Arabian Journal for Science and Engineering

https://doi.org/10.1007/s13369-020-04454-1

RESEARCH ARTICLE-BIOLOGICAL SCIENCES

Identification and Bioactivities of Two Endophytic Fungi Fusarium

fujikuroi and Aspergillus tubingensis from Foliar Parts of Debregeasia
salicifolia
Sobia Nisa1   · Nimra Khan1 · Waqas Shah2 · Maimoona Sabir1 · Wajiha Khan2 · Yamin Bibi3 · Muhammad Jahangir4 ·
Irshad Ul Haq1 · Sadia Alam1 · Abdul Qayyum5

Received: 20 November 2019 / Accepted: 28 February 2020

© King Fahd University of Petroleum & Minerals 2020

Abstract
Endophytic fungi isolated from medicinal plants are important for production of antibiotics. They can produce secondary
metabolites with diverse structures and activities. Debregeasia salicifolia is a plant of medicinal importance, and no report
exists regarding isolation of endophytic fungi from it. This study was focused to isolate and identify culturable endophytic
fungi from foliar parts of D. salicifolia and to determine their bioactivities. Molecular analysis resulted in identification of
Fusarium fujikuroi, Aspergillus tubingensis and Rhizopus oryzae based on specific internal transcribed spacer primer (ITS1/
ITS4). Our analysis revealed that all fungal endophytes possess antibacterial activity against Gram-negative and Gram-
positive bacteria. Remarkably, Rhizopus oryzae at a concentration of 5 mg/mL efficiently restricted the growth of ATCC
strain of E. coli in comparison with positive control ciprofloxacin. Rhizopus oryzae and F. fujikuroi at a concentration of
1000 µg/ml exhibited maximum antioxidant activity of 45% and 44%, respectively. They also showed antifungal activity
ranging from 60 to 75% against Aspergillus flavus and Aspergillus niger. Our analysis of the fungal extracts through GC–MS
indicated the presence of 21 compounds of diverse nature and structure. In conclusion, our study highlighted the potential
of D. salicifolia to host a plethora of fungal endophytes that secrete potentially therapeutic bioactive metabolites

Keywords  Debregeasia salicifolia · Endophytic fungi · ITS region · Bioactivities · GC–MS

1 Introduction poor hygienic conditions, increased number of immuno-

Development of resistance to available antibiotics by patho-
genic bacteria and fungi is one of most important problem of
global concern [1, 2]. Many factors are responsible for anti-
biotic resistance including inappropriate use of antibiotics,
* Sobia Nisa
sobia@uoh.edu.pk
1
Department of Microbiology, The University of Haripur,
Haripur, KPK, Pakistan
2
Department of Biotechnology, COMSATS University
Islamabad, Abbotabad Campus, Abbottabad, Pakistan
3
Department of Botany, PMAS Arid Agriculture University,
Rawalpindi, Pakistan
4
Department of Food Science and Technology, The University
of Haripur, Haripur, KPK, Pakistan
5
Department of Agronomy, The University of Haripur,
Haripur, KPK, Pakistan
compromised patients, and late diagnosis of infections [3].
So, the need to search new and more effective agents for
treating human diseases has been increased [4]. A number of
compounds have been isolated from medicinally important
plants and used for the treatment of human ailments [5].
Natural compounds are preferably used as pharmaceutical
agents due to their biological friendly nature [6]. Natural
compounds derived from plants, bacteria, and fungi have
been recently used to treat multidrug-resistant infectious
agents alone or in combination with antibiotics. The use
of antimicrobial agents of natural origin is advantageous
as they interact by making protein–protein interactions and
hence microbes rarely develop resistance against them.
Endophytes like bacteria and fungi colonize higher plant
tissues without damaging. They are considered as natural
reservoirs of diverse bioactive products [7, 8]. Bioactive
secondary metabolites like alkaloids, terpenoids, isocou-
marins, steroids, quinones, lignans, phenols, and lactones
have been isolated from endophytic bacteria and fungi [9,

