isolation, characterization and biological evaluation of...

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Microbiological Research 165 (2010) 199—210

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Isolation, characterization and biologicalevaluation of bioactive metabolites fromNocardia levis MK-VL_113

Alapati Kavithaa, Peddikotla Prabhakarb, Manchala Narasimhulub,Muvva Vijayalakshmia,�, Yenamandra Venkateswarlub,Karanam Venkateswara Raoa, Venkata Balaraju Subba Rajuc

aDepartment of Botany and Microbiology, Acharya Nagarjuna University, Guntur 522 510, IndiabOrganic Chemistry Division-I, Indian Institute of Chemical Technology, Hyderabad 500 007, IndiacUniversity of Texas at San Antonio, Texas, USA

Received 26 November 2008; received in revised form 29 April 2009; accepted 3 May 2009

KEYWORDSActinomycetes;Nocardia levis;Bioactivemetabolites;1-phenylbut-3-ene-2-ol

ee front matter & 2009micres.2009.05.002

ing author. Tel.: +91 086ess: [email protected]

SummaryAn Actinomycete isolate found to be prominent in the laterite soils of AcharyaNagarjuna University (ANU) Campus, Guntur was identified as Nocardia levis MK-VL_113 by 16S rRNA analysis. Cultural, morphological and physiological character-istics of the strain were recorded. Screening of secondary metabolites obtained from4-day old culture broth of the strain led to the isolation of two fractions activeagainst a wide variety of Gram-positive, Gram-negative bacteria and fungi. Thestructure of the first active fraction was elucidated using FT-IR, EI-MS, 1H NMR and13C NMR spectra and identified as 1-phenylbut-3-ene-2-ol which is first time reportedas a natural product. The compound exhibited good antimicrobial potential againstthe opportunistic and pathogenic bacteria and fungi. The antifungal activity of thestrain and its metabolite were further confirmed with in vitro and in vivo studies.Evidence for the antagonism of the strain against Fusarium oxysporum, causing wiltdisease in sorghum was demonstrated by the formation of inhibition zone in in vitroplate assay and reduction in the incidence of wilt of sorghum plants by using a greenhouse trial. Analysis of the rhizosphere soil extracts by high performance liquidchromatography also demonstrated the production of the compound by the strainunder in vivo conditions. As compared to the commercial fungicide mancozeb, thebioactive compound, 1-phenylbut-3-ene-2-ol was highly effective in controlling wiltof sorghum. Besides, the partially purified second fraction (PPF) subjected to gas

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A. Kavitha et al.200

chromatography–mass spectrometry revealed the presence of phenylethyl alcohol,dibutyl phthalate and 1,2-benzenedicarboxylic acid, 3-nitro.& 2009 Elsevier GmbH. All rights reserved.

Introduction

Development of multiple drug resistant microbesrevealed the need to search for new and novelantimicrobials (Wise 2008). Among the antibioticproducing microbes, the class Actinobacteria re-presents a broad range of valuable and prominentsources of pharmaceutically active metabolites inwhich, the members of the genus Streptomycesalone contributes more than half of the naturallyoccurring metabolites discovered up to date. Be-sides, 60% of antibiotics developed for agriculturalpurpose were isolated from the same genus (Berdy2005). However, the percent of discovery of newmetabolites from these common and ubiquitousactinomycetes has been declined (Kurtboke et al.1992). Hence, in the present era, search for rareactinomycetes has gained much importance inorder to enhance the rate of discovery of new andpotent antimicrobial agents (Mikami 2007). Ourcontinuous screening for new bioactive metabolitesfrom Actinomycete genera resulted in the isolationof a strain MK-VL_113 from laterite soil sample ofAcharya Nagarjuna University (ANU) Campus, Gun-tur which exhibited high antimicrobial potential.Therefore, in the present study, attempts havebeen made to study the taxonomic position of thestrain as well as the extraction, identification andbiological evaluation of its active metabolites.

Materials and methods

Microorganism

The actinomycete strain MK-VL_113 was isolatedfrom the laterite soil sample of ANU Campus byemploying soil dilution technique on glycerol-asparagine-salts agar medium. The strain wasmaintained on yeast extract–malt extract–dextrose(YMD) agar medium at 4 1C for further study(Williams and Cross 1971).

Taxonomic studies

Cultural, morphological, and physiological char-acteristics of the strain were studied according tothe standard procedures (Shirling and Gottlieb1966). Cultural characters of the strain were

recorded on International Streptomyces Project(ISP) media such as tryptone–yeast extract agar(ISP-1), YMD agar (ISP-2), oat meal agar (ISP-3),starch–inorganic-salts agar (ISP-4), glycerol–aspar-agine–salts agar (ISP-5), tyrosine agar (ISP-7) andnon-ISP media like nutrient agar and Czapek-Doxagar media (Dietz and Thayer 1980). The micro-morphology of the strain cultured on ISP medium 2at 37 1C for 5 days was examined under a lightmicroscope (Kageyama et al. 2004a, b). Utilizationof carbohydrates was tested in minimal mediumcontaining different carbon sources at 1% concen-tration (Isik et al. 1999). Tolerance of the strain tolysozyme, phenol, NaCl and the ability of the strainto produce different enzymes were tested inaccordance to standard protocols (Holding andCollee 1971; Gordon et al. 1974). The strain wasalso examined for its ability to produce H2S, indoleand acid (Poonwan et al. 2005). The sensitivity ofthe strain to different antibiotics was determinedby paper disc method which can also be used as ataxonomic aid to identify actinomycete genera(Goodfellow and Orchard 1974).

