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ISSN: 2410-8790 Mensah et al / Current Science Perspectives 1(3) (2015) 96-101 iscientic.org
www.bosaljournals/csp/ 96 editorcsp@bosaljournals.com
Article type:
Research article
Article history:
Received April 2015
Accepted May 2015
August 2015 Issue
Keywords:
Trichoderma koningii
Rhizopus stolonifer
Fusarium oxysporum
Antimicrobial
Secondary metabolites
Broth dilution assay
Fungi have been source organisms for drug discovery, but remain unexplored source
materials for natural product in Ghana. This study provides preliminary antimicrobial
susceptibility assessment of crude extracts of secondary metabolites derived from
select soil-habitat fungi. Pure cultures of Trichoderma koningii, Rhizopus stolonifer
and Fusarium oxysporum, isolated from soil, were fermented for secondary
metabolites in Sabouraud Dextrose Broth (SDB) for 21 days. Bioactive compounds
were isolated with ethylacetate and crude extracts screened for antimicrobial activities
against a panel of microorganisms including Staphylococcus aureus, Escherichia coli,
Pseudomanas aeruginosa, and Candida albican (fungus) in broth dilution assay.
Extracts displayed variable antimicrobial activities that were microorganism-specific
and comparatively low in relation to the strength of the activities displayed by the
clinically used standard agents Ciprofloxacin (antibacterial) and Ketoconazole
(antifungal). This study provides access to previously unexplored extract source
materials for anti-infectives.
© 2015 International Scientific Organization: All rights reserved.
Capsule Summary: Ethylacetate extracts of the secondary metabolites of pure cultured Trichoderma koningii, Rhizopus stolonifer
and Fusarium oxysporum displayed variable antimicrobial activities against a panel of four microbes.
Cite This Article As: John Kenneth Mensah, Charles Kwoseh, Nicholas Banahene, Samuel Aweinatey Atuilik, Daniel Oppong and
Malik Borigu. Assessment of the Antimicrobial Activities of the Secondary Metabolites Produced by Pure Cultured Trichoderma
koningii, Rhizopus stolonifer and Fusarium oxysporum. Current Science Perspectives 1(3) (2015) 96-101
INTRODUCTION
Fungi synthesize chemically diverse and often biologically active
compounds as defense weapons (Yi et al., 2009), as chemical
signals (Dufuor and Rao, 2011), as competition weapons (Cueto
et al., 2001) and as adaptive response chemical signals (Hay,
2009). Cultured soil fungi have, therefore, been productive
sources of drugs worldwide that includes cyclosporin,
pernicillins, and some statins (Butler, 2004). But fungi remain
unexplored source materials for natural product leads in Ghana.
In the continuing effort to identify fungal secondary metabolites
with medicinally-relevant anti-infective properties an evaluation
of crude extracts of select fungal cultures for potential
antimicrobial therapeutic leads has been undertaken.
Crude fungal secondary metabolites selected for antimicrobial
evaluation were derived from a broad fungal bio-diversity
spectrum that include genetically-diverse strains as Trichoderma
koningii, Rhizopus stolonifer and Fusarium oxysporum. All
selected fungi are soil-habitants and are amenable to standard
methods of culture in readily available media that support
secondary metabolism in vitro. Chosen strains are also proven
rich sources of media-specific biologically active secondary
metabolites including Beauvericin (Logrieco et al., 1999).
Besides Fusarium oxysporum and Trichoderma koningii the
Current Science Perspectives 1(3) (2015) 96-101
Assessment of the Antimicrobial Activities of the Secondary Metabolites Produced by
Pure Cultured Trichoderma koningii, Rhizopus stolonifer and Fusarium oxysporum
John Kenneth Mensah1,
*, Charles Kwoseh2
, Nicholas Banahene1
, Samuel Aweinatey Atuilik1
, Daniel Oppong1
and Malik
Borigu2
1
Department of Chemistry, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana
2
Department of Crop and Soil Sciences, College of Agriculture and Natural Resources, KNUST, Kumasi, Ghana
*Corresponding author’s E-mail: jkmensah75@yahoo.com
A R T I C L E I N F O A B S T R A C T

literature is silent about Rhizopus stolonifer ability to produce
anti-infective secondary metabolites.
