A potential Antimicrobial, Extracellular Enzymes, and Antioxidants resource: Endophytic fungi associated with medicinal plants

119 Attia et al. Int. J. Biosci. 2020 RESEARCH PAPER OPEN ACCESS A potential Antimicrobial, Extracellular Enzymes, and Antioxidants resource: Endophytic fungi associated with medicinal plants Eman A. Attia1, Bhim P. Singh2, Kavya Dashora3, Ahmed M. Abdel-Azeem1* 1Botany Department, Faculty of Science, University of Suez Canal, Ismailia 41522, Egypt 2Department of Biotechnology, Mizoram University, Mizoram-796004, India 3Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India Key words: Medicinal plants, Prospective isolates, Antibacterial, Antifungal, Antioxidant, Industrial enzymes, Saint Katherine, Egypt. http://dx.doi.org/10.12692/ijb/17.1.119-132 Article published on July 17, 2020 Abstract This study aimed for the production of antimicrobial, extracellular enzymes and antioxidants by endophytic teleomorphic Ascomycota associated with medicinal plants. A total of eleven teleomorphic species were isolated from four medicinal plant species. Chaetomium grande and Sordaria fimicola were the most frequently isolated species and represented by 12 (Chg1-Chg12) and 7 (Sf1-Sf7) isolates respectively. The minimum inhibitory concentration (MIC) of all the isolates was determined against nine reference strains of bacteria and fungi. Effectiveness of 100-300 µg/ ml DEMSO then in H2O of the ethyl acetate fractions of the most effective two isolates Chg5 and Sf3 on the tested reference strains revealed a different inhibitory effecst. Saturated disc of Streptomycin and Rifampin (0.165 mg/ml) were used for bacteria and amphotericin B and fluconazole were used for yeasts and fungi as a positive control. Enzymatically, Chg5 isolate considered as a resource of amylase, cellulase, protease, lipase, and chitinase. However, Sf3 isolates considered as a resource of amylase, laccase, and chitinase out of six screened enzymes. Total phenolics (TP), total flavonoids (TF) and antioxidant activity of the Sf3 and Chg5 extracs were measured. The TP values were expressed as milligram gallic acid equivalents per gram of dry extract of Sf3 and Chg5, which equal to 53.9±0.35 and 97.9±0.48 respectively. TF present in both Sf3 and Chg5 isolates extracts with values equal to 2.44±0.01 and 7±0.05 respectively expressed as routine equivalents. In vitro, the antioxidant activity of the extracts was investigated using DPPH radical-scavenging assay, and equal to 0.06% and 0.39% respectively in the extract of both taxa. * Corresponding Author: Ahmed M. AbdelAzeem  eman_moha[email protected] International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 17, No. 1, p. 119-132, 2020 120 Attia et al. Int. J. Biosci. 2020 Introduction Fungi from extreme environments are considered as good potential candidates for the isolation of new bioactive compounds (Chávez et al., 2015) and this interest has been reflected in the gradual increase of published articles reporting new compounds from these fungi in Egypt (Abdel-Azeem et al., 2012; Abdel-Azeem and Salem, 2012; Salem and AbdelAzeem,2014; Selim et al., 2014; Selim et al., 2016; Abdel-Azeem et al., 2016; Abdel-Azeem et al., 2018a). Out of about 1 million natural products around 25% are biologically active, of these, about 60% come from plants. (Newman and Cragg, 2007; Demain, 2014). Endophytic fungi are symbiotically associated biota of living plant tissues that induce symptomless disease to their hosts (Petrini, 1991, Cohen, 2006). In the beginning of this century pharmacological and pharmaceutical studies have been directed to endophytic fungi (Strobel and Daisy, 2003) e.g. antimicrobial, antitumor, anti-inflammatory, liver curative and antiviral activities (Ali et al., 2008; Liu et al., 2008. In Egypt, about 55 species and one variety of the genus Chaetomium have been recorded till now. (Blanchette et al., 2017; Abdel-Azeem et al., 2018b). Chaetomium has attracted the attention of researchers as an important genus in Ascomycota because of the variety of biological and biotechnological applications