Cyclosporine A from a nonpathogenic Fusarium oxysporum suppressing Sclerotinia sclerotiorum

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1 Journl of Applied Microbiology ISS RIGIAL ARTICLE Cyclosporine A from nonpthogenic Fusrium oxysporum suppressing Sclerotini sclerotiorum M.A. Rodríguez 1, G. Cbrer 2 nd A. Godes 1 1 Deprtmento de Biodiversidd y Biologí Experimentl, Fcultd de Ciencis Excts y turles, Universidd de Buenos Aires, C1428 EGA Buenos Aires, Argentin 2 Deprtmento de Químic rgánic, Fcultd de Ciencis Excts y turles, Universidd de Buenos Aires Buenos Aires, Argentin Keywords ntifungl ctivity, biocontrol, cyclosporine A, Fusrium oxysporum, Sclerotini sclerotiorum, soyben. Correspondence Mrí Alejndr Rodríguez, Deprtmento de Biodiversidd y Biologí Experimentl, Fcultd de Ciencis Excts y turles, Universidd de Buenos Aires, Intendente Güirldes 216, Ciudd Universitri, Pb. II, C1428EGA, Buenos Aires, Argentin. E-mil: rodrig@bg.fcen.ub.r 25/31: received 23 Mrch 25, revised 1 July 25 nd ccepted 7 July 25 doi:1.1111/j x Abstrct Aims: To evlute the ntgonistic ctivity of Fusrium oxysporum nonpthogenic fungl strin S6 ginst the phytopthogenic fungus Sclerotini sclerotiorum nd to identify the ntifungl compounds involved. Methods nd Results: The ntgonistic ctivity of Fusrium oxysporum strin S6 ws determined in vitro by dul cultures. The metbolite responsible for the ctivity ws isolted by chromtogrphic techniques, purified nd identified by spectroscopic methods s cyclosporine A. The ntifungl ctivity ginst the pthogen ws correlted with the presence of this metbolite by dilution ssy nd then quntified. Cyclosporine A cused both growth inhibition nd suppression of scleroti formtion. In greenhouse ssy, significnt increse in the number of surviving soyben (Glycine mx) plnts ws observed when S. sclerotiorum nd F. oxysporum (S6) were inoculted together when compred with plnts inoculted with S. sclerotiorum lone. Conclusion: Fusrium oxysporum (S6) my be good fungl biologicl control gent for S. sclerotiorum nd cyclosporine A is the responsible metbolite involved in its ntgonistic ctivity in vitro. Significnce nd Impct of the Study: Cyclosporine A hs not been previously described s n inhibitor of S. sclerotiorum. Its minimum inhibitory concentrtion (MIC) of Æ1 lg disc )1 mkes it suitble to use s biofungicide. In vivo experiments showed tht F. oxysporum (S6) is good cndidte for the biocontrol of S. sclerotiorum in soyben. Introduction Sclerotini sclerotiorum is soil-borne pthogen with wide host rnge including mny of economicl importnce (Bolnd nd Hll 1994). This pthogenic fungus produces significnt losses in soyben (Glycine mx), sunflower (Helinthus nnuus) nd lettuce (Lctuc stiv) crops (Zhou nd Bolnd 1998). Sclerotini species produce myceli nd scleroti (nmorph) nd potheci, sci nd scospores (teleomorph) (Kohn 1979). Scleroti re vible for more thn 5 yers (Le Tourneu 1979) nd my germinte by producing either myceli (myceliogenic germintion) or potheci (crpogenic germintion). During myceliogenic germintion, the hyphe grow towrds host roots nd hypocotyls cusing shoot wilt (Adms nd Ayers 1979). Severl methods of disese control such s grochemicls, use of resistnt cultivrs nd culturl prctices hve been used. However, none of them hs been completely successful (Adms nd Ayers 1979; Whipps nd Budge 199). Biocontrol my be n environmentlly friendly nd efficient lterntive to mnge pthogenic fungi (Hung 1992; Whipps 21). Some soils, clled suppressive soils, inhibit fungl myceli nd/or spore germintion, phenomenon known s soil fungistsis. In fct, there re severl diseses in which the pthogen cnnot develop nturlly becuse of the soil. Previous reports hve determined tht micro-orgnisms hve n importnt role s cusl gents of fungistsis, with their ction medited either by vilble crbon limittion or by production of ª 26 The Society for Applied Microbiology, Journl of Applied Microbiology 1 (26)

2 Cyclosporine A from nonpthogenic F. oxysporum suppressing S. sclerotiorum M.A. Rodríguez et l. ntifungl compounds (de Boer et l. 23). For this reson, suppressive soils re n importnt source of biocontrol gents. Severl studies hve used this nturl dvntge to develop effective biologicl control strtegies (Butt et l. 21; Whipps 21). Fusrium oxysporum is one of the most distributed species in soil-borne fungi communities, prticulrly in plnt rhizospheres (Gordon nd Mrtyn 1997), where pthogenic nd nonpthogenic strins my be found. Some strins of F. oxysporum hve shown the bility to suppress the growth of severl fungl plnt pthogens such s Phytophtothor erythroseptic nd Pythium ultimum (Prk 1963; Benhmou et l. 22) nd to ffect the germintion of S. sclerotiorum scleroti (Zzzerini nd Tosi 1985). ther species of Fusrium hve been evluted both ginst P. ultimum (Ishimoto et l. 24) nd s potentil biocontrol gent ginst S. sclerotiorum in the rhizosphere (Zhou nd Bolnd 