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10]. Endophytic fungi from medicinal plants have been
reported for antibacterial and antifungal activities [11–14].
Plants also serve as a reservoir of secondary metabolites.
Endophytic fungi could also produce metabolites similar to
or with more pronounced activity than that of their respec-
tive hosts [15].
Endophytic fungi are thought to have mutualistic asso-
ciations with plants. In this symbiotic relationship, plants
provide shelter and nutrients to the endophytes [16, 17]
while the host plants are protected against pathogens and
herbivores [18–20]. Moreover, endophytes are also reported
to support plant growth by producing phytohormones [21]
and increase their resistance to multiple abiotic stresses like
salinity and heavy metal toxicity [22, 23]. Studies on iso-
lation of bioactive metabolites from endophytic fungi can
help in identification of some novel compounds and that
can further be developed as antimicrobial agents to cope
antibiotic resistance.
Debregeasia salicifolia is an important plant with ethno-
botanical importance and is widespread in different parts of
West Asia and Western Himalayan. Its bark paste is useful
externally on forehead to relieve pain [24]. Traditionally, it
is used to treat several diseases like urinary tract infections,
bone fractures, boils, bloody diarrhea, carbuncles, pimples,
dermatitis, skin rash, eczema, and tumors [25]. Our group
reported the antibacterial and anticancer activity of D. sali-
cifolia previously [26, 27]. This plant has not been evaluated
for isolation of culturable endophytic fungi and their activi-
ties. In the present investigation, we evaluated the diversity
and bioactive potential of endophytic fungi colonizing D.
salicifolia.
2 Materials and methods
2.1 Collection and Surface Sterilization of Plant
Samples
Healthy leaves and stems of D. salicifolia were collected
from Abbotabad, Pakistan, and identification of the plant
material was done by using standard criteria. Samples were
washed under running tap water prior to surface sterilization.
Surface sterilization was done using 0.1% ­HgCl2 solution for
1–3 min, 70% (v/v) ethanol for 1 min, followed by 1% (v/v)
sodium hypochlorite for 5 min and immersion in 70% (v/v)
for 30 s. The sterilized segments were washed thrice with
autoclaved distilled water and dried on blotting paper. Stem
and leaf segments were cut using sterilized surgical blades
and shifted to potato dextrose agar (Oxoid) supplemented
with 100 mg/mL tetracycline to stop growth of bacteria.
Aliquots from final washed water were also plated on the
PDA plates to check the effectiveness of surface sterilization.
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Arabian Journal for Science and Engineering
The Petri plates were sealed with parafilm and incubated for
5–7 days at ­28◦C for initiation of fungal growth.
2.2 Development of Pure Culture
After 5–7  days of incubation, mycelial growth started
emerging from the explants, and they were subcultured to
get pure culture. Pure colonies were transferred on potato
dextrose agar slants and preserved at 4–5 °C for future use.
The preserved fungi were subcultured from time to time.
2.3 Morphological Identification of Endophytic
Fungi
The fungi were identified on morphological basis as previ-
ously described [28, 29]. Identification was performed by
noticing morphological parameters like colony diameter,
texture, front and reverse color of colony and microscopic
appearance of hyphae and reproductive structures.
2.4 Molecular Identification of Endophytic Fungi
DNA extraction was performed using phenol chloroform
method with little modification. For PCR amplification, 1
μL of diluted genomic DNA, primers (ITS-1 and ITS-4) and
Ampliqon (Taq polymerase) (Thermo Fisher Scientific) were
used to make the final reaction mixture. Preliminary denatur-
ation was done at 95 °C for 7 min followed by 35 cycles of
amplification, denaturation (95 °C, 30 s), annealing (58 °C,
45 s), and elongation (72 °C, 60 s) and the final elongation
was performed at 72 °C for 6 min. After the reaction, PCR
products purification and sequencing were performed com-
mercially through alpha genomics.
2.5 Phylogenetic Analysis of Isolated Fungal
Endophytes
For phylogeny of the isolated fungal endophytes, we
retrieved sequences of the close hits from NCBI and per-
formed multiple sequence alignment using Clustal W. The
aligned files were then exported in mega format. Neigh-
bor-joining tree was constructed in MEGAX using default
parameters and bootstrap values of 1000.
2.6 Production of Secondary Metabolites
For the production of secondary metabolites, 150 mL of
potato dextrose broth was prepared in 250-mL flasks and
autoclaved. Medium was inoculated with fungal culture and
incubated at 28 ± 1 °C in shaking incubator at 170 rpm for
4 weeks. Secondary metabolites were excreted by myce-
lium in their surrounding media. Mycelium was separated
from the culture media using muslin cloth. The crude
Arabian Journal for Science and Engineering
culture broth was filtered through Whatman filter paper and
extracted with ethyl acetate using separating funnel. The
ethyl acetate extract was dried by using rotary evaporator.
The extract was then stored at 4–5◦C for further use.
2.7 Antibacterial Activity
Crude fungal extracts were dissolved in dimethyl sulfox-
ide (DMSO) (5  mg/mL), and antibacterial activity was
determined against seven American Type Culture Collec-
tion (ATCC) bacterial strains, i.e., Staphylococcus aureus
(292013), Bacillus spizizenii (66330), Listeria monocy-
togenes (35152), Escherichia coli (25922), Klebsiella
pneumoniae (13883), Salmonella typhimrium (14028) and
Acinetobacter baumannii (19606) using agar well-diffusion
method. Bacterial strains were cultured overnight in nutri-
ent broth to get fresh culture for antibacterial activity. Wells
were aseptically made in the seeded media with sterile borer.
Ciprofloxacin solution was prepared by dissolving it in
DMSO to get 1 mg/mL concentration and used as a positive
control. Pure DMSO was used as a negative control. Fifty
microliters of the crude extract was poured in the wells and
incubated at 37 °C for 24 h. Finally, plates were observed
for zones of inhibition and their respective diameters were
measured.
For the determination of minimum inhibition concentra-
tion (MIC), bacterial strains were inoculated and incubated
overnight in nutrient broth. Then, the cultures were diluted
in physiological saline by ratio of 1:100 and the concentra-
tion of inoculum was finally adjusted as 1 × 106cells cfu/
ml. Two folds of CAMHB (cation-adjusted Mueller–Hinton
broth) medium was poured in each well of 96-well micro-
titer plate followed by the addition of adjusted inoculum.
Microtiter plate was incubated for 24 h at 37 °C. Triplicates
of each extract were carried out to measure the effective
concentration. After incubation, the optical density was
measured using microtiter plate reader at the wavelength of
600 nm. Minimum concentration which inhibits growth of
pathogenic bacteria was taken as the MIC.
2.8 Antifungal Activity
Two pathogenic fungal strains Aspergillus niger and
Aspergillus flavus were used to screen antifungal poten-
tial of the endophytic fungal extracts using agar tube dilu-
tion method [30]. Briefly, 5 mL of Sabouraud dextrose
agar media was poured in test tubes that were labeled and
autoclaved at 121 °C. Fungal extracts 200 µL (400 µg/
mL) were added to autoclaved media before solidifica-
tion. Sodium benzoate (2.4 µg/mL) was used as a positive
control, whereas pure DMSO was used as a negative con-
trol. Medium was mixed by shaking carefully and allowed
solidification in slanting position. Each tube was inocu-
lated with seven-day-old fungal strains. After inoculation,
these tubes were incubated for seven days at 27 °C. The
inhibition of fungal growth was determined by using the
following formula:
Inhibition of fungal growth = (lg c − lg t)∕ lg c × 100
where
lg t = linear growth of test sample
lg c = linear growth of control sample.
2.9 Antioxidant Activity by Using Free Radical
Scavenging Assay
2,2-Diphenyl-1-picrylhydrazyl hydrate (DPPH) free radi-
cal scavenging assay was used to determine the antioxidant
activity of fungal extracts. In antioxidant assay different
concentrations (1000, 500, 250, 125, 62.5 µg/mL) of each
sample were mixed with 2.85 mL of DPPH reagent (1 mg
of DPPH reagent/25 mL of dimethyl sulfoxide) in 15-mL
falcon tube. The reaction mixture was incubated in dark
for half an hour, and then absorbance was recorded at
517 nm by using spectrophotometer. Different concentra-
tions (1000, 500, 250, 125, 62.5 µg/mL) of ascorbic acid
were used as a positive control, and dimethyl sulfoxide was
used as a negative control [31].
The following formula was used to determine the DPPH
free radical scavenging assay.
(DPPH radical-scavenging activity(%) inhibition
[
= (control OD − sample OD)∕control OD)] × 100
where
control OD = absorbance without samples
sample OD = absorbance in the presence of samples.
2.10 Gas Chromatography–Mass Spectrometry
Analysis of Fungal Extracts
The crude extracts of samples were used to evaluate their
chemical composition using GC–MS. A 20-min run was
conducted from the initial temperature of 40 °C to the final
temperature of 250 °C. The spectrum was noted in the
range of 40–600 m/z. Peaks of various compounds eluted
from the column of GC were recorded along with their
retention time. Data were correlated with mass spectra of
the compounds, and database was searched for the similar
compounds with same retention time and molecular mass.
Bioactivities of already reported natural compounds were
also studied, and a comparison was made to correlate the
activities of fungal extracts with their constituents.
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 Arabian Journal for Science and Engineering

