MICROBIOLOGY LETTERS. Harzianic acid: a novel siderophore from Trichoderma harzianum. Introduction RESEARCH LETTER

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1 RESEARCH LETTER Hrzinic cid: novel siderophore from Trichoderm hrzinum Frncesco Vinle 1, Mrco Nigro 1,2, Krishnpilli Sivsithmprm 3, Gvin Flemtti 4, Emilio L. Ghislerti 4, Michelin Ruocco 1, Rosri Vrlese 2, Roert Mrr 2, Stefni Lnzuise 2, Ahmed Eid 2, Sheridn L. Woo 1,2 & Mtteo Lorito 1,2 1 CNR Istituto per l Protezione delle Pinte (IPP-CNR), Portici, Itly; 2 Diprtimento di Agrri, Universit degli Studi di Npoli Federico II, Portici, Itly; 3 School of Erth nd Environment, The University of Western Austrli, Perth, WA, Austrli; nd 4 School of Chemistry nd Biochemistry, The University of Western Austrli, Perth, WA, Austrli Correspondence: Frncesco Vinle, CNR Istituto per l Protezione delle Pinte (IPP-CNR), Vi Universit 133, Portici, Nples, Itly. Tel.: ; fx: ; e-mil: f.vinle@ipp.cnr.it Received 21 June 2013; revised 16 July 2013; ccepted 28 July Finl version pulished online 10 Septemer DOI: / Editor: Polin Grev Astrct Agriculture-relevnt microorgnisms re considered to produce secondry metolites during processes of competition with other micro- nd mcroorgnisms, symiosis, prsitism or pthogenesis. Mny different strins of the genus Trichoderm, in ddition to direct ctivity ginst phytopthogens, re well-known producers of secondry metolites nd compounds tht sustntilly ffect the metolism of the host plnt. Hrzinic cid is Trichoderm secondry metolite, showing ntifungl nd plnt growth promotion ctivities. This report demonstrtes the ility of this tetrmic cid to ind with good ffinity essentil metls such s Fe 3+, which my represent mechnism of iron soluilistion tht significntly lters nutrient vilility in the soil environment for other microorgnisms nd the host plnt. MICROBIOLOGY LETTERS Keywords secondry metolites; hrzinic cid; iron(iii); Trichoderm; siderophores. Introduction Trichoderm spp. re well-studied filmentous fungi commonly found in the soil community tht re widely mrketed s iopesticides, iofertilisers nd soil mendments, due to their ility to protect plnts, enhnce vegettive growth nd contin pthogen popultions (Vinle et l., 2008). To dte, hundreds of different Trichoderm-sed preprtions re commercilly used to protect nd/or increse the productivity of vrious crops (Lorito et l., 2010). One fctor tht contriutes to the eneficil iologicl ctivities exerted y Trichoderm strins is the wide vriety of secondry metolites tht they re le to produce (Sivsithmprm & Ghislerti, 1998; Reino et l., 2008). Fungl secondry metolites my e considered lrge nd heterogeneous group of smll molecules not directly essentil for growth, ut hving n importnt role in signlling, development nd interction with other orgnisms (Song et l., 2006; Mukherjee et l., 2012). Trichoderm metolites my ct y directly inhiiting the growth of pthogens, or y indirectly triggering the defence system in the host plnt, thus incresing disese resistnce, nd y promoting plnt growth (Vinle et l., 2012). The ccumultion of Trichoderm metolites vries ccording to the species or the strin used nd is relted to the iosynthesis nd iotrnsformtion rtes (Vinle et l., 2009). Siderophores re low-moleculr-weight metolites produced for scvenging iron from the environment nd hve n high ffinity for iron(iii) (Hider & Kong, 2010). Fe 3+ -chelting molecules cn e eneficil to plnts ecuse they cn soluilise the iron which is otherwise unville nd cn suppress the growth of pthogenic microorgnisms y depriving the pthogens of this necessry micronutrient (Leong, 1986). We hve recently purified from the culture filtrte of strin of Trichoderm hrzinum nitrogen heterocyclic compound nmed hrzinic cid (HA) nd demonstrted its growth promotion effect (Vinle et l., 2009). Growth of different fungl pthogens nd cnol seedlings ws shown to e ffected y tretments with HA in concentrtion-dependent mnner, suggesting role of this FEMS Microiol Lett 347 (2013) ª 2013 Federtion of Europen Microiologicl Societies. Pulished y John Wiley & Sons Ltd. All rights reserved