Phylogenetic analysis

Extraction of genomic DNA of the strain wasperformed according to the method described byRainey et al. (1996). The 16S rRNA gene wasamplified with primers forward (50-GAGTTTGATCCTGGCTCA-30) and reverse (50-ACGGCTACCTTGTTAC-GACTT-30). The amplified DNA fragment was sepa-rated on 1% agarose gel, eluted and purified usingQiaquick gel extraction kit (Qiagen, Germany). Thepurified PCR product was sequenced using the Big-Dye terminator kit ABI 310 Genetic Analyzer(Applied Biosystems, USA). Species related to thenew soil isolate (MK-VL_113) was identified byperforming a nucleotide sequence database searchusing BLAST program from GenBank. Sequence dataof related species were retrieved from GenBank.Nucleotide substitution rates (Knuc values) werecalculated (Kimura 1980) and the phylogenetic treewas constructed by using neighbour-joining method(Saitou and Nei 1987). The statistical significance ofthe tree topology was evaluated by bootstrapanalysis of sequence data using CLUSTALW software(Thompson et al. 1997).

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Isolation, characterization and biological evaluation of bioactive metabolites from N. levis 201

Fermentation

A loopful of well sporulated culture of the strainMK-VL_113 was inoculated into a 500ml Erlenmeyerflask containing 100ml of seed medium composedof yeast extract (0.4%), malt extract (1.0%),dextrose (0.4%) and CaCO3 (0.2%) with pH 7.2.The inoculated flask was incubated on rotary shaker(250 rpm) at 28 1C for 48 h. Seed culture (10% v/v)was transferred to production medium consisting of2% sucrose, 1% tryptone, 0.05% K2HPO4, 0.05% NaCland 0.001% FeSO4 � 7H2O (pH 6.5) and incubated onrotary shaker (250 rpm) at 30 1C for 96 h.

Isolation, purification and identification ofbioactive metabolites

The fermented broth collected at the end of 96 hwas centrifuged and the culture filtrate thusobtained was extracted twice with ethyl acetate.The solvent extracts were pooled and concentratedin vacuum to dryness and the resultant crude darkbrown residue was subjected to silica gel columnchromatography (25� 5 cm, Silica gel 60, Merck)using gradient solvent system of hexane:ethylacetate. Elutions collected from column chromato-graphy were concentrated and screened for anti-microbial activity against Gram-positive (Bacilluscereus MTCC 430) and Gram-negative (Escherichiacoli MTCC 40) bacteria and yeast (Candida albicansMTCC 183). Two active fractions exhibiting goodantimicrobial potential were selected for furtherstudy. The structure of the first active fractionpurified with HPLC semi-preparative column(250� 10mm, 5 mm using hexane:2-propanol (8:2v/v), scanned at UV 254 nm) was elucidated on thebasis of FT-IR, EI-MS, 1H NMR and 13C NMR spectraldata. Optical rotation of the pure compound wasmeasured on a Perkin-Elmer 241 polarimeter.

The components of the partially purified secondfraction (PPF) were analyzed on Agilent GC–MSsystem (GC: 5890 series II; MSD 5972). The fused-silica HP-5 capillary column (30m� 0.25mm, ID,film thickness of 0.25 mm) was directly coupled tothe MS. The carrier gas was helium with a flow rateof 1.2mlmin�1. Oven temperature was pro-grammed (50 1C for 1min, then 50–280 1C at a rateof 5 1C/min) and subsequently, held isothermallyfor 20min. The temperature of injector port wasmaintained at 250 1C and that of detector at 280 1C(Boussaada et al. 2008). The peaks of the obtainedcomponents in gas chromatography were subjectedto mass-spectral analysis. The spectra were ana-lyzed from the available library data, NIST MS

Search (version 2.0) (included with NIST’02 massspectral library, Agilent p/n G1033A).

Biological assays

The antimicrobial spectra of the bioactivecompounds produced by the strain were deter-mined in terms of minimum inhibitory concentra-tion (MIC) against a wide variety of Gram-positive,Gram-negative bacteria and fungi by using agarplate diffusion assay (Cappuccino and Sherman2002). Nutrient agar and Czapek-Dox agar werethe media prepared for the growth of bacteriaand fungi, respectively. The metabolite dissolvedin DMSO at concentrations ranging from 0 to1000 mg/ml was used to assay against the testbacteria such as B. cereus (MTCC 430), B. mega-terium (NCIM 2187), B. subtilis (MTCC 441),Corynebacterium diphtheriae (MTCC 116), E. coli(MTCC 40), Proteus vulgaris (ATCC 6380), Pseudo-monas aeruginosa (MTCC 424), P. flourescens (MTCC103), P. solanacearum (NCIM 5103), Serratia mar-cescens (MTCC 118), Staphylococcus aureus (MTCC96), S. epidermis (MTCC 120), Xanthomonas mal-vacearum (NCIM 2954), X. campestris (NCIM 2310)and fungi including Aspergillus flavus, A. niger,Alternaria alternata, C. albicans (MTCC 183),Curvularia maculans, C. lunata, Fusarium oxyspor-um (MTCC 218) and Penicillium citrinum. Theinoculated plates were examined after 24–48 h ofincubation at 37 1C for bacteria and 48–72 h at 28 1Cfor fungi. The lowest concentration of the bioactivemetabolite exhibiting significant antimicrobial ac-tivity against the test microbes was taken as theMIC of the compound.

