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Characterization of the Newly Isolated Antimicrobial Strain Streptomyces SCIENCE LETTERS 2015 | Volume 3 | Issue 3 | Pages 94-97 Research article Characterization of the newly isolated antimicrobial strain Streptomyces goshikiensis YCXU Muhammad Faheem1, Waseem Raza2*, Zhao Jun1, Sadaf Shabbir1, Nasrin Sultana1 1Institute of Soil Science, Chinese Academy of Sciences, Nanjing, PR China 2College of Resource and Environmental Sciences, Nanjing Agricultural University, 210095, Nanjing, PR China Abstract A rhizosphere bacterial strain coded as Streptomyces goshikiensis YCXU with broad spectrum antifungal activity was isolated from a cucumber field infested with Fusarium oxysporum f. sp. niveum. The strain YCXU showed antagonism to a broad range of phyto-pathogenic fungi and bacteria as well as strain YCXU produced volatile organic compounds that could reduce the fungal growth up to 40% compared to control, concluding that it can be used as biocontrol agent. Because of little information about the newly isolated strain, we further characterized the strain YCXU. The strain YCXU showed maximum growth on glucose containing yeast-malt extract (YME) medium at pH 7 and pink spores were produced after 7 days of incubation at 28°C. The strain YCXU exhibited nitrate reduction, melanin production, blood hemolysis, and casein, gelatin, starch, tyrosine, and hypoxanthine hydrolysis. This characterization will aid further research regarding the strains of S. goshikiensis. Key words: Characterization, growth, hydrolysis, Streptomyces goshikiensis YCXU. Received April 06, 2015 Revised May 29, 2015 Published online first June 30, 2015 *Corresponding author Waseem Raza Email [email protected] To cite this manuscript: Faheem M, Raza W, Jun Z, Shabbir S, Sultana N. Characterization of newly isolated antimicrobial strain Streptomyces goshikiensis YCXU. Sci Lett 2015; 3(3):94-97. Introduction actinomycetes have been found to form intimate The genus Streptomyces has been proposed by association with plants and colonize their internal Waksman and Henrico [1] as a term to encompass tissue. The actinomycetes inhabit a wide range of Gram positive, aerobic, spore-forming actinomycetes plants as either symbionts or parasites [15]. and is a unique source of novel antibiotics [2]. At Streptomyces strains were isolated from the present, the genus Streptomyces contains a large rhizosphere of different plant species in Italy and number of described species and nearly 600 were from maize in Brazil, which showed antagonistic validly published [3]. Most of the recently described activities against Gram positive bacteria and fungi Streptomyces species have been delineated using [16, 17]. polyphasic taxonomic approaches [4, 5]. Streptomyces goshikiensis YCXU was isolated Streptomycetes mostly represent a considerable from a healthy cucumber plant in a wilt diseased field proportion of the actinomycetes community in soils because of its strong antimicrobial activity against [6]. Streptomycetes are also widely distributed in a Fusarium oxysporum f. sp. niveum. Due to its little variety of natural and man-made environments, known characteristics, we decided to further constituting a significant component of the microbial characterize this strain for future use. Thus the aim of population in most environments [7]. They were our study was to characterize Streptomyces commonly believed to be intermediate between goshikiensis YCXU, so that we could further utilize bacteria and fungi [8]. this antifungal and antibacterial strain as an At the present time with several thousand of antagonist against different plant diseases efficiently. described microbial metabolites, new strategies must Material and methods be introduced into the screening programs to increase the chances of discovering the novel compounds [9]. Isolation of microbial strain The filamentous actinomycetes share significant Tenfold dilution series method was used to fraction of microbial metabolites and among them; isolate Streptomyces goshikiensis YCXU on PDA Streptomyces is most prolific genus and generally (potato infusion 200g, dextrose 20g, agar 15g in 1L synthesize a huge number of diverse natural of distilled water) medium from soil as an antifungal secondary metabolites e.g. platensimycin [10], strain against Fusarium oxysporum f. sp. niveum, daptomycin, streptomycin, the best known of which After isolation from plant rhizosphere, the strain was are antibiotics used worldwide as pharmaceutical and grown on YME media (yeast extract 4g/L, malt agricultural products [11, 12]. The actinomycetes extract 20g/L, glucose 4g/L, pH 7) and stored at - produce ~3,000 known antibiotics and 90% of those 20°C for future use. are from Streptomyces species [13, 14]. In addition, 94 1 SCIENCE LETTERS 2015 | Volume 3 | Issue 3 | Pages 94-97 