5

Mikologia Lekarska 2012, 19 (1): 5-11

Copyright 2012 Cornetis
www.cornetis.pl
ISSN 1232-986X
ORIGINAL ARTICLES / PRACE ORYGINALNE
Antimicrobial and antioxidant activity of endophytic
fungi isolated from ethnomedicinal plants
of the Sacred forests of Meghalaya, India
Przeciwbakteryjne i antyoksydacyjne dziaanie endophytic grzybw wyizolowanych
z ethnomedicinal rolin lasy Sacred Meghalaya, Indie
Ranjan Kumar Bhagobaty, S.R. Joshi
Microbiology Laboratory, Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Umshing, Shillong 793 022, Meghalaya, India
ABSTRACT
Introduction: Medicinal plants growing in the sacred forests of Meghalaya (20.1N-26.5N latitude and
85.49E-92.52E longitude), in the north-eastern region of India are used by the traditional medical prac-
titioners of the ethnic tribes of the region to treat a diverse range of diseases.
Aim of the study: The aim of the study was to elucidate the antimicrobial and antioxidant potency of the
most dominant fungal endophytes of some selected medicinal plants of the Sacred forests.
Material and methods: Isolation of the endophytes was done in water agar plates followed by subsequ-
ent pure culturing in potato dextrose agar plates. Molecular characterization of the endophytic fungal
isolates was done by sequencing their -tubulin gene. The antimicrobial properties of the culture broths
and the crude ethyl acetate extracts of endophytic fungal metabolites were ascertained as per standard
protocols against five human pathogens, namely, Bacillus cereus, Salmonella typhi, Escherichia coli, Staphy-
lococcus aureus, and Candida albicans. The total antioxidant power and free radical scavenging activity of
the metabolites was estimated using the ferric reducing antioxidant power (FRAP) and 1, 1-diphenyl-2-
-picrylhydrazyl (DPPH) free radical assays.
Results: Molecular characterization of the endophytes showed their relatedness to human pathogenic
fungi. Minimum inhibitory concentration of the ethyl acetate extracts against the test pathogens ranged
from 13-45 g/ml. The metabolites of the endophytic fungi also showed high anti-oxidant activity.
Conclusion: Endophytic fungi of the medicinal plants from the Sacred forests of Meghalaya, India are
potential treasure troves of novel antimicrobial molecules and antioxidants.
KEY WORDS: Medicinal plants; fungal endophytes; antimicrobial activity; FRAP; DPPH
STRESZCZENIE
Wprowadzenie: Lecznicze roliny rosnce w witych lasach Meghalaya (20,1N, 26,5 szerokoci geogra-
ficznej N i 85,49E-92.52E dugoci geograficznej), w pnocno-wschodniej czci Indii s wykorzystywane
przez tradycyjnych medykw z etnicznych plemion regionu, w leczeniu rnego rodzaju chorb.
Cel: Celem bada byo zbadanie przeciwbakteryjnego i antyoksydacyjnego dziaania grzybw endofi-
tycznych wybranych rolin leczniczych ze witych lasw.
Materia i metody: Izolacj endofitw przeprowadzono na poywce agarowej, a nastpnie przesiewano
hodowle na podoe dekstrozowo-ziemniaczane. Charakterystyka molekularna izolatw grzybw endo-
fitycznych polegaa na sekwencjonowaniu genu -tubuliny. Waciwoci przeciwbakteryjne metaboli-
tw grzybw endofitycznych (ekstrakty w octanie etylu) dla 5 patogenw ludzkich: Bacillus cereus, Sal-
monella typhi, Escherichia coli, Staphylococcus aureus i Candida albicans, zostay ustalone wg standardo-
wych protokow. Waciwoci antyoksydacyjne metabolitw grzybw endofitycznych zostay oszaco-
wane przez okrelenie zdolnoci redukcji jonow Fe3+ do jonow Fe2+, (metoda FRAP i metoda z uyciem
1-difenylo-2-picrylhydrazyl DPPH).
Wyniki: Charakterystyka molekularna endofitw wykazaa ich pokrewiestwo do grzybw patogen-
nych dla czowieka. Minimalne stenie hamujce ekstraktw metabolitw w octanie etylu w stosunku
do patogenw testw wahaa si od 13 do 45 mg/ml. Metabolity grzybw endofitycznych wykazay rw-
nie wysok aktywno antyoksydacyjn.
Wnioski: Endofityczne grzyby rolin leczniczych z witych lasw Meghalaya w Indiach s potencjaln
skarbnic nowych czsteczek przeciwbakteryjnych i antyoksydacyjnych.
SOWA KLUCZOWE: roliny lecznicze; grzybw endophytes; aktywnoci przeciwbakteryjnej; FRAP; DPPH
ADDRESS FOR CORRESPONDENCE:
Dr S. R. Joshi, Assoc. Prof.
