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Antimicrobial Activity Of Endophytic Fungi Isolated From Ocimum Sanctum Linn Desale Monali G.1 * and Bodhankar M. G.2 1
Research Fellow, Dept. of Microbiology, Bharati Vidyapeeth University Yashwantrao Mohite College, Pune- 411038 (India) 2
Dean, Faculty of Science, Bharati Vidyapeeth University, Yashwantrao Mohite College, Pune- 411038 (India) Contact Details for Corresponding Author :- Telephone : +917202584353 Mobile :+917588182021 Email –
[email protected]
ABSTRACT: The attention of scientific community has been drawn to the Endophytes due to their capacity to produce novel bioactive compounds. In the present study, the bioprospecting of fungal endophytes from Ocimum sanctum L. was studied and were screened for their antimicrobial potential. Use of different parts of the plant led to isolation of eighteen endophytic fungi. Significant antimicrobial potential was shown by Nigrospora oryzae isolated from the leaves of the plant under study. Antimicrobial potential was studied for the crude extracts of Nigrospora oryzae isolate with Hexane, Ethyl acetate and Methanol. The extract by ethyl acetate showed significant antimicrobial activity against E. coli, K. pneumonia, S. aureus. typhimurium,
S.
B. cereus and B. subtilis. The highest antimicrobial activity was shown against E. coli
followed by K. pneumoniae and S. aureus. The study helped to justify the traditional use of Ocimum sanctum L. against human pathogenic bacteria. It also confirmed that the antimicrobial activity is attributable to the presence of endophytic fungi in the plant. It also leads to the justification that the studies on isolation and identification of the bioactive compounds can be an important and crucial approach to search for novel natural products. Keywords: Endophyte, Nigrospora oryzae, Ocimum sanctum, antimicrobial activity, bioactive compounds.
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Every part of Ocimum sanctum L. has medicinal value for
INTRODUCTION The search for new and effective antimicrobial agents has become a necessity due to rise in the number of super resistant strains and failure of currently used antibiotics against them leading to the increased global health concern. Though the extracts of various medicinal plants offer an alternative, their indiscriminate exploitation for extraction of antimicrobial agents and limitations of plant resources due to various factors like requirement of large tracts of land
for
cultivation,
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environmental
competence
of
medicinal plants, seasonal specificity etc. are barriers in their large scale use. This has led to the worldwide interest in searching novel bioactive compounds having high effectiveness, negligible environmental impacts and low toxicity. Microbes are one of the
abundant sources of
novel chemo-types and pharmacophores from hundreds of years. The capacity of endophytes to produce novel bioactive compounds has drawn attention of the scientific community1,2,3,4. Endophytes are the microorganisms which are present in living tissues of various plants, establishing
various diseases. Leaves of this plant are useful in chronic fever, dysentery, and haemorrhage. It is widely used in Indian herbal medicines. It is very popular in the treatment of chronic bronchitis and cold and possesses antimicrobial and hepatoprotective activity16,17. Despite the therapeutic potential of medicinal plants from ancient time and that of Ocimum sanctum L. in particular, a systematic, scientific and rational approach to the traditional medical practice with modern system of medicine is not in vogue. Considering this lacuna, the identification of endophytic fungi has potential to establish a scientific basis for the traditional therapeutic uses of Ocimum sanctum. Hence, it is very important to search the antibacterial compounds of indigenous endophytic fungi from Ocimum sanctum L. which is intended to be carried out with a view of human health care and drug discovery.
