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<p>Vol.:(0123456789)1 3</p><p>Arabian Journal for Science and Engineering</p><p>https://doi.org/10.1007/s13369-020-04454-1</p><p>RESEARCH ARTICLE-BIOLOGICAL SCIENCES</p><p>Identification and Bioactivities of Two Endophytic Fungi Fusarium</p><p>fujikuroi and Aspergillus tubingensis from Foliar Parts of Debregeasia</p><p>salicifolia</p><p>Sobia Nisa1  · Nimra Khan1 · Waqas Shah2 · Maimoona Sabir1 · Wajiha Khan2 · Yamin Bibi3 · Muhammad Jahangir4 ·</p><p>Irshad Ul Haq1 · Sadia Alam1 · Abdul Qayyum5</p><p>Received: 20 November 2019 / Accepted: 28 February 2020</p><p>© King Fahd University of Petroleum & Minerals 2020</p><p>Abstract</p><p>Endophytic fungi isolated from medicinal plants are important for production of antibiotics. They can produce secondary</p><p>metabolites with diverse structures and activities. Debregeasia salicifolia is a plant of medicinal importance, and no report</p><p>exists regarding isolation of endophytic fungi from it. This study was focused to isolate and identify culturable endophytic</p><p>fungi from foliar parts of D. salicifolia and to determine their bioactivities. Molecular analysis resulted in identification of</p><p>Fusarium fujikuroi, Aspergillus tubingensis and Rhizopus oryzae based on specific internal transcribed spacer primer (ITS1/</p><p>ITS4). Our analysis revealed that all fungal endophytes possess antibacterial activity against Gram-negative and Gram-</p><p>positive bacteria. Remarkably, Rhizopus oryzae at a concentration of 5 mg/mL efficiently restricted the growth of ATCC</p><p>strain of E. coli in comparison with positive control ciprofloxacin. Rhizopus oryzae and F. fujikuroi at a concentration of</p><p>1000 µg/ml exhibited maximum antioxidant activity of 45% and 44%, respectively. They also showed antifungal activity</p><p>ranging from 60 to 75% against Aspergillus flavus and Aspergillus niger. Our analysis of the fungal extracts through GC–MS</p><p>indicated the presence of 21 compounds of diverse nature and structure. In conclusion, our study highlighted the potential</p><p>of D. salicifolia to host a plethora of fungal endophytes that secrete potentially therapeutic bioactive metabolites</p><p>Keywords Debregeasia salicifolia · Endophytic fungi · ITS region · Bioactivities · GC–MS</p><p>1 Introduction</p><p>Development of resistance to available antibiotics by patho-</p><p>genic bacteria and fungi is one of most important problem of</p><p>global concern [1, 2]. Many factors are responsible for anti-</p><p>biotic resistance including inappropriate use of antibiotics,</p><p>poor hygienic conditions, increased number of immuno-</p><p>compromised patients, and late diagnosis of infections [3].</p><p>So, the need to search new and more effective agents for</p><p>treating human diseases has been increased [4]. A number of</p><p>compounds have been isolated from medicinally important</p><p>plants and used for the treatment of human ailments [5].</p><p>Natural compounds are preferably used as pharmaceutical</p><p>agents due to their biological friendly nature [6]. Natural</p><p>compounds derived from plants, bacteria, and fungi have</p><p>been recently used to treat multidrug-resistant infectious</p><p>agents alone or in combination with antibiotics. The use</p><p>of antimicrobial agents of natural origin is advantageous</p><p>as they interact by making protein–protein interactions and</p><p>hence microbes rarely develop resistance against them.</p><p>Endophytes like bacteria and fungi colonize higher plant</p><p>tissues without damaging. They are considered as natural</p><p>reservoirs of diverse bioactive products [7, 8]. Bioactive</p><p>secondary metabolites like alkaloids, terpenoids, isocou-</p><p>marins, steroids, quinones, lignans, phenols, and lactones</p><p>have been isolated from endophytic bacteria and fungi [9,</p><p>* Sobia Nisa</p><p>sobia@uoh.edu.pk</p><p>1 Department of Microbiology, The University of Haripur,</p><p>Haripur, KPK, Pakistan</p><p>2 Department of Biotechnology, COMSATS University</p><p>Islamabad, Abbotabad Campus, Abbottabad, Pakistan</p><p>3 Department of Botany, PMAS Arid Agriculture University,</p><p>Rawalpindi, Pakistan</p><p>4 Department of Food Science and Technology, The University</p><p>of Haripur, Haripur, KPK, Pakistan</p><p>5 Department of Agronomy, The University of Haripur,</p><p>Haripur, KPK, Pakistan</p><p>http://orcid.org/0000-0002-4541-1490</p><p>http://crossmark.crossref.org/dialog/?doi=10.1007/s13369-020-04454-1&domain=pdf</p><p>Arabian Journal for Science and Engineering</p><p>1 3</p><p>10]. Endophytic fungi from medicinal plants have been</p><p>reported for antibacterial and antifungal activities [11–14].