2.11 Statistical Analysis

Data were obtained as mean of four replicates and analyzed

by two-factor factorial in completely randomized design
(CRD). Mean comparison (p < 0.05) was carried out using
Duncan multiple-range (DMR) test by M-Stat-C Statistical
software [32].

3 Results

3.1 Isolation and Identification of Fungal

Endophytes from D. Salicifolia

Five strains of endophytic fungi were isolated and identified

on the basis of colony morphology and microscopic exami-
nation (Table 1, Figs. 1, 2). Identification of three strains
(DS 1, DL2H, and DL3.2) was confirmed by molecular
analysis. These fungi were identified as Rhizopus oryzae
(DL2H), F. fujikuroi (DL3.2), and Aspergillus tubingensis
(DS 1). The results of DNA sequencing were submitted to
GenBank with the accession number of MN947248 for Rhiz-
opus oryzae (DL2H), MN947247 for F. fujikuroi (DL3.2),
and MN990210 for A. tubingensis (DS 1) (Table 2, Fig. 3).
3.2 Antibacterial Activity of Fungal Extracts
Extracts of isolated endophytic fungal species were sub-
jected to antibacterial activity against clinical isolates of
both Gram-positive and Gram-negative bacteria. Extracts
exhibited activity against both Gram-negative and Gram-
positive bacterial species with few exceptions. Extracts
of all fungal strains were active against tested bacteria.
Best activity was exhibited by DL2H (Rhizopus oryzae)
against E. coli with a zone of inhibition having a diameter
of 28.33 mm, followed by S. aureus and B. subtilis (25 and
23 mm), respectively (Fig. 4). DL3.2 (F. fujikuroi) inhib-
ited the growth of Listeria monocytogenes ATCC (35152),
Staphylococcus aureus ATCC (292013), Salmonella typh-
imurium ATCC (14028) and Bacillus spizizenii ATCC
(6633) with a zone of inhibition having diameter of 25, 24,
Fig. 1  Initiation of growth of fungal endophyte from leaf segment of
Debregeasia salicifolia 
24 and 23 mm, respectively. All the strains exhibited best
activity against E. coli and S. aureus, while least activity
was observed against A. baumanii. Minimum inhibitory
concentration of DS1 against B. subtilis, S. aureus and
E. coli was 0.312, 1.25 and 0.625 mg/mL, respectively.
Ethyl acetate extract of endophytic fungal isolate DL3.2
exhibited MIC values of 1.25, 2.5 and 5 mg/mL against B.
subtilis, E. coli and S. aureus, respectively.
3.3 Antioxidant Activity of Fungal Extracts
DL2H (Rhizopus oryzae) exhibited the highest radical
scavenging activity but still much lower than ascorbic
acid. Similarly, F. fujikuroi and A. tubingensis also exhib-
ited moderate antioxidant activity (Table 3). At 1000 µg/
ml the best antioxidant activity was observed by DL2H
that is 45.17% as compared to 87% as exhibited by ascor-
bic acid. Similarly, 43 and 41% free radical scavenging
activity was shown by fungal extract of F. fujikuroi.

Table 1  Colony morphology and microscopic presentation of isolated endophytic fungi

Endophytic fungi Media Colony presentation Background of colony Microscopic presentation

DS1 PDA Blackish in color with wrinkled mar- Blackish brown reverse Aseptate, branched, sporangium and
gins, raised smooth velvety surface spore forming
DS2 PDA Velvety green wrinkled convex Yellowish back with brownish Septate, branched conidiophores and
surface margins spore forming
DL2H PDA Fluffy, flat, smooth in texture with Orange back with yellowish margins. Aseptate, branched and conidiophores
white color with conidia
DL3.2 PDA Raised colony, smooth velvety wrin- Dark brownish back with blackish Aseptate, branched with dispersed
kled hard dark brown color margins conidia

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Arabian Journal for Science and Engineering

Fig. 2  Fungal endophytes
isolated from Debregeasia sali-
cifolia, where A; DS1, B; DS2,
C; DL3.2, D; DL2H

Table 2  Identification of S. no. Source Isolates 18S r RNA ampli- % similarity NCBI Accession no.
endophytic fungi on the base of fied region length
sequencing results
1 Leaf DL2H 600 bp 84% with Rhizopus oryzae MN947248
2 Leaf DL3.2 528 bp 99% with Fusarium fujikuroi MN947247
3 Stem DS 1 568 bp 95% with Aspergillus tubingensis MN990210

