Antimicrobial Activity of Secondary Metabolites of Fungi Isolated from Leaves of Bush Mango

Abstract

Antimicrobial activity of secondary metabolites of fungi isolated from leaves of Bush mango against some selected microorganisms (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Penicillium chrysogenum and Aspergillus fumigates) was carried out in this study. Agar well diffusion method was used to study the antimicrobial activity of the secondary metabolites and each of the secondary metabolites showed antagonist activity against the test organisms; two gram positive bacteria (Staphylococcus aureus and Bacillus subtilis), two gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two fungi (Penicillium chrysogenum and Aspergillus fumigatus). Findings from the research indicates that the studies could also pave a way for new therapeutic agents which can be used as potential drugs against the selected microorganisms or its infections.

Full text

Antimicrobial Activity of Secondary Metabolites of Fungi Isolated from
Leaves of Bush Mango
Chioma Nwakanma1*, Njoku EN2 and Pharamat T3
1Department of Environmental Management and Toxicology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria
2Department of Biological Sciences, Godfrey Okoye University, Enugu State, Nigeria
3Faculty of Science, Chulalongkorn University, Thailand
*Corresponding author: Chioma Nwakanma, Department of Environmental Management and Toxicology, Michael Okpara University of Agriculture, Umudike, Abia
State, Nigeria, Tel: +08032017146; E-mail: dr.nwakanmac@gmail.com
Rec date: Aug 23, 2016; Acc date: Oct 04, 2016; Pub date: Oct 06, 2016
Copyright: © 2016 Nwakanma C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Antimicrobial activity of secondary metabolites of fungi isolated from leaves of Bush mango against some
selected microorganisms (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis,
Penicillium chrysogenum and Aspergillus fumigates) was carried out in this study. Agar well diffusion method was
used to study the antimicrobial activity of the secondary metabolites and each of the secondary metabolites showed
antagonist activity against the test organisms; two gram positive bacteria (Staphylococcus aureus and Bacillus
subtilis), two gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two fungi (Penicillium
chrysogenum and Aspergillus fumigatus). Findings from the research indicates that the studies could also pave a
way for new therapeutic agents which can be used as potential drugs against the selected microorganisms or its
infections.
Keywords: Endophytes; Ecology; Antioxidants; Microorganisms;
Metabolites
Introduction
Cancers and various infectious diseases threaten worldwide human
health [1,2]. Endophytes are microorganisms that live in the
intercellular spaces of healthy host tissues without causing obvious
symptoms. Bioprospecting studies of endophytic microorganisms for
pharmaceutical and biotechnological purposes are fundamental for the
discovery of new substances for human therapeutics including
antibiotics, anti-malarials, and anticancer [3-5]. Microorganisms are
important sources of bioactive natural products with enormous
potential for the discovery of new molecules for drug discovery,
industrial use and agricultural applications [6-10]. In comparison to
other natural sources like plants, microorganisms are highly diverse
but narrowly explored. Studies based on estimation of microbial
populations have revealed that only about 1% of bacteria and 5% of
fungi have been characterized and the rest remain unexplored for their
contribution to the human welfare. In addition, more than 60% of the
anticancer and 70% of the antimicrobial drugs currently in clinical use
are natural products or natural product derivatives [11].
A study of the World Health Organization (WHO) revealed that
90% of the bacteria strains are resistant to drugs of rst choice.
Examples of the resistance problems on a global scale are the
methicillin resistant
Staphylococcus aureus
(MRSA), vancomycin
resistant
Enterococci
and
Enterobacteriaceae
producing beta-
lactamases. Drug resistance in bacteria has become a global concern
and the search for new antibacterial agents is urgent and ongoing [12].
e abuse and overuse of drugs especially antibiotics and
antimicrobials contributes to microbial drug resistance. Fox television
sent a 26-year-old assistant producer to four doctors’ oces in Denver,
Colorado, claiming that she did not feel well. Four out of four doctors
prescribed an antibiotic.
