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Effect of biopolymers on geldanamycin production andbiocontrol ability of Streptomyces melanosporofaciensstrain EF-76Nancy Clermont a , Geneviève Legault a , Sylvain Lerat a & Carole Beaulieu aa Centre SÈVE, Département de Biologie, Université de Sherbrooke, Sherbrooke, QC J1K2R1, CanadaPublished online: 27 Sep 2010.

To cite this article: Nancy Clermont , Genevive Legault , Sylvain Lerat & Carole Beaulieu (2010) Effect of biopolymers ongeldanamycin production and biocontrol ability of Streptomyces melanosporofaciens strain EF-76, Canadian Journal of PlantPathology, 32:4, 481-489, DOI: 10.1080/07060661.2010.512121

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Can. J. Plant Pathol. (2010), 32(4): 481–489

ISSN 0706-0661 print/ISSN 1715-2992 online © 2010 The Canadian Phytopathological SocietyDOI: 10.1080/07060661.2010.512121

Soilborne pathogens/Agents pathogènes telluriques

TCJP

Effect of biopolymers on geldanamycin production and biocontrol ability of Streptomyces melanosporofaciens strain EF-76

Antibiotic production in StreptomycesN. Clermont et al.

NANCY CLERMONT, GENEVIÈVE LEGAULT, SYLVAIN LERAT AND CAROLE BEAULIEU

Centre SÈVE, Département de Biologie, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada

(Accepted 27 July 2010)

Abstract: The biocontrol agent Streptomyces melanosporofaciens strain EF-76 produces geldanamycin, an ansamycin antibiotic which displays antagonistic activity towards several Gram-positive bacteria and fungi. Biopolymers [chitin, carboxymethylcellulose (CMC) and xylan] or N-acetylglucosamine were added to S. melanosporofaciens culture media and the effect of these additional carbon sources on geldanamycin production was analysed. Geldanamycin biosynthesis was only stimulated in the presence of 1 g L−1 of chitin. Higher concentrations of chitin, as well as the presence of CMC or xylan, did not improve antibiotic production. N-acetylglucosamine, the final degradation product of chitin, did not stimulate geldanamycin production at all concentrations tested. Nevertheless, the addition of chitin in culture media had no or a negative impact on the antagonistic activity of EF-76 towards the pathogen Streptomyces scabies. Furthermore, the effect of chitin, CMC and xylan on the biocontrol ability of EF-76 against S. scabies was tested on radish seedlings. None of the polymers affected the biocontrol efficiency of EF-76 under gnotobiotic conditions. However, the addition of CMC or xylan to plant growth medium protected radish seedlings against S. scabies infection.

Keywords: biological control, common scab, Streptomyces scabiei, Streptomyces scabies

Résumé: L’agent de lutte biologique, le Streptomyces melanosporofaciens souche EF-76, produit de la geldanamycine, un antibiotique de la classe des ansamycines qui présente une activité antagoniste envers plusieurs bactéries à Gram-positif ainsi qu’envers les champignons. Différents biopolymères [la chitine, la carboxyméthylcellulose (CMC) et le xylane] ou le N-acétylglucosamine ont été ajoutés aux milieux de culture du S. melanosporofaciens. L’effet de la présence de ces sources de carbone supplémentaires sur la production de geldanamycine a été analysé. La biosynthèse de la geldanamycine est stimulée seulement en présence de 1 g L−1 de chitine. Des concentrations plus élevées de chitine, ainsi que la présence de CMC ou de xylane n’ont pas permis d’améliorer la production d’antibiotique. L’ajout de N-acétylglucosamine, le produit de dégradation final de la chitine, n’a stimulé la production de geldanamycine à aucune des concentrations testées. Néanmoins, l’ajout de chitine au milieu de culture a des répercussions négatives ou n’a pas de répercussions sur l’activité antagoniste du EF-76 envers l’agent pathogène, Streptomyces scabies. De plus, les effets de la chitine, du CMC et du xylane sur l’activité antagoniste de la souche EF-76 envers le S. scabies ont été testés sur des plantules de radis. Aucun des polymères n’affecte l’efficacité de l’agent de lutte biologique EF-76 en conditions gnotobiotiques. Toutefois, l’ajout de CMC ou de xylane au milieu de croissance des végétaux confère une protection aux semis de radis contre l’infection du S. scabies.