The scientific rationale that undergirds this study is that the pool
of secondary metabolites that emerge from these diverse strains
will exhibit significant structural and substantial chemical
diversity and might even encompass novel chemotypes that are
potentially capable of exhibiting potent antimicrobial activities
towards the clinically problematic multi-drug resistant microbial
pathogens. And that success in this endeavor will certainly
provide clinicians with a powerful new treatment option against
problematic multi-drug resistant pathogens.
This study provides preliminary antimicrobial susceptibility
assessment of crude extracts of secondary metabolites derived
from three select soil-habitat fungi. Antimicrobial activity of the
extracts assayed by broth dilution methods against a panel of
microorganisms that include Staphylococcus aureus (gram
positive bacterium), Escherichia coli (gram negative bacterium),
Candida albicans (fungus) and Pseudomanas aeruginosa (gram
negative bacterium) were variable. Although extracts displayed
differential levels of activity that were substantially lower than
clinically used pure standard agents Ciprofloxacin (antibacterial)
and Ketoconazole (antifungal), this study provided access to
previously unexplored source of natural product extract materials
for anti-infectives.
MATERIALS AND METHODS
Chemicals
Nutrient broth, nutrient agar and Sabouraud broth were obtained
from Sigma Chemical Co. (St. Louis, MO, USA). Ethanol and
Methanol were obtained from Merck Chemical Supplies
(Damstadt, Germany). All reagents were of analytical grade.
METHODS
Sterilization of all flasks and media was accomplished by
autoclaving at 120 o
C for 30 min. All fermentation studies were
conducted in 500-mL Erlenmeyer flasks containing 100 mL of
Sabouraud media within the flasks closed with cotton plugs.
Culture and maintenance of microorganisms
The panel of microbes used for the assessment of antimicrobial
activity were obtained from ATCC (USA) and were stored in
liquid nitrogen in 20% glycerol. Microbes were maintained by
regular sub-culturing on nutrient agar (bacterial) and on potato
dextrose agar (PDA) (fungi) before storage at 4o
C prior to
experimental use.
Isolation of fungi from soil
Soil samples were obtained from farms located in KNUST
campus. About 1 g soil sample was suspended in 9 mL sterile
distilled water and the mixture vortexed vigorously. The soil
allowed to settle from the suspension and the supernatant was
filtered and then diluted by transferring 1 mL to a second test
tube containing 9 mL of sterile water. An aliquot (100 µL) of the
diluted filtrate was spread evenly on potato dextrose agar (PDA)
medium supplemented with 50 µg/mL chloramphenicol in a petri
dish and incubated at 37°C for 7 to 10 days. Individual fungal
colonies were removed and repeatedly sub-cultured until pure
fungal cultures were obtained.
Identification of fungi
Spores of the pure fungal culture were mounted on a microscope
slide and examined under a light microscope (Leica, Germany).
Fungi were identified via comparison of observed spore
morphological features and culture characteristics with published
notes available in the standard reference manual (Barnett and
Hunter, 1986).
Inoculation of media with fungal isolate
Sabouraud broth was sterilized by autoclaving at 120 ºC for 30
min. Sterilized media was inoculated under aseptic conditions
with a single isolated fungal colony selected from the pure
cultured fungi on PDA. Inoculated media was kept at room
temperature (27-28 ºC) with intermittent shaking for 21 days.
Extraction of secondary metabolites
Culture supernatant was filtered to separate mycelia from broth.
Filtrates were partitioned three times with equal volumes of
ethylacetate. The organic fractions were combined, dried over
anhydrous sodium sulphate, filtered and reduced to dryness on a
rotavap. Dried residues were stored at -20 ºC until needed for
bioactivity studies. Yield of extracts ranged from 250 mg to 350
mg.
Quality control
To assure that extracts were derived solely from the cultured
fungi of interest, an aliquot of the fermentation culture was
pipetted and its contents cultured on PDA. Fungal strain were
authenticated after microscopic visualization. All cultures were
found to contain only the fungi of interest and harbored no
additional microbe.
