of C. globosum and the anal-ysis of different compounds present in methanolic, hexane, and chloroform extracts through GC-MS.

Isolation and Identification of the Endophytic Fungus.
The endophytic fungus (C. globosum) isolated from the roots of U. indica was collected from the botanical garden of Shoolini University, (Himachal Pradesh), India. U. indica belongs to the family of Liliaceae U. indica was identified by Professor Sunil Puri Shoolini University, Solan, Himachal Pradesh, India. Roots of U. indica were collected in December 2021. Now, the voucher specimen is deposited in BSI Solan. Morphological and molecular identifications of the isolate (MCC 9353) were carried out further for phylogenetic analysis. The isolation of the fungus from the roots was done on a PDA medium (Potato Dextrose Agar).

Morphological
Identification. The morphology of the isolated fungus was studied under the microscope through the scotch tape method [9]. The morphological identification was authenticated by the plant tissue culture Lab, Department of biology and biotechnology, Shoolini University, District Solan, Himachal Pradesh. The cut tape is about 2-inches long and touches the tape to a fungal colony in a culture dish opened in a biological safety cabinet. Use a second applicator stick to press the tape down onto the mycelial surface, lift the tape flag with attached fungal elements from the colony and lower it onto a glass slide, add drops of cotton blue dye, and then cover it with a coverslip. The completed mount is now ready for microscopic study.

Molecular Identification.
For molecular identification of the fungus, DNA extraction was done. For genomic DNA extraction, around 5 g of fungal mycelia were used. The extraction was done according to [10]. Internal transcribed spacer (ITS) primers were used for the amplification of nuclear ribosomal DNA. The nuclear ribosomal DNA ITS of the fungal isolates were amplified using the forward primer, ITS 1-forward primer (5 ′ -AAACCATTGGTGAA CGTTA-3 ′ ) and ITS 4 reverse primer (3 ′ ACCGAGGTCAC CTTG-5 ′ ) [11]. The final reaction volume was 25 μL, containing 2 μg of genomic DNA, Primers 0.1-0.5 μL of 2× PCRBioTaq Mix, 0.4 μL. PCR was done by using a Thermal cycler (Applied Biosystems, India), PCR cycle occurs at 35 cycles programmed for denaturation of 5 min at 94°C, denaturation of 45 s at 94°C, annealing of 45 s at 55°C, and extension of 120 sec at 72°C. PCR products were separated using a 1% agarose gel in 1× TAE buffer (90 mM Tris-acetate and 2 mM EDTA, pH 8.0), stained with ethidium bromide (1 μg/mL) and recognized using Gel doc. PCR products were sent to Eurofins Bangalore, India, for sequencing.

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BioMed Research International 2.4. Extract Preparation of C. globosum. Crude extracts of C. globosum were prepared according to [12] to fifty mL of potato dextrose broth (PDB) being inoculated with two discs (8 mM in size) of C. globosum grown on Potato Dextrose Agar (PDA) plates. The flasks were then incubated for 5 days at 25°C as stationary cultures. After the growth of fungal, biomass was filtered through Whatman filter paper no. 1.
The filtrate obtained was extracted twice with an equal volume of chloroform, hexane, and methanol to obtain three different extracts. The solvent layer was evaporated using a hot air oven at 40°C [13].
2.5. Antagonistic Activity against Different Pathogens. C. globosum antagonistic effects against different pathogenic fungi including Fusarium oxysporum, Rosellinia necatrix, Cladosporium xanthochromaticum, and Sclerotinia sclerotiorum were evaluated using the dual-culture technique [2]. In this method, 2 mM-sized discs of the culture of C. globosum (5day-old culture) and the same-sized discs of pathogenic fungi were placed on opposite sides of 90 mM petri plates containing PDA. As controls, only pathogens are used. The experiment was repeated three times, and the culture was incubated for seven days at 25°C. After 7 days of incubation, the area between the two colonies at the interaction point was measured, the growth of colonies of the tested fungi and antagonist was observed and photographed, antagonistic activity was repeated thrice, and mean standard deviation was observed for the results [2].

