ORIGINALRESEARCH published:09October2015 doi:10.3389/fmicb.2015.01081 Diversity and functions of volatile organic compounds produced by Streptomyces from a disease-suppressive soil VivianeCordovez1,2,VictorJ.Carrion1,DesalegnW.Etalo1,RolandMumm3,4,HuaZhu5, GillesP.vanWezel1,5andJosM.Raaijmakers1,5* 1DepartmentofMicrobialEcology,NetherlandsInstituteofEcology(NIOO-KNAW),Wageningen,Netherlands,2Laboratoryof Phytopathology,WageningenUniversity,Wageningen,Netherlands,3PlantResearchInternational,BusinessUnitBioscience, WageningenUniversityandResearchCentre,Wageningen,Netherlands,4CentreforBiosystemsGenomics,Wageningen, Netherlands,5MolecularBiotechnology,InstituteofBiology,LeidenUniversity,Leiden,Netherlands In disease-suppressive soils, plants are protected from infections by specific root Editedby: pathogensduetotheantagonisticactivitiesofsoilandrhizospheremicroorganisms.For StéphaneHacquard, MaxPlanckInstituteforPlant mostdisease-suppressivesoils,however,themicroorganismsandmechanismsinvolved BreedingResearch,Germany inpathogencontrolarelargelyunknown.OurrecentstudiesidentifiedActinobacteriaas Reviewedby: themostdynamicphyluminasoilsuppressivetothefungalrootpathogenRhizoctonia MikaTapioTarkka, solani. Here we isolated and characterized 300 isolates of rhizospheric Actinobacteria HelmholtzCentreforEnvironmental Research-UFZ,Germany from the Rhizoctonia-suppressive soil. Streptomyces species were the most abundant, TomislavCernava, representing approximately 70% of the isolates. Streptomyces are renowned for the GrazUniversityofTechnology,Austria productionofanexceptionallylargenumberofsecondarymetabolites,includingvolatile *Correspondence: JosM.Raaijmakers, organic compounds (VOCs). VOC profiling of 12 representative Streptomyces isolates DepartmentofMicrobialEcology, bySPME-GC-MSallowedamorerefinedphylogeneticdelineationoftheStreptomyces NetherlandsInstituteofEcology isolates than the sequencing of 16S rRNA and the house-keeping genes atpD and (NIOO-KNAW),Droevendaalsesteeg 10,6708PBWageningen, recA only. VOCs of several Streptomyces isolates inhibited hyphal growth of R. solani Netherlands and significantly enhanced plant shoot and root biomass. Coupling of Streptomyces [email protected] VOC profiles with their effects on fungal growth, pointed to VOCs potentially involved Specialtysection: in antifungal activity. Subsequent assays with five synthetic analogs of the identified Thisarticlewassubmittedto VOCsshowedthatmethyl2-methylpentanoate,1,3,5-trichloro-2-methoxybenzeneand PlantBioticInteractions, asectionofthejournal the VOCs mixture have antifungal activity. In conclusion, our results point to a potential FrontiersinMicrobiology role of VOC-producing Streptomyces in disease suppressive soils and show that VOC Received:27July2015 profiling of rhizospheric Streptomyces can be used as a complementary identification Accepted:22September2015 tooltoconstructstrain-specificmetabolicsignatures. Published:09October2015 Citation: Keywords:Actinobacteria,SPME-GC-MS,antifungalactivity,plantgrowthpromotion,suppressivesoil CordovezV,CarrionVJ,EtaloDW, MummR,ZhuH,vanWezelGPand RaaijmakersJM(2015)Diversityand Introduction functionsofvolatileorganic compoundsproducedby Disease-suppressive soils are soils in which plants are effectively protected from infections by Streptomycesfroma specific root pathogens due to antagonistic activities of soil and rhizosphere (micro)organisms disease-suppressivesoil. Front.Microbiol.6:1081. (Hornby, 1983; Weller et al., 2002). This phenomenon has been described worldwide, but doi:10.3389/fmicb.2015.01081 the responsible (micro)organisms and underlying mechanisms are largely unknown for most FrontiersinMicrobiology|www.frontiersin.org 1 October2015|Volume6|Article1081 Cordovezetal. Streptomycesvolatiles:diversityandfunctions suppressive-soils (Weller et al., 2002; Mendes et al., 2011; Materials and Methods Chapelle et al., 2015). In recent studies, we identified the microbiome of a soil suppressive to Rhizoctonia solani, an SelectiveIsolationofActinobacteria economically important soil-borne fungal pathogen of many Actinobacteria were isolated from the rhizosphere (roots with cropsincludingsugarbeet,potato,andrice(Mendesetal.,2011; adhering soil) of sugar beet plants grown in a soil suppressive Chapelle et al., 