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Abdulhadi et al (2020): Detection and activity of Endophytic fungi September 2020 Vol. 23 Issue 13B
Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.231384
Molecular detection and antimicrobial activity of Endophytic fungi
isolated from a medical plant Rosmarinus officinalis
Shimal Yonuis Abdulhadi, Ghazwan Qasim Hasan* and Raghad NawafGergees
Department of Biology, College of Education for pure science, Mosul University, Mosul, Iraq.
*For correspondence. E-mail: ghazwan.qasim@yahoo.com.
ABSTRACT
Endophytes are tiny organisms present in living tissues of distinct plants and have been extensively studied for their
endophytic microbial complement. Roots of Rosmarinus officinalis were subjected to the isolation of endophytic
fungi and screened for antimicrobial activity against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and
Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria. Genomic DNA from
active fungal strain of Trichoderma harzianum was isolated, and the internal transcribed spacer (ITS) region was
ampliﬁed using ITS4 and ITS5 primers and sequenced for genetic inference in fungus. The crude extract of T.
harzianum isolate with Ethyl acetate was showed significant antimicrobial activity against P. aeruginosa, S. aureus,
K. pneumonia, B. subtilis and E. coli. The antimicrobial activity was highest against P. aeruginosa at concentration of
40µg/ml, followed by S. aureus and K. pneumonia at the same concentration. The lowest antimicrobial activity was
against by S. aureus at concentration of 60µg/ml. The current study is confirmed that the antimicrobial activity is due
to bioactive compounds founded in endophytic fungi.
Key words: Rosmarinus officinalis, Endophytic fungi, antimicrobial
How to cite this article: Abdulhadi SY, Hasan GQ, Gergees RN (2020): Molecular detection and antimicrobial activity of
Endophytic fungi isolated from a medical plant Rosmarinus officinalis, Ann Trop Med & Public Health; 23(S13B): SP231384.
DOI: http://doi.org/10.36295/ASRO.2020.231384
INTRODUCTION
Rosmarinus officinalis, commonly known as Rosemary, is a member of mint family Lamiaceae. The name
"rosemary" derives from Latin ros marinus ("dew of the sea") (Room, 1986). Plant roots are not only colonized
by mycorrhizal bacteria and rhizobial, but also by endophytic fungi (Girlanda and Luppi, 2006). The endophytic
fungi, do not cause obvious disease, but colonize different plant tissues. For example, can improve host growth,
provide the host with some nutrients, convey stress tolerance, and many other benefits. In contrast to endophytic
growth in the ground plant organs, growing within the roots has frequently been found to be extensive (Stone et
al., 2000, Schulz and Boyle, 2005). The interaction between the plant and endophyte be based on the disposition
of host and fungus and the environmental conditions, this relationship could be neutral, mutualistic or
antagonistic. Some endophytic fungi may adopt mycorrhizal functions and gives the plants opportunity to
competitive other organisms such as insect pests, herbivores, or bacteria (Carroll, 1988; Hawksworth, 1991).
Other endophytes can be pathogenic to the host when conditions are unsuitable (Schulz et al. 1999). The
biodiversity of root endophytic communities differs depending on the type of vegetation , environmental factors
and the interactions between microorganisms. About 80% of endophytic fungi produce secondary metabolites
that active biologically in vitro and have fungicidal, antibacterial and herbicidal activities (Schulzetal.2002). For
instance, Fusarium spp. colonize both the roots and shoots in many hosts, and synthesis a number of toxins
including beauvericin, a cyclic hexadepsipeptide to protect infected plants against herbivory insects (Kuldau
and Yates 2000; Miller, 2001). Moreover, fungal root endophytes produce metabolites toxic to the nematode
Meloido gyneincognita. For instance, both phomalactone are produced by Verticillium chlamydosporium
(Khambay et al., 2000), and secondary compounds present in the culture ﬁltrate of F. oxysporum decreased the
mobility of the nematode within10 minutes after exposure (Hallmann and Sikora, 1996).
In addition, several root endophytic fungi synthesis antimicrobial metabolites (Schulz et al., 2002), e.g. the
antibacterial and antifungal metabolites synthesized in vitro by Cryptosporiopsis sp. from Larix decidua (Schulz
et al., 1995), which help to protect the host from other organisms. In addition, the endophytic Neotyphodium
spp. can be stimulated in shoots of the host after the damage, to increase production of mycotoxins as a kind of
adaptive (Bultman and Murphy, 2000); the same effect can be occurred in the injured roots which colonized by