Microbiology Laboratory,
Department of Biotechnology
and Bioinformatics,
North-Eastern Hill University
Shillong 793-022, India
tel.: +91 364 272 24 05
fax: +91 364 255 00 76
e-mail: srjoshi2006@yahoo.co.in
Mikologia Lekarska 2012, 19 (1)
6
Bhagobaty R.K., Joshi S.R.
Antimicrobial and antioxidant activity of endophytic fungi isolated from ethnomedicinal plants of the Sacred forests of Meghalaya, India
Introduction
Endophytic fungi have been shown to be a promising source
of new natural bioactive agents [1]. Several crude extracts from
different culture broths have shown antimicrobial activity against
pathogenic fungi, bacteria, and yeasts; cytotoxic activity on
human cell line; anti-herpes simplex virus type 1 activity (anti-
-HSV); and antimalarial activity against the protozoan Plasmodium
falciparum [2-6]. Strobel and Daisy [7] reported that plants gro-
wing in unique environmental settings and having ethnobotani-
cal uses with extreme age or interesting endemic locations gene-
rally produce novel endophytic microorganisms, of which the
secondary metabolites are usually unique and may have applica-
bility in medicine. The present investigation therefore focused on
investigating the antimicrobial and antioxidant activities of the
endophytic fungi isolated from five selected ethnomedicinal
plants growing in the virgin Sacred Groves of Meghalaya, India,
which had not been previously screened for the presence of fun-
gal endophytes.
Material and methods
Isolation and characterization of endophytic fungi
from medicinal plants
Five selected ethnomedicinal plants, namely, Potentilla fulgens,
Osbeckia stellata, Osbeckia chinensis, Camellia caduca, and Schima
khasiana were collected from Nongkrem, Cherrapunji, Mawph-
lang, and Umsaw sacred forests spread over the state of Megha-
laya, India [8]. About 100 surface sterilized root and stem pieces
of each of the 5 plants were air dried and flamed before removing
the outer layers. Two-centimeter-long pieces of these roots and
stems were placed on petri plates containing water agar as descri-
bed by Strobel et al. (1996) [9] and incubated at 24C for 7 days.
After incubation for 7 days, hyphal tips of developing fungi were
aseptically removed and placed on potato dextrose agar (PDA),
according to the procedure described by Strobel et al. (2005) [10].
The endophytic fungal isolates were stained with aniline blue, as
per the standard protocol described by Cappucino and Sherman
(1992) [11], and morphologically identified with the help of
a Motic phase contrast trinocular research microscope model
BA-450PH (Feintechnik, Germany). Molecular characterization of
the most dominant endophytic fungal isolates, i.e., RS07PF(1),
RS07OS, RS07OC, RS07CC, and RS07SK from each of the 5 selected
medicinal plants was carried out by PCR amplification of the
-tubulin gene using the universal primers btub3 and btub4r, as
per the protocol described by Huang et al. (2009) [12]. The ampli-
cons obtained were sequenced in an Applied Biosystems 3700
Genetic Analyzer with BigDye Terminator ver. 3.1. Alignments and
phylogenetic analyses were performed using MEGA4 software
(Tamura et al., 2007) [13].
Extraction of the endophytic fungal metabolites
Endophytic fungal isolates, i.e., RS07PF1, RS07OS, RS07OC,
RS07CC, and RS07SK, were cultured in liquid potato dextrose
medium (Himedia, India). After 2 weeks of growth in the liquid
culture medium, the fungal broth was centrifuged at 13000 rpm
at 4C and filtered through a bacterial filter (0.2 microns). Equal
volumes of fungal culture broth and organic solvents (ethyl ace-
tate and acetonitrile) were taken in a 500-ml Erlenmeyer flask, and
metabolites were extracted by vigorous shaking. Extraction was
repeated 3 times, and all the resultant ethyl acetate and acetoni-
trile fractions were pooled to a total fraction of about 100 ml for
each endophytic fungal isolate. The fractions were then concen-
trated in a rotary evaporator (RE-300; Stuart, UK) at 50C at 40 rpm
to a final volume of 20 ml for each of the endophytic fungal isola-
tes. They were then lyophilized in a lyophilizer (Scanvac, Denmark)
for storage and subsequent use.
Antimicrobial activity assay
The antimicrobial potential of the extracted metabolites [14]
was tested against 4 strains of human pathogenic bacteria obta-
ined from Microbial Type Culture Collection (MTCC), Chandigarh,
India, namely, Bacillus cereus MTCC 430, Salmonella typhi MTCC
733, Escherichia coli MTCC 118, and Staphylococcus aureus MTCC
740 and 1 strain of human pathogenic yeast, i.e., Candida albicans
MTCC 227. The antimicrobial assays were performed using the Kir-
by-Bauer disk diffusion technique (Bauer et al., 1966) [15]. Fifty
microliters of ethyl acetate and fungal culture broth were inocu-
lated separately into agar wells. The susceptibility of the test
pathogenic microorganisms to known antibiotics was also tested
using the standard protocols described by Clinical and Laborato-
ry Standards Institute (CLSI), 2008. Minimum inhibitory concen-
trations (MICs) of the crude ethyl acetate extracts of the endophy-
tic fungi were determined using the protocols described by Sette
et al. (2006) [16] with some minor modifications.
