Purification and biological evaluation of the metabolites produced by Streptomyces sp. TK-VL_333

Research in Microbiology 161 (2010) 335e345 www.elsevier.com/locate/resmic

Purification and biological evaluation of the metabolites produced by Streptomyces sp. TK-VL_333 Alapati Kavitha a, Peddikotla Prabhakar b, Muvva Vijayalakshmi a,*, Yenamandra Venkateswarlu b a b

Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur 522 510, India Organic Chemistry Division-I, Indian Institute of Chemical Technology, Hyderabad 500 007, India Received 10 December 2009; accepted 30 March 2010 Available online 18 April 2010

Abstract An Actinobacterium strain isolated from laterite soils of the Guntur region was identified as Streptomyces sp. TK-VL_333 by 16S rRNA analysis. Cultural, morphological and physiological characteristics of the strain were recorded. The secondary metabolites produced by the strain cultured on galactoseetyrosine broth were extracted and concentrated followed by defatting of the crude extract with cyclohexane to afford polar and non-polar residues. Purification of the two residues by column chromatography led to isolation of five polar and one non-polar fraction. Bioactivity- guided fractions were rechromatographed on a silica gel column to obtain four compounds, namely 1H-indole-3-carboxylic acid, 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde, 4-(4-hydroxyphenoxy) butan-2-one and acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester from three active polar fractions and 8-methyl decanoic acid from one non-polar fraction. The structure of the compounds was elucidated on the basis of FT-IR, mass and NMR spectroscopy. The antimicrobial activity of the bioactive compounds produced by the strain was tested against the bacteria and fungi and expressed in terms of minimum inhibitory concentration. Antifungal activity of indole-3-carboxylic acid was further evaluated under in vitro and in vivo conditions. This is the first report of 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde, 4-(4-hydroxyphenoxy) butan-2-one, acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester and 8-methyl decanoic acid from the genus Streptomyces. Ó 2010 Elsevier Masson SAS. All rights reserved. Keywords: Actinobacteria; Streptomyces sp. TK-VL_333; Bioactive metabolites; 1H-indole-3-carboxylic acid; 2,3-Dihydroxy-5-(hydroxymethyl) benzaldehyde; Acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester

1. Introduction Nature acts as a prominent reservoir for new and novel therapeutics. By employing sophisticated techniques in various screening programs, the rate of discovery of natural compounds exceeds 1 million. Out of 22, 500 biologically active compounds that have been extracted thus far from microbes, 45% are produced by Actinobacteria, 38% by fungi and 17% by unicellular bacteria (Demain and Sanchez, 2009). Members of the class Actinobacteria, and especially Streptomyces spp. have long been recognized as prolific sources of useful bioactive metabolites, providing more than half of the naturally occurring * Corresponding author. Tel.: þ91 (0) 863 2293189; fax: þ91 (0) 0863 2293378. E-mail address: [email protected] (M. Vijayalakshmi). 0923-2508/$ - see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2010.03.011

antibiotics discovered to date and continuing to be a source of new bioactive metabolites (Berdy, 2005). Unfortunately, the emergence of drug-resistant pathogens and the increase in diseases affecting the immune system have greatly intensified the need to investigate new bioactive metabolites for potential pharmaceutical and industrial applications (Demain and Sanchez, 2009; Wise, 2008). The search for new antimicrobials has not been limited to the medicinal field, but also extends to crop protection. Development of fungicide-resistant plant pathogens as well as excessive and indiscriminate use of synthetic agrochemicals has led to ecological imbalances in soil and human health (Thind, 2008). Therefore, the search for alternatives to chemical control of plant pathogens, such as biological control, has gained momentum in recent years. Several approaches, including cultural, genomic and metagenomic analysis, have been employed for the isolation of

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novel bioactive compounds. In cultural methods, potent bioactive metabolite-producing Actinobacteria strains were isolated by pretreatment of soil samples collected from deserts (Hozzein et al., 2008), caves (Nakaew et al., 2009), marine (Fenical and Jensen, 2006) and other unexplored terrestrial areas (Sait et al., 2002; Wu et al., 2009) with calcium carbonate (El-Nakeeb and Lechevalier, 1963), dry heat (Nonomura and Ohara, 1969), phenol (Hayakawa et al., 2004), irradiation with microwaves (Bulina et al., 1997), sodium dodecyl sulfate and yeast extract (Yamamura et al., 2007), etc. followed by plating them on various selective culture media like Gauze I agar (Zakharova et al., 2003), Kuster agar (Vargas et al., 2009) and humic acid vitamin agar incorporated with antibiotics, viz., trimethoprim, nalidixic acid (Hayakawa, 2008) etc. On the other hand, ‘genome mining’ aims at the genes that encode tailoring enzymes from natural product biosynthesis pathways and serve as indicator genes for identification of strains that have the genetic potential to produce novel natural products (Hornung et al., 2007). Metagenomics is a new powerful tool for identification of uncultured microbial genes encoding for bioactive compounds (Gillespie et al., 2002), new polyketide synthases (Courtois et al., 2003) and even new functions like a membrane-associated proteolytic system (Beja et al., 2000). In our continuous quest for new bioactive metabolites, Actinobacterium strain TKVL_333 having good antimicrobial potential was selected among fifty isolated strains from the soils of the Guntur region unexplored for Actinobacteria using a CaCO3-based approach. The present study reveals the fermentation, isolation, characterization and biological evaluation of the metabolites produced by strain TK-VL_333 along with its taxonomic study. 2. Materials and methods 2.1. Isolation Actinobacterium strain TK-VL_333 found to be predominant in laterite soils of the Guntur region was isolated on asparagineeglucose agar medium incubated for 7 days at 37  C using a soil dilution technique. The strain was maintained on yeast extract-malt extract-dextrose (YMD) agar medium at 4  C for further study (Williams and Cross, 1971). 2.2. Taxonomic studies Cultural characters of strain TK-VL_333 were studied on International Streptomyces Project (ISP) and non-ISP media (Dietz and Thayer, 1980). The micromorphology of the strain cultured on ISP medium 2 at 37  C for 4 days was examined under scanning electron microscopy (model JOEL-JSM 5600) at various magnifications (7500 and 18,000) (Bozzola and Russell, 1999). Utilization of carbon sources by the strain was carried out according to the method of Gottlieb (1961). Tolerance of the strain to lysozyme, phenol, NaCl and the ability of the strain to produce different enzymes, H2S, indole and acid were tested in accordance with standard protocols