1998). Little is known bout the ntgonism relted with ntifungl metbolite production by nonpthogenic F. oxysporum (Frvel et l. 23). As prt of our continued interest in the studies on biocontrol fungl gents ginst the fungl plnt pthogen S. sclerotiorum, suppressive soils from fields cropped with soyben were investigted. A nonpthogenic strin of F. oxysporum with ntgonist ctivity ws isolted. The gol of this study ws to purify nd identify the compound responsible for tht ctivity. two different culture medi: mlt extrct gr (MEA) nd potto dextrose gr (PDA). Two ssys were conducted: F. oxysporum (S6) ws inoculted 48 h before nd t the sme time s the pthogen. The first ssy detected the presence of ntifungl metbolites, wheres the second ssessed the effectiveness of its ntgonistic cpcity s the ntgonist strin showed growth rte less thn tht of the pthogen. In both tests two 4-mm dimeter plugs were inoculted 4Æ5 cm prt in 9 cm dimeter Petri dishes. In ssys in which plugs were used, they were excised from the leding edge of n ctively growing colony of ech fungus (Whipps 1987). Control dishes were inoculted with the pthogen strin on ech of the medi being ssessed. A fctoril design ws used with four tretments per ssy: S. sclerotiorum vs F. oxysporum (S6) in MEA, S. sclerotiorum lone in MEA, S. sclerotiorum vs F. oxysporum (S6) in PDA, S. sclerotiorum lone in PDA. Three replictes were included for ech confronttion. All Petri dishes were kept t 25 C in drkness nd the experiment ws repeted twice. The width of the inhibition zone ws determined nd the percentge of rdil growth inhibition (%RGI) ws clculted s: %RGI ¼½ðr c r d Þ=ðr c 1ÞŠ where r c is the control pthogen colony rdius; r d is the pthogen colony rdius in the dul culture (Melgrejo et l. 1985). Mterils nd methods Fungl strins An rgiudol vertic soil [totl C: 3Æ2, ph 5Æ95 (1 : 2Æ5 V:V), : Æ26, P extr. mg kg )1 24Æ15] of soyben field (Slto, Buenos Aires, Argentin) with ptches of suppressive nd nonsuppressive soils ginst Sclerotini wilt, ws used for fungl isoltion. Sclerotini sclerotiorum strin (BAFC 225) ws isolted from scleroti of infected soyben plnts in the nonsuppressive soil. The ntgonistic strin ws isolted from the suppressive soil with soil prticle wshing method (Prkinson 1994) nd ws selected ccording to its ntgonistic behviour in dul cultures confronting S. sclerotiorum (BAFC 225). By using culture chrcteristics nd spore morphology, the ntgonistic strin ws identified s F. oxysporum (Booth 1971; elson et l. 1983). Dul cultures Sclerotini sclerotiorum (BAFC 225) nd F. oxysporum (S6) were confronted in dul cultures in Petri dishes, on bservtions by light microscopy After 1 dys of inocultion, squres of 1 mm 1 mm from the pthogen colonies in contct with the inhibition zone in MEA were removed. Ech squre ws stined with cotton blue nd observed by light microscopy. Assessment of Fusrium oxysporum (S6) s mycoprsite on scleroti Twenty-five dys old scleroti of 2 3 mm dimeter, obtined from culture on MEA, were employed in two different experiments for mycoprsitism evlution: Scleroti spore inocultion Forty scleroti were submerged in suspension of F. oxysporum (S6) spores in sterile wter in reltion of Æ sp ml )1 scleroti )1 for 5 min. Hlf of the scleroti (2) were plnted in Petri dish contining sterile snd nd the other hlf in Petri dish contining sterile soil. For control tretments, only sterilized distilled wter ws used (Whipps nd Budge 199). We performed four tretments with two replictes ech: (i) inoculted scleroti in snd, (ii) control scleroti in 576 ª 26 The Society for Applied Microbiology, Journl of Applied Microbiology 1 (26)

3 M.A. Rodríguez et l. Cyclosporine A from nonpthogenic F. oxysporum suppressing S. sclerotiorum snd, (iii) inoculted scleroti in soil nd (iv) control scleroti in soil. Substrtes were moistened up to field cpcity with sterile wter. All Petri dishes were kept t 25 C for 28 dys in drkness. Ech ssy ws performed twice. From ech Petri dish the following vlues were recorded percentge of: recovered scleroti (%RS), colonized scleroti (%CS), infected scleroti by the ntgonist (%IS) nd vible scleroti (%VS). Ech prmeter ws clculted s: %RS ¼ n rs/(n ps 1); %CS ¼ n cs/(n ps 1); %IS ¼ n is/(n ts 1); %VS ¼ n vs/(n ts 1),where n rs is the number of recovered scleroti; n ps is the number of plnted scleroti; n cs is the number of colonized scleroti; n is is the number of infected scleroti; n ts is the number of scleroti rndomly tken from ech Petri dish nd n vs is the number of vible scleroti. For the lst two vlues (%IS nd %VS), five scleroti were rndomly tken from ech Petri dish, rinsed with wter, superficilly sterilized with 7% sodium hypochlorite for 3 min nd finlly wshed with sterile distilled wter. They were dried on sterile filter pper nd plnted on MEA with ntibiotics (streptomycin Æ5% nd chlorotetrcyclin Æ25%). Effect of the Fusrium oxysporum colony on the