of this species in different areas (Abdel-Azeem et al., 2016). To the best of our knowledge, more than 200 compounds, associated with unique and diverse structural types have been isolated and chemically identified from the genus Chaetomium (Fujimoto et al.,2004; Jiao et al.,2004; Bashyal et al.,2005; Kobayashi et al.,2005; Ding et al.,2006; Isham et al.,2007). Genus Sordaria Ces. & De Not. Includes approximately 10 species, except for S. humana and S. fimicola that are cosmopolitan species and are frequent in soil, while the remaining species have a coprophilic habitat (Guarro et al., 2012). Different secondary metabolites e.g. sordarin, sordarin B, sordariol, neosordarin, tricyclic uronic acid, immunosuppressive constituents were studied from different species of Sordaria by several investigators. (Hauser and Sigg, 1971; Bouillant et al., 1989; Schneider et al., 1995; Okada et al.,1995; Fujimoto et al., 1999; Davoli et al., 2002; Weber et al., 2005). Only 3 species of Sordaria were recorded in Egypt (Moustafa and Abdel-Azeem., 2011). Some important medicinal plant species were widely distributed in arid Sinai, Egypt. It has been reported to possess many medicinal properties. (Hanafi and Abdel-Wahab., 2000; Hameed et al., 2015). Some endophytic fungi can produce the same bioactive compounds as their host plants (Stierle et al., 1993; Zhao et al., 2010. Due to the previous demonstration, further researches on medicinal plants and the isolated endophytes must be done to fill the gap in this area, to conserve the medicinal plant as possiple, and to employ the isolated endophytes as a resources of prospective valuable components, Thus, this study was accompanied to isolate diffetent species of endophytes from arid Sinai, Egypt., and to to evaluate antioxidant, antimicrobial activities and valuable enzymes of the endophytic fungi as a prospective sources. Materials and methods Study area, sampling, and isolation of endophytic fungi One hundred samples from the four plant species under investigation were collected from Wadi Itlah (28°58'72.3" N, 33°92'01.7" E, 1385 m above sea level), Wadi Tala (28°34'02.3" N, 33°55'55.8" E, 1450–1670 m above sea level, Wadi El-Arbaein (28°54'54" N, 33°55'36" E, 1385-1859 m above sea level) and Gebel Ahmar (28°52'83" N, 33°61'83" E, 1892 m above sea level) respectively. Aerial parts (25 sample/ plant) from Artemisia herba-alba Asso; Chiliadenus montanus (Vahl) Brullo; Origanum syriacum L.; and Verbascum sinaiticum Benth. were sampled according to (Salem and Abdel-Azeem., 2014). For isolation of endophytic microfungi, small parts of the shoot system of each plant were surfacesterilized according to (Abdel-Azeem and Salem 121 Attia et al. Int. J. Biosci. 2020 .,2012) Sterilized pieces were plated out on 400 plates of different isolation media (Potato Dextrose Agar, Czapek’s Yeast Extract Agar, Malt Extract Agar, Czapek’s Yeast Extract Agar) supplemented with bacteriostatic Rosebengal and bacteriocidal chloramphenicol and incubated at 28 oC. Phenotypic identification Phenotypic identification of recovered microfungi was primarily based on the relevant identification keys for Penicillium (Pitt, 1979); Aspergillus (Klich, 2002). dematiaceous hyphomycetes (Ellis 1971,1976). Fusarium (Leslie and Summerell, 2006) different taxa (Domsch et al., 2007) ascomycetes of soil. (Guarro et al., 2017) Chaetomium and Alternaria (Simmons, 2007) Taxonomic position, assignments and name corrections of all recovered taxa were checked against the Index Fungorum website database (Kirk, 2018). Molecular identification As potent isolates, Chg5 and Sf3 were molecularly identified. DNA of single spore culture was extracted by adapted chloroform procedure (Arenz and Blanchette, 2011) the internal transcribed spacer (ITS) region of ribosomal DNA was targeted for PCR amplification with the primers ITS1 and ITS4 for large subunit amplification. PCR amplifications were done using Amplitaq Gold PCR Master-mix (Applied Biosystems, Foster City, CA) and 1 ml of template DNA using the following parameters: 94 oC for 5 min, 35 cycles of 94 