(Holding and Collee, 1971). In addition, the sensitivity of the strain to different antibiotics was determined by the paper disc method (Cappuccino and Sherman, 2004). Extraction of genomic DNA of the strain was performed according to the method described by Rainey et al. (1996). 16S rRNA gene was amplified with primers forward (50 and reverse (50 TGCCGTCGCTCCTCAGCGTC-30 ) 0 TGCATGTGTTTAGGGCCTGA-3 ). The amplified DNA fragment was separated on 1% agarose gel, eluted and purified using the Qiaquick gel extraction kit (Qiagen, Germany). The purified PCR product was sequenced using the Big-Dye terminator kit ABI 310 Genetic Analyzer (Applied Biosystems, USA). The phylogenetic position of the isolated strain (TK-VL_333) was assessed by performing a nucleotide sequence database search using the BLAST program from NCBI GenBank. Sequence data of related species were retrieved from NCBI GenBank. Nucleotide substitution rates (Knuc values) were calculated (Kimura, 1980) and the phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei, 1987). The statistical significance of the tree topology was evaluated by bootstrap analysis of sequence data using CLUSTAL W software (Thompson et al., 1997). 2.3. Fermentation, extraction, purification and structural elucidation of bioactive metabolites For isolation and identification of bioactive metabolites, a loopful culture of Streptomyces sp. TK-VL_333 was cultivated in YMD broth (seed broth) and incubated on a rotary shaker (250 rpm) at 30  C. After 48 h of incubation, the seed culture (10% v/v) was transferred to the optimized fermentation medium consisting of 2% galactose, 1% tyrosine, 0.05% K2HPO4, 0.05% NaCl and 0.001% FeSO4$7H2O with pH adjusted to 7.0 (Kavitha and Vijayalakshmi, 2009). The culture filtrates (25 L) obtained after cultivation of the strain in 2.5 L conical flasks for 96 h were extracted twice with ethyl acetate and concentrated to dryness under a vacuum at 35  C. The crude extract (1.8 g) thus obtained was defatted with cyclohexane to give polar and non-polar residues. The resulting red-colored polar residue was chromatographed on a silica gel column (25  5 cm, Silica gel 60, Merck, Mumbai, India) using the gradient solvent system of CHCl3:MeOH (100e30:70 v/v). Three fractions collected at different eluent (CHCl3:MeOH) conditions (first at 80:20 v/v; second at 70:30 v/v and third at 60:40 v/v) showed antimicrobial activity against the Firmicute (Bacillus cereus), the Gammaproteobacteria (Escherichia coli) and the yeast (Candida albicans). On the other hand, the non-polar residue of the crude extract was subjected to silica gel column (25  5 cm, silica gel 60, Merck, Mumbai, India) using a gradient solvent system of hexane:ethyl acetate (100e70:30 v/v). Elutions were collected and rechromatographed on a silica gel column (25  5 cm, Silica gel 100, Merck, Mumbai, India) to yield one major fraction (fourth). The structure of the compounds was elucidated and confirmed on the basis of FT-IR (Fourier transform infra-red; model: Thermo Nicolet Nexus 670