Mots clés: contrôle biologique, gale commune, Streptomyces scabiei, Streptomyces scabies

Introduction

Actinomycetes produce over 10 000 bioactive secondarymetabolites, including antimicrobial and antitumour

compounds, immunosuppressants and enzyme-inactivatingcompounds. Of these, about 7600 are produced by Strep-tomyces species (Olano et al., 2008). Streptomycetes are

Correspondence to: Carole Beaulieu. E-mail: carole.beaulieu@usherbrooke.ca

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Gram-positive, sporulating bacteria that represent a highproportion of the soil microbial biomass (Atlas & Bartha,1993). Members of this genus are essentially saprophytes andtheir main function in soil ecosystems is the decomposition ofcomplex organic polymers through the production of a widevariety of extracellular enzymes.

Several streptomycetes exhibit the capacity to enhanceplant growth as well as to lessen or prevent the harmfuleffects of plant pathogenic microorganisms (Lahdenperä,1991; Doumbou et al., 2001b; Tokala et al., 2002;Simao-Beaunoir et al., 2009). Mechanisms involved inthe plant disease suppression include antibiosis (Rothrock& Gottlieb, 1984; Sturz et al., 2004; Islam et al., 2005;Kinsella et al., 2009; Recep et al., 2009), production of lyticenzymes (Valois et al., 1996; Chamberlain & Crawford,1999; Verma et al., 2007), degradation of plant toxiccompounds (Doumbou et al., 2001b) and elicitation ofplant defence mechanisms (Trotel-Aziz et al., 2006;Mohamed et al., 2007; Ongena et al., 2007; Hase et al.,2008). Biological control products containing livingStreptomyces cells as active ingredients are commerciallyavailable (Gracia-Garza et al., 2003; Elmer, 2006;Hiltunen et al., 2009).

Streptomyces melanosporofaciens Arcamone et al.strain EF-76 (Doumbou et al., 2001a) was isolated frompotato tuber (Faucher et al., 1992) and shown to be anefficient biocontrol agent against both raspberry root rotcaused by Phytophthora fragariae Hickman var. rubi(Valois et al., 1996) and common scab of potato causedby Streptomyces scabies (Thaxter) Lambert & Loria(Beauséjour et al., 2003). This strain also inhibits thegrowth of a wide range of bacteria and fungi (Toussaintet al., 1997) and its antagonistic property is due togeldanamycin production (Toussaint et al., 1997). Amutant strain deficient in geldanamycin biosynthesis lostits ability to protect potato tubers against common scab(Agbessi et al., 2003). Several strains producinggeldanamycin seem to be efficient agents for biocontrol(Trejo-Estrada et al., 1998; Beauséjour et al., 2003).

Geldanamycin (C29H40N2O9) is a type-I polyketidecompound belonging to the ansamycin antibiotic class.This aromatic compound, which contains a benzoquinonenucleus (Sasaki et al., 1970), was first isolated fromStreptomyces hygroscopicus var. geldanus (DeBoer et al.,1970). Geldanamycin exhibits a wide variety of biologi-cal properties, including antibacterial, antifungal, herbi-cidal and antitumor activities (Sasaki et al., 1970; Heisey& Putnam, 1986; Neckers et al., 1999). The mode ofaction of geldanamycin as an antimicrobial compoundhas yet to be elucidated.

Both abiotic and biotic factors affect the production ofsecondary metabolites by microorganisms (Milner et al.,

1996; Duffy & Défago, 1999; Hwang et al., 2002). Forexample, the addition of alfalfa seedling exudates to theculture medium of Bacillus cereus improved kanosamineproduction by 300%. Soil-dwelling streptomycetes canhydrolyse complex natural biopolymers; thus, these poly-mers could influence the production of bioactive moleculesby these organisms. For instance, N-acetylglucosamine(GlcNAc), a degradation product of chitin, modulatesantibiotic biosynthesis and development in Streptomycescoelicolor (Rigali et al., 2008) and suberin promotes theproduction of secondary metabolites in Streptromycesscabies (Lerat et al., 2010).

