Novel bioactive actinomycetes from a niche biotope, Loktak Lake, in ...

Suchitra Sanasam et al. / Journal of Pharmacy Research 2011,4(6),1707-1710

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

Novel bioactive actinomycetes from a niche biotope, Loktak Lake, in Manipur, India Suchitra Sanasam, Salam Nimaichand, Debananda Ningthoujam Microbial Biotechnology Research Laboratory (MBRL),Department of Biochemistry,Manipur University,Canchipur, Imphal 795 003,India

Received on: 11-02-2011; Revised on: 16-03-2011; Accepted on:21-04-2011 ABSTRACT Falling in the Indo-Burma biodiversity hotspot, Manipur is a promising area for microbial diversity studies. As a part of our ongoing research on actinomycete diversity of Manipur, sediments from various depths of the Loktak Lake – the largest freshwater lake in NE India - were sampled for isolation, bioactivity screening and characterization of the potent bioactive actinomycete isolates. We have obtained a total of 207 actinomycete isolates from the sediment samples of Loktak Lake. Based on the results of primary screening, 23 isolates were selected for secondary screening. Nine (9) strains showed significant antibacterial or broad spectrum antimicrobial (antibacterial and antifungal) activities. Phylogenetic analysis indicated that a majority of them were Streptomyces and nocardioform actinomycetes. Five lake isolates (LS1-81, LS1-88, LS1-117, LS1-128, and LS1-10C) were characterized as Streptomyces spp, 3 (LS1-145, LSCH-7, LSCH-31) belong to Pseudonocardia and 1 strain (LSCH-6B) was identified as Nocardiopsis sp. Of these, LS1-81 and LSCH-7 are possibly novel species of Streptomyces and Pseudonocardia respectively. These prospective novel actinomycete strains also exhibited significant antimicrobial activities. LS1-81 showed potent activities against Micrococcus luteus, Bacillus subtilis and Aspergillus niger and LSCH-7 against Staphylococcus aureus, Micrococcus aureus, Bacillus subtilis and Aspergillus niger. This study highlights the promise of discovering novel bioactive actinomycetes in underexplored niche biotopes such as lake sediments. Keywords: Microbial diversity, Actinomycete, Loktak Lake, Sediments, Streptomyces. INTRODUCTION Actinomycetes especially Streptomyces spp. are rich sources of bioactive natural products with potential applications as pharmaceuticals and agrochemicals (2,3,28). They are prolific producers of secondary metabolites such as antibiotics, herbicides, pesticides and anticancer agents (4, 39, 43). More than 6000 compounds are produced by Streptomyces spp. and many of them have commercial importance as anti-infectives (antibiotics, antiparasitic and antifungal agents), anticancer or immunosuppressant agents (50).

As exploration of actinomycetes from terrestrial sources are nearly exhausted, there is the imperative need to access various unexplored and underexplored habitats including lakes, pristine forests, cave soils etc. (51, 20, 59 60, 22, 17). There is also urgent need for new antimicrobials and antifungal agents, and reports of actinomycetes from freshwater habitats producing bioactive compounds warrant more studies on aquatic actinomycetes (38, 42). In our continuing study of bioactive actinomycetes in various habitats in Manipur, part of Indo-Burma Biodiversity Hotspot (29), we had isolated a large number of actinomycetes esp. Streptomyces spp. from lake sediments of Loktak Lake, the largest freshwater lake in Eastern India (35,46,31,32,33). The aim of the present investigation was to screen actinomycetes isolated from Loktak Lake for antagonistic activities against bacteria and fungi of medical and agricultural importance and characterize the bioactive strains. MATERIALS AND METHODS Media and Reagents Microbiological media were obtained from Himedia (Mumbai, India) or prepared in the Lab according to standard protocols. All other reagents and chemicals were of the highest grade available. Loktak Lake Loktak Lake is one of the largest freshwater lakes in Eastern India. It has been covered under Ramsar convention in 1990 (35). The lake’s ecology is in precarious state due to shallowing by siltation and pollution by incoming streams and rivers and from human activities and agricultural runoff. About 40% of the lake is covered by different types of weeds, both floating and submerged. It is composed of a continuous mass of floating phumdi (floating biomass of putrefying vegetation) occupying an area of 40 sq.km. Its condition worsened after launching of a hydroelectric project in the lake about 30 years ago. Sampling Sampling was done from the beds of the Loktak Lake. The sediment samples were collected from about 4-5 feet depth below the water surface and placed in