Antioxidant assays
Ferric reducing antioxidant power (FRAP) assay
The total antioxidant activity of the endophytic fungal extracts
was tested using the FRAP assay, as described by Szllsi and
Varga (2002) [17]. The relative activities of samples were assessed
by comparing their activities with that of a known antioxidant, i.e.,
L-ascorbic acid.
1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical assay
To different concentrations of a sample in methanol (0.5 ml
each) 1 ml of a methanolic solution of 0.2 mM DPPH (Sigma
Aldrich, Germany) was added. After mixing thoroughly, the mixtu-
re was allowed to stand in the dark for 30 min and the absorban-
ce at 523 nm was measured using methanol for the baseline cor-
rection. The results were then compared with those of the control
prepared as above but without any sample (Rukachaisirikul et al.,
2007) [18]. Radical scavenging activity was expressed as a percen-
tage and was calculated using the following formula:
% Scavenging = [(A control A sample)/A control] 100.
A: Absorbance
Results
Isolation and characterization of endophytic fungi
Morphologically isolates RS07PF1, RS07OS, and RS07OC were
identified to be Talaromyces flavus, Mortierella hyalina, and Paeci-
lomyces variabilis, whereas RS07CC and RS07SK were identified to
be 2 distinct species of Penicillium. The evolutionary position of
the 5 most dominant endophytic fungi based on partial -tubulin
gene sequence similarity is shown in figure 1.
Antimicrobial activity of endophytic fungal culture broths
The inhibition zone size ranged from 10 to 32 mm in all the fun-
gal metabolites assayed (tab. I). The group of fungi isolated from
P. fulgens, O. Stellata, and O. chinensis (i.e., Talaromyces flavus, Mor-
7
Bhagobaty R.K., Joshi S.R.
Przeciwbakteryjne i antyoksydacyjne dziaanie endophytic grzybw wyizolowanych z ethnomedicinal rolin lasy Sacred Meghalaya, Indie
tierella hyalina, and Paecilomyces variabilis) showed potent antimi-
crobial activity against S. aureus with a zone of inhibition of 11, 13.5,
and 13.5 mm, respectively (tab. I). All the endophytic fungal cultu-
re broths inhibited the growth of the pathogenic yeast C. albicans
(tab. I). The maximum antimicrobial activity against B. cereus was
shown by the Penicillium sp. isolated from C. caduca, whereas the
highest antimicrobial activity against the other gram-positive
pathogenic bacteria S. aureus was shown by M. hyalina and P. varia-
bilis at 13.50 mm (tab. I). Except P. variabilis (isolated from Osbeckia
chinensis), all the other endophytic fungal culture broths showed
growth inhibiting activity on the gram-negative pathogenic E. coli.
The maximum inhibitory effect on E. coli was shown by T. flavus iso-
lated from P. fulgens with an inhibition zone of 14.1 mm. Only T. fla-
vus and P. variabilis showed inhibitory activity against the other
gram-negative pathogen, i.e., S. typhi. The highest activity among
the two was shown by P. variabilis with an inhibition zone size of
Fig. 1. Evolutionary position of the five endophytic fungal isolates with other related fungal species based on -tubulin gene sequence similarity
Ryc. 1. Pozycja ewolucyjna piciu izolatw grzybw endofitycznych wraz z innymi powizanymi gatunkami grzybw na podstawie podobiestwa sekwencji genu -tubuliny
Syncephalastrum racemosum(gb AY944811.1)
Saccharomyces cerevisiae(emb V01296.1)
Isolate RS07OS
Talaromyces stipitatus(refXM002341495.1)
Penicillium marneffei(ref XM002151381.1)
Metarhizium anisopliae(gb AY995134.1)
Chaetomidium pilosum(gb FJ666372.1)
Triangularia tanzaniensis(gb AY780143.1)
Aspergillus chrysogenum(emb X72789.1)
Monosiga brevicollis(ref XM 001743918.1)
Jakoba libera(gb AF267184.1)
Phycomyces blakesleeanus(gb AY944795.1)
Isolate RS07CC
Isolate RS07SK
Isolate RS07OC
Isolate RS07PF1
72
100
99
35
48
43
43
33
95
96
99
100
100
83
0.05
Kuzuhaea moniliformis(gb AY944821.1)
Table I: Antimicrobial activity of culture broths of the endophytic fungal metabolites
Tabela I: Aktywno przeciwbakteryjna i przeciwgrzybicza podoa pynnego zawierajcego metabolity grzybw endofitycznych
Host plant
Rolina gospodarz
Endophytic fungi*
Grzyby
endofityczne