scleroti of Sclerotini sclerotiorum A 4-mm dimeter plug of the ntgonist ws centrlly inoculted in 6-cm dimeter Petri dish contining MEA. After 7 dys, eight scleroti were plced equidistnt from ech other round 2 cm of the centre of the colony. For the control tretment, eight scleroti were kept in sterile Petri dishes with sterile filter pper. All Petri dishes were kept t 25 C in drkness. Ech procedure ws performed in triplicte. Three hrvesting conditions were considered: 15, 3 nd 45 dys fter scleroti incorportion, employing different Petri dishes for ech tretment. Three scleroti from ech Petri dish were rndomly smpled fter 15 nd 3 dys, nd two of them, fter 45 dys. Percentge of colonized scleroti (%CS), infected scleroti by F. oxysporum (S6) (%IS) nd vible scleroti (%VS) were recorded. They were clculted s in ). For %IS nd %VS, hrvested scleroti were rinsed nd sterilized s bove. A fctoril design ws employed. Production of nonvoltile metbolites Petri dishes contining MEA were covered with boiled sterilized cellophne (5 mm dimeter). A plug (4 mm dimeter) from F. oxysporum (S6) colony ws centrlly inoculted. The pltes were incubted t 25 C in drkness. After 3 dys, the cellophne nd the colony were removed. A 4-mm plug of pthogen, excised from 4-dy-old culture, ws plced t the centre of the plte. The sme experiment ws repeted but without inocultion of F. oxysporum (S6) s control. A fctoril design ws used with two tretments: pthogen growing with nd without the presence of ntgonist exudtes in MEA. The pltes were incubted t 25 C for 15 dys in drkness, nd the dimeters of the pthogen colonies were mesured every dy (Whipps 1987). Three replictes were mde for ech tretment, nd the experiment ws repeted twice. For fungicidl or fungisttic ctivity determintion, the ssy ws repeted gin, but the pltes were incubted t 25 C only for 4 dys fter S. sclerotiorum plugs were plced onto the plte. Then, S. sclerotiorum inocul were re-inoculted onto MEA nd the percentge of vible plugs ws determined (Dennis nd Webster 1971; Jckson et l. 1991). This procedure ws mde in triplicte nd repeted twice. Antifungl ctivity during the ntgonist growth The ntgonist strin F. oxysporum (S6) ws cultured in mlt extrct broth (MEB) under sttic condition t 25 C nd the efficcy of nonvoltile metbolites present in the medi ws studied (Fuhrmnn 1994). A 4-mm plug of F. oxysporum (S6), tken from the growing edge of MEA culture ws used to inoculte 5 ml Erlenmeyer flsks contining 2 ml of MEB. Time of incubtion ws determined to relte ntifungl ctivity of exudtes nd ntgonistic strin growth, by mesuring mycelium dry weight t 1, 3, 5, 7 nd 1 dys fter inocultion. Individul 2 ml cultures were vcuum-filtered (Whtmn o. 1 filter pper) nd the mycelium ws oven-dried t 7 C for 48 h. Ech smple time ws mde in duplicte. Petri dishes were prepred with the broth (filtered by Millipore filter Æ2 lm) incorported into fresh MEA (1% concentrtion v/v). A fctoril design ws employed with two tretments: colony growing with or without F. oxysporum (S6) exudtes corresponding to ech hrvesting time. The experiment ws performed in duplicte. Control dishes were prepred by using filtered culture medium (MEB t 1% concentrtion in MEA). A 4-mm pthogen plug ws centrlly inoculted in ech Petri dish nd incubted t 25 C. After 4 dys of incubtion, the percentge of pthogen colony growth inhibition (%GI) ws clculted s: %GI ¼ [(D 1 ) D 2 ) 1]/D 1, where D 1 is the dimeter of control pthogen colony nd D 2 is the dimeter of the pthogen colony when the ntgonist exudtes were dded. Twenty dys fter the inocultion, the number of scleroti nd dry weight per scleroti were determined for the pthogen colony growing in both tretments. Also, morphologicl chnges in the colony were described. ª 26 The Society for Applied Microbiology, Journl of Applied Microbiology 1 (26)

4 Cyclosporine A from nonpthogenic F. oxysporum suppressing S. sclerotiorum M.A. Rodríguez et l. Fermenttion A 5-mm plug of the F. oxysporum colony on MEA ws used to inoculte 25 ml Erlenmeyer flsks contining 1 ml of MEB. After week, this culture ws employed to inoculte 4 l Erlenmeyer flsk contining 1 l of MEB. Incubtion ws crried out t 25 C for 21 dys under sttic conditions. 1 H nd 13 C MR spectr were recorded on Bruker AM-5 instrument (Bruker, Billeric, MA, USA) t 5Æ1 MHz for 1 H nd t 125Æ13 MHz for 13 C MR. pticl rottions were mesured on Perkin Elmer 343 polrimeter (Perkin Elmer, Wellesley, MA, USA). FAB + /MS ws recorded t the Wshington University Resource for Biomedicl nd Bio-orgnic Mss Spectrometry. Deprtment of Chemistry, St. Louis, M. Extrction nd isoltion of the ctive compound The culture (1 l) ws filtered nd the filtrte prtitioned with EtAc. The myceli ws wshed with distilled wter nd extrcted with EtH nd EtAc. Both orgnic extrct medi nd myceli were evported to dryness. The myceli extrct ws vcuum-chromtogrphed on reversed phse silic gel by using H 2 nd mixtures of H 2 -MeH of incresing polrity. The ctive frction ws further chromtogrphed on HPLC [Column: YMC C 18, 5 lm, 22Æ5 2Æ5 cm; Elunt: MeH-H 2 85 : 15 (v/v); RI nd UV (215 nm) detection], yielding pure ctive cyclosporine A (6 mg). The medium extrct ws treted in the sme wy to yield cyclosporine A (5 mg). Cyclosporine A. [ D ] ¼ )192 (MeH, c ¼ Æ113; )189 (Trber et l. 1987)) FAB + MS (mtrix: 3-BA): m/z 122 (M + H) +. 