*Corresponding author. Debananda Ningthoujam Department of Biochemistry Manipur University Imphal 795 003,India Tel phone and fax number: 0385 2435 089(O); 09862 027 271(Mob); 0385 2435 145/813(Fax). E-mail:[email protected]

polyethylene bags, sealed tightly and stored in a refrigerator before processing. Pretreatment Sediment samples were air-dried at ambient temperature. The air-dried samples were crushed and sieved. The sieved samples were then treated with 10% CaCO3 for a week and then used for isolation of actinomycetes. Isolation of lake actinomycetes Actinomycetes were isolated using Starch Casein Nitrate Agar (SCNA, 23) and ColloidalChitinAgar(18)supplementedwith 50µg/ml each of nystatin and cycloheximide by plating of serially diluted samples (10 -2 – 10 -7 ). Putative actinomycete isolates were subcultured to get pure cultures and maintained on Bennett’s agar slants (21) at 280C and 40C and as spore suspensions in 20% (v/ v) glycerol at -200C (57). Antimicrobial assay Test organisms The test bacteria used were the Gram positive organisms Staphylococcus aureus (MTCC 96), Micrococcus luteus (MTCC 106), and Bacillus subtilis (MTCC 121), and the Gram negative bacteria Escherichia coli (MTCC 739) and Pseudomonas species (DN1) and the test fungi used were Candida albicans (MTCC 227) and Aspergillus Niger (MTCC 1344). All the reference strains were procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India except for DN1 (30) which is a strain isolated in our laboratory. Initial antimicrobial assay was carried out using the cross-streak technique (40, 56). Actinomycete isolates which showed inhibition of >50% against the test organisms in the primary screening were further subjected to secondary screening by Kirby Bauer method (5) against the above test organisms. The actinomycete strains showing good antimicrobial activities were subjected to phenotypic and genotypic characterization. Phenotypic and Genotypic characterization Phenotypic features refer to all the expressed characteristics such as morphological, physiological and biochemical properties. The various morphological, physiological and biochemical characterization tests were carried out using the standard procedures (47). The micromorphology (Carl Zeiss microscope, AxioScope A.1, Germany, magnification 600X) of the spore chains were recorded using 14 day old cultures grown on Streptomyces agar. The cultural properties of the strain were evaluated according to the guidelines of the International Streptomyces Project (ISP) as described by Shirling & Gottlieb (47). 16S rDNA amplification and sequencing were carried out as described by Kim et al. (22). The almost complete 16S rDNA sequence was submitted to EzTaxon server (11) which contains manually curated databases of type strains of prokaryotes for sequence analysis. Related strains were selected for alignment by CLUSTAL W program and phylogenetic analyses were done according to the neighbor-joining method (45) using the MEGA version 4.1 (52). To determine the support of each clade, bootstrap analysis was performed with 1000 replications (16).

Journal of Pharmacy Research Vol.4.Issue 6. June 2011

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Suchitra Sanasam et al. / Journal of Pharmacy Research 2011,4(6),1707-1710 Table 1 Secondary Screening profile of the selected isolates exhibiting good antimicrobial activity in primary screening Test isolates

MTCC 121

Gram negative bacteria MTCC 739 DN1*

Yeast/fungi MTCC 227

MTCC 1344

Standard antibiotic discs

Test organisms Gram positive bacteria MTCC 96 MTCC 106 Inhibition zone (in mm diameter) Erythromycin16 Penicillin-G18

Amikacin-18

Streptomycin-18

Rifampicin -12

Amphotericin-B16

Nystatin13

LS1-2 LS1-11 LS1-81 LS1-83 LS1-88 LS1-93 LS1-117 LS1-122 LS1-125 LS1-128 LS1-134 LS1-140 LS1-145 LS1-155 LS1-172 LSCH-2 LSCH-6B LSCH-7 LSCH-8 LSCH-10C LSCH-11 LSCH-23 LSCH-31

12± 0.75 21± 0.58 13±1.04 15±0.29 22±0.76 24±0.58 23±0.58

14± 0.75 17± 0.5 14± 0.5 22± 0.5 13 ± 1.0 15±0.75 14± 0.75 17± 0.58 19±0.76 11±0.5 14±0.1 15±0.76 18±0.5 17±1.0 13±0.58 18±0.5 13±0.58 15±0.29 18±1.0

