87 Arab Univ. J. Agric. Sci., Ain Shams Univ., Cairo, 14(1), 87-103, 2006 NUMERICAL STUDY ON SOME ACTINOMYCETES ISOLATED FROM BURULLOS LAKE IN EGYPT [6] 1 2 Abu-Elela , Gihan M. and Nevin, B. Ghanem

ABSTRACT

Twenty nine actinomycetes isolates were isolated from Burullos Lake and char- acterized taxonomically for 62 phenotypic traits including morphological; biochemi- cal, nutritional, substrate utilization and anti-microbial activities. The results were analyzed by numerical techniques using the simple matching coefficient (SSM) and UPGMA clustering. At 54% similarity level, the majority of the isolates were grouped into six phena (A, B, C, D, E and F). Only two isolates were grouped sepa- rately and formed two single clusters at the same level of similarity. A representative isolate from each phenon was identified. The isolates were found to be Streptover- ticillum morookaense, Nocardia brasiliensis, Streptomyces alanosinicus, Streptomy- ces globosus and Streptomyces gancidicus

Keywords: Burullos Lake, Actinobacteria, Numerical taxonomy, Fresh water habi- tats

INTRODUCTION growth in aquatic habitats but can never- theless be recovered readily from fresh Class Actinobacteria (high G+C con- water, sea water and sediments samples tent, gram positive bacteria) has been (McCarthy and Williams, 1990). proposed by Stackebrandt et al (1997) Several studies demonstrated by culti- and includes members with unparalleled vation independent methods revealed that ability to produce diverse secondary me- the freshwater actinobacteria are present tabolites (Mellouli et al 2003). They rep- in a wide spectrum of ecologically differ- resent an ubiquitous group of microbes ent and globally distributed freshwater widely distributed in natural ecosystem habitats (Glöckner et al 2000; including soils (Xu et al 1998), marine Lindström and Leskinen, 2002; Hahn sediments (Ghanem et al 2000, Sabry et et al 2003). Zwart et al (2002) recently al 2004) and fresh water (Hahn et al identified 34 putative phylogenetic clus- 2003). Actinomycetes are not adapted for ters of bacteria which seem to contain

1- National institute of Oceanography and Fisheries Alexandria. Egypt 2- Botany Department, Faculty of Science, Alexandria University, Egypt

(Received November 26, 2005) (Accepted January 2, 2006)

88 Gihan Abu-Elela and Nevin Ghanem typical freshwater inhabitants, 5 of the 34 MATERIAL AND METHODS clusters were affiliated with the class Ac- tinobacteria. Actinomycetes isolates Distribution of Actinobacteria in fresh water has been reported by Glöckner et Twenty nine actinomycetes isolates al (2000) who performed in situ hybridi- were selected from a total of 130 isolates zation. On the other hand, Pernthaler et previously isolated from sediments and al (2001) successfully enriched a phylo- water samples of Lake Burullous (Abu- type of one freshwater cluster of actino- Elela et al 2004). They were maintained bacteria in a continuous culture system. on a basal medium prepared with 1 liter Thus, despite the high number of cells of of Lake water of the following composi- actinobacteria observed in fresh water tion (g/l): soluble starch, 10.0; KNO3, samples no representatives of the fresh- 2.0; K2HPO4, 2.0; MgSO4 .7H2O, 0.05; water actinobacterial lineages have been CaCO2, 0.02; FeSO4, 0.01 and agar, 20.0 isolated so far (Zwart et al 2002). Zwart at 28 ºC (Küster and Williams, 1964). et al (2003) found that actinobacterial Culture stocks were maintained at -20 ºC cluster ACK-M1 is being well represent- in starch nitrate broth with 20% glycerol ed in most fresh water habitats. They de- (Kieser et al 2000). tected this cluster in all of the 81 lakes screened (from Belgium, The Nether- Characterization and numerical analy- lands, Denmark, Sweden and Norway), sis using reverse line blot hybridization. In the most recent published data (Van der Numerical analysis was not used for a Gucht et al 2005) the ACK-M1 (Actino- direct taxonomical purpose but also to bacteria) was the most representative in facilitate data handling and strains group- each of the studied lakes. These data con- ing. A total of 62 characters were coded firm those previously reported (Crump et as negative (0) or positive (1). The simple al 1999; Urbach, et al 2001). matching coefficient (SsM) (Sokal and Up to our Knowledge, few studies Michener, 1958) and the Jaccard coeffi- dealt with the distribution of class Ac- cient (Sj) (Sneath, 1957) were used and tinobacteria in aquatic habitats in Egypt clustering was achieved by unweighted (Al-Diwany and Cross 1978; Ghanem pair group average linkage (UPGMA), et al 2000). Lake Burullos is shallow Sneath and Sokal, (1973) and Sneath, brackish water that serves as fishery re- (1979). The computations were per- sources and reservoirs for drainage water. formed by using SYSTAT-PC program V It is contaminated with anthropogenic 7 (Wilkinson et al 1992) on an IBM materials (Abu-Elela et al 2004). There- computer. fore, this study is an attempt to evaluate The selected isolates were phenotypi- the distribution of members of class Ac- cally characterized as described by tinobacteria in Lake Burullous using phe- Pridham and Gottlieb (1948); Shirling notypic, physiological and biochemical and Gottlieb, (1966) and Tresner et al data, depending on numerical taxonomic (1968). All tests were carried out at 28ºC procedures. for up to 3 weeks.