Antifungal spectrum of the strain MK-VL_113under in vitro and in vivo conditions

In vitro screeningAgar streak method was employed for initial

screening of the strain against the wilt pathogen,F. oxysporum. At one end of the YMD agar plate, thestrain MK-VL_113 was streaked horizontally andincubated at 28 1C for 5 days. This was done toallow the culture to be established on the agarsurface and to sporulate prior to inoculation of theplates with the test fungus. An actively growingculture of F. oxysporum was placed about 6 cmaway from the strain and the inoculated plateswere incubated at 28 1C for 4 days. YMD platecontaining F. oxysporum alone served as a control.The degree of antagonism was expressed bymeasuring the inhibition zone in terms of distance

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between the strain and the test fungus in dualculture (Taechowisan et al. 2005).

Green house studiesBioefficacy of antagonistic strain MK-VL_113

against F. oxysporum was evaluated on sorghum(Sorghum bicolor (L.) Moench) plants using theprocedure described by Singh and Reddy (1979)with slight modifications. The experiment wasconducted in polyethylene bags containing 500 gof autoclaved soil and includes five differenttreatments such as (i) soil inoculated with thepathogen alone, (ii) soil inoculated with theantagonistic alone, (iii) simultaneous inoculationof the soil with the pathogen and antagonist, (iv)soil initially treated with the antagonist and after 4days of incubation inoculated with the pathogenand (v) uninoculated soil that served as control.

The pathogen grown on Czapek-Dox and that ofthe antagonist on YMD agar was used for soilinoculation. Antagonism of the strain againstF. oxysporum was tested by raising the surfacesterilized sorghum seeds in the bags and theincidence of wilted plants was recorded after 15days. Data are the means of 10 plants pertreatment and a result of three trials in greenhouse experiments. The results are statisti-cally analyzed with one-way analysis of variance(ANOVA) and the standard deviations of the meanvalues are calculated.

Detection of the active fraction in soil inoculatedwith antagonist followed by pathogen treatment

In addition, the sterilized soil pre-treated withthe antagonist followed by pathogen treatment wasalso tested for the production of the active fractionby the antagonist under in vivo conditions while theuninoculated soil served as control. For the detec-tion of active fraction, rhizosphere soil samples(500 g) were collected, soaked in ethyl acetate for12 h and the obtained solvent layer was extractedwith the same solvent. It was then concentratedunder vacuum to obtain a crude residue (50mg)which in turn subjected to silica gel columnchromatography (25� 5 cm, Silica gel 60, Merck)using the gradient solvent system of hexane:ethylacetate. Basing on Rf values, elutions were col-lected and concentrated. As per procedure ofDecker et al. (1990), the presence of the activecompound in the partially purified extract (10mg)was further analyzed by using HPLC semi-preparativecolumn (250� 10mm, 5m using hexane:2-propanol(8:2 v/v)) scanned at UV 254nm.

Antifungal spectrum of the first activefraction under in vitro and in vivo conditions

In vitro screeningThe antifungal spectrum of the first active

fraction obtained from the strain was furtherconfirmed by in vitro and in vivo studies againstthe wilt causing pathogen, F. oxysporum in sor-ghum. Its activity was compared with that ofcommercially available systemic fungicides likemancozeb and carbendazim. The conidial suspen-sions of F. oxysporum grown on Czapek-Doxagar at 30 1C for 10 days were treated with activefraction, mancozeb and carbendazim to givefinal concentrations of 0, 1, 10, 50, 100, 200 and500 mg/ml. After incubation for 24 h at 28 1C,conidial germination was examined microscopicallyin five replicates (Hwang et al. 2001). Resultsare statistically analyzed by two-way ANOVA andthe standard deviations of the mean values arecalculated.

Green house studiesThe antifungal activity of the active fraction was

also evaluated in vivo for its ability to suppressFusarium wilt on sorghum plants in a growth room.Stock solutions of the antifungal compound wereprepared by dissolving the active fraction, manco-zeb and carbendazim individually in water+metha-nol (95:5), which in turn diluted to give differentconcentrations of 0, 50, 100, 200, 500, 700 and1000 mg/ml. Seeds of sorghum were sown in plasticbags containing steam sterilized soil drenched withantifungal solution (30ml). Three-day-old seedlingsof sorghum were inoculated with conidial suspen-sions of F. oxysporum (105 spores/ml) by using soildrench method (Hwang et al. 2001). Diseaseseverity on sorghum plants was rated after 15 daysof inoculation based on the percent of wiltedplants. Data were statistically analyzed by two-wayANOVA and the standard deviations of the meanvalues are calculated.

Results and discussion

Taxonomy of the strain

Cultural and physiological characteristics of thestrain are recorded in Table 1. The strain exhibitedgood growth on ISP-1, ISP-2, ISP-5 and ISP-7 whilethe growth was moderate in ISP-3, ISP-4 andnutrient agar media. Soluble pigment productionby the strain was not found on the culturemedia tested except melanoid pigmentation on

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Table 1. Cultural and physiological characteristics ofNocardia levis MK-VL_113.