12±0.29 13± 0.58 18 ± 1.0 15± 0.58 20± 0.5 15± 0.05 15±0.58 19±0.58 12±0.29 -

-

11± 0.58 11±1.15 -

16± 0.57 15± 1.0 16± 0.76 11±0.5 17±0.76 14±0.5 17±1.52 15±0.5

19± 0.58 15± 0.58 16± 0.5 13± 0.58 17±0.58 13±0.58 15±0.58 19±0.29 15±1.0 23±0.29 14±0.5 18±0.58

Table 2 Biochemical and physiological tests of the bioactive actinomycete isolates

series (LSCH-1 to 35).

Name of the test

LS 181

LS 1 88

LS1 117

LS1 128

LS1 145

LSCH 6B

LSCH 7

LSCH 10C

LSCH 31

Gram’s staining Production of diffusible pigment Growth at 40 C 150C 250C 300C 370C 420C 600C Growth at pH range 5.2 7.0 8.0 9.0 10. 0 Growth in the presence of 2% NaCl 5% NaCl 7% NaCl 10% NaCl Degradation of Adenine 0.5% Guanine 0.05% Tyrosine 0.5% Xanthine 0.4% Hydrolysis of Casein Starch Urea Biochemical tests Catalase activity Oxidase activity Methyl Red (MR) Voges Proskauer (VP) Citrate utilization Indole production Nitrate reduction Gelatin liquefaction H2S production

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ + + -

+ + + + + -

+ + + + + -

+ + + + + -

+ + + -

+ + + + -

+ + -

+ + + + + -

+ + -

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + +

+ + + +

+ + + -

+ + + -

+ + + +

+ + + -

+ + + -

+ + + w

+ + + -

+ -

+ + -

+ + -

+ -

+ + -

+ + +

-

+ + -

-

Antimicrobial assay Of 207 isolates subjected to primary screening, 23 (11.1%) showed an inhibition zone of more than 50% against one or more of the test pathogens. These isolates were then shortlisted for secondary screening (Table 1). Of 23 strains subjected to secondary screening, 9 (39.1%) isolates (LS1-81, LS1-88, LS1-117, LS1-128, LS1-145, LSCH6B, LSCH-7, LSCH-10C and LSCH-31) showed good antimicrobial activities with inhibition zones diameters of 17 mm or more against one or more of the test organisms. Among these bioactive isolates, 3 (LS1-88, LS1-117, LS1-145) were found to be purely antibacterial and 6 (LS1-81, LS1-128, LSCH-6B, LSCH-7, and LSCH-10C) had broad antimicrobial activities. Of these 6 isolates with broad spectrum activities, only LS1-128 was found to be antibacterial and anti-yeast, while the remaining 5 isolates were active against gram positive bacteria and fungi. Studies on screening of bioactive actinomycetes from freshwater habitats have been reported by some workers. Elliah et al. (14) studied bioactive actinomycetes from Krishna River sediments of Andhra Pradesh. Of 30 morphologically distinct actinomycete isolates screened by cross streak method, 16 (53.3%) had excellent antagonistic properties. From detailed submerged fermentation studies, it was found that 12 isolates (40.0%) exhibited antibacterial, 9 (30%) antifungal and 5 (16.6%) both antibacterial as well as antifungal activities. Singh et al. (41) isolated 37 actinomycetes from phoomdi in Loktak Lake in Manipur, India. Twentyone (21) isolates showed antimicrobial activities against test microorganisms in primary screening. Of these, 12 were found to have broad spectrum (antibacterial and antifungal) activities.

+ -

+ -

+ -

+ + +

+ + +

+ + +

+

-

+

+ + -

+ + + -

+ + + -

+ + + -

+ + -

-

+ + -

+ + + -

Phenotypic and genotypic characterization Phenotypic characteristics of the bioactive strains and their growth morphologies on different ISP and other actinomycete specific media are shown in Tables 2-3. The gross morphologies on SCNA media and their micromorphologies are shown in Figure 1. Phylogenetic analyses characterized these bioactive isolates as belonging to the genera Streptomyces, Nocardiopsis and Pseudonocardia. Five strains were identified as Streptomyces species. LS1-81 was most closely related to Streptomyces pactum (similarity index 98.718%), LS1-88 to Streptomyces sindenensis (similarity index 99.677%), LS1-117 to Streptomyces tritolerans (similarity index 99.464%), LS1-128 to Streptomyces sindenensis

