Isolation, Identification and Optimal Culture Conditions of Streptomyces albidoflavus C247 Producing Antifungal Agents against Rhizoctonia solani AG2-2

Abstract

Streptomyces albidoflavus C247 was isolated from the soil of the Gyeongsan golf course in Korea. Physiological, biochemical and 16S rDNA gene sequence analysis strongly suggested that the isolate belonged to Streptomyces albidoflavus. Preliminary screening revealed that the isolate was active against fungi and bacteria. Self-directing optimization was employed to determine the best combination of parameters such as carbon and nitrogen source, pH and temperature. Nutritional and culture conditions for the production of antibiotics by this organism under shake-flask conditions were also optimized. Maltose (5%) and soytone (5%) were found to be the best carbon and nitrogen sources for the production of antibiotics by S. albidoflavus C247. Additionally, 62.89% mycelial growth inhibition was achieved when the organism was cultured at 30℃ and pH 6.5. Ethyl acetate (EtOAc) was the best extraction solvent for the isolation of the antibiotics, and 100 µg/ml of EtOAc extract was found to inhibit 60.27% of the mycelial growth of Rhizoctonia solani AG2-2(IV) when the poison plate diffusion method was conducted.

Numerous antibiotics have been isolated from a variety of microorganisms; however, studies are still being conducted to identify novel antibiotics effective against pathogenic fungi and bacteria. The number and species of microbes in soil vary directly in response to environmental conditions such as nutrient availability, soil texture, and type of vegetation cover (Atlas and Bartha, 1998). Filamentous Acitomycetes are known to have the ability to produce a wide variety of secondary metabolites. Indeed, each strain of Actinomycetes likely has the genetic potential for the production of 10 to 20 secondary metabolites (Bentley et al., 2002; Sosio et al., 2000).

Actinomycetes are useful biological tools for the production of antimicrobials against fungi and bacteria (Okami and Hotta, 1988). In general, Streptomyces are saprophytic and are commonly associated with soils, where they contribute significantly to the turnover of complex biopolymers and antibiotics (Woese, 1987). However, in the past two decades there has been a decline in the discovery of new lead compounds from common soil-derived actinomycetes. As a result, the cultivation of various taxa of actinomycetes has become a major focus in the search for the next generation of pharmaceutical agents (Mincer et al., 2002).

Additionally, Streptomyces are especially prolific (Locci, 1989; Nolan and Cross, 1998). The problems of drug resistance, patient sensitivity and an inability to control certain infectious diseases have driven a continuous search for new antibiotics worldwide. To combat multidrug resistant organisms, it is essential to develop new antimicrobial compounds or antibiotics.

Marten et al. (2001) reported that Rhizovit^R from Streptomyces rimosus was used to control a wide range of fungi including Pythium spp., Phytophthora spp., Rhizoctonia solani, Alternaria brassicola, and Botrytis sp. In addition, Liu et al. (2004) reported that S. rimosus showed a high antagonistic activity against Fusarium solani, F. oxysporum f. sp. cucumarinum, Verticillium dahliae, R. solani, Fulvia fulva, Botrytis cinerea, A. alternata, Sclerotinia sclerotiorum and Bipolaris maydis.

In the present study, we reported the media and culture conditions of S. albidoflavus C247 and the extraction methods for the isolation of antibiotics.

Materials and Methods

Soil sample and isolation of actinomycetes

A soil sample was collected from a golf course in Korea. Five grams of soil were then suspended in 45 ml of sterile double distilled water, and the suspension was then serially diluted to 10^-5. Next, aliquots (100 µl) were spread on TSA and actinomycetes isolation agar (MTCC, 1994). To minimize the fungal and bacterial growth, actidione (20 mg/l) and nalidixic acid (100 mg/l) were added. The plates were then incubated at 27℃ for 7~14 days, after which actinomycetes were selected based on their morphology and then cultured on ISP4 medium. The stock culture was preserved in 15% (v/v) glycerol at -20℃ (Maniatis et al., 1989).

Microorganism and its maintenance

The common turfgrass pathogen, Rhizoctonia solani AG2-2(IV) (KACC 40152), was used as a test organism in this study. The organism was stored at 4℃ in tubes containing potato dextrose agar (PDA) for routine use. For long term preservation, it was stocked in glycerol solution at -70℃.

Screening of antibiotic producing actinomycetes

The antagonistic effect and antibiotic production by the isolate was evaluated using a slightly modified version of the dual culture method (Landa et al., 1997). Isolated actinomycetes were streaked on a 4 cm wide TSA plate and incubated for 48 h at 27℃. A mycelial disk (5mm in diameter) of R. solani AG 2-2 (IV) was then inoculated on the plate and incubated for 72 h at 27℃. The the mycelial growth of Rhizoctonia solani AG2-2 (IV) towards (a) and away (b) from the actinomycetes were then measured and used to calculate the percentage growth inhibition rate (IR %) as follows:

IR (%) = 100 - (a/b × 100)

Molecular and morphological characterization

Strain C247 was identified through morphological, physiological and biochemical analysis as well as 16S rDNA gene sequence analysis.