Endophytes, microorganism that reside in the tissue of living plants,
are relatively understudied and potential source of novel natural
products for exploitation in medicine, agriculture and industries [13].
e resistance of pathogenic microorganisms to drugs and antibiotics
has become a major challenge in the health sector leading to reduction
in drug eectiveness and economic wastage. is challenge has once
again stirred up a need in scientic research and the discovery of new
more eective antimicrobial metabolites becoming a major research
interest. In Recent years, the isolation of endophytic fungi and
screening of antimicrobial activity has gained more attention [3].
Endophytic fungal
isolates
Initial weights
of flasks (gram)
Final weight of
flasks (gram)
Weight of
secondary
metabolite (gram)
EDF1 157.26 158.673 1.413
EDF2 191.24 192.5 1.26
EDF3 166.101 167.331 1.23
EDF4 162.91 164.85 1.94
EDF5 161.162 162.682 1.52
Key: EDF= Endophytic fungi
Table 1: Weight of secondary metabolite extracted.
e increase in the resistance of drugs by infectious pathogens as
well as undesirable eects of certain antimicrobial agents indicates that
there is an urgent need for novel and eective bioactive compounds
with fresh modes of action which is what this study is aimed at
Journal of Next Generation
Sequencing & Applications
Nwakanma, et al., Next Generat Sequenc & Applic
2016, 3:3
DOI: 10.4172/2469-9853.1000135
Research Article OMICS International
Next Generat Sequenc & Applic, an open access journal
ISSN: 2469-9853
Volume 3 • Issue 3 • 1000135

achieving stated that during the last 20 years [9,10]. It has been
observed that much of the wealth of microbial biodiversity with novel
biochemistry and secondary metabolite production resides in plant
tissues.
is increased the interest in such microorganisms, termed as
endophytes, with the discovery of an endophytic fungus, from
Taxus
brevifolia
, producing the billion-dollar anti-cancer drug, taxol [14].
Endophytes are metabolically more active than their free counterparts
due to their specic functions in nature and activation of various
metabolic pathways to survive in the host tissues [5,9,15]. However, the
sequencing of the taxadiene synthase gene from the taxol producing
endophyte revealed that endophytes possess biosynthetic pathways
independent of the plant host [16]. is implies that microorganisms
have much more biosynthetic prociency than previously thought.
us, microorganisms may be screened for a wide range of biological
activities and explored for useful chemical entities consistently
produced by them. e discovery of novel anti-microbial metabolites
from endophyte is an important alternative to overcome the increasing
levels of drug resistance by plants and human pathogens [17]. e
microbial resources of green plants particularly the endophytic
populations remain completely unexplored. us, an investigation is
carried out to isolate and characterize the endophytic microorganisms
form bush mango leaves (
Irvingia gabonensis
) and explore their
bioactive potential and also isolate new leads which will assist in drug
discovery, industrial and agriculture improvement.
Irvingia gabonensis
is a species of African trees in the genus
Irvingia
. A research by showed that the possess medicinal properties
including antimicrobial eects against
Escherichia coli
and
Staphylococcus aureus
[18]. He also reported that the plant showed
antioxidant activity [19].
Leaf and root extracts have documented inhibitory activity against
several bacteria and fungi [20,21]. In Cameroon, preparation mainly
from the bark are used to treat hernia and yellow fever and as an
antidote for poisoning and kernels of
Irviginia gabonensis
are used to
treat diabetes [18]. According to preparations from the bark are
rubbed on to the body to relieve pains and are applied to sores and
wound and against toothache and they are also taken to treat diarrhea
[22]. In their research reported that the potential mechanism of action
includes membrane disruption by terpenoids and inactivation of
microbial adhesion, enzymes and cell envelope transport proteins by
ellagic acid-like compounds [21].
