title of the project biodiversity of khumbu region: population

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

2

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).

3

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

Bioactive metabolites Source Antibiotics Others Total

"inactive" metabolites

Non-actinomycete bacteria Actinomycetes Fungi

1400 (12%)

79001 (66%) 2600 (22%)

240 (9%)

12201 (40%) 1540 (51%)

1640 (11%)

91201 (61%) 4140 (28%)

2000-5000

8000-10,000 15,000-25,000

Total mocroorganisms

11,900 (100%)

3000 (100%)

14,900 (100%)

25,000- 40,000

Lichens Algae Higher plants Terrestrial animals Marine animals

150 700 5000 500 1200

200-500 800-900

25,000-35,000 10,000-15,000

1500-2000

¬ 1000 1000-2000 500,000-800,000 200,000-300,000

2000-3000 Total higher Organisms

7500

35,000-50,000 > 1,000,000

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

4

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 Pattern Arabinose Fucose Galactose Madurose Xylose A + + B +

Chemotype I II III IV V VI VII VIII L-DAP + Meso-DAP + + + DABA + Aspartic Acid

Va

Glycine + + + Lysine + V Ornithine + + Arabinose + Galactose + V 1 2

3 4 5 6 7 8

5

C D + + E +

6

Table IV Taxonomic Classification of Order Actinomycetales Suborder Family Genus Micromonosporineae

Micromonosporaceae

Micromonospora, Actinoplanes, Catellatospora, Couchioplanes, Catenuloplanes, Pilimelia Dactylosporangium

Frankineae Frankiaceae Sporichthyaceae Geodermatophilaceae Microsphaeraceae Acidothermaceae

Frankia Sporichthya Geothermatophills, Blastococcus Microsphera Acidohermus

Pseudonocardineae Pseudonocardiaceae Pseudonocardia, Actinopolyspora, Actinosynnema, Amycolatopsis, Kibdelosporium, Kutzneria, Lentzea, Saccharomonospora, Saccharopolyspora, Saccarothrix, Streptoalloteichus, Thermocrispum.

Streptomycineae Streptomycetaceae Streptomyces Corynebacteriuam Nocardiaceae

Gordoniaceae Mycobacteriaceae Dietziaceae Tsukamurellaceae Corynebacteriaceae

Nocardia, Rhodococcus. Gordonia Mycobacterium Dietzia Tsukamurella Corynebacterium, Turicella

Micrococcineae Micrococcaceae Brevibacteriaceae Cellulomondaceae Dermabacteraceae Intrasporangiaceae Jonesiaceae Microbacteriaceae Promicromonosporaceae

Micrococcus, Arthrobacter, Kocuria, Nesterenkonia, Rorhia, Renibacterium, Stomatococcus Brevibacterium Cellulomonas, Oeskovia, Rarobacter Dermatobacter, Brachybacterium Intrasporangium, Sanguibacter, Terrabacter Jonesia Microbacterium, Agrococcus, Agromyces, Aureobacterium, Clavibacter, Curtobacterium, Rathaybacter Promicromonospora

Actinomyineae Actinomycetaceae Actinomyces, Mobiluncus, Arcanobacterium Propionibacterianeae

Propionibacteraceae Propionibacterium, Luteococcus, Microlunatus, Propioniferax

Streptosporangineae

Streptosporangiaceae Thermomonosporaceae Nocardiopsaceae

Streptosporangium, Herbidospora, Microbispora, Microtetraspora, Planobispora, Planomonospora Thermomonospora, Actinomadura, Spirillospora Nocardiopsis

Glycomycineae Glycomycetaceae Glycomyces

7

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.

8

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).

9

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.

Place Height in meters

Numbers of soil samples

Numbers of isolates

Frequency (isolates/soil sample)

Lukla 2660 107 157 1.48 Phading 2640 25 16 0.640 Jorsela 2837 24 20 0.833 Namche 3446 19 10 0.526 Sagarmatha national park

3446

45

56

1.244

Tangboche 3867 19 13 0.684 Tangboche

to Pangboche 3867-3901

103

82

0.796

Pheriche 4243 53 15 0.283 Thukla

to Lobuche

4500-5000

24

0

Nil

Lobuche

5000-5300 156

92*

0.589

* 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 ) .

10

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

Lukla Phading Jorsale Namche Sagarmatha national park

Tanboche Tanboche To Panboche

Pheriche Lobuche

Yellow 17 3 - - 5 4 4 - 24 yellowish 17 - - - 1 1 4 2 - Yellowish white

2 - - - - - - 1

Creamy 64 7 6 7 11 - 34 9 35 White 13 - - 1 14 5 3 1 12 Dirty white

14 - 6 - 3 1 4 - 4

Pinkish white

3 - 1 - - - - - -

Brownish 3 1 - - - - 1 - - Brown 4 - - - - - - - 2 Black 9 3 5 2 13 2 25 3 1 Blackish - - - - 9 - - - - Reddish 8 - 1 - - - 2 - 1 Green 1 1 - - - - - - - Greenish 1 1 1 - - - 2 - 2 Blue 1 - - - - - 3 - - Total 157 16 20 10 56 13 82 15 82 Table VI (B) Diversity within White variety of actinomycetes. Yellowish white 3 White 49 Dry white 32 Pinkish white 4 MORPHOLOGICAL CHARACTERIZATION OF ACTINOMYCETES