Table 3 Colony morphology of the bioactive isolates

Table 4 Growth morphologies of the bioactive isolates on different ISP media and other actinomycete specific media

Isolates

Media

LS1-81 LS1-88 LS1-117 LS1-128 LS1-145 LSCH-6B LSCH-7 LSCH-10C LSCH-31

Colony Morphology Shape Margin

Elevation

Concentric Round Round Concentric Round Round Round Round Round

Raised Raised Raised Flat Convex Convex Convex Convex Raised

Undulate Smooth Smooth Ciliate Smooth Smooth Smooth Smooth Smooth

Isolate LS1-81

LS1-88

LS1-117

LS1-128

LS1-145

LSCH-6B

LSCH-7

LSCH-10C LSCH-31

ISP1

RESULTS Isolation of Lake Actinomycetes A total of 207 putative actinomycete isolates were isolated from the sediment samples of Loktak Lake. Of these 172 isolates were obtained from SCNA medium. The isolates obtained from SCNA were coded as LS1 series (LS1-1 to 172). 35 isolates obtained from the colloidal chitin agar were coded as LSCH

AM White Pale Cream Cream Cream Off white Sandal wood Sandal wood G r e y Sandal wood SM Cream White White Light Yellow Light Brown Light Brown Light Brown Cream Light Brown ISP2 AM White Cream Cream Cream Light Cream Cream Cream Off White Cream SM Light Brown Cream Off White Off White Brown Deep Brown Brown White Brown ISP3 AM Light Grey G r e y Off white Off white Sandal wood Sandal wood Sandal wood G r e y Sandal wood SM Brown Light yellow Yellow Cream Light brown Light Brown Brown Brown Brown ISP4 AM Cream Off White Off White Off White Light Cream Cream Cream Grey Cream SM Brown Cream Cream Yellow White White White Brown White ISP5 AM White Off White Off White White Sandal wood Sandal wood Sandal wood White Sandal wood SM Cream Fluorescent Fluorescent Cream Sandal wood Sandal wood Sandal wood Cream Sandal wood ISP6 AM Pale cream Pale cream Pale cream Pale Cream Cream Cream Cream Off White Cream SM Light Brown Light Brown Cream Off White Brown Brown Brown Grey Brown ISP7 AM Off White Cream Cream Pale Cream Off White Cream Cream Grey Off White SM Brown Off White Cream Off White Cream Cream Brown Grey Brown SCNA AM White Off White Off White Off White Cream Cream Pale Cream G r e y Cream SM White Yellow Light Yellow Red dish Light Cream Off White Cream Yellow Cream SA AM Light Grey White White Pale Cream Cream Cream Cream Off White Cream SM Brown Yellow Yellow White Light Brown Brown Brown White Brown TSA AM Pale cream Cream Pale Cream Sandal wood Pale Cream Off White Off White Pale cream Off White SM Cream Brown White Cream Brown Brown Brown Cream Brown N.B.à ISP- International Streptomyces Project, SA – Streptomyces Agar, TSA- Tryptone Soya Agar, AM- Aerial mycelium, SM- Substrate mycelium ,P.G. – Poor Growth


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Suchitra Sanasam et al. / Journal of Pharmacy Research 2011,4(6),1707-1710

T

95 Streptomyces olivaceus NBRC 12805 (AB249920) T

57

Streptomyces pactum NBRC 13433 (AB184398) LS1-81

50

LS1-117 57

T

72

Streptomyces vinaceusdrappus NRRL 2363 (AY999929)

89

LSCH-10C 89

T

Streptomyces enissocaesilis NRRL B-16365 (DQ026641) T

Streptomyces tritolerans DAS 165 (DQ345779) 100

80 Streptomyces tendae ATCC 19812T (D63873) T

Figure 1: Gross Morphological feature and micromorphologies of the bioactive actinomycetes (LS1-81, LS1-88, LS1-117, LS1-128, LSCH 145, LSCH-6B, LSCH-7, LSCH-10C and LSCH31)

Streptomyces badius NRRL B-2567 (AY999783) 100 Streptomyces parvus NBRC 3388T (AB184756) T

89

55

Streptomyces sindenensis NBRC 3399 (AB184759) LS1-88

(similarity index 99.927%) and LSCH-10C to Streptomyces plicatus (similarity index 99.701%). Of the 3 Pseudonocardia strains obtained, LS1-145 was found to be most closely related to Pseudonocardia carboxydivorans (similarity index 99.762%), LSCH7 to Pseudonocardia alni (similarity index 97.728%), LSCH-31 to Pseudonocardia alni (similarity index 99.423%). The isolate (LSCH-6B) was found to be most closely related to Nocardiopsis prasina (similarity index 99.423%).