The aim of this study was to analyse the effects ofthree biopolymers [chitin, carboxymethylcellulose(CMC) and xylan] on both geldanamycin production andthe biocontrol efficiency of S. melanosporofaciens strainEF-76.

Materials and methods

Bacterial strains and culture media

Streptomyces melanosporofaciens strain EF-76 and S. sca-bies strain EF-35 were isolated from common scab lesionson potato tubers in Quebec, Canada (Faucher et al., 1992).Strain EF-76 was grown in Yeast Malt Extract medium(YME; Pridham et al., 1956–1957) or in a minimal medium(MM) consisting of 0.5 g K2HPO4, 0.2 g MgSO4·7H2O,0.01 g FeSO4·7H2O, 0.01 g CaCl2·7H2O, 0.1 g (NH4)2SO4,0.5 g L-asparagine, 4 g D-mannitol, 4 μg ZnCl2, 20 μgFeCl3·7H2O, 1 μg CuCl2·2H2O, 1 μg MnCl2·4H2O, 1 μgNa2B4O7·10H2O and 1 μg (NH4)6Mo7O24·4H2O per L.When appropriate, these media were supplemented withadditional carbon sources. S. scabies strain EF-35 pro-duces thaxtomin A, a plant toxic compound (Beausé-jour et al., 1999). This strain induces common scab onpotato tuber and inhibits the growth of radish seed-lings (Goyer & Beaulieu, 1997). Strain EF-35 wasgrown in Trypticase Soy Broth (TSB; Fisher Scien-tific, Ottawa, ON). Both strains were grown at 30 oC.They were conserved as spore suspension in a 20% (v/v)glycerol solution at −20 ºC.

Chitinolytic, cellulolytic and xylanolytic activities in Streptomyces melanosporofaciens EF-76

To assay chitinase, cellulase or xylanase activity, aninoculum of S. melanosporofaciens was prepared asfollows. One hundred microlitres of EF-76 spores [2.8× 108 colony-forming units (CFU) mL−1)] were inocu-lated into 25 mL of YME and incubated with shaking(300 rpm) for 48 h. The culture was then centrifuged

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(2500 g, 10 min) and the pellet was washed twice with0.85% NaCl, pH 7.2. The pellet was finally resus-pended in five volumes of fresh MM. A fraction of thissuspension (200 μL) was used as inoculum and wasadded to 20 mL of MM supplemented with 1 g L−1 ofchitin, CMC or xylan (Sigma-Aldrich, St-Louis, MO).Cultures were grown for five days.

Chitinase, cellulase or xylanase activity associatedwith culture supernatants was determined as describedby Fayad et al. (2001). Briefly, a 0.5% (w/v) chitinsuspension, a 0.1% (w/v) carboxymethylcellulose(CMC) solution or 0.1% (w/v) suspension of xylan in a50 mmol L−1 sodium acetate buffer (pH 5.5) was sub-jected to enzymatic hydrolysis in the presence of cul-ture supernatants. The cellulase assay was carried outat 37 ºC for 30 min, whereas the chitinase and thexylanase assays were carried out at 50 ºC for 60 minand 10 min, respectively. Chitinase, cellulase andxylanase activities were estimated by the amount ofreducing sugars released from the polymers. One unitof chitinase, cellulase or xylanase was defined as theamount of enzyme that released 1 μmol of GlcNAc,glucose or xylose per min under the assay conditions.This experiment was repeated twice in two independ-ent replicates.