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For morphological characterization, Chemotaxonomic study was per- the following media were used: Krass- formed by the detection of dia- linikov SRL agar, Glycerol- nitrate agar, minopimelic acid (DAP) isomers and Czapek- Dox agar after slight modifica- diagnostic sugars according to Becker et tion, Glycerol- asparagine agar and Inor- al (1964) and Lechevalier et al (1977) in ganic salts- starch agar (Shirling and AL-Azhar University Fermentation Bio- Gottlieb, 1966). technology and Applied Microbiology Additional phenotypic characteriza- Center. Identification was carried out in tion was performed using the standard comparison to reference strains. procedures, catalase and urease produc- tion were detected, melanin production Scanning electron microscopy according to Shirling and Gottlieb, Electron micrographs were made with (1966), nitrate reduction (Williams et al Jeol model ISM 5300 scanning electron 1983), sulphide precipitation (Cowan, microscope (SEM) operating at 15 KeV 1974) and indole production (Molin and (Molitoris et al 1996). Trenstrom, 1986). Lecithinase was per- formed on egg-yolk medium according to RESULTS AND DISCUSSION the method of Nitsch and Kutzner, (1969), Lipase (Elwan et al 1977), prote- In a previous survey on the distribu- ase (Ammar et al 1991), pectinase, am- tion of Actinobacteria in Lake Burullos, ylase (Ammar et al 1998) production 130 isolates were obtained (Abu-Elela et was investigated. Also Degradation ac- al 2004). Based on colony morphology, tivities of some substance such as starch, 29 isolates were selected which represent cellulose, chitin, gelatin and tyrosine different morphotypes. In this study, we were performed according to Nonomura aimed to cluster those isolates for the and Ohara (1969). purpose of identification and classifica- The antagonistic activity of the tested tion. isolates against Escherichia coli HP101, Actinomycetes isolates were studied Staphylococcus aureus ATTC 29523 and for 62 characters listed in Table (1). Bacillus subtilis was determined as de- Analysis of the 62 characters using the scribed by Goodfellow et al (1990). simple matching coefficient (SSM) and Zones of inhibition were scored as posi- UPGMA clustering yielded the den- tive results after 24 h at 28C°. Resistance dogram in Fig. (1). Data showed that the against phenol (0.002, 0.01 w/v), crystal majority of the isolates were grouped at violet (0.0001, 0.001 w/v), and sodium 54% similarity level, into six phena (A, azide (0.01w/v) was tested. Antibiotic B, C, D, E and F). Only two isolates were resistance was examined as the ability to grouped separately and formed two single grow on medium supplemented with clusters at this level. antibiotics one at a time using gentamy- cin (998 μg.ml-1), erythromycin (804 Phenon A: The four isolates grouped in μg.ml-1), tetracycline (990 μg.ml-1) and phenon A possess several features that cefixine (997 μg.ml-1) according to Wil- are common to members of genus Strep- liams et al (1983). toverticillium. It is thus suggested that the

Arab Univ. J. Agric. Sci., 14(1), 2006 90 Gihan Abu-Elela and Nevin Ghanem

Table 1. Comparison of the frequencies of positive and negative characters for the six clusters of actinomycetes obtained by numerical taxonomy analysis