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spectrophotometer with NaCl optics), EIMS/ESIMS (Electron ionization mass/electron spray ionization mass; model: micromass VG-7070H, 70 eV spectrophotometer) and nuclear magnetic resonance (1H and 13C NMR; model: Varian Gemini 200 and samples were made in CCl4/CDCl3 þ DMSO using tetramethyl silane as internal standard) spectroscopy. 2.4. Biological assays The antimicrobial spectra of the compounds were tested in terms of minimum inhibitory concentration (MIC) against the test bacteria like B. cereus (MTCC 430), Bacillus megaterium (NCIM 2187), Bacillus subtilis (MTCC 441), Corynebacterium diphtheriae (MTCC 116), E. coli (MTCC 40), Proteus vulgaris (ATCC 6380), Pseudomonas aeruginosa (MTCC 424), Pseudomonas solanacearum (NCIM 5103), Serratia marcescens (MTCC 118), Staphylococcus aureus (MTCC 96), Staphylococcus epidermis (MTCC 120), Xanthomonas malvacearum (NCIM 2954), Xanthomonas campestris (NCIM 2310) and fungi including Aspergillus flavus, Aspergillus niger, Alternaria alternata, C. albicans (MTCC 183), Curvularia maculans, Curvularia lunata, Epidermophyton floccosum (MTCC 145), Fusarium oxysporum (MTCC 218) and Penicillium citrinum by using the agar well diffusion assay (Cappuccino and Sherman, 2004). The compounds dissolved in DMSO at different concentrations ranging from 0 to 1000 mg/ml were employed for antimicrobial assay. The lowest concentration of the bioactive metabolite exhibiting antimicrobial activity against the test organisms was taken as the MIC of the compound (Kavitha et al., 2009). The antagonism of Streptomyces sp. TK-VL_333 against phytopathogenic fungi like F. oxysporum was tested through the agar streak method (Taechowisan et al., 2005a). The strain was streaked radially at one end of the YMD agar plate followed by incubation at 30  C. After the growth of the strain for 5 days, the plate was inoculated with the test fungus at a distance of 6 cm away from the strain and incubated for 4 days at 30  C. Simultaneously, the test fungus alone grown on YMD plate served as a control. The inhibition zone in between the strain and the test fungus revealed the antagonistic nature of the strain. The efficacy of the strain towards the wilt pathogen F. oxysporum was further confirmed by conducting a greenhouse trial (Singh and Reddy, 1979) in which polyethylene bags containing 500 g of autoclaved soil were employed for five different kinds of treatment: (i) soil inoculated with the pathogen alone; (ii) soil inoculated with the antagonist alone; (iii) simultaneous inoculation of the soil with the pathogen and antagonist; (iv) soil initially incubated with the antagonist for 4 days inoculated with the pathogen; and (v) uninoculated soil that served as control. The pathogen and strain TK-VL_333 grown individually on YMD agar were employed for soil inoculation. The antagonistic nature of the strain was tested by raising surface-sterilized sorghum seeds in the bags and the incidence of the wilted plants was recorded after 15 days. The experiment was done in triplicate and the results are the means of 10 plants per treatment. Data were statistically evaluated with one-way analysis of variance

337

(ANOVA) and standard deviations of the mean values were analyzed. Among the bioactive metabolites, 1H-indole-3-carboxylic acid produced by the strain showed strong inhibition against the growth of F. oxysporum causing wilt of Sorghum bicolor. Therefore, its antifungal potential was further tested by in vitro and in vivo studies against F. oxysporum, and bioefficacy was compared with that of commercially available systemic fungicides like mancozeb and carbendazim. The conidial suspensions of F. oxysporum grown on Czapek-Dox agar at 30  C for 10 days were treated with different concentrations, i.e. 0, 1, 10, 50, 100, 150, 200 and 500 mg/ml of 1H-indole-3-carboxylic acid, mancozeb and carbendazim. The effect of these compounds on conidial germination was recorded. After 48 h of incubation at 28  C, conidial germination was examined microscopically in 3 replicates (Hwang et al., 2001). In a greenhouse trial, seeds of sorghum sown in plastic bags containing steam-sterilized soil were drenched with antifungal solution (30 ml) prepared by dissolving the compounds individually in water þ methanol (95:5) to give different concentrations such as 0, 50, 100, 200, 400, 500, 700 and 1000 mg/ml. Sixty seedlings of sorghum (three-days-old) were inoculated with conidial suspensions of F. oxysporum (105 spores/ml) by using the soil drench method (Hwang et al., 2001). Disease severity on sorghum plants was rated after 15 days of inoculation based on the percent of wilted plants. The results were statistically analyzed by two-way ANOVA and standard deviations of the mean values were evaluated.

3. Results and discussion 3.1. Taxonomy of the strain Cultural characteristics of strain TK-VL_333 are presented in Table 1. It exhibited good growth on ISP-1, ISP-2, ISP-3, ISP-4, ISP-5, ISP-7, malt extract and maltose tryptone agar media. Growth was moderate on nutrient agar, while it was poor on Sabouraud agar media. The color of the aerial mycelium appeared white, while that of substrate mycelium went from light to dark brown. The strain produced reddish brown pigment on ISP-2, ISP-4 and ISP-5 and exhibited Table 1 Cultural characteristics of Actinobacterium strain TK-VL_333. Culture media

Growth

Pigment production

a

Good Good Good Good Good Good Good Good Moderate Poor

eb Reddish brown e Reddish brown Reddish brown Melanin e e e e

ISP-1 ISP-2 ISP-3 ISP-4 ISP-5 ISP-7 Malt extract agar Maltose tryptone agar Nutrient agar Sabouraud’s agar a b

International Streptomyces Project media. No production.