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

11

Table VII Identification of Actinomycetes IDENTIFIED SPECIES

LOBUCHE

TANGBOCHE

TANGBOCHE TO PANGBOCHE

TOTAL

New (Probably) 3 1 - 4 Streptomyces spp 38 7 33 78 Micromonospora spp 5 - - 5 Intrasporangium spp 7 - - 7 Streptoverticillium spp

12 3 22 37

Saccharopolyspora spp

5 - - 5

Nocardiopsis spp 2 - - 2 Nocardia spp 2 - - 2 Catellospora spp 2 - - 2 Actinosynema spp 1 - 1 2 Ketasatospora spp 2 - 1 3 Streptosporangium spp

- 1 - 1

Strepoericillium spp 1 - - 1 Streptoalloteichus spp - - 1 1 Amycolata - - 1 1 Actinopolyspora spp 1 - 2 3 Kibdellosporium spp 1 - 1 2 Ampularia spp - 1 - 1 TOTAL 82 13 62 157 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).

12

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

Incubation S.N Soil sample number

4ºC 27ºC

Colour of spore chain

Colour of mycelium

Remarks

1 Lob 7.4 + + Yellow Yellow Streptomyces spp 2 Lob 7.3 + + Yellow Creamy Intrasporangium spp 3 Lob 8.5 + + Yellow Yellow Nocardiopsis spp 4 Lob 15.1 + + Creamy Yellow Kitasatospora spp 5 Lob 13 + - White White Not identified 6 Lob 12.2 + + Creamy Dirty white Streptomyces spp 7 Lob 11.6 + + Creamy Creamy Not identified 8 Lob 11.5 + + Yellowish

white Creamy Streptomyces spp

9 Lob 11.4 + + White Creamy Nocardia spp 10 Lob 8.4 + + Yellow Yellow Streptoverticillium spp 11 Lob 28.2 + + Yellowish Yellow Saccharopolyspora/Streptomyces 12 Lob 8.1 + + Yellow Yellow Streptoverticillium spp 13 Lob 10.1 + + White Creamy Streptoverticillium spp 14 Lob 52.2 + + Yellow Yellow Micromonospora/Streptoverticilliu

m/Actinoplans 15 Lob 52.1 + + Grey Brown Catellospora/Streptomyces/ 16 Lob 55 + + White Yellowish

white Saccharopolyspora/Streptomyces

17 Lob 66.2 + - White Dirty white Not identified 18 Lob 102.1 + - Grey Brownish Not identified 19 Lob 102.2 + - Dirty white White Not identified 20 Lob 102.3 + - Dirty white White Not identified 21 Lob 109.1 + - Yellow Creamy Not identified 22 Lob 109.2 + - Brown Reddish Not identified 23 Lob 138 + - Yellowish Purple Not identified

13

24 Lob 149 + - Bluish Bluish Not identified 25 Lob 147 + - White Dirty white Not identified 26 Lob 8.15 + - Dirty white Yellow Not identified

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.

REFERENCES Bergey's manual of determinative bacteriology (2000), Actinomycetales, 9th edition. Charter, KF (1984) Morphological and Physiological differentiation in Stre[ptomyces. In microbial Development, ed. Losiek, L. Shapiro, pp 89-115. Cold Spring Harbour: Cold Spring Harbour. Crawford DL (1988) Biodegradation of agricultural and urban wastes. In Goodfellow, M., Williams, ST and Mordarski, M. (eds), Actinomycetes in Biotechnology.Academic Press, London, pp. 433-439. Goodfellow, M and O' Donnell, AG (1989) Search and discovery of industrially significant actinomycetes. In: Baumberg, S., Hunter, I.S. and Rhodes, P.M. (eds.) Microbial Products: New Approaches, Cambridge University Press, Cambridge, pp 343-383. Holt, JG (1989) Bergey's manual of systematic bacteriology, vol 4, ed. S.T. Williams and M.E. Sharpe, Baltimore, Md: Williams and Williams. Hopwood DA, Buttner MJ, Bibb MJ, Kieser T and Charter KF (2000) Antibiotic production by Streptomyces. Practical Streptomyces Genetics.1, 1-42. Hourinouchi S and Beppu T (1992) Autoregulatory Factors and Communication in Actinomycetes. Ann,. Rev. Microbiol, 46:377-98. Huntzens JLM (1972) Aminoacid composition of humic acid like polymers produced by Streptomycetes and of humic acids from pasture & arable land. Soil Biol. Biochem., 4 : 339-345. Kononova MM (1966) Soil Organic Matters. Its nature, its role in soil formation & in soil fertility. 2 nd. English Ed. Pergamon Press.