Materials and Methods
e plants were collected with tweezers, hand gloves, plastic bags
and containers from a eld outing and survey plan. e rational
selection of host plant is crucial to increase the chances of isolation of
novel microorganisms which may produce new bioactive compounds
[23]. erefore, it is necessary to understand the methods and
rationale used to provide the best opportunities to isolate novel
endophytic microorganisms as well as ones making novel bioactive
products.
e plant sample was identied and authenticated by a taxonomist
in the department of botany University of Nigeria Nsukka, Enugu
state, Nigeria. e study area was Amorji-Nike village in Enugu-East
local government area of Enugu State, Nigeria. e convenient
sampling technique used was simple random selection method. is
was used in order to give the dierent plant leaves an opportunity to
represent therapeutic values of the plant vegetation within the
geographical locations of the study area.
Endophytic fungal
isolates
Inhibition zone (mm) (mean ± SD)
EC SA BS PA AF PC
EDF1 9.50 ± 0.50 8.50 ± 1.50 13.50 ± 1.50 3.50 ± 0.50 10.50 ± 0.50 4.50 ± 0.50
EDF2 0.00 ± 0.00 3.00 ± 1.00 12.00 ± 2.00 9.00 ± 1.00 9.50 ± 0.5 11.50 ± 0.50
EDF3 2.00 ± 1.00 2.50 ± 0.50 0.00 ± 0.00 2.50 ± 0.50 9.50 ± 0.50 7.50 ± 1.50
EDF4 3.00 ± 1.00 3.50 ± 0.50 0.00 ± 0.00 1.50 ± 0.50 11.50 ± 0.50 10.50 ± 0.50
EDF5 1.50 ± 0.50 2.50 ± 0.50 4.00 ± 1.00 3.00 ± 1.00 13.00 ± 2.00 16.50 ± 2.50
Ciprofloxacin 7.00 ± 0.00 5.50 ± 0.50 15.00 ± 0.00 12.50 ± 0.50 - -
Fluconazole - - - - 17.50 ± 0.50 15.50 ± 0.50
Water - - - - - -
Keys: BS=B. subtilis, SA=S. aureus, EC=E. coli, PA=P. aeruginosa, PC=P. chrysogenum DF=Endophytic fungi and AF=A. fumigatus
Table 2: Inhibition diameters (mm) ± Standard error.
e population of this study identied about sixteen dierent
colonies of the endophytic fungi. To determine the sample size of the
study, ve distinct colonies based on morphological observation were
selected by the researcher to form a 31.25% ratio of the population of
the study. Sample collection were done through the help of a highly
renowned herbal products specialists and a plant specialist to acquire
information and also help in identifying the plants. For preparation of
the potato dextrose agar, the laboratory bench was cleaned with cotton
wool soaked in ethanol, this is done to avoid contamination and
enhance aseptic conditions. 3.9 g of potato dextrose agar powder was
weighed into a 250 ml conical ask and then dissolved with 150 ml of
distilled. e media was homogenized by agitating and then sterilized
by autoclaving at 121°C for 15 minutes, aer which it was aseptically
poured into sterile Petri dishes and allowed to gel. Also, preparation of
nutrient broth, sabour and dextrose agar and Mueller Hinton agar was
Citation: Nwakanma C, Njoku EN, Pharamat T (2016) Antimicrobial Activity of Secondary Metabolites of Fungi Isolated from Leaves of Bush
Mango. Next Generat Sequenc & Applic 3: 135. doi:10.4172/2469-9853.1000135
Page 2 of 6
Next Generat Sequenc & Applic, an open access journal
ISSN: 2469-9853
Volume 3 • Issue 3 • 1000135

also carried out in the laboratory. For isolation of endophytic fungi, the
leaves were washed under running tap water for 10 minutes.
e leaves were cut into small pieces and sterilize in series with
sterile distilled water for 1 minute, 70% ethanol for 1 minute, 1.0%
sodium hypochlorite (NaOCl) for 2 minutes and further cleaned by
passing through two sets of sterile distilled water for 5 minutes and
allowed to surface dry under sterile conditions. e sterile samples
were placed on the surface of potato dextrose agar (PDA) plates
supplemented with 1 g/ml of streptomycin to suppress bacterial growth
and contamination.