oC for 1 min, 50 oC for 1 min, 72 oC for 1 min, and a final extension step of 5 min at 72 oC. PCR amp icons were visualized on a 1 % agarose gel using SYBR green 1 (Life Technologies, Grand Island, NY) prestain and a Dark Reader DR45 Trans illuminator (Clare Chemical Research, Denver, CO). Primers used for PCR were used for sequencing reactions on automated DNA sequencer (Model 3100; Perkin Elmer Inc/Applied Bio systems–Bioneer, South Korea), according to the manufacturers protocol. Consensus sequences were assembled using Geneious 9.0 (Kearse et al., 2012). And compared to those in Gen Bank using BLASTn for identification. Extraction of active metabolites Isolates of Ch. grande and S. fimicola under investigation were grown on Oat Meal Agar at 28°C for 15 days. Each taxon was prepared by inoculation in 2L Erlenmeyer flasks containing 1L autoclaved potato dextrose broth and shaking at 180 rpm at 28°C for 21 days. The fermentation broth of each species was filtered and fresh mycelia were washed three times with distilled water and stored in a freezer. Ethyl acetate was used for the extraction of active metabolites. The filtrate was extracted three times with equal volumes of ethyl acetate and collected separately (aqueous and organic phases). The frozen mycelia were ground and extracted three times in the organic solvent, and combined with organic extracts of the filtrate and evaporated till dryness under reduced pressure according to the procedures outlined by (Salem and Abdel-Azeem 2014) After evaporation, the dried extract was stored in darkness in a refrigerator until further use. For antimicrobial studies, a freshly prepared solution of solid metabolites was applied through re-suspension in the DMSO solution and water. Antimicrobial screening The following test microorganisms were used for screening of antimicrobial activities: Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 6629, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Shigella flexneri ATCC 12022, Salmonella typhimurium ATCC 14028, Candida albicans ATCC10231, Aspergillus brasiliensis ATCC 16404 and Aspergillus niger ATCC 16404. The bacterial and fungal pathogens were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The agar well diffusion method was used to test the antimicrobial effects of the tested fungal isolates extract against the tested pathogenic microbes, via measuring the diameter of the inhibition zone. Concisely, the hole-plate diffusion method consisted of performing a uniform spread of the tested isolates suspension (about 100 μL, corresponding to Mc Farland standard 2) on Potato dextrose agar plate followed by the making of wells of 6 mm diameter on labeled positions using cork borers 122 Attia et al. Int. J. Biosci. 2020 and filling particular wells with 100 μL (corresponding to 100, 200 and 300 μg/ ml of DEMSO then repeated by water as a solvent per well of Chg5 and Sf3 prepared extract). Saturated disc of Streptomycin and Rifampin (0.165 mg/ml) were used for bacteria and also amphotericin B and fluconazole were used for yeasts and fungi as positive control while Water and DMSO served as the negative control (Valgas et al., 2007). The plates were incubated at 37°C and inhibition zones were observed after 24 h for bacteria and C. albicans and 72 h for fungi. Triplicates of each sample were used. Both antibacterial and antifungal activities were determined by measuring the diameter of the inhibition zone mean value ± standard error. Extracellular enzymes Both taxa were screened for production of six extracellular enzymes by plate assay method and were assessed by placing 5 mm mycelial plugs on solid media with substrates specific to the respective enzyme, starch for amylase, carboxymethyl cellulose for cellulase, guiacol for laccase, olive oil for lipase, colloidal chitin for chitinase and gelatin for protease respectively (Kouker and Jaeger 1987; Maria et al., 2005; Sharaf et al., 2012; Pavithra et al., 2012). After incubation at room temperature for 5 days, the plates were examined for the presence of a clear zone in the agar around the