Zone of inhibition [mm] of the culture broth / Strefa inhibicji [mm] podoa pynnego
Bacteria / Bakterie Yeast / Drode
Gram +ve Gram ve
Bc Sa Ec St Ca
Potentilla fulgens Talaromyces flavus 11.00a1.00 14.17b1.26 10.00a1.00 23.00c1.00
Osbeckia stellata Mortierella hyalina 13.50a0.50 10.00b1.00 18.83c1.61
Osbeckia chinensis Paecilomyces variabilis 13.50a0.50 32.67b1.15 23.33c4.04
Camellia caduca Penicillium sp. 16.93a0.90 11.73b0.25 17.03a1.05
Schima khasiana Penicillium sp. 11.00a1.00 12.00a1.00 20.33b0.58
Positive control (methyl alcohol)
Kontrola dodatnia (alkohol metylowy)
9.67ab0.58 9.67ab0.58 8.60a0.53 8.67a0.58 10.67b1.15
Negative control (potato dextrose broth medium)
Kontrola ujemna (podoe pynne dekstrozowo-ziemniaczane)

No antimicrobial activity / Brak aktywnoci przeciwbakteryjnej i przeciwgrzybiczej
Bc: Bacillus cereus (MTCC 430); Sa: Staphylococcus aureus (MTCC 740); Ec: Escherichia coli (MTCC 116); St: Salmonella typhi (MTCC 733); Ca: Candida albicans (MTCC 227)
Size of the agar wells: 8 mm; * morphological identity. Values are represented as MeanSD of 3 replicates. Means having different superscripts (a, b, c) differ significantly between
bacteria/yeast
Wielko dokw agarowych: 8 mm; * identyfikacja morfologiczna. Wartoci s reprezentowane jako redniaSD z 3 powtrze. rednie majce rnicy si grny indeks (a, b, c) rni si znaczco
midzy bakteriami/grzybami
Mikologia Lekarska 2012, 19 (1)
8
Bhagobaty R.K., Joshi S.R.
Antimicrobial and antioxidant activity of endophytic fungi isolated from ethnomedicinal plants of the Sacred forests of Meghalaya, India
32.6 mm. All the endophytic fungal culture broths in the study
showed antimicrobial activity against the pathogenic yeast strain,
with the highest being shown by Penicillium sp. isolated from
S. khasiana with an inhibition zone size of 23.3 mm (tab. I).
Antimicrobial activity of ethyl acetate extracts of the endophytic
fungal culture broths
All the ethyl acetate fractions of the crude endophytic fungal
metabolites showed antimicrobial activity against all the test
pathogens (tab. II). The ethyl acetate extract of the Penicillium sp.
isolated from C. caduca showed the highest antimicrobial activity
against B. cereus (21.3 mm). The highest antimicrobial activity for
S. aureus was exhibited by extract of P. variabilis isolated from
O. chinensis with an inhibition zone size of 29.7 mm (tab. II). The
highest antimicrobial activity for E. coli and S. typhi, the 2 gram-ne-
gative pathogenic bacteria used in the present study, was exhibi-
ted by the ethyl acetate extract of the Penicillium sp. (13.8 mm)
isolated from C. caduca and in combination by the same isolate
and M. hyalina isolated from O. stellata, respectively (32.5 mm)
(tab. II). Extracts of P. variabilis showed the highest microbicidal
activity against the pathogenic yeast strain under study with
a zone of inhibition of 17 mm (tab. II).
Antimicrobial susceptibility profile of the test pathogens
The susceptibility pattern of the test pathogens was ascerta-
ined for 9 antibiotics, namely, ampicillin, chloramphenicol, cipro-
floxacin, erythromycin, gentamicin, methicillin, streptomycin,
tetracycline, and kanamycin (tab. III). All the test pathogens were
resistant to ampicillin and methicillin. On the other hand, all the
pathogens showed sensitivity to ciprofloxacin and intermediate
sensitivity to kanamycin. B. cereus and S. typhi showed intermedia-
te sensitivity to chloramphenicol, whereas E. coli and C. albicans
were sensitive to it. S. aureus was resistant to chloramphenicol
(tab. III). All the test microorganisms showed resistance to eryth-
romycin, with the lone exception of E. coli, that was sensitive to it.
Both gram-positive bacterial test pathogens, i.e., B. cereus and
S. aureus were resistant to gentamicin whereas the gram-negative
group, i.e., E. coli, was sensitive and S. typhi showed intermediate
inhibition with gentamicin. Out of all the test pathogens, S. aureus
and E. coli were sensitive to streptomycin, whereas all others showed
intermediate susceptibility. S. typhi and C. albicans were resistant to
tetracycline, while B. cereus, S. aureus, and E. coli showed interme-
diate susceptibility (tab. III).