2015).PhyloChip-based metagenomics detected toR.solani.Thesoilwaspreviouslycollectedin2003and2004 morethan33000bacterialandarchaealtaxaintherhizosphere fromanagriculturalsugarbeetfieldclosetothetownofHoeven, ofsugarbeetseedlingsgrownintheRhizoctonia-suppressivesoil the Netherlands (51◦35′10′′N 4◦34′′44′E). For the collection of and revealed bacterial groups consistently associated with the Actinobacteria from the rhizosphere, sugar beet seeds (cultivar diseasesuppressivestate.Amongthetop10%ofmostdynamic Alligator) were sown in square PVC pots containing 250 g taxa (i.e., taxa relatively more abundant in suppressive than in of field soil with an initial moisture content of 10% (v/w). non-suppressive soil), Actinobacteria were the most dynamic Plants were grown in a growth chamber (24◦C/24◦C day/night phylumfoundintherhizosphereofsugarbeetseedlingsgrowing temperatures; 180µmol light m−2 s−1 at plant level during 16 inthesuppressivesoil. h/d; 70% relative humidity) and watered weekly with standard Actinobacteria are ubiquitously found in nature and the Hoaglandsolution(macronutrientsonly).After3weeksofplant phylum comprises more than 500 formally described species growth,1gofsugarbeetrootswithadheringsoilwassuspended (Goodfellow, 2012; Labeda et al., 2012). Many Actinobacteria in 5mL of potassium-phosphate buffer (pH 7.0). Samples were are multicellular bacteria with a complex life cycle and are vortexedandsonicatedfor1min.Toenrichfordifferentgenera renowned for the production of an exceptionally large number of Actinobacteria, a number of treatments were applied to ofbioactivemetabolites(Claessenetal.,2014).Membersofthe the soil suspension (Supplementary Table S1). Single colonies genus Streptomyces produce over 10000 secondary metabolites, were picked based on the morphology and purified on fresh including volatile organic compounds (VOCs) (Bérdy, 2005; agar plates. Isolates were stored in glycerol (20%, v/v) at −20 Hopwood, 2007; van Wezel et al., 2009). Approximately 1000 and−80◦C. microbial VOCs have been identified to date (Piechulla and Degenhardt, 2014). Although the production of VOCs by CharacterizationofActinobacteria microorganismsisknownformanyyears(ZollerandClark,1921; All300Actinobacterialisolateswerecharacterizedby16SrRNA StotzkyandSchenck,1976),itisonlysincethelastdecadethat gene sequencing. PCR amplifications were conducted using an increasing number of studies have reported on the diversity primers8F(5′-AGAGTTTGATCCTGGCTCAG-3′)and1392R andpotentialfunctionsofthesecompounds.TheblendofVOCs (5′- ACGGGCGGTGTGTACA - 3′) or 27F (5′- GAGTTTGAT released by microorganisms is diverse and complex. Microbial CCTGGCTCAG-3′)and1492R(5′-ACCTTGTTACGACGAC VOCsbelongtodifferentclassesofcompoundssuchasalkenes, TT - 3′) (Lane, 1991; Deangelis et al., 2009). For obtaining alcohols, ketones, terpenes, benzenoids, aldehydes, pyrazines, DNA, bacterial cells were disrupted by heating at 95◦C for acids, esters, and sulfur-containing compounds (Effmert et al., 10min. For spore forming isolates, cells were disrupted in the 2012). The same VOCs can be found for different, often microwave at 650 W for 30s in TE buffer. Suspensions were unrelated,microorganismsbutsomeVOCsareuniquetospecific centrifugedat13000rpmfor10min.Aftercentrifugation,2µlof microorganisms (Schulz and Dickschat, 2007; Garbeva et al., thesupernatantswereusedforthePCRreactions.PCRproducts 2014). Microbial VOCs display versatile functions: they inhibit were purified and sequenced at Macrogen Inc. Isolates were bacterial and fungal growth, promote or inhibit plant growth, characterized based on sequence identity with 16S rRNA gene trigger plant resistance and attract other micro- and macro- sequences in the Greengenes database (McDonald et al., 2012) organisms(Ryuetal.,2003,2004;Vespermannetal.,2007;Kai (http://greengenes.lbl.gov/). et al., 2009; Verhulst et al., 2009; Bailly and Weisskopf, 2012; Hagaietal.,2014;Schmidtetal.,2015).Furthermore,VOCshave CouplingStreptomycesIsolatestoOTUs been proposed to function as signaling molecules in inter- and DetectedbyPhyloChip intra-specificinteractionsandincell-to-cellcommunication.To 16S rRNA gene sequences of 173 Streptomyces isolates were date,however,thenaturalfunctionsofmicrobialVOCsandtheir compared with the 16S rRNA gene sequences of Streptomyces