1 H MR (CDCl 3 ): 1-MeBmt: d 5Æ46 (H-2), 5Æ34 (H-6, 7), 3Æ8 (H-3), 3Æ5 (CH 3 ), 2Æ39 nd 1Æ67 (H-5), 1Æ63 (H-4, 8), Æ72 (H-9); 2-Abu: d 7Æ93 (H), 5Æ3 (H-2), 1Æ69 (H-3), Æ86 (H-4); 3-Sr: d 4Æ73 (H-2), 3Æ39 (CH 3 ), 3Æ2 (H-2); 4-MeLeu: d 5Æ33 (H-2), 3Æ1 (CH 3 ), 2Æ nd 1Æ6 (H-3), 1Æ43 (H-4), Æ94 (H- 5), Æ88 (H-6); 5-Vl: d 7Æ46 (H), 4Æ66 (H-2), 2Æ41 (H- 3), 1Æ6 (H-4), Æ89 (H-5); 6-MeLeu: d 4Æ99 (H-2), 3Æ25 (CH 3 ), 2Æ5 (H-3), 1Æ75 (H-4), 1Æ4 (H-3), Æ93 (H-5); Æ84 (H-6); 7-Al: d 7Æ64 (H), 4Æ51 (H-2), 1Æ35 (H-3); 8-Al: d 7Æ16 (H), 4Æ83 (H-2), 1Æ26 (H-3); 9-MeLeu: d 5Æ7 (H-2), 3Æ11 (CH 3 ), 2Æ11 (H-3), 1Æ32 (H-4), 1Æ27 (H-3), Æ96 (H-5), Æ88 (H-6); 1-MeLeu: d 5Æ7 (H-2), 2Æ7 (CH 3 ), 2Æ9 (H-3), 1Æ49 (H-4), 1Æ25 (H-3), 1Æ2 (H-5), 1Æ1 (H-6); 11-MeVl: d 5Æ15 (H-2), 2Æ71(CH 3 ), 2Æ15 (H-3), 1Æ (H-4), Æ85 (H-5). 13 C MR (CDCl 3 ): MeBmt: d 129Æ6 nd 126Æ2 (C-6,7), 74Æ7 (C-3), 58Æ8 (C-2), 36Æ (C-4), 35Æ6 (C-5), 17Æ9 (C-8), 16Æ7 (C-9); Abu: d 48Æ8 (C-2), 24Æ9 (C-3), 9Æ8 (C-4); Sr: d 5Æ3 (C-2); MeLeu: d 55Æ5 (C-2), 36Æ (C-3), 24Æ8 (C-4), 23Æ4 (C-5), 21Æ1 (C-6); MeVl: d 48Æ3 (C-2), 29Æ (C-3), 2Æ2 (C-5), 18Æ7 (C-4); Vl: d 55Æ4 (C-2), 31Æ1 (C-3), 19Æ8 (C-4), 18Æ4 (C-5); Me- Leu: d 55Æ3 (C-2), 37Æ4 (C-3), 25Æ3 (C-4), 23Æ8 (C-5), 21Æ9 (C-6); Al: d 48Æ6 (C-2), 15Æ9 (C-3); Al: d 45Æ1 (C-2), 18Æ1 (C-3); MeLeu: d 48Æ3 (C-2), 39Æ (C-3), 24Æ6 (C-4), 23Æ6 (C-5), 21Æ8 (C-6); MeLeu: d 57Æ5 (C-2), 4Æ7 (C-3), 24Æ5 (C-4), 23Æ8 (C-5), 23Æ3 (C-6); C: 173Æ7, 173Æ6, 173Æ5, 173Æ4, 171Æ6, 171Æ1 ( 2), 17Æ4, 17Æ3, 17Æ1, 17Æ. Antifungl ctivity of the frctions nd MIC determintion The ntifungl ctivity for bioguided frctiontion ws determined by the gr diffusion method by using 1 lg of smple per disc ginst S. sclerotiorum (Strobel et l. 1999). After isoltion nd purifiction of the metbolite, the minimum inhibitory concentrtion (MIC) ws determined by using the sme method. Different concentrtions of the metbolite were employed: Æ5, Æ1, Æ2, Æ5, 1Æ, 5Æ, 1Æ nd 2Æ lg of smple per disc. MIC ws determined s the minimum ssyed concentrtion t which zone of inhibition ws obtined. Cyclosporine A quntifiction Fusrium oxysporum (S6) strin ws cultured s bove; plug ws incorported in 5-ml flsk with 2 ml MEB. The flsks were incubted in sttic conditions t 25 C in the drkness. A dily hrvest of two flsks ws mde. Every smple ws filtered, extrcted nd ph nd dry weight mycelium were mesured. The medium ws subjected to n SPE crtridge (C18, 6 ml), wshed with H 2 (1 ml) nd eluted with MeH (1 ml). The mycelium ws extrcted with EtH (2 ml) nd treted with ultrsound for 2 min. After 24 h it ws vcuum-filtered. The ctive orgnic frctions (frction eluted with MeH from medium f1- nd the mycelium extrct f2-) were dried nd weighed. Then, 2 ll of MeH : DMS 1 : 1 nd 1 : 3 ws dded to f1 nd f2, respectively. F2 ws filtered through n inorgnic membrne filter (Æ2 lm, Anotop, Whtmn). Both medium nd mycelium smples were nlysed by HPLC to determine cyclosporine A concentrtion. Concentrtions were mesured by reversed phse HPLC (Pinncle II, C8, 5 lm, 15 4Æ6 mm, Restek) in liner grdient (1 ml min )1 flow rte) with MeH : H 2 (3:7)toMeH 1% in 15 min monitoring the bsorbnce t 22 nm. A Gilson chromtogrph with Rheodyne injector nd diode rry detector ws employed. Biocontrol cpcity in greenhouse experiment A greenhouse experiment ws conducted by using Asgrow 549 soyben seeds. The F. oxysporum (S6) inoculum ws 578 ª 26 The Society for Applied Microbiology, Journl of Applied Microbiology 1 (26)

5 M.A. Rodríguez et l. Cyclosporine A from nonpthogenic F. oxysporum suppressing S. sclerotiorum dded to the stem-psteurized soil s mss of mycelium nd spores growing on boiled rice t concentrtion of 1Æ3 1 6 CFU g )1 (colony-forming units per grm of soil: CFU g )1 ). Pregerminted seeds were plnted in 25- ml pots contining 2 ml of stem-psteurized soil inoculted with the ntgonist strin. After 3 dys, the plnts nd the inoculted soil were trnsferred to 6-ml pots contining 3 ml of inoculted soil with S. sclerotiorum inoculum (concentrtion: 15%, w/v), contining pthogen myceli nd scleroti. This inoculum ws produced in sterile polythene bgs contining rice : brn : wter (2 : 2 : 1; V : V : V) s substrte inoculted with 5-mm pthogen plugs (six per 35 g of substrte) nd incubted for 2 dys t C in the drkness. A completely rndomized fctoril design ws used. We performed four tretments with four replictes ech: pthogen only (tretment S), ntgonist only (tretment F), pthogen + ntgonist (tretment S + F) nd control (C) with