14

Kuster E (1967) The actinomycetes. In: " Soil Biology", eds. Burges (A.) & Raw (F.), 111-124. Academic Press, London. Kuster E (1968) Taxonomy of soil actinomycetes & related organisms. In : " Ecology of soil bacteria", eds. Gray (TRG) and Parkinson (D.), 322-336. Liverpool University Press, Liverpool. Kuzner HJ (1968) Uber die Bildung Von Huminstoffen durch Streptomyceten. Landwirtsch. Forsch, 21:48- 61. McCarthy AJ (1987) Lignocellulose degrading actinomycetes. FEMS Microbiol.Rev.,46, 145-163. McCarthy AJ, Williams ST (1992) Actinomycetes agents of biodegradation in the environment - a review.Gene, 115, 189-192. National Science Board (1989). Loss of Biological Diversity: a global requiring international solution. National Science Board, Washington DC. Nisbet LJ and FM Fox (1991) The importance of microbial biodiversity to biotechnology, In, The biodiversity of microorganisms and invertebrates: its role in sustainable Agriculture, ed. D.L. Hawksworth, pp229-224, CAB International. Vining L (1990) Functions of Secondary Metabolites, Ann. Rev. Microbiol, 44: 395-427. Williams ST & Cross T (1971) Actinomycetes. In: Norris, J.R., Robbins, D. W. (eds), Methods in microbiology, vol.4. London, 295-334, Academic Perss, NewYork. Wong HC, Ting Y, Lin HC, Reichert F, Myambo K, Watt KWK, Toy PL, Drummond RJ (1991) Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus. J. Bacteriol., 173, 6849-6858.

APPENDIX

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

sample number

Isolate number

Colour of the spore chain

Colour of the mycelium

Remarks/genera (if identified)

1 1. Lu1 White. Rough and dry

Yellowish Not identified

2 2. Lu2 White. Rough and dry

Yellowish Not identified

3 3. Lu3 Brown, Dirty white Not identified

15

chalky 4 4. Lu4 (1) Brown,

chalky Dirty white Contaminated

5 5 Lu5 Brown (creamy)

Dirty white Not identified

6/7 No actinomycetes found

6 8 Lu8(2) Creamy Creamy Not identified 7 8 Lu8(4) White Dirty white Not identified 8 8. Lu8(5) White Yellowish white Not identified 9 8. Lu8(7) Deep

brown, rough

Brown Not identified

10 8 Lu8(8) Dirty white (grey)

Dirty creamy Not identified

11 8. Lu8(9) Creamy Creamy Not identified 12 8. Lu8(11) Black

centered Black Not identified

13 8. Lu8(12) White White Not identified 14 8. Lu8(13) White White Not identified 15 8. Lu8(14) White-deep

brown Dirty white Not identified

16 9. Lu9(1) Greenish white

Smoky Not identified

17 9. Lu9(3) Greenish white

Smoky Not identified

18 10. Lu10(2) White Creamy Not identified 19 10. Lu10(4) Yellowish Yellowish white Not identified 20 11. Lu 11(2) Bluish Creamy Not identified 21 11. Lu 11(5) Bluish Yellowish Not identified 22 11. Lu11(6) Yellowish Yellowish Not identified 12 No actinomycetes found 23 13. Lu 13 (1) Deep

brown Brown Not identified

24 13. Lu13(2) Bluish Yellowish Not identified 25 14. Lu14(1) Grey Creamy Not identified 26 14. Lu 14(3) Bluish Creamy Not identified 27 15. Lu 15 (2) Bluish Creamy Not identified 28 15. Lu 15(3) Bluish Creamy Not identified 29 15. Lu15(4) Creamy Creamy Not identified 30 16. Lu16(2) Bluish Yellowish Not identified 31 17. Lu 17(5) Bluish

white Creamy Not identified

32 18. Lu18(2) Black Brown Not identified 33 18. Lu 18(3) Creamy Yellowish Not identified

16

34 18. Lu 18(8) Bluish white

Creamy Not identified

35 19. Lu19(2) Bluish white


20/21 No actinomycetes found 36 22. Lu 22(2) White Creamy Not identified 37 22 Lu 22(3) Yellowish Yellowish Not identified 38 23. Lu 23(4) Yellowish Yellowish Not identified 39 24 No actinomycetes found 40 25. Lu25 White Creamy Not identified

26/27 No actinomycetes found 41 28. Lu 28(3) Yellowish Creamy Not identified 29/30 No actinomycetes found 42 31. Lu 31(1) Grey Dirty white Not identified 43 31. Lu 31(3) Black


44 32. Lu32(1) Brown centered with white margin

Brownish Not identified

33 No actinomycetes found 45 34 Lu34 (1) Yellowish Creamy Not identified 46 34. Lu 34(2) White White Not identified 47 35. Lu35(1) White White Not identified 48 35. Lu 35(3) White Creamy Not identified 49 35. Lu35(4) Yellowish


50 36. Lu 36 Black Black Not identified 51 37. Lu 37(2) Creamy Creamy Not identified 52 38. Lu 38 (1) Dirty white Creamy Not identified 53 38. Lu 38(2) Yellowish Creamy Not identified 54 38. Lu 38(3) White Yellow Not identified 55 39. Lu 39(2) Pinkish

brown Black Not identified

56 39. Lu39(3) Creamy Dirty white Not identified 57 40. Lu 40 Creamy Creamy Not identified 58 40. Lu 40(1) Ash Brown Not identified 59 40. Lu40(3) Brown Brown Not identified 60 40. Lu 40(5) Fade brown Yellowish Not identified 61 40. Lu 40(9) White Yellowish Not identified 62 41. Lu 41(2) Yellowish Creamy Not identified 63 41. Lu 41(3) Yellowish Yellowish Not identified 42/43 No actinomycetes found 64 44. Lu44(3) Brown

