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 diarrhea, 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. DNA-based techniques have increasingly become the tool of choice for understanding the genetic diversity and phylogeny of F. oxysporum [9, 10]. Of particular importance is the rDNA internal transcribed spacer (ITS) region sequences which are largely used for identification and phylogenetic analysis of fungi [11]. However, there is meager information on phylogenetic study of F. oxysporum those exist as saprotrophs, pathogens and endophytes. Therefore, in the present study an attempt was made for phylogenetic placement of an endophytic fungus identified as F. oxysporum considering other isolates those exist as endophytes, saprophytes and pathogens and to assess its antimicrobial potential against some clinically significant microorganisms.
MATERIALS AND METHODS
COLLECTION OF SAMPLES
Plant samples were collected randomly in February 2007 from Similipal Biosphere Reserve (SBR), Orissa of eastern India. Samples were taken from four individual healthy plants by cutting off the rhizome 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. The excess water was dried under laminar airflow chamber. Then, with a sterile scalpel, outer tissues were removed and the inner tissues of 0.5cm size were carefully dissected and placed on petri-plates containing different mycological media. The media were supplemented with streptomycin sulphate (100mg/L) to suppress bacterial growth. The plates were incubated at 25±2oC until fungal growth appeared. The plant segments were observed once a day for the growth of endophytic fungi. Hypal tips growing out the plated segments were immediately transferred into PDA slant and maintained at 4oC. All the isolates were evaluated for their antimicrobial activity. An endophytic fungus displaying good antimicrobial activity was selected for further study. A living culture of the isolate has been maintained in the Department of Botany, North Orissa University, with accession no. AC30.
METABOLITE EXTRACTION AND DETERMINATION OF ANTIMICROBIAL ACTIVITY
The fungus was cultivated on Potato dextrose broth (Himedia) by placing agar blocks of actively growing pure culture (3mm in diameter) in 250ml Erlenmeyer flask containing 100ml of the medium. The flask was incubated at 25±1oC for 3 weeks with periodical shaking at 150 rpm. After the incubation period, the fermentation broth of the fungus was filtered through sterile cheesecloth to remove the mycelia mats. Metabolite was extracted by solvent extraction procedure using ethyl acetate as organic solvent. Equal volume of the filtrate and ethyl acetate was taken in a separating funnel and shaken vigorously for 10 min. The solution was then allowed to stand, where the cell mass got separated and the solvent so obtained was collected. Ethyl acetate was evaporated and the resultant compound was dried in vacuum evaporator using MgSO4 to yield the crude metabolite. The crude extract was then dissolved in Dimethyl sulphoxide (DMSO) for antimicrobial bioassay. Antimicrobial activity was determined by agar well diffusion assay [12] against eight common human pathogens that include three Gram positive bacteria i.e. Staphylococcus epidermidis, Bacillus subtilis, Staphylococcus aureus, three Gram negative bacteria i.e. Klebsiella pneumoniae, Shigella flexneri, Escherichia coli and two fungal pathogens i.e. Candida albicans and Candida tropicalis. All the test pathogens were obtained from Institute of Microbial Technology (IMTECH), Chandigarh, India. The metabolite was dissolved in dimethyl sulphoxide (DMSO) to give a concentration of 1mg ml-1. Each well was loaded with 100µl of the metabolite. The plates were incubated at 35±1oC for 24 h and the zone of inhibition in diameter (mm) was recorded. The experiment was replicated three times.
DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION (MIC)
Minimum inhibitory concentration was determined by broth micro-dilution assay technique in 96 wells micro-titer plates as described by Eloff [13] with slight modification. Overnight broth cultures of the each test organisms were seeded into the wells (90μl) and crude metabolite (10μL) was added in each well at decreasing concentration starting from 1000μg ml-1 to 75μg ml-1. Three wells were inoculated for a given concentration. The plates were incubated for 24 h at 35±1oC and Triphenyl tetrazdium chloride (TTC) was used as microbial growth indicator. MIC was determined as the least concentration of the crude metabolite that inhibited the growth of the test organisms.
ISOLATION OF GENOMIC DNA, PCR AMPLIFICATION AND SEQUENCING
The fungus was cultured on potato dextrose agar medium and small amount of the mycelia was suspended in 40µL MQ water. The suspended culture was added with 160µL of NaOH (0.05M) and mixed well. The samples were incubated on dry bath for 45 min at 60°C and vortex intermittently. Then 12µL of Tris-HCL (0.01M) was added and the mixture was diluted up to 100 fold. From the diluted extract 6µL was used for PCR. The PCR was set up using the following components: 2.5µL Buffer (10X), 1.5µL MgCl2 (25mM), 2.5µL dNTPs (2mM), 0.2µL Taq polymerase (5U), 1.0µL primer F (5µM), 1.0µL primer R (5µM) and 6.0µL DNA from diluted extract. The PCR condition was run in such a way, where initial denaturation was at 94°C for 3 min. Denaturation, annealing and elongation were done at 96°C for 10 sec, 55°C for 10 sec and 72°C for 30 sec respectively in 45 cycles. Final extension was done at 72°C for 10 min and hold at 4°C forever. After the PCR cycle, 2µL of the product was used to check on 1% agarose gel. It was then purified using quick spin column and buffers (washing buffer and elution buffer) according to the manufacturer's protocol (QIA quick gel extraction kit Cat No. 28706). DNA sequencing was performed using an Applied Biosystem 3130xl analyzer.
PHYLOGENETIC INVESTIGATION
Phylogenetic placement of the fungus and its possible evolutionary relationship with other F. oxysporum isolates those exist as endophytes, saprophytes and pathogens was carried out considering their 18S ribosomal RNA gene sequences using MEGA4.0 software [14].