Diagnostic TLC
To determine the number of possible compounds in each extract,
a 20 µL of each ethylacetate extract was dissolved in 100 µL
chloroform. An aliquot 10 µL of the resulting solution was
spotted at the origin of a 10- by 5-cm TLC glass plate coated
with silica gel (Fisons Scientific silica gel S/0790/53,
Loughborough England) that was approximately 500 µm in
thickness. The spots were allowed to dry and eluted with
chloroform for 60 min. Developed plates were air-dried and
product spots were visualized with iodine vapor. Rf values of
visible spots were estimated.
Basic phytochemical screening

The ethylacetate extract was assayed for its phytochemical
constituents using protocols previously described (Trease and
Evans, 1984).
Alkaloids: Acidified solution of alkaloid produce a white-
yellowish precipitate upon the addition of a few drops of Mayer’s
reagents. Extracts were initially heated on a boiling water bath
with 2% hydrochloric acid. The mixture was allowed to cool and
few drops of Mayer’s reagent added. A yellow precipitate
indicated the presence of alkaloids.
Terpenoids and steroids: To a small amount of the extract (2
mg) was added (250 µL of acetic anhydride and 250 µL of
chloroform). Concentrated sulphuric acid (1 mL) was added
slowly and a red-violet color indicated the presence of terpenes
while a green-bluish color was indicative of the presence of
steroids.
Flavonoids: To a small amount of the extract was added 1.5 mL
of 50% methanol and the solution warmed. Metal magnesium
was then added followed by 5-6 drops of concentrated
hydrochloric acid. The formation of a red color indicated the
presence of flavonoids while an orange color indicated the
presence of flavones.
Tannins: To 0.5 mL of an aqueous solution of the extract was
added 1- 2 drops of ferric chloride solution. A blue color
indicated the presence of gallic tannins while a greenish-black
color indicated the presence of catecholic tannins.
Coumarins: To 500 µL of an aqueous solution of the extract was
added 750 µL of 10 % NaOH. The formation of a yellow colour
is indicative of the presence of coumarins.
Saponins: To 500 µL of an aqueous solution of the extract was
added 1 mL of 1% sodium bicarbonate and the solution shaken.
A persistent froth is indicative of the presence of saponins.
Glycosides: To a small amount of the extract (2.0 mg) in a test
tube was added few drops of acetic anhydride. To the resulting
solution was added 2-3 drops of concentrated sulphuric acid. A
blue-green color shows the presence of glycosides.
Antimicrobial assay
Broth dilution assay was performed using a modification of the
protocol described by Murray et al., 1999. Briefly, Escherichia
coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853),
Staphylococcus aureus (ATCC 25923) and Candida albicans
(strain NCPF 113; National Collection of Type Cultures,
London, England) were grown in Mueller-Hinton agar and broth
(Difco Laboratories). The microbial culture was inoculated in the
nutrient broth (MH broth) and incubated at 37o
C for 24 h. Then a
20 mL fresh broth was seeded with 0.25 mL of the 24-hour broth
cultures and a two-fold serial dilution method was performed as
described below. The dried fungal extract was dissolved in 85%
methanol to obtain a 20 mg/mL solution that was sterilized by
filtration through a 0.45 μm membrane filter. A 0.2 mL solution
of the material was added to 1.8 mL of the seeded broth to obtain
the first dilution. A 1 mL of this dilution was further diluted with
1 mL of the seeded broth to produce the second dilution, and the
process was repeated until seven dilutions were obtained (10
mg/mL, 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL,
0.3125 mg/mL and 0.15625 mg/mL). Tubes containing only
seeded broth and 85% methanol were kept as controls. All tubes
were incubation for 24 h at 37o
C. Ciprofloxacin and fluconazole
(at different concentration in Table 2) were used as positive
controls. All determinations were made in triplicate and the data
was expressed as mean±standard deviation. MICs (expressed
either in µg/mL or mg/mL) were taken as the lowest extract
concentrations that showed complete growth inhibition and were
represented by the last tube with no visible violet color from the
addition of 0.1 mL of MTT.