3.4 Antifungal Activity of Fungal Extracts
Ethyl acetate extract of endophytic fungi was tested for anti-
fungal activity by using tube dilution method. Overall, the
fungal extract from DL2H (Rhizopus oryzae) was highly
active against Aspergillus flavus followed by Aspergillus
niger with 73.3% and 62.5% of activity. DL3.2 (F. fujikuroi)
also exhibited significant activity against Aspergillus flavus
with 60% of inhibition (Table 4).
3.5 GC–MS Analysis of Fungal Extracts
GC–MS analysis of ethyl acetate extract of endophytic
fungi revealed the presence of bioactive compounds. The
presence of several compounds with known bioactivity was
found in the extracts of each of the 4 fungi analyzed, and
references are provided in Tables 5, 6, 7 and 8 describing
where compounds observed in this study were previously
identified. The compounds showed resemblance with the
natural products of plant and fungal origin. The analysis of
GC–MS data revealed that most of them were derivatives
of volatile compounds like esters, ethers, alkaloids and phe-
nolic compounds.
4 Discussion
Medicinal plants are known to harbor fungal endophytes
of great medicinal potential that include, Torreya mairei,
Fusarium sp., Pestalotiopsis jesteri, Chloridium sp. and
Penicillium microspora [7]. In order to explore the poten-
tial of D. salicifolia as a host of important endophytic

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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)
MEGA X. Bootstrap values are shown at respective node

Fig. 4  Antibacterial activity of 35 DS1

endophytic fungi against ATCC
bacterial strains. Vertical bars 30 DS2
Zones of Inhibion (mm)

indicate mean (n = 4) ± standard

DH2H
deviation (SD) 25
DL3.2
20
Ciprofloxacin
15

10

Bacterial s
rains

fungi, we carried out an exploratory study to identify any fujikuroi and A. tubingensis) endophytic fungal strains in
endophytic fungi with potential to synthesize medici- D. salicifolia.
nally important natural products. Remarkably, our results The isolated fungi belonged to the phylum Ascomycota
revealed the presence of three (Rhizopus oryzae, F. and Zygomycota. Elsewhere, Ascomycetes, Deuteromycetes,

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Table 3  Antioxidant activity Fungal extract Concentrations

of endophytic fungi extracts at
different concentrations 1000 µg/ml 500 µg/ml 250 µg/ml 125 µg/ml 62.5 µg/ml

DS1 21.10% 19.93% 15.58% 13.89% 11.98%

DL2H 45.17% 34.99% 34.25% 23.54% 22.79%
DS2 44.74% 41.19% 39.77% 38.06% 1.42%
DL3.2 43.75% 41.47% 41.33% 39.77% 26.13%
Ascorbic acid control 87% 87% 85% 83% 51%

Table 4  Antifungal activity of endophytic fungi isolated from Debre- phytotoxic secondary metabolites with herbicidal activity
geasia salicifolia  [5]. Similarly, Fusarium proliferatum (MTCC 9690) and A.
Fungal extracts Antifungal activity of fungal extracts in  % tubingensis (strain AN103) have been isolated from the bark
(4 mg/ml) of Dysoxylum binectariferum and branches of apple plant
Aspergillus niger Aspergillus flavus
(Malus domestica), respectively [54, 55]. The occurrence
DS1 42.5% 6.6% of endophytic Rhizoctonia like fungi has also been reported
DS2 41.25% 43.3% that is primarily considered as plant pathogenic fungi [56].
DL2H 62.5% 73.3% Fungal endophytes are reported to harbor genes encoding
DL3.2 60% 26.6% antibacterial compounds and involved in antioxidant activi-
ties. Consistent with other reports, our data revealed that F.
proliferatum isolated from Dysoxylum binectariferum and
and Basidiomycetes have been shown to reside inside plants’ Syzygium cordatum possess cytotoxic, antibacterial and anti-
tissues and endophytes [52, 53]. The occurrence of fungi oxidant activities [54, 57]. Moreover, Fusarium sp. has been
inside plants is widespread, a trait that is present in many reported as one of the most dominant fungal genera isolated
fungal species. For instance, F. fujikuroi has been isolated from Dracaena cambodiana and Aquilaria sinensis, with
from the Brazilian Pampa biome that is able to produce significant antimicrobial activity. This genus is comprised

Table 5  Major constituents of fungal extract DS1, as indicated by GC–MS analysis

S. no. Retention time Compound name Mol. weight Formula Biological activity

1 4.87 3,7-Diacetamido-7H-S-triazolo[5,1-C]-S- 223 C7H9O2N7 Antibacterial and antifungal activity is

triazole reported [33, 34]
2 5.30 1,6-D deoxy-L-mannitol 150 C6H14O4 Antibacterial and antipyretic activities of its
derivative are reported [35]
3 6.02 2-methyl-9-beta-D-ribofuranosyl hypoxan- 282 C11H14O5N4 Antifungal and antibacterial activity is
thine reported [36]
4 29.66 CIS-9,10-epoxyoctadecan-1-ol 284 C18H36O2 Antioxidant and antibacterial activity is
reported [37]

Table 6  Major constituents of fungal extract DS2, as indicated by GC–MS analysis

S. no. Retention time Compound name Mol. weight Formula Biological activity

1 3.66 Propanoic acid, 2-hydroxy-, pentyl ester 160 C8H16O3 Cytotoxic activity of its derivative is reported
[38]
2 4.49 2-Methyl-9-beta-D-ribofuranosyl hypox- 282 C11H14O5N4 Antibacterial and antifungal activity is
anthine reported [39]
3 5.24 2-vinyl-9-[beta-D-ribofuranosyl] hypoxan- 294 C12H14O5N4 Antibacterial and antifungal activity is
thine reported [40]
4 6.8 Silane, trichlorodocosyl 442 C22H45CI3Si Antibacterial, anti-inflammatory, and antitu-
mor activity is reported [41]
5 6.8 2-Tridecanol 200 C13H28O Antibacterial activity of its derivative is
reported [42].
6 8.89 Methoxy acetic acid, 2-pentadecyl ester 300 C18H36O3 Antimicrobial activity is reported [41]

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Table 7  Major constituents of fungal extract DL2H, as indicated by GC–MS analysis