modesofactionremainlargelyunknown(Kaietal.,2009;Kim OTUs previously identified by PhyloChip-based metagenomic etal.,2012;Schmidtetal.,2015). analysis as the top 10% of most abundant taxa associated Here we studied the diversity and functions of VOCs withdiseasesuppressiveness(Mendesetal.,2011).Phylogenetic produced by different Streptomyces from the rhizosphere of analysis was performed with Muscle in MEGA6 (Tamura sugar beet seedlings grown in a Rhizoctonia-suppressive soil. et al., 2013) and iTOL (Letunic and Bork, 2011) (http:// We first isolated and characterized 300 Actinobacteria. As itol.embl.de/). A Neighbor-joining consensus tree (Saitou and Streptomyces represented almost 70% of all isolates, subsequent Nei, 1987) with 1000 bootstrap replicates (Felsenstein, 1985) VOC analyses, phylogeny, antifungal activity and plant growth was constructed using Tamura-Nei model (Tamura and Nei, assays were conducted with this group of Actinobacteria. By 1993) with gamma distribution. A total of 11 isolates, which coupling SPME-GC-MS and hierarchical clustering of VOC were closely related to the isolates detected by PhyloChip, profiles, we identified VOCs potentially involved in antifungal was selected to study the composition of emitted VOCs and activity. their in vitro effects on fungal and plant growth. Streptomyces FrontiersinMicrobiology|www.frontiersin.org 2 October2015|Volume6|Article1081 Cordovezetal. Streptomycesvolatiles:diversityandfunctions lividans1326(Cruz-Moralesetal.,2013)wasusedasareference datawerethensubjectedtomultivariatestatisticalanalysis.One- strain. way ANOVA was performed with GeneMaths XT Version 2.11 (AppliedMaths,Belgium)toidentifyVOCssignificantlydifferent CharacterizationofSelectedStreptomyces fromthecontrol(mediumonly)[p < 0.05;withfalsediscovery Isolates rate (FDR) correction]. After that, hierarchical cluster analysis The11Streptomycesisolateswerecharacterizedbasedoncolony (HCA) using Pearson’s correlation coefficient with UPGMA morphology and by sequence analysis of the house-keeping algorithmwasperformed. genes recA (recombinase A) and atpD (ATP synthase subunit B). These genes were amplified and sequenced as previously VOC-mediatedAntifungalActivity described(Guoetal.,2008).PartialsequencesofrecA(500bp), The effect of Streptomyces VOCs on the growth of the fungus atpD (423 bp), and 16S rRNA (516 bp) genes of Streptomyces R. solani was investigated using the bottoms of two 90-mm- were concatenated to yield an alignment of 1439 sites. A diameter Petri dishes allowing physical separation between the concatenatedphylogenetictreesupplementedwithsequencesof bacteria and the fungus. One bottom contained a Streptomyces Streptomycesstrainswithasequencedgenome(NCBIdatabase) isolate on GA medium, previously incubated at 30◦C for 4 was constructed using UPGMA with the Tamura-3 parameter days.TheotherbottomcontainedaplugofR.solanimycelium calculationmodelwithgammadistributionand1.000bootstrap on 1/10th Tryptone Soy Agar (TSA, Oxoid). Both Petri dishes replicates. All sequences were deposited to GenBak and have were sealed facing each other and incubated at 25◦C with the been assigned to accession numbers: KT60032-KT600042 (16S Petri dish containing the Streptomyces on the bottom to avoid rRNAgene),KT600043-KT600053(recAgene),andKT600054- spores transferring to the plate with the fungus. As a control, KT600064(atpDgene). the Petri dish containing R. solani was exposed to a Petri dish containing GA medium only. Fungal growth inhibition was CollectionandAnalysisofStreptomycesVOCs calculatedbymeasuringtheradialgrowthofthefungalhyphae FortrappingtheVOCs,theStreptomycesisolateswereinoculated after 1, 2, and 3 days of incubation. Percentage of inhibition individually in 10ml sterile glass vials containing 2.5ml of was calculated as [(diameter of fungus in control − diameter GA medium (Zhang, 1990) with three replicates each. Vials offungusexposedtoVOCs)*100/diameteroffungusincontrol] containingmediumonlyservedascontrols.Allvialswereclosed for each of the 3 replicates. Student’s t-Test was performed to andincubatedat30◦C.After7days,VOCsfromtheheadspace determine statistically significant differences compared to the of each vial were collected by solid phase microextraction control(p<0.05,n=3). (SPME) with a 65-mm