Growth and pigment productionISP-1a G, NSPISP-2 G, NSPISP-3 M, NSPISP-4 M, NSPISP-5 G, NSPISP-7 G, MNutrient agar medium M, NSPCzapek-Dox –

Utilization of carbon sources and acid productionFructose Mo, +Galactose G, +Glucose G,+Glycerol Mo, +Inositol G, +Lactose G, +Maltose G, +Mannitol G, +Sorbitol G, +Sucrose G, �Xylose G, �

Tolerance toLysozyme +Phenol (0.1%) �

NaCl Up to 7%

Production ofMelanoid pigments +Catalase +Chitinase +Nitrate reductase +Protease +Urease +H2S +Indole +

Antibiotic sensitivity (mg/ml)Amikacin (30) SAmoxicillin (10) RAmpicillin (10) RBacitracin (10) SCephoxitin (30) RCiprofloxacin (5) SClindamycin (2) RCloxacillin (1) RColistin (10) RCo-Trimazine (25) RFuroxone (100) RGentamicin (10) SKanamycin (300) SMethicillin (5) RMetronidazole (5) SNeomycin (30) SNitrofurantoxin (300) SPolymyxin-B (300) SRifampicin (5) SStreptomycin (10) R

Table 1. (continued )

Antibiotic sensitivity (mg/ml)Tetracycline (30) SVancomycin (30) S

+, positive result; �, negative result; R, resistant; S, sensitive;NSP, no soluble pigment; M, melanin; G, good; Mo, moderate.aInternational Streptomyces Project (ISP) media.


ISP medium 7. Micromorphology of the strain grownon ISP medium 2 showed white aerial myceliumwith light orange colored substrate myceliumthat fragments into rod-shaped elements, which isthe characteristic feature of Nocardia (Hoshinoet al. 2004). In the carbohydrate assimilation test,carbon sources such as galactose, glucose, inositol,lactose, maltose, mannitol, sorbitol, sucrose andxylose were utilized by the strain efficiently. Itshowed NaCl tolerance up to 7% and could produceenzymes such as catalase, chitinase, nitrate re-ductase, protease, tyrosinase and urease. It alsoshowed various other biochemical activities likeH2S, indole and acid production. It showed resis-tance to different antibiotics like amoxicillin,ampicillin, cephoxitin, clindamycin, cloxacillin,colistin, co-trimazine, furoxone, methicillin andstreptomycin while it exhibited sensitivity toamikacin, bacitracin, ciprofloxacin, gentamicin,kanamycin, metronidazole, neomycin, nitrofuran-toxin, polymyxin-B, rifampicin, tetracycline andvancomycin.

The phylogenetic position of the strain wasdetermined by amplifying 16S rRNA region andsequence of the strain was examined by BLASTanalysis. The results revealed that the strainbelongs to the genus Nocardia, the suborderCorynebacterineae of the family Nocardiaceae.The 16S rRNA genome sequence of the strainshowed 100% similarity with that of N. levis(Fig. 1), thereby the strain MK-VL_113 was identi-fied as N. levis and the 16S rRNA sequence wassubmitted to Genbank with an accession numberFJ209734. This is the first report of N. levis fromsoil environment.

Isolation, purification and identification ofbioactive metabolites

Chemical analysis of the secondary metabolitesobtained from 4-day-old culture broth of the strainled to the isolation of two fractions active againstGram-positive (B. cereus) and Gram-negative(E. coli) bacteria and yeast (C. albicans).

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Figure 1. Phylogenetic tree derived from 16S rRNA gene sequences showing the relationship between strain MK-VL_113and species belonging to the genus Nocardia was constructed by using neighbour-joining method. Bootstrap values areexpressed as percentages of 1000 replications. Bar, 0.005 substitutions per nucleotide position.

OH

Figure 2. Molecular structure of 1-phenylbut-3-ene-2-ol.


Physico-chemical properties and structuralelucidation of bioactive compounds

The active principle of the first fraction collectedafter HPLC purification appeared as yellowish solid,

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Figure 3. GC-spectra of a partially purified second fraction (PPF) of N. levis MK-VL_113. By means of mass spectralanalysis, the compounds (C1–C3) with retention time 13.39, 15.13 and 22.59 in PPF are identified as phenylethylalcohol, dibutyl phthalate and 1,2-benzenedicarboxylic acid, 3-nitro, respectively.


which was soluble in CHCl3, DMSO and MeOH. Thestructure of the pure bioactive compound wasanalyzed on the basis of FT-IR, EI-MS, 1H NMR and13C NMR. The IR absorption maxima Vmax at 3399and 1689 cm�1 suggested the presence of a hydro-xyl residue and a double bond in the structure. Inelectron impact (EI) mass spectra, the compoundshowed molecular ions at m/z are 57 (40), 83 (70),85 (53), 149 (15) and 171 (42) inferring a molecularweight of 149 (M+H+) and 171 (M+Na adduct). The1H NMR (CDCl3, 300MHz) of the pure bioactivecompound displayed 11 proton signals at 7.30d(Ar-H, 5H, m), 5.75d (1H, m), 5.10d (2H, J ¼ 1.5 Hz,7.55 Hz, dd), 4.65d (1H, J ¼ 6.043 Hz, t) and 2.50d(2H, J ¼ 6.79 Hz, t). While 13C NMR (CDCl3, 75MHz)depicted 10 peaks at 144.30d, 134.52d, 128.43d,127.56d, 125.84d, 118.78d, 73.37d and 44.05d.Optical rotation of the pure bioactive compoundwas determined as [a]589

27¼ �12.1 (c. 2.5, CHCl3).