Antifungal Activity of Different Extracts of C. globosum.
The antifungal activity of all the extracts of C. globosum was performed using the poison food technique [14,15]. Briefly, each extract at 20 mg/mL of extract dissolved by dimethylsulfoxide (DMSO), 10% was added to sterilize the PDA medium. Pathogenic fungal discs of 6 mM in diameter were placed in the centers of petri dishes. Plates without extracts and 5 mg/mL of hygromycin were used as negative and positive controls, respectively. After placing the fungal disc, the plates were incubated at 28 ± 2°C for 7-8 days.
Then, the circular growth of mycelium was calculated. The growth results were compared with the negative control. The experiment was repeated three times, and the mean of the readings was taken for further calculations. The percent inhibition was calculated using the following formula: where L, C, and T are the percent inhibition, colony radius in the control plate, and the radial growth of the pathogen in the presence of C. globosum extracts, respectively [15].

Antioxidant
Activity of Different Extracts of C. globosum. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay was done to check the antioxidant capacity of methanol, chloroform, and hexane extracts of the fungus according to Joshi et al. [16], with some modifications. Briefly, a stock solution of DPPH (0.1 mM) was prepared in methanol and kept in the dark at room temperature for 2 hours. Different concentrations of extracts (20-160 μg/ mL) were mixed with 150 μL of DPPH for checking the radical scavenging activity of them and kept in dark for 30 min at room temperature. Absorbance was measured at 517 nm using Systronics UV-vis double beam spectrophotometer 2205. DPPH free radical scavenging activity was expressed as the percentage inhibition of each extract and by calculating the IC 50 value. The antioxidant activity was calculated using the following formula equation: where Ao and A1 are the absorbance of the blank and test/ positive control, respectively.

GC-MS Analysis.
Crude extracts (i.e., hexane, methanol, chloroform) of the C. globosum fungus were further analyzed for GC-MS, which is used for compound detection. GC-MS was carried out by using a Thermo Trace 1300GC coupled with a Thermo TSQ 800 Triple Quadrupole MS with a column (30 * 0.25 mM,0.25 μM). The samples were injected in split mode as 10 : 1. Different steps are involved in the reaction to carry out different compounds present in the extracts; the initial temperature is 60°C for 3 minutes, the oven temperature is 280°C at an increased rate of 15°C for 19 minutes, injection port temperature is 260°C for 1 minute, helium used for 1 mL, the flow rate is 1 min, and ionization voltage is 70 eV. MS scans at speeds ranging from 50 to 650 m/z. The identification of each compound was based on the comparison of the mass spectra (MS) spectra computer matching with standard reference databases NIST Ver.2.1 MS.

Statistical Analysis.
All the experiments were observed in triplicate, and the obtained data were analyzed statistically with the help of GraphPad Prism 5.02 software, and the results are presented as mean ± standard deviation.

Isolation and Characterization of Endophytic Fungus.
After isolation, identification of the fungus strain was done. Molecular identification was done by rDNA sequencing of the ITS region, and morphological identification was done through the scotch tape method. The sample was identified as one of the Chaetomium species previously mentioned in the literature. Figure 1 is the picture of the microorganism observed and photographed under 100×. This identification shows the 99% similarity of the isolate with the fungus identified. Then, the fungus was deposited in NCIM Pune to get an accession number (MCC9354). The phylogenetic relationship of the isolate with its related fungus is shown in Figure 1. The isolated endophytic fungus belongs to the phylum Ascomycota [17]. C. globosum was isolated from different parts of the medicinal plants earlier, such as Moringa oleifera [18], Amaranthus viridis [19], and barnyard grass [7]. [20] isolated 10 isolates of C. globosum from different medicinal plants and identified these isolates by morphological and molecular methods. For the first time, we isolated the endophytic fungus C. globosum from the   BioMed Research International roots of U. indicia. This is the first report of the isolation of C. globosum from this plant.