71 LS1-128 100

LSCH-6B T

99 Nocardiopsis prasina DSM 43845 (X97884) T

Nocardiopsis metallicus KBS6 (AJ420769) LSCH-7

Two strains, LS1-81 and LSCH-7, could possibly be novel species of Streptomyces and Pseudonocardia respectively. Further analyses including DNA-DNA hybridization with the respective type strains and other polyphasic studies will be necessary for definitive taxonomic placements of the above strains which is beyond the scope of the present study. Though Streptomyces is the prolific genus for bioactive metabolite production, our study indicates great promise of finding rare actinomycetes producing bioactive metabolites in underexplored biotopes of Manipur such as lakes. This study also indicates the promise of aquatic habitats for exploration of novel bioactive strains of actinomycetes. Terkina et al. (54, 55) observed predominance of Micromonospora in Lake Baikal sediments and Streptomyces in Lake Baikal water. Similar findings were also found by Rifaat (42) for Nile River. In contrast to these earlier findings, our results indicate the dominance of Streptomyces in the Loktak lake sediments. This may, however, be a reflection of the trophic status of the contrasting lakes; whereas Lake Baikal is an oligotrophic lake,

T

99

Pseudonocardia autotrophica IMSNU 20050 (AJ252824) 98

98

T

Pseudonocardia carboxydivorans Y8 T (EF114314) Pseudonocardia alni DSM 44104 (Y08535) 97

T

Pseudonocardia antarctica DVS 5a1 (AJ576010) 100 LS1-145

58

LSCH-31

Bifidobacterium bifidum YIT 4039T (AB437356) 0.02

Figure 2:Phylogenetic relationship of the bioactive Loktak actinobacteria with its closest homologs.


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27. 28.

CONCLUSION Apart from Streptomyces, bioactive actinomycetes belonging to the genera Pseudonocardia and Nocardiopsis were also isolated from Loktak lake sediments. Though Streptomyces still seem to be the major bioactive metabolite producer, our findings corroborate the increasing prominence of rare actinomycetes in the search for bioactive metabolites. The present study indicates that Loktak Lake can be a promising source of bioactive actinomycetes. Further characterization of the Loktak lake actinomycete strains and their bioactive metabolites shall be the target of our future studies.

29. 30. 31. 32.

ACKNOWLEDGEMENTS The authors acknowledge the kind help of Prof. Rup Lal, Department of Zoology, University of Delhi, Delhi (INDIA), Dr. S. Mayilraj, Actinomycete Section, MTCC, IMTECH, Chandigarh (INDIA) and Dr. S. Shivaji, Scientist, CCMB, Hyderabad (INDIA) in 16S rDNA sequencing of the bioactive actinomycete strains. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25. 26.