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. The sequence data was then assembled and submitted at the NCBI Genbank with accession number GU056168. Based on BLAST search of ribosomal RNA gene sequence, the fungus was found to be closest homolog to Fusarium oxysporum. The crude metabolite of the fungus displayed considerable antimicrobial activity against some clinically significant pathogens (Table 1). The metabolite showed effective inhibition against shigella flexneri (31.3 mm) followed by Staphylococcus epidermidis (19.6 mm) and Escherichia coli (17.6 mm). Very less inhibitory zone was observed against Staphylococcus aureus (08.6 mm). Fungal pathogens i.e. Candida albicans (12 mm) and Candida tropicalis (11.3 mm) were moderately effective against the metabolite. The minimum inhibitory concentration (MIC) ranged from 76.6µg ml-1 to 376.6 µg ml-1 with lowest and highest value determined against Shigella flexneri and Staphylococcus aureus respectively (Table 1). F. oxysporum is a soil borne facultative parasite that exist both as pathogenic and saprophytic forms. Recently, it has also been isolated as endophyte from several plant species with diverse biological activity [15, 7]. In the present investigation the antimicrobial producing endophytic F. oxysporum was isolated from A. calamus rhizomes. A similar result with antibacterial activity was also reported from endophytic F. oxysporum isolated from Dioscorea zingiberensis rhizomes [8]. In many instances metabolites extracted from endophytic F. oxysporum has displayed only antibacterial activity [16, 8]. But effective inhibition of both Gram positive and Gram negative bacteria and moderate antifungal activity suggest the current metabolite to be broad spectrum in nature. Such results agree with some studies that fungal endophytes isolated from medicinal plants show antimicrobial activities and support the assumption that endophytic fungi are rich source of functional bioactive metabolites [17]. The colonization frequency of genus Fusarium was found to be very high in A. calamus rhizomes as encountered in our previous study [18]. Such results with high colonization of the genus Fusarium were also obtained from several plant species [8, 16, 19]. This suggests allopatric existence of endophytic Fusarium based on different geographical regions and ecological niches and their ubiquity as endophyte among plant species.
Occurrences of F. oxysporum in different forms suggest existence of genetic diversity within this species and molecular methods can be used to detect such variation [20]. Several researchers have reported genetic diversity within pathogenic and non pathogenic isolates of F. oxysporum and of endophytic isolates from various plants [21, 22]. Yet little is known about their phylogeny considering isolates those exist in different forms. In this study, an attempt was made to place an endophytic F. oxysporum isolated from A. calamus rhizomes with other isolates those exist as pathogens, saprophytes and endophytes considering their 18S ribosomal RNA gene sequences (Table 2). Phylogenetic tree was generated using maximum parsimony method. The generated tree showed four clades and one independent lineage which have descended from a saprophytic F. oxysporum ancestor (Figure 2). Clade I showed two coevolved saprophytic isolates which have radiated into two major clusters giving rise to all forms of F. oxysporum strains. Clade II displayed one endophytic and three saprophytic isolates with a common ancestral origin those diverged into two subclades. Clade III showed one endophytic isolate (the fungus under investigation) and two pathogenic strains. This clade depicted possible evolution of endophytic strain from pathogenic form and retaining its pathogenicity. The co-existence of pathogenic and endophytic strain is well supported by their bootstrap values which favor possible emergence of endophyte from pathogenic forms. Clade IV comprised of different F. oxysporum isolates which clustered into a major group. This clade also indicated co-existence of pathogenic and endophytic strains which have given rise to different forms of F. oxysporum. Based on the phylogenetic analysis we hypothesize that pathogenic and endophytic strains of F. oxysporum might have evolved from a saprophytic ancestor and pathogenic strains have given rise to endophytic forms. Such transformation could be supported from the fact that there is a great deal of genetic relatedness between pathogenic and non-pathogenic F. oxysporum isolates, which has led some researchers to conclude that particular pathogenic isolates might have evolved from non-pathogenic strains by mutations involving a few loci [23, 24]. Further, the emergence of endophytic forms from pathogenic strains may be attributable from the fact that some nonpathogenic isolates have been considered to evolve from pathogenic strains through loss of virulence due to induce mutation in their genome [24]. This has been demonstrated by some researchers where mutational changes in arginine biosynthesis gene have been observed to reduce pathogenicity in Fusarium oxysporum f.sp. melonis [25]. Moreover, studies have also indicated that endophytes become saprotrophs at host senescence [26]. Such findings led us to conclude transformation in F. oxysporum into various forms seems to be natural phenomenon which may be governed by different cultural and environmental conditions. This also supports the assumption that F. oxysporum is phylogenetically diverse and genetic variations within this species complex could arise based on different geographical locations and ecological habitats. In our present study, we have considered 18S rRNA gene to derive phylogenetic relationship of F. oxysporum isolates, which seems to have some merit because it is believed that this gene is more conserved evolutionarily [27]. However, consideration and analyses of other genes from different loci may provide more information on phylogenetic relationship of F. oxysporum isolates those exist as pathogens, saprophytes and endophytes. The result of this study has shown that fungal endophytes associated with ethnomedicinal plants could be an important source of therapeutic agents; giving a less chance of exploitation on these plant resources as microbial source for medicinally important substance could be found. It will also give an opportunity to discover novel metabolites from endophytic Fusarium spp. which is ubiquitous in plants and known to produce diverse bioactive substances. Moreover, the present study would provide information about phylogenetic relationship of F. oxysporum those exist in different forms which is still less studied.
REFERENCES
