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International Journal of Research in Pharmacy and Biosciences Volume 3, Issue 3, March 2016, PP 19-31 ISSN 2394-5885 (Print) & ISSN 2394-5893 (Online)

New Records of and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain

Qais Yusuf M. Abdullah1, Maher Ali. Al-Maqtari2, Ola, A A. Al-Awadhi3 Abdullah Y. Al-Mahdi 4 Department of Biology, Section Faculty of Science, Sana'a University1, 3, 4 Department of Chemistry, Biochemistry Section, Faculty of Science, Sana'a University2

ABSTRACT Actinomycetes are ubiquitous soil-dwelling saprophytes known to produce secondary metabolites may of which are . 20 soil samples were collected from three different sites at high altitude environments surrounding Sana'a city, which ranged from 2300-3000 m above sea level as a prime source of promising native rare actinomycetes. 516 actinomycetes isolates were isolated in pure culture and five selective pretreatment isolation methods. Out of 232 isolates, 26 actinomycetes showing good activity. The identification of 26 selected actinomycetes based on cultural morphology, physiology and biochemical characterization. From the preceding , thirteen actinomycetes were recorded for the first time from Yemeni soil of these: four non-Streptomyces namely (Intrasporangium sp., Nocardiodes luteus, Sporichthya polymorpha and Streptovirticillium cinnamoneum) and nine Streptomyces namely (S. anulatus, S. celluloflavus, S. cellulosae, S. chromofucus, S. erythrogriseus, S. flavidvirens, S. flavissimus, S. globosus and S. griseoflavus). The results of this study suggested that the soil of high mountains such as Sana'a Mountains could be an interesting source to explore new strains that recorded for the first time in Yemen, Sana'a City. Keywords: Sana'a Higher Mountain Soils, Actinomycetes, isolation methods, identification, new records