Phenon A B C D E F Character No. of 4 3 6 5 3 6 isolates Growth on Krasslinikov SRL agar 100 100 100 0 33 83 Glycerol-asparagine agar 100 100 100 100 100 100 Glycerol –nitrate agar 100 100 100 100 100 100 Czapek-Dox agar 100 100 100 100 100 100 Inorganic salts –starch agar 75 100 100 100 100 100 Substrate mycelium Yellow brown 50 67 33 80 0 33 Reddish brown 25 0 50 0 67 50 Blue 25 0 0 0 0 0 Violet 0 33 17 20 33 17 Aerial mycelium Gray yellowish pink 0 33 0 0 67 0 Pink gray 0 67 17 0 0 0 Gray 0 0 67 80 0 50 Whitish gray 50 0 0 0 0 0 Blue 25 0 0 0 0 0 Violet 0 0 17 20 0 17 Yellow 25 0 0 0 33 33 Diffusible pigment Unpigmented 0 100 100 80 67 50 Reddish brown 0 0 0 20 33 0 Yellowish brown 100 0 0 0 0 50 Growth at ºC 20 50 100 100 100 100 100 30 100 100 100 100 100 100 40 100 100 100 100 100 100 50 25 0 0 0 0 17 Growth at pH 5 50 100 50 20 0 50 6 75 100 100 100 100 100 7 100 100 100 100 100 100 8 75 100 100 100 100 100 9 75 67 100 100 100 100

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Table 1. continued

Phenon A B C D E F Character No. of 4 3 6 5 3 6 isolates Utilization of D-Glucose 75 100 83 80 100 100 D-Fructose 75 67 100 40 33 100 L-Arabinose 0 0 50 0 67 50 Lactose 75 0 100 100 100 50 Raffinose 50 0 67 20 0 50 Rhamnose 50 33 67 40 67 83 Mannitol 25 33 67 80 67 67 Galactose 25 33 50 60 33 50 Xylose 25 33 67 80 67 100 Growth in presence of NaCl % 0 100 100 100 100 100 100 4 100 100 100 100 100 100 7 75 33 50 20 33 50 10 0 0 0 0 0 0 Degredation of Starch 1% 100 100 100 100 100 100 Cellulose 1% 50 67 100 0 0 100 Chitin 0.25% 0 0 33 0 0 0 Gelatin 0.4% 0 0 0 0 0 67 Tyrosine 0.1% 100 67 100 100 67 100 Enzyme activities Catalase 25 67 33 100 100 50 Urease 50 67 0 40 0 50 Nitrate reduction 50 100 17 40 33 67 Sulfide precipitation 25 0 0 0 33 50 Melanin production 0 0 0 100 33 0 Antibiosis against E.coli HP101 0 33 17 0 33 50 S.aureus ATCC 29523 25 0 100 20 33 83 B.subtilis 0 0 17 0 67 100

Arab Univ. J. Agric. Sci., 14(1), 2006 92 Gihan Abu-Elela and Nevin Ghanem

Table 1. continued

Phenon A B C D E F Character No. of 4 3 6 5 3 6 isolates Resistance to Phenol (0.002 w/v) 100 100 100 100 100 100 Phenol (0.01w/v) 0 0 0 0 0 0 Crystal violet (0.0001 w/v) 50 100 100 80 100 100 Crystal violet (0.001 w/v) 25 67 33 40 100 100 Gentamycin at (998 μg.ml-1) 0 67 0 0 0 17 Erythromycin (804 μg.ml-1) 0 0 0 0 0 17 Tetracycline (990 μg.ml-1) 0 0 0 0 0 0 Cefixine (997 μg.ml-1) 0 0 0 0 0 0

