Biodiversity of Actinomycetes of Lobuche in Mount Everest I

Biodiversity of actinomycetes in Lobuche Area (5000m-5300m) of Khumbu Region situated at the base of Mt. Everest has been studied. Aqueous suspensions of soil were applied to Starch-Casein Agar medium and incubated at 27⁰C up to 10 days. Chalky dried colonies were streaked on Starch-Casein agar and isolated in the same medium. For morphological identification cover slip technique was used and the identification was done according to Bergey's Manual of Determinative Bacteriology, 2000. From 120 soil samples collected from Lobuche, 68 isolates of actinomycetes were obtained and 42 of them belonged to genera: Catellospora, Intrasporangium, Kibdelosporium, Kitasatospora, Micromonospora, Nocardia, Saccharopolyspora, Streptomyces and Streptoverticilium; at least one isolate could be a novel species as it had unexpectedly large and highly branched chain of conidia. The isolates were very diverse in the color of their spores [white 33% (Chalky white 82%, Greenish white 5%, Bluish white 7%, Reddish white 2.5% and Chocolate white 4%), Yellow 21.5%, Creamy 16%, Brown 11%, Grey 6.5%, Black 5% and Ash 7%].

Key words: Isolation, characterization, colour, morphology, novel actinomycetes
INTRODUCTION

Actinomycetes population has been identified as one of the major groups of the soil population (Kuster, 1968). They are gram-positive organisms and assumed to be the transition group between fungi and bacteria. They belong to the order Actinomycetales (Superkingdom: Bacteria, Phylum: Firmicutes, Class: Actinobacteria, Subclass: Actinobacteridae). According to Bergey's Manual actinomycetes are divided into eight diverse families: Actinomycetaceae, Mycobacteriaceae, Actinoplanaceae, Frankiaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae (Holt, 1989) and they comprise 63 genera (Nisbet and Fox, 1991). Based on 16s rRNA classification system they have recently been grouped in ten suborders: Actinomycineae, Corynebacterineae, Frankineae, Glycomycineae, Micrococineae, Micromonosporineae, Propionibacterineae, Pseudonocardineae, Streptomycineae and a large members of actinomycetes are still remained to be grouped (www.ncbi.nlm.nih.gov). Actinomycetes have characteristic biological aspects such as mycelial forms of growth that accumulates in sporulation and the ability to form a wide variety of secondary metabolites including most of the antibiotics. Complex morphological development in the genera is phenotypically related to secondary metabolism (Hourinouchi and Beppu, 1992). Among bacteria, the actinomycetes are noteworthy as antibiotic producers, making three quarters of all known products; the Streptomyces are especially prolific. The antibiotics from actinomycetes are diverse in chemical structure. In contrast, antibiotics from the unicellular bacteria are more commonly peptides or modified peptides (Vining, 1990).

The study site belongs to Khumbu region, which is largely situated at the lower part of Mt. Everest base camp used by expedition teams. Results obtained from such a study can be of relevance to molecular biologists, ecologists and evolutionists. Moreover, this study can possibly reveal actinomycetes species that produce novel antibiotics. It is anticipated that efforts for the isolation, characterization and the study on actinomycetes can be a milestone for the discovery of antibiotics and novel species of actinomycetes.

During October 9 - 22, 2001 altogether 575 soil samples were collected from 6-10 cm depth into sterile plastic bags from Mt. Everest regions (Fig. 1A); 106 samples from Lukla (2660m), 25 from Phagding(2640 m), 24 from Jorsale (2730 m), 12 from Phakding to Namche (2730m-3200m), 45 from Namche National Park Area (3446m), 15 from Sangboche (3700m), 19 from Namche to Tengboche (3446m-3867m), 103 from Tengboche to Pangboche (3867m-3901m), 53 from Pangboche to Pheriche(3901m-4243m), 31 from Thukla to Lobuche (4243m-5000 m) and 120 from from Lobuche area (5000m-5300m) collected. Soils samples were transported to RLABB and were air dried in room temperature.