INTRODUCTION Actinomycetes usually defined as Gram-positive bacteria that have high G+C (>55%) content in their DNA (Embley and Stackebrandt, 1994). It characterized by forming branching filaments with hyphae and asexual spores. In addition, they were considered an intermediate group between bacteria and fungi but now are recognized as prokaryotic organisms (Lechevalier and Lechevalier, 1967; and Goodfellow and Williams, 1983). Most soil actinomycetes are neutrophils, growing between pH 5.0 and 9.0 with an optimum close to neutrality. In addition, most soil actinomycetes are strict aerobes (Goodfellow and Williams, 1983). Moreover they are important in soil biodegradation and humus formation (Stach and Bull, 2005), and produce several volatile substances like geosmin responsible for the characteristic “wet earth odor” (Wilkins, 1996). Non-Streptomyces is non-taxonomic term which called also rare actinomycetes (non-Streptomyces = rare actinomycetes). Non-Streptomyces defined as actinomycetes other than Streptomyces sp., which are less frequent in terrestrial soil (Okazaki, 2006 and Schwartz and Schwartz, 1979). Non- Streptomyces growth normally slower than Streptomyces and requires complicated procedure for isolation, preservation and cultivation in some genera (Lazzarini et al., 2000). Therefore, they were regarded as less exploited microorganism. It might represent a unique source of novel biologically active compounds (Baltz, 2006). The most influence factors in distribution and density of non-Streptomyces in the terrestrial such as high mountains environment were texture, pH, temperature and salinity (Hop et al., 2011) and organic carbon and total nitrogen (Ghanem et al., 2000). For this reason, using pretreatment of soil samples, by both drying and heating, for stimulating the isolation of spores of rare actinomycetes (Naikpatil and Rathod, 2011). *Address for correspondence [email protected] International Journal of Research in Pharmacy and Biosciences V3● I3● March 2016 19 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” Recently, rare actinomycetes have been shown to be an important source of novel and useful (Lazzarini et al., 2000). Nowadays, most of isolation of non-Streptomyces actinomycetes that isolated from high altitude ecosystems, such as Micromonospora sp., Saccharomonospora sp., Streptosporangium sp., Nocardia sp. and Dactylosporangium sp. (Kim et al., 1998) Streptoverticillium sp. and Nocardia sp. (Katsifas et al., 1999). Nocardiopsis sp., Nocardia sp., Amycolatopsis sp., Actinomadura sp., Nonomuraea sp., Microtetraspora sp and Pseudonocrdia sp. (Hozzein et al., 2004). Actinoplanes sp. (Gadelhak et al., 2005). In addition to, Micromonospora sp., Intrasporangium sp. and Streptosporangium sp. (Gurung et al., 2009). Saccharothrix sp. and sp. (Bharti et al., 2010). As well as Kitasatosporia sp. (Jeffrey et al., 2011). Micromonospora sp., Saccharothrix sp., Streptosporangium sp. and Cellulomonas sp. (Ding et al., 2012). Besides, Nocardia sp., Pseudonocardia sp., Nocardiopsis sp., Micromonospora sp., Rhodococcus sp. and Streptosporangium sp. (George et al The present research was performed to isolate specific Streptomyces and Non Streptomyces actinomycetes colonies from high altitude environments surrounding Sana'a for the first time. Such environment not studied yet in Yemen and attracted for exploring for new strains records in Yemen. MATERIALS AND METHODS Collection of Samples Twenty soil samples were collected on January 2011 to March 2012 from three different locations in high altitude environment surrounding Sana'a basin 2300-3000 m above sea level. Soil samples were collected at a depth of 5-10 cm after removing approximately 3 cm of the soil surface. Pretreatment of Samples Actinomycetes were isolated using both selective and non-selective methods. Samples were passed through 2 mm sieve filter to remove gravel and debris (Hayakawa and Nonomura, 1989). In this study, we employed five selective methods for isolation rare actinomycetes (non-Streptomyces) from soil as follows:  Samples were air-dried at room temperature for seven days. This helps in decreasing the population of gram-negative bacteria (Jeffrey et al., 2008).  Dry heat in hot air oven at 55ºC for 15 min (Baskaran et al., 2011).  Moist heat treatment. Soil suspensions were heated at 50°C for 10 min in a water bath (Sasiavam et al., 2008).  Samples were air-dried at room temperature for 7 days after that the samples were keeping 50oC for 10 min (Pisano et al., 1986).  Samples were air-dried at room temperature for 7 days after that Soil suspensions were heated at 50°C for 10 min in a water bath (Ambarwati et al., 2012). Isolation of Actinomycetes Method Actinomycetes were isolated from soil using serial dilution method on starch casein agar plate's incubation at 28°C for 7-10 days, actinomycetes colonies were purified by streak plate technique on cultivation medium (Kuster & Williams, 1964). Identification of Most Active Actinomycetes Morphological Studies The isolates characterized of by morphological studies according to Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994). Physiological Characteristics Growth parameters were studies first, the salt tolerance, isolates were grown in different concentrations of sodium chloride (0,2,4,6,8 and 10%) (Tresner et al., 1968). Second, isolates were incubated at different temperature range from 24-60 oC. Third, the ability to grow at range of pH range from 3-11(Kokare et al., 2004).