S. no. Retention time Compound name Mol. weight Formula Biological activity

1 3.5 4-Octadecene-1,3-diol, 2-amino 299 C18H37O2N Antibacterial activity of its derivative is

reported [43]
2 3.5 Benserazide 257 C10H15O5N3 Antibacterial and antifungal activity of its
derivative is reported [44]
3 3.68 Tetra acetyl-D-xylonic nitrile 343 C14H17O9N Antibacterial and antifungal activity is
reported [33, 34]
4 3.68 2-Methyl-9-beta-D-ribofuranosyl hypoxan- 282 C11H14O5N4 Antibacterial and antifungal activity is
thine reported [40]
5 3.68 3,7-Diacetamido-7H-S-triazolo[5,1-C]-S- 223 C7H9O2N7 Antibacterial and antifungal activity is
triazole reported [45]

Table 8  Major constituents of fungal extract DL3.2, as indicated by GC–MS analysis

S. no. Retention time Compound name Mol. weight Formula Biological activity

1 8.69 Methoxy acetic acid, pentadecyl ester 300 C18H36O3 Antimicrobial activity is reported [46]
2 10.28 12-Methyl-E,E-2, 13-octadecadien-1-OL 280 C19H36O Antimicrobial activity is reported [47]
3 11.57 Phytol, acetate 338 C22H42O2 Antimicrobial and antioxidant activities is
reported [48]
4 12.19 2-Dodecen-1-YL(-) succinic anhydride 266 C16H26O3 Antioxidant and antimicrobial activity was
reported [49]
5 12.90 Octadecanal 268 C18H36O Anti-inflammatory activity of its derivatives is
reported [50]
6 13.89 1-Hexyl-2-nitrocyclohexane 213 C12H23O2N Antioxidant, antimicrobial and anti-inflammatory
activity is reported [51]

of more than 70 endophytic species capable of producing a
wide array of active metabolites [58]. Similarly, extract of
Rhizopus oryzae isolated from Crocus sativus L. (Saffron)
inhibited all bacteria tested. However, contrary to our find-
ings different levels of antibacterial activity were recorded
with maximum activity against Luteibacter sp. followed by
Stenotrophomonas sp., Pantoea sp., Pseudomonas putida,
E. coli and Bacillus sp. [59].
Extracts isolated from fungal endophytes are reported
to possess antioxidant activity [60]. Our results supported
that concept as the extracts we obtained from four fungal
endophytes showed antioxidant activity to a variable extent.
Our findings are consistent with previously published data
regarding fungal endophyte P. citrium that exhibited signifi-
cant antioxidant potential [61].
In an effort to identify and characterize the bioactive
compounds of fungal extracts, we took advantage of
GC–MS analysis, which indicated the presence of esters,
alcohols, alkanes, amines and their derivatives in the fun-
gal extracts. These findings are in accordance with the
previous reports where extra-cellular production of vola-
tile hydrocarbons by F. solani isolated from Taxus baccata
has been detected [62]. Similarly, volatile hydrocarbons
metabolites have also been reported in some endophytic
fungi with antimicrobial activity against human and
plant pathogenic bacteria and fungi [63]. Very interest-
ingly, 2-vinyl-9-[beta-D-ribofuranosyl] hypoxanthine was
detected in ethyl acetate extract of Rhizopus oryzae. It
has previously been reported from the methanolic fruit
extract of Citrus aurantifolia [39]. Production of bioactive
compounds in endophytic organisms can be linked with
the medicinal properties of plants, and plants may have
a role in initiating production of these metabolites due
to specific environment offered to fungal strains [64, 65].
Our results are in accordance with the previous findings
that endophytic fungi can produce volatile organic com-
pounds extractable with ethyl acetate and can be analyzed
by GC–MS [66]. Findings of study are also in agreement
with previous reports that majority of volatile organic
compounds produced by the endophytic fungi comprises
a mixture of volatile components that generally has a syn-
ergistic or an additive effect thus enhances their bioac-
tivity against pathogenic microbes [67]. The antifungal
activity exhibited by endophytic fungi can be linked to the
presence of extracellularly secreted antifungal compounds
that may contribute to the protection systems of the plant
against pathogens as organic compounds from fungal
endophytes are being used as biological control agents for
preventing deterioration of crops, fruits and vegetables. In
future, there is scope to use these organic compounds in