neither ntgonist nor pthogen. The control plnts received the sme substrtes t the sme proportion, but without the inoculum. The plnts grew in greenhouse for month, until they fruited. The experiment ws conducted twice. Percentge of surviving plnts, shoot length, dry weight of roots nd shoots were recorded. b Sttisticl nlysis Anlysis of vrince ws performed t the significnce level of P <Æ5. When pproprite, mens were seprted by using Tukey s test (P <Æ5). Results Dul cultures The experiments on ntgonist ctivity showed tht F. oxysporum (S6) significntly inhibited pthogen growth on both medi ssessed (MEA nd PDA) with differences on inhibition hlo width whether it ws plnted simultneously or 48 h erlier. An inhibition zone ws obtined on both culture medi (Tble 1), but in PDA, the ntgonist grew to the edge of the pthogen colony, mking Figure 1 Dul cultures where F. oxysporum (S6) ws inoculted 48 h before S. sclerotiorum (fter 1 dys): () on mlt extrct gr (MEA); (b) on potto dextrose gr (PDA). the inhibition zone significntly smller thn on MEA (Fig. 1). The gretest inhibition percentges were found on PDA (Tble 1) where there ws less necrosis of the pthogen colony compred to MEA (Fig. 1). The %RGI nd the inhibition zone remined stble during the experiments. The colour of the F. oxysporum (S6) colony differed in the two medi. n PDA it ws pink-white (5YR 8/1- Munsell Color Co. 1954), nd violet (5YR 6/1 Munsell Color Co. 1954) when the colony confronted the pthogen. n MEA it ws pink (1R 5/4 Munsell Color Co. 1954) nd red pigment ws relesed into the med- Tble 1 Percentge of rdil growth inhibition (%RGI), percentge of scleroti formtion inhibition (%SFI), width of inhibition zone (Iz) for S. sclerotiorum in dul cultures with F. oxysporum (S6) on MEA nd PDA in simultneous inocultion nd deferred inocultion. Vlues represent the mens for ech tretment with the stndrd devition MEA PDA %RGI %SFI Iz (mm) %RGI %SFI Iz (mm) Simultneous inocultion 59Æ44 ± 6Æ12 4Æ75 ± 26Æ68 2Æ ± Æ8 78Æ33 ± 2Æ79 51Æ5 ± 8Æ81 Æ3 ± Æ5 Deferred inocultion 67Æ88 ± 1Æ11 4Æ76 ± 7Æ9 3Æ5 ± Æ6 77Æ22 ± 4Æ21 42Æ68 ± 16Æ89 Æ5 ± Æ6 ª 26 The Society for Applied Microbiology, Journl of Applied Microbiology 1 (26)

6 Cyclosporine A from nonpthogenic F. oxysporum suppressing S. sclerotiorum M.A. Rodríguez et l. ium. This pigment ws of more intense colour (1R 4/ 6- Munsell Color Co. 1954) when the ntgonist colony confronted S. sclerotiorum nd ppered severl dys before tht in the control colony. This difference in behviour between the control colony nd the confronted colony ws not observed on PDA. bservtions by light microscopy The mycelium of S. sclerotiorum in contct with the inhibition zone showed mrked necrosis on MEA (Fig. 1). Mycelium ltertions included incresed brnching, reduced length of brnches, grnultion, retrction of the plsmlemm nd collpse of cytoplsm (Fig. 2). o differences were found in either of the inocultion times. Production of nonvoltile metbolites The cellophne test showed tht the ntgonistic strin produced diffusible metbolites with ntifungl ctivity ginst S. sclerotiorum. Also, diffusion of the red pigment from the F. oxysporum (S6) colony ws observed. The S. sclerotiorum inoculum showed mrked melniztion nd mycelium did not develop throughout the 15 dys of the ssy. After 6 dys, the pthogen strin plnted in fresh MEA begn to grow. Evlution of the cpcity of Fusrium oxysporum s mycoprsite on Sclerotini sclerotiorum scleroti When spores were used for inocultion, ll the scleroti were recovered from both snd nd soil. At the end of the ssy, differences were found between substrte. The F. oxysporum (S6) strin ws ble to colonize the scleroti superficilly only in soil nd the ntgonist ws re-isolted only from 6Æ67% of them. Also, there ws slight reduction of the scleroti vibility (Tble 2). When the scleroti were plnted in the centre of the F. oxysporum colony, there ws nonsignificnt increse of scleroti infection, but the vibility decresed (Tble 3). In fct, percentges of vible scleroti were significntly lower fter 3 nd 45 dys. Reltionship between growth nd ntgonistic ctivity A mrked reduction in the growth rte of the pthogen ws obtined when the F. oxysporum (S6) culture filtrte ws dded on the medi (Fig. 3). The percentge of growth inhibition (%GI) of the pthogen colony ws significntly lrger when the filtrte from 7-dy-old culture (d7) of F. oxysporum (S6) ws used. Figure 4 shows the growth inhibition percentges obtined long the time (GI%) with F. oxysporum (S6) exudtes culture. Also, there ws significnt reduction in the number of scleroti produced nd their verge dry weight ws significntly lrger when filtrtes from d7 nd d1 were dded Tble 2 Mycoprsitism of S. sclerotiorum scleroti by F. oxysporum (S6) inoculted s spore suspension nd incubted in snd or soil s substrte nd evluted s: percentge of recuperted scleroti (%RS), percentge of colonized scleroti (%CS), percentge of infected scleroti (%IS) nd percentge of vible scleroti (%VS). In ech substrte two tretments were employed: F. oxysporum (S6) spore inoculted scleroti nd control scleroti (without F. oxysporum inocultion). There were no significnt differences between tretments (AVA P < Æ5) Snd Soil Inocultion with S6 spores Control Inocultion with S6 spores Control Figure 2 Altertions of the S. sclerotiorum mycelium: () edge of the colony in the control; (b) mycelium in contct with F. oxysporum (S6) inhibition zone on MEA, collpsed cytoplsm nd brnched hyphe. Scle br: 1 lm. %RS 1Æ 1Æ 1Æ 1Æ %CS Æ Æ 26Æ67 Æ %IS Æ Æ 6Æ67 Æ %VS 1Æ 1Æ 93Æ33 1Æ 58 ª 26 The Society for Applied Microbiology, Journl of Applied Microbiology 1 (26)