MICs of the crude ethyl acetate extracts
The MICs of the crude ethyl acetate extracts ranged from 13 to
45 g/ml (tab. IV). Against B. cereus the extracts from M. hyalina iso-
lated from O. stellata showed the least MIC, i.e., 27 g/ml. It also sho-
wed a low MIC value for the other gram-positive bacterial patho-
gen, S. aureus. However, the best result was given by the endophy-
tic fungi derived from O. chinensis with a MIC value of 22.3 g/ml.
The ethyl acetate extracts of the metabolites from M. hyalina also
showed the lowest MIC values for E. coli, S. typhi, and the yeast
pathogen C. albicans with values of 34.67, 13.33, and 28.33 g/ml,
respectively (tab. IV). The ethyl acetate fractions obtained from
M. hyalina were found to be the most potent in terms of the MIC
values amongst all the endophytic fungal isolates (tab. IV).
Antioxidant assays
The antioxidant potential of the fermentation broths of the
endophytic fungal isolates was tested using the FRAP and DPPH
assays (tab. V). In both the assays, M. hyalina isolated from the
plant O. stellata showed a high antioxidant activity with a FRAP
value of 1.316 and percentage free radical scavenging activity of
its fermentation broth at 79.7% (tab. V). However, interestingly
enough, a FRAP value for the Penicillium sp. isolated from S. Kha-
siana was 1.417, which was the highest amongst the fungal isola-
tes tested (tab. V). The DPPH percentage free radical scavenging
activity of the same fungi was measured to be 65.6%, which was
the lowest in comparison to the free radical scavenging activity
shown by the other endophytic fungal species (tab. V).
Discussion
The inhibition zone size ranged from 10 to 32 mm among all
the fungal metabolites assayed (tab. I), which is comparable to
Table II: Antimicrobial activity of the ethyl acetate extracts of the endophytic fungal metabolites
Tabela II: Aktywno przeciwbakteryjna i przeciwgrzybicza ekstraktw acetyloetylowych metabolitw grzybw endofitycznych
Host plant
Rolina gospodarz
Endophytic fungi *
Grzyby endofityczne
Zone of inhibition [mm] of ethyl acetate extract of the fungal culture broth
Strefa inhibicji [mm] ekstraktw acetyloetylowych podoa pynnego
Bacteria / Bakterie Yeast / Drode
Gram +ve Gram ve
Bc Sa Ec St Ca
Potentilla fulgens Talaromyces flavus 17.60a0.53 15.33b0.58 11.33c1.15 13.87d0.23 15.37b0.55
Osbeckia stellata Mortierella hyalina 17.33a0.58 17.33a0.58 13.70b0.61 32.57c2.50 14.93b0.12
Osbeckia chinensis Paecilomyces variabilis 13.80a0.35 29.70b1.57 12.33a0.58 21.00c1.00 17.07d1.10
Camellia caduca Penicillium sp. 21.30a0.52 16.67b0.58 13.83c0.29 32.57d2.50 13.70c0.61
Schima khasiana Penicillium sp. 19.27a1.62 14.00b0.00 13.53b0.81 25.07c0.90 13.00b1.00
Positive control (ethyl acetate) / Kontrola dodatnia (octan etylu) 8.67a0.58 8.67a0.58 8.67a0.58 8.67a0.58 9.00a1.00
Negative control (potato dextrose broth medium)
Kontrola ujemna (podoe pynne dekstrozowo-ziemniaczane)

No antimicrobial activity / Brak aktywnoci przeciwbakteryjnej i przeciwgrzybiczej
Bc: Bacillus cereus (MTCC 430); Sa: Staphylococcus aureus (MTCC 740); Ec: Escherichia coli (MTCC 116); St: Salmonella typhi (MTCC 733); Ca: Candida albicans (MTCC 227)
Size of the agar wells: 8 mm; * morphological identity. Values are represented as MeanSD of 3 replicates. Means having different superscripts (a, b, c) differ significantly between
bacteria/yeast
Wielko dokw agarowych: 8 mm; * identyfikacja morfologiczna. Wartoci s reprezentowane jako redniaSD z 3 powtrze. rednie majce rnicy si grny indeks (a, b, c) rni si znaczco
midzy bakteriami/grzybami
9
Bhagobaty R.K., Joshi S.R.