Based on these spectral data, the first activefraction was identified as 1-phenylbut-3-ene-2-olwith a molecular formula C10H12O (Fig. 2).

The partially purified second fraction subjectedto GC–MS analysis revealed the presence of threepeaks noted at the retention time of 13.39 (C1),15.13 (C2) and 22.59 (C3) with M+ at m/z 122, 278and 166 g/mol of molecular weights, respectively(Fig. 3). By means of available library data, C1, C2

and C3 in PPF were identified as phenylethylalcohol, dibutyl phthalate and 1,2-benzenedicar-boxylic acid, 3-nitro, respectively.

Biological assays

Testing the minimum inhibitory concentration ofthe bioactive metabolites

The antimicrobial activities of the bioactivecompounds were tested by agar plate diffusionassay. The two active fractions were found to beactive against a wide variety of test organisms forwhich the MIC values ranged from 10 to 250 mg/ml(Table 2). Among the bacteria tested, P. flourescensand S. epidermis exhibited high sensitivity towards1-phenylbut-3-ene-2-ol followed by B. cereus,P. solanacearum and S. aureus. C. albicans is highlysensitive to the compound when compared withother test fungi. Components of PPF showed goodantimicrobial activity against S. aureus, S. epider-mis, B. subtilis, C. albicans and A. niger.

Antifungal spectrum of N. levis MK-VL_113 underin vitro and in vivo conditions

The strain showed promising inhibitory effect onthe growth of F. oxysporum as indicated by thediameter of the inhibition zone (20mm) in in vitro

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Table 2. Antimicrobial activities of 1-phenylbut-3-ene-2-ol and PPF produced by Nocardia levis MK-VL_113.

Test organism MIC (mg/ml)

1-phenylbut-3-ene-2-ol PPFa Antibioticsb

BacteriaBacillus cereus 15 75 75B. megaterium 50 100 50B. subtilis 90 25 50Corynebacterium diptheriae 25 50 50Escherichia coli 25 50 25Proteus vulgaris 75 90 25Pseudomonas aeruginosa 60 75 50P. flourescens 10 50 50P. solanacearum 15 50 50Serratia marcescens 50 100 25Staphylococcus aureus 15 20 50S. epidermis 10 25 50Xanthomonas malvacearum 50 75 50X. campestris 50 75 50YeastCandida albicans 15 25 50Filamentous fungiAspergillus flavus 50 75 2A. niger 25 50 5Alternaria alternata 200 250 7Curvularia maculans 150 25 10C. lunata 100 200 12Fusarium oxysporum 20 200 5Penicillium citrinum 25 75 2

aPPF – partially purified second fraction.bTetracycline against bacteria, Griseofulvin against yeast and Carbendazim against fungi.

Table 3. Antifungal spectrum of Nocardia levis MK-VL_113 against Fusarium oxysporum under in vivo conditions.

Sl.no.

Treatment Percent of wiltedsorghum plants

Percent reduction inwilting

1. Soil inoculated with the pathogen alone 10070 0702. Soil inoculated with the antagonist alone 070 0703. Simultaneous inoculation of the soil with the pathogen and

antagonist53.2971.26 46.7171.26

4. Pre-treatment of the soil with the antagonist followed by pathogeninoculation after 4 days

30.070.81 70.070.81

5. Uninoculated soil 070 070

Average of three trials with 10 plants per treatment (7SD).Data were statistically analyzed by one-way ANOVA and found to be significant at 5%.


plate assay. In vitro studies conducted earlier alsoindicated the antagonistic action of actinomycetesto the fungal pathogens causing plant diseases(Crawford et al. 1993; Taechowisan et al. 2005,2009). Fravel (1988) noticed that several actino-mycetes which showed inhibitory activity under invitro conditions were also antagonistic to fungalpathogens in in vivo studies.

Results regarding the antagonism of the strainagainst Fusarium wilt of sorghum in soil are

recorded in Table 3. It was found that simultaneousinoculation of the soil with the antagonist and thepathogen reduced the Fusarium wilt of sorghum upto 46.7% as compared to inoculation with pathogenalone. The reduction in the incidence of wilt waseven up to 70% when the soil was pre-treated withthe antagonist prior to inoculation with the patho-gen. This may be due to the elaboration ofbioactive metabolites by the strain before theestablishment of pathogen. The plants in untreated

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Figure 4. HPLC chromatogram illustrating the production of 1-phenylbut-3-ene-2-ol in rhizosphere extracts of soil pre-treated with Nocardia levis MK-VL_113 followed by Fusarium oxysporum inoculation.


soil as well as soil treated with the antagonist alonedid not exhibit wilt symptoms and remainedhealthy. Cao et al. (2004) and Rizk et al. (2007)also reported the control of plant diseases by usingantagonistic actinomycetes. Ours is the first reportshowing N. levis MK-VL_113 as biocontrol agentagainst Fusarium wilt of sorghum. The data wereanalyzed by one-way ANOVA and the differencesbetween the treatments were statistically signifi-cant at 5%.