Microscopic study were prepared and black color around colonies was considered as positive [23]. Morphological studies were done under high resolution light microscope and individual colony Salt utilization assay pictures were taken with mycelial structures. The Citrate, private, benzoate and tartrate salts mycelia of strain YCXU were stained with aniline utilization were also examined by following Pridham crystal violet dye and were observed under light and Gottlieb [24] method with slight modification. For microscope to confirm its mycelial structures like this, TYG medium (tryptone 3g/L, yeast extract 5g/L, other Streptomyces strains. glucose 5g/L, and agar 20g/L) was prepared with Morphological identification on different media respective salts. Bacterial growth in media was considered as positive. Morphological characters were noticed on different media including tryptone yeast media Antifungal and antibacterial activity assay (TYG), Luria-Bertani (LB) media, potato dextrose The liquid culture (100 ml) of strain YCXU was agar (PDA) media, Czapek media, Bennets agar centrifuged (12000×g) after 7 days growth at 28oC in media, soil agar media, inorganic starch salt agar YME medium and antimicrobial compounds were media, potato carrot (PC) agar media, Gauss media extracted twice from cell free liquid culture with two and International Streptomyces Projects (ISP) 1, 2, 3, volumes of ethyl acetate. The ethyl acetate was dried 4, 5, 6, 7 media [18]. Bacterial colony color and in rotatory evaporator and residues were dissolved in 1 growth habits were recorded after 3 and 7 days. ml of methanol, which was used to evaluate its Degradation activity assay antagonistic activity against fungal and bacterial strains. From the PDA medium plates, an 8 mm agar The degradation of adenine, tyrosine, plug was removed and filled with 100 µl of cell free hypoxanthine, xanthine and casein were detected by liquid culture after filtering through 20 µm filter and in the method of Washington and Koneman [19]. Basal the middle an 8 mm agar plug taken from the edge of medium was prepared (beef extract 3g/L, peptone actively growing fungal pathogen strain Fusarium 5g/L and agar 15g/L) with 1% of each compound. oxysporum f. sp. niveum was placed. For antibacterial Transparent zones present around bacterial colonies activity, bacterial pathogen Ralstonia solanacearum were considered as positive. Gelatin hydrolysis was overnight cultures were spread on the PDA medium. detected in basal medium amended with 1% gelatin Plates were incubated at 28oC and inhibition zones [20] and starch degradation was detected in the same were determined after three days for fungal strain and basal medium amended with 1% starch by flooding after two days for bacterial strain. plates with iodine solution [21]. Blood agar hydrolysis analysis was done on blood agar plates Antifungal volatile compounds assay (Chengdu Rich Science Company Co. Ltd). For antifungal volatile compounds production Nitrate reduction and hydrogen sulfide assay, divided plates were used. Both compartments of production assay plates were added with PDA medium. One compartment was inoculated with a plug of freshly For nitrate reduction test, nitrate broth was grown Fusarium oxysporum f. sp. niveum and other prepared (beef extract 0.3g/L, peptone 0.4g/L, compartment was inoculated with newly isolated protease peptone no. 3 0.1g/L and potassium nitrate bacterium YCXU. The plates were sealed with 0.1g/L) and color change to pink or red was an Parafilm and fungal growth was measured on daily indicator of the presence or absence of nitrite. basis. Two control treatments were included: one was Hydrogen sulfide production was tested by preparing containing Escherichia coli DH5ά in place of strain sulfide-indole-motility (SIM) medium (pancreatic YCXU and second without any bacterial inoculation. digest of casein 2g/L, beef extract 6.1g/L, ferrous ammonium sulfate 0.2g/L and agar 3.5 g/L) and Results and Discussion production of black precipitates was considered as positive for hydrogen sulfide reduction [22]. Streptomyces goshikiensis YCXU was Gram- positive, non-motile and aerobic like other Melanin pigment production Actinobacteria. Streptomyces species produced For melanin pigment production, tyrosine-casein mycelia with the aerial hyphae and mobility is agar plates (L-tyrosine 1g/L, casein hydrolysate achieved by the dispersion of spores [25]. Single 2.5g/L, sodium nitrate 1g/L and agar 20g/L; pH 7) colony (Fig.1A) and stained mycelia (Fig.1B) were 95 SCIENCE LETTERS 2015 | Volume 3 | Issue 3 | Pages 94-97 showed the best result and the characteristic color on this media was in-between light grayish yellowish brown and grayish yellowish pink as described by Niida [26]. The strain YCXU was able to produce antimicrobial compounds that
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