20 International Journal of Research in Pharmacy and Biosciences V3 ● I3 ● March 2016 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” Biochemical Characteristics Many characteristic were studied. Different carbon and nitrogen sources was carried out according to (Pridham and Gottlieb, 1948 and Lambert and Loria, 1989). The ability of the isolates to produce different enzymes as Amylase and Gelatinase (Collins et al., 1995). Casease ( Kanavade,2003) ,Catalase (Schumann et al., 1997), Cellulase (Bradner et al., 1999).Chitinase (Rowbotham and Cross, 1977), Lipase (Deshmukh, 1997), Pectinase (Hankin and Anagnostakis, 1975), and Urease (Gordon et al., 1974). Isolates were tested for Antibiotic sensitivity and antimicrobial activities by agar diffusion method according to the method of (Bauer et., al 1966). The ability of the isolate to motile and produce hydrogen sulphide determined (Clarke, 1953). Nitrate reduction was performed according to the method of (Gottlieb, 1961). Chemotaxonomy Determination of Diaminopimelic acid (DAP) and sugar pattern was carried out according to (Becker et., al 1965, and Staneck and Roberts, 1974). Probabilistic Identification of Bacteria Software Isolates of actinomycetes were identified using PIB Win (Probabilistic identification of bacteria) software (Bryant, 1993). RESULTS Isolation of Actinomycetes The current study detected 516 isolates of actinomycetes that were isolated from twenty soil samples collected from three extreme environments of high altitude at 2200 m above sea level in Sana'a basin Yemen (Table, 1). Table1. Twenty collected samples from different elevation of high altitude ecosystem surrounding in Sana'a basin. Samples Elevation (m) Ecosystem no. of total colonies 1 2260 mountain 69 2 2260 basin 64 3 2220 basin 179 4 2260 basin 245 5 2240 basin 190 6 2280 mountain 142 7 2280 basin 195 8 2210 basin 262 9 2260 basin 246 10 2280 basin 150 11 2220 basin 29 12 2300 mountain 175 13 2220 valley 206 14 2400 valley 66 15 2298 valley 129 16 2210 basin 268 17 2640 mountain 33 18 2260 basin 22 19 2300 basin 176 20 2306 basin 95 Total count 2941 These actinomycetes were isolated using five selective pretreatment isolation methods. Out of 516 isolated actinomycetes, 232 isolated actinomycetes were selected for primary screening (Table, 2). Twenty-six isolates were collected the most active actinomycetes that selected for further studies. The Streptomyces sp. contributed the largest spectrum of species among twenty-two isolates and the remaining four were identified as non-Streptomyces species International Journal of Research in Pharmacy and Biosciences V3● I3● March 2016 21 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” Table2. Non-Streptomyces and Streptomyces species isolated from three different high altitudes soils ecosystem and different elevation in Sana'a. Sr. no Isolate No. Group Identified species Ecosystem 1 NSA 201 Streptovirticillium cinnamoneum basin 2 NSA 202 Non- Intrasporangium sp. basin Streptomyces 3 NSA 203 species Sporichthya polymorpha valley 4 NSA 204 Nocardiodes luteus basin 5 SAS 101 Streptomyces erythrogriseus basin

6 SAS 102 basin 7 SAS 103 Streptomyces atroolivaceus basin 8 SAS 104 Streptomyces griseoflavus basin 9 SAS 105 Streptomyces Streptomyces chromofucus basin 10 SAS 106 species mountain 11 SAS 107 mountain

12 SAS 108 Streptomyces flavidvirens basin

13 SAS 109 Streptomyces exfoliates basin 14 SAS 110 valley 15 SAS 111 Streptomyces flavissimus mountain 16 SAS 112 Streptomyces celluloflavus basin 17 SAS 113 Streptomyces filipinensis mountain 18 SAS 114 basin

19 SAS 115 Streptomyces celluloflavus basin

20 SAS 116 Streptomyces glaucescens basin 21 SAS 117 valley 22 SAS 118 Streptomyces anulatus valley 23 SAS 119 basin 24 SAS 120 Streptomyces cyaneus basin 25 SAS 121 Streptomyces cyaneus basin

26 SAS 122 Streptomyces flaveolus valley Microscopic Methods Micro-morphological characteristics of isolated actinomycetes studied using coverslip method. All isolates were non-acid fast stain. The results of microscopic features showed that, nineteen isolates have chains of spores with different morphology as shown in (Table 3), NSA 201, SAS 105, SAS 106, SAS 107, SAS 108, NSA 203, SAS 110, SAS 111, SAS 113, SAS 114, SAS 115, SAS 116, SAS 117, SAS 118, SAS 119, SAS 120, SAS 121, NSA 204 and SAS 122. Electron microscope examination for isolates (NSA 203 and NSA 201) show smooth spores. Table3. Colonial morphology characteristics for isolated non-Streptomyces actinomycetes and Streptomyces sp. Iso Isolate S Shape SSize: mm c Consistency Edges Structur El Elevation Visible in: hrs late hape ize: mm y dges e evation sible in: hrs *SAS101 R 2 *H *E O *C 48 **NSA20 R <1 *D E O *El 24 SAS1 102 R 2 D E O *F 48 SAS 103 R <1 H E O F 48 SAS 104 R 3 H E O C 48 SAS 105 R 5 H E O C 48 NSA 202 R >1 H E O C 72 SAS 106 I 4 D *U O C 72 SAS 107 R <1 H E O F 48 SAS 108 R 6 D E O C 48 SAS 109 R 2 D E O C 48 NSA 203 R >1 D E O F 48 SAS 110 R 2 D E O C 96 SAS 111 R 4 H E O El 48 SAS 112 R 2 D E O F 72