MATERIALS AND METHODS Isolation and Identification of Endophytic Fungus
mutual relationship without apparently any symptoms of diseases5,6 Many endophytes produce bioactive metabolites
The sterilization protocol of Petrini et al.(1986)18 was
with important applications. Endophytes have proved to be
followed for fungal isolation with minor modifications.
the promising sources of biologically active products which
Various parts of Ocimum sanctum viz. leaf, stem and roots
are of interest for specific health care applications. 7,8,9
were first rinsed, immersed in 75% ethanol followed by
Endophytic fungal strains are also found to be potentially
NaOCl (1 to 13% depending upon type of tissue) and then
useful in the production of pigments, bioactive metabolites,
with 75% ethanol. All the segments were then washed
immuno-suppressants, anticancer compounds and bio-
three to four times with sterile distilled water and allowed
control agents.10,11,12
to surface-dry on sterilized filter paper. The efficiency of surface sterilization procedure was checked for each
Therefore, study of various traditionally used medicinal
segment of tissue following the imprint method of Schulz
plants for their ability to host endophytic fungi having
et al,(1993)19. All the segments of each plant part were
antimicrobial potential have been undertaken world-wide.
placed
However, since this is a
recent development, the
supplemented with chloramphenicol (50 µg/mL) to inhibit
endophytes though relatively less studied as compared to
bacterial growth. The hypal tips were then transferred to
their other counterparts, are potential sources for novel
fresh PDA for obtaining pure cultures.
on
potato
dextrose
agar
(PDA)
medium
natural products. Ocimum sanctum L. has been used by the practitioners of traditional systems of medicine for curing
For the morphological identification, sporulating structures
various ailments and conditions due to its substantial
of endophytic fungi were considered as diagnostic features.
therapeutic potential and
Among several endophytic isolates, Nigrospora oryzae
widespread occurrence in
India.13,14,15
showed significant antimicrobial activity against all six bacterial pathogens and hence was selected for further O.R.A.| 18 | A M E R I C A N I J
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study. The selected fungus was induced to sporulate by
and inoculated with standardized inoculum. About 6 mm
inoculating in potato dextrose agar medium and identified
diameter of wells were prepared and filled with 100 μl of
morphologically as well as by ITS rDNA sequence
endophytic
analysis.
Chloramphenicol and equal quantity of DMSO was used
fungal
extract.
Standard
antibiotic
like
as a positive and negative control respectively. All the Isolation of Secondary Metabolites
experiments were carried out in triplicates. Results were
Isolation of secondary metabolites from liquid media was carried out by the method described by Choudhary et al. (2004)20. Filtration technique was used to separate mycelia
recorded as zone of inhibition in mm. Process
Optimization
for
Production
of
Active
Metabolites
of the endophytic fungi. Organic solvents like hexane, ethyl acetate and methanol were used for the isolation of
For this process, different parameters like temperature, pH
secondary metabolites. The mycelia were soaked in
and incubation period were optimized with respect to
methanol for 7 to 8 days and the methanolic extract was
fungal biomass and crude metabolite production in shake
collected.
The culture filtrate was then extracted three
culture condition. The mycelial mats were filtered and
times with equal volume of Hexane and Ethyl acetate. Each
dried at 500C until constant weight was obtained. The final
solvent was subjected to liquid - liquid extraction for 3 to 4
fungal biomass was recorded in mg/100mL. The crude
0
times. After evaporation of the organic extracts at 50 C
metabolite production was studied at three different
under reduced pressure, solid residues were obtained. It
temperatures (250C, 270C and 300C), pH (3,5 and 7) and
was then used for the evaluation of antimicrobial activity
two incubation periods (15 and 21 day).
by dissolving it into DMSO. Isolation of genomic DNA, PCR amplification and Test Microorganisms
sequencing
Antibacterial activity of crude metabolite isolated from the
Fungal genomic DNA was isolated using gene O-spin
selected
against
Microbial DNA isolation kit (GeneOmbio technologies,
pathogenic bacteria using agar well diffusion method. Six
Pune, India). The ITS1, ITS2 and inverting 5.8S coding
bacteria viz. Gram positive B. subtilis (NCIM No. 2063), S.
rDNA were amplified using Universal ITS rDNA typing
aureus (NCIM No. 2079 ) , B. cereus (NCIM No. 2155)
primers ITS1 and ITS4 in standard PCR reaction. After
and Gram negative E. coli (NCIM No. 2345), K.
amplification, products were purified by using a gene O-
pneumoniae (NCIM No. 2706) and S. typhimurium (NCIM
spin PCR product purification kit and were directly
No.2501) were used for antimicrobial activity. 0.5
sequenced using an ABI PRISM Big Dye Terminator V3.1
McFarland standard suspension was used for this assay.