Geldanamycin production kinetics

EF-76 inoculum was prepared as described above. Fiftymicrolitres of inoculum were added to 15 mL of YME sup-plemented with or without 1 g L−1 of chitin, CMC orxylan. Cultures were incubated with shaking (300 rpm) forfive days at 30 °C. Fractions of these cultures were sam-pled periodically to monitor both geldanamycin production(supernatant) and bacterial growth (pellet). Bacterial cellswere recovered by centrifuging the culture (2500 g, 10 min).Geldanamycin was extracted from culture supernatantsthree times successively by adding a volume of chloro-form corresponding to one-third of the supernatantvolume. The chloroform fractions were pooled andevaporated. Geldanamycin was then separated by thinlayer chromatography on glass plates precoated with0.25 mm of silica gel 60, using chloroform:methanol(95:5) as the mobile phase. The intensity of the yellowcompounds having a Rf of 0.47 and of geldanamycinstandards of known concentrations was monitoredusing a scanner (Epson, Toronto, ON) and processedwith the Image Quant 5.0 program.

DNA was extracted from the corresponding cell pelletsaccording to Kieser et al. (2000) to estimate bacterialgrowth. DNA was quantified by spectrophotometry as

described by Alvi et al. (1995). This experiment wasrepeated twice in three independent replicates.

Geldanamycin biosynthesis in the presence of chitin or N-acetylglucosamine

EF-76 inoculum was prepared as described above. Twohundred microlitres of this inoculum were added to 50 mLYME supplemented with chitin or N-acetylglucosamine(GlcNAc). These carbon sources were added at the fol-lowing concentrations: 0 g L−1, 1 g L−1, 2 g L−1, 5 g L−1

or 10 g L−1. Cultures were incubated with shaking(300 rpm) for 60 h at 30 °C and then centrifuged at 2500 g,for 10 min. Bacterial pellets were dried at 80 oC for 24 h toestimate bacterial growth. Geldanamycin was extractedfrom culture supernatants with chloroform as describedabove, dissolved in acetonitrile and quantified by HighPerformance Liquid Chromatography (HPLC) using aProStar Varian liquid chromatograph instrument (Missis-sauga, ON) equipped with a μBondapack C18 column(10 μm particle size, 3.9 mm × 300 mm). Geldanamycinwas eluted from the column with a 55–85% acetonitrilegradient for 10 min at a flow rate of 1 mL min−1 and theantibiotic was monitored at 306 nm using a UV detector. Astandard curve was established using dilutions of knownquantities of purified geldanamycin. The experiment wasrepeated twice, with three replicates per treatment.

Antagonism assay against Streptomyces scabies

The inhibitory effect of geldanamycin on S. scabies growthwas demonstrated as follows. A filter paper disc placed inthe centre of a YME plate was soaked with 25 μl of a 1 mgmL−1 geldanamycin solution in methanol. In the controltreatment, the paper disc was soaked with methanol only.These plates were covered with soft agar (0.6%) containing106 spores of S. scabies EF-35 and incubated for 72 h. Pres-ence of growth inhibition zones were then recorded.

The ability of S. melanosporofaciens EF-76 to inhibit thegrowth of S. scabies EF-35 was tested on YME and MMplates supplemented with chitin at concentrations varyingbetween 0 and 10 g L−1. EF-76 was streaked in the centre ofthese plates using 15 μL inoculum prepared as describedabove. The plates were incubated for five days and werethen covered with soft Trypticase Soy Agar medium (TSA;Fisher Scientific, Ottawa, ON) (0.3% agar) inoculated withS. scabies EF-35 (500 μl of an overnight culture in 4 ml ofsoft TSA). The plates were incubated for an additional 72 hand the diameter of antibiosis zones was recorded. Five rep-lications per treatment were carried out and the experimentwas repeated three times.

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Biocontrol assay on radish seedlings

The biocontrol assay against the pathogenic agent S.scabies EF-35 was tested on radish seedlings. Radish(Raphanus sativus L.) seeds ‘Cherry Belle’ were sterilizedby soaking them for 5 min successively in 70% (v/v) etha-nol and twice in 10% (v/v) commercial bleach containing0.1% (w/v) of sodium dodecyl sulphate (SDS). The seedswere then rinsed three times with sterilized distilled water.Fifty seeds were germinated in the dark on Petri dishescontaining 1.5% agar for 40 h at 30 ºC. Seedlings weretransferred to plant growth pouches (Mega International,West St. Paul, MN) containing 10 mL of sterile water.