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melanin pigmentation on ISP-7. As depicted in Fig. 1, sporophore morphology of the strain was of a spiral type, which may be placed in the spira group of the family Streptomycetaceae and the genus Streptomyces. These results are in close agreement with the findings of Pridham et al. (1958) and Williams et al. (1983). Table 2 depicts physiological and biochemical characteristics of strain TK-VL_333. The strain utilized D-fructose, Dgalactose, D-glucose, D-xylose, glycerol, mannitol, starch and sucrose as carbon sources indicating its wide pattern of carbon assimilation. It exhibited salt tolerance up to 7% that may be placed in the intermediate group of salt tolerance, as suggested by Tresner et al. (1968). Production of melanoid pigments on ISP-7 medium and enzymes such as amylase, L-asparaginase, catalase, cellulase, chitinase, nitrate reductase, protease and urease by the strain was noticed. It was found positive for biochemical tests like H2S, indole and citrate utilization. Kampfer et al. (1991) suggested that all these tests are indispensable tools for classification of Actinobacteria. It showed sensitivity to a variety of antibiotics, but was resistant to ampicillin, cephoxitin, cloxacillin, colistin, co-trimazine, deoxycycline hydrochloride, methicillin, nalidixic acid, nitrofurantoxin, rifampicin and trimethoprim, suggesting that bioactive compounds produced by the strain may be responsible for the resistance of the strain to these antibiotics. The phylogenetic position of Streptomyces sp. TK-VL_333 was established by amplifying the 16S rRNA region and the

Table 2 Physiological characteristics of Streptomyces sp. TK-VL_333. Utilization of carbon sources D-fructose

Antibiotic sensitivity (mg/ml)

D-xylose Glycerol Inositol Lactose Maltose Mannitol Sorbitol Starch Sucrose

P P P P P W W W P W P P

Tolerance to NaCl

Up to 7%

Production of Melanoid pigments Amylase L-asparaginase Catalase Cellulase Chitinase DNAase Lipase Nitrate reductase Protease RNAase Urease H2S Indole Citrate utilization

þ þ þ þ þ þ þ þ þ þ þ þ

D-glucose D-galactose

Amikacin (30) Amoxicillin (10) Ampicillin (10) Bacitracin (10) Cephoxitin (30) Ciprofloxacin (5) Clindamycin (2) Cloxacillin (1) Colistin (10) Co-Trimazine (25) Deoxycycline hydrochloride (30)

S S R S R S S R R R R

Erythromycin (15) Furoxone (100) Gentamicin (10) Kanamycin (30) Methicillin (5) Metronidazole (5) Nalidixic acid (30) Neomycin (30) Nitrofurantoxin (300) Oxytetracycline (30) Penicillin-G (10) Polymyxin-B (300) Rifampicin (5) Roxithromycin (30) Streptomycin (10) Tetracycline (30) Trimethoprim (5) Vancomycin (30)

S S S S R S R S R S S S R S S S R S

P, positive; W, weak; þ, positive result; -, negative result; R, resistant; S, sensitive.

sequence of the strain (705 bases) was examined by BLAST analysis. The 16S rRNA genome sequence of the strain showed 100% identity with that of Streptomyces tendae (Fig. 2); hence, strain TK-VL_333 was assigned to the S. tendae cluster and the 16S rRNA sequence was submitted to the NCBI GenBank with accession number FJ877150.

3.2. Extraction, purification, identification and structural elucidation of bioactive metabolites produced by Streptomyces sp. TK-VL_333

Fig. 1. Scanning electron microscope images of Streptomyces sp. TK-VL_333.

The fermented broth obtained after culturing the strain in the optimized culture medium composed of 2% galactose, 1% tyrosine, 0.05% K2HPO4, 0.05% NaCl and 0.001% FeSO4$7H2O with initial pH adjusted to 7.0 for 96 h was extracted with ethyl acetate and concentrated under a vacuum to yield a red-colored crude extract. It was later defatted with cyclohexane to separate polar and non-polar residues which in turn subjected to a silica gel column using CHCl3:MeOH (70:30 v/ v) and hexane:ethyl acetate (80:20 v/v) as the eluting solvent system, respectively. Three different fractions collected from polar residue and one from a non-polar fraction of the crude extract were active against B. cereus, E. coli and C. albicans. Further purification of these fractions through a silica gel

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Fig. 2. Phylogenetic tree derived from 16S rRNA gene sequences showing the relationship between strain TK-VL_333 and species belonging to the genus Streptomyces was constructed using the neighbor-joining method. Bootstrap values are expressed as percentages of 1000 replications. Bar, 0.005 substitutions per nucleotide position.

