INTRODUCTION

Infectious diseases rank next to cardiovascular diseases (CVDs) as the leading cause of death worldwide. Emergence of new and resistant fungal pathogens, increasing incidence in immunocompromised patients and environmental and health problems of chemical fungicides dictate the search for novel antifungal agents for human diseases and plant protection (Sharma and Kumar, 2009; Guo et al., 2008; Augustine et al., 2005). Candida sp. cause 6.2% of human infections, ranking as the 4th most prevalent infectious agent (Cowen et al., 2009).

The search for new antifungal agents (AFAs) has been rather slow (Gupte et al., 2002). This is because molds and fungi being eukaryotic, it is difficult to find selective agents targeting fungal metabolism without significant toxicity to humans (Georgopapadakou and Walsh, 1994) and also, till recently, the antifungal market was considered too small to warrant development of new AFAs (Georgopapadakou and Walsh, 1996). Currently used AFAs are either highly toxic (amphotericin B) or pathogens have acquired resistance (azoles). Hence, there is an urgent demand for new antifungal agents and drugs.

Novel lead compounds usually come from natural sources especially plants and microorganisms (Bevan et al., 1995; Cragg et al., 1997; Nolte et al., 1997; Shadomy, 1987). Actinomycetes are the most prolific producer of antibiotics, accounting for about 80% of known antibiotics (Berdy, 2005) As terrestrial sources are getting exhausted, survey is increasingly directed towards underexplored habitats, niche ecosystems, or extreme biotopes supplemented by novel selective media and/or pretreatment methods for isolation of rare actinomycetes or novel strains of Streptomyces. Manipur holds great promise for such bioprospecting studies as it is largely unexplored for actinomycetes though it is rich in biodiversity, being part of Indo-Burma Hotspot (Myers et al., 2000; Ningthoujam et al., 2009). In our continuing search for bioactive actinomycetes from various biotopes in Manipur, we now have a substantial collection of 172 lake, 75 forest, 50 river, 35 cave, 25 endophytic and 10 salt spring isolates.

The present study reports the results of screening randomly selected 35 isolates from our collection for anticandidal activity, identification of the bioactive strains and initial data on fermentation conditions for antifungal production.

MATERIALS AND METHODS

Isolation
Soil and sediment samples were obtained from various niche habitats in Manipur such as Loktak lake, Bishnupur district and Nambul river, Imphal West district, Shirui jungle, Shirui hills and other forests in Ukhrul district, Salt spring at Shikhong and other sites, Thoubal district, Kangkhui cave, Ukhrul district, Manipur, India. Most of these samplings were done in the period Jan-Oct, 2008. Short-term preservation, pretreatment of the samples and subsequent isolation and preservation of actinomycetes were performed at MBRL, Department of Biochemistry, Manipur University Canchipur, India.

Actinomycetes were isolated from soil and sediment samples collected from various biotopes in Manipur by dilution plating on Starch Casein Nitrate Agar (SCNA) (Kuster and Williams, 1964) and Chitin Agar (Hsu and Lockwood, 1975). After incubation at 28°C for up to 3 weeks, actinomycete colonies were purified by subculturing. Pure colonies were preserved in Bennett’s agar slants at 4°C.

Test Pathogens
Candida albicans (MTCC 227), Curvularia oryzae (MTCC 2605), Fusarium oxysporum (MTCC 287) and Pyricularia oryzae (MTCC 1477) were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. Bipolaris oryzae was procured from Life Science Department, Manipur University, Imphal, India.

Screening
Primary screening was done by cross-streak method (Madigan et al., 1997) and secondary screening by Kirby-Bauer method (Bauer et al. 1966). For primary screening actinomycete isolates are grown on SCNA and indicator fungi are grown on Sabouraud Dextrose Agar (SDA). Actinomycetes were streaked in the middle of the plate and allowed to grow for about 3 days and then test fungi are streaked perpendicular to the actinomycetes and further incubated for 48-72 h at 30°C and inhibition profiles were then measured.

For secondary screening, actinomycete strains were grown in SCN broth and inoculated in wells (6 mm diameter) punched in SDA plates containing lawns of test fungi and inhibition zones were measured after 48-72 h of incubation at 30°C.

Antagonism against phytopathogenic fungi was assayed by dual culture (Yuan and Crawford, 1995). Percent inhibition was measured by the formula:

where, A is diameter of fungal growth in control plate and B is diameter of fungal growth in experimental plate.