The effect of each biopolymer (chitin, CMC or xylan)and of S. melanosporofaciens EF-76 alone on symptomsinduced by S. scabies EF-35 was analysed. Furthermore,combination of a polymer and the biocontrol agent wasalso tested. Each biopolymer was added to growthpouches at a concentration of 1 g L−1. Growth poucheswith or without biopolymers were inoculated with S.melanosporofaciens EF-76 and/or S. scabies EF-35.Inocula were prepared by growing strain EF-76 or EF-35for 48 h in YME or TSB, respectively. Cultures werecentrifuged at 2500 g for 10 min and the pellets wereresuspended in four volumes of water. This bacterial sus-pension was added to growth pouches at a final concen-tration of 1:500. Radish seedlings were grown for fivedays under a 16 h photoperiod in a growth chambermaintained at 21 ºC.

Liquid in the growth pouches inoculated with EF-76was recovered to quantify geldanamycin as describedabove. Root length of radish seedlings was determinedusing MacRHIZO & WinRHIZO Image Analysis Systemfor Root Measurement version 2002c (Regent Instru-ments, Quebec City, QC). An experimental unit consistedof a growth pouch containing six seedlings; three repli-cates of each treatment were performed and the experi-ment was repeated three times.

Statistical analysis

Statistical analyses were carried out using the GLMprocedure of the SAS 9.1 statistical package (SAS Insti-tute Inc., Cary, NC) and performed with two-wayANOVA with treatment and repetition as main factors. Aposteriori comparisons were made using least significantdifference (LSD) tests.

Results

Extracellular enzymatic activities of S. melanosporofaciens EF-76

Chitinolytic, cellulolytic and xylanolytic activities weredetected after 24, 48, 72 h of growth in the EF-76 superna-tant of cultures containing chitin, CMC and xylan,respectively (Fig. 1). After five days of growth, chitino-lytic, cellulolytic and xylanolytic activities reached 0.28,0.02 and 0.85 U mL−1, respectively.

Effect of chitin, carboxymethylcellulose and xylan on geldanamycin production by S. melanosporofaciens EF-76

Growth of S. melanosporofaciens EF-76 and geldanamy-cin production were determined in YME supplementedwith or without 1 g L−1 of chitin, CMC or xylan. The ana-lysis of variance revealed variability between repeats(P < 0.05). However, different repetitions were analysedseparately (one-way ANOVA) and generated analogoussignificant differences among treatments. Consequently,results of one repetition are presented but are representativeof a general pattern. After a 24 h incubation period, S.melanosporofaciens EF-76 growth was significantlyhigher (P < 0.001) in the medium supplemented with chitinthan in media containing no or other polymers (Fig. 2a).No significant difference in growth was observedamongst treatments on subsequent days. After both two

Fig. 1. Enzymatic activities (± SD) associated with culture filtrates of Streptomyces melanosporofaciens EF-76 grown in the presence ofchitin (diamond), CMC (square) and xylan (triangle), respectively.

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and three days of growth, geldanamycin production(expressed per mg of bacterial DNA) was significantlyhigher in the medium supplemented with chitin than inthe other media tested (Fig. 2b). In the YME mediumsupplemented with chitin, geldanamycin level reached146 μg mg−1 DNA after two days of incubation, whereas46–68 μg/mg DNA of geldanamycin were produced inthe non-amended YME as well as in the YME mediasupplemented with CMC or xylan. Geldanamycin pro-duction reached a maximum level (342 μg mg−1 DNA)after three days of growth in the YME medium supple-mented with chitin.