column yielded one compound (T1) from the first fraction, two (T2, T3) from the second and one (T4) from the third of the polar residue. On the other hand, one compound (T5) was extracted from the non-polar component (fourth) by employing silica gel column chromatography. The entire flow chart showing extraction and purification of bioactive metabolites from the culture filtrate of Streptomyces sp. is presented in Fig. 3. Compound T1 was obtained as light-brown amorphous powder. It was completely soluble in DMSO, MeOH and partially in CHCl3. The IR spectrum exhibited absorption bands at Vmax 3388.4 cm1 and 1700.7 cm1 indicating NH and C]O groups in the structure respectively (Supplementary Fig. 1A). EIMS analysis of the compound gave a molecular ion at m/z 184.0 [M þ Na]þ (Supplementary Fig. 1B). The 1H NMR (CDCl3þDMSO, 200 MHz) spectrum of compound T1 showed 4 signals at 10.2 (1H, br, s), 7.64 (1H, d, J ¼ 8.7 Hz), 7.40 (1H, d, J ¼ 8.5 Hz) and 7.14 (4H, m) (Supplementary Fig. 1C), while 13C NMR (CDCl3þDMSO, 50 MHz) depicted 9 signals at 174.39 (eCOOH), 136.11 (C-9), 127.10 (C-2), 123.58 (C-8), 121.39 (C-5), 118.88 (C-6), 118.5 (C-7), 111.42 (C-4) and 107.66 (C-3) (Supplementary Fig. 1D). Based on the above spectral data, bioactive compound T1 was identified as 1H-indole-3-carboxylic acid (Fig. 4A) with the molecular formula C9H7O2N.

Compound T2 appeared as a pale yellow solid which was completely soluble in DMSO and MeOH and partially in CHCl3. The IR absorption bands at Vmax 3449.5 cm1 and 2923.87 cm1 suggested the presence of OH groups and Vmax 1637.9 cm1 indicated a CHO group in the structure, respectively (Supplementary Fig. 2A). EIMS analysis of the compound displayed a molecular ion at m/z 192 [M þ H]þ (Supplementary Fig. 2B). Analysis of the 1H NMR (CDCl3 þ DMSO, 200 MHz) spectrum of the compound revealed 4 signals at 9.61 (1H, s), 7.16 (1H, d, J ¼ 3.39 Hz), 6.48 (1H, d, J ¼ 3.58 Hz) and 4.69 (2H, s) (Supplementary Fig. 2C), whereas 13C NMR (CDCl3þDMSO, 50 MHz) exhibited 7 signals at 200.48 (eCHO), 155.96 (C-2), 148.41 (C-3), 146.63 (C-5), 139.90 (C-6), 132.47 (C-4) and 57.73 (eCH2, C-7) (Supplementary Fig. 2D). Compound T2 was identified and confirmed as 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde (Fig. 4B) with the molecular formula C8H8O4 by employing the above spectral data. Compound T3 is a light yellow solid and was found to be completely soluble in DMSO and MeOH, but partially in CHCl3. The IR spectrum indicated the presence of C]O and CeO groups in the structure by showing absorption bands at Vmax 1637.9 cm1 and 1218.9 cm1 respectively (Supplementary Fig. 3A). In EIMS analysis, the compound exhibited a molecular ion at m/z 180 [M]þ (Supplementary

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Fermentation broth (25 L)

Culture filtrate

Cell mass (Discarded)

Extracted with ethyl acetate Crude extract (1.8 g) Defatted with cyclohexane

Polar residue (1.3 g)

Non-polar residue (500 mg)

Silica gel column chromatography (Eluted with CHCl3: MeOH)

Silica gel column chromatography (Eluted with hexane: ethyl acetate)

Biologically active fractions screened against bacteria and yeast

First (300 mg)

Second (200 mg)

Third (150 mg)

Rechromatographed on silica gel column

Rechromatographed on silica gel column

(eluted with CHCl3: MeOH)

(eluted with CHCl3: MeOH)

*T1 T2 (100 mg) (15 mg)

T3 (13 mg)

Fourth (200 mg)

Rechromatographed on silica gel column

Rechromatographed on silica gel column

(eluted with CHCl3: MeOH)

T4 (20 mg)

(eluted with hexane: ethyl acteate)

T5 (24 mg)

Fig. 3. Flow chart illustrating the extraction and purification of bioactive metabolites from Streptomyces sp. TK-VL_333. On the basis of spectral analysis, T1 was identified as 1H-indole-3-carboxylic acid, T2 as 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde, T3 as 4-(4-hydroxyphenoxy) butan-2-one, T4 as acetic acid-2hydroxy-6-(3-oxo-butyl)-phenyl ester and T5 as 8-methyl decanoic acid.

Fig. 3B). The 1H NMR (CDCl3þDMSO, 200 MHz) spectrum of the compound depicted 5 signals at 7.04 (2H, d, J ¼ 8.08 Hz), 6.71 (2H, d, J ¼ 8.06 Hz), 3.78 (2H, t, J ¼ 5.88 Hz), 2.76 (2H, t, J ¼ 5.90 Hz) and 2.14 (3H, s) (Supplementary Fig. 3C) while 13C NMR (CDCl3 þ DMSO, 50 MHz) inferred 8 signals at 187.53 (C-20 , eC¼O) 147.3 (C4), 134.83 (C-1), 130.14 (C-3, C-5), 115.44 (C-2, C-6), 63.82 (C-40 ), 38.26 (C-30 ) and 29.69 (C-10 ) (Supplementary Fig. 3D). On the basis of the above spectral information, compound T3 was analyzed as 4-(4-hydroxyphenoxy) butan-2-one (Fig. 4C) with the molecular formula C10H12O3. Compound T4 was a yellow solid found to be completely soluble in DMSO, MeOH and partially in CHCl3. Its IR spectrum showed absorption bands at Vmax 3447.5 cm1, 1717.02 cm1 and 1216.2 cm1 assignable to hydroxyl, C]O and CeO groups in the structure respectively (Supplementary Fig. 4A). EIMS analysis of the compound showed a molecular ion at m/z 223 [M þ H]þ (Supplementary Fig. 4B). Analysis of the 1H NMR (CDCl3 þ DMSO, 200 MHz) spectrum of the compound informed 4 signals at 7.25 (3H, m), 2.96 (2H, t,