Characterization of Bioactive Strains
Phenotypic characterization of bioactive strain was performed as per ISP protocols and standard procedures (Shirling and Gottlieb, 1966; Holt et al., 1994). Genotypic characterization was done according to standard procedures with the help of CCMB, Hyderabad, India.

Fermentation
Several standard microbiological media such as Nutrient Broth (NB), Streptomyces Broth (SB), Tryptic Soy Broth (TSB), SCN broth and LB broth were tested to find out the most suitable medium for shake flask fermentation of the bioactive strains and inhibition zones tested at different times of incubation by Kirby-Bauer method (Bauer et al., 1966).

RESULTS AND DISCUSSION

Isolation
Several actinomycete isolates were obtained from various biotopes in Manipur by selective isolation (data not shown).

Screening
Out of 35 randomly chosen isolates, 2 strains obtained from Nambul river in Imphal, Manipur, India, NRP1-14 (Fig. 1) and NRP1-26 were found to be antagonistic against C. albicans in cross-streak test. This was also confirmed by disc diffusion assay (Kirby-Bauer method) after shake-flask fermentation in SCN broth. Based on relative potency (Fig. 2), NRP1-14 was selected for further studies (Table 1).

Characterization of Bioactive Strain
NRP1-14 was subjected to phenotypic characterization (Table 2, 3). Based on the morphological, physiological and biochemical characteristics and genotypic and phylogenetic analysis with the help of Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India, the strain was identified as Streptomyces mutabilis NRP1-14 (Fig. 3) based on analysis of the phylogenetic tree with related strains obtained from BLAST search, using neighbor joining method.

Fig. 1:	NRP1-14

Fig. 2:	Anticandidal activity of NRP1-14

Table 1:	Time course of bioactivity of Streptomyces mutabilis NRP1-14
#Results are averages of 3 replicates

Fermentation
Among various media tested for bioactive metabolite production by NRP1-14 in submerged fermentation, SCN broth (pH 7.2, 28⁰C, 150 rpm) was found to be the best.

Table 2:	Major phenotypic characteristics of Streptomyces mutabilis NRP1-14
+: Positive, w: Weak positive, -: Negative

Table 3:	Growth morphology of NRP1-14 on different media
SA: Streptomyces agar, ISP: International streptomyces project

Fig. 3:	Phylogenetic tree of NRP1-14

Table 4:	Biocontrol activities of Streptomyces mutabilis NRP1-14
bResults are averages of 3 replicates

Biocontrol Activities of NRP1-14
Streptomyces mutabilis NRP1-14, interestingly, also shows promising antagonistic activities against several important fungal pathogens of rice (Table 4) besides inhibiting Candida albicans.

The strain Streptomyces mutabilis NRP1-14 has been shown to exhibit promising bioactivities against C. albicans and several plant pathogenic fungi. It was isolated from a river soil sample. Cross (1981) reported that freshwater habitats are fruitful for isolation of antibiotic-producing actinomycetes. Manipur abounds in lakes, streams, rivers and other wetlands and freshwater habitats. Reports on freshwater actinomycetes are however, scanty compared to that of marine actinomycetes except for some publications on lake actinomycetes (Terkina et al., 2006).

Recent reports on antifungal Streptomyces species are predominantly of biocontrol strains for agricultural use as compared to anticandidal strains for potential medical applications (Han et al., 2008). To our knowledge, this is the first report of a Streptomyces mutabilis strain antagonistic to C. albicans. The few reports on bioactive strains of Streptomyces mutabilis are about antibacterial or anticoccidal activities (Fehr et al., 1977).

CONCLUSION

Streptomyces mutabilis NRP1-14 reported in this study shows significant bioactivities against both Candida albicans and several plant pathogenic fungi. Further studies are needed to explore its potential for possible medical and agricultural applications. Characterization of the bioactive metabolite (s) of this strain and optimization of its production must now be done. This will be the focus of our further investigations.

ACKNOWLEDGMENT

The authors wish to gratefully acknowledge the kind help of Dr S. Shivaji, Scientist, CCMB, Hyderabad, for help in 16S rDNA sequencing of NRP1-14 and Mr. S. Nimaichand, Ph. D student, MBRL, Department of Biochemistry, Manipur University, Imphal for help in phylogenetic analysis.