</p><p>Plants also serve as a reservoir of secondary metabolites.</p><p>Endophytic fungi could also produce metabolites similar to</p><p>or with more pronounced activity than that of their respec-</p><p>tive hosts [15].</p><p>Endophytic fungi are thought to have mutualistic asso-</p><p>ciations with plants. In this symbiotic relationship, plants</p><p>provide shelter and nutrients to the endophytes [16, 17]</p><p>while the host plants are protected against pathogens and</p><p>herbivores [18–20]. Moreover, endophytes are also reported</p><p>to support plant growth by producing phytohormones [21]</p><p>and increase their resistance to multiple abiotic stresses like</p><p>salinity and heavy metal toxicity [22, 23]. Studies on iso-</p><p>lation of bioactive metabolites from endophytic fungi can</p><p>help in identification of some novel compounds and that</p><p>can further be developed as antimicrobial agents to cope</p><p>antibiotic resistance.</p><p>Debregeasia salicifolia is an important plant with ethno-</p><p>botanical importance and is widespread in different parts of</p><p>West Asia and Western Himalayan. Its bark paste is useful</p><p>externally on forehead to relieve pain [24]. Traditionally, it</p><p>is used to treat several diseases like urinary tract infections,</p><p>bone fractures, boils, bloody diarrhea, carbuncles, pimples,</p><p>dermatitis, skin rash, eczema, and tumors [25]. Our group</p><p>reported the antibacterial and anticancer activity of D. sali-</p><p>cifolia previously [26, 27]. This plant has not been evaluated</p><p>for isolation of culturable endophytic fungi and their activi-</p><p>ties. In the present investigation, we evaluated the diversity</p><p>and bioactive potential of endophytic fungi colonizing D.</p><p>salicifolia.</p><p>2 Materials and methods</p><p>2.1 Collection and Surface Sterilization of Plant</p><p>Samples</p><p>Healthy leaves and stems of D. salicifolia were collected</p><p>from Abbotabad, Pakistan, and identification of the plant</p><p>material was done by using standard criteria. Samples were</p><p>washed under running tap water prior to surface sterilization.</p><p>Surface sterilization was done using 0.1% HgCl2 solution for</p><p>1–3 min, 70% (v/v) ethanol for 1 min, followed by 1% (v/v)</p><p>sodium hypochlorite for 5 min and immersion in 70% (v/v)</p><p>for 30 s. The sterilized segments were washed thrice with</p><p>autoclaved distilled water and dried on blotting paper. Stem</p><p>and leaf segments were cut using sterilized surgical blades</p><p>and shifted to potato dextrose agar (Oxoid) supplemented</p><p>with 100 mg/mL tetracycline to stop growth of bacteria.</p><p>Aliquots from final washed water were also plated on the</p><p>PDA plates to check the effectiveness of surface sterilization.</p><p>The Petri plates were sealed with parafilm and incubated for</p><p>5–7 days at 28◦C for initiation of fungal growth.</p><p>2.2 Development of Pure Culture</p><p>After 5–7  days of incubation, mycelial growth started</p><p>emerging from the explants, and they were subcultured to</p><p>get pure culture. Pure colonies were transferred on potato</p><p>dextrose agar slants and preserved at 4–5 °C for future use.</p><p>The preserved fungi were subcultured from time to time.</p><p>2.3 Morphological Identification of Endophytic</p><p>Fungi</p><p>The fungi were identified on morphological basis as previ-</p><p>ously described [28, 29]. Identification was performed by</p><p>noticing morphological parameters like colony diameter,</p><p>texture, front and reverse color of colony and microscopic</p><p>appearance of hyphae and reproductive structures.