Statistical Analysis
All data were reported as the mean±standard deviation of the set
of triplicates determinations as estimated using Microsoft Excel
software. Student’s t test was used to compare the data, and all
tests were considered statistically significant at p < 0.05.
RESULTS AND DISCUSSIONS
Phytochemical Screening
Fungi constitute outstanding source organisms for antimicrobial,
antioxidants, anticancer, antidiabetic and immunosuppressive
drug leads. This study assessed the antimicrobial activities of the
ethylacetate extracts of the fermentation culture of three fungal
species:Trichoderma koningii, Rhizopus stolonifer and Fusarium
oxysporum.
All three fungi are uniformly characterized by saprophytic
feeding within soil-habitat. Trichoderma koningii is a saprophyte
that produces diverse bioactive compounds used for competitive
inhibition against other fungi and pathogenic microorganisms
within its milleu (Song et al., 2006). Rhizopus stolonifer is a
cosmopolitan filamentous fungi with mycelia that persist in soil
and in other moist environments and that enables the
biosynthesis of diverse bioactive compounds within its resident
environment (Hernandez-Lauzardo et al., 2006). Fusarium
oxysporum is a common saprophyte and a pervasive plant
endophyte that is a proven rich source of biologically active
secondary metabolites (Liu, 2011).
While the contemporary literature reveals that the examined
species are rich sources of bioactive secondary metabolites, none
of the reported metabolites were obtained via fermentation in
Sabouraud media and none has been examined for antimicrobial
activities against the specific tested panel of microbial strains (Lu
and Shen, 2004; Hsu and Koh, 2011).
TLC analysis revealed extracts that are mixtures of at least
two compounds with distinct Rf values (Table 1). Since the TLC
spots were specific for the stationary and mobile phase used, the
possibility exists that more spots representative of other bioactive
compounds would be revealed under better resolution provided
by a different mobile phase or by another stationary phase.

Trichoderma koningii showed four evenly spaced spots.
Rhizopus stolonifer extract revealed two spots representative of
compounds that were highly polar and that congregated close to
the baseline of the TLC plate. Fusarium oxysporum revealed a
pattern of TLC separation that was similar to that of Rhizopus
stolonifer with minimal movement of the two spots from the
baseline (Table 1).
Phytochemical test revealed that all three extracts contain
flavonoids, terpenoids, saponins and tannins. Extracts from
Trichoderma koningii and Rhizopus stolonifer contain the
additional presence of alkaloids and reducing sugars. Only
Rhizopus stolonifer extract tested positive for anthraquinones
(Table 1). The presence of the observed phytochemicals is often
predictive of bioactivity of the constituent compound.
Inhibitory effect of the extracts on microbial growth was
assessed via broth dilution assay and means minimal inhibitory
concentrations (MIC), in the mg/mL range, are provided in Table
2. Each extract showed inhibitory activity against more than one
test microorganism.
Test microbes were differentially susceptible to the
inhibitory effect of the extract and showed variations in broth
dilution MICs that appear to be test microorganism-specific.
Trichoderma koningii extract was only weakly inhibitory on
Staphylococcus aureus. Inhibitory bioactivity of Rhizopus
stolonifer on Staphylococcus aureus was on a level that was
Table 1: Thin layer chromatography (TLC) report and phytochemical contents of the ethylacetate extract of the fungal
culture
Fungi Number of spots from
TLC and Rf values
Phytochemicals present in extract
Trichoderma koningii Four
Rf: 0.27, 0.40, 0.51 and
0.92
Alkaloids, Reducing Sugars, Terpenoids, Saponins and Tannins
and Steroids.
Rhizopus stolonifer
Two
Rf: 0.115 and 0.29
Alkaloids, Reducing Sugars, flavonoids, Anthraquinones,
Terpenoids and Saponins.
Fusarium oxysporum
Two
Rf: 0.04 and 0.13
Flavonoids, Terpenoids, Saponins and Tannins.