Detection of 1-phenylbut-3-ene-2-ol in soilinoculated with antagonist followed by pathogentreatment

The incidence of Fusarium wilt on sorghum plantswas greatly reduced in the soil pre-treated with theantagonist followed by pathogen inoculation.Therefore, the rhizospere soils collected from thistreatment were extracted with ethyl acetate andthe production of 1-phenylbut-3-ene-2-ol by theantagonist under in vivo conditions was detected byusing silica gel column chromatography followedby HPLC with reference to that of active compound

isolated from the fermented broth of the strain.These results clearly demonstrate the elaborationof 1-phenylbut-3-ene-2-ol by the strain under invivo conditions (Fig. 4), whereas in the extracts ofuninoculated soil samples, presence of 1-phenyl-but-3-ene-2-ol was found to be negative. Hence, itindicates that the compound was produced by thestrain itself and these findings are in consistentwith Thomashaw et al. (2008) who studied thesignificance of bioactive metabolites produced bythe microbes in their native habitats.

Antifungal spectrum of 1-phenylbut-3-ene-2-olunder in vitro and in vivo conditions

The antifungal spectrum of the bioactive com-pound, 1-phenylbut-3-ene-2-ol was further evalu-ated under in vitro and in vivo conditions. As shownin Fig. 5, 1-phenylbut-3-ene-2-ol inhibited theconidial suspension of F. oxysporum under invitro conditions at a concentration of 200 mg/mlwhile the fungicides such as mancozeb and carben-dazim exhibited inhibitory action at a level of500 and 50 mg/ml, respectively. The efficacy of

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0

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Concentration of antifungal compound (µg/ml)

Inhi

bitio

n of

spo

re g

erm

inat

ion

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1-phenylbut-3-ene-2-ol

MancozebCarbendazim

50020010050101

Figure 5. Antifungal spectrum of the metabolite, 1-phenylbut-3-ene-2-ol isolated from Nocardia levis MK-VL_113 against Fusarium oxysporum under in vitroconditions (values are means of five replicates7SD).Statistically significant at 5%.

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lant

s)

1-phenylbut-3-ene-2-ol

Mancozeb

Carbendazim

100070050020010050

Figure 6. Antifungal spectrum of the metabolite, 1-phenylbut-3-ene-2-ol isolated from Nocardia levis MK-VL_113 against Fusarium oxysporum under in vivoconditions in comparison with mancozeb and carbenda-zim (values are means of three replicates7SD). Statis-tically significant at 5%.


1-phenylbut-3-ene-2-ol, mancozeb and carbendazimon Fusarium wilt disease of sorghum was evaluatedin vivo under a green house trial (Fig. 6). In sorghumplants, Fusarium wilt is often characterized bywilting followed by the death of the affected plant.Stems and veins of diseased plants have discoloredvascular tissue, usually yellow or brown. However,in the present study, 1-phenylbut-3-ene-2-ol, man-cozeb and carbendazim were effective in control-ling Fusarium wilt at concentrations of 500, 700and 100 mg/ml, respectively. The efficacy of thebioactive compound, 1-phenylbut-3-ene-2-olagainst the wilt disease was found superior tomancozeb but less effective than carbendazim.

Hence, it is clear from the present study, that1-phenylbut-3-ene-2-ol produced by N. levisMK-VL_113 plays a prominent role in controllingthe wilt causing pathogen, F. oxysporum.

The genus Nocardia remains as an outstandingresource for the isolation of novel and potentbioactive metabolites. Among the Nocardia spp.,the strain N. brasiliensis was known to producebrasilidine A and EDDA possessing good antimicro-bial potential (Imai et al. 1997; Kobayashi et al.1997). Nothramicin, a new anthracycline antibioticwas recorded from Nocardia sp. MJ896-43F17(Tsuda et al. 1996). The strain N. brasiliensis IFM0089 was reported to produce new cytotoxicantibiotics, brasiliquinones A, B and C (Momoseet al. 1998). Three new benzenoid compoundsnamed as nocarasins A, B and C were isolated fromN. brasiliensis IFM 0677 (Tsuda et al. 1999).Immunosuppressive agents such as 32-memberedmacrolides with a tetrahydropyrone and a 2-deoxypyranose and a novel tricyclic metabolite,brasilicardin A were obtained from N. brasiliensisIFM0406 (Komatsu et al. 2005). Erythromycin E andnargeninus were isolated from N. brasiliensis IFM0466 and N. otitidiscaviarum IFM 0986, respectively(Mikami et al. 1999). Other Nocardia spp. such as N.asteroides IFM 0959 and N. asteroides SCRC-A2359elaborated new antitumor substances like aster-obactin (Nemoto et al. 2002) and amamistatins Aand B (Kokubo et al. 2000). A clinical isolate of N.transvalensis IFM 10065 was reported to producenovel antifungal antibiotics like transvalencin Aand transvalencin Z (Mukai et al. 2006). A newangucyclinone antibiotic, chemomicin A was ex-tracted from the culture broth of N. mediterraneisubsp. kanglensis 1747-64 (Sun et al. 2007).Bioactive metabolites such as chrysophanol 8-methyl ether, asphodelin-4,70-bichrysophanol, jus-ticidin B and ayamycin-1,1-dichloro-4-ethyl-5-(4-nitro-phenyl)-hexan-2-one were isolated fromNocardia sp. ALAA 2000 (El-Gendy et al. 2008). Twonovel antibiotics, neocitreamicins I and II, wereextracted from the fermented broth of Nocardiastrain G0655 (Peoples et al. 2008).