</p><p>2.4 Molecular Identification of Endophytic Fungi</p><p>DNA extraction was performed using phenol chloroform</p><p>method with little modification. For PCR amplification, 1</p><p>μL of diluted genomic DNA, primers (ITS-1 and ITS-4) and</p><p>Ampliqon (Taq polymerase) (Thermo Fisher Scientific) were</p><p>used to make the final reaction mixture. Preliminary denatur-</p><p>ation was done at</p><p>95 °C for 7 min followed by 35 cycles of</p><p>amplification, denaturation (95 °C, 30 s), annealing (58 °C,</p><p>45 s), and elongation (72 °C, 60 s) and the final elongation</p><p>was performed at 72 °C for 6 min. After the reaction, PCR</p><p>products purification and sequencing were performed com-</p><p>mercially through alpha genomics.</p><p>2.5 Phylogenetic Analysis of Isolated Fungal</p><p>Endophytes</p><p>For phylogeny of the isolated fungal endophytes, we</p><p>retrieved sequences of the close hits from NCBI and per-</p><p>formed multiple sequence alignment using Clustal W. The</p><p>aligned files were then exported in mega format. Neigh-</p><p>bor-joining tree was constructed in MEGAX using default</p><p>parameters and bootstrap values of 1000.</p><p>2.6 Production of Secondary Metabolites</p><p>For the production of secondary metabolites, 150 mL of</p><p>potato dextrose broth was prepared in 250-mL flasks and</p><p>autoclaved. Medium was inoculated with fungal culture and</p><p>incubated at 28 ± 1 °C in shaking incubator at 170 rpm for</p><p>4 weeks. Secondary metabolites were excreted by myce-</p><p>lium in their surrounding media. Mycelium was separated</p><p>from the culture media using muslin cloth. The crude</p><p>Arabian Journal for Science and Engineering</p><p>1 3</p><p>culture broth was filtered through Whatman filter paper and</p><p>extracted with ethyl acetate using separating funnel. The</p><p>ethyl acetate extract was dried by using rotary evaporator.</p><p>The extract was then stored at 4–5◦C for further use.</p><p>2.7 Antibacterial Activity</p><p>Crude fungal extracts were dissolved in dimethyl sulfox-</p><p>ide (DMSO) (5 mg/mL), and antibacterial activity was</p><p>determined against seven American Type Culture Collec-</p><p>tion (ATCC) bacterial strains, i.e., Staphylococcus aureus</p><p>(292013), Bacillus spizizenii (66330), Listeria monocy-</p><p>togenes (35152), Escherichia coli (25922), Klebsiella</p><p>pneumoniae (13883), Salmonella typhimrium (14028) and</p><p>Acinetobacter baumannii (19606) using agar well-diffusion</p><p>method. Bacterial strains were cultured overnight in nutri-</p><p>ent broth to get fresh culture for antibacterial activity. Wells</p><p>were aseptically made in the seeded media with sterile borer.</p><p>Ciprofloxacin solution was prepared by dissolving it in</p><p>DMSO to get 1 mg/mL concentration and used as a positive</p><p>control. Pure DMSO was used as a negative control. Fifty</p><p>microliters of the crude extract was poured in the wells and</p><p>incubated at 37 °C for 24 h. Finally, plates were observed</p><p>for zones of inhibition and their respective diameters were</p><p>measured.</p><p>For the determination of minimum inhibition concentra-</p><p>tion (MIC), bacterial strains were inoculated and incubated</p><p>overnight in nutrient broth. Then, the cultures were diluted</p><p>in physiological saline by ratio of 1:100 and the concentra-</p><p>tion of inoculum was finally adjusted as 1 × 106cells cfu/</p><p>ml. Two folds of CAMHB (cation-adjusted Mueller–Hinton</p><p>broth) medium was poured in each well of 96-well micro-</p><p>titer plate followed by the addition of adjusted inoculum.</p><p>Microtiter plate was incubated for 24 h at 37 °C. Triplicates</p><p>of each extract were carried out to measure the effective</p><p>concentration. After incubation, the optical density was</p><p>measured using microtiter plate reader at the wavelength of</p><p>600 nm. Minimum concentration which inhibits growth of</p><p>pathogenic bacteria was taken as the MIC.