colony, indicating extracellular enzyme activity. Amylase and protease activities were detected by staining the plates with Lugol’s solution and Coomassie brilliant blue solution, respectively. Relative enzyme activity (RA) was calculated according to (Krishnan et al., 2014). Total phenolic, flavonoid and antioxidant activity Total phenolic content of organic extracts of both taxa was estimated using Folin-Ciocalteau reagent based assay using Gallic acid as standard (Oliveira et al., 2008). The total flavonoid content was determined by the aluminum chloride colorimetric method (Chang et al., 2002) the percentage of antioxidant activity of both isolates was assessed by 1, 1 diphenyl-2-picryl hydrazyl (DPPH) free radical assay according to (Shiban et al., 2012). Statistical analysis Statistical analyses were performed using the IBMSPSS statistical software version 23. A one-way analysis of variance was used to determine whether a significant difference existed between the treated groups and the positive control. Data were expressed as mean values of three replicates and differences were considered statistically significant if P < 0.05. Ethics statement All participants gave their verbal, written and informed consent to participate in the study after they were verbally read all the elements of written consent. Samples were collected under the permission of the Saint Katherine Protectorate (permit no. 15/2017) for scientific purposes and no endangered species were involved in the study. All relevant data are within the paper. Results and discussion Study area, sampling, and isolation of endophytic fungi During the present study of endophytic fungi hosted four plant species in Saint Katherine protectorate we recovered 387 CFU from all plant segments. Our observations showed that 46 species belonging to 20 genera were isolated and assigned to 2 phyla, 3 classes, 7 orders, and 10 families. Order Eurotiales accommodates the greatest range of species (16 species) followed by, Hypocreales and Pleosporales (9 species each) and Sordariales (8 species). Other orders were represented by one or two species. Chiliadenus montanus and Origanum syriacum are by far the richest plant by showing a spectrum of 25 species, followed by Artemisia herba-alba (24species). Verbascum sinaiticum considered the poorest plant by recorded 16 species. Regarding sociability of endophytic fungal species, i.e., association with specific host plant, the results showed that while some taxa are likely able to exist on more than one plant species such as Alternaria alternata, Chaetomium globosum, and Nigrospora oryzae, others showed clear tendency for a restricted 123 Attia et al. Int. J. Biosci. 2020 occurrence, like Sordaria fimicola which restricted to Origanum syriacum and Chaetomium grande to Verbascum sinaiticum. The ordination showed that some species are not specific in their occurrences such as Chaetomium globosum, Aspergillus niger, Alternaria alternate, Trichoderma harzianum and Penicillium chrysogenum which are common to a wide range of plants. Table 1. Table 1. Total count of fungal taxa recovered from plants under investigation on isolation media at 28°C. Species Artemisia herba-alba Chiliadenus montanus Origanum syriacum Verbascum sinaiticum Total Teleomorphic taxa Chaetomium bostrychodes Zopf. 0 2 3 0 5 Ch. brasiliense Bat. & Pontual 1 0 0 0 1 Ch. globosum Knuze 2 3 1 1 7 Ch. grande Asgari & Zare 0 0 0 12 12 Ch. nigricolor L.M. Ames 0 1 2 0 3 Ch. piluliferum J. Daniels 3 0 0 0 3 Ch. senegalense L.M. Ames 0 1 2 0 3 Emericella nidulans (Eidam) Vuill. 1 0 3 0 4 Eurotium chevalieri L. Mangin 0 1 0 0 1 Microascus trigonosporus C.W. Emmons & B.O. Dodge 1 0 1 0 2 Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. 0 0 9 0 9 Anamorphic taxa Acremonium alternatum Link 1 0 0 1 2 A. murorum (Corda) W. Gams 0 0 2 1 3 A. rutilum W. Gams 1 0 0 2 3 Alternaria alternata (Fr.) Keissl. 5 5 5 3 18 A. atra (Preuss) Woudenb. & Crous 5 2 4 4 15 A. botrytis (Preuss) Woudenb. & Crous 0 1 0 0 1 Alternaria tenuissima (Kunze) Wiltshire 0 2 1 0 2 Aspergillus alliaceus Thom & Church 2 0 0 1 3 A. candidus Link 0 0 2 0 2 A. flavus Link 2 10 14 0 26 A. fumigatus Fresen. 