TITLE OF THE PROJECT

BIODIVERSITY OF KHUMBU REGION: POPULATION STUDY OF ACTINOMYCETES.

COLLABORATORS

1. EV K2- CNR, ITALY

2. Royal Nepal Academy of Science and Technology (RONAST)

3. Research Laboratory for Agricultural Biotechnology and    Biochemistry (RLABB)

PROJECT LEADER

        Prof. Dr. Vishwanath P. Agrawal.

INVESTIGATORS

Deepak Singh

Yogan Khatri

   Arishma Singh

SUMMARY

Altogether 461 actinomycetes isolates were obtained from 575 soil samples collected from Khumbu region situated at the base of Mt. Everest, but due to time constraint only 157 actinomycetes belonging to Lobuche, Tangboche and Pangboche could be classified in different genera on the basis of morphology. Morphological identification of the isolates showed the presence of highest percentage of Streptomycetes (» 50%) followed by Streptoverticillium (» 24%) in soils samples. The study showed a high degree of biodiversity among actinomycetes population in terms of distribution of color of spore. Psychrophilic actinomycetes were studied only in Lobuche isolates. It was found that 16 isolates were able to grow at both 4ºC and 27ºC; 14 belong to different genera. In addition 11 isolates were true psychrophilic as they could grow only at 4ºC after an incubation period of 45 days. It was found that four isolates, three from the Lobuche and one from Tangboche, had no resemblance with the morphological structure of actinomycetes illustrated in Holt, JG (1989) Bergey's manual of systematic bacteriology, vol 4, ed. S.T. Williams and M.E. Sharpe, Baltimore, Md: Williams and Williams and hence may be novel.

INTRODUCTION

Actinomycetes were originally considered to be an intermediate group between bacteria and fungi but are now recognized as prokaryotic. The gram-positive bacteria include two major branches: the low G+C organisms, containing genera such as Bacillus, Closdridium, Staphylococcus and Streptococcus; and the high G+C (> 55%) organisms referred to as the actinomycetes. The name "Actinomycete" derives from the Greek aktis (a ray beam) and mykes (fungus) and was given to these organisms from initial observations of their morphology.

The majority of the actinomycetes are free living, saprophytic bacteria found widely distributed in soil, water and colonizing plants. Actinomycetes population has been identified as one of the major group of soil population (Kuster 1968), which may vary with the soil type. These organisms  participate in the turn over of the soil components, especially in the transformation of organic compounds (Konova 1966, Kuster 1967, Kuzner 1968, Huntzens 1972). In soil they are involved in the decomposition and mineralization cycles with the production of extracellular enzymes, such as cellulases, chitinases, and lignin peroxidases. Since they can decompose complex mixtures of polymers in dead plant, animal and fungal material (Mc Carthy 1987; Crawford 1988; Wang et al. 1991), they have important role in soil biodegradation by recycling of nutrients associated with recalcitrant polymers (McCarthy and Williams, 1992).

The actinomycetes are noteworthy as antibiotic producers, making three quarters of all known products; the Streptomyces are especially prolific and can produce a great many antibiotics and other class of biologically active secondary metabolites (Table I ) and cover around 80% of the total antibiotic product, with other genera trailing numerically: Micromonospora is the runner-up with fewer than one-tenth as many as Streptomyces. If we include secondary metabolites with biological activities other than antimicrobial, actinomycetes are still out in front, with over 60%; Streptomyces spp. accounting for 80% of these (Hopwood, et al., 2000).

Table I   Approximate numbers of secondary metabolites produced by different groups of organisms as in 1994

¹ In each category, nearly 80% were found in Streptomyces and 20% in other actinomycetes.

MORPHOLOGICAL CHARACTERIZATION

The majority of genera of actinomycetes during differentiation result in the formation of specialized cell types and produce characteristic mycelium of hyphae. Considerable morphological diversity exists within the group with the formation of an extensive mycelium of substrate and aerial hyphae, the latter bearing specialized spores or sporangia. Certain genera such as Mycobacterium and Corynebacterium have less well-developed or nonexistent mycelial stages with the formation of only rods and cocci.

TAXONOMY

Actinomycetes 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 comprise 63 genera (Nisbet and Fox, 1991).