Isolation and enumeration of actinomycetes were performed by soil dilution plate technique using Starch-Casein Agar (Starch 10, casein 0.3, KNO₃ 2, NaCl 2, K₂HPO₄ 2, Mg SO₄.7H₂0 0.05, CaCO₃ 0.02, FeSO₄. 7 H₂0 0.01 and agar 18 g/l).

One gram of dried soil was taken in 9 ml of distilled water and agitated vigorously and preheated at 50⁰ C for 0.5 hour. Different aqueous dilutions, 10^-3, 10^-5 and 10^-7 of the suspension were applied onto plates and 20 ml of melted medium at around 50⁰C was added to it. After gently rotating, the plates were incubated at 27⁰C for 10 days. Selected colonies (rough, chalky) of actinomycetes were transferred from mixed culture of the plates onto respective agar plates (Waksman, 1961) and incubated at 27⁰C for 7 days. Plates containing pure cultures were stored at 4⁰C until further examinations. For long storage purified actinomycetes were grown in starch – casein broth for 7 days. To it glycerol was added to a final concentration of 15% (v/v) and stored at –20⁰C (Maniatis et.al, 1984).

Purified isolates of actinomycetes were identified up to the genera level by comparing their morphology of spore-bearing hyphae with entire spore chain and structure of spore chain with the actinomycetes morphologies as described in Bergey's, 2000. This was done by using cover- slip method in which individual cultures were transferred to the base of cover slips buried in starch-casein agar (Williams and Cross, 1971; Singh, 1999) for photomicrographs. Colors of spores were visually estimated by using a color – chart.

From 120 soil samples collected from Lobuche, 68 isolates of actinomycetes were obtained. Morphological examination of 42 isolates clearly indicates that these isolates (Fig.1) belong to the following genera: Streptomyces family Streptomycetaceae [spore chain with coiling and branching] (Fig. 1B&1C), Micromonospora family Micromonosporaceae [clusters of spore chain, single conidia on substrate mycelia] (Fig. 1D), Intrasporangium family Streptomycetaceae [spores within the mycelium: sub-terminal vesicles] (Fig. 1E), Catellospora [no sporangia; short chains of conidia formed protruding from the surface of the colonies] (Fig. 1F), Kibdelosporium [aerial mycelium with curled hyphae] (Fig.1G), Saccharopolyspora [very long chains of conidia on the aerial mycelium] (Figure not given), Streptoverticilium [whorls of straight chain of conidia formed] (Figure not given), Kitasatospora [Streptomyces type of morphology] (Figure not given), Nocardia [morphology ranging from fugacious substrate mycelium only to Streptomyces-like] (Figure not given) and Actinopolyspora [long chains of conidia on the aerial mycelium] (Figure not given), and but more evidence is required for their unequivocal classification into genus. So far it was found that one isolate had unexpectedly large and highly branched chain of conidia (Fig. 2) - a morphology that did not match with actinomycetes morphologies given in Bergey’s, 2000.

Fig 1. Exploration of Actinomycetes in Lobuche. A. Sketch of Base Region of Mt. Everest, B-F Photomicrographs (x100) of some Actinomycetes Isolates. Photomicrographs were taken following Cover- Slip method given in “Material and Methods”.

The isolates had different colours: white 33%, Yellow 21.5%, Brown 11%, Grey 6.5%, Creamy 16%, Black 5% and Ash 7%. When the white colonies were analysed for their shades, chalky white type of colonies were found in high percentage; Chalky white 82%, Greenish white 5%, Bluish white 7%, Reddish white 2.5% and Chocolate white 4%.

The difference in colours and heterogeneous morphology of isolates reveals that there is a high degree of diversity among actinomycetes in Lobuche.

ACKNOWLEDGEMENTS

We are grateful to CNR EV-K² CNR, Italy and Royal Nepal Academy of Science and Technology for financial support. We thank Central Dept. of Microbiology, Tribhuvan University for photomicrographs. We appreciate Arishma Singh for her assistance in research and Yogan Khatri for help in computer work.

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[1] Deepak Singh has applied for registration for PhD in Central Department of Microbiology, Tribhuvan University and this paper is based on part of his research for PhD.