Anderson, AS and Wellington, EMH (2001). The taxonomy of Streptomyces and related genera. Int J Syst Evol.Microbiol. 51: 797-814. http://ijs.sgmjournals.org/cgi/reprint/51/3/797 Atta, HM (2009). An antifungal agent produced by Streptomyces olivaceiscleroticus AZSH514. World Appl Sci J. 6: 1495-1505.http://idosi.org/wasj/wasj6(11)/7/pdf Atta, HM, Dabour, SM, and Desoukey, SG (2009). Sprasomycin antibiotic production by Streptomyces sp. AZ-NIOFD1: taxonomy, fermentation, purification and biological activitites. American-Eurasian J. Agric. Environ. Sci. 5: 368-377.http://idosi.org/aejaes/jaes5(3)11.pdf Atta, MA and Ahmad, MS (2009). Antimycin-A antibiotic biosynthesis produced by Streptomyces sp. AZ-AR-262: taxonomy, fermentation, purification and biological activities. Australian J Bas Sci 3: 126-135.http://www.insinet.net/ajbas/2009/126-135.pdf Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Truck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 45: 493-496.http:// www.ncbi.nlm.nih.gov/pubmed/5325707 Berdy, J. (2005). Bioactive microbial metabolites. J. Antibiot. 58: 1-26.http://www.nature.com/ ja/journal/v58/n1/abs/ja2005a.html Berdy, J. (1995). Are actinomycetes exhausted as a source of secondary metabolites? In Proceedings of the 9th International Symposium on the Biology of actinomycetes, part I, pp. 323.New York: Allerton. Biodiversity hotspots (2009). URL: www.biodiversityhotspots.org (Accessed on October 30, 2009). Bonjar, S. (2004). Broadspectrin, a novel antibacterial from Streptomyces sp. Biotechnol. 3: 126-130. http://scialert.net/.. Chen, CN, Huang, CH, Lee, HH, Tsai, HH and Kirby, R (2002). Once the circle was broken: dynamism and evolution of Streptomyces chromosomes. Trends Genet. 18: 522-529. Chun, J., Lee, J.-H., Jung, Y., Kim, M., Kim, S., Kim, B. K. & Lim, Y. W. (2007). EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol. 57, 2259-2261.http://ijs.sgmjournals.org/cgi/content/full/ 57/10/2259 Conservation International (2009). URL: www.conservation.org (Accessed on October 30, 2009). Cross, T. (1981). Aquatic actinomycetes: a critical survey of the occurrence, growth, and role of actinomycets in aquatic habitats. J. Appl. Bacteriol. 50: 397-423. http://www.ncbi.nlm.nih.gov/ pubmed/7019182 Elliah, P., Raju KVV Bapi, SN, Adinarayana, K, Adinarayana, G, Saisha, V, Madhavi, S. and Premkumar, J. (2002). Bioactive actinomycetes from Krishna river sediments of Andhra Pradesh. Hindusthan Antibiotics Bulletin 44: 1-4. http://www.ncbi.nlm.nih.gov/pubmed/ 15061588 Euzeby, JP (2009) List of prokaryotic names with standing in nomenclature. Last full update: October 08, 2009(URL: http://www.bacterio.net) (Accessed on October 30, 2009). Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the boothstrap. Evolution. 39:783-791. http://www.jstor.org/stable/pdfplus/2408678.pdf Goodfellow, M and Haynes, JA (1984). Actinomycetes in fresh water sediments. In: OrtizOrtiz L, Bojalil, LF, Yakoleff, V(eds). Biological, biochemical and biomedical aspects of actinomycetes. Academic Press, New York, London, pp. 453-472. Hsu, S. C. and Lockwood, J.l. (1975). Powdered chitin agar as a selective medium for enumeration of Actinomycetes in water and soil. Applied Microbiol. 29 : 422-426. Jensen, PR, Dwight, R., and Fenical, W ( 1991). Distribution of actinomycetes in near-shore tropical marine sediments. Appl. Environ. Microbiol. 57: 1102-1108.. http://aem.highwire.org/ cgi/content/abstract/57/4/1102 Jiang, C and Xu, L.(1996). Diversity of aquatic actinomycetes in lakes of the middle plateau, Yunnan, China. Appl. Environ. Microbiol. http://aem.highwire.org/cgi/content/abstract/62/1/ 249 Jones, K.L. (1949). Fresh isolates of actinomycetes in which the presence of sporogenous aerial mycelia is a fluctuating characteristics.J.Bacteriol. 57: 141-145.http:// www.ncbi.nlm.nih.gov/pmc/articles/PMC385488/pdf/jbacter00630- 0027.pdf Kim, S.B., Falconer, C., Williams, E. & Goodfellow, M. (1998). Streptomyces thermocarboxydovorans sp. nov. and Streptomyces thermocarboxydus sp. nov., two moderately thermophilic carboxydotrophic species from soil. Int J Syst Bacteriol. 48: 59-68.http:// ijs.sgmjournals.org/cgi/content/abstract/48/1/59 Kuster, E. & Williams, S.T. (1964). Selection of media for isolation of Streptomyces. Nature. 202: 928-929.http://adsabs.harvard.edu//abs/1964Nature.202..928K Kuznetsov, SI (1970). Microflora of lakes and its geochemical activity. Lenigrad: Nauka 1-9. Lakshmipathy, DT and Kannabiran, K.(2009). A morphological, biochemical and biological studies of halotolerant Streptomyces sp isolated from saltpan environment. Amer J. Inf. Dis. 5: 207-213. http://www.akademik.unsri.ac.id/download/journal/files/scipub/ajid53207213.pdf Lango, Z., Toth, LG, Kovacs, L, and Cruz-Pizarro, L.(1999). Identification and biochemical characterization of Streptomyces galbus from the sediment of the high mountain lake La Caldera(Sierra Nevada, Spain). Folia Microbiologia 44: 651-656. http://www.springerlink.com/ content/r727672127pn8t34/

33.