polydimethylsiloxane-divinylbenzene fiber(Supelco,Bellefonte,USA). AntifungalActivityofSyntheticVOCs Streptomyces VOCs were analyzed by GC-MS (Agilent Methylbutanoate(≥98%),methyl2-methylpentanoate(≥98%), GC7890A with a quadrupole MSD Agilent 5978C). VOCs methyl 3-methylpentanoate (≥97%), 1,3,5-trichloro-2-methoxy were thermally desorbed at 250◦C by inserting the fiber for benzene(99%),and3-octanone(≥98%)wereobtainedatSigma- 2min into the hot GC injection port. The compounds released Aldrich. All VOCs were dissolved in methanol with final were transferred onto the analytical column (HP-5MS, 30m × concentrations ranging from 1M to 1nM (10-fold dilutions). 0.25mm ID, 0.25µm—film thickness) in splitless mode. The Assays were performed using a standard 90mm-diameter Petri temperature program of the GC oven started at 45◦C (2- dish with the fungal plug on 1/10th TSA medium on top and min hold) and rose with 10◦C min−1 to 280◦C (3-min hold). withasterilepaperfilter(1.5×1.5cm)onthebottom.Twenty Mass scanning was done from 33 to 300 m/z with a scan microlitersofeachVOCdissolvedinmethanolwereappliedon time of 2.8 scans s−1. GC-MS raw data were processed thepaperfilter,plateswereimmediatelysealedandincubatedat by an untargeted metabolomics approach. MetAlign software 25◦C. Radial hyphal growth of the fungus was measured after (Lommen and Kools, 2012) was used to extract and align the 1 and 2 days of exposure to single or a mixture of the 5VOCs mass signals (s/n = 3). MSClust was used to remove signal and compared to control (empty top of a Petri dish). To check redundancy per metabolite and to reconstruct compound mass whetherthesolventitselfhadanyeffectongrowthofthefungus, spectra as previously described (Tikunov et al., 2012). VOCs R. solani was also exposed to methanol alone. Student’s t-Test were tentatively annotated by comparing their mass spectra was performed to determine statistically significant differences withthoseofcommercial(NIST08)andin-housemassspectral comparedtothecontrol(p<0.05,n=3−5). libraries.Linearretentionindices(RI)ofVOCswerecalculated as previously described (Strehmel et al., 2008) and compared VOC-mediatedPlantGrowthPromotion withthoseintheliterature.VOCsselectedforinvitroantifungal To determine whether Streptomyces VOCs had an effect on assays [methyl butanoate (≥98%), methyl 2-methylpentanoate plant growth, Arabidopsis thaliana seedlings were exposed to (≥98%), methyl 3-methylpentanoate (≥97%), 1,3,5-trichloro- the VOCs emitted by the different isolates. A. thaliana seeds 2-methoxy benzene (99%) and 3-octanone (≥98%)] were (wild-typeCol-0)weresurfacesterilizedaspreviouslydescribed confirmedwithauthenticreferencestandardsobtainedatSigma- (van de Mortel et al., 2012) and sown on 90-mm-diameter Aldrich. Processed VOC data were log transformed and auto- Petri dishes containing 50ml of 0.5X Murashige and Skoog scaledusingtheaverageasanoffsetandthestandarddeviation medium(MurashigeandSkoog,1962)supplementedwith0.5% asscale[rawvalue-average(offset)/SD(scale)].Logtransformed (w/v) sucrose. The 90-mm-diameter Petri dishes were placed FrontiersinMicrobiology|www.frontiersin.org 3 October2015|Volume6|Article1081 Cordovezetal. Streptomycesvolatiles:diversityandfunctions insidea145-mm-diameterPetridish,sealedandincubatedina climatechamber(21◦C/21◦Cday/nighttemperatures;180µmol light m−2 s−1 at plant level during 16 h/d; 70% relative humidity).After7days,35-mm-diameterPetridishescontaining Streptomyces isolates growing on GA medium (previously incubatedat30◦Cfor1week)wereaddedtothe145-mmPetri dishes with the A. thaliana seedlings. Plates were sealed and keptat21◦C.After14days,plantfreshweightwasdetermined. Inaddition,plantdryweightwasmeasuredafterdryingshoots and roots overnight in an incubator at 65◦C. Student’s t-Test was performed to determine statistically significant differences compared to the control treatment (plants exposed to medium only). Results DiversityofActinobacteriaIsolatedfrom SuppressiveSoil Using PhyloChip-based metagenomic analyses, we previously described the diversity of the bacterial community associated withtherhizosphereofsugarbeetplantsgrowninaRhizoctonia- suppressive soil (Mendes et al., 2011). Actinobacteria were prominently more represented in the suppressive soil than in the non-suppressive (conducive) soil. Bacterial