(Yellowish) White Not identified

17

65 45. Lu 45(1) White Creamy Not identified 66 45. Lu 45(2) White Creamy Not identified 67 45. Lu45 (3) White Creamy Not identified 68 45. Lu 45 (4) White Creamy Not identified 69 45. Lu 45(5) Yellowish


70 45. Lu45 (6) Black Black Not identified 71 46. Lu 46(1) White Creamy Not identified 72 46. Lu 46(7) White Creamy Not identified 47-50 No actinomycetes found 73 51. Lu 51(1) Yellowish Yellow Not identified 74 52. Lu52 Yellowish Yellow Not identified 53-54 No actinomycetes found 75 55. Lu55 White Yellow Not identified 76 56. Lu56 Yellowish Creamy Not identified 78 57. Lu57 Creamy Yellowish Not identified 79 58. Lu58 Yellowish Yellow Not identified 80 59. Lu59 Yellowish Yellow Not identified 60-62 No actinomycetes found 81 63 Lu63(1) White Creamy Not identified 82 64. Lu64 (2) Creamy Creamy Not identified 83 65 Lu 64 (3) Yellowish Yellow Not identified 84 66 Lu 65 (1) Creamy Yellowish Not identified 85 67. Lu 67(1) Ash Dirty white Not identified 86 67. Lu 67(2) Ash with

white margin

Pinkish Not identified

87 68. Lu 68(1) Creamy emerged in medium


88 68. Lu68(2) White Yellowish Not identified 89 68. Lu 68(3) Ash Creamy Not identified 90 69. Lu 69(2) Black Black Not identified 91 70. Lu70 Creamy Creamy Not identified 92 71. Lu 71(1) Yellowish Creamy Not identified 93 71. Lu 71(2) Yellowish Creamy Not identified 94 71. Lu 71(3) White Creamy Not identified 95 71. Lu 71(4) Yellowish Creamy Not identified 96 71. Lu 71(7) White Yellow Not identified 97 72. Lu72(1) Ash Creamy Not identified 98 72. Lu72(2) Ash Creamy Not identified 99 73. Lu73(1) Yellowish Yellowish Not identified 100 73. Lu73(2) Yellowish Creamy Not identified 101 74. Lu74(2) Emerged

Creamy Creamy Not identified

18

102 74. Lu 74(5) Dirty white Reddish Not identified 103 74. Lu 74(6) Creamy Creamy Not identified 104 74. Lu 74(7) Creamy Creamy Not identified 105 75. Lu75(1) Creamy Creamy Not identified 106 75. Lu 75(2) White Creamy Not identified 107 75. Lu75(3) White Reddish Not identified 108 76. Lu76(1) Yellow Yellow Not identified 109 76. Lu76(2) Yellow Yellow Not identified 77 Contaminated 110 78. Lu 78 (1) White Pinkish white Not identified 111 78. Lu 78(2) White Pinkish white Not identified 112 79 Not actinomycetes

found 113 80. Lu 80(1) White Creamy Not identified 114 80. Lu80(2) Yellow Creamy Not identified 115 81. Lu 81(3) Creamy Creamy Not identified 116 82. Lu82(2) White Creamy Not identified 117 83 Lu 83 (1) White

embedded White Not identified

118 84. Lu 84(2) White Creamy Not identified 119 84 Lu84(3) White Creamy Not identified 120 86 Lu 86 (1) Yellow Yellow Not identified 121 87. Lu87(1) White Creamy Not identified 88-93 No actinomycetes found 122 94. Lu 94(3) Black Black Not identified 123 94. Lu94(5) Chocholaet


124 94. Lu 94(6) Yellow Yellow Not identified 125 95. Lu 95(1) White

embedded White Not identified

126 95. Lu 95(2) White embedded

White Not identified

127 96. Lu96(2) Yellow Yellow Not identified 128 96. Lu96(3) Creamy Creamy Not identified 129 96. Lu96(4) White Yellow Not identified 130 96. Lu 96(5) White Creamy Not identified 131 96. Lu96(7) White Yellow Not identified 132 97. Lu97(1) Ash Brown Not identified 133 97. Lu97(2) Creamy Creamy Not identified 134 97. Lu97(6) Chocolate

white Yellow Not identified

135 97. Lu97(7) Ash Ash Not identified 136 98. Lu98(1) Small

white Reddish Not identified

137 98. Lu98(2) Small Reddish Not identified

19

white 138 98. Lu98(3) Small

white Reddish Not identified

139 98. Lu98(3) Small white

Reddish Not identified

140 99. Lu99(1) White Reddish Not identified 141 99. Lu 99(3) Ash Black Not identified 142 99. Lu99(4) Yellow Yellow Not identified 143 99. Lu99(5) Black


144 100. Lu100(1) White White Not identified 145 100. Lu

100(2) White embedded


146 100. Lu100(3) White White Not identified 147 101. Lu101(1) Brownish

white White Not identified

148 101. Lu101(3) White Creamy Not identified 149 101. Lu101(4) Reddish

white Red Not identified

151 101. Lu 101(5)

White White Not identified

152 101. Lu 101(6)