2 124 F. Vinle et l. metolite in plnt growth regultion, s well s in the ntgonism ginst phytopthogenic gents. In this work, we demonstrte tht HA hs Fe 3+ -chelting ctivity. This secondry metolite strongly inds essentil metls such s iron, thus representing previously unrecognised siderophore. Mterils nd methods Microil strin nd culture conditions Trichoderm hrzinum strin M10 ws mintined on potto dextrose gr (PDA; SIGMA, St Louis, MO) slnts t room temperture nd sucultured imonthly. Genomic DNA ws isolted to nlyse riosoml DNA. Using PCR pproch were mplified frgments contining the internl trnscried spcer 1 (ITS1-prtil sequence), the 5.8S riosoml RNA gene nd the internl trnscried spcer 2 (ITS2-complete sequence), using primers SR6R (5 -AAGTAGAAGTCGTAACAAGG-3 ) nd LR1 (5 -GGT TGGTTTCTTTTCCT-3 ). The PCR products were gel electrophoresed for quntifiction nd ssessment of PCR specificity nd finlly sequenced. Sequence nlysis of the internl trnscried spcers (ITS) of the rdna indicted 99% similrity with GenBnk sequences of T. hrzinum confirming the identity of this species. Ten 7-mm dimeter plugs of M10 strin otined from ctively growing mrgins of PDA cultures were inoculted to 5-L conicl flsks contining 1 L of sterile potto dextrose roth (PDB; SIGMA). The sttionry cultures were incuted for 21 dys t 25 C. The cultures were filtered under vcuum through filter pper (Whtmn No. 4, Brentford, UK). Extrction nd isoltion of HA The filtered culture roth (2 L) ws cidified to ph 4 with 5 M HCl nd extrcted exhustively with ethyl cette (EtOAc). The comined orgnic frction ws dried (N 2 SO 4 ) nd evported in vcuo t 35 C. The red residue recovered ws dissolved in CHCl 3 nd extrcted three times with 2 M NOH. HA ws then precipitted with 2 M HCl. The solid ws recovered (135 mg), soluilised nd sujected to RP-18 vcuum chromtogrphy (20 g), eluting with grdient of methnol (MeOH) : H 2 O: CH 3 CN (1 : 8 : 1 to 10 : 0 : 0). After the seprtion, 45 mg of pure HA ws collected. The homogeneity of pure pooled products ws verified y nlyticl reversephse TLC (glss precoted silic gel 60 RP-18 pltes Merck Kieselgel 60 TLC silic gel 60 RP-18 F254s, 0.25 mm) using 3 : 4 : 3 CH 3 CN MeOH H 2 O s eluent (Rf of HA: 0.3). The compounds were detected on TLC pltes using UV light (254 or 366 nm) nd/or y sprying the pltes with 5% (v/v) H 2 SO 4 in EtOH followed y heting t 110 C for 10 min. UV, IR, 1 H NMR, 13 C NMR nd HR-FABMS were identicl to those reported y Sw et l. (1994). ESMS (+) m/z [M 2 + N] +, [M + K] +, [M + N] +, [M + H] +, [M + H C 7 H 18 ] +. 1 H nd 13 C NMR spectr were recorded with Vrin 400 instrument operting t 400 ( 1 H) nd 125 ( 13 C) MHz, using residul nd deuterted solvent peks s reference stndrds. CAS gr plte ssys The method to detect siderophore production ws previously descried y Schwyn & Neilnds (1987). Ornge hlos round the colonies, growth on chrome zurol S (CAS) pltes, re indictive of siderophore ctivity. CAS solution ws lso used for detection of siderophore production in culture filtrte (50 ll of culture ws dded to 950 ll of CAS solution, nd fter reching equilirium, the sornce ws mesured t 630 nm). The CAS ssy ws lso used to test the chelting properties of 10 3 M HA solution in methnol. The CAS ssy ws modified ccording to Milgres et l. (1999) to test the ility of strin M10 to produce iron-inding compounds eventully voiding the growth inhiition cused y the toxicity of the CAS lue gr medium. Petri dishes (10 cm in dimeter) were prepred with the mlt extrct gr (MEA) medium. After solidifiction, the medium ws cut into hlves, one of which ws replced y CAS lue gr. The hlves contining culture medium (MEA) were inoculted with M10 plugs. The pltes were incuted t 25 C for 6 dys. Pseudomons fluorescens strin CHA0 (cteril strin collection of prof A. Zoin University of Nples) ws used s positive control (Murhofer et l., 1994; Yourd et l., 2007). Iron-inding ffinity of HA To mesure the iron-inding ffinity of HA, the method of Kufmnn et l. (2005) ws used with some modifictions. Stock solutions of ferric chloride (10 mm) nd HA (10 mm) were prepred with 4 : 1 MeOH/0.1 M NOAc uffer solution (ph 7.4). Aliquots of oth stock solutions were diluted, nd the sornces of the formed complexes were mesured t 290 nm in triplicte in the presence nd sence of EDTA (10 mm nd sturted solution). LC/MS of HA Fe(III) complex The Fe(III)-inding properties of the HA were investigted y dding 100 ll of Fe(III) chloride solution ª 2013 Federtion of Europen Microiologicl Societies. Pulished y John Wiley & Sons Ltd. All rights reserved FEMS Microiol Lett 347 (2013)

3 Hrzinic cid, T. hrzinum siderophore 125 (10 mm) to 100 ll of 10 mm HA in MeOH. The solution turned red nd ws directly infused into the LC/MS system t 5 ll min 1 using syringe pump. Full scns in the rnge m/z were performed on Bruker 6340 ion trp mss spectrometer equipped with n electrospry ionistion source (ESI) nd operting in the positive ion mode. High-resolution spectr were recorded using Wters Allince e2695 HPLC connected to Wters LCT Premier XE mss spectrometer with n ESI. Smples were injected using the onord injector in 10 ll injection volumes nd eluted with 20% cetonitrile/ wter t flow rte of 0.3 ml min 1 to the time-of-flight mss spectrometer. For the HA Fe(III) complex, positive ESI-HRMS found m/z ([C 19 H 27 NO 6 -FeCl 2 ] + requires ). Plnt growth promotion ssy Tomto (Solnum lycopersicum cv. Rom) seeds were surfce sterilised using 70% EtOH for 2 min, followed y 2% NClO for 2 min, thoroughly wshed with sterile distilled wter, then plced on mgent ox contining hlfstrength Murshige nd Skoog slt (MS) medium (ICN Biomedicls, Irvine, CA) contining 1% gr nd 1.5% sucrose, djusted to ph 5.7 nd vernlised for 2 dys t 4 C in the drk. Medi with iron concentrtions different from the stndrd 50 lm were prepred ccording to Murshige & Skoog (1962), except tht defined mounts of Fe(III) were dded (diluted 1/10 nd 1/100 corresponding to 5 nd 0.5 lm, respectively). Sterile solutions of HA were dded to the sustrte efore the gr solidifiction to hve finl concentrtions of 100, 10 nd 1 lm. Untreted sustrtes were used s controls. Seedlings were grown in growth chmer (16-h photoperiod); the temperture ws mintined t 25 1 C with reltive humidity of 65 75%. Growth of stem height ws mesured dily. Ech tretment consisted of 20 replictes, nd the experiment ws repeted four times. At the end of ech experiment, the whole plnts were dried nd weighed. Dt from the experiments were comined s sttisticl nlysis determined homogeneity of vrince (P 0.05). Plnt tissues ( mg) were ground with liquid N 2, minerlised ccording to Beinert (1978) nd the iron contents were determined using tomic sorption nlysis. Results Purifiction nd identifiction of HA Trichoderm hrzinum M10 ws grown in PDB, nd the culture filtrte ws extrcted with ethyl cette, from which HA ws purified s descried ove. The moleculr mss of this compound, s determined y electrospry ionistion mss spectrometry, ws 365 D, nd its pttern corresponded to tht of 2-hydroxy-2-[4-(1-hydroxy-oct- 2,4-dienylidene)-1-methyl-3,5-dioxo-pyrrolidin-2-ylmethyl]-3-methyl-utyric cid (Fig. 1) descried y Sw et l. (1994) nd Vinle et l. (2009). The HA structure ws confirmed y nucler mgnetic resonnce. Iron(III)-inding ctivity of M10 nd HA The ssy descried y Schwyn & Neilnds (1987) nd Milgres et l. (1999) ws used for the detection of siderophores relesed in the sustrte y M10 strin. The fungus grew on CAS lue gr nd the iron(iii)-chelting compounds, excreted y the microorgnism nd diffused through the medium producing colour chnge from lue to ornge. Purified HA decolorises CAS lue gr, indicting tht it could form complex with Fe(III) (Fig. 2). While the free compound in queous solution is ple ornge, ddition of Fe(III) results in the ppernce of red colour, indicting tht n iron complex is formed. Fig. 1. Chrcteriztion of HA Fe(III) complex y LC/MS. () HA nd its mss spectrum; () mss spectrum of HA Fe(III). FEMS Microiol Lett 347 (2013) ª 2013 Federtion of Europen Microiologicl Societies. Pulished y John Wiley & Sons Ltd. All rights reserved