diversity detected by the PhyloChip used in the aforementioned study is displayed in Figure1A. To select as many Actinobacterial isolates as possible, several pre-treatments of the rhizospheric soil and different selective media were used for their isolation (SupplementaryTableS1).Atotalof300Actinobacterialisolates wereobtainedandcharacterizedby16SrRNAgenesequencing. Based on the sequence similarities (95–100%) to the 16S rRNA gene sequences available in the Greengenes database (used as reference in the PhyloChip analyses), 18 different genera of Actinobacteria were identified. These were Streptomyces, Microbacterium, Rhodococcus, Micromonospora, Microbispora, Kribbella, Pseudonocardia, Cellulomonas, Mycobacterium, Actinoplanes, Arthrobacter, Actinomadura, Amycolaptosis, Nocardioides, Nonomureae, Streptosporangium, Micrococcus, FIGURE1|Top10%mostdynamicbacterial(andarchaeal)phyla and Rothia (Figure1B). The genus Streptomyces was the most detectedbyPhyloChipanalysisoftherhizospheremicrobiomeof sugarbeetseedlingsgrowninRhizoctonia-suppressivesoil(piechart abundant,representing69%ofallisolatesandatleast25different A,adaptedfromMendesetal.,2011).DiversityofActinobacteria(piechart speciesbasedon16SrRNAgenesequences(Figure1C). B)andofStreptomycesspecies(piechartC)isolatedfromtherhizosphereof sugarbeetseedlingsgrowninRhizoctonia-suppressivesoil(thisstudy). PhylogeneticAnalysisofStreptomycesIsolates ToselectStreptomycesisolatesforVOCandfunctionalanalyses, 16S rRNA gene sequences of the Streptomyces isolates (n = the different Streptomyces isolates than based on 16S sequences 173) obtained in this study were compared with those of the only. However, closely related but phenotypically different representativeStreptomycesOTUs(n = 430)originallydetected isolates, like Streptomyces strains W75.5 and W126 (Figure3), by PhyloChip (Mendes et al., 2011). A phylogenetic tree was could not be distinguished based on these three molecular constructedusingthesesequencesandthesequencesofdifferent markers. Streptomyces type strains (Figure2). This comparison led to the selection of 11 isolates (Figure3). We then constructed VOCProfilingofStreptomycesIsolates phylogenetic trees with these 11 isolates, their closest type For the 12 Streptomyces isolates (11 rhizosphere isolates and strains,otherStreptomycesspecieswithsequencedgenomesand referencestrainS.lividans1326)grownonGAmediumandthe the reference strain Streptomyces lividans 1326 (Supplementary medium alone (control), a total of 536VOCs were detected in Figure S1A). Additionally, we sequenced the house-keeping the headspace. Out of these, 381VOCs that were significantly genesatpDandrecA(SupplementaryFigureS1B).Concatenation different (ANOVA, p < 0.05) and detected at intensities at ofatpD,recA,and16Ssequencesallowedabetterresolutionof leasttwiceashighasinthecontrolwereconsideredforfurther FrontiersinMicrobiology|www.frontiersin.org 4 October2015|Volume6|Article1081 Cordovezetal. Streptomycesvolatiles:diversityandfunctions FIGURE2|Neighbor-joiningphylogenetictreebasedon16SrRNAgenesequencesoftheStreptomycescollectionobtainedinthisstudy(inblue), StreptomycesdetectedbyPhylochipanalysis(inpink),andStreptomycestypestrains(ingreen).StreptomycesisolatesselectedforVOCanalysisare indicatedinbold. analyses. The diversity of VOCs produced by the different strains were able to significantly retard the growth of R. solani. Streptomyces isolates is shown in Supplementary Table S2 and Streptomyces strains W47 and W214 were the most inhibitory. highlighted in the heat-map (Figure4). The VOCs detected Whenexposedfor2daystotheVOCsproducedbytheseisolates, belong to diverse classes of compounds such as alcohols, radial hyphal growth was reduced by 57 and 41%, respectively aldehydes, carboxylic acids, esters, ketones, sulfur compounds, (Figure6A). and several terpenes (Supplementary Table S2). Most VOCs Additionally, we tested whether Streptomyces VOCs could were found to be specific for some Streptomyces isolates and promote plant growth. To that end, we exposed 7-day-old 45VOCs were found to be commonly produced by all isolates A. thaliana seedlings to VOCs from each of the isolates and tested. Geosmin (trans-1,10-dimethyl-trans-9-decalol, RI 1423; determined root and shoot biomass. After 2 weeks of exposure