7 M.A. Rodríguez et l. Cyclosporine A from nonpthogenic F. oxysporum suppressing S. sclerotiorum Tble 3 Mycoprsitism in scleroti incorported into F. oxysporum (S6) colony (tretment FS6) nd in scleroti control over time evluted s: percentge of colonized scleroti (%CS), percentge of infected scleroti (%IS) nd percentge of vible scleroti (%VS). In ech substrte two tretments were employed: F. oxysporum (S6) spore inoculted scleroti nd control scleroti (without F. oxysporum inocultion). Different letters indicte significnt differences between tretments (AVA Tukey test P < Æ5) Dy 15 Dy 3 Dy 45 FS6 Control FS6 Control FS6 Control %CS 83Æ33 b Æ 1Æ b Æ 1Æ b Æ %IS 11Æ11 Æ 11Æ11 16Æ67 Æ %VS 88Æ9 1Æ 44Æ44 b 1 5Æ b 1Æ () Mycelium dry weight (mg) Time (dys) %GI S. sclerotiorum colony dimeter (cm) Time (dys) (b) Cyclosporine A concentrtion µg ml 1 (medium) µg mg 1 (mycelium) Time (dys) Figure 3 Antifungl ctivity nd cyclosporine A production: () F. oxysporum (S6) growth curve in mlt extrct medium broth (MEB) mesured s dry weight of mycelium (() nd ntifungl ctivity presented by the medium s percentge of growth inhibition (%GI) of the S. sclerotiorum colony on MEA when tht medium ws dded t concentrtion of 1% (v/v) ( ). (b) Cyclosporin A quntifiction, nlysed by HPLC nd mesured s microgrms (lg) of compound per millilitre of medium (d) nd microgrms of compound per milligrm of dry mycelium (s) over time. Also, ph ws determined t ech hrvesting dy ( ). Brs indicte stndrd devitions of the men. to the growing medi of S. sclerotiorum (Fig. 5). Pthogen colonies showed noticeble ltertions such s reduction of eril mycelium with irregulr borders nd slight melniztion. Also, white structures such s immture scleroti were observed ph Figure 4 Growth curve of the pthogen S. sclerotiorum on solid medium mesured s colony dimeter over time with the ddition of 1% (v/v) of the filtrte obtined F. oxysporum (S6) culture t different dys: d1 (1 dy old culture) ( ); d3 (3 dys old culture) ((); d5 (5 dys old culture) ( ); d7 (7 dys old culture) (d); d1 (1 dys old culture) (s); control ( ). Brs indicte stndrd devitions of the men. Extrction nd isoltion of the ntifungl compound The prtition of the crude extrcts of myceli nd medium of F. oxysporum (S6), guided by ntifungl ctivity ginst S. sclerotiorum, yielded pure compound (MIC Æ1 lg disc )1 ). The isolted compound exhibited four signls s doublets between 7Æ nd 8Æ ppm, eleven multiplets between 4Æ4 nd 5Æ8 ppm, seven methyl groups ttched to nitrogen between 2Æ7 nd 3Æ5 ppm, besides other signls in the 1 H MR ( 1 H nucler mgnetic resonnce) spectrum. Both this fct nd the presence of 11 crbonyl groups in the 13 C MR spectrum suggested tht the pure compound ws peptide of 11 mino cids, seven of them -methylted, s shown by 1 H MR. The twodimensionl (2D) MR experiments, HETCR (Heteronucler Chemicl Shift Correltion) nd CSY H,H (homonucler correltion spectroscopy) llowed to estblish the connectivities between protons nd crbons directly ttched, nd between vicinl protons, respectively. A 2D-J experiment ws employed to discriminte ª 26 The Society for Applied Microbiology, Journl of Applied Microbiology 1 (26)

8 Cyclosporine A from nonpthogenic F. oxysporum suppressing S. sclerotiorum M.A. Rodríguez et l. () 45 nº scleroti (b) Scleroti dry weight (mgrs) b d1 d3 d5 d7 d1 Control Tretment d1 d3 d5 d7 d1 Control Tretment b b b b () (ppm) H-1 (b) (ppm) Figure 5 () umber per plte nd (b) dry weight of scleroti formed in the S. sclerotiorum colony on mlt extrct gr. F. oxysporum (S6) ws grown in mlt extrct medium broth (MEB). After 1, 3, 5, 7 nd 1 dys, F. oxysporum medi ws filtered nd dded t concentrtion of 1% to mlt extrct gr (MEA) (v/v). Different letters indicte significnt differences between tretments (nov Tukey test P < Æ5). Brs indicte stndrd devitions of the men. between overlpped proton signls. All these dt determined the presence of four MeLeu, two Al, nd one MeBmt, Abu, Sr, Vl nd MeVl. As these mino cids re present in cyclosporine A (CyA), this compound ws isolted from commercil smple of SndimmunÒ (ovrtis) in order to compre 1 H nd 13 C MR spectr. Both the spectr (Fig. 6) nd the opticl rottion were