Przeciwbakteryjne i antyoksydacyjne dziaanie endophytic grzybw wyizolowanych z ethnomedicinal rolin lasy Sacred Meghalaya, Indie
Table III: Susceptibility profile of the test pathogenic microorganisms against known antimicrobial agents
Tabela III: Profil wraliwoci patogennych mikroorganizmw na znane czynniki antymikrobowe
Antibiotic / Antybiotyk Disk
content*
in mcg
Zawatrto
leku
w patku
[mcg]
Symbol Diameter of the zone of inhibition [mm]# / rednica strefy inhibicji [mm]#
Pathogenic bacteria
Bakterie patogenne
Pathogenic Yeast
Drode patogenne
Gram +ve Gram ve Ca
Bc Sa Ec St
Ampicillin / Ampicylina 10 A (R) 8.00a0.00 (R) (R) 8.20a0.20 (R) 10.33b0.58 (R)
Chloramphenicol / Chloramfenikol 30 C 13.60a0.53 (IM) 10.33b0.58 (R) 22.17c0.21 (S) 13.60a0.53 (IM) 18.60d0.53 (S)
Ciprofloxacin / Cyprofloksacyna 5 Cf 23.87a0.42 (S) 23.67a0.58 (S) 22.57a0.49 (S) 22.60a2.16 (S) 23.07a0.12 (S)
Erythromycin / Erytromycyna 15 E 7.47a0.50 (R) 7.47a0.50 (R) 40.17b1.56 (S) 7.47a0.50 (R) 10.33c0.58 (R)
Gentamicin / Gentamycyna 10 G 12.00a0.00 (R) 10.00b0.00 (R) 22.00c1.00 (S) 12.67a0.58 (IM) 19.47d0.50 (S)
Methicillin / Metycylina 5 M 8.67a0.58 (R) 8.67a0.58 (R) 8.67a0.58 (R) (R) (R)
Streptomycin / Streptomycyna 10 S 11.67a0.58 (IM) 17.37c0.64 (S) 15.13d1.01 (S) 12.67ab0.61 (IM) 13.00b0.00 (IM)
Tetracycline / Tetracyklina 30 T 20.53a0.76 (IM) 19.90a0.17 (IM) 19.90a0.17 (IM) 15.00b0.10 (R) 14.13c0.42 (R)
Kanamycin / Kanamycyna 30 K 15.00a0.10 (IM) 15.00a0.10 (IM) 16.00b0.00 (IM) 13.97c0.15 (IM) 13.93c0.12 (IM)
No antimicrobial activity; * Concentration of antimicrobial agent in the discs is as per the performance standards for antimicrobial disk susceptibility tests of Clinical and Laboratory
Standards Institute (CLSI), 2008.
# Inference of the susceptibility pattern based on the zone size interpretative chart supplied by Himedia Laboratories Pvt. Limited, Mumbai (India), along with the antibiotic discs.
R = Resistant; IM = Inter mediate sensitivity; S = Sensitive
Bc: Bacillus cereus (MTCC 430); Sa: Staphylococcus aureus (MTCC 740); Ec: Escherichia coli (MTCC 116); St: Salmonella typhi (MTCC 733); Ca: Candida albicans (MTCC 227). Antimicrobial
susceptibility test discs of Himedia Laboratories Pvt. Limited, Mumbai (India), were used for the tests and assay was carried out as per the standard procedures described by the
manufacturer. Values are represented as MeanSD of 3 replicates. Means having different superscripts (a, b, c) differ significantly between bacteria/yeast
Brak aktywnoci przeciwbakteryjnej i przeciwgrzybiczej; * Stenia czynnikw antymikrobowych s zgodne ze standardem okrelonym przez Clinical and Laboratory Standards Institute
(CLSI), 2008.
# Okrelono jako R = oporne IM = o poredniej wraliwoci; S = wraliwe zgodnie z instrukcj Himedia Laboratories Pvt. Limited, Mumbai (India)
Bc: Bacillus cereus (MTCC 430); Sa: Staphylococcus aureus (MTCC 740); Ec: Escherichia coli (MTCC 116); St: Salmonella typhi (MTCC 733); Ca: Candida albicans (MTCC 227). Wartoci s reprezentowane
jako redniaSD z 3 powtrze. rednie majce rnicy si grny indeks (a, b, c) rni si znaczco pomidzy bakteriami/grzybami
Table IV: Minimum inhibitory concentrations [g/ml] of the crude ethyl acetate extracts of the endophytic fungal metabolites
Tabela IV: Minimalne stenie hamujce [g/ml] ekstraktw acetyloetylowych metabolitw grzybw endofitycznych
Pathogens
Patogeny
MTCC accession no.
Nr szczepw
MICs of ethyl acetate extracts of endophytic fungal metabolites [g/ml]
MIC ekstraktw acetyloetylowych metabolitw grzybw endofitycznych [g/ml]
aC
Tf* Mh* Pv* Psp1* Psp2*
B. cereus MTCC 430 29.670.58 27.000.00 44.930.12 25.900.17 28.002.65 11.003.61
S. aureus MTCC 740 35.670.58 27.000.00 22.332.52 30.002.00 37.670.58 10.331.53
E. coli MTCC 116 44.930.12 34.670.42 48.971.00 37.670.58 37.670.58 6.002.00
S. typhi MTCC 733 39.330.58 13.332.89 30.330.58 18.000.00 22.332.52 8.671.15
C. albicans MTCC 227 36.801.06 28.332.89 37.670.58 41.331.15 44.334.04 6.001.73
a
C: Chloramphenicol used as positive control; * Morphological identity; Tf: Talaromyces flavus; Mh: Mortierella hyalina; Pv: Paecilomyces variabilis; Psp1: Penicillium sp. isolated from Camellia
caduca; Psp2: Penicillium sp. isolated from Schima khasiana.
a
Chloramfenikol jako kontrola dodatnia * identyfikacja morfologiczna; Tf: Talaromyces flavus; Mh: Mortierella hyalina; Pv: Paecilomyces variabilis; Psp1: Penicillium sp. izolowane z Camellia
caduca; Psp2: Penicillium sp. izolowane z Schima khasiana.