Effect of various concentrations of chitin and N-acetylglucosamine on geldanamycin production by Streptomyces melanosporofaciens EF-76

Chitin concentration showed a significant impact ongeldanamycin production by S. melanosporofaciens(P < 0.001). Once again, the addition of chitin at aconcentration of 1 g L−1 significantly stimulated pro-duction of the antibiotic (Fig. 3a). When chitin wasadded at concentrations varying between 2 g L−1 and10 g L−1, antibiotic production significantly decreasedcompared with control medium (Fig. 3a). About 200 mgof geldanamycin per g of dried cells were extractedfrom control supernatants. Similar experiments werecarried out with GlcNAc, the monomer constituting thechain of chitin. Indeed, antibiotic production decreasedwhen GlcNAc was added at concentrations higher than2 g L−1 (Fig. 3b).

Fig. 2. Effect of polymers on Streptomyces melanosporofaciensEF-76 growth (a) and geldanamycin production (b) (expressed permg of bacterial DNA). The control treatment consisted of a YMEculture medium (black bars). This medium was amended with 1 g l−1

of chitin (hatched bars), CMC (white bars) or xylan (grey bars).Bars accompanied by an asterisk represent values that significantlydiffered from the control treatment (P < 0.05, ANOVA).

Fig. 3. Effect of various concentrations of chitin (a) andN-acetylglucosamine (b) on geldanamycin production (± SD) byStreptomyces melanosporofaciens EF-76. Bars accompanied bythe same letter did not significantly differ (P < 0.05, ANOVA).

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Antagonism ability of Streptomyces melanosporofaciens EF-76 against Streptomyces scabies EF-35

Geldanamycin, the predominant antibiotic produced by S.melanosporofaciens EF-76, efficiently inhibited the growthof Streptomyces scabies EF-35 as illustrated in Fig. 4. Theeffect of chitin on the antagonistic activity of S. melanosporo-faciens EF-76 was tested against S. scabies strain EF-35 inrich and MM media (Table 1). The presence of chitin reducedantagonistic activity of EF-76 against EF-35 in both media.In MM, the growth inhibition zone was significantly reduced(P < 0.001) in the presence of chitin at all concentrationstested. In rich medium, the effect of chitin on the antagonismwas less pronounced and was only observed significantly(P < 0.001) when chitin was added at 2 g L−1 and 10 g L−1.

Effect of plant polymers on the biocontrol ability of Streptomyces melanosporofaciens EF-76

The effect of plant polymers on the biocontrol ability of EF-76 was tested on radish seedlings infected with S. scabies, a

pathogen that inhibits the growth of most monocot and dicotseedlings (Leiner et al., 1996). Inoculation of radish seed-lings with EF-35 significantly reduced root length. Radishroot length in the non-inoculated treatment reached 12.6 cm(Fig. 5). Inoculation of radish seedlings with S. scabiesreduced root length to 8.3 cm (Fig. 5). In contrast to S. sca-bies, inoculation of EF-76 increased radish root length to19.3 cm (Fig. 5). The biocontrol agent EF-76 protected rad-ish seedlings against the deleterious effects of S. scabies. Nosignificant difference was observed between the root lengthof radish seedlings inoculated with EF-76 alone or in combi-nation with EF-76 and EF-35 (Fig. 5).

In the absence of bacteria, no biopolymer significantlymodified plant growth (Fig. 5). When added in the pres-ence of S. scabies strain EF-35, CMC and xylan con-ferred a significant level of protection against thepathogenic agent (Fig. 5). Indeed, seedling root lengthwas similar for these treatments and the non-inoculatedcontrol. No polymer significantly modified the biocontrolefficiency of EF-76 (Fig. 5).

Fig. 4. Effect of geldanamycin on the growth of Streptomyces sca-bies EF-35. a, The paper disk was soaked with methanol in the con-trol treatment. b, A zone of growth inhibition was observed around thedisk soaked with geldanamycin solution (25 μl at 1 mg mL−1).

Table 1. Effect of chitin amendments on the antagonistic ability of Streptomyces melanosporofaciens EF-76 against Streptomyces scabies EF-35 in minimal (MM) and complex (YME) media.