Brownish white


153 101. Lu101(7) Greenish yellow

Green Not identified

102 No actinomycetes found 154 103. Lu 103 Yellow Yellowish Not identified 155 104. Lu104 Ash Greenins Not identified 156 105. Lu105 Creamy

embedded Creamy Not identified

106 No actinomycetes found 157 107. Lu

107(2) Small white

greenish Not identified

20

Appendix II Isolation and Characterization of Actinomycetes from soil samples collected from Phagding ( Height: 2640 Mtrs)

Soil sample number

Isolate number




1 No actinomycetes found 2 Ph2.1 Bluish

white Yellow Not identified

2 Ph2.3 White Creamy Not identified 2 Ph2.4 White black Not identified 2 Ph2.5 Ash Black Not identified 2 Ph2.6 Ash Brown Not identified 3 Ph3.1(GA) .white White Not identified 4/5 No actinomycetes found 6 Ph6.1(GA) Black spot

in the center and ash in the edge

Greenish Not identified

6 Ph6.2(GA) .Chocolate White Not identified 7 Ph7.3(GA) .Dirty


7 Ph7.4(GA) .white White Not identified 8/9 No actinomycetes found 10 Ph10.1(GA) Creamy Yellow Not identified 10 Ph10.3(GA) Ash Black Not identified 10 Ph10.4(GA) .Dirty

White Creamy Not identified

11-13 No actinomycetes found 14 Ph14.2(GA) yellow Creamy Not identified 15-17 No actinomycetes found 18 Ph18.6(GA) White Yellow Not identified 19-24 No actinomycetes found 25 Ph25(GA) Deep

brown Green Not identified

21

Appendix III Isolation and Characterization of Actinomycetes from soil samples collected from Jorsale ( Height: 2837 Mtrs)

Soil sample number

Isolate number




1-3 No actinomycetes 4 J4.1(GA) White

and red Reddish Not identified

4 J4.2(GA) White Creamy Not identified 4 J4.3(GA) Pinkish

white Pinkish Not identified

5-10 No actinomycetes 11 J11.1(SCA) Ash Blackish Not identified 11 J11.2(SCA) White Greenish Not identified 11 J11.3(SCA) Ash Black Not identified 12-15 No actinomycetes 16 J16.1(SCA) Dirty

white Dirty white

Not identified

16 J16.2(SCA) Ash Dirty white

Not identified

16 J16.3(SCA) Ash Blackish Not identified 17-19 No actinomycetes 20 J20.1(GA) Dirty

white Black Not identified

20 J20.2(GA) Dirty white

Black Not identified

21 No actinomycetes 22 J22.1(SCA) Brown Creamy Not identified 22 J22.2(SCA) White Creamy Not identified

22 J22.3(SCA) Yellow Creamy Not identified 22 J22.4(SCA) White Creamy Not identified 23 No actinomycetes 24 J24.1(AS) Dirty


24 J24.2(AS) Ash Dirty white

Not identified


Not identified

22


Not identified


Not identified

23

Appendix IV Isolation and Characterization of Actinomycetes from soil samples collected from Namche ( Height: 3446 Mtrs)

Soil sample number

Isolate number




1 No actinomycetes 2 N2(As) Small


3-9 No actinomycetes 10 N10.1 Ash Black Not identified 10 N210.1(As) Chalky


10 N10.3(As) Pin headed whitish


10 N10.4(As) Chalky Creamy Not identified 10 N10.5(As) Spreaded


10 N10.6(As) Chalky Creamy Not identified 10 N10.7(As) Chalky


11 No actinomycetes 12 N12.3(Sc) White White 13 No actinomycetes 14 N14.4(As) Chalky Creamy Not identified 15-19 No actinomycetes

24

Appendix V Isolation and Characterization of Actinomycetes from soil samples collected from Sagarmatha National Park ( Height: 3446 Mtrs)

SN Soil sample number

Isolate number




1 1 S1.1(SCA) White creamy Not identified 2 1 S1.3(SCA) Chalky


3 1 S1.1(AS) Ash Black Not identified 4 1 S1.1(GA) White

and red Black Not identified

5 1 S1.2(AS) Ash Dirty white

Not identified

6 1 S1.2(GA) Dirty white

Dirty white

Not identified

7 1 S1.3(AS) Dirty white

Dirty white

Not identified

8 1 S1.3(GA) Ash Black Not identified 9 1 S1.4(AS) Ash Black Not identified 10 1 S1.4(GA) Dirty


11 1 S1.5(AS) Black Black Not identified 12 1 S1.5(GA) Dirty


13 2 S2.1(SCA) White White Not identified 14 2 S2.2(SCA) White White Not identified 15 2 S2.3(SCA) Ash White Not identified 16 2 S2.1(AS) Yellow Creamy Not identified 17 2 S2.2(AS) Dirty










25


black Not identified

23 3 S3.1(GA) Yellow Yellow Not identified 24 3 S3.2(GA) Yellow Yellow Not identified 25 4 S4.1 White White Not identified 26 4 S4.2 Creamy Creamy Not identified 27 5 S5.1(GA) Pinkish

white yellow Not identified

28 5 S5(SCA) Creamy White Not identified 6 No actinomycetes found 29 7 S7.1(SCA) White White 30 7 S7.2(SCA) Dirty