e paralm wrapped Petri dishes were incubated at 25°C ± 2°C till
the fungal mycelia starts growing from the samples. When the growth
started, a pure culture of a particular endophytic fungi was isolated
and inoculated on a fresh media. Solid state fermentation was carried
out using the following procedures:100 g of rice and 200 ml of water
were poured in 1000 ml conical ask and then autoclaved at 121°C at
15 psi for 1 hour and allowed to cool. 3 mm diameter agar blocks
containing the test fungi was inoculated in the conical ask containing
the already autoclaved rice and it was incubated at 25°C to 27°C for 21
days. Aer the 21 days of incubation, the secondary metabolite was
extracted using ethyl acetate. 500 ml of ethyl acetate was poured into
the conical ask containing the fermented rice and endophytic fungi. A
glass rod was used to break the clumps formed during the incubation
and it was le to stand for 24 hours. Aer 24 hours, it was ltered with
mushlin cloth into a beaker of known weight.
e ethyl acetate ltrate in the beaker was allowed to air dry. Aer
air drying, the crude extract residue was then dissolved in dimethyl
sulfoxide (DMSO) and stored at 4°C and used as stock solution for
antimicrobial assay and phytochemical analysis. e test organisms
used were obtained from the microbiology laboratory section and
mycology laboratory section of the University of Nigeria Teaching
Hospital Ituku-ozala, Enugu State, Nigeria. e test organism used
were six and they include two gram positive bacteria (
Staphylococcus
aureus
and
Bacillus subtilis
), two gram negative bacteria
(
Pseudomonas aeruginosa
and
Escherichia coli
) and two fungi
Aspergillus
and
Penicillium
. e agar well diusion method described
by with modication was used to evaluate the antimicrobial and
antifungal activity against the test microorganisms [24].
e test organisms were spread aseptically using a cotton swab on
the surface of the already prepared Mueller Hinton agar for the
bacteria and the fungi was aseptically inoculated on the already
prepared Sabouraud dextrose agar. All culture plates were allowed to
dry for about ve (5) minutes and four wells were made on the agar
using a 6 mm sterile cork borer. Two wells were lled with 200 µl of
100 mg/ml concentration of the extract, other well was lled with 200
µl of 50 mg/ml concentration of the positive control and the last well
was lled with 200 µl of the negative control. e plates were kept on
the work bench to allow the agents diuse into the agar and incubated
accordingly. Ciprooxacin and uconazole were used as the positive
controls for the antibacterial and antifungal evaluation respectively
while distilled water was used as the negative control. e Mueller
Hinton agar plates were incubated at 37°C for twenty-four (24) hours
and the Sabouraud dextrose agar plates were incubated at 27°C for two
(2) days. e antimicrobial activities were evaluated by measuring the
diameter of the inhibition zones in millimeters and the readings were
recorded. e experiment was replicated twice and an average of two
independent reading for each microorganism was used. e
antimicrobial activities were evaluated by measuring the diameter of
the inhibition zones in millimeters and the readings were recorded.
e experiment was replicated twice and an average of two
independent reading for each microorganism was used.
Results
A total of sixteen distinct colonies of the endophytic fungi were
isolated from healthy leaves of Irvingia gabonensis. Each of the isolated
sixteen distinct colonies were subcultured into potato dextrose agar
plates. From the sixteen distinct colonies of the endophytic fungi
subcultured, ve of the isolates (31.25%) were fermented and their
extracts were used for antimicrobial assay. e weight of the secondary
metabolites produced and extracted from the endophytic fungi aer
the 21 days of fermentation is shown in Table 1. From the table, it can
be observed that the endophytic fungi in extract EDF4 produced more
secondary metabolites than others while the endophytic fungi in
extract EDF3 produced the least amount of secondary metabolite as
shown below (Table 1).
e antimicrobial activities of crude extract of the secondary
metabolite isolated from the endophytic fungi showed a board
spectrum and eective antibacterial and antifungal activity.
e crude extracts of ve endophytic fungi exhibited a wide variety
of antimicrobial activities against six tested microorganisms. Each of
the endophytic fungi produced bioactive compounds that exhibited
antimicrobial activity against at least one test microorganisms used
(Table 2).