identicl for both compounds. For these resons, the isolted compound ws unmbiguously identified s cyclosporine A (Fig. 7). Cyclosporine A quntifiction The production of cyclosporine A initited on dy 5 nd the mount of cyclosporine A reched mximum on dys 7 nd 8, the sme s the ntifungl ctivity (Fig. 3b). The mximum production found in medium nd in mycelium ws 3Æ88 lg ml )1 nd 18Æ1 lg mg )1, respectively. Associted with tht lrgest metbolite production, there ws medium ph decrese. Figure 6 1 H MR spectr of cyclosporine A from F. oxysporum (S6) () nd commercil smple (b). MeLeu-1 MeLeu-9 Al-8 H Figure 7 Cyclosporine A. MeVl-11 Al-7 Assessment of the biocontrol cpcity of the ntgonist strin (greenhouse experiments) H The greenhouse experiments showed reduction in the percentge of disesed soyben plnts when they were pretreted with the ntgonistic strin (Fig. 8). The percentge of surviving plnts ws significntly lower for plnts inoculted with the pthogen (tretment S) thn H MeLeu-6 H MeBmt-1 Vl-5 H Abu-2 Sr-3 MeLeu ª 26 The Society for Applied Microbiology, Journl of Applied Microbiology 1 (26)

9 M.A. Rodríguez et l. Cyclosporine A from nonpthogenic F. oxysporum suppressing S. sclerotiorum % survivl soyben plnts for plnts in which S. sclerotiorum ws inoculted with F. oxysporum (S6) (tretment S + F). Under the experimentl conditions, the presence of F. oxysporum (S6) lone (tretment F) did not ffect the percentge of surviving plnts. At the end of the experiment, there were significnt differences in shoot length nd in dry weight of shoots of surviving plnts. In plnts under tretment F, shoot length ws significntly lrger (Tukey unequl ; P <Æ5). Shoot dry weight ws significntly lower in plnts from tretment S + F thn in control plnts (tretment C) (Tukey unequl ; P <Æ5). Shoot dry weight in plnts from tretment F ws intermedite between vlues for tretments C nd S + F nd did not differ significntly from them (Tukey unequl ; P < Æ5). There ws no significnt difference in the dry weight of roots in ny cse (Fig. 9). Discussion c 77 5 b CS S S + F F Tretment Figure 8 Biocontrol experiments with S. sclerotiorum (pthogen) nd F. oxysporum (S6) (biocontrol gent) in soyben plnt under greenhouse conditions. Percentge of surviving plnts for the four tretments: plnts with F. oxysporum (S6) inocultion (F); with S. sclerotiorum (S); plnts with F. oxysporum (S6) nd S. sclerotiorum inocultion (F + S) nd control [without F. oxysporum (S6) or S. sclerotiorum inocultion]. Different letters indicte significnt differences between tretments (nov Tukey test P < Æ5). Brs indicte stndrd devitions. Fusrium oxysporum is well known for its bility to grow nd sporulte profusely in soil, when lrge mount of nutrients re vilble, overcoming soil fungistsis (Ppvizs 1985). It hs the bility to colonize the rhizosphere nd roots of different plnt species (Frvel et l. 23) nd it is present with high frequency in suppressive soyben soils ginst S. sclerotiorum (Rodríguez 24). ur in vitro ssys showed tht the F. oxysporum (S6) strin hs mrked ntgonistic cpcity ginst the pthogen S. sclerotiorum, producing significnt reduction () Shoot length (cm) (b) 3 Dry weight (g) b C S S + F F Tretment b 1 63 b C S S + F F Tretment Figure 9 Growth prmeters in the greenhouse experiments: length ( () nd dry weight of shoots n nd dry weight of roots (b) of surviving plnts in the biocontrol experiments with F. oxysporum (S6) confronting S. sclerotiorum in four tretments. Plnts inoculted with F. oxysporum (S6) (F); S. sclerotiorum (S); F. oxysporum (S6) nd S. sclerotiorum (S + F) nd control [without F. oxysporum (S6) or S. sclerotiorum inocultion]. Different letters (nov Tukey test P <Æ5) indicte significnt differences between tretments. Brs indicte stndrd devitions. in the growth in ll the experimentl conditions ssyed. Potentilly the in vitro tests, such s dul cultures, could indicte the potentil of some orgnisms to produce chemicls or to ct s mycoprsites, nd could be used to delinete the rnge of vribles from which both optiml use of the ntgonist nd control of certin pthogens cn be obtined (Whipps 1987). Antgonist strins of Trichoderm hrzinum nd Coniothyrium minitns hve shown ntgonistic effect in in vitro ssys (Whipps 1987) nd different isoltes of these species hve been evluted s potentil biocontrol gents of S. sclerotiorum nd other species in this genus (Zhou nd Bolnd 1998). F. oxysporum (S6) showed inhibition of mycelium growth nd scleroti production, so it my therefore be considered s potentil biocontrol gent. Hyphl ltertions hve been described in dul cultures of S. sclerotiorum with different Fusrium species (Zzzerini nd Tosi 1985), but the microscopic ltertions were less evident thn the ones we found in our experiments. The melniztion present in the pthogen mycelium in dul cultures involves the deposition of polymers nd phenolic nd/or indolic compounds in the tissues ª 26 The Society for Applied Microbiology, Journl of Applied Microbiology 1 (26)