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development of sprays that can be used for sanitization of
public places and to treat fungal infections [68].
5 Conclusions
Our findings indicate that endophytic fungi from D. sali-
cifolia have the potential to produce phytochemicals with
biological activity and are pharmaceutically important. The
medicinal properties of D. salicifolia may be a consequence
of its endophytic microorganisms to produce biologically
active compounds. Furthermore, it is important to separate
and characterize active compounds from endophytic fungi
in order to discover new drugs with antibacterial activity
and to determine their mechanism of action. In future, our
focus will be to isolate bioactive compounds from endo-
phytic fungi extract using column chromatography and to
characterize them using NMR.
Acknowledgements  The authors are grateful to Higher Education
Commission Pakistan for financial support (NRPU#4695) during this
research work under National Research Program for Universities.
References
1. Monowar, T.; Rahman, M.; Bhore, S.; Raju, G.; Sathasivam, K.:
Silver nanoparticles synthesized by using the endophytic bacte-
rium pantoeaananatis are promising antimicrobial agents against
multidrug resistant bacteria. Molecules 23(12), 3220 (2018)
2. Proia, L.; Anzil, A.; Subirats, J.; Borrego, C.; Farre, M.; Llorca,
M.; Balcázar, J.L.; Servais, P.: Antibiotic resistance along an
urban river impacted by treated wastewaters. Sci. Total Environ.
628, 453–466 (2018)
3. Subramani, R.; Narayanasamy, M.; Feussner, K.: Plant-derived
antimicrobials to fight against multi-drug-resistant human patho-
gens. 3 Biotech 7, 172 (2017)
4. Cioch, M.; Satora, P.; Skotniczny, M.; Semik-Szczurak, D.; Tarko,
T.: Characterisation of antimicrobial properties of extracts of
selected medicinal plants. Pol. J. Microbiol. 66, 463–472 (2017)
5. Daniel, J.J.; Zabot, G.L.; Tres, M.V.; Harakava, R.; Kuhn, R.C.;
Mazutti, M.A.: Fusariumfujikuroi: a novel source of metabolites
with herbicidal activity. Biocatal. Agric. Biotechnol. 14, 314–320
(2018)
6. Waheed, A.; Bibi, Y.; Nisa, S.; Chaudhary, F.M.; Sahreen, S.;
Zia, M.: Inhibition of human breast and colorectal cancer cells
by Viburnum foetens L. extracts in vitro. Asian Pac. J. Trop. Dis.
3(1), 32–36 (2013)
7. Gouda, S.; Das, G.; Sen, S.K.; Shin, H.S.; Patra, J.K.: Endophytes:
a treasure house of bioactive compounds of medicinal importance.
Front. Microbiol. 7, 1538 (2016)
8. Molina, G.; Pimentel, M.R.; Bertucci, T.C.; Pastore, G.M.: Appli-
cation of fungal endophytes in biotechnological processes. Chem.
Eng. Trans. 27, 289–294 (2012)
9. Radic, N.; Strukelj, B.: Endophytic fungi: the treasure chest of
antibacterial substances. Phytomedicine 19(14), 1270–1284
(2012)
10. Deshmukh, S.K.; Verekar, S.A.; Bhave, S.V.: Endophytic fungi:
a reservoir of antibacterials. Front. Microbiol. 8(5), 715 (2014)
11. Liang, H.; Xing, Y.; Chen, J.; Zhang, D.; Guo, S.; Wang, C.:
Antimicrobial activities of endophytic fungi isolated from Ophi-
opogon japonicus (Liliaceae). BMC Complement. Altern. Med.
12(238), 1–6 (2012)
12. Gherbawy, Y.; Gashgari, R.: Molecular characterization of
endophytic fungi from Calotropis procera plants in Taif region
(Saudi Arabia) and their antifungal activities. Plant. Biosyst.
148(6), 1085–1092 (2014)
13. Idris, A.; Ietidal, A.; Idris, M.: Antibacterial activity of endo-
phytic fungi extracts from the medicinal plant Kigeliaafricana.
Egypt. Acad. J. Biol. Sci. 5(1), 1–9 (2013)
14. Bhardwaj, A.; Sharma, D.; Jodan, N.; Agrawal, P.K.: Antimicro-
bial and phytochemical screening of endophytic fungi isolated
from spikes of Pinus roxburghii. Arch. Clin. Microbiol. 6(3),
1–9 (2015)
15. Venieraki, A.; Dimou, M.; Katinakis, P.: Endophytic fungi resid-
ing in medicinal plants have the ability to produce the same or
similar pharmacologically active secondary metabolites as their
hosts. Hell. Plant Protect. J. 10, 51–66 (2017)
16. Liarzi, O.; Bucki, P.; Miyara, S.B.; Ezra, D.: Bioactive volatiles
from an endophytic Daldinia cf. concentrica isolate affect the
viability of the plant parasitic nematode Meloidogyne javanica.
PLoS ONE 11(12), e0168437 (2016)
17. Debbab, A.; Aly, A.H.; Proksch, P.: Endophytes and associated
marine derived fungi ecological and chemical perspective. Fun-
gal Divers. 57(1), 45–83 (2012)
18. Clay, K.: Fungal endophytes of grasses: a defensive mutualism
between plants and fungi. Ecology 69(1), 10–16 (1988)
19. Qin, S.; Feng, W.W.; Wang, T.T.; Ding, P.; Xing, K.; Jiang,
J.H.: Plant growth-promoting effect and genomic analysis of
the beneficial endophyte Streptomyces sp. KLBMP 5084 iso-
lated from halophyte Limonium sinense. Plant Soil 416(1–2),
117–132 (2017)
20. Singh, L.P.; Gill, S.S.; Tetuje, N.: Unravelling the role of fungal
symbionts in plant abiotic stress tolerance. Plant. Signal. Behav.
6, 175–191 (2011)
21. Hoffman, M.T.; Gunatilaka, M.K.; Wijeratne, K.; Gunatilaka,
L.; Arnold, A.E.: Endohyphal bacterium enhances production of
indole-3-acetic acid by a foliar fungal endophyte. PLoS ONE 8(9),
e73132 (2013)
22. Deng, Z.; Cao, L.; Huang, H.; Jiang, X.; Wang, W.; Shi, Y.; Zhang,
R.: Characterization of Cd-and Pb-resistant fungal endophyte
Mucor sp. CBRF59 isolated from rapes (Brassica chinensis) in a
metal-contaminated soil. J. Hazard. Mater. 2(3), 717–724 (2011)
23. Khan, A.L.; Waqas, M.; Hussain, J.; Al-Harrasi, A.; Lee, I.J.:
Fungal endophyte Penicillium janthinellum LK5 can reduce cad-
mium toxicity in Solanum lycopersicum (Sitiens and Rhe). Biol.
Fert. Soils 50(1), 75–85 (2014)
24. Negi, C.S.; Nautiyal, S.; Dasila, L.; Rao, K.S.; Maikhuri, R.K.:
Ethnomedicinal plant uses in a small tribal community in a part
of central Himalaya, India. J. Hum. Ecol. 14(1), 23–31 (2003)
25. Almubayedh, H.; Ahmad, R.: Ethnopharmacological uses, phy-
tochemistry, biological activities of Debregeasia salicifolia: a
review. J. Ethnopharmacol. 231(1), 179–186 (2019)
26. Bibi, Y.; Nisa, S.; Chaudhary, F.M.; Zia, M.: Antibacterial activity
of some selected medicinal plants of Pakistan. BMC Complement.
Altern. Med. 11(1), 52–63 (2011)
27. Nisa, S.; Bibi, Y.; Waheed, A.; Zia, M.; Sarwar, S.; Ahmed, S.;
Chaudhary, M.F.: Evaluation of anticancer activity of Debregea-
sia salicifolia extract against estrogen receptor positive cell line.
Afr. J. Biotechnol. 10(6), 990–995 (2011)
28. Dugan, F.M., Dugan, F.M.: The identification of fungi: an illus-
trated introduction with keys, glossary, and guide to literature. The
American Phytopathological Society Publications, USA (2017).
https​://doi.org/10.1094/97808​90545​041
13