31 7 S7.4(SCA) White White Not identified 8 No actinomycetes found 32 9 S9.2 Creamy yellowish Not identified 33 10 S10.2 Ash Black Not identified 11/12 No actinomycetes found 34 13 S13.1(AS) White White Not identified 35 13 S13.2(AS) White White Not identified 14 No actinomycetes found 36 15 S15.1(GA) Ash Black Not identified 37 16 S16.1 Ash Black Not identified 17-20 No actinomycetes found 38 21 S21.1(AS) Ash Blackish Not identified 39 21 S21.2(AS) Blackish Blackish Not identified 40 21 S21.3(AS) Blackish Blackish Not identified 41 21 S21.4(AS) Blackish Blackish Not identified 22 No actinomycetes found 42 23 S23.1(AS) Blackish Blackish Not identified 43 23 S23.2(AS) Blackish Blackish Not identified 44 23 S23.3(AS) Blackish Blackish Not identified 45 23 S23.4(AS) Creamy Creamy Not identified 24 No actinomycetes found 46 25 S25.1(AS) Dirty




48 25 S25.3(AS) Black Black at the edge

Not identified

49 25 S25.4(AS) Small white


26

colony, 50 25 S25.5(AS) Dirty


26-34 No actinomycetes found 51 35 S35.3 Yellow Yellow Not identified 52 36 S36(GA) Pinkish

white yellow Not identified

53 37 S37.2(SCA) White Creamy Not identified 54 37 S37.4(SCA) Ash white Not identified 38-40 No actinomycetes found 55 41 S41.3(AS) Dirty

white creamy Not identified

42/43 No actinomycetes found 56 44 S44.1(AS) Blackish Blackish Not identified 45 No actinomycetes found

27

Appendix VI Isolation and Characterization of Actinomycetes from soil samples collected from Tengboche ( Height: 3867 Mtrs)


Isolate number




1 1 T1.1(As) Dirty white White Streptoverticillium spp 2 1 T1.2(As) Chalky white White Ampularia/Actinoplans 3 2/3 No actinomycetes 4 4 T4.1 Dirty white White Probably nobel 5 4 T4.2 Chalky white Yellow Streptomyces spp 6 4 T4.3(Sc) Chalky white Yellow Streptomyces spp 7 4 T4.4(Sc) Chalky white Yellow Streptomyces spp 8 5/6 No actinomycetes 9 7 T7.1(As) White Dirty white Streptoverticillium spp 10 7 T7.3 White Yellow Streptomyces spp 11 7 T7.4(As) White White Streptoverticillium spp 12 7 T7.5(As) White White Streptosporangium spp 13 8/9 No actinomycetes 14 10 T10 White Yellowish Streptomyces spp 15 11 No actinomycetes 16 12 T12.2 Yellowish Black Streptomyces spp 17 12 12.3 White Black Streptomyces spp 18 13-19 No actinomycetes

28

Appendix VII Isolation and Characterization of Actinomycetes from soil samples collected from Tengboche to Pangboche (Height: 3867 - 3901 Mtrs ) SN Soil

sample number

Isolate number




1 1 PTT1.3(G) Dirty white

Black Streptomyces spp

2 1 PTT1.4(G) White White Streptomyces spp 3 1 P1.9(GA) Ash Black Streptomyces spp 4 1 P1.10(GA) Reddish Reddish Streptomyces spp 5 1 P1.11(GA) Dirty

white Dirty white

Streptoverticillium spp

6 1 P1.13(GA) White Brownish Streptoverticillium spp

7 1 P1.14(GA) Grey Dirty white

Streptomyces

8 1 P1.16(GA) White Creamy Streptoverticillium spp

9 1 P1.17(GA) White Black Streptoverticillium spp



12 1 PTT1.29 Dirty white

Black Strerptoverticillium spp

13 1 PTT1.30 Dirty white

Black Streptoverticillium spp

14 1 PTT1.31 Ash Black Strerptoverticillium spp

15 1 PTT1.32 Ash Black Streptoverticillium spp

16 1 PT1.33(G) White Black Streptomyces spp 17 1 PT1.34(G) White Black Streptomyces spp 18 1 P1.39(GA) Dirty

white Black Streptomyces spp

19 2 PTT2.2 White Creamy Streptomyces spp 20 2 PTT2.3(G) Dirty

white Dirty white

Streptomyces spp

2 No actinomycetes found

21 4 PTT4.3 Reddish white

Reddish Streptomyces spp

29

22 4 PTT4.5 Ash Greenish Streptoverticillium spp

5-7 No actinomycetes found

23 8 PTT8.1 White Yellowish Streptoverticillium spp

24 9 P9.1(GA) Blackish white

Black Kitasatospora /Actinoplans

25 9 P9.2(GA) Blackish white


26 9 PTT9.4 Brown Greenish Streptomyces spp 27 10 PTT10.1 White Creamy Streptomyces spp 28 10 PTT10.2 White Dirty

white Streptomyces spp

29 11 PTT11.3 Creamy Creamy Streptomyces spp 12 No actinomycetes

found 30 13 PTT13.1 White Creamy Streptomyces spp 31 13 PTT13.2 Chalky


32 13 PTT13.2 White Creamy Not identified 33 13 PTT13.3 Faded



34 18 P18.1(GA) Yellow Yellow Actinopolyspora/ Kitasatospora

35 18 PTT18.2 Yellow Yellow Streptoverticillium spp

36 18 PTT18.1 Yellowish Creamy Steptomyces spp 37 19 PTT19.1 Yellow Yellow Actinopolyspora/