</p><p>2.8 Antifungal Activity</p><p>Two pathogenic fungal strains Aspergillus niger and</p><p>Aspergillus flavus were used to screen antifungal poten-</p><p>tial of the endophytic fungal extracts using agar tube dilu-</p><p>tion method [30]. Briefly, 5 mL of Sabouraud dextrose</p><p>agar media was poured in test tubes that were labeled and</p><p>autoclaved at 121 °C. Fungal extracts 200 µL (400 µg/</p><p>mL) were added to autoclaved media before solidifica-</p><p>tion. Sodium benzoate (2.4 µg/mL) was used as a positive</p><p>control, whereas pure DMSO was used as a negative con-</p><p>trol. Medium was mixed by shaking carefully and allowed</p><p>solidification in slanting position. Each tube was inocu-</p><p>lated with seven-day-old fungal strains. After inoculation,</p><p>these tubes were incubated for seven days at 27 °C. The</p><p>inhibition of fungal growth was determined by using the</p><p>following formula:</p><p>where</p><p>lg t = linear growth of test sample</p><p>lg c = linear growth of control sample.</p><p>2.9 Antioxidant Activity by Using Free Radical</p><p>Scavenging Assay</p><p>2,2-Diphenyl-1-picrylhydrazyl hydrate (DPPH) free radi-</p><p>cal scavenging assay was used to determine the antioxidant</p><p>activity of fungal extracts. In antioxidant assay different</p><p>concentrations (1000, 500, 250, 125, 62.5 µg/mL) of each</p><p>sample were mixed with 2.85 mL of DPPH reagent (1 mg</p><p>of DPPH reagent/25 mL of dimethyl sulfoxide) in 15-mL</p><p>falcon tube. The reaction mixture was incubated in dark</p><p>for half an hour, and then absorbance was recorded at</p><p>517 nm by using spectrophotometer. Different concentra-</p><p>tions (1000, 500, 250, 125, 62.5 µg/mL) of ascorbic acid</p><p>were used as a positive control, and dimethyl sulfoxide was</p><p>used as a negative control [31].</p><p>The following formula was used to determine the DPPH</p><p>free radical scavenging assay.</p><p>where</p><p>control OD = absorbance without samples</p><p>sample OD = absorbance in the presence of samples.</p><p>2.10 Gas Chromatography–Mass Spectrometry</p><p>Analysis of Fungal Extracts</p><p>The crude extracts of samples were used to evaluate their</p><p>chemical composition using GC–MS. A 20-min run was</p><p>conducted from the initial temperature of 40 °C to the final</p><p>temperature of 250 °C. The spectrum was noted in the</p><p>range of 40–600 m/z. Peaks of various compounds eluted</p><p>from the column of GC were recorded along with their</p><p>retention time. Data were correlated with mass spectra of</p><p>the compounds, and database was searched for the similar</p><p>compounds with same retention time and molecular mass.</p><p>Bioactivities of already reported natural compounds were</p><p>also studied, and a comparison was made to correlate the</p><p>activities of fungal extracts with their constituents.</p><p>Inhibition of fungal growth = (lg c − lg t)∕ lg c × 100</p><p>(DPPH radical-scavenging activity(%) inhibition</p><p>=</p><p>[</p><p>(control OD − sample OD)∕control OD)] × 100</p><p>Arabian Journal for Science and Engineering</p><p>1 3</p><p>2.11 Statistical Analysis</p><p>Data were obtained as mean of four replicates and analyzed</p><p>by two-factor factorial in completely randomized design</p><p>(CRD). Mean comparison (p < 0.05) was carried out using</p><p>Duncan multiple-range (DMR) test by M-Stat-C Statistical</p><p>software [32].</p><p>3 Results</p><p>3.1 Isolation and Identification of Fungal</p><p>Endophytes from D. Salicifolia</p><p>Five strains of endophytic fungi were isolated and identified</p><p>on the basis of colony morphology and microscopic exami-</p><p>nation (Table 1, Figs. 1, 2). Identification of three strains</p><p>(DS 1, DL2H, and DL3.2) was confirmed by molecular</p><p>analysis. These fungi were identified as Rhizopus oryzae</p><p>(DL2H), F. fujikuroi (DL3.2), and Aspergillus tubingensis</p><p>(DS 1). The results of DNA sequencing were submitted to</p><p>GenBank with the accession number of MN947248 for Rhiz-</p><p>opus oryzae (DL2H), MN947247 for F. fujikuroi (DL3.2),</p><p>and MN990210 for A. tubingensis (DS 1) (Table 2, Fig. 3).</p><p>3.2 Antibacterial Activity of Fungal Extracts</p><p>Extracts of isolated endophytic fungal species were sub-</p><p>jected to antibacterial activity against clinical isolates of</p><p>both Gram-positive and Gram-negative bacteria. Extracts</p><p>exhibited activity against both Gram-negative and Gram-</p><p>positive bacterial species with few exceptions. Extracts</p><p>of all fungal strains were active against tested bacteria.