SupplementaryTableS2)wasoneofthesecommonVOCs.HCA toStreptomycesVOCs,nonegativeeffectsonplantgrowthwere of the VOC profiles resulted in a similar clustering of the 12 observed. Ten out of 12 isolates significantly increased shoot Streptomyces isolates as the clustering based on the different biomass, and 8 significantly increased root biomass compared molecular markers (Figure5). In contrast to the molecular to the control (Figure6B). S. lividans 1326, and Streptomyces markers,however,VOCprofilingalloweddifferentiationbetween strainsW47andW62ledtothelargestincreaseinplantbiomass, closelyrelatedStreptomycesisolatessuchasStreptomycesstrains whereas Streptomyces strains W214 and 3A41 did not increase W75.5andW126aswellasStreptomycesstrainsW47andW214. shootandrootbiomass. EffectofStreptomycesVOCsonFungaland IdentificationofStreptomycesVOCs PlantGrowth ContributingtoAntifungalActivity To test the antifungal activity of VOCs produced by the Since Streptomyces strains W47 and W214 are phylogenetically Streptomyces isolates from disease suppressive soil, hyphal closelyrelatedandbothshowedstrongantifungalactivity,these growthofR.solaniwasmeasuredduringexposuretoVOCsfrom isolates were selected to identify VOCs with activity against R. each of the isolates. In the control, fungal hyphae reached the solani.ScreeningofVOCswithpotentialantifungalactivitywas edgeoftheagarplatesafter2daysofincubation.AllStreptomyces computedwithOne-wayANOVA[p<0.05;withfalsediscovery FrontiersinMicrobiology|www.frontiersin.org 5 October2015|Volume6|Article1081 Cordovezetal. Streptomycesvolatiles:diversityandfunctions FIGURE3|CharacterizationofStreptomycesisolatesusedinthisstudy.Speciesnamesarebasedon16SrRNAgenesequencecomparisonusingthe Greengenesdatabase.Picturesdepict4–7day-oldisolatesgrownonGAmedium.*S.lividans1326referstoJohnInnesCentercollectionnumberandcorresponds toS.lividans66(Hopwoodetal.,1983). rate (FDR) correction] and a fold change >2 using the peak and W214. Subsequently, different concentrations of these five intensityofVOCsfromW214/controlandW47/control.Forthe VOCs were used to test their inhibitory effect on hyphal selection of VOCs for in vitro antifungal activity, three criteria growth of R. solani (Figure7C). The VOC 1,3,5-trichloro-2- wereused:(1)matchfactorandreversematchfactorhigherthan methoxy benzene completely inhibited radial hyphal growth of 850, (2) reliable annotation based on retention indices and, (3) R. solani at concentrations of 1M and 100mM (Figure7D). availabilityofpure(synthetic)referencecompounds. Exposure to this VOC led to melanization of R. solani hyphae A comparison of the VOC profiles of Streptomyces strains (Figure7E). The VOC methyl 2-methylpentanoate reduced W47 and W214 with the control (medium only) pinpointed fungal growth by 47 and 25% after 1 and 2 days of exposure, VOCs potentially involved in antifungal activity (Figure7A). respectively. Additionally, a mix of the 5 synthetic VOCs, A total of 96VOCs were shared between these two isolates; each at a final concentration of 200mM, inhibited hyphal 65 and 7VOCs were unique for Streptomyces strains W47 growth by 58 and 42% after 1 and 2 days of exposure, andW214,respectively(Figures7A,B).SincebothStreptomyces respectively. strains W47 and W214 showed antifungal activity, we looked TofurtherdetermineiftheantifungalVOC1,3,5-trichloro-2- into the VOCs detected for both strains. We selected five methoxybenzeneistypicallyfoundforStreptomycesisolatesthat commonVOCs(methylbutanoate,methyl2-methylpentanoate, inhibit hyphal growth of R. solani, we determined the relative methyl3-methylpentanoate,1,3,5-trichloro-2-methoxybenzene, amounts of this VOC produced by each of the 12 Streptomyces and 3-octanone) which could be reliably annotated based on isolates tested in this study. The results show that production RI and mass spectral similarity and which were commercially of this VOC is widespread among the 12 Streptomyces isolates. available as authentic reference standards. The identity of Moreover,apositivenonlinearcorrelationwasfoundbetweenthe these compounds was verified by analyzing pure standards by percentageofhyphalgrowthinhibitionandtheabundance(peak the GC-MS and comparing their mass spectra and RI with intensity)of1,3,5-trichloro-2-methoxybenzenedetectedforthe those of the VOCs detected