1 0 0 0 1 A. japonicus Saito 0 0 1 0 1 A. niger Tiegh. 8 28 31 11 87 A. terreus Thom 1 11 0 0 12 A. versicolor (Vuill.) Tirab. 0 0 2 0 2 Cladosporium cladosporioides (Fresen.) G.A. de Vries 2 3 2 0 7 Curvularia lunata (Wakker) Boedijn 0 0 5 0 5 Drechslera australiensis Bugnic. ex M.B. Ellis 0 0 0 2 2 D. hawaiiensis Bugnic. Ex Subram. & B.L. Jain 0 1 2 1 4 D. spicifer (Bainier) Arx 0 1 0 0 1 Embellisia phragmospora (Emden) E.G. Simmons 0 0 0 1 1 Fusarium oxysporum Schltdl. 8 0 4 2 14 F. solani (Mart.) Sacc. 0 5 0 0 5 Mucor hiemalis Wehmer 8 0 0 0 8 Nigrospora oryzae (Berk. & Broome) Petch 5 21 10 10 46 Paecilomyces variotii Bainier 0 2 0 0 2 Penicillium brevi-compactum Dierckx 0 4 4 0 8 P. chrysogenum Thom 6 6 4 3 19 P. citrinum Thom 5 0 0 0 5 P. notatum Westling 0 2 0 0 2 P. rubrum Stoll 2 0 0 0 2 Sarocladium strictum (W. Gams) Summerb. 0 1 0 0 1 Stachybotrys chartarum (Ehrenb.) S. Hughes 1 2 4 0 7 Trichoderma harzianum Rifai 1 2 0 0 3 T. viride Pers. 3 4 3 7 17 124 Attia et al. Int. J. Biosci. 2020 The sequences obtained from C. grande and S. fimicola isolates were 542 and 553 bp in length respectively. C. grande and S. fimicola sequences were checked against the NCBI database using the BLAST homology search. Native C. grande and S. fimicola were deposited in the GenBank database under accession number MF787599 and MF787600 respectively. Antimicrobial activity It was declared that the crude ethyl acetate extracts (100 μL) of both taxa Sf3 and Chg5 (corresponding to 100, 200 and 300) μg/ ml of DEMSO per well revealed an efficiently in suppressing the growth of eight tested isolates with variable strength. As indicated in Table (2). The mean inhibition zone according to the Sf3 effect ranged from 1.37 to 2.50 on S.aureus, 2.10-2.40 on E. coli, 2.50-2.67 on B. cereus. 8.50-9.00 on S. flexneri, 3.33-3.60 on S. Typhimurium, 2.27-3.27 on C. albicans, 0.00-2.33 on A. brasiliensis, and 0.00-2.47 on A. niger indicating a remarkable antimicrobial effect when compared with that of the positive control of bacteria and fungi, According to Chg5 isolate effect the mean inhibition zone ranged from1.27 to 1.47 on S.aureus, 1.47-1.77 on E. coli, 2.07-2.60 on B. cereus. 8.50-9.03 on S. flexneri, 3.47-3.73 on S. Typhimurium, 2.30-2.57 on C. albicans,0.00-2.60 on A. brasiliensis, and 0.002.37 on A. niger, revealed a remarkable antimicrobial effect when compared with that of the positive control of bacteria and fungi, however, the both isolate crude solvent extract had no effect on K. pneumoniae Table 2. Antimicrobial effects of Chaetomium grande and Sordaria fimicola fungi against different bacterial and fungal species. Treatments S. aureus E. coli K. pneumoniae B. cereus S. flexneri S. yphimurium C. albicans A. brasiliensis A. niger A1 1.37±0.09bc 2.10±0.06de 0.00±0.00 a 2.50±0.06de 8.50±0.29d 3.33±0.33cd 2.27±0.15c 0.00±0.00a 0.00±0.00a A2 2.10±0.06f 2.50±0.06ghi 0.00±0.00 a 2.30±0.12cd 8.90±0.21d 4.67±0.33e 3.53±0.29e 0.00±0.00a 0.00±0.00a A3 2.50±0.06g 2.40±0.06fgh 0.00±0.00 a 2.67±0.03e 9.00±0.26d 3.60±0.23cd 3.27±0.13e 2.33±0.18d 2.47±0.07d B1 1.27±0.03b 1.47±0.24b 0.00±0.00 a 2.07±0.07b 8.50±0.29d 3.47±0.29cd 2.30±0.15c 0.00±0.00a 0.00±0.00a B2 1.57±0.15d 2.13±0.09def 0.00±0.00 a 3.07±0.07f 8.67±0.33d 3.80±0.20d 3.20±0.15e 0.00±0.00a 0.00±0.00a B3 1.47±0.09cd 1.77±0.09c 0.00±0.00 a 2.60±0.12e 9.03±0.27d 3.73±0.35d 2.57±0.13cd 2.60±0.06e 2.37±0.07cd C1 0.00±0.00a 2.13±0.09def 0.00±0.00 a 2.17±0.09bc 2.50±0.15b 3.37±0.09cd 0.00±0.00a 0.00±0.00a 0.00±0.00a C2 0.00±0.00a 2.20±0.12def 0.00±0.00 a 2.17±0.09bc 2.77±0.03b 3.50±0.06cd 0.00±0.00a 0.00±0.00a 0.00±0.00a C3 0.00±0.00a 2.33±0.07efg 0.00±0.00 a 2.30±0.06cd 2.80±0.10b 3.60±0.06cd 2.67±0.03d 2.63±0.07e 2.27±0.15c D1 2.10±0.06f 2.50±0.06ghi 0.00±0.00 a 2.17±0.09bc 2.40±0.06b 3.07±0.07c 0.00±0.00a 0.00±0.00a 0.00±0.00a D2 2.10±0.06f 2.70±0.06i 0.00±0.00 a 2.13±0.03bc 2.47±0.03b 3.07±0.07c 0.00±0.00a 0.00±0.00a 0.00±0.00a D3 2.40±0.06g 2.63±0.03hi 0.00±0.00 a 2.63±0.09e 2.47±0.03b 3.23±0.09cd 2.37±0.09cd 1.87±0.03c 