Discussion
e antimicrobial activity of the crude ethyl acetate extract of the
secondary metabolite isolated from dierent fungi were evaluated with
the aim of discovering novel bioactive compounds of biomedical
importance. Previous studies have shown that several extracts from
endophytic fungi exhibited antimicrobial activity [24]. Endophytic
fungal species are now considered as exciting novel sources for
obtaining new bioactive compounds and have been reported from
several hosts [25-27]. Five of the endophytic fungi isolated from leaves
of
Irvingia gabonensis
have signicantly inhibited and showed
antagonist activities against the representative gram positive bacteria,
gram negative bacteria and lamentous fungi.
is results correlated with the ndings of other reports where they
reported the antimicrobial activity of endophytes [26-31]. 16 out of 203
endophytic isolates showed antimicrobial activity with a wider action
spectrum inhibiting gram-positive and gram-negative and fungi [32].
In their research also reported that 5 out of 21 isolates showed broad
antagonistic activity against the all the test organisms [33]. Some of the
zones of inhibition by these endophytic fungi are very much
comparable to the standard antibiotics. It has been reported that
Pseudomonas aeruginosa
have developed drug resistance towards
many antibiotics [34]. In this study all the ve isolates inhibited
Pseudomonas aeruginosa
, with extract EDF2 showing the highest
inhibition zone diameter of 9 mm. Other extracts also showed a wide
variety of antimicrobial activity on the bacteria test organisms. EDF1
exhibited antimicrobial eect against all the test organisms used. e
maximum zone of inhibition recorded was 13.5 mm against
Bacillus
subtilis
. EDF2 was found to be active against all test organisms except
on
Escherichia coli
. e maximum zone inhibition was 12 mm (Figure
1).
e eect of the endophytic fungal extract on EDF1 and EDF2 is
comparable to the antibacterial standard ciprooxacin which showed
Citation: Nwakanma C, Njoku EN, Pharamat T (2016) Antimicrobial Activity of Secondary Metabolites of Fungi Isolated from Leaves of Bush
Mango. Next Generat Sequenc & Applic 3: 135. doi:10.4172/2469-9853.1000135
Page 3 of 6
Next Generat Sequenc & Applic, an open access journal
ISSN: 2469-9853
Volume 3 • Issue 3 • 1000135

an inhibition zone diameter of 15 mm.
Bacillus subtilis
was resistant to
extracts of EDF3 and EDF4 while they showed their antimicrobial
eect against the other ve test organisms used. 9.5 mm and 11.5 mm
respectively were the highest inhibition zone diameter recorded from
the antimicrobial activity of the two extracts aforementioned and it
was against a lamentous fungus
Aspergillus fumigatus
.
Figure 1: Antimicrobial activity of the secondary metabolites extracted from the fungi.
EDF5 showed a wide range of antimicrobial activity against all the
test organisms used. e highest inhibition zone diameter of 16.5 mm
was recorded against
Penicillum chysorgenum
, which is comparable to
the inhibition by the antifungal standard Fluconazole (15.5 mm). is
result corresponds with the reported research results from dierent
individuals. According to several preliminary studies have been
reported on screening of endophytic fungi from medicinal plants for
production of antimicrobial activities found that more than 50% of
isolates displayed antimicrobial activity against at least one tested
microorganisms.
ey also reported that 56% of endophytic fungi from
Dracaena
cambodiana
(
Agavaceae
) inhibited growth of at least one of the test
organisms and 8% showed broad spectrum inhibition [35]. Crude
extracts of nine endophytic fungi (75%) isolated from
Adenocalymma
alliaceum
Miers showed antibacterial potential against one or more
clinical human pathogen [36]. Similarly, reported that the compound
Nectriapyrone isolated from the endophytic fungi
Colletotrichum
gloeosporioides
and
Glomerella cingulate
showed a strong
antimicrobial activity against
Staphylococcus aureus
,
Escherichia coli,
Candida albicans, Trypanosoma cruzi, Leishmania tarentllae
and
Human T leukemia cell. Also, the isolated twelve endophytic fungi
from the healthy leaves of
Madhuca longifolia L
. for antimicrobial
activity [37]. ey reported that the ethyl acetate extract of
Colletotrichum gloeosporioides
had an eective antimicrobial activity
against
Staphylococcus aureus
,
Escherichia coli
and
Candida albicans
.