22 International Journal of Research in Pharmacy and Biosciences V3 ● I3 ● March 2016 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” SAS 113 R 4 H E O F 72 SAS 114 R 2 D E O F 72 SAS 115 R 4 D E O C 48 SAS 116 I 1 H U O F 48 SAS 117 R 3 D E O El 72 SAS 118 R 2 H E O El 48 SAS 119 I 2 D U O El 48 SAS 120 R 2 H E O C 72 SAS 121 R <1 H E O F 72 NSA 204 R <1 H E O C 48 SAS 122 R 1 D E O El 48 *SAS= Streptomyces sp., **NSA= Non-Streptomyces. R= Regular, I= Irregular, H= Hard, D= Dry, E= Entire, U= Undulate, O= Opaque, C= Convex, El= Elevated, F= Flat Physiological Characteristics Physiological characteristics of isolated actinomycetes from different habitat soils collected from high mountain ecosystem in Sana'a were as following: Growth in Different Temperature Growth of isolated actinomycetes in different temperature contrast as follows: All isolates grew at 37oC and 24oC whereas six isolates (SAS 106, SAS 109, SAS 110, SAS 115, SAS 117 and SAS 122) grew at 45oC and 60oC. In addition to four isolates (SAS 107, SAS 108, SAS 114 and SAS 116) grew at 45oC and failed to grow at 60oC as showed in (Figure, 1). Growth with Different NaCl Concentration Growth of isolated actinomycetes with different NaCl concentration was as follows: all isolates could grow in 0.0% NaCl except SAS 101 as well as all isolates could grow in 2.0% NaCl except two isolates (SAS 101 and NSA 202). All isolates could grow in 4.0% NaCl. However, six isolates could tolerate 6.0% NaCl (SAS 101, NSA 201, SAS 105, SAS 106, NSA 203 and SAS 119). In addition to SAS 101 and NSA 203 could tolerate 8.0% and 10.0% NaCl; also NSA 201 can grow in 8.0% NaCl but cannot in 10.0% NaCl (Figure, 2). Growth with Different pH Growth of isolated actinomycetes with different pH contrast as follows: all actinomycetes isolates could grow at pH 5, 9, and 11 except one isolate (NSA 202). Whereas at pH 3 eleven isolates could not grow (SAS 103, NSA 202, SAS 108, SAS 109, SAS 111, SAS 112, SAS 113, SAS 114, SAS 115, SAS 116 and NSA 204) as shows in (Figure, 3).

Fig1. Growth of 26 actinomycetes isolates at different temperature.

Fig2. Growth of actinomycetes isolates at different NaCl concentration. International Journal of Research in Pharmacy and Biosciences V3● I3● March 2016 23 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain”

Fig3. Growth of 26 actinomycetes isolates at different pH. Enzymes Activity Ability of isolated actinomycetes to produce enzymes was varied. All isolates hydrolyzed starch except NSA 204 and SAS 111. The isolates could not hydrolyze neither casein nor chitin. All isolates produced lipase except seven isolates (NSA 202, SAS 116, SAS 118, SAS 119 and NSA 204). SAS 105 and SAS 113 could not produce gelatinase. Whereas three isolates could not produce cellulose (NSA 201, NSA 202 and SAS 119). But all isolates produce urease. In addition to twelve isolates could produce pectinase (SAS 101, SAS 102, SAS 103, SAS 105, SAS 106, SAS 107, NSA 203, SAS 111, SAS 116, SAS 117, SAS 121 and NSA 204) whereas four isolates could not produce catalase (NSA 202, SAS 109, SAS 120 and SAS 122) showed in (Table 4). Table4. Screening of 9 enzymes activity of isolated actinomycetes. Isolate Amylase Casease lipase Gelatinase Cellulase Urease Chitinase Pectinase Catalase SAS 101 + - + + + + - + + NSA 201 + - + + - + - - + SAS 102 + - + + + + - + + SAS 103 + - + + + + - + + SAS 104 + - + + + + - - + SAS 105 + - + - + + - + + NSA 202 + - - + - + - - - SAS 106 + - + + + + - + + SAS 107 + - + + + + - + + SAS 108 + - + + + + - - + SAS 109 + - + + + + - - - NSA 203 + - + + + + - + + SAS 110 + - + + + + - - + SAS 111 + - + + + + - + + SAS 112 - - + + + + - - + SAS 113 + - + - + + - - + SAS 114 + - + + + + - - + SAS 115 + - + + + + - - + SAS 116 + - - + + + - + + SAS 117 + - + + + + - + + SAS 118 + - - + + + - - + SAS 119 + - - + - + - - + SAS 120 + - + + + + - - - SAS 121 + - + + + + - + + NSA 204 - - - + + + - + + SAS 122 + - + + + + - - -