endophytic fungi. Furthermore, the fungal roots can synthesis biologically active secondary compounds in both
in vitro and in vivo (in planta), which able to be antagonistic against other organisms such bacteria, and lead to
suppress the disease; it could also play a vital role in keeping a balance of antagonism between endophyte and
host.
In the other side, many endophytic fungi have shown antimicrobial activity against the human pathogenic
bacteria which the current paper about. For instance, the isolated endophytic fungi (Phomopsis sp.) from Vitex
negundo L leaves with ethyl acetate showed significant antimicrobial activity against, S. typhimurium, E. coli,
B. subtilis, K. pneumoniae, S. aureus, and B. cereus (Desale and Bodhankar, 2013). The ethyl acetate extract of
endophytic fungi Colletotrichum gloeosporioides showed potential inhibition against E. coli, S. aureus, S.
typhimurium, B. cereus and P. aeruginosa; again the ethyl acetate extract of Fusarium oxysporum has the same
effect against previous bacteria except P. aeruginosa (Ramesha and Srinivas, 2014). Endophyte fungi strains
isolated from the roots of Sonneratia griffithii Kurz with ethyl acetate and butanol have shown higher
antibacterial activity against S. aureus and E. coli comparing with other parts (Handayani et al., 2017).
Endophyte fungi isolated from leaves of Bush mango showed antibacterial activity against some pathogenic
bacteria such as S. aureus, B. subtilis, E. coli and P. aeruginosa (Nwakanma et al ., 2016). The ethyl acetate
extracts of mycelia and filtrates isolated from Vaccinium dunalianum var. Urophyllum revealed a significant
inhibit of the growth of pathogenic bacteria such as B. subtilis, S. aureus, P. vulgaris, L. monocytogenes and
Salmonella bacteria (Tong et al ., 2018).
MATERIALS AND METHODS
Plant samples collection:
Samples of R. officinalis plants were collected from Mosul city, north of Iraq during a week in September 2019. The
roots were excised from vegetable parts using a sterile knife and brought to the laboratory in sterile polythene bags.
Isolation of endophytic fungi:
The roots were washed with water several times to remove the soil and the impurities sticking to it. The roots were cut
into small pieces (5 mm × 2 mm) using sterile blade and washed with sterile distilled water thrice and allowed to
surface dry under aseptic conditions (Nithya and Muthumary, 2010). Then samples were dipping into 96% ethanol for
one minute and then in 2% sodium hypochlorite for three minutes and 70% ethanol for one minute, then washed with
sterile water twice and allowed to surface dry by placing them into sterile filter papers. The samples were placed on
Potato Dextrose Agar (PDA) plates with adding 50 mg/L tetracycline to inhibit the bacterial growth and incubated at
28ºC for 14 days. The hyphal tip of growing out from root tissue was conveyed to fresh PDA plates amended with 50
mg/L tetracycline. The surface sterilization process was confirmed for efficacy by taking 0.5 ml of washing water from
the last step and spreading it on the Petri dishes containing PDA medium, the plates were incubated in the same steps
above. The absence of any fungal colony is an indication of the efficiency of the surface-sterilization process.
Identification of endophytic fungi:
Morphological identification:
Several fungal isolates were appeared. The most common fungi selected was T. harzianum as shown in Figure1.
Figure1: Culture plate of T. harzianum which was marked by the green color.
DNA extraction, PCR ampliﬁcation and molecular detection:

The T. harzianum were grown in 100 ml Potato Dextrose Broth (PDB) for 10 days at 25º C. The Mycelia were
collected from the PDB and washed with distilled water and ground using sterile pestle and mortar with liquid nitrogen
until dry powder was obtained. 1.0 g DNA of fresh mycelium was extracted (Saghai Maroof et al., 1984). The nucleic
acid was extracted using the cetyl Trimethyl Ammonium Bromide (cTAB) method (O'Donnell et al., 1997). The
internal transcribed spacer (ITS) region was ampliﬁed by polymerase chain reaction using the universal primers ITS4
(5′- TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAG TAAAAGTCGTAACAAGG-3′) (Shweta et al., 2010).
The 590- bp DNA fragment PCR products was checked by electrophoresis on a 1 % agarose with ethidium bromide
and visualized using a UV transilluminator. The fragment was eluted and purified using and the Agarose Gel DNA
Extraction Kit (Takara, Japan). Sequencing of ITS product was sent to macrogen company/ South Korea
https://dna.macrogen.com/#. A basic local alignment search tool (BLAST) analysis was carried out in the NCBI
database, the results showed a 100% matches between the current sequences and deposited sequences in Genebank
(Accession: MH363810.1).
Fungal Isolates and Culturing Conditions:
The T. harzianum isolates were obtained from Rosmarinus officinalis roots growing in Mosul city north of Iraq.
For cultivation of fungi, five mycelia plugs taken from actively growing colony margin using cork borer No. 2
(5- mm diameter) were inoculated into a 500 ml conical flasks containing 100 ml PDB and incubated at 25°C
for 14 days.
Extraction of secondary metabolites:
After incubation the culture filtrate was separated from the fermentation medium using a rotary vacuum evaporator,
and filtered through a filter paper type Whaitman No.1, then the pH was adjusted to 3 by adding some drops of 0.1 N
HCL. In order to extract the bioactive compounds, the equal amount of ethyl acetate was added to the culture filtrate
and allowed to stand for one day using Separating funnel. The organic phase was collected in petri dishes and was
evaporated to dryness at room temperature. The dry extract was collected in sterile and tightly closed flasks and put in
the refrigerator until use.
Antimicrobial activity by agar well diffusion method
Endophytic fungi isolated from a medical plant of R. officinalis was subjected to screening for antimicrobial activity
against the human pathogenic bacteria Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia,
Bacillus subtilis, and Escherichia coli. The crude extract of T. harzianum was dissolved in Dimethyl sulfoxide
(DMSO) at different concentrations of 20 µg/ml, 40 µg/ml and 60 µg/ml ; poured into the 5-mm diameter well made
in Petri dishes containing 2% nutrient agar (Peptone-5 g, Beef Extract-3 g, NaCl-5 g, distilled water-1000 ml, Agar
powder-20 g, pH 7.0). The cultures were kept for 24 hours at 28 °C for the antimicrobial metabolite diffusion and
thereafter they were incubated at an appropriate temperature for the growth of test-microorganisms. The zone of
inhibition was measured in mm (Visalakchi and Muthumary, 2009). Furthermore, Gentamicin (40 µg/mL) and used as
positive control, while Distilled water was used as the negative control (Table 1).
Results
DNA extraction and ITS sequence analysis
The DNA was extracted using cTAB method, the ITS region was amplified by PCR using the universal primers ITS4
and ITS5. The product was examined by electrophoresis in 1% agarose gels (Figure 2). The puriﬁed fragment was sent
for sequencing. The sequence data of the ITS region was compared with deposited sequences in NCBI (Accession:
MH363810.1) using (BLAST) analysis. The studied ITS sequence result showed a homology of 100% with deposited
sequences (Figure 3).

1kb
500bp
100bp
1 2
Figure 2: Amplified ITS fragments of T. harzianum. Agarose gel image showing: Lane (1) 100 bp DNA ladder, lane
(2) ITS PCR product amplified with ITS4 forward and ITS5 reverse primers, Fragment size around 650 bp.
Figure 3: Multiple alignments of ITS sequences of T. harzianum with deposited sequences in Genebank
(Accession: MH363810.1
HPLC analysis
1 ml of culture filtrate of T. harzianum extracted by ethyl acetate was used for high-performance liquid
chromatography analysis according to (Mradu et al ., 2012), the process of diagnosing phenolic compounds was
carried out in the laboratories of the Ministry of Science and Technology / Environment and Water Department after
acid hydrolysis

a
process by HPLC model (SYKAM) Germany. Column is 18-ODS and dimensions 4.6 mm* 25 cm. The mobile phase
is (Methanol: D.W. : acetic acid) (85:13:2), samples were detected at a wavelength 360 nm at a flow rate of 1 ml per
minute. The HPLC analysis of the fungal extracts revealed the presence of many natural products which may be
responsible for the antimicrobial activities they elicit. Five compounds apiginine, catechine, gallic acid, keamferol and
qurcetine were identified as the major compounds (Figure 4).