Actinomycetes were originally classified according to their morphology. Subsequent analysis of chemotaxonomic markers has assisted in the detection of genera and differentiation of species in some groups. On the basis of cell wall analysis actinomycetes family has been divided into eight types (Table II). Actinomycetes from the types II-IV can be further distinguished by their whole organism sugar pattern (Table III). A whole array of taxonomic tools has been used to define genera and supergeneric groups of actinomycetes (Goodfellow and O’Donnell  1989), but partial sequence analysis of 16 s rRNA is the most significant. Based on 16s rRNA classification system they have recently been grouped in ten suborders: Actinomycineae, Corynebacterineae, Frankineae, Glycomycineae, Micrococineae, Micromonosporineae, Propionibacterineae, Pseudonocardineae, Streptomycineae and Streptosporangineae (Table IV) and a large members of actinomycetes are still remained to be grouped (www.ncbi.nlm.nih.).

ECOLOGY

Actinomycetes are cosmopolitan in nature and found in most environments. Soil is colonized by actinomycetes hyphae, which can form aerial mycelia bearing hydrophobic spores dispersed by air, water, and microarthropods. Some groups such as the actinoplanes are adapted for water -mediated dispersion and produce motile zoospores within desiccation-resistant sporangia.

Table II  Cell Wall Chemotypes with illustrating Families

Table II Cell wall Chemotypes with illustrating Families

Note: 1 Streptomycetaceae, Nocardiaceae, 2 Micromonosporaceae, 3 Dermatophilaceae, Mycobacteriaceae, Thermomonosporaceae, Streptosporangiaceae, Frankiaceae, 4 Nocardiaceae, Mycobacteriaceae, Corynebacteriaceae, Pseudonocardiaceae, 5 Actinomycetacea, 6 Cellulomonadaceae, Micrococcaceae, Microbacteriaceae, Actinomycetaceae, 7 Microbacteriaceae, 8 Cellulomodaceae, Microbacteriaceae.

DAP: Diaminopimelic acid, DABA: Diaminobutyric acid,  ^aV: Variable amount.

Table III Whole organism sugar pattern of Actinomycetes from cell wall Chemotypes II-IV

Table IV   Taxonomic Classification of Order Actinomycetales

MATERIALS AND METHODS

COLLECTION OF THE SOIL SAMPLES

In the present study, soil samples were collected from a number of places from Khumbu Region (Fig. 1) in Mt. Everest Region in Nepal. In total 575 soil samples were collected. Soils were taken from 6-10 cm depth into sterile plastic bags, sealed and transported to lab.. Soil samples were air dried to reduce the number of vegetative bacterial cells and fungal hyphae (Williams and Cross, 1971).

ISOLATION OF ACTINOMYCETES COLONIES

Isolation and enumeration of actinomycetes were performed by soil dilution plate technique by Williams and Cross, 1971 using (i) 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) (ii) Glycerol Arginine Agar (Glycerol 12.5, Arginine 1.0, NaCl 1.0, K₂HPO₄ 1.0, Mg SO₄.7H₂O 0.5 Fe₂ (SO₄)₃. 6 H₂O 0.01, CuSO₄. 5 H₂O 0.001, ZnSO₄. 7 H₂O 0.01, MnSO₄. H₂O 0.01 and Agar 15 g/l) and (iii) Alanine-Starch Agar medium (Yeast Extract 1.0, L- Arginine, 0.2, L- Alanine 0.5, NaCl 2.5, Na₂SO₄ 10 and Agar 20 g/l ).

 Isolation of Mesophilic Actinomycetes: One gram of dried soil was dissolved in 9 ml of distilled water, agitated vigorously,  heated at 50⁰ C for 0.5 hour and then diluted to 10^-3. One ml of  the resulting suspension was applied onto plates and 20 ml of melted medium at around 50⁰C was added to it. After gently mixing the soil suspension, the plates were incubated at 27⁰C for 15 days. Selected colonies (rough, chalky) of actinomycetes  were transferred from mixed culture of the plates onto new agar plates (Waksman, 1961) and incubated at 27⁰C for 7 days. Plates containing pure cultures were stored at 5⁰C until further examinations.

Isolation Of Cold Tolerant Actinomycetes: One gram of dried soil was dissolved in 9 ml of distilled water and agitated vigorously and aqueous dilution of 10^-3 of the suspension was spread over the sterile medium and incubated at 4⁰C for 45 days. Actinomycetes colonies  were transferred onto another plate and incubated in the condition as mentioned above.

PURIFICATION OF ACTINOMYCETES

Cultures of actinomycetes contaminated by bacteria, fungi and other actinomycetes were purified by streak plate method. The observed actinomycetes colonies were picked up either by straight wire or by loop according to the condition. The picked specks of the colonies were streaked over the dried medium divided into sectors radially and furher incubated. Actinomycetes colonies found to be contaminated with fungi were again transferred to another sterile medium.