Molecular Identification of the Fungal Sample Based on
Its Region-Specific Primers. The growth of the fungus was observed after 4-5 days of inoculation. For identification at the molecular level, the total genomic DNA was isolated as per the protocol given by [21]. A PCR was performed. The PCR cycle occurs every 35 cycles and the following steps are involved in the completion of this cycle: denaturation at 94°C for 5 min, again denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec, extension at 72°C for 120 sec, and again extension at 72°C for 10 min. For PCR genomic DNA 1-2 μg, primers forward and reverse 0.1-0.5 μM, TE buffer100 μM, Mg 2+ ,1.5 mM, and dNTP 100 mM can be seen in Figure 2. DNA bands (PCR products) in the gel were shown. The results revealed that molecular confirmation of C. globosum using the universal primers ITS1 and ITS4,  Figure 5: Free radical scavenging activity of different fungal extracts (HE represents hexane extract; ME represents methanol extract; CL represents chloroform extract) and the standard drug (AC represents ascorbic acid).

BioMed Research International
which had an amplicon of 500-560 bp, [22] also reported that with the combination of universal primers, ITS1 and ITS4, sequences amplicon sizes of 500-560 bp were observed. After BLAST analysis, the sample showed 100% similarity with Chaetomium spp. strain CBS 105.40 with accession number MH856051.1. Further phylogenetic analysis was done by MEGA 7.0.9. Figure 3 shows the similarity percentage of C. globosum, and Figure 2 shows the phylogenetic relationship of the fungus.

Antagonistic Effects of C. globosum.
Antagonistic activity of C. globosum against different phytopathogenic fungi through a bicultural method such as Fusarium oxysporum, Sclerotinia sclerotiorum, Rosellinia necatrix, and Cladosporium xanthochromaticum has been shown in Figure 3. In the control plate, pathogenic fungi grow faster and remarkably form larger colony diameters with a mean of 9.97 cm; while in bicultural plates, these pathogenic fungi form small colonies with a mean diameter of 4 cm for Fusarium oxysporum, 3.8 cm for Cladosporium xanthochromaticum, 3.2 cm for Sclerotinia sclerotiorum, and 3.9 cm for Rosellinia necatrix. The highest inhibition of mycelial growth of pathogens against Chaetomium globosum was shown by Fusarium oxysporum as (80.4%), followed by Rosellinia necatrix (78.32%), Cladosporium xanthochromaticum (68.69%), and Sclerotinia sclerotiorum (60.66%). The antagonism effect of Chaetomium globosum was reported earlier by [2] showed inhibition against the pathogens in the bicultural method F. graminearum, 50.2% and S. sclerotiorum, 78.9%. Strongest inhibition has been displayed against S. sclerotiorum, i.e., 78.9% which is high as compare to our results where the highest inhibition of 80.4% was shown against Fusarium oxysporum as compare to Sclerotinia sclerotiorum which is 60.66%. Antagonistic activity of Chaetomium globosum through biculture test against Fusarium oxysporum (67.25%) has been previously reported by Phong et al. [23], and our study displays the antagonistic activity of 80.4% against Fusarium oxysporiu, i.e., which is higher as compared to their study.      [25]. The results of this assay are shown in Table 1. All the extracts at 500 μg/mL showed moderate to high antifungal activity against all the pathogens. Methanolic extract showed maximum inhibition against all pathogens as compared to the chloroform and hexane extracts [26]. Extracts were found to be effective against all tested fungal isolates. Antifungal activity of ethanol, methanol, and butanol extracts of C. globosum was studied previously by [27]; results of the study stated that hexane extract shows less inhibition as compared to ethyl acetate and ethanol extracts, showing 80% inhibition against S. sclerotiorum. The isolated fungus shows outstanding antifungal activity against different pathogens. Chaetomium globosum on PDA plates shows potent antagonistic activity against different pathogens that result in the exposition of positive biological activities [28]. Hexane extract of Chaetomium globosum has been reported as an antifungal against Sclerotiorum and Botrytis cinerea [29]. Strong antifungal activity of C. globosum against Fusarium oxysporum which causes fusarium wilt of tomato [30]. Inhibition of Sclerotinia sclerotiorum by solvent extract of C. globosum, i.e., hexane, methanol, and ethyl acetate extract were shown by Kumar et al. [31].Though we find polar extract, i.e., methanol and chloro-form were more active than hexane against Fusarium oxysporum, Rosellinianecatrix, Cladosporium xanthochromaticum, and Sclerotinia sclerotiorum. Our study also shows less inhibition in hexane extract as compared to methanol, and chloroform extracts show 80-90% inhibition. The graphical representation of shows inhibition of all the extracts is shown in Figure 4.