Chitin concentration (g L−1)

Inhibition zone (cm)

MM YME

0 1.13 ± 0.09a 1.37 ± 0.12a1 1.03 ± 0.11b 1.27 ± 0.17b

2 0.97 ± 0.09c 1.18 ± 0.09c

5 0.93 ± 0.10c 1.27 ± 0.09b

10 0.93 ± 0.07c 1.13 ± 0.08c

Notes: Inhibition zone values (within a column) that are accompanied bythe same letter did not significantly differ.

Fig. 5. Effect of polymers on the biocontrol ability of Streptomyces melanosporofaciens EF-76 against the pathogen S. scabies EF-35 inoculated onradish seedlings. Root length (± SD) of radish seedlings was measured after five days of growth in water (black bars) or in the presence of 1 g L−1 ofchitin (hatched bars), CMC (white bars) or xylan (grey bars) in combination with different bacterial conditions. Bars accompanied by a sameletter did not significantly differ (P < 0.05, ANOVA).

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The addition of polymers or the pathogen did notsignificantly modify geldanamycin production in growthpouches (data not shown). In these conditions, S. melano-sporofaciens EF-76 produced an average of 1.2 μg ofgeldanamycin per pouch.

Discussion

Recent studies suggest that biopolymers, which are abun-dant carbon sources for actinomycetes in natural environ-ments, play a critical role in monitoring the globalnutritional status (Colson et al., 2008) and in determiningthe onset of antibiotic biosynthesis (Rigali et al., 2008).The effect of such natural polymers (chitin, CMC, xylan)on geldanamycin biosynthesis in S. melanosporofaciensstrain EF-76 was thus tested. Although chitin was not thesole biopolymer hydrolysed by EF-76, as both cellulolyticand xylanolytic activities were detected in this strain, chi-tin was the only biopolymer tested that significantlyincreased geldanamycin production in EF-76. When chitinwas added at a concentration of 1 g L−1, an increase ingeldanamycin biosynthesis was observed after two andthree days of growth but not later. It thus seems that theeffect of chitin is only short-term. Chitin assimilation dur-ing the bacterial growth may explain this observation sinceaddition of chitin at concentrations lower than 1 g L−1

caused no significant difference in geldanamycin produc-tion by strain EF-76 in YME medium (data not shown).

Chitin is composed of N-acetylglucosamine residuesand consequently, provides additional carbon and nitro-gen sources for chitinolytic bacteria. The chitinolyticactivity of EF-76 might explain the improved growth rateobserved in the presence of chitin at the early growthphase. Chitinolytic activity would generate an increase inthe amount of GlcNAc. This sugar is a high-energy mole-cule involved in glycolysis and nitrogen metabolism. Italso acts as a precursor of peptidoglycan synthesis(Rigali et al., 2006). Nevertheless, the increased geldan-amycin biosynthesis in the presence of 1 g L−1 chitincould not simply be explained by an improved growthrate. Indeed, the ratio between antibiotic production andbiomass also increased when chitin was added at thisconcentration to the growth medium. A modulation ofchitinase gene expression might affect the activity of reg-ulatory genes controlling antibiotic synthesis. Colson etal. (2007) suggested that a chitinase promoter bindingprotein could also interact with genes encoding regula-tors involved in functions other than chitinase geneexpression.

As chitin is a long-chain polymeric polysaccharide ofβ-1,4 GlcNAc, one could have expected that the additionof the GlcNAc monomer would also have a positive

effect on geldanamycin biosynthesis. However, antibioticsynthesis was not improved in the presence of this monomerat all concentrations tested. The presence of chitin or itsmonomer had different effects on S. coelicolor secondarymetabolism as well (Colson et al., 2008). In S. coelicolor,addition of GlcNAc into complex media could blockdevelopment and antibiotic production, whereas chitinhad no effect on either process (Colson et al., 2008).