2.43±0.09d Streptomycin 0.00±0.00a 1.93±0.03cd 1.73±0.03 c 2.47±0.03de 3.80±0.06c 2.17±0.03b 0.00±0.00a 0.00±0.00a 0.00±0.00a Riphambin 1.87±0.03e 0.00±0.00a 0.87±0.03b 3.10±0.06f 3.73±0.03c 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a Fluconazole 0.00±0.00a 0.00±0.00a 0.00±0.00 a 0.00±0.00a 0.00±0.00a 0.00±0.00a 2.50±0.06cd 1.57±0.03b 1.93±0.03b Amphotercin 0.00±0.00a 0.00±0.00a 0.00±0.00 a 0.00±0.00a 0.00±0.00a 0.00±0.00a 1.83±0.03b 3.73±0.03f 3.93±0.03e ANOVA F-ratio 285.477 121.821 159.867 159.552 353.331 62.130 152.984 4642.912 723.489 p-value <0.001** <0.001** <0.001** <0.001** <0.001** <0.001** <0.001** <0.001** <0.001** A1, A2, A3 S. fimicola solvent extract (100, 200. 300) µg, B1, B2, B3 Ch. grande solvent extract at (100, 200. 300) µg. C1, C2, C3 S. fimicola aqueous extract at (100, 200. 300) µg, D1, D2, D3 Ch. grand aqueous extract at (100, 200. 300) µg Positive antibacterial control 1(Rifampin), Positive antibacterial control 2(Streptomycin) Antifungal positive control 1(Fluconazole), Antifungal positive control 2(Amphotercin). Mean ±ST. error estimated by one way ANOVA. Values are means ± standard error (SE). Means within the same row or column with different superscripts are significantly different. Also in the present study, It was demonstrated that the crude aqueous extracts(100 μL) of both taxa Sf3 and Chg5 (corresponding to 100, 200 and 300 μg/ ml of H2O per well revealed an efficiently in suppressing the growth of seven and eight isolates out of the tested nine isolates by Sf3 and Chg5 respectively with variable strength. As indicated in Table (2). egarding the Sf3 isolate effect, the mean inhibition zone ranged from 2.13-2.33 on E. coli, 2.17-2.30 on B. cereus. 2.50-2.80 on S. flexneri, 3.37-3.60 on S. 125 Attia et al. Int. J. Biosci. 2020 Typhimurium, 0.00-2.67 on C. albicans,0.00-2.63 on A. brasiliensis, and 0.00-2.27 on A. niger indicating a notable antimicrobial effect when compared with that of the positive control of bacteria and fungi, however, no effect had shown on S. aureus and K. pneumonia. Regarding the Chg5 isolate the mean inhibition zone ranged from from1.27 to 1.47 on S.aureus 2.50-2.63 on E. coli, 2.17-2.63 on B. cereus. 2.40-2.47 on S. flexneri, 3.07-3.23 on S. Typhimurium, 0.00-2.37 on C. albicans,0.00-1.87 on A. brasiliensis, and 0.00-2.43 on A. niger indicating a notable antimicrobial effect when compared with that of the positive control of bacteria and fungi, however, no effect had shown on K. pneumoniae. Regarding the in vitro study, the results manifested that both the solvent and the aqueous extract of Sf3 and Chg5 isolates gained a strong antimicrobial activity against the tested pathogenic bacteria and fungi in a dose-dependent manner. In comparing the tested extract with the standard positive control antibiotic Rifampin and Streptomycin and the Antifungal positive control Fluconazole and Amphotericin it revealed their significant antimicrobial activities. The increasing prevalence of fungi and bacteria that resistant to some drug, lead to search for new natural components that have a potent antimicrobial effects and low sides effects on humans. The variety of the biologically active components (novel bioactive secondary metabolites) with potential employment in the medical and agricultural fields extracted from endophytes led to extensive focus and research on these organisms. (Bilal et al., 2018). Table 3. Relative enzyme activities (RA) of Chaetomium grande and Sordaria fimicola. Fungal strain Enzymes Amylase laccase Cellulase Protrinase Lipase Chitinase Ch. Grande 1.6 - 1.5 1.4 0.9 1.3 S. fimicola 1.9 1.2 - - - 1.9 From the given data it was demonstrated that both taxa Sf3 and Chg5 exhibited different strengths of antimicrobial activities which may be attributed to the extraction procedure culture condition and the test strain used for the antimicrobial analysis (Hoffman et al., 2008). In many studies Chaetomium is genus of ascomycete reported to produce a lot of bioactive compounds as reported by Zhangb et al., (2012); Wang et al., (2017). Studying the secretion of