J ¼ 7.55 Hz), 2.68 (2H, t, J ¼ 7.55 Hz) and 2.17 (6H, s) 13 C NMR (Supplementary Fig. 4C), whereas (CDCl3 þ DMSO, 50 MHz) displayed 10 signals at 176.64 (C30 ), 168.0 (C-100 ), 148.52 (C-2), 140.18 (C-1), 128.54 (C-6), 128.25 (C-3, C-5), 126.35 (C-4), 35.22 (C-20 ), 30.90 (C-40 ) and 30.63 (C-10 , C-200 ) (Supplementary Fig. 4D). From the above spectral information, compound T4 was characterized as acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester (Fig. 4D) with the molecular formula C12H14O4. Compound T5 is a yellow solid which was completely soluble in CHCl3, DMSO and MeOH. The IR absorption maxima at Vmax 1711.0 cm1 indicated the presence of a C] O group in the structure (Supplementary Fig. 5A). ESIMS analysis of the compound suggested a molecular ion at m/z 186 [M þ Na]þ (Supplementary Fig. 5B). The 1H NMR (CDCl3, 200 MHz) spectrum of the compound T5 displayed 5 signals at 2.32 (2H, t, J ¼ 7.55 Hz), 1.99 (1H, m), 1.61 (2H, m) and 1.20e1.30 (10H, m) and 0.9e0.82 (6H, m) (Supplementary Fig. 5C). 13C NMR (CDCl3, 50 MHz) showed 11 signals at 180.20 (C-1), 34.17 (C-2), 32.03 (C-3), 29.79 (C-

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O 4 5

1 6

HO

OH

3

8

4

7

5

OH

3 6

2

HO 2

9 N H 7

1

CHO

A

B O 5 6

4

6

O 4'

1

5

3'

2'

1'

4'

O

2

2' 1'

1

3

O

2

4

3'

1"

OH

2"

O

3

C

D O 5

HO

1

2

3

7 8

4

9 10

6 8'

E Fig. 4. Molecular structures of A: 1H-indole-3-carboxylic acid (T1), B: 2,3dihydroxy-5-(hydroxymethyl) benzaldehyde (T2), C: 4-(4-hydroxyphenoxy) butan-2-one (T3), D: Acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester (T4) and E: 8-methyl decanoic acid (T5).

4), 29.55 (C-5), 29.47 (C-6), 29.38 (C-7), 29.19 (C-8), 24.76 (C-9), 22.30 (C-10) and 14.27 (C-80 ) (Supplementary Fig. 5D). Using the above spectral data, compound T5 was elucidated as 8-methyl decanoic acid (Fig. 4E) with the molecular formula C11H22O2. 3.3. Biological assays The antimicrobial profile of the bioactive compounds in terms of MIC is shown in Table 3. Among the five bioactive compounds isolated from Streptomyces sp., 1H-indole-3carboxylic acid (T1) exhibited good antimicrobial activity followed by acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester (T4) and 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde (T2). Of the opportunistic and pathogenic Gram-positive bacteria tested, S. aureus and S. epidermis were highly sensitive to metabolites T1, T2, T4 and T5. Among the Gramnegative bacteria, E. coli and X. campestris showed high sensitivity to T1, T2, T3 and T4. As compared to tetracycline (positive control), T1 was more active against several bacteria tested, whereas compounds T2 and T4 exhibited good activity against a few bacteria. In other cases, tetracycline exhibited good antibacterial activity over the metabolites produced by the strain. MIC values of compounds T1, T2, T3, T4, T5 and tetracycline (positive control) ranged from 10 to 45 mg/ml, 15 to 55 mg/ml, 45 to 125 mg/ml, 15 to 55 mg/ml, 30 to 95 mg/ml and 25 to 75 mg/ml respectively.