</p><p>Best activity was exhibited by DL2H (Rhizopus oryzae)</p><p>against E. coli with a zone of inhibition having a diameter</p><p>of 28.33 mm, followed by S. aureus and B. subtilis (25 and</p><p>23 mm), respectively (Fig. 4). DL3.2 (F. fujikuroi) inhib-</p><p>ited the growth of Listeria monocytogenes ATCC (35152),</p><p>Staphylococcus aureus ATCC (292013), Salmonella typh-</p><p>imurium ATCC (14028) and Bacillus spizizenii ATCC</p><p>(6633) with a zone of inhibition having diameter of 25, 24,</p><p>24 and 23 mm, respectively. All the strains exhibited best</p><p>activity against E. coli and S. aureus, while least activity</p><p>was observed against A. baumanii. Minimum</p><p>inhibitory</p><p>concentration of DS1 against B. subtilis, S. aureus and</p><p>E. coli was 0.312, 1.25 and 0.625 mg/mL, respectively.</p><p>Ethyl acetate extract of endophytic fungal isolate DL3.2</p><p>exhibited MIC values of 1.25, 2.5 and 5 mg/mL against B.</p><p>subtilis, E. coli and S. aureus, respectively.</p><p>3.3 Antioxidant Activity of Fungal Extracts</p><p>DL2H (Rhizopus oryzae) exhibited the highest radical</p><p>scavenging activity but still much lower than ascorbic</p><p>acid. Similarly, F. fujikuroi and A. tubingensis also exhib-</p><p>ited moderate antioxidant activity (Table 3). At 1000 µg/</p><p>ml the best antioxidant activity was observed by DL2H</p><p>that is 45.17% as compared to 87% as exhibited by ascor-</p><p>bic acid. Similarly, 43 and 41% free radical scavenging</p><p>activity was shown by fungal extract of F. fujikuroi.</p><p>Table 1 Colony morphology and microscopic presentation of isolated endophytic fungi</p><p>Endophytic fungi Media Colony presentation Background of colony Microscopic presentation</p><p>DS1 PDA Blackish in color with wrinkled mar-</p><p>gins, raised smooth velvety surface</p><p>Blackish brown reverse Aseptate, branched, sporangium and</p><p>spore forming</p><p>DS2 PDA Velvety green wrinkled convex</p><p>surface</p><p>Yellowish back with brownish</p><p>margins</p><p>Septate, branched conidiophores and</p><p>spore forming</p><p>DL2H PDA Fluffy, flat, smooth in texture with</p><p>white color</p><p>Orange back with yellowish margins. Aseptate, branched and conidiophores</p><p>with conidia</p><p>DL3.2 PDA Raised colony, smooth velvety wrin-</p><p>kled hard dark brown color</p><p>Dark brownish back with blackish</p><p>margins</p><p>Aseptate, branched with dispersed</p><p>conidia</p><p>Fig. 1 Initiation of growth of fungal endophyte from leaf segment of</p><p>Debregeasia salicifolia</p><p>Arabian Journal for Science and Engineering</p><p>1 3</p><p>3.4 Antifungal Activity of Fungal Extracts</p><p>Ethyl acetate extract of endophytic fungi was tested for anti-</p><p>fungal activity by using tube dilution method. Overall, the</p><p>fungal extract from DL2H (Rhizopus oryzae) was highly</p><p>active against Aspergillus flavus followed by Aspergillus</p><p>niger with 73.3% and 62.5% of activity. DL3.2 (F. fujikuroi)</p><p>also exhibited significant activity against Aspergillus flavus</p><p>with 60% of inhibition (Table 4).</p><p>3.5 GC–MS Analysis of Fungal Extracts</p><p>GC–MS analysis of ethyl acetate extract of endophytic</p><p>fungi revealed the presence of bioactive compounds. The</p><p>presence of several compounds with known bioactivity was</p><p>found in the extracts of each of the 4 fungi analyzed, and</p><p>references are provided in Tables 5, 6, 7 and 8 describing</p><p>where compounds observed in this study were previously</p><p>identified. The compounds showed resemblance with the</p><p>natural products of plant and fungal origin. The analysis of</p><p>GC–MS data revealed that most of them were derivatives</p><p>of volatile compounds like esters, ethers, alkaloids and phe-</p><p>nolic compounds.</p><p>4 Discussion</p><p>Medicinal plants are known to harbor fungal endophytes</p><p>of great medicinal potential that include, Torreya mairei,</p><p>Fusarium sp., Pestalotiopsis jesteri, Chloridium sp. and</p><p>Penicillium microspora [7]. In order to explore the poten-</p><p>tial of D. salicifolia as a host of important endophytic</p><p>Fig. 2 Fungal endophytes</p><p>isolated from Debregeasia sali-</p><p>cifolia, where A; DS1, B; DS2,</p><p>C; DL3.2, D; DL2H</p><p>Table 2 Identification of</p><p>endophytic fungi on the base of</p><p>sequencing results</p><p>S. no. Source Isolates 18S r RNA ampli-</p><p>fied region length</p><p>% similarity NCBI Accession no.