Optimization for maximum antibiotic production

Various media were used to determine the optimal media and culture conditions for the growth and production of antibiotics. LB medium was supplemented with different carbon and nitrogen sources, after which 5 ml of mycelial suspension was inoculated into the medium (100 ml in 500 ml Erlenmeyer flask) and incubated on a rotary shaker at 150 rpm and 28℃ for 5 days. The effects of various temperature (25, 30, 35 and 40℃), pH (5.0, 5.5, 6.0, 6.5, 7.0 and 8.0) and incubation time (48, 72, 96 and 120 h) on the growth and antibiotic production were then evaluated.

Organic solvent extraction

For isolation of the antibiotics, the culture was stopped when it reached the maximum activity. The culture broth (5 liter) was centrifuged and the supernatant containing antibiotics was extracted using solvents such as hexane, chloroform, ethyl acetate and dichloromethane. The solvents were added to the culture supernatant at a ratio of 1 : 3 (v/v) and then shaken vigorously for 20 min. This process was repeated three times. The solvent layer was then collected and evaporated to dryness in a vacuum evaporator at 40℃, after which it was stored at 4℃.

In vitro antimicrobial test

The crude extract was dissolved in methanol (MeOH) and then added to a molten PDA plate. Freshly grown mycelial discs (7 mm in diameter) of R. solani AG2-2(IV) were then placed in the centre of the plates and incubated at 27 ± 2℃. Plates were prepared in triplicate for each treatment. Positive and negative controls (MeOH) were also included. The mycelial growth of R. solani AG2-2(IV) was recorded after 3 to 5 days. Measurement of the colony diameter was conducted according to the growth rate of the fungal pathogen. The inhibitory activity of each treatment was expressed as the percentage growth inhibition when compared to the negative control (0%) using following calculation:

where, DC = the diameter of the control colony and DT = the diameter of the treated fungal colony (Pandey et al., 1982).

Results

Isolation and identification

Strain C247 was isolated from the soil and found to have antimicrobial activity against the turfgrass pathogen, R. solani AG2-2(IV). The isolate was aerobic, gram positive and had areal mycelia with sporangium (sporophore). During culture, grayish red pigment diffused into the surrounding medium (Table 1). The strain was also mesophilic and catalase positive, but did not produce oxidase or hydrogen sulphide. The strain also did not produce amylase, protease or gelatinase (Table 2). Based on the morphological, physiological, and biochemical results, isolate C247 was found to be a member of the genus Streptomyces, and 16S rDNA gene sequence analysis suggested that it was most closely related to Streptomyces albidoflavus (Fig. 1 and 2).

Table 1.

Cultural characteristics of Streptomyces albidoflavus C247

ISP: International Streptomyces Project medium; ISP 4-G: ISP-4 medium with glucose; ISP 4-Y: ISP-4 medium with yeast extract; GYM: Glucose, yeast and malt extract agar; LBA: Luria broth agar; TSA: Tryptic soy agar; PDA: Potato dextrose agar.

^a+: poor; ++: moderate; +++: good.

^bISCC-NBS color centroid system.

Streptomyces albidoflavus C247 was incubated at 27 ± 2℃ for 14 days.

Table 2.

Characteristics of Streptomyces albidoflavus C247

All of the above experiments were performed according to Bergey's Manual of Systematic Bacteriology.

+: positive; -: negative.

Fig. 1.

Morphological micrograph of isolate C247 after 14 days of culture on ISP4 medium. A; Light microscopic observation (× 400), B; Scanning electron micrographs (SEM).

Fig. 2.

The 16S rDNA sequences of isolate C247.

Screening of antibiotics

Preliminary screening revealed that Streptomyces albidoflavus C247 was an excellent producer of antifungal and moderate antibacterial compounds (Table 3 and 5). The antagonistic effect of isolate C247 was most evident on R. solani AG2-2(IV). The EtOAc extract of S. albidoflavus C247 inhibited the mycelial growth of various isolates of R. solani by 65 to 75% (Fig. 3 and Table 4).

Table 3.

Inhibitory effect of Streptomyces albidoflavus C247 on the mycelial growth of phytopathogenic fungi grown in dual culture on TSA

The S. albidoflavus A247 were streaked (35 mm) on one side of a TSA plate, and 48 hr after incubation at 27℃ a mycelial disk (5 mm in diameter) of the test fungus was placed 35 mm from the antagonistic bacteria. The plates were then incubated at 27℃.

Table 5.

Antibacterial susceptibility test of EtOAc extract of S. albidoflavus C247

MIC. Minimum inhibitory concentration.

Fig. 3.