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The bioactive compounds exhibited good antifungal activity against dermatophytes like C. albicans and E. floccosum, for which MIC values recorded included 10 and 20 mg/ ml (T1), 30 and 45 mg/ml (T2), 65 and 70 mg/ml (T3), 25 and 40 mg/ml (T4), 55 and 75 mg/ml (T5) and 50 and 60 mg/ml (griseofulvin). The compounds T1, T2 and T4 showed superior antifungal activity against the dermatophytes tested when compared to that of griseofulvin (positive control). Among the filamentous fungi tested, A. niger and F. oxysporum were sensitive to metabolites of the strain as well as to the commercial fungicide carbendazim (positive control). Carbendazim showed high antifungal activity when compared to T1, T4 and T2. MIC values of the compounds, i.e. T1 (15e150 mg/ml), T2 (20e175 mg/ml), T3 (85e400 mg/ml), T4 (20e160 mg/ml), T5 (50e230 mg/ml) and carbendazim (2e12 mg/ml) were recorded. S. tendae represents a prominent source of production of novel bioactive compounds. It has been reported to produce nucleoside peptide antibiotics such as nikkomycins Z, X, J and I (Dahn et al., 1976). Fiedler et al. (1994) recorded a new naphthoquinone antibiotic, juglomycin Z from S. tendae Tu 901/8c, active against Gram-positive and Gram-negative bacteria and fungi. Other bioactive compounds reported from S. tendae include echoserine (Blum et al., 1995), dioxolides, anhydroshikimate, para-hydrobenzamide (Blum et al., 1996) and cervimycins A-D (Herold et al., 2005). However, in the present study, production of five bioactive metabolites, namely 1H-indole-3-carboxylic acid (T1), 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde (T2), 4-(4-hydroxyphenoxy) butan-2one (T3), acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester (T4) and 8-methyl decanoic acid (T5) by Streptomyces sp. TKVL_333 closely related to S. tendae cluster was recorded. Aldridge et al. (1971) isolated indole-3-carboxylic acid from culture filtrates of Lasiodiplodia theobromae. Indole-3carboxylic acid has been isolated and characterized from Streptomyces sp. Act80115 by Shaaban et al. (2008) through electrospray ionization mass spectra. Indole-3-carboxylic acid-related metabolites, indole-3-carboxylic acid, 2-desoxythymidin and 5-dimethylallylindole-3-carboxylic acid were recorded from Streptomyces sp. B8000 (Poumale et al., 2006) and Streptomyces sp. MS239 (Motoshashi et al., 2008) respectively. To our knowledge, information regarding the natural occurrence of the compounds 2,3-dihydroxy-5(hydroxymethyl) benzaldehyde (T2), 4-(4-hydroxyphenoxy) butan-2-one (T3), acetic acid-2-hydroxy-6-(3-oxo-butyl)phenyl ester (T4) and 8-methyl decanoic acid (T5) from microorganisms is not yet reported. In the present study, five bioactive compounds extracted from strain TK-VL_333 showed antimicrobial activity against opportunistic and pathogenic bacteria and fungi, and ours is the first report of isolation and characterization of compounds T2, T3, T4 and T5 from Actinobacteria. Streptomyces sp. TK-VL_333 as an antagonistic agent against the wilt pathogen F. oxysporum was determined by the agar plate method. A clear zone of inhibition (30 mm) was noted between Streptomyces sp. TK-VL_333 and F. oxysporum, suggesting the antagonistic nature of the strain. The

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Table 3 Antimicrobial activities of bioactive metabolites produced by Streptomyces sp. TK-VL_333. Test organism

MIC (mg/ml) 1H-indole-3carboxylic acid

2,3-Dihydroxy-5(hydroxymethyl) benzaldehyde

4-(4Hydroxyphenoxy) butan-2-one

Acetic acid-2hydroxy-6-(3-oxobutyl)-phenyl ester

Anta

8-Methyl decanoic acid

Bacteria Bacillus cereus B. megaterium B. subtilis Corynebacterium diphtheriae Escherichia coli Proteus vulgaris Pseudomonas aeruginosa P. solanacearum Serratia marcescens Staphylococcus aureus S. epidermis Xanthomonas campestris X. malvacearum

25 20 30 40 25 40 40 30 45 10 15 25 35

35 35 40 55 35 50 55 40 50 20 15 35 45

45 60 85 100 60 70 75 70 125 50 50 55 75

30 25 35 50 30 45 50 40 55 15 20 30 40

45 55 95 70 50 65 65 60 75 35 30 70 60

75 50 50 50 25 25 50 50 25 50 50 50 50

Yeast Candida albicans Epidermophyton floccosum

10 20

30 45

65 70

25 40

55 75

50 60

40 20 150 125 80 15 20

45 20 175 145 95 25 30

90 85 400 350 210 95 100

45 25 160 140 85 20 30

85 60 230 175 125 65 50

2 5 7 10 12 5 2

Filamentous fungi Aspergillus flavus A. niger Alternaria alternata Curvularia maculans C. lunata Fusarium oxysporum Penicillium citrinum a

Antibiotic: tetracycline against bacteria, griseofulvin against yeast and carbendazim against fungi.

degree of antagonism of the strain towards the wilt pathogen analyzed through greenhouse trials is shown in Table 4. Simultaneous treatment of the soil with the pathogen and strain reduced the incidence of Fusarium wilt on sorghum plants up to 53.3% when compared to that of plants raised in soil inoculated with the pathogen alone. A nearly 80% drastic reduction in wilt occurrence was noted in sorghum plants pretreated with antagonist followed by pathogen inoculation. This may be due to the elaboration of bioactive metabolites by the strain prior to the establishment of the pathogen. On the other hand, sorghum plants raised in soils inoculated with antagonist alone/untreated soils appeared healthy.

Majority of Actinobacteria found in soils belong to the genus Streptomyces (Goodfellow and Simpson, 1987) and most of them act as potent antagonists against a variety of phytopathogenic fungi by elaborating various bioactive compounds (Prapagdee et al., 2008). In the present study, Streptomyces sp. TK-VL_333 showed inhibitory activity against Fusarium wilt. Likewise, Yuan and Crawford (1995) recorded the antifungal activity of Streptomyces lydicus against Pythium ultimum and Rhizoctonia solani. Anitha and Rebeeth (2009) reported the inhibition of fungal pathogens like F. oxysporum, Fusarium solani, R. solani and A. alternata by Streptomyces griseus using the dual culture method.