isolates are different species of this genus that it could not utilize raffinose and in (Table 1). SEM observation of the repre- this respect, it was suggested that the iso- sentative strain (Fig. 2A) showed pheno- late is closely related to Streptoverticilli- typic features clearly related to the refer- um morookaense as recommended by the ence strain Streptoverticillium mo- international Keys (Williams et al 1989 rookaense (Hensyl, 1994) the characters and Hensyl, 1994). shared with both strains are shown in (Table 2) aerial mycelia are gray while Phenon B: This phenon contained three substrate mycelia are yellow brown, the isolates grouped at 55% similarity level. aerial mycelia consisted of long, straight They had morphological and biochemical filaments of conidia showing side characteristics similar to members of no- branches at regular intervals and whitish cardioform actinomycetes (Lechevalier, to gray spore masses. The cell wall hy- 1989). drolysates contained LL-DAP with no Selected isolate from phenon B (Fig.2 diagnostic sugars. Spores are not motile. B) was similar to the reference strain No- Absence of melanin pigments but gray cardia brasiliensis in having pink gray yellowish brown diffusible pigments was aerial mycelium and the aerial mycelium produced. Several carbon sources were moderately fragmented into a chain of utilized (Table 2). Positive results were conidia. The cell wall hydrolysate con- recorded with urease, nitrate reductase tained meso-DAP with arabinose and and capable degrading starch, tyrosine, galactose as diagnostic sugars. The exper- xanthine and esculin .Our isolate differs imental strain shared some properties only from the reference strain in the fact with N. brasiliens in being non motile,

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Fig. 1. Simplified dendrogram showing the relationships among phena based on the Ss µ - UPGMA analysis.

Arab Univ. J. Agric. Sci., 14(1), 2006 94 Gihan Abu-Elela and Nevin Ghanem

Table 2. Characteristic features of the identified actinomycetes isolates

Characteristic St.m N.b S.a S.g S.r S.gn Morphological

characters Substrate yellow brown Light yel- reddish yellowish reddish gray mycelium low brown brown brown brown yellowish brown Aerial gray Pink gray gray gray gray gray mycelium yellowish pink Spore chain Long straight Aerial spiral spiral and spiral spiral /conidia filament of conidial rectiflexible conidia showing chain at regular intervals side branches Diffusible gray yellowish - - - - - pigments brown Melanin - - + - - - production Cell wall hydrolysate Diaminopimelic LL-DAP meso-DAP LL-DAP LL-DAP LL-DAP LL-DAP acid Sugar pattern nd arabinose & nd nd nd nd galactose Utilization of carbohydrates D-glucose + + + + + + D-fructose + + + - + + L-arabinose - - + + + + Lactose - - + + + - Raffinose - - + - - - Rhamnose + - + - + + Mannitol + - + - + + Galactose + + + - + + Xylose + + + + + + Sucrose + - - - - - Trehalose nd + + nd + + Maltose + - + + + - Cellobiose + nd nd - nd nd Sodium citrate + + - - - + Sodium acetate + nd nd + nd nd Ribose - - - nd - - Mannose nd + nd nd + +

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Table 2. Continued

Characteristic St.m N.b S.a S.g S.r S.gn Utilization of amino-acids L-cycteine + - - + - + L-valine + + - - + + L-alanine + + + + + - L-phenyl-alanine + - - - - - L-arginine + + + + + + L-tryptophane + - - + - - Biochemical activities Catalase - - - + - - Urease + + + + + + Nitrate reductase + + + + + + Sulfide precipitation + - - - + - Indole test ------Degradation of Starch + + + + + + Cellulose - - + - - + Chitin ------Gelatin - - - - - + Tyrosine + + - + + + Xanthin + + + + + + Esculin + + + + - + Degradation of Protein + + + - + + Lipid + + + - + + Pectin + nd + - nd nd Lecithin + + + nd + - Antibiosis against E.coli - - + - + + S.aureus - - - - + + B.subtilis - - + - - +

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Table 2. Continued

Characteristic St.m N.b S.a S.g S.r S.gn

Resistance to Phenol (0.01w/v) ------

Phenol (0.002 w/v) + + + + + +

Crystal violet (0.001 w/v) - + - - + + Crystal violet (0.0001 w/v) + + + + + +

Sodium azide (0.01 w/v) - - - - - +

Gentamycin (998 μg.ml-1) - + - - - - Erythromycin (804 μg.ml-1) ------

Tetracycline (990 μg.ml-1) ------

Cefixine (997 μg.ml-1) ------

St.m : Streptoverticillum morookaense N.b : Nocardia brasiliensis S.a : Streptomyces alanosinicus S.g : Streptomyces globosus S.r : Streptomyces rubber S.gn : Streptomyces gancidicus nd : not detected DAP : Diaminopimelic acid

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Fig. 2. Scanning electron micrographs of representative strains from each phena Streptoverticillum morookaense (A); Nocardia brasiliensis (B); Streptomyces alanosinicus (C); Streptomyces globosus (D); Streptomyces ruber (E ); Streptomyces gancidicus (F)