10 Cyclosporine A from nonpthogenic F. oxysporum suppressing S. sclerotiorum M.A. Rodríguez et l. (osnchuk nd Csdevll 23) protecting cells ginst physicl nd biologicl stress, nd incresing hyphl resistnce to cell wll-degrding enzymes (Butler et l. 21). In our experiments, the melniztion produced in the S. sclerotiorum colonies might be becuse of the response of the pthogen ginst toxic metbolite produced by F. oxysporum (S6). This response my be more efficient in MEA thn in PDA, where less melniztion nd greter %RGI ws obtined, suggesting tht it could be relted to the less efficient protection of the pthogenic colony. This fct would indicte tht the nutritionl conditions could be involved in the protection response of the pthogen. Both mycoprsitic cpcity nd superficil coloniztion with symptoms of softening in some strins of different Fusrium species hve been reported (Zzzerini nd Tosi 1985). In this sense, our results showed differences with those isoltes. Fusrium oxysporum (S6) colonized the scleroti superficilly, but did not produce the softening mentioned bove. In both mycoprsitim evlution ssys, F. oxysporum (S6) reduced the vibility of this resistnt structure in the soil, so it could be used to reduce this importnt inoculum source. The cellophne test provided evidence of the presence of nonvoltile metbolite tht cn diffuse into the medium nd tht completely inhibits the pthogen myceli growth. The dilution ssy using ntgonist exudtes provides n dequte tool to confirm the diffusible nture of nonvoltile metbolites (Hdcek nd Greger 2) nd ntifungl ctivity (Hostettmnn 1991). The dilution ssy ws lso used to demonstrte the possible role of ntibiosis in biocontrol (Frvel 1988), which is welldocumented phenomenon in soil-borne ntgonist fungi (Whipps 21). The dilution experiments showed tht the 1% dilution (v/v) produced significnt reduction of pthogen growth nd number of scleroti. The metbolite responsible for the ntifungl ctivity ws identified s cyclosporine A, which is well known s n immunosuppressor (Crlile et l. 21). This is the first time tht the ntifungl ctivity of cyclosporine A ws described ginst the phytopthogenic fungus S. sclerotiorum. However, ntifungl ctivity of cyclosporine A ginst other fungl strins such s some species of the gener Aspergillus, Curvulri, Trichophyton, Rhodotorul nd eurospor (Dreyfuss et l. 1976, Cruz et l. 2) hs been linked to the inhibition of chitin synthesis (Dreyfuss et l. 1976). The ntimicrobil ctivity of cyclosporine A hs been relted to the intrcellulr receptor cyclophilin A (Foor et l. 1992). The complex cyclophilin A cyclosporine A might work s potent inhibitor of different proteins, importnt in clcineurin metbolism (Breuder et l. 1994). In fct, the cyclosporine A cyclophilin A complex hs been linked with clcineurin inctivtion, involved in the infection of Mgnporthe grise. Clcineurin is required for ppressorium morphogenesis nd the regultion hyphl growth nd development in this phytopthogenic fungus (Viud et l. 22). The cyclosporine A ntifungl ctivity spectr hve been compred with those of polioxins (Dreyfuss et l. 1976), which re employed s fungicides for griculture (Monghn nd Tkcz 199). The purifiction nd identifiction of the metbolite could improve our understnding on the mechnism involved in this system. The production of cyclosporine A ws quntified both in medium nd mycelium, showing correltion between metbolite concentrtion nd ntifungl ctivity. The gretest concentrtion ws in the mycelium. This method could llow us to investigte the effect of different growth conditions to rech greter ntifungl ctivity from the strin, through metbolite quntifiction. The significnt reduction in the number of scleroti nd their vibility would gretly reduce the pthogen inoculum source, thus mking F. oxysporum (S6) nd cyclosporine A potentil biocontrol sources. The soyben plnts inoculted only with the ntgonist strin showed significnt increse of shoot length nd no symptoms of disese. For this reson, this strin my be considered s nonpthogenic. Considering the reduction in the number of disesed plnts when the ntgonist strin ws inoculted together with the pthogen, we concluded tht F. oxysporum (S6) strin my be suitble biocontrol gent. Acknowledgements We thnk Dr Jorge A. Plermo, LAAIS-MR (CI- CET-FCE, UBA) for the MR spectr, Wshington University Resource for Biomedicl nd Bio-orgnic Mss Spectrometry for the mss spectr, UMYMFR (CI- CET-FCE, UBA), CICET, APCYT nd Universidd de Buenos Aires for prtil finncil support. We re grteful to R.L. Peterson (University of Guelph) nd nonymous reviewers of our mnuscript for improving the lnguge nd for useful remrks. References Adms, P.B. nd Ayers, W.A. (1979) Ecology of Sclerotini species. Phytopthology 69, Benhmou,., Grnd, Ch. nd Goulet, A. 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