b
c

e
d

g h
i j
f

k
Figure 4: The HPLC data and result tables of compounds detected in T. harzianum extracted by ethyl acetate: (a)
HPLC chromatogram showing the detection of sample plenolic; (b) apiginine; (c) catechine; (d) gallic acid; (e)
kaemferol; (f) quercetin (g) Chemical structure of apiginine (C15H10O5) https://en.wikipedia.org/wiki/Apigenin ; (h)
Chemical structure of catechine (C15H14O6) https://en.wikipedia.org/wiki/Catechin; (i) Chemical structure of gallic acid
(C7H6O5) https://en.wikipedia.org/wiki/Gallic_acid; (j) Chemical structure of kaemferol (C15H10O6)
https://en.wikipedia.org/wiki/Kaempferol; and (k) Chemical structure of quercetin (C15H10O7)
https://en.wikipedia.org/wiki/Quercetin .
Antimicrobial activity
Different fungal isolates were obtained from the roots of a medical plant R. officinalis, but the most frequency fungi
selected was T. harzianum. Identification of this endophyte was carry out based on morphology where culture plate of
was marked by the green color, also by molecular identification of the most common region which is ITS region using
universal primers ITS4 and ITS5 in a standard PCR reaction. The crude extracted from T. harzianum roots and isolate
with ethyl acetate was screened for its antimicrobial potential. This extract showed significant antimicrobial activity
against
P. aeruginosa, S. aureus, , K. pneumonia, B. subtilis, and E. coli. The antimicrobial activity was highest against P.
aeruginosa (35mm) at a concentration of 40µg/ml, followed by S. aureus (25mm) and K. pneumonia (25mm) at the
same concentration (Table 1). The second- highest antimicrobial activity was at a concentration of 20 µg/ml against P.
aeruginosa (25mm), followed by K. pneumonia (20mm) and B. subtilis (20mm). The lowest antimicrobial activity was
against E. coli at all concentrations used in this study (Table 1). These antimicrobial activities might be due to some
bioactive secondary metabolites founded in Entophytic fungi T. harzianum such as apiginine, catechine, gallic acid,
kaemferol and quercetin. These results are in agreement with (Schulz et al, 2002) that several root endophytic fungi
synthesis antimicrobial metabolites, also these findings are in corroborate with the previous work that the endophytes
have shown the presence of phenolic compounds and flavonoids (Ramesha and Srinivas, 2014) which are known to
have strong antimicrobial activities.

