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Phylogenetic Placement of an Endophytic Fungus Fusarium oxysporum
Isolated from Acorus calamus Rhizomes with Antimicrobial Activity
Bibhuti Prasad Barik1, *Kumananda Tayung2, Prema Narayan Jagadev3, Sushil Kumar Dutta4
1
Department of Bioinformatics, North Orissa University, Baripada-757003, Orissa, India
2
Department of Botany, North Orissa University, Baripada-757003, Orissa, India
3
Department of Bioinformatics, Orissa University of Agriculture and Technology,
Bhubaneswar- 751003, Orissa, India
4
Cell and Molecular Biology Laboratory, Department of Zoology, North Orissa University, Baripada, Orissa India
Email: kumanandbotnou@rediffmail.com
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ABSTRACT
An endophytic fungus was isolated from Acorus calamus rhizomes, which is an important medicinal plant. Colonial
morphological trait and microscopic observation revealed Fusarium sp. in taxonomy. Based on 18S rRNA gene
sequence the fungus was identified as Fusarium oxysporum. The crude metabolite of the fungus displayed
considerable antimicrobial activity against some clinically significant microorganisms. The metabolite was highly
effective against both Gram positive and Gram negative microorganisms and moderately effective against fungal
pathogens. The fungus was placed phylogenetically which revealed that it has evolved from saprophytic ancestor
and co-exists with a pathogenic strain. Phylogenetic tree was generated using maximum parsimony method to
establish relationship of the fungus with other F. oxysporum isolates those existing in different forms.
Key words: Endophytic fungus, antimicrobial activity, 18S rRNA gene, F. oxysporum, Phylogenetic investigation
INTRODUCTION
Increasing global health concern due to failure of currently used antibiotics to many super resistant strains have
necessitated the search for new and effective antimicrobial agents. Natural products from microorganisms have been
the primary source of antibiotics, but with the increasing acceptance of herbal medicine as an alternative form of
health care, screening of medicinal plants for active compounds has become very popular. In fact, many common
prescribed drugs, anticancer and antimicrobial agents that are in current use are of plant origin [1]. However,
indiscriminate exploitation of these plant resources have rapidly declined its population and threatened their
existence. It is now known that plants serve as a reservoir of some untold number of microbes known as endophytes
[2], which are defined as microbes that colonized inner healthy plant tissues without causing any disease symptoms.
Some of these endophytes have produced important bioactive metabolites for therapeutic applications. More
recently it has also been found that endophytes colonizing medicinal plants could produce same bioactive natural
product(s) or derivatives that are more bioactive than those of their respective hosts [3].
Acorus calamus Linn. (family Araceae) commonly known as “sweet flag” is a well known medicinal plant. The
rhizomes are considered to possess anti-spasmodic, carminative and anthelmintic properties and also used for
treatment of epilepsy, mental ailments, chronic diarohea, dysentery, bronchial catarrh, intermittent fevers and tumors
[4]. Study on fungal endophytes from A. calamus rhizomes results into isolation of an endophytic fungus identified
as Fusarium oxysporum with antimicrobial activity. F. oxysporum is a widely distributed soil inhabiting fungus,
which is known to be phylogenetically diverse. Most strains assigned to this species are saprotrophic. Some
nonpathogenic strains have been used as biological control agents [5], while others have veterinary and/or medical
significance [6]. Besides, several F. oxysporum isolated as endophytes from medicinal plants have been reported to
produce metabolites with anticancer and antimicrobial activity [7, 8]. The occurrence of F. oxysporum in different
forms is often confusing and methods are needed to distinguish such closely related species.
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MATERIALS AND METHODS
COLLECTION OF SAMPLES
Plant samples were collected randomly during February 2007 from Similipal Biosphere Reserve (SBR), Orissa of
eastern India. Samples were taken from four individual healthy plants by cutting off the rhizomes with an ethanol
disinfected sickle. The materials were placed in polybags and transported to the laboratory within 12h and stored at
4°C until isolation procedure was completed.
ISOLATION OF ENDOPHYTIC FUNGI
Each sample was washed thoroughly in running tap water and air dried before it was processed. The materials were
then surface sterilized by immersing them sequentially in 70% ethanol for 3min and 0.5% NaOCl for 1min and
rinsed thoroughly with sterile distilled water.
RESULTS AND DISCUSSION
The fungus was isolated from Acorus calamus rhizome, which is an important ethnomedicinal plant of Similipal
Biosphere Reserve, India. The colonial morphological trait of the isolate was white cottony mycelia, turning into
purple red. Microscopic observation of the fungus revealed production of both macro and microconidia. The
macroconidia were septate, dorsi-ventrally curve and sickle shaped (Figure 1). The above mentioned characteristics
revealed the fungus to be Fusarium sp. in taxonomy. Genotypic identification was carried out by considering the
18S rDNA sequence. The rDNA sequence of 18S ribosomal RNA gene was amplified and the PCR product was
bidirectionally sequenced using forward (ITS4) and reverse (ITS5) primers.
REFERENCES
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Cragg, G.M., D.J. Newman, and K.M. Snader. 1997. Natural products drug discovery and development. Journal
of Natural Product 60: 52–60.
Bacon C.W., and J.F. White. 2000. Microbial endophytes. Marcel Decker, Inc., New York.
Strobel, G.A, 2002. Microbial gifts from the rainforest. Canadian Journal of Phytopathology. 24:14-20.
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