4 126 F. Vinle et l. Chrcteristion of Fe(III) HA complex To investigte the formed Fe(III) HA complexes, experiments were crried out dding FeCl 3 to solution of HA. The uncomplexed HA molecule is detected s [M + H] + (m/z 366.2), [M + N] + (m/z 388.2), [M + K] + (m/z 404.2) nd [M 2 + N] + (m/z 753.3; Fig. 1). After dding Fe(III), the spectrum showed dditionl signls t m/z nd m/z corresponding to two 1 : 1 Fe(III)- contining complexes, which were ssigned the following signls sed on the chloride isotope pttern, [M H + Fe (III) + Cl 2 + H] + (m/z 491.1) or [M 2H + Fe (III) + Cl + H] + (m/z 455.1), s determined y isotopic distriution. The resulting mss spectrum is depicted in Fig. 1. High-resolution spectrum of the Fe(III) HAcomplex showed signls t m/z ([C 19 H 27 NO 6 FeCl 2 ] + requires ), confirming the 1 : 1 HA Fe complex. The pprent inding constnt (K d, pp ) ws determined to mesure the ffinity of HA for iron (III). These experiments were performed using previously descried protocol sed on the competition etween HA nd EDTA for iron nd the detection of the complexes y mesuring the chrcteristic sorption (Wng et l., 2002). The loss of Fe(III) HA sornce t 340 nm upon ddition of EDTA ws used to clculte the () () (c) Fig. 2. CAS-lue gr plte ssys: () plte mended with 10 ll of HA 10 3 M; () plte inoculted with Trichoderm hrzinum M10; (c) plte inoculted with Pseudomons fluorescens strin CHA0 (used s positive control). equilirium constnt, presuming the formtion of 1 : 1 Fe HAcomplex, ccording to the following eqution: EDTA Fe þ HA ¼ EDTA þ Fe HA K eq ¼½EDTAŠ½Fe HAŠ=½HAŠ½EDTA FeŠ ¼ K d;edta =K d;ha : Using the known ffinity of EDTA for Fe(III) ( M), we were le to determine the reltive ffinity (K d,pp ) of HA for Fe 3+ to e M. Plnt growth promotion The ctivity of HA s plnt growth promoter ws evluted y nlysing the effect on seed germintion nd mesuring lengths, fresh or dry weights of tomto seedlings treted with metolite solutions s compred to controls. Seed germintion ws significntly ffected y HA pplictions. In prticulr, when 100 or 10 lm HA solutions were used, the percentges of germinted seeds were 4 5 times higher thn in control (Tle 1). Shoot nd root growth of tomto seedlings ws enhnced y tretments with HA used t different concentrtions. The highest increse of shoot nd root length (+76% nd +66%, respectively) ws registered using 10 lm of HA compred with control (Fig. 3). A promotion effect on oth fresh nd dry plnt weights ws oserved using HA t 10 nd 1 lm. Conversely, higher concentrtion of HA (100 lm) did not significntly ffect plnt weights or seedling lengths compred with the untreted control (Tle 1, Fig. 3). To further chrcterise the role of HA in iron vilility, plnts were supplemented with low concentrtions of iron (5 nd 0.5 lm). Root length of tomto seedlings grown in medi contining the lowest iron concentrtion ws improved y tretments with oth 10 nd 1 lm HA; conversely, no significnt effect ws detected in shoot growth (Fig. 4) or in plnt weight in comprison with control (dt not shown). However, in Tle 1. Growth promotion of tomto seedlings treted with HA t different concentrtions fresh nd dry weights 12 dys fter tretment; germintion 2 dys fter tretment Tretment Fresh weight (mg) SD Dry weight (mg) SD %of Germintion SD Control HA 100 lm , HA 10 lm HA 1 lm c 1.9 Vlues re mens of 10 replictes. SD: stndrd devition. Vlues with the sme letter do not differ significntly (P < 0.05). ª 2013 Federtion of Europen Microiologicl Societies. Pulished y John Wiley & Sons Ltd. All rights reserved FEMS Microiol Lett 347 (2013)