antimicrobial components by promising endophytic fungi gained a lot of attention due to the increasing resistance rate of pathogenic bacteria and fungi to the most antibiotics and antifungal and also as a result of the serious side effects of both the synthetic antibacterial and the antifungal. (Hoffman et al., 2008). The current study will be concerned with the effect of some endophytic fungal extract on some pathogenic bacteria and fungi, which give inhibitory effects against them with different degrees. this confirmed by Ananda et al. (2012) who reported that the endophytes become novel sources of antimicrobial components and the beneficial role of these endophytes may be due to increasing the resistance of plant host immunity to the different pathogenic fungi and bacteria. Noteworthy there are many studies reported with the antimicrobial compounds produced by endophytes in cultures that were active against plant and human pathogenic microorganisms (Pandey at al., 2004; Ogundare et al., 2006 Chareprasert et al., 2006). With the finding of our study, it was previously reported by Ibrahim et al (2018) that the naturally extracted fungal metabolites possessed remarkable antibacterial, antifungal, antioxidant, radical scavenging. 126 Attia et al. Int. J. Biosci. 2020 Extracellular enzymatic potential Both endophytes Chaetomium grande and Sordaria fimicola display an extracellular enzymatic potential with a variable degree as demonstrated in Table 3. Where Chaetomium grande had the activity of Amylase RA= 1.6, Cellulose RA= 1.5, Protease RA=1.4, Lipase RA= 0.9 and Chitinase RA=1.3 secretion. Sordaria fimicola had the potential of Amaylase RA=1.9 Laccase RA=1.2 and Chitinase RA= 1.9 secretion. From the given data it was declared that both taxa Sf3 and Chg5 exhibited different strengths of enzymatic activity, and so it considered as rich sources of enzymes that are used in the large scale in medicine and industry. Knowledge of the types, amounts, and characteristics of enzymes produced by the endophytic fungi would help for industrial requirements. Studied taxa must gain attention because they considered a rich important origin of enzymes that are more stable at different temperature ranges and diverse pH than enzymes derived from plants and animals (Mari et al., 2005) and it is helpful in agriculture, industries (food processing, production of beverages, textiles and leather industry) and human health. Table 4. Total phenolic compounds, flavonoids and reducing power as antioxidant activities of Chaetomium grande and Sordaria fimicola. Fungal strains Reducing power Total phenolic mg/mL Flavonoids mg/ml Chaetomium grande 0.06% 53.9±0.35 2.44±0.01 Sordaria fimicola 0.39% 97.9 ±0.48 7.0±0.05 Recently, enzymes producer endophytic fungi have become very fascinating resources of some valuable enzymes that used in a lot of applications in life sciences. Amylase has an industrial and agricultural use, and active in a wide range of temperatures and pH, Proteases are used in medical application especially in diabetic patient’s treatment (Ananda et al., 2012). The extracellular cellulase used in industrial applications of the paper industry and lipase used fats as energy sources (Amirita et al., 2012). Endophytic fungi differ in their ability to secrete enzymes, and their activity is controlled by many factors as climatic states and geographical places. Total phenolic, flavonoid and antioxidant activity Total phenolics (TP), total flavonoids (TF) and the antioxidant activity of the Sf3 and Chg5 isolates extracts were represented in Table 4. The TP values were expressed as milligram gallic acid equivalents per gram of dry extract of Sf3 and Chg5 isolates which equal to 53.9±0.35 and 97.9±0.48 respectively. TF present in both Sf3 and Chg5 isolates extract with a value equal to 2.44±0.01 and 7±0.05 respectively expressed as routine equivalents. In the present study, in vitro antioxidant activity of the extracts was investigated using DPPH radical-scavenging assay, and equal to 0.06% and 0.39% in both taxa respectively. Antioxidants have to pay attention