IDENTIFICATION OF ISOLATES (MORPHOLOGICAL EXAMINATION)

The sterilized cover slips were carefully inserted at an angle of about 45 degree into solidified medium in petridish, until about half of the cover slip was buried in the medium. The isolates were inoculated along the line where the medium meets the upper surface of the cover slip. After incubation for 7 - 10 days, the cover slip was carefully removed and placed downwards on the slide and directly examined under the microscopic field (x 100) (Williams and Cross, 1971). Purified isolates of actinomycetes were identified up to the genera level by comparing their morphological characteristics:  their spore-bearing hyphae, color of the aerial mycelium and color of the soluble pigments were described as illustrated in Bergey's Manual (2000).

RESULTS

Soil samples were collected during October 10 to 21, 2001 from Lukla (2660m) to Upper Lobuche (5300m) in the Mt. Everest Region. Altogether 461 actinomycetes (including true psychrophilic) were isolated from 575 soil samples (Appendix I-X).

Population density of actinomycetes (isolates/soil sample) was higher in Lukla (1.48) followed by Sagarmatha National Park(1.244), Jorsale (0.833), Tanagboche to Pangboche (0.796), Tangboche (0.684), Phading (0.640), Lobuche (0.589), Namche (0.526) and Pheriche (0.283)  (Table V).

Table V Actinomycetes Population Density of Soil collected collected  from different areas of Khumbu region.

* Includes 10 true psychrophiles (Table VIII)

 Study of diversity of actinomycetes in reference with colour of spore and mycelium in Khumbu region indicated that white colored actinomycetes represent the highest population ( Table VI A and B ) .

Table VI (A) Diversity in Actinomycetes on the basis of colour of mycelium in different area of Khumbu region.

Table VI  (B) Diversity within White variety of actinomycetes.

MORPHOLOGICAL CHARACTERIZATION OF ACTINOMYCETES

Morphological characterization of actinomycetes was only done for the isolates obtained from Lobuche, Tengboche and Tengboche to Pangboche (Table VII ).

Table VII  Identification of Actinomycetes

Lobuche: The examination indicated that the isolates belonged to the genera Streptomyces, Micromonospora, Intrasporangium and  Streptoverticillium. Moreover, some isolates showed good resemblances with Catellospora, Kibdelosporium, Kitasatospora, Actinomsynemma, Nocardia, Saccharopolyspora and Nocardiopsis but more evidence is required for their unequivocal classification into genus. In addition three isolates had a morphology that did not match with any actinomycetes morphologies (Holt, 2000). Among those isolated genera, Streptomyces were found in the highest percentage (44%), followed by Streptoverticillium (15%), Saccharopolyspora (8%), Micromonospora and Intrasporangium (7% each).

Tengboche : Among  13 actinomycetes from the Tengboche soil samples, the morphological examination showed the highest percentage of Streptomyces (43%), followed by Streptoverticillium (23%), Streptosporangium (15%),   Ampularia or Actinoplanes (8%)  and 8%

of unidentified genera of actinomycetes;  one isolate had a morphology that did not match with any actinomycetes morphologies (Holt,  2000).

Pangboche : Out of the total 82 Pangboche actinomycetes isolates, 63 were examined morphologically where Streptomyces were (52 %) followed by Streptoverticillium (34%), Actinosynnema or Kitasatospora (3%), Actinopolyspora (3%) and 3% unidentified actinomycetes.

COLD TOLERANT ACTINOMYCETES

Generally, the generation time of the cold tolerant organism is longer, therefore, some of the isolates showed growth only after the incubation of three months in 1 to 4⁰ C. Most of the cold tolerant isolates were with clean white colored spore chain. Study of psychrophilic  actinomycetes can be very  important in  evolutionary biology.

Psychrophilic actinomycetes were studied only from the Lobuche isolates. It was found that 15 isolates can grow at both the temperature of 4ºC and 27ºC. But 11 of them were true psychrophilic and can grow only at 4ºC.It is illustrated in Table VIII.

Table VIII  Diversity of Psychrophilic Actinomycetes isolated from Lobuche samples

ACKNOWLEDGEMENTS

We are grateful to EV-K2-CNR, Italy and Royal Nepal Academy of Science and Technology (RONAST) for financial support. We thank Central Dept. of Microbiology, Tribhuvan University for photomicrographs. We are thankful to Mr. Rajendra Aryal who was a member of the expedition team during collecting the soil sample from Khumbu Region.  

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APPENDIX

Appendix I Isolation and Characterization  of Actinomycetes from soil samples collected from Lukla (  Height: 2660 Mtrs)