Table V: Antioxidant activity of the endophytic fungal metabolites
Tabela V: Antyoksydacyjna aktywno metabolitw grzybw endofitycznych
Host plant
Rolina gospodarz
Endophytic fungi *
Grzyby endofityczne
FRAP value M/L
FRAP warto M/L
% free radical scavenging activity
% aktywno wolnych rodnikw
Potentilla fulgens Talaromyces flavus 0.881a0.006 76.698a0.266
Osbeckia stellata Mortierella hyalina 1.316b0.015 79.762b0.251
Osbeckia chinensis Paecilomyces variabilis 0.314c0.003 74.074c0.064
Camellia caduca Penicillium sp. 1.025d0.025 71.090d1.012
Schima khasiana Penicillium sp. 1.417e0.001 65.652e0.565
Ascorbic acid control # 2.000f 64.000f
Values are represented as MeanSD of 3 replicates / Wartoci s reprezentowane jako redniaSD z 3 powtrze
# 1000 M solution / 1000 M roztwr
* Morphological identity / Identyfikacja morfologiczna
Mikologia Lekarska 2012, 19 (1)
10
Bhagobaty R.K., Joshi S.R.
Antimicrobial and antioxidant activity of endophytic fungi isolated from ethnomedicinal plants of the Sacred forests of Meghalaya, India
that previously reported in the case of endophytic fungi of Chine-
se medicinal plants [19]. The fermentation broth of endophytic
fungal species isolated from P. fulgens, O. stellata, and O. chinensis
did not show any antimicrobial activity against pathogenic gram-
-positive B. cereus, whereas both the endophytic isolates from
C. caduca and S. khasiana (both belonging to the plant genera
Theaceae) showed inhibition of B. cereus (16.93 and 11 mm,
respectively) (tab. I). Interestingly, these isolates did not show any
antimicrobial effect on S. aureus, the other gram-positive test
pathogen in the study. The maximum antimicrobial activity aga-
inst B. cereus was shown by the Penicillium sp. isolated from
C. caduca, whereas the highest antimicrobial activity against the
other gram-positive pathogenic S. aureus was shown by M. hyali-
na and P. variabilis at 13.50 mm (tab. I). In a similar study on the
endophytic fungi of Taxus mairei, Cephalataxus fortunei, and Tor-
reya grandis, from the Fujian province of China, it was reported
that among all the endophytic fungi isolated, the genus Paecilo-
myces showed the highest positive rate of antitumor and antifun-
gal activity [3]. The crude culture broth of endophytic fungi must
have potent antimicrobial activity as a whole or should possess
high concentrations of active principles that result in the exhibi-
tion of positive biological activities by the fungal strains [20]. It is
therefore likely that once the concentration of the active princi-
ples in an extract is enhanced, its antimicrobial potency also incre-
ases. This was shown to be the case for our isolates, since all the
ethyl acetate fractions of the crude endophytic fungal metaboli-
tes showed antimicrobial activity against all the test pathogens
(tab. II). Further, among the endophytic fungal isolates, ethyl ace-
tate extracts of the culture broth of the Penicillium sp. isolated
from C. caduca showed the highest antimicrobial activity against
a majority of the test pathogens, namely, B. cereus (21.3 mm),
E. coli (13.8 mm), and S. typhi (32.5 mm). The fungal genera Acre-
monium, Aspergillus, Fusarium, and Penicillium are regarded as
creative species based on their ability to produce several bioac-
tive metabolites [21]. The activity shown by the ethyl acetate
extracts of the two Penicillium species in the present study there-
fore indicates that these species may be very prolific producers of
antimicrobial compounds. However, the ability to produce anti-
microbials among isolates of the same species may vary, as the
Penicillium sp. isolated from C. caduca in the present study showed
an inhibition zone of 32.5 mm against S. typhi, while the other
Penicillium sp. isolated from S. khasiana produced an inhibition
zone of 25 mm against the same pathogen (tab. II).
Molecular characterization of the partial -tubulin gene reve-
aled the evolutionary position of the five isolates with fungal
sequences available in Genbank (NCBI) (fig. 1). Interestingly, the
endophytic fungal isolates showed homology to clinically signifi-
cant human pathogenic fungi namely Syncephalastrum racemo-
sum (gb AY944811.1) and Penicillium marneffei (ref XM002151381.1)
(fig. 1). However, it is to be noted that the endophytic fungal iso-
lates in the present study demonstrated cryptic morphological
characters and as such have been referred to in this paper only by
their isolate numbers and morphological identity [8].