However, in the present study, the soil isolate,N. levis MK-VL_113 was found to produce a potentbioactive compound, 1-phenylbut-3-ene-2-ol whichis not yet reported as a natural product. This is thefirst report of 1-phenylbut-3-ene-2-ol from N. levisMK-VL_113. In addition, the compounds such asphenylethyl alcohol, dibutyl phthalate and 1,2-benzenedicarboxylic acid, 3-nitro identified byusing GC–MS analysis were also not yet reportedfrom the genus Nocardia. The present investigationalso reveals the efficiency of the metabolite,1-phenylbut-3-ene-2-ol produced by N. levis

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MK-VL_113 as a bioactive compound against avariety of opportunistic and phytopathogenic bac-teria and fungi and also as a good potential agentfor controlling Fusarium wilt.

Acknowledgements

The authors A.K., P.P. and M.N. are grateful toIndian Council of Medical Research (ICMR), NewDelhi, India, Department of Biotechnology (DBT),New Delhi, India and Council of Scientific andIndustrial Research (CSIR), New Delhi, India, re-spectively, for providing financial assistance.

References

Berdy J. Bioactive microbial metabolites. J Antibiot2005;58:1–26.

Boussaada O, Ammar A, Saidana D, Chriaa J, Chraif I,Daami M, Helal AN, Mighri Z. Chemical compositionand antimicrobial activity of volatile components fromcapitula and aerial parts of Rhaponticum acaule DCgrowing wild in Tunisia. Microbiol Res 2008;163:87–95.

Cao L, Qui Z, You J, Tan H, Zhou S. Isolation andcharacterization of endophytic Streptomyces strainsfrom surface-sterilized tomato (Lycopersicon esculen-tum) roots. Lett Appl Microbiol 2004;39:425–30.

Cappuccino JG, Sherman N. Microbiology: A LaboratoryManual. Harlow: Benjamin; 2002. pp. 263–264.

Crawford DL, Lynch JM, Whipps JM, Ousley MA. Isolationand characterization of actinomycete antagonists of afungal root pathogen. Appl Environ Microbiol 1993;59:3899–905.

Decker H, Walz F, Bormann C, Zahner H, Fiedler HP.Metabolic products of microorganisms. 255+. Nikko-mycins Wz and Wx, new chitin synthetase inhibitorsfrom Streptomyces tendae. J Antibiot 1990;43:43–8.

Dietz A, Thayer DW. Actinomycete taxonomy. Society forIndustrial Microbiology Special Publication, number 6,1980. p. 26–31.

El-Gendy MMA, Hawas UW, Jaspars M. Novel bioactivemetabolites from a marine derived bacterium Nocar-dia sp. ALAA 2000. J Antibiot 2008;61:379–86.

Fravel DR. Role of antibiosis in the biocontrol of plantdisease. Annu Rev Phytopathol 1988;26:75–91.

Goodfellow M, Orchard VA. Antibiotic sensitivity of somenocardioform bacteria and its value as a criterion fortaxonomy. J Gen Microbiol 1974;83:375–87.

Gordon RED, Barnett A, Handerhan JE, Pang CNN.Nocardia coeliaca, Nocardia autotrophica, and thenocardin strain. Int J Syst Bacteriol 1974;24:54–63.

Holding AJ, Collee JG. Routine biochemical tests.Methods Microbiol 1971;6A:1–31.

Hoshino Y, Mukai A, Yazawa K, Uno J, Ishikawa J, Ando A,Fukai T, Mukami Y. Transvalencin A, a thiazolidine zinccomplex antibiotic produced by a clinical isolate ofNocardia transvalensis. I. Taxonomy, fermentation,

isolation and biological activities. J Antibiot 2004;12:797–802.

Hwang BK, Lim SW, Kim BS, Lee JY, Moon SS. Isolation andin vivo and in vitro antifungal activity of phenylaceticacid and sodium phenylacetate from Streptomyceshumidus. Appl Environ Microbiol 2001;67:3739–45.

Imai T, Yazawa K, Tanaka Y, Mikami Y, Kudo T, Suzuki T, AndoA, Nagata Y, Graefe U. Productivity of antimicrobialsubstance in pathogenic actinomycetes Nocardia brasi-liensis. Microbiol Cult Coll 1997;13:103–8.

Isik K, Chun J, Hah YC, Goodfellow M. Nocardiasalmonicida nom. rev., a fish pathogen. Int J SystBacteriol 1999;49:833–7.

Kageyama Y, Poonwan N, Yazawa K, Mikami Y, NishimuraY. Nocardia asiatica sp. nov., isolated from patientswith nocardiopsis in Japan and clinical specimens fromThailand. Int J Syst Evol Microbiol 2004a;54:125–30.

Kageyama Y, Yazawa K, Nishimura Y, Mikami Y. Nocardiainohanensis sp. nov., Nocardia yamanashiensis sp. nov.and Nocardia niigatensis sp. nov., isolated fromclinical specimens. Int J Syst Evol Microbiol 2004b;54:563–9.

Kimura M. A simple method for estimation of evolutionaryrate of base substitutions through comparative studiesof nucleotide sequences. J Mol Evol 1980;16:111–20.

Kobayashi J, Tsuda M, Nemoto A, Tanaka Y, Yazawa K,Mikami Y. Brasilidine A, a new cytotoxic isobitrilecontaining indole alkaloid from the actinomyceteNocardia brasiliensis. J Nat Prod 1997;60:719–20.