Table 1: Antibacterial activity of phenolic extract by ethyl acetate of Trichoderma harizanum. Zone of inhibition
(Diameter in mm).
DISCUSSION
There is no doubt that Endophytic fungi are important microorganisms that are widespread in different environments,
which is a source of secondary metabolites. It possesses effectiveness against many human pathogens (Desale and
Bodhankar., 2013, Ramesha and Srinivas, 2014., Handayani et al., 2017). Plant roots are colonized by endophytic
fungi (Girlanda and Luppi, 2006). These organisms do not harm the host, but have many benefits to the host such as
improving host growth, supply the host with nutrients, etc. (Stone et al., 2000, Schulz and Boyle, 2005). Some
endophytic fungi produce metabolites compounds that active biologically against fungi, bacteria, and herbicids (Schulz
et al., 2002). The secondary metabolites contain active chemical groups such as flavonoids, phenols, alkaloids,
terpenoids, steroids, quinines, etc (Tran et al., 2010, Aly et al., 2010, Muthu et al., 2011 ). In this study the T.
harzianum isolates were obtained from Rosmarinus officinalis roots to test its ability to produce secondary metabolites
that are effective against bacteria, and to determine the optimal concentrations of fungi activity and its production of
secondary metabolism using agar disc diffusion method, as it is a rapid test method and shows whether the fungus has
the ability to produce effective compounds outside the hypha that inhibits bacterial growth. The efficacy of fungal
discs in inhibiting bacterial growth may be attributed to the ability of the fungus to produce effective metabolites
compounds outside cells which inhibit the growth of a wide range of bacteria and other microorganisms (Kaul et al.,
2012, Jain and Pundir., 2011). The difference in the effectiveness of the fungal species may be due to the difference in
the secreted substances and their vital effect, or it may be due to the different components of the extract, such as mono
and twin turbines, as well as different phenolic compounds, because different compounds have different diffusion rates
which may be responsible for the differences in the inhibitory areas obtained during testing the effectiveness of the
materials against sensitive organisms (Irobi et al., 1996). Here, many human pathogenic bacteria were used such as P.
aeruginosa, S. aureus, Klebsiella pneumoniae, Bacillus subtilis and Escherichia coli which considered a dangerous
species, and as a result of the mutations occurrence continuously; they became resistant to antibiotics. Therefore, it is
necessary to constantly search for new types of antibiotics that are more effective against pathogenic bacteria,
Endophytic fungi are among the most important sources of antibiotics (Mekawey, 2010, Fischbach, 2009). In order to
obtain secondary metabolites in abundant quantities, the fungus was grown in a liquid food medium (PDB) at 25 ° C
and using a vibrator for a 10-day incubation period, which is the ideal conditions for fungi growth (Ritchie et al .,
2009, Bhattacharyya and Jha., 2011), as it is known that the process of producing secondary metabolites occurs during
the final stages of growth (Calvo et al., 2002), therefore the fungal isolates were extracted after 10 days. The current
results are also consistent with previous studies on other fungal groups (Rosa et al., 2003, Peláez et al., 1998). The
process of extracting secondary metabolites is an important stage in obtaining bioactive compounds, when extracting
the crude compounds from the filtrate of fungal isolates; the appropriate solvent must be chosen depending to the
nature of the extracted substance. Here, ethyl acetate was used in the extraction process, as it mixes with water in a low
percentage by 33%, in addition to its low boiling point about 77 °C, therefore the process of removing it by
evaporation is easy (El-Naggar., 2001). The extraction process depended on reducing the pH of the filtrate of fungal
S. No Microorganisms Distilled
water
Gentamycin 20ϻg/ml 40ϻg/ml 60ϻg/ml
40ϻg/ml
(-ve control)
1. Pseudomonas
aeuroginosa
-
( +ve
control)
23 25 35 12
2. Staphylococcus aureus - 15 19 25 10
3. Klebsiella pneumonia - 17 20 25 15
4. Bacillus subtilis - 22 20 22 16
5. Escherichia coli - 14 18 20 11

isolates to pH = 3, to convert the active substance (antagonist) extracted into ionic form, thus reducing its solubility in
the aqueous solution and facilitating its extraction by ethyl acetate (Boon., 1988, Endo et al., 1986). In the present
study, HPLC analysis of ethyl acetate extracts of T. harzianum extract reveald the presence of phenolic compounds
(Table 4). Our results are consistent with those described by Ramesha and Srinivas, (2014) that the endophytes have
phenolic compounds and flavonoids with displaying antimicrobial activities against pathogenic bacteria. This result is
also consistent with (Desale and Bodhankar, 2013, Nwakanma et al., 2016, Tong et al., 2018, Mekawey, 2010,
Fischbach, 2009 ) which confirmed that most fungal extracts have a inhibitory effect on bacteria and that fungi are an
important source of antibiotics. The difference in the effectiveness of extracted compounds against bacteria may be
due to several factors, including the differences in cellular structure of bacteria, as well as some of inhibitory
substances are working on a specific site which is the target site. For example, many bacterial inhibitors are inhibiting
the important steps of peptidoglycan production, which is a main component of the cell wall (Ghannoum and Rice.,
1999). However, E. coli bacteria showed resistance against the secondary metabolites at all concentrations used, this
may be due to the difference in structure of the cell wall between the Gram-negative and Gram-positive bacteria, as the
wall of negative bacteria is characterized by low permeability to metabolites due to the presence of the outer layer,
which prevents the access of the antibiotics to the target area (Yoshimura and Nikaido., 1982), also the Gram-negative
bacteria wall contains lipopolysaccharides, lipoprotein and phospholipids compounds which maybe making it less
permeable and resistant to antibiotics and other compounds (Wu., 2014).
CONCLUSION
The current study revealed that the endophytic fungi T. harzianum isolated from the roots of a medical plant R.
officinalis is effective alternative sources of antimicrobial drugs, with a diversified chemical composition and vital
effectiveness against other microorganisms.
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