</p><p>1 Leaf DL2H 600 bp 84% with Rhizopus oryzae MN947248</p><p>2 Leaf DL3.2 528 bp 99% with Fusarium fujikuroi MN947247</p><p>3 Stem DS 1 568 bp 95% with Aspergillus tubingensis MN990210</p><p>Arabian Journal for Science and Engineering</p><p>1 3</p><p>fungi, we carried out an exploratory study to identify any</p><p>endophytic fungi with potential to synthesize medici-</p><p>nally important natural products. Remarkably, our results</p><p>revealed the presence of three (Rhizopus oryzae, F.</p><p>fujikuroi and A. tubingensis) endophytic fungal strains in</p><p>D. salicifolia.</p><p>The isolated fungi belonged to the phylum Ascomycota</p><p>and Zygomycota. Elsewhere, Ascomycetes, Deuteromycetes,</p><p>Fig. 3 A neighbor-joining tree: depicting the phylogenetic relationship of DS 1, 2 H and 3.2. The tree has been constructed using (ITS region)</p><p>MEGA X. Bootstrap values are shown at respective node</p><p>Fig. 4 Antibacterial activity of</p><p>endophytic fungi against ATCC</p><p>bacterial strains. Vertical bars</p><p>indicate mean (n = 4) ± standard</p><p>deviation (SD)</p><p>0</p><p>5</p><p>10</p><p>15</p><p>20</p><p>25</p><p>30</p><p>35</p><p>Zo</p><p>ne</p><p>s o</p><p>f I</p><p>nh</p><p>ib</p><p>i�</p><p>on</p><p>(m</p><p>m</p><p>)</p><p>Bacterial s</p><p>rains</p><p>DS1</p><p>DS2</p><p>DH2H</p><p>DL3.2</p><p>Ciprofloxacin</p><p>Arabian Journal for Science and Engineering</p><p>1 3</p><p>and Basidiomycetes have been shown to reside inside plants’</p><p>tissues and endophytes [52, 53]. The occurrence of fungi</p><p>inside plants is widespread, a trait that is present in many</p><p>fungal species. For instance, F. fujikuroi has been isolated</p><p>from the Brazilian Pampa biome that is able to produce</p><p>phytotoxic secondary metabolites with herbicidal activity</p><p>[5]. Similarly, Fusarium proliferatum (MTCC 9690) and A.</p><p>tubingensis (strain AN103) have been isolated from the bark</p><p>of Dysoxylum binectariferum and branches of apple plant</p><p>(Malus domestica), respectively [54, 55]. The occurrence</p><p>of endophytic Rhizoctonia like fungi has also been reported</p><p>that is primarily considered as plant pathogenic fungi [56].</p><p>Fungal endophytes are reported to harbor genes encoding</p><p>antibacterial compounds and involved in antioxidant activi-</p><p>ties. Consistent with other reports, our data revealed that F.</p><p>proliferatum isolated from Dysoxylum binectariferum and</p><p>Syzygium cordatum possess cytotoxic, antibacterial and anti-</p><p>oxidant activities [54, 57]. Moreover, Fusarium sp. has been</p><p>reported as one of the most dominant fungal genera isolated</p><p>from Dracaena cambodiana and Aquilaria sinensis, with</p><p>significant antimicrobial activity. This genus is comprised</p><p>Table 3 Antioxidant activity</p><p>of endophytic fungi extracts at</p><p>different concentrations</p><p>Fungal extract Concentrations</p><p>1000 µg/ml 500 µg/ml 250 µg/ml 125 µg/ml 62.5 µg/ml</p><p>DS1 21.10% 19.93% 15.58% 13.89% 11.98%</p><p>DL2H 45.17% 34.99% 34.25% 23.54% 22.79%</p><p>DS2 44.74% 41.19% 39.77% 38.06% 1.42%</p><p>DL3.2 43.75% 41.47% 41.33% 39.77% 26.13%</p><p>Ascorbic acid control 87% 87% 85% 83% 51%</p><p>Table 4 Antifungal activity of endophytic fungi isolated from Debre-</p><p>geasia salicifolia</p><p>Fungal extracts</p><p>(4 mg/ml)</p><p>Antifungal activity of fungal extracts in  %</p><p>Aspergillus niger Aspergillus flavus</p><p>DS1 42.5% 6.6%</p><p>DS2 41.25% 43.3%</p><p>DL2H 62.5% 73.3%</p><p>DL3.2 60% 26.6%</p><p>Table 5 Major constituents of fungal extract DS1, as indicated by GC–MS analysis</p><p>S. no. Retention time Compound name Mol. weight Formula Biological activity</p><p>1 4.87 3,7-Diacetamido-7H-S-triazolo[5,1-C]-S-</p><p>triazole</p><p>223 C7H9O2N7 Antibacterial and antifungal activity is</p><p>reported [33, 34]</p><p>2 5.30 1,6-D deoxy-L-mannitol 150 C6H14O4 Antibacterial and antipyretic activities of its</p><p>derivative are reported [35]</p><p>3 6.02 2-methyl-9-beta-D-ribofuranosyl hypoxan-</p><p>thine</p><p>282 