The test pathogens used in this study were resistant to stan-
dard antibiotics such as ampicillin and methicillin. They also sho-
wed resistance to erythromycin, with the lone exception of E. coli,
that was sensitive to it (tab. III). Our results with the crude prepa-
rations of the endophytic fungal culture broths and their ethyl
acetate extracts indicate that these fungi have a comparatively
high antimicrobial activity against the tested human bacterial and
yeast pathogens. The MICs of the crude ethyl acetate extracts of
the endophytic fungal metabolites ranged from approximately 13
to 45 g/ml (tab. IV). Wang et al. (2008) [22], while studying the
effect of 6 bioactive compounds of Penicillium sp. endophytic on
Hopea hainanensis against 3 human pathogenic fungi Candida
albicans, Trichophyton rubrum, and Aspergillus niger, reported that
the bioactive compounds 2-4 and 6 inhibited the growth of
C. albicans with MICs of 40.0, 20.0, 50.0, and15.0 g/ml, respecti-
vely, and the compound 6 showed growth inhibition against
A. niger with MICs of 40.0 g/ml. Similar MIC ranges were also
reported by Sette et al. (2006)[15], while screening for the bioac-
tive endophytic fungal extracts against Salmonella choleraesuis
(CBMAI 484), Staphylococcus aureus (CBMAI 485), Pseudomonas
aeruginosa (CBMAI 489), and against 4 different Escherichia coli
serotypes. The minimal inhibitory concentration (MIC) for the fun-
gus extracts varied from 0.025 to 1.0 mg/ml, thereby demonstra-
ting the antimicrobial potential of some of these fungi. Aligiannis
et al. (2001) [23] proposed a classification for plant materials
based on MIC results as: strong inhibitors: MIC up to 0.5 mg/ml;
moderate inhibitors: MIC between 0.6 and 1.5 mg/ml; and weak
inhibitors: MIC above 1.6 mg/ml. However, there is no agreement
on what MIC values are acceptable for extracts when compared
with standards; therefore, some authors consider only the activi-
ty comparable to antibiotics, while others consider even higher
values [16].
The FRAP value of the Penicillium sp. isolated from S. khasiana
was 1.417, which is the highest amongst the fungal species tested
(tab. V). The DPPH or percentage free radical scavenging activity
of the same fungi was measured to be 65.6%, which was the
lowest in comparison to the free radical scavenging activity
shown by the other endophytic fungal species. This can be possi-
bly explained by the fact that the FRAP assay primarily measures
only the hydrophilic antioxidants present in a sample, while the
DPPH assay is more sensitive to antioxidants that are soluble in
organic solvents, especially alcohols [24]. Although both DPPH
and FRAP are electron transfer (ET) based assays, both of them
employ different chromogenic redox reagents with different stan-
dard potentials. The DPPH assay, acting by radical reduction, uses
preformed radicals and determines the decrease in absorbance,
while the FRAP assay measures the formed ferrous ions by incre-
ased absorbance. The total antioxidant power as determined by
the FRAP assay as an integrated parameter of antioxidants pre-
sent in a complex sample is often more meaningful to evaluate
health beneficial effects because of the cooperative action of
antioxidants [25].
Our results with both the antioxidant assays clearly indicate
that the endophytic fungal isolates obtained from the traditional-
ly used ethnomedicinal plants of Meghalaya possess different
groups of antioxidants that are comparable or even better in
terms of their potency with standard antioxidants and may be
useful as microbial cell factories for the production of antioxidants
in the near future. In a previous study, we have reported the DNA
damage protective activity of the crude fermentation broths of
endophytic fungi isolated from P. fulgens and O. stellata [26].
Although records of antimicrobial activity of endophytes isolated
from different gymnosperms of North East India are available [27,
28], to the best of our knowledge, this is the first report of the anti-
microbial potency of endophytic fungi isolated from the ethno-
medicinal plants of the sacred groves of Meghalaya, India. The
results of the present study further strengthen our view that the
11
Bhagobaty R.K., Joshi S.R.
Przeciwbakteryjne i antyoksydacyjne dziaanie endophytic grzybw wyizolowanych z ethnomedicinal rolin lasy Sacred Meghalaya, Indie
endophytic fungi residing in the medicinal plants of one of the
highest rainfall receiving areas in the world, i.e., Meghalaya (India),
are potential treasure troves of novel antimicrobial molecules and
antioxidants.
Conflict of Interest: None
Acknowledgments: The authors are thankful to N. K. Bhagobaty, IASST,
Guwahati, for the help provided with the statistical analysis of the results. The
grant received from UGC-UPE Biosciences Programme of our university for
meeting laboratory requirements for the present study is also acknowledged.
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