H2S Production Test

H2S production was studied for isolated actinomycetes. Most of the isolates were not H2S producer except four isolates (SAS 101, SAS 102, SAS 110 and NSA 204) (Figure 4). Motility Test Motility was studied for isolated actinomycetes. Most of them were non-motile except three isolates were motile isolates (SAS 103, NSA 203 and SAS 115) (Figure 4).

24 International Journal of Research in Pharmacy and Biosciences V3 ● I3 ● March 2016 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” Nitrate Reduction Test Nitrate reduction was studied for isolated actinomycetes. Thirteen isolates have ability to reduce nitrate (SAS 101, NSA 201, SAS 102, SAS 103, SAS 105, SAS 108, SAS 109, NSA 203, SAS 114, SAS 115, SAS 117, SAS 118, and SAS 122). Other isolates could not reduce nitrate (Figure 4).

Fig5. H2S production, Nitrate reduction and motility tests for 26 isolated actinomycetes during this investigation. Chemotaxonomy Cell wall was analyzed for all isolates. Amino acids were L-DAP and glycine and no detected diagnostic sugars (Plate, 1).

Amino Acid analysis Sugar analysis Plate1. Amino acids and sugar analysis of cell wall. All identified actinomycetes were gram positive, aerobic, non-acid alcohol fast, LL-DAP cell wall amino acid and did not have diagnostic sugars. The most active actinomycetes against tested pathogenic fungi were 26 actinomycete isolates. Which can be classified four strains of actinomycetes identified as non-Streptomyces species and 22 isolates of actinomycetes identified as Streptomyces species. Four genera belong to 25 and 22 group according to Bergey's Manual of Determinative Bacteriology (1994). Group 22: (genus: Nocardiodes sp.). Group 25: Streptomyces sp. and related genera.Genus: Intrasporangium sp., Sporichthya sp., Streptovirticillium sp., Streptomyces sp. Non-Streptomyces Species Only four isolated actinomycetes that identified as non-Streptomyces (rare actinomycetes) species namely: Intrasporangium sp., Nocardiodes sp., Sporichthya sp., and Streptovirticillium sp. They characterized by the following characterizations: they cannot grow at 45oC, utilized glucose, galactose, lactose, mannitol, xylose, arabinose, rhamnose, fructose and ribose. Acid produce from glucose, and cannot produce acid from lactose, dextrin and xylose. Also, can utilized aspargine, proline, glycine and histidine. They produced gelatinase and urease, and cannot produce casease.

International Journal of Research in Pharmacy and Biosciences V3● I3● March 2016 25 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” They are sensitive to Gentamycin, , and Novobiocin. Cell wall amino acids were LL-DAP and glycine. No sugar was found in whole cells. The taxonomic levels of isolated and identified non-Streptomyces species classification (Table, 5) according to Bergey's Manual of Determinative Bacteriology (1994). Table5. Classification of identified non-Streptomyces species according to Bergey's Manual of Determinative Bacteriology (1994). Phylum Class Order Family Genus & Species Actinobacteria Actinomycetales Intrasporangium sp. Plate 2 Actinobacteria Actinobacteria Actinomycetales Nocardioidaceae N. luteus Plate 3 Actinobacteria Actinobacteria Actinomycetales Sporichthyaceae S. polymorpha Plate 4 Actinobacteria Actinobacteria Actinomycetales S. cinnamoneum Plate 5

Plate2. Colony of NSA 202 isolate was identified as Plate3. Colony of NSA 204 isolate that identified as N. Intrasporangium sp. luteus.

A. Colonies of S. polymorpha B. Electron microscope im ge (8000×) of spore chain of S. polymorpha

Plate4. NSA 203 isolate which was identified as S. polymorpha.

A.Colonies of S. cinnamoneum B. Electron microscope image (8000×) of spore chain of S. cinnamoneum.