C11H14O5N4 Antifungal and antibacterial activity is</p><p>reported [36]</p><p>4 29.66 CIS-9,10-epoxyoctadecan-1-ol 284 C18H36O2 Antioxidant and antibacterial activity is</p><p>reported [37]</p><p>Table 6 Major constituents of fungal extract DS2, as indicated by GC–MS analysis</p><p>S. no. Retention time Compound name Mol. weight Formula Biological activity</p><p>1 3.66 Propanoic acid, 2-hydroxy-, pentyl ester 160 C8H16O3 Cytotoxic activity of its derivative is reported</p><p>[38]</p><p>2 4.49 2-Methyl-9-beta-D-ribofuranosyl hypox-</p><p>anthine</p><p>282 C11H14O5N4 Antibacterial and antifungal activity is</p><p>reported [39]</p><p>3 5.24 2-vinyl-9-[beta-D-ribofuranosyl] hypoxan-</p><p>thine</p><p>294 C12H14O5N4 Antibacterial and antifungal activity is</p><p>reported [40]</p><p>4 6.8 Silane, trichlorodocosyl 442 C22H45CI3Si Antibacterial, anti-inflammatory, and antitu-</p><p>mor activity is reported [41]</p><p>5 6.8 2-Tridecanol 200 C13H28O Antibacterial activity of its derivative is</p><p>reported [42].</p><p>6 8.89 Methoxy acetic acid, 2-pentadecyl ester 300 C18H36O3 Antimicrobial activity is reported [41]</p><p>Arabian Journal for Science and Engineering</p><p>1 3</p><p>of more than 70 endophytic species</p><p>capable of producing a</p><p>wide array of active metabolites [58]. Similarly, extract of</p><p>Rhizopus oryzae isolated from Crocus sativus L. (Saffron)</p><p>inhibited all bacteria tested. However, contrary to our find-</p><p>ings different levels of antibacterial activity were recorded</p><p>with maximum activity against Luteibacter sp. followed by</p><p>Stenotrophomonas sp., Pantoea sp., Pseudomonas putida,</p><p>E. coli and Bacillus sp. [59].</p><p>Extracts isolated from fungal endophytes are reported</p><p>to possess antioxidant activity [60]. Our results supported</p><p>that concept as the extracts we obtained from four fungal</p><p>endophytes showed antioxidant activity to a variable extent.</p><p>Our findings are consistent with previously published data</p><p>regarding fungal endophyte P. citrium that exhibited signifi-</p><p>cant antioxidant potential [61].</p><p>In an effort to identify and characterize the bioactive</p><p>compounds of fungal extracts, we took advantage of</p><p>GC–MS analysis, which indicated the presence of esters,</p><p>alcohols, alkanes, amines and their derivatives in the fun-</p><p>gal extracts. These findings are in accordance with the</p><p>previous reports where extra-cellular production of vola-</p><p>tile hydrocarbons by F. solani isolated from Taxus baccata</p><p>has been detected [62]. Similarly, volatile hydrocarbons</p><p>metabolites have also been reported in some endophytic</p><p>fungi with antimicrobial activity against human and</p><p>plant pathogenic bacteria and fungi [63]. Very interest-</p><p>ingly, 2-vinyl-9-[beta-D-ribofuranosyl] hypoxanthine was</p><p>detected in ethyl acetate extract of Rhizopus oryzae. It</p><p>has previously been reported from the methanolic fruit</p><p>extract of Citrus aurantifolia [39]. Production of bioactive</p><p>compounds in endophytic organisms can be linked with</p><p>the medicinal properties of plants, and plants may have</p><p>a role in initiating production of these metabolites due</p><p>to specific environment offered to fungal strains [64, 65].</p><p>Our results are in accordance with the previous findings</p><p>that endophytic fungi can produce volatile organic com-</p><p>pounds extractable with ethyl acetate and can be analyzed</p><p>by GC–MS [66]. Findings of study are also in agreement</p><p>with previous reports that majority of volatile organic</p><p>compounds produced by the endophytic fungi comprises</p><p>a mixture of volatile components that generally has a syn-</p><p>ergistic or an additive effect thus enhances their bioac-</p><p>tivity against pathogenic microbes [67]. The antifungal</p><p>activity exhibited by endophytic fungi can be linked to the</p><p>presence of extracellularly secreted antifungal compounds</p><p>that may contribute to the protection systems of the plant</p><p>against pathogens as organic compounds from fungal</p><p>endophytes are being used as biological control agents for</p><p>preventing deterioration of crops, fruits and