for Streptomyces strains W47 12isolates(Figures7F,G). FrontiersinMicrobiology|www.frontiersin.org 6 October2015|Volume6|Article1081 Cordovezetal. Streptomycesvolatiles:diversityandfunctions FIGURE4|Hierarchicalclusterandheat-mapanalysesofVOCprofilesoftheselectedStreptomycesisolates.ColumnsrepresentthreereplicateVOC measurementsofeachofthe12isolatesandthemediumalone(control).RowsrepresentthedifferentVOCs(green,lowabundance;red,highabundance),severalof whichwereputativelyannotated(seeSupplementaryTableS2). FrontiersinMicrobiology|www.frontiersin.org 7 October2015|Volume6|Article1081 Cordovezetal. Streptomycesvolatiles:diversityandfunctions FIGURE5|(A)Phylogenetictreeofconcatenatedpartialsequencesof16SrRNA,atpDandrecAgenesof11Streptomycesisolatesfromthe Rhizoctonia-suppressivesoilandthereferencestrainS.lividans1326.ThetreewasconstructedusingUPGMAmethodandTamura-3parametercalculationmodel withgammadistributionand1000bootstrapreplicates.(B)Hierarchicalclusteranalysis(HCA)ofStreptomycesVOCswithUPGMAmethodandPearson’scorrelation coefficient.DifferentcolorsindicatedifferentclustersofisolatesbasedonVOCprofiles. Discussion different streptomycetes. Most of the 381VOCs detected for the different streptomycetes from the Rhizoctonia-suppressive The production of VOCs by microorganisms is known for soil were found to be specific for some isolates whereas fewer several decades. Only recently an increasing number of studies VOCswerefoundtobecommonlyproducedbyallisolates.The reported on the chemical diversity and possible functions of bestknownVOCsfromstreptomycetesare2-methylisoborneol this group of microbial compounds (Schmidt et al., 2015). (MIB)andtrans-1,10-dimethyl-trans-9-decalol(geosmin)which In comparison to plant VOCs, knowledge about the natural are responsible for the characteristic musty or earthy smell of functions of microbial VOCs is still limited (Bitas et al., 2013). moist soils (Gerber, 1968; Jiang et al., 2007). Our results also Here we studied the diversity and activities of VOCs produced show that these VOCs are widely produced by Streptomyces by different streptomycetes from a Rhizoctonia-suppressive isolates from the rhizosphere of sugar beet plants grown in soil. Rhizoctonia-suppressive soil. Geosmin was detected for all VOC profiling has been extensively used for food flavoring isolates, whereas MIB was detected for eight isolates. Members and aroma as well as indicators of fungal growth in buildings of the Streptomyces genus differ greatly in their morphology, and in post-harvest management (Morath et al., 2012). More physiology, and biochemical characteristics (Anderson and recently,VOCchemotypingallowednotonlytoidentifyspecies- Wellington,2001).Taxonomicdelineationofthisgenusremains andstrain-specificVOCsbutalsotostudysoilmicrobialactivity complex and leads to over- or under-classified groups. Current and shifts in microbial community compositions (McNeal and approaches for classification of Streptomyces as well as other Herbert, 2009; Müller et al., 2013; Trefz et al., 2013). We prokaryotes rely on genetic and phenotypic traits, mainly on showed that VOC profiling can be used for chemotyping 16S rRNA gene sequences. This molecular marker, however, FrontiersinMicrobiology|www.frontiersin.org 8 October2015|Volume6|Article1081 Cordovezetal. Streptomycesvolatiles:diversityandfunctions FIGURE6|Inhibitionoffungalgrowthafter1and2daysofexposuretoStreptomycesVOCs(A)andgrowthofArabidopsisthalianaseedlingsafter2 weeksofexposuretoStreptomycesVOCs(B).Thecontrolsaredisplayedingreenandisolateswiththestrongestantifungalactivityinred.Barsrepresent standarderrorsofthemeanof3independentbiologicalreplicates.Asterisksindicateastatisticaldifferenceascomparedtocontrols(exposedtomediumonly)using Student’st-Test(p<0.05,n=3).Picturesofantifungalactivityandplantgrowthpromotionweremadeafter3and14daysofexposure,respectively. is not always sufficient to discriminate between closely related isolates inhibited hyphal growth, with Streptomyces strains species and between strains of a given species (Girard et al., W47 and W214 showing the strongest inhibitory effect. Given 2013). We showed that concatenation of atpD, recA, and that these streptomycetes were obtained from a Rhizoctonia- 16S rRNA gene sequences displayed a better phylogenetic suppressive