Antagonistic activity of isolate C247. A, Antagonistic activity of isolate C247 against R. solani AG2-2(IV); B, Normal growth of R. solani AG2-2(IV) on TSA.

Table 4.

Antifungal activity of EtOAc extracts (100 µg/ml PDA) of Streptomyces albidoflavus A247 against 8 isolates of Rhizoctonia solani AG2-2(IV)

Effect of media and nutrients

Two thirds strength tryptic soy broth (TSB) was found to be a favorable basal media for the growth and production of antimicrobial materials from S. albidoflavus C247. Culture supernatant from C247 cultured in 10, 20 and 30 gm/l TSB induced 50.94, 59.03 and 49.58% mycelial growth inhibition, respectively (Fig. 4).

Fig. 4.

Effect of tryptic soy broth (TSB) concentration on the production of antibiotics by Streptomyces albidoflavus C247. S. albidoflavus C247 was cultured in 3 different concentration of TSB (A, 10 g/l; B, 20 g/l; C, 30 g/l) for 72 hours at 30℃ and the cell-free filtrates were then collected and subjected to aseptic 0.4 µm-filtration. A A freshly growing R. solani AG2-2(IV) mycelial disk was placed on the center of a TSA plate containing 10% (v/v) of each culture filtrate. D; A normal TSA plate culture of R. solani AG2-2(IV), Control.

To screen for a suitable carbon and nitrogen source, strain C247 was incubated in basal medium containing various carbon and nitrogen sources. Each of the following carbon sources was added to the basal medium (LB broth) at a concentration of 1 to 9% (w/v): maltose, fructose, corn starch, sucrose, glycerol, water soluble starch, glucose, galactose, lactose and dextrin. Various nitrogen sources including urea, glycine, soytone, beef extract, peptone, malt extract, tryptone, yeast extract and poly peptone were also added to the basal medium at concentrations of 1 to 9% (w/v).

Maltose and sucrose resulted in the highest antibiotic production, with maltose inducing 74.33% growth inhibition and sucrose inducing 70.67% (Fig. 5). Soytone was favorable for the cell growth and production of antibiotics (Fig. 6). When compared with inorganic nitrogen sources, organic nitrogen sources gave a relatively higher production of antibiotics.

Fig. 5.

Effect of carbon sources on cell growth and production of antibiotics by Streptomyces albidoflavus C247.

Fig. 6.

Effect of nitrogen sources on cell growth and production of antibiotics by Streptomyces albidoflavus C247.

Effect of initial pH and temperature

Strain C247 was cultivated in the above described medium at different initial pHs (pH 3.5 to 8.5) and temperatures (25 to 40℃). The results revealed that the optimal pH and temperature were 6.5 at 30℃, respectively, and the corresponding inhibition rate was 62.89% (Fig. 7). It is believed that this occurred because strain C247 has adapted to the slightly acidic soil from which it was isolated.

Fig. 7.

Effect of initial pH and culture temperature on the production of antibiotics by Streptomyces albidoflavus C247.

Solvent extracts

Hexane, ethyl acetate, dichloromethane and chloroform extracts of strain C247 were tested against R. solani AG2-2(IV). All extracts showed antifungal activity against the test pathogen and EtOAc extract was effective against 8 isolates of R. solani AG2-2(IV) isolated from diseased turf grass (Table 4). The minimum inhibitory concentration (MIC) of the crude EtOAc extract against Bacillus subtilis IFO 3026 was o.25 mg/ml (Table 5). Treatment with 10 and 100 µg/ml of the crude EtOAc extracted resulted in 30.88 and 60.27% inhibition of the mycelial growth of R. solani AG2-2(IV), respectively (Fig. 8).

Fig. 8.

Effect of solvents used for the extraction of antibiotics from Streptomyces albidoflavus C247.

Discussion

In the present study, the optimal conditions for the growth of isolate C247 were found to be pH 6.5 and 30℃. Additionally, maltose (5%) and soytone (5%) were found to be the best carbon and nitrogen sources. These results were similar to the findings of a study conducted by Sujatha et al. (2005). Additionally, it has been reported that environmental factors such as temperature, pH and incubation have a profound influence on antibiotic production (Iwai et al., 1973). In this study, the extracellular products (ECPs) of S. albidoflavus C247 showed potent antimicrobial activity against various pathogenic fungi and bacteria (Table 4 and 5). Furthermore, the MIC of the ethyl acetate extracts against tested bacteria indicated that it is a broad spectrum antibiotic (Sujatha et al., 2005). Finally, the efficiency with which the EtOAc extract inhibited the mycelial growth of turfgrass pathogens clearly indicates that S. albidoflavus C247 is an effective antagonist against turfgrass pathogens.

In conclusion, the actinomycete isolated in this study was found to be a potential antimicrobial agent and the optimal media, nutrients and culture conditions promoted the antifungal activities of S. albidoflavus C247.