Although the addition of 1 g L−1 of chitin appeared toslightly improve antibiotic production in EF-76, no sucheffect was detected in S. coelicolor (Colson et al., 2008).However, different methods were used to monitor antibi-otic biosynthesis in both species. In S. coelicolor, antibi-otic production was visually determined by estimatingthe pigmentation level associated with culture media.Small differences in the production of pigmented antibi-otics by S. coelicolor would be unlikely to have beendetected using such a method.

Although chitin induced an increase in geldanamycinbiosynthesis in complex medium under specific condi-tions, this improved production of geldanamycin did nottranslate to a higher level of antagonism towards S.scabies. Addition of chitin had a low impact on the antag-onism between EF-76 and S. scabies both in rich andpoor culture media, suggesting that differences in theamount of geldanamycin produced in vitro only moder-ately influenced the interaction between the pathogen andthe antagonist microbe. The in vitro antagonism againstS. scabies was nevertheless the weakest at chitin concen-trations inhibiting the most geldanamycin biosynthesis.Although no geldanamycin was detected in a complexmedium supplemented with 10 g L−1 of chitin, the antag-onistic activity was only reduced by 20% under the sameconditions. This relatively high antagonistic activitymight be due to the fact that strain EF-76 produces anti-microbial compounds other than geldanamycin (Agbessiet al., 2003).

Chitin did not confer an improved biocontrol capacityto S. melanosporofaciens EF-76 under the conditionsused in the bioassay and this might be explained by thefact that geldanamycin biosynthesis was not modified bythe presence of chitin in the plant growth pouches. Theamount of geldanamycin produced under the bioassayconditions is very low compared with the quantity ofantibiotic associated with a rich culture medium. The poornutritional status of the bioassay conditions (bioassays wereperformed with water) only allowed scarce bacterialgrowth as the sole carbon and nutrients sources were sup-plied by root exudates. Under these conditions, resourcesto support the production of secondary metabolites werelikely limited. Nevertheless, EF-76 efficiently protectedradish seedlings against S. scabies in the presence or

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absence of chitin. Similar results were obtained in thepresence of CMC alone and xylan alone. This protectionis likely due to the fact that these plant constituents couldact as elicitors of various plant defence systems (Walkeret al., 1994; Belien et al., 2006; Kaku et al., 2006). Chitinderivatives are also recognized as elicitors of plantdefence mechanisms since chitin is the main cell wallcomponent of most fungi (Kaku et al., 2006). While xylanaseand cellulase genes are detected in S. scabies genome,strain EF-35 did not grow on chitin (data not shown). Thesystem could thus have lacked the chitin degradation prod-ucts necessary for the elicitation of defence mechanisms.

Interestingly, S. melanosporofaciens EF-76 promotedradish growth even in the absence of the pathogen. Thebiocontrol capacity of EF-76 against common scab ofpotato (Agbessi et al., 2003; Beauséjour et al., 2003) andraspberry root rot (Valois et al., 1996) has been previ-ously demonstrated, but EF-76 was not shown to promotegrowth of potato and raspberry in the absence of pathogens.However, radish seedlings might be more sensitive to plantgrowth promoting factors secreted by EF-76 than tubers ormature plants. EF-76 like several other actinomycetes mightthus be recognized as a plant growth-promoting organism(Doumbou et al., 2001b; Hamdali et al., 2008).

Although chitin did not improve the biocontrol activityof EF-76 under gnotobiotic conditions, the possibilitythat the polymer might be useful in agricultural practicescannot be excluded. Under field conditions, chitin mightconfer a selective advantage to EF-76 by facilitating itsestablishment in the soil as shown with chitosan, thedeacetylated form of chitin (Prévost et al., 2006). As inother streptomycetes, biosynthesis of secondary metabo-lites, such as geldanamycin, by the biocontrol agent S.melanosporofaciens is affected by the presence of chitin.Nevertheless, the relationships between chitin supply,secondary metabolism and antagonistic activity in actino-mycetes is complex and appears to depend on severalbiotic and abiotic factors.

Acknowledgements

We thank Isabelle Madore for technical assistance. Thiswork was supported by grants from the Natural Sciencesand Engineering Research Council (NSERC) of Canada.

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