Plate5. Colony of NSA 201 isolate was identified as S. cinnamoneum. DISCUSSION High altitude mountain habitation is likely to harbor unusual types of microorganisms while poorly studied habitation increases chance of finding novel microorganisms (Gurung et al., 2009). The identified isolates obtained during this investigation can be classified into two categories: the first one is 4 (15%) non-Streptomyces species identified as following: S. cinnamoneum, Intrasporangium sp., S. polymorpha, and N. luteus.

26 International Journal of Research in Pharmacy and Biosciences V3 ● I3 ● March 2016 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” The second category is the genus Streptomyces species, which was the dominant and contributed the largest spectrum of species among 22 (85%) isolates namely: S. erythrogriseus, S. globosus, S. atroolivaceus, S. griseoflavus, S. chromofucus, S. flavidvirens, S. exfoliates, S. flavissimus, S. filipinensis, S. celluloflavus, S. cellulosae, S. diastaticus, two isolates of each of the following: S. flaveolus, S. anulatus, S. celluloflavus and S. glaucescens and three isolates of S. cyaneus. Isolation of actinomycetes has always been faced with difficulties in comparison to their competitors like other bacteria and fungi. This may be due to their long incubation period (Gurung et al., 2009). In this respect, previous studies reported that Streptomyces species were the most dominant actinomycetes inhabited high altitude like study of (Ahmed, 2003; Al-Mahdi, 2005; Al-Mekhlafi, 2007; Gurung et al., 2009; Raja et al., 2010; Varghese et al ; Kumar et al., 2013; Jiang et al., 2013 and Al Zubairy, 2014). (Ahmed, 2003) collected soil samples from Sanaʼa, Taiz and Hadramot Identified isolates were belonging to Streptomyces sp. (Al-Mahdi, 2005) found the majority of isolated actinomycetes were belonging to Streptomyces sp. (76%). The other isolates were Micromonospora sp., Catellatospora sp., Actinomadura sp., Streptoverticillium sp., Saccharopolyspora sp., Kitasatospora sp., Nocardia sp., Thermoactinomyces sp. and Actinopolyspora sp., which isolated actinomycetes from soil samples collected from Sanaʼa, India, Hodeida, Aden, Makah and Jeddah (Al-Mekhlafi, 2007) Identified actinomycetes were belonging to Streptomyces sp. which isolated from four regions (Sanaʼa, Taiz, Ibb and Hodeida) (Gurung et al. 2009) found that seventy-nine actinomycetes isolated from soils of Kalapatthar mountain (5545 m), Mount Everest region. Out of them 27 actinomycetes isolates were identified as Streptomyces (16 isolates), Micromonospora (2 isolates), Intrasporangium (1 isolate), and Streptosporangium (1 isolate). Also (Raja et al. 2010) isolated actinomycetes from the soil of Rothang hill, Himachal Prethesh, India at a distance of 4061 km from the sea level. They found a total six actinomycetes were isolated from the soil sample and identified as Intrasporangium sp., Dactylsporangium sp., Micromonospora sp., Streptoverticillium sp. and two Streptomyces sp., However (Abbas , 2009) found that 82 actinomycetes were isolated from different site of Iraqi soil’s at a distance of 3611 m from the sea level. All isolates were found to be belonging to genus Streptomyces. In addition to (Varghese et al ) studied actinomycetes from high altitude grassland soils of Eravikulam National Park in Idukki District, Kerala, India at an altitude of 1900-2400 m above MSL. They identified (36) isolates revealed that most of the isolates belonged to the genus Streptomyces (17) followed by Nocardia (9), Micromonospora (3), Pseudonocardia (2), Streptosporangium (2), Nocardiopsis (2) and Saccharomonospora (1). Whereas (Kumar et al., 2013) isolated actinomycetes from valleys (below 700 m), mid hills (800- 2000 m) and high hills (above 2000 m) in Uttarakhand, India. They observed a total of 512 actinomycetes were isolated which were found to belong the 14 different genera of actinomycetes (Streptosporangium, Actinomadura, Nocardia, Nocardiodes, Saccharopolyspora, Thermoactinomycetes, Amycolatopsis, Micromonospora, Microbispora, Intrasporangium, Planobispora, Nocardiopsis and Geodermatophilus). Mainly the genus Streptomyces was dominant in all the soil samples. Another study of (Jiang et al. 2013) found that 782 actinomycetes strains isolated from Emei Mountain western China .The highest peak is 3099 m. Selected 86 strains of them were identified most of them belong to Streptomyces genus and the other were Actinomadura, Dactylosporangium, Micrococcus, Micromonospora, Mycobacterium, Nocardia, Nonomuraea, Promicromonospora, While