Table 2: Minimum inhibitory concentration (MIC) obtained from the agar diffusion assay of the ethylacetate extract of the
fungal culture and of the standard drugs used as positive controls
Fungi Test organism Agar diffusion assay
MIC (mg/mL) OF
EXTRACT
Agar diffusion assay MIC
(mg/mL) OF ciprofloxacin
Agar diffusion assay
MIC (mg/mL) OF
fluconazole
Trichoderma
koningii
Staphylococcus aureus 3.0 0.031 -
Pseudomonas aeruginosa 2.4 1.0 -
Escherichia coli 1.2 0.062 -
Candida albicans 2.4 - 1.0
Rhizopus
stolonifer
Pseudomonas aeruginosa No effect 1.0 -
Fusarium
oxysporum
Pseudomonas aeruginosa No effect 1.0 -

similar to that of Trichoderma koningii. In contrast, Fusarium
oxysporum extract was more bioactive on Staphylococcus aureus
than on the other microbes. Extracts from Rhizopus stolonifer
and Fusarium oxysporum showed no inhibitory activity on
Pseudomonas aeruginosa at the assayed concentration and
extracts from Trichoderma koningii were inhibitory on
Pseudomonas aeruginosa only at a higher concentration (2.4
mg/mL). Extracts from Fusarium oxysporum had a strongest
antibacterial activity against E. Coli. Similarly, Trichoderma
koningii extracts exhibited maximum growth inhibition against
E. Coli in contrast to extracts of Rhizopus stolonifer that
displayed the least inhibitory activity against E. Coli.
Extracts showed antifungal activities on Candida albicans as
well. The most potent antifungal activity against Candida
albicans was shown by Rhizopus stolonifer extract. Trichoderma
koningii extract had better fungicidal effect on Candida albicans
than Fusarium oxysporum extract although both inhibitions were
observed at higher MICs values. In all cases, bioactivity of the
standard drugs (antibacterial Ciprofloxacin and antifungal
Fluconazole) used as positive controls were higher with MICs at
lower concentrations (at least 2-fold to at most a 100-fold) than
that of the extracts.
Reports abound about antimicrobial compounds produced by
specific strains of Fusarium, the only endophytic fungi among
the list (Liu, 2011) and by several strains of Trichoderma (Song
et al., 2006). Antimicrobial extracts from Rhizopus stolonifer
have not been reported in the literature.
Although bioactivity was minimal with MICs above the range of
clinically useful concentrations, the possibility that one or more
synthetic analogs of the constituent compounds might exhibit
potent antimicrobial activity should spur further research
designed to derive useful anti-infective medicinal agents from the
extracts of these cultured fungi. Consequently, the next phase of
this research study would be to purify each fungal extract to
single pure bioactive constituents. Using activity-guided
fractionation of the active constituents with agar diffusion assay
as the primary screens, the fraction(s) that contain the antibiotic
activity can be isolated. Structure determination using NMR, IR
and mass spectroscopy can be conducted on the isolated active
fractions. Analog synthesis of the most promising active pure
compounds can then be accomplished and analogs can then be
screened for their ability to inhibit specific microorganisms in
vitro and in vivo. The possibility for the development of potent
antimicrobial compounds using the bioactive extract constituents
as templates should stimulate further research into the
bioactivities of fungal extracts.
CONCLUSION
Ethyl acetate extracts of the secondary metabolites of pure
cultures of Trichoderma koningii, Rhizopus stolonifer and
Fusarium oxysporum displayed variable antimicrobial activities
against Staphylococcus aureus (gram positive bacterium),
Escherichia coli (gram negative bacterium), Pseudomanas
aeruginosa (gram negative bacterium) and Candida albican
(fungus). Although the three fungal source organisms are
extensively studied, their secondary metabolites have not been
thoroughly examined for antimicrobial activities against the
panel of tested microbes. The levels of antimicrobial activities
displayed were substantially lower than clinically used pure
standard agents Ciprofloxacin (antibacterial) and Ketoconazole
(antifungal). This study increased the repertoire of natural
product sources available for investigation in Ghana and
provided access to previously unexplored source of extract
materials for anti-infectives.
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