29. Nisa, H.; Azra, N.; Irshad, K.A.; Mohd, N.; Bhat, S.; Nazi, R.:
Isolation and identification of endophytic fungi from Artemisia
scoparia (Asteraceae). Int. J. Theor. Appl. Sci. 10(1), 83–88
(2018)
30. Bibi, Y.; Nisa, S.; Zia, M.; Waheed, A.; Ahmed, S.; Chaudhary,
M.F.: The study of anticancer and antifungal activities of Pista-
cia integerrima extract in vitro. Indian J. Pharm. Sci. 74(4),
375–379 (2012)
31. Naz, R.; Ayub, H.; Nawaz, S.; Islam, Z.U.; Yasmin, T.; Bano,
A.; Wakeel, A.; Zia, S.; Roberts, T.H.: Antimicrobial activity,
toxicity and anti-inflammatory potential of methanolic extracts
of four ethnomedicinal plant species from Punjab, Pakistan.
BMC Complement. Altern. Med. 17, 302 (2017)
32. Bibi, Y.; Naeem, J.; Zahara, K.; Arshad, M.; Qayyu, A.: In vitro
antimicrobial assessment of selected plant extracts from Paki-
stan. Iran. J. Sci. Technol. Trans. A Sci. 42(1), 267–272 (2018)
33. Hussein, H.M.; Hameed, I.H.; Ibraheem, O.A.: Antimicrobial
activity and spectral chemical analysis of methanolic leaves
extract of Adiantum capillus-veneris using GC-MS and FTIR
spectroscopy. Int. J. Pharm. Phytochem. Res. 8(3), 369–385
(2016)
34. Devakumar, J.; Keerthana, V.; Sudha, S.S.: Identification of
bioactive compounds by gas chromatography-mass spectrom-
etry analysis of Syzygium jambos (L.) collected from western
ghats region Coimbatore, Tamilnadu. Asian J. Pharm. Clin. Res.
10(1), 364–369 (2017)
35. Kadhim, M.J.; Mohammed, G.J.; Hussein, H.M.: Analysis of
bioactive metabolites from Candida albicans using (GC-MS)
and evaluation of antibacterial activity. Int. J. Pharma. Clin.
Res. 8(7), 655–670 (2016)
36. Subavathy, P.; Thilaga, R.D.: GC-MS analysis of bioactive com-
pounds from whole body tissue methanolic extract of Cypraea
arabica. World J. Pharm. Res. 5(6), 800–806 (2016)
37. Singh, D.; Rathod, V.; Singh, A.K.: Phytochemical analysis of
the crude extracts of endophytic fungus, Alternaria sp. from
the medicinal plant Euphorbia hirta (L.). Int. J. Green Chemist.
Bioprocess 5(2), 14–20 (2015)
38. Mohammed, G.J.; Hameed, I.H.; Kamal, S.A.: Analysis of
methanolic extract of Fusarium chlamydosporum using GC-MS
technique and evaluation of its antimicrobial activity. Indian J.
Public Health Res. Dev. (2018). https​://doi.org/10.5958/0976-
5506.2018.00214​.0
39. Hameed, R.H.; Shareefi, E.A.; Hameed, I.H.: Analysis of metha-
nolic fruit extract of Citrus aurantifolia using gas chromatog-
raphy-mass spectrum and FT-IR techniques and evaluation of
its anti-bacterial activity. Indian J. Public Health Dev. 9(5),
480–486 (2018)
40. Nicoletti, R.; Fiorentino, A.: Plant bioactive metabolites and
drugs produced by endophytic fungi of Spermatophyta. Agri-
culture 5(4), 918–970 (2015)
41. Mujeeb, F.; Bajpai, P.; Pathak, N.: Phytochemical evaluation,
antimicrobial activity, and determination of bioactive compo-
nents from leaves of Aegle marmelos. Bio. Med. Res. Int. 2014,
11 (2014)
42. Rouis-Soussi, L.S.; Ayeb-Zakhama, A.E.; Mahjoub, A.; Flamini,
G.; Jannet, H.B.; Harzallah-Skhiri, F.: Chemical composition
and antibacterial activity of essential oils from the Tunisian
Allium nigrum L. EXCLI J. 13, 526–535 (2014)
43. Kraub, J.; Bracher, F.: Pharmacokinetic enhancers (boosters)—
escort for drugs against degrading enzymes and beyond. Sci.
Pharm. 86, 43 (2018)
44. Rudramurthy, S.M.; Colley, T.; Abdolrasouli, A.; Ashman, J.;
Dhaliwal, M.; Kaur, H.; Armstrong-James, D.; Strong, P.; Rape-
port, G.; Schelenz, S.; Ito, K.: In vitro antifungal activity of a
novel topical triazole PC945 against emerging yeast Candida
auris. J. Antimicrob. Chemother. 74(10), 2943–2949 (2019)
13
Arabian Journal for Science and Engineering
45. Khatua, S.; Pandey, A.; Biswas, S.J.: Phytochemical evalua-
tion and antimicrobial properties of Trichosanthes dioica root
extract. J. Pharm. Phytochem. 5(5), 410–413 (2016)
46. Agboke, A.A.; Attama, A.A.: Bioactive components and anti-
bacterial activities of n-hexane extract of Moringa oleifera root
bark on clinical isolates of methicilin resistant Staphylococcus
aureus. Int. J. Curr. Res. Chem. Pharm. Sci. 3(3), 1–9 (2016)
47. Wei, L.S.; Wee, W.; Siong, J.Y.F.; Syamsumir, D.F.: Charac-
terization of anticancer, antimicrobial, antioxidant properties
and chemical compositions of Peperomia pellucida leaf extract.
Acta Med. Iran. 49(10), 670–674 (2011)
48. Segueni, N.; Zellagui, A.; Boulechfar, S.; Derouiche, K.;
Rhouati, S.: Essential oil of Hertia cheirifolia leaves: chemical