Arab Univ. J. Agric. Sci., 14(1), 2006 98 Gihan Abu-Elela and Nevin Ghanem lacking melanin pigments, positive for flexibles and spirals is not clear (Wil- urease and nitrate reductase, degraded liams and Wellington 1980; Williams et starch, tyrosine, xanthine and esculin, in al 1989). The determination of the color addition to the number of carbon sources of the spore mass is also not easy utilized (Table 2). So it could be stated (Kutzner, 1981), especially with respect that actinomycete members of this phe- to spore color and other morphological non are closely related to Nocardia criteria. These traditional characters, brasiliensis. which are often difficult to determine (Williams and Wellington 1980; Phena C, D, E, and F: It is widely ac- Kutzner, 1981), are inadequate for clas- cepted that the genera Streptomyces and sification and identification. Carbon Streptoverticillium are closely related. source utilization tests have been found in Both have cell-wall I (Lechevalier 1989), earlier studies to be characters with great a high content of GC in their DNA differentiation potential. The utilization (Pridham and Tresner 1974) and high of sucrose, L-arabinose, inositol, manni- similarities in DNA homology tol, rhamnose and raffinose was recom- (Kroppenstedt et al 1981), and are lysed mended as an aid to species differentia- by the same phages (Wellington and tion in the International Streptomyces Williams 1981). Additionally, they con- Project (Shirling and Gottlieb, 1972) tain similar menaquinones, fatty acids and these tests have been used in several and polar lipids (Lechevalier 1989). The identification schemes (Nonomura, only support for distinguishing between 1974; Szabo et al 1975). The phenetic Streptomyces and Streptoverticillum has and diversity of streptomycetes are major come from DNA-DNA pairing studies problem and as pointed out by Kampfer (Gladek et al 1985). and Kroppenstedt (1991), streptomy- The presence of LL-diaminopimelic cetes taxonomy is still developing. acid in the cell wall of isolates belonging to phena C, D, E and F with no diagnostic Phenon C: The 6 isolates grouped in sugars is consistent with the proposal that phenon C were clustered at 65% similari- these strains belong to the genus Strepto- ty level (Fig.1).The chemotaxonomic myces and related genera (Williams et al properties and morphological characteris- 1989; Hensyl 1994). As pointed out by tics of our experimental isolates are con- Williams et al (1983), there is no simple, sistent with the reference strain Strepto- rapid procedure for objective identifica- myces alanosinicus (gray series) Wil- tion of streptomycetes. The problems of liams et al (1989). Both possess the same streptomycetes identification are largely a cell wall chemotype; produce gray aerial reflexion of the difficulties in streptomy- mycelia carrying gray spiral and rectiflex- cetes taxonomy. Morphological or pig- ible spore chains (Fig. 3C). No observed mentation characteristics were not in- motility and melanin pigments not pro- cluded in streptomycetes taxonomy by duced. Both utilized arabinose, fructose, Kampfer et al (1991). It is generally ac- galactose, glucose, mannitol, raffinose cepted that more than one category of and xylose but no utility to sucrose (Table spore chains can be observed in the same 2). species and the distinction between recti-