5 Hrzinic cid, T. hrzinum siderophore 127 these growing conditions, iron levels incresed in plnts treted with HA, prticulrly when 1 lm HA ws used (Fig. 4). Discussion The interction of Trichoderm spp. with plnts confers severl enefits for the ssocited host tht include (1) the suppression of phytopthogens using direct ntgonistic mechnisms (i.e. ntiiosis, mycoprsitism, competition for nutrient nd spce); (2) plnt growth promotion; (3) enhnced nutrient vilility nd uptke nd (4) induction of plnt host resistnce (Hrmn et l., 2004). Length (cm) Shoot Root, Control HA 100 μm HA 10 μm HA 1 μm Tretments Fig. 3. Growth promotion of tomto seedlings treted with 100, 10 or 1 lm HA, mesured s shoot nd root lengths 12 dys fter tretment. Vlues re mens of 20 replictes. Brs: Stndrd devition. Vlues with the sme letter do not differ significntly (P < 0.05). In ddition, some Trichoderm strins produce compounds tht cn cuse sustntil chnges in the metolism of the host plnt. The involvement of secondry metolites in the ility of Trichoderm spp. to ctivte plnt defence mechnisms nd regulte plnt growth hs een investigted (Vinle et l., 2008). HA is one of these nturl products tht shows ntifungl nd plnt growth promoting ctivities (Vinle et l., 2009). In the present study, we investigted the Fe +3 -chelting properties of HA. HA, isolted y RP-18 vcuum chromtogrphy from 2 M NOH 2 M extrct, showed the sme 1 H nd 13 C prmeters s those previously reported for 2-hydroxy-2- [4-(1-hydroxy-oct-2,4-dienylidene)-1-methyl-3,5-dioxopyrrolidin-2-ylmethyl]-3-methyl-utyric cid, compound elonging to the chemicl clss of tetrmic cids. The nturlly occurring tetrmic cid derivtives hve ttrcted significnt ttention ecuse these metolites hve wide distriution nd hve een found to disply remrkle diversity of iologicl ctivities plying significnt role in ecologicl interctions. These ctivities my e enhnced significntly through the cheltion of the tetrmic cid nucleus with metl ions (importnt for trnsport cross memrnes in iologicl tissues). It ws found tht in some cses the metl complexes otined reveled higher iologicl ctivity thn their lignds (Ghislerti, 2003; Schoert & Schlenk, 2008; Athnsellis et l., 2010). Both the living microorgnism nd the isolted metolite HA when tested in the CAS lue gr pltes cused colour chnge of the sustrte from lue to ornge indicting tht the iron(iii)-inding properties of the fungus is relted lso to the production of the tetrmic Length (cm) () Root length Shoot length c d Iron in plnt (100 μg Fe g 1 DW) 0 Control HA 10 um HA 1 um Control HA 10 um HA 1 um Tretments Tretments () c Fig. 4. Plnt growth promotion ctivity of HA in iron deficient conditions. () Growth of tomto seedlings supplemented with low concentrtion of iron (0.5 lm) nd treted with 10 or 1 lm HA, mesured s shoot nd root lengths, 12 dys fter tretments. () Anlysis of iron content in tomto seedlings supplemented with low concentrtion of iron (0.5 lm) nd treted with 10 or 1 lm HA, 12 dys fter tretments. Vlues re mens of 20 replictes. Brs: Stndrd devition. Vlues with the sme letter do not differ significntly (P < 0.05). FEMS Microiol Lett 347 (2013) ª 2013 Federtion of Europen Microiologicl Societies. Pulished y John Wiley & Sons Ltd. All rights reserved

6 128 F. Vinle et l. Tle 2. Affinity constnts of cheltors for Fe(III) Compound EDTA Desferrioxmine Pyoverdin Pyochelin HPD HA K d (M) registered). This finding supports the hypothesis tht HA ctively influences the growth of Trichodermcolonised plnts. The isoltion nd ppliction of ioctive compounds produced y eneficil microorgnisms responsile for the desired positive effects on plnts my e promising lterntive to the use of living ntgonists. cid derivtive. Moreover, the LC MS nlysis of HA Fe +3 solution showed dditionl signls t nd m/z corresponding to 1 : 1 chloride-contining complex, [M H + Fe(III) + Cl 2 + H] + (m/z 491.1) or [M 2H + Fe(III) + Cl + H] + (m/z 455.1). As chloride is coordinting lignd for iron, it is possile tht the chloride nion is directly ound to the metl (Cudle et l., 1994). The vlue of K d of Fe HA complex ( M) my e directly compred with tht of other cheltors showing 1 : 1 Fe : lignd stoichiometry, such s desferrioxmine, EDTA, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (HPD), pyoverdin nd pyochelin (Kufmnn et l., 2005). As shown in Tle 2, HA hs lower ffinity to Fe(III) thn desferrioxmine, pyoverdin nd HPD, while it hs stronger ffinity for iron (III) thn EDTA nd pyochelin. This dt suggest tht HA could compete for ville iron in solution nd provide method for iron soluilistion. Microil siderophores re used s iron-chelting gents tht cn regulte the vilility of iron in the plnt rhizosphere. It hs een ssumed tht competition for iron in the rhizosphere is controlled y the ffinity of the siderophore for iron. The importnt fctors, which prticipte, re concentrtion of vrious types of siderophores, kinetics of exchnge nd vilility of Fe complexes to microorgnisms s well s plnts. Siderophores produced y eneficil gents my hve importnt effects on oth microil nd plnt nutrition. Fe 3+ -siderophore complexes cn e recognised nd tken up y severl plnt species, nd this process is considered crucil for plnt iron uptke, prticulrly in clcreous soils (Shrm et l., 2003). Trichoderm metolites my help the plnt to withstnd pthogens y oth promoting the growth nd development of root nd shoot systems, nd stimulting the defence mechnisms (Vinle et l., 2008). In the present work, we confirm possile involvement of HA in plnt growth regultion proly ecuse of its Fe(III)-inding ctivity. In fct, HA ffected the germintion of tomto seeds nd improved the growth of the seedlings even in iron-deficient conditions (increment of iron concentrtion into the plnts ws lso Acknowledgements The uthors thnk Dvide Stellitno, Gelsomin Mgniello nd Alerto Pscle for their technicl ssistnce. The uthors thnk Antonio De Mrtino for the iron concentrtion mesurements. Work y the uthors F.V. nd M.R. ws supported y dedicted grnt from the Itlin Ministry of Economy nd Finnce to the Ntionl Reserch Council for the project Innovzione e Sviluppo del Mezzogiorno Conoscenze Integrte per Sosteniilit ed Innovzione del Mde in Itly Agrolimentre Legge n. 191/2009. Work y the uthors M.N., R.V., R.M., S.L., A.E., S.L.W. nd M.L. hs een supported y the following projects: S.Re.V.Pr.O., CA.SVI.PR.OLI. nd MI.P.RE.VEGE. ; PRIN MIUR 2008 prot. 2008SNPNC2 prot. 2008WKPAWW. The uthors lso cknowledge the fcilities, scientific nd technicl ssistnce of the Centre for Microscopy, Chrcteristion & Anlysis t the University of Western Austrli. References Athnsellis G, Igglessi-Mrkopoulou O & Mrkopoulos J (2010) Tetrmic nd tetronic cids s scffolds in ioinorgnic nd ioorgnic chemistry. Bioinorg Chem Appl 2010: Beinert H (1978) Micro methods for the quntittive determintion of iron nd copper in iologicl mteril. Methods Enzymol 54: Cudle MT, Stevens RD & Crumliss AL (1994) Electrospry mss spectrometry study of 1 : 1 ferric dihydroxmtes. Inorg Chem 33: Ghislerti EL (2003) Bioctive tetrmic cid metolites. Studies in Nturl Products Chemistry, Vol. 28/1 (Att-ur Rhmn, ed.), pp Elsevier, Amsterdm. Hrmn GE, Howell CR, Vitero A, Chet I & Lorito M (2004) Trichoderm species opportunistic, virulent plnt symionts. Nt Rev Microiol 2: Hider RC & Kong X (2010) Chemistry nd iology of siderophores. Nt Prod Rep 27: Kufmnn GF, Srtorio R, Lee SH, Rogers CJ, Meijler MM, Moyss JA, Clphm B, Brogn AP, Dickerson TJ & Jnd KD (2005) Revisiting quorum sensing: discovery of dditionl chemicl nd iologicl functions for 3-oxo-N-cylhomoserine lctones. P Ntl Acd Sci USA 102: ª 2013 Federtion of Europen Microiologicl Societies. Pulished y John Wiley & Sons Ltd. All rights reserved FEMS Microiol Lett 347 (2013)

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