34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61.

Locci, R. (1989). Streptomyces and related genera. In Bergeys Manual of Systematic Bacteriology, vol.4, pp.2451-2508, Williams, ST, Sharpe, ME and Holt, JG(eds), Williams & Wilkins, Baltimore, USA. Manteca, A, Alvarez, R, Salazar, N, Yague, P, and Sanchez, J(2008). Mycelium differentiation and antibiotic production in submerged culture of Streptomyces coelicolor. Appl. Environ. Microbiol. 74: 3877-3886.http://aem.asm.org/cgi/reprint/74/12/3877 Myers, N, Mittermeier, RA, Mittermeier, CG, da Fonseca, GAB, and Kent, J(2000). Biodiversity hotsopts for conservation priorities. Nature 403: 853-858DOI: 10.1038/35002501 PMID: 10706275 URL: http://web.ipb.ac.id/~mujizat/jurnal/Myers%20et%20al%202000.pdf Ningthoujam, D.S. and Shovarani, N. (2008). Isolation and Characterization of a Pseudomonas aeruginosa Strain DN1 degrading p-Nitrophenol Research Journal of Microbiology. 3 (5): 345351. Ningthoujam, D.S., Sanasam, S. and Nimaichand, S. (2009). Screening of Actinomycetes Isolates from Niche Habitats in Manipur for Antibiotic Activity. American Journal of Biochemistry and Biotechnology, 5: 221-225. Ningthoujam, D., Sanasam, S. and Nimaichand, S. (2009). A Streptomyces sindenensis Strain LS1-128 Exhibiting Broad Spectrum Antimicrobial Activity. Research Journal of Biological Sciences, 4: 1085-1091. Ningthoujam, D.S., Kshetri, P., Sanasam, S. and Nimaichand, S. (2009). Screening, Identification of Best Producers and Optimization of Extracellular Proteases from Moderately Halophilic Alkalithermotolerant Indigenous Actinomycetes. World Applied Sciences Journal, 7: 907-916. Ningthoujam, D.S., Sanasam, S., Tamreihao, K. and Nimaichand, S. (2009). Antagonistic activities of local actinomycete isolates against rice fungal pathogens. African Journal of Microbiology, 3: 737-742. Ningombam, B and Bordoloi, S, B(2007). Amphibian fauna of Loktak Lake, Manipur, India with ten new records for the State. Zoos’ Print Journal 22: 2688-2690. Doi: X PMID: X URL: http://www.zoosprint.org/ZooPrintJournal/2007/May/2688-2690.pdf Okami, Y. & Hotta, K. (1988). Search and discovery of new antibiotics. In Actinomycetes in Biotechnology, pp. 33-67. Edited by M. Goodfellow, S.T. Williams & M. Mordarski. London: Academic Press. Okami, Y. (1986). Marine microorganisms as a source of bioactive agents. Microbial Ecol. 12: 65-78. http://springerlink.com/content/1416484325082067/fulltext.pdf Omura, S (1986). Philosophy of new drug discovery. Microbiol. Rev. 50: 259-274.http:// www.ncbi.nlm.nih.gov/pmc/articles/PMC373071/pdf/microrev00054/0037.pdf Osada, H. (1998). Actinomycetes: how fascinating microorganisms. Actinomycetol. 12: 8588 http://www.jstage.jst.go.jp/articles/saj/12/2/85/_pdf Oskay, M., Tamer, A.U. and Azeri, C. (2004). Antibacterial activity of some actinomycetes isolated from farming soils of Turkey. Afr. J. Biotechnol.39: 441-446. Singh, L.S., Baruah, I. and Bora, T.C. (2006). Actinomycetes of Loktak habitat: Isolation and screening for antimicrobial activities. Biotechnology. 5(2): 217-221. Rifaat, HM (2003). The biodiversity of actinomycetes in the river Nile exhibiting antifungal activity. J. Mediterranean Ecol. 4: 5-7.http://www.jmecology.com/pdf/2003/3-4/01rifaat.pdf Rowbotham, TJ and Cross, T (1977). Ecology of Rhodococcus coprophilus and associated actinomycetes in fresh water and agricultural habitats. J. Gen. Microbiol. 