Table 4 Antifungal spectrum of Streptomyces sp. TK-VL_333 against F. oxysporum under in vivo conditions. S.No.

Treatment

Percent of wilted sorghum plants

1. 2. 3. 4.

Soil inoculated with the pathogen alone Soil inoculated with the antagonist alone Simultaneous inoculation of the soil with the pathogen and antagonist Soil incubated with the antagonist for 4 days followed by pathogen inoculation Uninoculated soil

100 0 46.6 20.0

5.

Average of 3 trials with 10 plants per treatment (SD). Data were statistically analyzed by one-way ANOVA and found to be significant at 5%.

   

0 0 0.92 0.81

00

Percent reduction in wilting 0 0 53.3 80.0

   

0 0 0.92 0.81

00

Inhibition of conidial germination (%)

A. Kavitha et al. / Research in Microbiology 161 (2010) 335e345

120

1H-indole-3-carboxylic aicd Mancozeb Carbendazium

100 80 60 40 20 0 0 1 10 50 100 150 200 500 Concentration of antifungal compound (µg/ml)

Fig. 5. Antifungal spectrum of the metabolite 1H-indole-3-carboxylic acid isolated from Streptomyces sp. TK-VL_333 against F. oxysporum under in vitro conditions (values are means of three replicates  SD). Statistically significant at 5%.

The bioactive metabolite 1H-indole-3-carboxylic acid obtained from Streptomyces sp. TK-VL_333 showed good antifungal activity against F. oxysporum causing wilt of S. bicolor. Therefore, its antifungal potential was further tested by in vitro and in vivo studies against F. oxysporum. As shown in Fig. 5, 1H-indole-3-carboxylic acid inhibited conidial germination of F. oxysporum under in vitro conditions at a concentration of 150 mg/ml, and the fungicides, mancozeb and carbendazim showed inhibitory activity at a level of 500 mg/ml and 50 mg/ml respectively. Under in vivo conditions, the bioefficacy of the compound against Fusarium wilt of sorghum was checked in a greenhouse trial in comparison with that of systemic fungicides. The compounds 1H-indole-3carboxylic acid, mancozeb and carbendazim were effective in controlling Fusarium wilt at concentrations of 400 mg/ml, 700 mg/ml and 100 mg/ml respectively (Fig. 6). Matsuda et al. (1998) noted the suppression of bacterial wilt in tomato caused by Ralstonia solanacearum with 3indole propionic acid. The metabolites of different Actinobacteria species have been reported to be active against Fusarium spp. and other plant pathogens. Soil-borne phytopathogens such as F. oxysporum, Fusarium moniliforme, Fusarium semitectum, F. solani and R. solani were highly sensitive to 2-methylheptyl isonicotinate produced by

Streptomyces sp. 201 (Bordoloi et al., 2002). Zhong et al. (2004) reported that wuyiencin isolated from Streptomyces hygroscopicus var. wuyiensis inhibited conidial germination of Botrytis cinerea. Secondary metabolites such as 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4phenylcoumarin produced by Streptomyces aureofaciens CMUAc 130 showed antifungal activity against phytopathogenic fungi (Taechowisan et al., 2005b). In the present study, the efficacy of the bioactive compound indole-3-carboxylic acid against the wilt pathogen was found to be better than that of mancozeb, but less active than carbendazim. Bioactive compounds, because of their natural origin, are biodegradable and they do not leave toxic residues or byproducts to contaminate the environment (http://www.ias. ac.in/currsci/jan25/articles22.htm-pesticides), whereas commercial fungicides such as carbendazim pose severe toxicity to humans, plants and animals (Mantovani et al., 1998). In order to provide tools for integrated agriculture, biological control offers an eco-friendly alternative to the use of synthetic fungicides for controlling plant diseases, and the bioactive compound, 1H-indole-3-carboxylic acid produced by the strain may be used to control Fusarium wilt. From the present investigation, it is apparent that the five bioactive compounds obtained from Streptomyces sp. TK-VL_333 have antimicrobial potential against a wide variety of bacteria, yeast and filamentous fungi. Acknowledgements A senior research fellowship awarded by Indian Council of Medical Research (ICMR), New Delhi, India to AK is gratefully acknowledged. The authors (PP and YV) are indebted to the Department of Biotechnology (DBT), New Delhi, India for providing financial support.

Appendix. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.resmic.2010.03.011.

120

Disease severity (Percent of wilted plants)

343

1H-indole-3-carboxylic acid 100

Mancozeb Carbendazim

80 60 40 20 0 0

50 100 200 400 500 700 1000 Concentration of antifungal compound (µg/ml)

Fig. 6. Antifungal spectrum of the metabolite 1H-indole-3-carboxylic acid isolated from Streptomyces sp. TK-VL_333 against F. oxysporum, causing Fusarium wilt in sorghum plants (60) under in vivo conditions in comparison with mancozeb and carbendazim (values are means of three replicates  SD). Statistically significant at 5%.

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