Antioxidant Activity.
In the present study, all the crude extracts of C. globosum showed significant DPPH radical scavenging activity. The antioxidant potential of different extracts was examined at different concentrations (20,40,80, and 160 μg/mL). Methanol extracts showed remarkable antioxidant activity ranging from 55 to 70% inhibition, while the hexane and chloroform extracts showed a narrow spectrum of radical scavenging activity ranging from 30 to 50% inhibition ( Figure 5). The standard drug, ascorbic acid, showed 97% inhibition radical scavenging activity. Antioxidant capacity was evaluated as IC 50 values were also examined, the lower the IC 50 value, the higher the scavenging activity, IC 50 of methanolic extract was 37:61 ± 1:37, and the chloroform extract was 40:82:±3:60, hexane extract was 45:20 ± 2:54, and IC 50 of ascorbic acid was 50 ± 1:2. DPPH   [18]. At 20-100 μg/mL concentration, percentage inhibition at lower concentration, i.e., 20 μg/mL was 30.59% inhibition, our chloroform extract shows 34.5% inhibition at 20 μg/mL, which expressively increased by the increase in the concentration of chloroform extract. The IC 50 value was 45 μg/mL, whereas our result shows a 40.82 μg/mL IC 50 value which is almost closer to their results. Antioxidant DPPH scavenging activity of polysaccharide produced by C. globosum was checked by [1] at 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/mL, activity showed 83.08% inhibition. In 2014, Awad et al. [32] studied the antioxidant activity of petroleum ether, ethyl acetate, diethyl ether, chloroform, and ethyl acetate extracts; all extracts shows 53, 81.9, and 93.9% inhibition at 10 mg, 50 mg, and 100 mg concentration. Results stated that as compared to other extracts, petroleum ether and ethyl acetate extract showed the highest antioxidant activity. There are lots of metabolites present in the extract of fungi; and because of the presence of these metabolites, extract shows the strongest antioxidant activity. In the current study, the polar extract, i.e., methanol and chloroform showed 55-70% inhibition and 30-50% inhibition scavenging potential, whereas the nonpolar extract, i.e., hexane showed 40% scavenging potential for DPPH.
This conforms with the previous investigation done by [35] on numerous endophytic fungi. Bioactive compounds present in the methanolic, chloroform, and hexane extracts have been analyzed by using GC-MS which displayed the existence of several compounds that might be responsible for bioactivities. The total compound was identified at 56.2% in chloroform extract, 54.72% in hexane extract, and 65% in methanol extract.

Conclusion
The present work is the first comprehensive study of the antioxidant and antifungal activities of Chaetomium globosum isolated from Urginea Indica. Different extracts (methanol, hexane, and chloroform) were prepared from the C. globosum fungus and checked for antifungal activities. Methanol and chloroform extracts showed good antifungal activity as compared to hexane extract. The antioxidant activity of all three extracts was measured by the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, methanol and chloroform extract showed the highest scavenging potential hexane extract showed the lowest scavenging potential for DPPH.
In addition, GC-MS analysis was also done to check the presence of various bioactive compounds in different extracts of Chaetomium globosum. This study concluded that the presence of bioactive compounds in fungi helps in the inhibition of pathogens causing disease in plants. Further studies are required to study the biocontrol activity of C. globosum against several plant diseases and to elucidate its mechanism of action in disease control programs. Endophytic C. globosum possesses

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.