soil suggests that VOCs may contribute to disease delineation of the different streptomycetes than 16S rRNA suppressiveness.Thissuggestionneedstobefurtherinvestigated gene sequences alone, although closely related isolates could in situ but fits well with one of the initial hypotheses of not be distinguished. We revealed that VOC profiling allowed Lockwood(Lockwood,1977)forthepotentialroleofmicrobial discriminationofStreptomycesisolatesthatarephylogenetically VOCs in soil fungistasis. To provide more conclusive proof close but phenotypically different, such as Streptomyces strains of the role of these Streptomyces VOCs in disease suppression W75.5/W126andW47/W214. in the soil ecosystem, specific soil bioassays are needed where ThegenusStreptomycesiswell-knownfortheproductionof the VOC producers and the pathogen are physically separated. several antifungal and antiviral compounds and accounts for However, there are several technical limitations to accomplish 80% of the currently available antibiotic compounds (Watve this. First, the strains used here are rhizospheric bacteria etal.,2001).StreptomycesalsoproducesVOCswhichreducethe that need to be positioned in their ecological context (the incidence and/or the severity of several plant diseases caused rhizosphere) to provide meaningful results. Given the need for by fungi and cause morphological abnormalities in different the localization of the Streptomyces strains in the rhizosphere fungi (Moore-Landecker and Stotzky, 1973; Wan et al., 2008; where also the pathogen colonizes and infects, it has not Boukaew et al., 2013; Wang et al., 2013; Wu et al., 2015). beenpossibleyettophysicallyseparatetheStreptomycesstrains VOCs produced by the streptomycetes tested here exhibited from the fungal pathogen. This is due in part to the prolific antifungal and plant growth promoting properties. Several growth of this particular fungus. The physical separation in FrontiersinMicrobiology|www.frontiersin.org 9 October2015|Volume6|Article1081 Cordovezetal. Streptomycesvolatiles:diversityandfunctions FIGURE7|(A)VOCprofilesofStreptomycesstrainsW47andW214comparedtocontrol(mediumonly).(B)VenndiagramforcommonanduniqueVOCsproduced byStreptomycesstrainsW47andW214.(C)Experimentalset-upforinvitroantifungalactivityassaywithsyntheticVOCs.(D)Invitroantifungalactivitywithsynthetic VOCsat1M[control,methanol,VOC1(methylbutanoate),VOC2(methyl2-methylpentanoate),VOC3(methyl3-methylpentanoate),VOC4(1,3,5-trichloro-2-methoxy benzene),VOC5(3-octanone)].MethanolwasusedtodiluteallVOCs.Barsrepresentstandarderrorsofthemeanof3independentreplicates.Asterisksindicate statisticaldifferencescomparedtocontrolaccordingtoStudent’st-Test(p<0.05,n=3).(E)Fungalgrowthafterexposureto1,3,5-trichloro-2-methoxybenzene.(F) Abundanceof1,3,5-trichloro-2-methoxybenzeneproducedbydifferentStreptomycesisolatesbasedonGC-MSpeakintensities.(G)Nonlinearrelationshipbetween fungalgrowthinhibitionandabundanceof1,3,5-trichloro-2-methoxybenzene. situ is needed to exclude a possible role of mechanisms other Severalstudieshavedescribedantifungalactivitybybacterial than VOCs. An alternative approach would be to generate VOCs, however, few have identified single or blends of site-directed mutants of the Streptomyces strains that do not VOCs responsible for the antifungal activity (Kai et al., 2007; produce one or more of the specific VOCs identified in Wang et al., 2013). For Pseudomonas, six VOCs (cyclohexanal, this study. Comparison of the activity of these mutants with decanal,2-ethyl1-hexanol,nonanal,benzothiazole,anddimethyl their wildtype strains would then more conclusively resolve trisulfide) were found to inhibit mycelial growth and sclerotial the role of specific VOCs in disease suppression in situ. For germination of Sclerotinia sclerotiorum at tested volumes of this alternative approach, however, we have not yet been 100 and 150µl (Fernando et al., 2005). Regarding VOCs able to generate mutants as many environmental Streptomyces produced by Streptomyces species, butanone (methyl vinyl species/strains are not or very difficult to access for genetic ketone) and dimethyl disulfide were described to inhibit the modification. sporegerminationinCladosporiumcladosporioidesandmycelial FrontiersinMicrobiology|www.frontiersin.org 10 October2015|Volume6|Article1081