kit. (Applied
endophytic
fungus
was
screened
Bio system, USA). The sequences were
analysed using sequencing analysis 5.2 software. BLAST Antimicrobial Activity
analysis was performed at BlastN site at NCBI server
The modified agar well diffusion method was carried out for primary screening of fungal extract against human
(http://www.ncbi.nlm.nih.gov/BLAST). Result and Discussion
pathogenic bacteria. A loopful of bacterial culture was inoculated in 5 ml of Muller Hilton broth and incubated at 0
37 c for 24 hours.
Total of 150 segments from Ocimum sanctum L. were
After incubation, standard inoculum
processed to investigate the presence of endophytic fungi.
7
was prepared by inoculating 100 μl (10 CFU/ml) of fresh
A total of 18 endophytic fungi from all the segments of
bacterial culture into soft agar and mixed properly. Plates
different parts of Ocimum sanctum L. were isolated as
were prepared with 15 ml of Muller Hilton Agar medium
summarised in Table 1. O.R.A.| 19 | A M E R I C A N I J
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Table 1 : Data showing Endophytic Fungi Isolated from
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species showing antibacterial properties, the highest
different Parts of Ocimum sanctum L. Amongst all endophytic fungi, 4 exhibited prominent Site
of
Number of samples
Isolation
Number
of
Fungi Isolated
antibacterial activity, 6 exhibited moderate antibacterial activity while 8 did not exhibit any activity. Among the species showing antibacterial properties, the highest
Leaves
50
6
Stems
50
8
Roots
50
4
Total number
150
18
of isolates
inhibitory activity was exhibited by Nigrospora oryzae. The fungus was identified morphologically as well as by molecular analysis. The antimicrobial activity was highest with ethanol extract against E. coli (20mm) followed by K. pneumoniae (18mm) and S. aureus (12mm). The methanol and hexane extracts showed moderate activity against all the bacterial pathogens (Table no 2).
Amongst all endophytic fungi, 4 exhibited prominent antibacterial activity, 6 exhibited moderate antibacterial
The growth of fungal isolate was studied on potato dextrose
activity while 8 did not exhibit any activity. Among the
broth aerobically at stationary phase by optimizing various parameters like temperature, pH and incubation time with respect to fungal biomass and crude metabolite production. Maximum growth was recorded at 250C at pH 5 on 21st day
of
incubation
(
Table
no.
4,
5
&
6
)
Table 2 : Antibacterial Activity Of Various Extracts Of Nigrospora oryzae.
Fig.1 Colony morphology of Nigrospora oryzae
E. coli Fig.2 Conidiospore of Nigrospora oryzae. O.R.A.| 20 | A M E R I C A N I J Volume 1 2014 Issue 3 AUG-SEP AIJCSR
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Fig. 5:- Effect of pH and Incubation time on the growth and production of crude metabolite at 270 C in PD broth
K. pneumonia
Fig. 6: Effect of pH and Incubation time on the growth and production of crude metabolite at 300 C in PD broth Conclusions S. aureus
The medicinal plant Ocimum sanctum harbours diverse
Figure 3. Antimicrobial activity of Nigrospora oryzae
species of endophytic fungi21. The stem of Ocimum
against selected bacterial pathogens
sanctum L. harbours the largest number of endophytic fungi as compared to roots and leaves. Nigrospora oryzae. identified morphologically as well as by molecular analysis, exhibited the most significant inhibitory activity against all the test human pathogens. Further studies on isolation of these antimicrobial compounds from selected endophytic fungi and identification of bioactive compounds can be a crucial approach to search of novel and innovative source of natural products. ACKNOWLEDGEMENT
Fig. 4: Effect of pH and Incubation time on the growth and production of crude metabolite at 250 C in PD broth
The authors are thankful to Agharkar Research Institute, Pune and GeneOmbio Technologies Pvt. Ltd. Pune for services related to identification of fungi. O.R.A.| 21 | A M E R I C A N I J
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