to their effects in preventing disease due to their oxidative stress, moreover, the epidemiological studies display that reactive oxygen and nitrogen species could damage the human body. Therefore it is important to increase the intake of antioxidants in the human diet. But it was demonstrated that the synthetic antioxidants might exhibit toxicity with carcinogenic potential and also has a low efficiency than natural antioxidants. (Lob et al., 2010). So it is critical to obtain natural antioxidant with low cost. In the present study it was detected a phenolic and flavonoids compounds in the Sf3 and Chg5 isolates extracts, Which are recognized to achieve several functions in plants, and may display many pharmacological beneits as effective agents in prevention and treatment of various diseases (Shehab 127 Attia et al. Int. J. Biosci. 2020 et al., 2015). Also, the scavenging capability of DPPH free radical is commonly used to explore the antioxidant property of the tested extracts (. Moukette et al., 2015). Our finding was confirmed with other studies that Endophytic fungi are previously reported as resources of ample bioactive compounds and secondary metabolites. These bioactive compounds and secondary metabolites have applications in biological control. Thus, there is an urgent to extract and identify natural components that have more economical and effective antioxidants characters (Mathew and Abraham, 2006, Kumar and Chattopadhaya, 2007 Chandra and Arora, 2009) Oduntan et al., 2017. Conclusion The present study declared that the endophytic teleomorphic Ascomycota Chaetomium grande and Sordaria fimicola hosted medicinal plants in Saint Katherine Protectorate, Egypt extract acts as an antimicrobial agent. The principal mechanisms of action may rely on its antioxidant potential. Thus, the extracted natural components are recommended as antimicrobial agents against some pathogenic fungi and bacteria. The extracted natural components considered as another source of safe antioxidants for integration into some foodstuffs and supplements and also in preventing many free radical-mediated diseases and used in healthy cosmetics. Beside that they acquire an enzymatic activity, which recommended to be used in biotechnology. The present study recommended further and continuous research on endophytic fungi to detect, purify and identify more of the bioactive compounds. Conflicts of interest The authors have no conflicts of interest to disclose. References Abdel-Azeem AM, Blanchette RA, Held BW. 2018b. New record of Chaetomium grande Asgari & Zare (Chaetomiaceae) for the Egyptian and African mycobiota. Phytotaxa 343(3), 283-288. Abdel-Azeem AM, Omran MA, Mohamed RA. 2018a. Evaluation of the curative probability of bioactive metabolites from endophytic fungi isolated from some medicinal plants against paracetamolinduced liver injury in mice. LAP LAMBERT Academic Publishing. ISBN: 978-613-9-89820-6. Abdel-Azeem AM, Salem FM. 2012. Biodiversity of laccase producing fungi in Egypt. Microsphere 3(6), 900–920. Abdel-Azeem AM, Salem FM, Mohamed HM, Rashad HM, Mohamed RM, Khalil WF. 2012. Bioprospecting of Egyptian fungi for ecological and medical applications. TWAS-ARO 8th meeting, Bibilotheca Alexandrina, December 30-31. Availableat: http://www.bibalex.go.eg/CSSP/Presentations/Attac hments/Fatma%20Salem/play.htm Abdel-Azeem AM, Zaki SM, Khalil WF,Makhlouf NA, Farghaly LM. 2016. Antirheumatoid activity of secondary metabolites produced by endophytic Chaetomium globosum. Frontiers in Microbiology 7(1477), 1-11. Aly AH, Edrada-Ebel R, Wray V, Muller WEG, Kozytska S, Hentschel U. 2008. Bioactive metabolites from the endophytic fungus Ampelomyces sp. Isolated from the medicinal plant Urospermum picroides. Photochemistry 69, 1716– 1725. http://dx.doi.org/10.1016/j.phytochem.02.013. Amirita A, Sindhu P, Swetha J, Vasanthi NS, Kannan KP. 2012. Enumeration of endophytic fungi, from medicinal plants and screening of extracellular enzymes. World Science and Technology 2, 13-9. Ananda K, Pavithra LN, Sathish L. 2012. Antimicrobial and enzyme activity of Endophytic