Kokubo S, Suenaga K, Shinohara C, Tsuji T, Uremura D.Structures of Amamistatins A and B, novel growthinhibitors of human tumor cell lines from Nocardiaasteroides. Tetrahedron 2000;56:6435–40.

Komatsu K, Tsuda M, Tanaka Y, Mikami Y, Kobayashi J. SARstudies of brasilicardin A for immunosuppressive andcytotoxic activities. Bioorg Med Chem 2005;13:1507–13.

Kurtboke DI, Chen CF, Williams ST. Use of polyvalentphage for reduction of Streptomycetes on soil dilutionplates. J Appl Bacteriol 1992;72:103–11.

Mikami Y, Yazawa K, Neomoto A, Komaki H, Tanaka Y,Grafe U. Production of erythromycin E by pathogenicNocardia brasiliensis. J Antibiot 1999;52:201–2.

Mikami Y. Biological work on medically importantNocardia species. Actinomycetologica 2007;21:46–51.

Momose I, Kinoshita N, Sawa R, Naganawa H, Iinuma H,Hamada M, Takeuchi T. Nothramicin, a new anthracy-cline antibiotic from Nocardia sp. MJ896-43F17. JAntibiot 1998;51:130–5.

Mukai A, Fukai T, Matsumoto Y, Ishikawa J, Hoshino Y,Yazawa K, Harada K, Mikami Y. Transvalencin Z, a newantimicrobial compound with salicylic acid residuefrom Nocardia transvalensis IFM 10065. J Antibiot2006;59:366–9.

Nemoto A, Hoshino Y, Yazawa K, Ando A, Mikami Y,Komkaki H, Tanaka Y, Grafe U. Asterobactin, a newsiderophore group from Nocardia asteroides. J Anti-biot 2002;55:593–7.

Peoples AJ, Zhang Q, Millett WP, Rothfeder MT, PescatoreBC, Madden AA, Ling LL, Moore CM. Neocitreamicins I

ARTICLE IN PRESS


and II, novel antibiotics with activity against methi-cillin-resistant Staphylococcus aureus and vancomy-cin-resistant Entercocci. J Antibiot 2008;61:457–63.

Poonwan N, Mekha N, Yazawa K, Thunyaharan S,Yamanaka A, Mikami Y. Characterization of clinicalisolates of pathogenic Nocardia strains and relatedactinomycetes in Thailand from 1996 to 2003. Myco-pathologica 2005;159:361–8.

Rainey FA, Rainey NW, Kroppenstedt RM, Stackebrandt E.The genus Nocardiopsis represents a phylogeneticallycoherent taxon and a distinct actinomycete lineage:proposal of Nocardiopsiaceae fam. nov. Int J SystBacteriol 1996;46:1088–92.

Rizk M, Rahman TA, Metwally H. Screening of antagonisticactivity in different Streptomyces species againstsome pathogenic microorganisms. J Biol Sci 2007:1418–23.

Saitou N, Nei M. The neighbor-joining method: a newmethod for reconstructing phylogenetic trees. Mol BiolEvol 1987;4:406–25.

Shirling EB, Gottlieb D. Methods for characterization ofStreptomyces species. Int J Syst Bacteriol 1966;16:313–40.

Singh RS, Reddy CS. Suppression of damping-off of tomatoand seedling blight of chick pea and sugarbeet bystrains of Streptomyces diastaticus. Indian Phyto-pathol 1979;32:374–7.

Sun CH, Wang Y, Wang Z, Zhou JQ, Jin WZ, You HG, ZhaoLX, Si SY, Li X. Chemomicin A: a new angucyclinoneantibiotic produced by Nocardia mediterranei subsp.kanglensis 1747–64. J Antibiot 2007;60:211–5.

Taechowisan T, Chuaychot N, Chanaphat S, Wanbanjob A,Tantiwachwutikul P. Antagonistic effects of Strepto-myces sp. SRM1 on Collectotrichum musae. Biotech-nology 2009;8:86–92.

Taechowisan T, Lu C, Shen Y. Secondary metabolites fromendophytic Streptomyces aureofaciens CMUAc130 andtheir antifungal activity. Microbiology 2005;151:1691–5.

Thomashaw LS, Bonsall RF, Weller DM. Detection ofantibiotics produced by soil and rhizosphere microbesin situ. In: Karlovsky P, editor. Secondary metabolites insoil ecology. Berlin, Heidelberg: Springer; 2008. p. 23–6.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F,Higgins DG. The Clustal X windows interface: flexiblestrategies for multiple sequence alignment aided byquality analysis tools. Nucleic Acids Res 1997;24:4876–82.

Tsuda M, Nemoto A, Komaki H, Tanaka Y, Yazawa K,Mikami Y, Kobayashi J. Nocarasins A–C, and brasilino-lide D, new metabolites from the actinomyceteNocardia brasiliensis. J Nat Prod 1999;62:1640–2.

Tsuda M, Sato H, Tanaka Y, Yazawa K, Mikami Y, Sasaki T,Kobayaashi J. Brasiliquiones A–C, a new cytotoxicbenz[a]anthraquinone with an ethyl group at C-3 fromactinomycete Nocardia brasiliensis. J Chem SocPerkins Trans 1996;1:1773–5.

Williams ST, Cross T. Isolation, purification, cultivationand preservation of actinomycetes. Methods Microbiol1971;4:295–334.

Wise R. The worldwide threat of antimicrobial resistance.Curr Sci 2008;95:181–7.

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