composition, antibacterial and antioxidant activities. J. Mater.
Environ. Sci. 8(2), 551–556 (2017)
49. Altameme, H.J.; Hameed, I.H.; Abu-Serag, N.A.: Analysis of
bioactive phytochemical compounds of two medicinal plants,
Equisetum arvense and Alchemila valgaris seeds using gas chro-
matography-mass spectrometry and fourier-transform infrared
spectroscopy. Malays. Appl. Biol. 44(4), 47–58 (2015)
50. Dinesh, M.G.; Subbarayan, R.; Rallapalli, S.; Kansrajh, C.;
Kalaivani, R.: Terminalia bellerica leaf extracts induce apop-
tosis in Hep G2 cells and regulates cell cycle progression by
inducing G2/M cell cycle arrest. Indian J. Res. Pharm. Biotech-
nol. 2(1), 1044 (2014)
51. Pawlwska, J.; Wilk, M.; Sliwinska-Wyrzychowska, A.; Mętrak,
M.; Wrzosek, M.: The diversity of endophytic fungi in the
above-ground tissue of two Lycopodium species in Poland.
Symbiosis 63(2), 87–97 (2014)
52. Tawfike, A.F.; Tate, R.; Abbott, G.; Young, L.; Viegelmann, C.;
Schumacher, M.; Diederich, M.; Edrada-Ebel, R.: Metabolomic
tools to assess the chemistry and bioactivity of endophytic
Aspergillus strain. Chem. Biodivers. 14(10), e1700040 (2017)
53. Kumara, P.M.; Zuehlke, S.; Priti, V.; Ramesha, B.T.; Shweta,
S.; Ravikanth, G.; Vasudeva, R.; Santhoshkumar, T.R.; Spitel-
ler, M.; Shaanker, R.U.: Fusarium proliferatum, an endophytic
fungus from Dysoxylum binectariferum Hook. f, produces
rohitukine, a chromane alkaloid possessing anti-cancer activ-
ity. Antonie Van Leeuwenhoek 101, 323–329 (2012)
54. Mohamed, H.A.; Ebrahim, W.; Peterson, A.M.; Ozkaya, F.C.;
Proksch, P.: Tensidols A and B from Aspergillus tubingensis
strain and their biological activity. Mycol. Phytopatol. 53(4),
223–228 (2019)
55. Zakaria, L.; Izham, M.; Jamil, M.; Anuar, I.S.M.: Molecular
characterization of endophytic fungi from roots of wild banana
(Musa acuminata). Trop. Life Sci. Res. 27(1), 153–162 (2016)
56. Dame, Z.T.; Silima, B.; Gryzenhout, M.; Van Ree, T.: Bioactive
compounds from the endophytic fungus Fusarium proliferatum.
Nat. Prod. Res. 30(11), 1301–1304 (2016)
57. Gong, L.J.; Guo, S.X.: Endophytic fungi from Dracaena cam-
bodiana and Aquilaria sinensis and their antimicrobial activity.
Afr. J. Biotechnol. 8(5), 731–736 (2009)
58. Summerell, B.A.; Leslie, J.F.: Fifty years of Fusarium: how
could nine species have ever been enough? Fungal Divers. 50,
135–144 (2011)
59. Chamkhi, I.; Sbabou, L.; Aurag, J.: Endophytic fungi isolated
from Crocus sativus L. (saffron) as a source of bioactive second-
ary metabolites. Pharmacogn. J. 10(6), 1143–1148 (2018)
60. Gunasekaran, S.; Sathiavelu, M.; Arunachalam, S.: In vitro anti-
oxidant and antibacterial activity of endophytic fungi isolated
from Mussaenda luteola. J. Appl. Pharm. Sci. 7(8), 234–238
(2017)
61. Hulikere, M.M.; Joshi, C.G.; Nivya, T.; Ananda, D.; Raju, N.G.:
Antiangiogenic and antioxidant activity of endophytic fungus iso-
lated from seaweed (Sargassum wightii). Asian J. Biochem. 11(4),
168–176 (2016)
Arabian Journal for Science and Engineering
62. Chakravarthi, B.V.S.K.; Das, P.; Surendranath, K.; Karande, A.A.;
Jayabaskaran, C.: Production of paclitaxel by Fusarium solani
isolated from Taxus celebica. Bio. sci. 33, 259–267 (2008)
63. Tayung, K.; Barik, B.P.; Jha, D.K.; Deka, D.C.: Identification and
characterization of antimicrobial metabolite from an endophytic
fungus, Fusarium solani isolated from bark of Himalayan yew.
Mycosphere 2(3), 203–213 (2011)
64. Kaul, S.; Gupta, S.; Ahmed, M.; Dhar, M.K.: Endophytic fungi
from medicinal plants: a treasure hunt for bioactive metabolites.
Phytochem. Rev. 11, 487–505 (2012)
65. Kusari, P.; Kusari, S.; Spiteller, M.; Kayser, O.: Endophytic fungi
harbored in Cannabis sativa L.: diversity and potential as bio-
control agents against host plant-specific phytopathogens. Fungal
Divers. 60, 137–151 (2013)
66. Abdel-Motaal, F.F.; Nassar, M.S.; El-Zayat, S.A.; El-Sayed, M.A.;
Ito, S.I.: Antifungal activity of endophytic fungi isolated from
Egyptian henbane (Hyoscyamus muticus L.). Pak. J. Bot. 42(4),
2883–2894 (2010)
67. DiFranscesco, A.; Ugolini, L.; Lazzeri, L.; Mari, M.: Production
of volatile organic compounds by Aureobasidium pullulans as a
potential mechanism against post-harvest fruit pathogens. Biol.
Control 81, 8–14 (2015)
68. Deshmukh, S.K.; Gupta, M.K.; Prakash, V.; Saxena, S.: Endo-
phytic fungi: a source of potential antifungal compounds. J. Fungi
(2018). https​://doi.org/10.3390/jof40​30077​
13