vegetables. In</p><p>future, there is scope to use these organic compounds in</p><p>Table 7 Major constituents of fungal extract DL2H, as indicated by GC–MS analysis</p><p>S. no. Retention time Compound name Mol. weight Formula Biological activity</p><p>1 3.5 4-Octadecene-1,3-diol, 2-amino 299 C18H37O2N Antibacterial activity of its derivative is</p><p>reported [43]</p><p>2 3.5 Benserazide 257 C10H15O5N3 Antibacterial and antifungal activity of its</p><p>derivative is reported [44]</p><p>3 3.68 Tetra acetyl-D-xylonic nitrile 343 C14H17O9N Antibacterial and antifungal activity is</p><p>reported [33, 34]</p><p>4 3.68 2-Methyl-9-beta-D-ribofuranosyl hypoxan-</p><p>thine</p><p>282 C11H14O5N4 Antibacterial and antifungal activity is</p><p>reported [40]</p><p>5 3.68 3,7-Diacetamido-7H-S-triazolo[5,1-C]-S-</p><p>triazole</p><p>223 C7H9O2N7 Antibacterial and antifungal activity is</p><p>reported [45]</p><p>Table 8 Major constituents of fungal extract DL3.2, as indicated by GC–MS analysis</p><p>S. no. Retention time Compound name Mol. weight Formula Biological activity</p><p>1 8.69 Methoxy acetic acid, pentadecyl ester 300 C18H36O3 Antimicrobial activity is reported [46]</p><p>2 10.28 12-Methyl-E,E-2, 13-octadecadien-1-OL 280 C19H36O Antimicrobial activity is reported [47]</p><p>3 11.57 Phytol, acetate 338 C22H42O2 Antimicrobial and antioxidant activities is</p><p>reported [48]</p><p>4 12.19 2-Dodecen-1-YL(-) succinic anhydride 266 C16H26O3 Antioxidant and antimicrobial activity was</p><p>reported [49]</p><p>5 12.90 Octadecanal 268 C18H36O Anti-inflammatory activity of its derivatives is</p><p>reported [50]</p><p>6 13.89 1-Hexyl-2-nitrocyclohexane 213 C12H23O2N Antioxidant, antimicrobial and anti-inflammatory</p><p>activity is reported [51]</p><p>Arabian Journal for Science and Engineering</p><p>1 3</p><p>development of sprays that can be used for sanitization of</p><p>public places and to treat fungal infections [68].</p><p>5 Conclusions</p><p>Our findings indicate that endophytic fungi from D. sali-</p><p>cifolia have the potential to produce phytochemicals with</p><p>biological activity and are pharmaceutically important. The</p><p>medicinal properties of D. salicifolia may be a consequence</p><p>of its endophytic microorganisms to produce biologically</p><p>active compounds. Furthermore, it is important to separate</p><p>and characterize active compounds from endophytic fungi</p><p>in order to discover new drugs with antibacterial activity</p><p>and to determine their mechanism of action. In future, our</p><p>focus will be to isolate bioactive compounds from endo-</p><p>phytic fungi extract using column chromatography and to</p><p>characterize them using NMR.</p><p>Acknowledgements The authors are grateful to Higher Education</p><p>Commission Pakistan for financial support (NRPU#4695) during this</p><p>research work under National Research Program for Universities.</p><p>References</p><p>1. 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Fungi</p><p>(2018). https ://doi.org/10.3390/jof40 30077</p><p>https://doi.org/10.3390/jof4030077</p><p>Identification and Bioactivities of Two Endophytic Fungi Fusarium fujikuroi and Aspergillus tubingensis from Foliar Parts of Debregeasia salicifolia</p><p>Abstract</p><p>1 Introduction</p><p>2 Materials and methods</p><p>2.1 Collection and Surface Sterilization of Plant Samples</p><p>2.2 Development of Pure Culture</p><p>2.3 Morphological Identification of Endophytic Fungi</p><p>2.4 Molecular Identification of Endophytic Fungi</p><p>2.5 Phylogenetic Analysis of Isolated Fungal Endophytes</p><p>2.6 Production of Secondary Metabolites</p><p>2.7 Antibacterial Activity</p><p>2.8 Antifungal Activity</p><p>2.9 Antioxidant Activity by Using Free Radical Scavenging Assay</p><p>2.10 Gas Chromatography–Mass Spectrometry Analysis of Fungal Extracts</p><p>2.11 Statistical Analysis</p><p>3 Results</p><p>3.1 Isolation and Identification of Fungal Endophytes from D. Salicifolia</p><p>3.2 Antibacterial Activity of Fungal Extracts</p><p>3.3 Antioxidant Activity of Fungal Extracts</p><p>3.4 Antifungal Activity of Fungal Extracts</p><p>3.5 GC–MS Analysis of Fungal Extracts</p><p>4 Discussion</p><p>5 Conclusions</p><p>Acknowledgements</p><p>References</p>