eighteen strains were unidentified. (Al Zubairy, 2014) isolated actinomycetes from Sanaʼa, Dhammar, Ammran and Mahwit She Identified most of the isolates were belonging to the genus Streptomyces sp. Other studies mentioned that rare non-Streptomyces were the dominant in highland ecosystem as study of (George et al., 2012 and Al-Hulu et al., 2012). International Journal of Research in Pharmacy and Biosciences V3● I3● March 2016 27 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” (George et al. 2012) studied 52 actinomycetes isolated from various mountain plantation sites at Kerala in India, which spreads along Manimala River basin (Mundakkayam–Peruvanthanam region) (75-450 m above MSL) and Pamba river basin (Ranni Forest division) (440 m above MSL). The common isolates were identified as Streptomyces, Nocardia, Pseudonocardia, Nocardiopsis, Micromonospora, Rhodococcus and Streptosporangium. About 33% of isolates were identified as various species of Nocardia. (Al-Hulu et al. 2012) isolated twenty isolates of actinomycetes from Babylon province in Iraq. of these 4 isolates were belonged to Streptomyces sp. which represents (20%) of all recovered actinomycetes organisms. The number of actinomycetes isolated was varied in the sampling areas; this could be due to the differences in the chemical composition of the soil (Tian et al., 2004) and this may be partly attributed to the geographical location of the soil. High diversity of actinomycetes may depend on the ability of these organisms to survive and compete in this extreme environment. The survival of the microorganisms in such harsh and challenging habitation might be due to their adaptation to the environment and ability to produce resistant structures like spores (Gurung et al., 2009). In Yemen, fewer studies were focus on isolation of actinomycetes from different soil environments. During this investigation recorded 13 actinomycetes for the first time in Yemen, namely: Intrasporangium sp., N. luteus, S. polymorpha, S. anulatus, S. celluloflavus, S. cellulosae, S. chromofucus, S. erythrogriseus, S. flavidvirens, S. flavissimus, S. globosus, S. griseoflavus and S. cinnamoneum. In this respect some of previous species were isolated from different high altitudes such as Intrasporangium sp. isolated from Kalapatthar mountain (5545 m), Mount Everest region (Gurung et al., 2009), N. luteus isolated from valleys (below 700 m), mid hills (800-2000 m) and high hills (above 2000 m) in Uttarakhand, India (Kumar et al., 2013), S. anulatus isolated from different rhizospheric semi-arid soils collected in Algeria (Aouar et al., 2012), S. erythrogriseus and S. globosus from different regions of Iran ( Tabrizi et al., 2013), S. chromofucus and S. flavissimus from different areas in Greek territory (Katsifas et al., 1999), S. griseoflavus from Chiang Mai province, Northern Thailand (Thampayak et al., 2008). CONCLUSIONS Higher mountains in Sana'a are very rich in actinomycetes and differ according to the soil site and topography. Soil conditions in high mountains are extremely variable and reflect a diversity of geologic topographic, vegetation condition that influence soil formation and composition of microorganisms. The identification of the rare actinomycetes isolated in this study was attempted using morphological, physiological, biochemical characterization showed that the occurrence and distribution of rare actinomycetes species were less frequent than the Streptomyces. Whereas identified actinomycetes include 4 non-Streptomyces and the dominant Streptomyces sp. with 22 isolates. Isolated actinomycetes have good growth on (SCA); can utilize glucose as a carbon source and histidine as a source of nitrogen. Analyzing of cell wall showed that amino acids were L-DAP. New species and genera records of actinomycetes in Republic of Yemen as Intrasporangium sp., N. luteus, S. polymorpha, S. anulatus, S. celluloflavus, S. cellulosae, S. chromofucus, S. erythrogriseus, S. flavidvirens, S. flavissimus, S. globosus, S. griseoflavus and S. cinnamoneum. These significant results make the new record strains suitable for further investigation and industrial application exploitation. ACKNOWLEDGEMENTS The authors are very grateful to Sana'a University, Faculty of Science, Department of Biology, for help in analytical analysis and providing Infrastructure facilities. Authors are also grateful to Al.saeed Foundation REFERENCES

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