e fungal test organisms (
Aspergillus fumigatus
and
Penicillum
chysorgenum
) were the most susceptible to the compounds produced
by all ve endophytic fungal isolates.
e maximum zone of inhibition by the endophytic fungal isolates
for
Aspergillus fumigatus
was 13 mm diameter, which is comparable to
the inhibition by the antifungal standard Fluconazole (17.5 mm). Also
for
Penicillum chysorgenum
the maximum inhibition zone diameter
was 16.5 mm which is also comparable to antifungal standard
uconazole (15.5 mm). e extract EDF1 showed the highest level of
inhibition against a gram positive bacteria (
Bacillus subtilis
) and it also
showed the highest level of inhibition against a gram negative bacteria
(
Escherichia coli
). According to the dierence in the
in vitro
activity
among the various crude extracts could be due to the production of
either a broad spectrum antimicrobial compound, or several
compounds with dierent activities [38]. In their research explained
that dierences in level of inhibition by the endophytic fungi could be
due to the expected dierent modes of action, the level of isolate
inoculation and activity of the individual biochemical constituent of
the respective isolates [39]. Also in his research concluded that the
dierences in levels of antagonism are dependent on concentration of
the active substance [40].
ese active endophytic fungi might be the future for antibiotics
production. From this study, it was discovered that these ve
endophytic fungal isolates exhibited at least ve of the test
microorganisms used including a lamentous fungi
Penicillium
Citation: Nwakanma C, Njoku EN, Pharamat T (2016) Antimicrobial Activity of Secondary Metabolites of Fungi Isolated from Leaves of Bush
Mango. Next Generat Sequenc & Applic 3: 135. doi:10.4172/2469-9853.1000135
Page 4 of 6
Next Generat Sequenc & Applic, an open access journal
ISSN: 2469-9853
Volume 3 • Issue 3 • 1000135

chrysogenum
which is interesting because according to a research by
they reported the all the six endophytic fungal isolates from Tulsi
inhibited all the test organisms except
Penicillium chrysogenum
[41].
e present study leads to the need of further in depth studies on these
isolated bioactive endophytic fungal isolates. Many are able to produce
quite a good amount of antimicrobial compounds tested in
preliminary test. Further growing those in large scale, modifying
culture conditions like changing pH, changing growth media and
supplying some stimulants might help in getting better production of
the particular bioactive compound and enzyme [42]. Furthermore, the
best proved active isolates should be identied using available methods
to place these fungi in the fungal kingdom. In conclusion, further
investigation may yield novel compounds with practical applications in
a variety of biotechnological areas, with countless useful drugs as
important therapeutics options for innumerable disease.
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Citation: Nwakanma C, Njoku EN, Pharamat T (2016) Antimicrobial Activity of Secondary Metabolites of Fungi Isolated from Leaves of Bush
Mango. Next Generat Sequenc & Applic 3: 135. doi:10.4172/2469-9853.1000135
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Next Generat Sequenc & Applic, an open access journal
ISSN: 2469-9853
Volume 3 • Issue 3 • 1000135

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Citation: Nwakanma C, Njoku EN, Pharamat T (2016) Antimicrobial Activity of Secondary Metabolites of Fungi Isolated from Leaves of Bush
Mango. Next Generat Sequenc & Applic 3: 135. doi:10.4172/2469-9853.1000135
Page 6 of 6
Next Generat Sequenc & Applic, an open access journal
ISSN: 2469-9853
Volume 3 • Issue 3 • 1000135