100: 231-240.http:/ /mic.sgmjournals.org/cgi/reprint/100/2/231.pdf Saadoun, I and Gharaibeh, R (2003). The Streptomyces flora of Badia region of Jordan and its potential as a source of antibiotics against antibiotic-resistant bacteria. J Arid Environ. 53: 365-371. Saitou, N. and Nei, M. (1987). The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 4: 406-425.http://mbe.oxfordjournals.org/cgi/reprint/4/ 4/406.pdf Sanasam, S. and Ningthoujam, D. (2005). Diversity of actinomycetes in selected soils of Manipur and their antibiotic potential. J. Assam Sci. Soc. 45: 44-47. Shirling E.B. and Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol. 16:313-340. http://ijs.sgmjornals.org/cgi/content/abstract/163/313 Sponga, F., Cavaletti, L., Lazzarini, A. and Bofghi, A. (1999). Biodiversity and potentials of marine-derived microorganisms. J. Staneck, J.L. and Roberts G.D. (1974). Simplified Approach to identification of aerobic actinomycetes by Thin-Layer Chromatography. Appl. Microbiol. 28:226-231.http://aem.asm.org/ cgi/content/abstract/28/2/226 Takahashi, YO and Omura, S. (2003). Isolation of new actinomycete strains for the screening of new bioactive compounds. J. Gen. Appl. Microbiol. 49: 141-154.http://www.jstage.jst.go.jp/ article/jgam/49/3/141/_pdf Takizawa, M, Colwell, RR, and Hill, RT (1993). Isolation and diversity of actinomycetes in the Chesapeake Bay. Appl. Environ. Microbiol. 59: 997-1002.http://aem.asm.org/cgi/reprint/59/4/ 997.pdf Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 24:1596-1599. http:// mbe.oxfordjournals.org/cgi/reprint/24/8/1596.. Tanaka, Y, and Omura, S. (1990) Metabolism and products of actinomycetes- An introduction.Actinomycetol.4, 13-14http://www.jstage.jst.go.jp/article/saj/4/1/13/_pdf Terkina, IA, Parfenova, VV, and Ahn, TS (2006). Antagonistic activity of actinomycetes of Lake Baikal. Appl. Biochem. Microbiol. 42: 173-176. http://www.springerlink.com/index/ 5103500479034026.pdf Terkina, IA, Drukker, VV, Perfenova, VV, and Kostornova, TY (2002). The biodiversity of actinomycetes in Lake Baikal. Microbiol. 71: 404-408. http://www.springerlink.com/index/ 1EV9CJPJXH2CHJJA Waksman, S.A, (1961). The Actinomycetes: Classification, Identification and Description of Genera and Species. Baltimore: The Williams and Wilkins Company. 2: 61-292. Wellington, E.M.H. and Williams, S.T. (1978). Preservation of actinomycete inoculum in frozen glycerol. Microbios. Letters., 6: 151-157. Yamamura, H., Hayakawa, M., Nakagawa, Y., and Iimura, Y. (2003). Species diversity of nocardiae isolated from lake and moat sediment samples. Actinomycetol. 17: 44-46.http: wwww. jstage. jst.go.jp/article/saj/17/2/44/_pdf Xu, LH, Jiang, Y, Li, WJ, Wen, ML, Li, MG, and Jiang, CL (2005). Streptomyces roseoalbus sp. nov., an actinomycete isolated from soil in Yunnan, China. Atonie van Leewenhoek 87: 189-194.http:/ /www.springerlink.com/content/h57535h026250j43?? Xu, LH, Li, QR, and Jiang, CL, (1996). Diversity of soil actinomycetes in Yunnan, China. Appl. Environ. Microbiol. 62: 244-248. http://aem.asm.org/cgi/reprint/62/1/244 Zarandi, ME, Shahidi Bonjar, GH, Padasht Dehkaei, F, Ayatollahi Moosavi, SA, Rashid Farokhi, P, and Aghighi, S (2009). Biological control of rice blast (Magnaporthe oryzae) by use of Streptomyces sindenensis isolate 263 in greenhouse. Amer. J. Appl. Sci. 6: 194-199. http://www.doaj.org/ doaj?func=abstract&id=422751(or http://www.scipub.org/fulltext/ajas/ajas61194-199.pdf)

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