Kitasatospora 38 19 PTT19.2 Yellow Yellow Actinosynnema spp 39 19 P9.1(GA) Yellowish Creamy Streptomyces spp 40 20 P20.1(GA) White Creamy Streptomyces spp 41 21 PTT21.1 Yellow Creamy Not identified 42 21 PTT21.2 Yellow Creamy Not identified 42 21 PTT21.3 Ash Creamy Not identified 22-23 No actinomycetes

found 43 24 P24.3(GA) White Creamy Not identified 44 25 P25.4(GA) Ash Black Not identified 45 25 P25.5(GA) Ash Black Not identified 46 25 P25.6(GA) Ash Black Not identified 47 25 P25.7(GA) Ash Black Not identified

30

48 25 P25.8(GA) Ash Black Not identified 49 26 PTT26.1 White Creamy Not identified 50 26 PTT26.2 White Creamy Not identified 51 26 PTT26.3 White Creamy Not identified 52 26 PTT26.4 White Creamy Not identified 53 26 PTT26.5 White Black Not identified 54 26 PTT26.7 White Yellowish Not identified 55 26 PTT26.8 White Yellowish Not identified 56 27 PTT27.1 Chalky

white Creamy Streptoverticillium

spp 57 27 PTT27.2 Yellowish Creamy Streptoverticillium

spp 58 27 PTT27.3 Yellow Creamy Streptoalloteichus

spp 59 27 PTT27.4 Ash Black Streptomyces spp 60 27 PTT27.5 Chalky


61 27 PTT27.15 Chalky white

White Streptomyces spp

62 27 P27.16(G) Chalky white

Black Streptoverticillium spp

63 27 PTT27.17 White White Streptomyces spp 64 28 PTT28.1 Ash Black Streptoverticillium

spp 65 28 PTT28.2 Ash Black Streptoverticillium

spp 66 28 PTT28.3 White Creamy Streptoverticillium

spp 67 29 PTT29.1 Cottony


68 29 PTT29.4 Cottony white


69 29 PTT29.5 Pink Creamy Amycolata 70 29 PTT29.6 White Yellowish Streptoverticillium

spp 71 29 PTT29.9 Chalky

white Dirty white

Streptomyces spp


72 38 PTT38.1 Ash Creamy Streptomyces spp 73 38 PTT38.2 White

margin Creamy Streptoverticillium

spp 74 38 PTT38.3 White Creamy Streptoverticillium

spp 39-67 No actinomycetes

31

found 75 68 PTT68.1 White Creamy 69-78 No actinomycetes

found 76 79 PTT79.1 Ash Creamy Streptomyces spp 77 79 PTT79.2 Ash Creamy Streptomyces spp 80-92 No actinomycetes

found 78 93 PTT93.1 White

spreaded Creamy Streptomyces spp

79 93 PTT93.2 White embadded


80 94 PTT94.1 Bluish Bluish Streptomyces spp 95-101 No actinomycetes

found 81 102 PTT102.2 Bluish Bluish Streptomyces spp 82 103 PTT103.1 Bluish Bluish Streptomyces spp

32

Appendix VIII Isolation and Characterization of Actinomycetes from soil samples collected from Pheriche (Height: 4243 Mtrs)


Isolate number





1 4 Pr4.1 Creamy Creamy Not identified 2 4 Pr4.2 Creamy Creamy Not identified 5 No

actinomycetes found

3 6 Pr6.1 Yellow Yellow Not identified 4 6 Pr6.2 Ash Black Not identified 5 6 Pr6.3 Yellow Yellow Not identified 6 6 Pr6.4 Dark ash Creaqmy Not identified 7 6 Pr6.5 transparent Creamy Not identified 8 6 Pr6.6 Ash Black Not identified 9 6 Pr6.7 Ash Creamy Not identified 10 6 Pr6.8 Ash Black Not identified 11 7 Pr7.4 Creamy Creamy Not identified 12 8 Pr8.1 White Creamy Not identified 13 8 Pr8.2 White White Not identified 9-15 No

actinomycetes found

14 16 Pr16.1 Creamy embedded



15 38 Pr38 Yellow Creamy Not identified 39-53 No

actinomycetes found

33

Appendix IX Isolation and Characterization of Actinomycetes from soil samples collected from Thukla to Lobuche (Height: 4500-5000Mtrs) Number of Samples: 22 Number of Isolates: Nil

34

Appendix X Isolation and Characterization of Actinomycetes from soil samples collected from Lobuche (Height: 5000 - 5300 Mtrs) SN Soil

sample number

Isolate number




1 1. Lob1(1) Small white


2 1. Lob1(2) Small white

Creamy Streptomyces spp

3 2. Lob2(2) White Creamy Streptomyces spp 4 3. Lob3(1) Grey Greenish Streptomyces spp 4 No actinomcetes found 5 5. Lob5(2) grey Creamy Miccromonospora 6 6 Lob 6.2 White Creamy Streptomyces 7 7. Lob7(1) Small

white Creamy Streptomyces

8 7. Lob7(3) Yellow Creamy Intrspornioum 9 7. Lob7(4) Yellow Yellow Streptomyces 10 8. Lob7 (9) Yellow Yellow Micromono/Streptoverticillium

