Antimicrobial activities of endophytic fungi isolated from the aquatic plant
Nymphaea nouchali (Nymphaeaceae) endemic to Sri Lanka
Introduction
There are high demands to search new clinically effective antibacterial and antifungal
agents. As infectious pathogenic bacterial strains are becoming multi drug resistant to
currently available antibiotics, the effectiveness of most of the antibacterial agents will be
reduced in next decay. Even now some antibiotics are useless for certain infections such
as Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcus and
Vancomycin resistant S. aureus and it is currently a global issue.1 Most fungal infections of
humans are not life threatening. However in immune compromised groups of individuals,
such as patients with AIDS, cancer patients undergoing chemotherapy and organ
transplanted patients, some fungal infections can be life threatening. Presently there are
only limited numbers of antifungal agents to treat them. 2 Therefore, developing new anti-
microbial compounds is of importance.
Endophytic fungi which reside symbiotically inside plants are considered as an
outstanding source of novel bioactive natural products because there are so many of them
occupying millions of unique biological niches growing in different types of environments.
Recent researches have shown that fungal endophytes are prolific producers of
biologically active secondary metabolites.3,4
Sri Lanka is considered as a biodiversity hotspot comprised of a rich biological diversity of
plant species with a high degree of endemism. Endophytic fungi inhabiting the endemic
plant species of Sri Lanka represent an almost completely uninvestigated and untapped
and a significant and unique resource for the discovery of new biologically active natural
products.
Methodology
The endemic aquatic plant Nymphaea nouchali (Nil manel) was selected to isolate
endophytic fungi and plant specimens were collected from the Udugampola,Gampaha
District in Sri Lanka. Healthy leaves, stems, sepals and petals from different plants were
randomly collected and brought to the laboratory in sterile bags and processed within
three hours after sampling. Plant materials were surface sterilized using sterile distilled
water, 70% Ethanol and 5% NaOCl solutions. Then plant parts were cut into 0.5 cm
fragments and placed on dilute Malt Yeast Agar (dMYA) plates at room temperature for
several days.5 After about 7days, endophytic fungi emerged from the samples and they
were sub cultured on Potato Dextrose Agar (PDA) plates to obtain pure cultures. Pure
fungal cultures were sorted according to their outer appearance for further studies.
1
From the isolates, 24 (leaves-10, stem-5, sepals-5 and petals-4) morphologically distinct
endophytic fungi were selected and each fungal species was cultured on 06 PDA plates
(120 mm×20 mm) for 03 - 05 weeks and the culture media together with the fugal mycelia
were extracted in to ethyl acetate and concentrated under reduced pressure to obtain
crude organic extracts. Molecular identification of isolated fungi is in progress.
Anti-bacterial bio-assays of crude fungal extracts were performed using the disc diffusion
method against Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC
9027), Escherichia coli (ATCC 35218) and Bacillus cereus (ATCC 11778) at 300 µg/disc.
Those were active at 300 µg/disc, were then tested at 50 µg/disc. Gentamycin was used
as the positive control.6
The anti-fungal bio assays of crude fungal extracts were performed using the disc
diffusion method against Fusarium oxysporum, Rigidoporus microporus, Colletotrichum
gloeosporioides, and Aspergillus niger at 300 µg/disc. Ketoconazole and Itraconazole
were used as the positive controls.6
Results and Discussion
The results of antibacterial bioassays indicated that 9 fungal extracts out of the 24
inhibited the growth of S. aureus and B. cereus at 300 µg/disc (Table 01). The diameter of
06 inhibition zones out of 09 were greater than 10 mm (including disk diameter of 6 mm)
indicating prominent antibacterial activity. Among them RDNM-01 and RDNM-18 were
active against all tested bacterial strains (both Gram positive and negative) at both 300
and 50 µg/disk. RDNM-04,06 and 22 were active only against tested Gram positive
bacteria at both 300 and 50 µg/disk (Table 02). RDNM-13,20,21 and 23 were active only
at 300 µg/disk for Gram positive bacteria.
Only 4 crude extracts out of 24 extracts showed antifungal activity (Table 01). The
promising activities were seen in the extracts of RDNM- 01 and RDNM-18 against F.
oxysporum and R. microporus. None of the extracts were active against A. niger and C.
gloeosporioides. The fungal extracts of RDNM-01, 18 and 22 show both antibacterial and
antifungal (antimicrobial) activities. Among them RDNM-01 and 18 possess considerable
antimicrobial activities when compare to the positive control.
2
Table 01. Antimicrobial activity of the extracts of endophytic fungi at 300 µg/disc of plant
Nymphaea nouchali (Nil manel)
Identification Origin Antibacterial activity Antifungal activity
No. of Fungi (Inhibition zone diameter - mm)
S. aureus B. cereus P. E. coli R. F.
aeruginosa microporus oxysporum
RDNM-01 Sepals 17 22 08 15 + +
RDNM-04 Leaves 16 19 - - - -
RDNM-06 Leaves 14 15 - - - -
RDNM-13 Stem 10 11 - - - -
RDNM-15 Petals - - - - + -
RDNM-18 Leaves 25 27 08 20 + +
RDNM-20 Leaves 10 12 - - - -
RDNM-21 Stem 08 08 - - - -
RDNM-22 Sepals 12 14 - - + -
RDNM-23 Leaves 09 07 - - - -
+ve control 25 27 17 22 +++ +++
+ inhibit the growth tested fungi
Table 02. Antibacterial activity of the extracts of endophytic fungi at 50 µg/disc of plant
Nymphaea nouchali (Nil manel)
Identification Antibacterial activity
No. of Fungi (Inhibition zone diameter - mm)
S. aureus B. cereus P. aeruginosa E. coli
RDNM-01 12 12 07 08
RDNM-04 09 12 - -
RDNM-06 10 14 - -
RDNM-18 15 16 08 09
RDNM-22 07 08 - -
+ve control 27 32 19 22
References
1. Antimicrobial resistance “global report on surveillance 2014”, World Health
Organization, 2014.
2. Denning, D.W.; Echinocandin antifungal drugs, Lancet, 2003, 362, 1142-1151.
3. Selim K.A., El-Beih A.A., AbdEl-Rahman T.M., Biology of Endophytic Fungi, Current
Research in Environment & Applied Mycology, 2012, 31-82.
4. Gunathilaka, L.; Natural products from Plant-Associated microorganism: Distribution,
structure diversity, bioactive and Implications of their occurance. J. Nat. Prod., 2006, 69,
509-526.
5. Ashour, M., Yehia., H.M., Proksch, P.; Utilization of Agro-industrial by-products for
production of bioactive natural products from endophytic fungi. J. Nat. Prod., 2011, 04,
108-114.
6. Performance standards for antimicrobial disk susceptibility test, Approved standard,
Ninth Edition, Clinical and laboratory standard institute, 2006.
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