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Phenon D: Five isolates are grouped in sugars. No motility. Absence of melanin. this phenon at similarity level 55%. The Both of them were able to use arabinose, strain selected from this phenon showed fructose, glucose, mannitol, galactose, phenotypic characteristics (Fig. 2D) and xylose where as they were unable to closely related to the reference strain utilize raffinose and sucrose (Table 2). Streptomyces globosus. Their aerial my- celia bear spiral and rectiflexible (straight Conclusion to flexuous) spore chains forming gray spore masses. The cell wall contained The data obtained from examined LL-DAP with no sugars; absence of mo- fresh water habitat in Burullous Lake tility; glucose, L-arabinose, lactose and clearly showed that the Burullous Lake xylose were utilized by the selected strain has its own distinct actinobacteria com- but can not utilized fructose, galactose, munity. The lake is characterized by high mannitol, raffinose, and sucrose. (Table representation of Streptomycetes species 2). On the basis of the previous collected from different series, and fewer numbers data and in view of the comparative study of nocardioforms were observed. of the recorded properties of the tested strain in relation to closet strain, the rec- ommended identification may be closely REFERENCES related to S. globosus.. Abu-Elela, G.M.; N.B. Ghanem and M. Phenon E: At 60% similarity level this Okbah (2004). Occurrence and distribu- phenon included three isolates. As report- tion of some actinomycetes groups in ed in Table (1) the representative isolate Burullous Lake. Bull. Fac. Sci. Assiut (Fig. 2 E) was closer to the reference Univ. 33 (2-d): 133-146. strain Streptomyces rubber, having gray- Al-Diwany, L.J. and T. Cross (1978). ish-yellowish pink spore masses, the Ecological studies on nocardioforms and spore chains were spiral, and the substrate other actinomycetes in aquatic habitats. mycelia were reddish brown. Both Zentralbl. Bacteriol. Parasitenkd. Infek- strains have the same cell wall chemo- tionskr. Hyg. Abt. 1, Suppl. 6: 153-160. type. No detection to motility or melanin Ammar, M.S.; S.S. El-louboudy and production. Both of them utilized glu- U.M. Abdul-Raouf, (1991). Purification cose, D- fructose, L-arabinose, mannitol, and properties of mesophilic protease(s) rhamnose and xylose (Table 2). produced by Bacillus anthracis S-44. isolated from a temple in Aswan. Al- Phenon F: At 55% similarity level, six Azhar Bull. Sci., 2(1): 325-338. isolates constituted this phenon, the se- Ammar, M.S.; M. El-Esawey; M. Yas- lected isolate and the reference strain sin and Y.M. Sherif (1998). Hydrolytic Streptomyces gancidicus shared in most enzymes of fungi isolated from certain characters. Their spore masses were in Egyptian Antiquities objects while utiliz- the gray series with gray yellowish brown ing the industrial wastes of Sugar and substrate mycelia. Spore chains are spi- Integrated Industries Company (SIIC). rals (Fig. 2F). Their cell hydrolysates Egypt. J. Biotechnol. 3: 60-90. contained LL-DAP with no diagnostic

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Xu, L.H.; Y.Q. Tiang; L.F. Zhang; Zwart, G.; E.J. Van Hannen; M.P. L.X. Zhao and C.L. Jiang, (1998). Kamst-Van Agterveld; K. Van der Streptomyces thermogriseus, a new spe- Gucht; E.S. Lindström; J. Van cies of the genus Streptomyces from soil, Widhelen; T. Lauridsen; M.P. Crump; lake and hot-spring. Int. J. Syst. Bacteri- S.K. Han and S. Declerck, (2003). Rap- ol. 48: 1089-1093. id screening for freshwater bacterial Zwart, G.; M.P. Crump; M.P. Kamst- groups by using reverse line blot hybridi- Van Agterveld; F. Hagen and S.K. zation. Appl. Environ. Microbiol. 69: Han, (2002). Typical freshwater bacteria: 5875-5883. an analysis of available16S rDNA gene sequences from plankton of lakes and rivers. Aquat. Microbiol. Ecol. 28:141- 155. جملة احتاد اجلامعات العربية للدراسات والبحوث الزراعية ، جامعة عني مشس ، القاهرة ،41)4( ، 401-78 ، 6002 دراسة عددية عن بعض االكتينوميسيتات المعزولة من بحيرة البرلس بمصر [6] جيهان ابو العال1 - نيڤين بهاء االدين غانم2 1- معهد علوم البحار و المصايـد – االسكندريـة - مصر 2- قسم النبات - كلية العلوم - جامعة االسكندرية - مصر

تمم عمم ٢٩ سممة ت تمت ممى م ممى عممت ت عممممت ك مممممت ٦ عممممات مممم ف ممممد مالكتيم يسيتات ن رس بيات بحيمر م بمر ع تين مف متيين عمن هم ل م عمات تم أ ريممت ممما رمسممت ع يمممت ت ممميفيت بعممم تعريممع ع ممت ثتممت كمم عممت كامممت مختبارها فى ٦٢ فت ت ميفيت مشمت تت عتمى م ع الت م عرفت هى م شممممممك م رف مممممم ى م م مممممم م مممممم م ى -Streptoverticillum morookaense, Nocar dia brasiliensis, Streptomyces alanosin- م خممم مل م فسممميت يت تممم تحتيممم م متممما -icus, Streptomyces globosus and Strepto مح مممما يا باسممممتخ م م دممممرن م ع يممممت عممممم myces gancidicus ٤٥% ممن م ت اثمم مم من علمم م عمم الت

تحكيم: أ. م سي أح ـ ا ـح أ. ثريا عب م فتاح برى

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