/Catellospora 11 8. Lob8(1) Yellow Yellow Strerptoverticillium 12 8. Lob8(4) Yellow Yellow Streptoverticillium 13 8. Lob8(5) Yellow Yellow Similar to Nocardiopsis 14 8. Lob8(6) Yellow Yellow Saccharapolyspora 15 8. Lob8(7) Yellow Yellow Intrasoprangium 16 8. Lob8(8) Yellow Yellow Intrasporangium 17 8. Lob8(9) Yellow Yellow Intrasporangium 18 8. Lob8(10) Yellow Yellow Intrasporangium/catello

/Actinopoly 19 8. Lob8(11) Yellow Yellow Kitasatospora 20 8. Lob8(12) Yellow Yellow

might be New 21 8. Lob8(13) Yellow Yellow Intrasporangium 22 8. Lob8(14) Yellow Yellow Actinopolyspora

/kitasatospora 23 8. Lob8(16) Yellow Yellow Saccharopolyspora 9 No actinomcetes found 24 10. Lob10(1) White Creamy Streptoverticillium 25 10. Lob10(2) Brown Brown Streptomyces spp 26 11. Lob11(1) Ash Creamy Streptomyces spp 27 11. Lob11(2) Yellow Creamy Streptomyces spp 28 11. Lob11(3) Bluish Creamy Streptomyces spp

35

white 29 11. Lob11(4) White Creamy Nocardia spp 30 11. Lob11(5) Yellowish


31 11. Lob11(6) Creamy Creamy Streptomyces spp 32 12. Lob12(1) White Creamy Streptomyces spp 33 12. Lob12(2) White Dirty


34 12. Lob12(3) Brown Black Streptomyces spp 35 13. Lob13(1) Greenism

Yellow Yellow Micromonospora

14 No actinomcetes found 36 15. Lob15(1) Creamy Yellow Actinopolyspora 37 15. Lob15(2) Yellow Creamy Streptomyces 38 15. Lob15(3) White Creamy Streptomyces spp 39 15. Lob15(4) White Creamy Streptomyces spp 40 16. Lob16(1) Yellow Yellow Saccharopolyspora 41 16. Lob16(2) Yellow Yellow Strepoericillum 17 No actinomcetes found 42 18. Lob18(1) White Creamy Srteptomyces 19 No actinomcetes found 43 20. Lob20(1) Dirty

white Reddish white

Streptomyces

44 20. Lob20(2) White Creamy Streptomyces spp 45 Lob20(3) White Creamy Streptomyces spp 46 20. Lob20(4) White Creamy Streptomyces spp 47 20. Lob20(5) Light

brown Creamy Streptomyces spp

48 21. Lob21 Grey Creamy Micromonospora spp 49 Lob25.7 White Creamy Streptomyces spp 50 Lob25.8 Small

White Creamy Streptomyces spp

51 22-27 No actinomycetes found 52 28. Lob28(1) Grey Dirty


53 28. Lob28(2) Yellowish Yellow Saccharopolyspora/ Streptomyces

29 No actinomycetes found 54 30 Lob30(As) Yellow Yellow Streptomyces spp 31-35 No actinomycetes found 55 36 Lob36 Yellow Yellow Streptoverticillium 56 Lob42.4 Yellowish White Probably new 57 37-49 No actinomycetes found 58 50. Lob50 Creamy Creamy Catellospora spp 51 No actinomycetes found

36

59 52. Lob52(1) Grey Brown Catellospora/ Streptomyces

60 52. Lob52(2) Yellow Yellow Micromono/Streptoverticillium /Actinopolyspora

53-54 No actinomycetes found 61 55 Lob55 White Yellowish

white Saccharopolyspora spp

62 56-58 No actinomycetes found 63 59 Lob 59 White Creamy Streptoverticillium spp 64 60. Lob60(1) Chalky

white White Streptoverticillium spp

65 60. Lob60(2) Yellow Greenish yellow

Streptoverticillium spp

66 60. Lob60(3) Grey Grey Actinosynema 67 60. Lob60(4) White Creamy

white Streptomyces

68 61. Lob61 Chalky white

White Streptoverticillium

69 62. Lob62 White White Streptoverticillium 70 63. Lob63 White Creamy

white Streptomyces

71 64. Lob64 Chalky white

Creamy white

Streptomyces

72 65. Lob65 Yellow Yellow Streptoverticillium 66-93 No actinomcetes found 73 94 Lob 94 White White Streptomyces 66-102 No actinomycetes found 74 103 Lob103.1 Chalky

white White Streptomyces spp

75 103 Lob103.2 Chalky white


76 103. Lob103.3 White White Streptomyces spp 104-106 No actinomycetes found 77 107 Lob 107 White White Streptomyces spp 104-

106. Lob107(As) White Dirty

white Streptoverticillium

78 110. Lob110 White Creamy white

Streptomyces

111-136 No actinomycetes found 79 137 Lob 137.1 Chalky

white Creamy white

Nocardiops/s/spirillospora/ Streptosporangium

80 137 Lob137.2 Chalky white

White Probably a new

138-141 No actinomycetes found 81 142 Lob142 Chalky White Kibdellosporium

37

white Streptoalloteichus 82 142 Lob 142.3 Yellow Yellow Intrasporangium 143-156 No actinomycetes found

title of the project biodiversity of khumbu region: population

Documents