Effect of germicidal UVC light on fungi isolated from grapes and raisins

Transcription

1 Letters in pplied Miroiology ISSN ORIGINL RTICLE Effet of germiidl UVC light on fungi isolted from grpes nd risins. Vlero 1, M. egum 2, S. L. Leong 2,. D. Hoking 2,. J. Rmos 1, V. Snhis 1 nd S. Mrín 1 1 Food Tehnology Deprtment, University of Lleid, UTPV-CeRT. v., Lleid, Spin 2 CSIRO Food Siene ustrli, North Ryde, NSW, ustrli Keywords fungi, grpes, risins, survivl, ultrviolet. Correspondene Soni Mrín, Food Tehnology Deprtment, University of Lleid, v llde Rovir Roure 191, Lleid, Spin. E-mil: smrin@tel.udl.es 27 32: reeived 11 Jnury 27, revised nd epted 3 pril 27 doi:1.1111/j x x strt ims: To exmine how UVC ffets the different gener of fungi ommonly isolted from grpes, with the im of understnding hnges in myoiot during grpe ripening nd possile pplitions for preventing grpe dey during storge. Methods nd Results: Spores of spergillus ronrius, spergillus niger, Cldosporium herrum, Peniillium jnthinellum nd lternri lternt (etween 1 25 spores plte gr) were UVC irrdited for (ontrol), 1, 2, 3, 6, 3 nd 6 s. Pltes were inuted t 25 C nd olonies were ounted dily up to 7 dys. lternri lternt nd spergillus ronrius were the most resistnt fungi. Conidil germintion in these speies ws redued y pprox. 25% fter 1 s of exposure, ompred with greter thn 7% redution for the remining speies tested. Peniillium jnthinellum spores were the most suseptile t this wvelength. UVC exposures of 3 s prevented growth of ll isoltes studied, exept for lternri lternt. Conlusions: UVC irrdition plys mjor role in seleting for prtiulr fungi tht dominte the myoiot of drying grpes. Signifine nd Impt of the Study: The UVC irrdition of hrvested grpes ould prevent germintion of ontminnt fungi during storge or further dehydrtion. Introdution Grpes re suseptile to inset ttk nd to fungl diseses, espeilly grey rot, downy mildew nd lk rot. Dmged grpes re vulnerle to further diseses suh s summer unh rot, whih my e used y spergillus niger, lternri tenuis, Cldosporium herrum, Rhizopus rrhizus, Peniillium spp. nd other fungi. Fungl invsion depends on grpe mturity. lternri, Cldosporium, otrytis nd Rhizopus re ommon t erly verison, wheres spergillus nd Peniillium re more frequently found t hrvest nd during sun drying (Mgnoli et l. 23; ellí et l. 24; Vlero et l. 25). spergillus ronrius nd spergillus niger re know to produe ohrtoxin (OT) in grpes nd risins (r et l. 23; ellí et l. 24). Chnges in the myoiot of grpes re ffeted y wether, solr irrdition, rising tempertures nd dereses in the wter tivity of erries due to inresing erry sugr ontent. Grpes efore hrvest nd espeilly during drying, re exposed for long periods to sunlight. Ultrviolet rdition from the sun is divided into three wvends: UV, UV nd UVC. Ultrviolet rdition hs een known for mny yers to ffet miro-orgnisms. UVC (1 28 nm) is highly germiidl nd is ommonly used for sterilistion of surfes, wter nd ir; however, quntifition of these effets hs een diffiult. Levetin et l. (21) reported tht UVC rdition pplied to ir-onditioning systems signifintly redued the inidene of Cldosporium spp. nd spergillus versiolor. Green et l. (24) reported 35 nd 54 mj m )2 [UV dose (mj m )2 ) = Irrdine (lw m )2 ) Æ Irrdition time (s)æ1 )3 ] s the doses of ultrviolet germiidl irrdition ( nm) neessry to intivte 9% of the spores of spergillus flvus nd spergillus fumigtus, ª 27 The uthors 238 Journl ompiltion ª 27 The Soiety for pplied Miroiology, Letters in pplied Miroiology 45 (27)

2 . Vlero et l. UVC on fungi from grpes nd risins respetively. Jun et l. (23) modelled the effet of pulsed UV-light on the intivtion of spergillus niger spores in orn mel nd reported tht for 1 s tretment time, 3 m of distne from the UV stroe nd with 38 V input gve 4Æ9 log 1 redution of spergillus niger. In ontrst, Nigro et l. (1998) found tht irrditing grpe erries with UVC light hd no effet on filmentous fungi nd even inresed the inidene of yests nd teri. The effet of solr rdition on some gener of irorne fungi, suh s Cldosporium, Peniillium, lternri, Epioum or spergillus mong others, ws studied y Ulevičius et l. (24), who found tht spergillus niger ws the speies most ommonly isolted fter tretment. Given these findings, we exmined how the most energeti wvelength from sunlight, UVC, ould ffet the different gener of fungi ommonly isolted from grpes, with the im of understnding hnges in myoiot during grpe ripening nd possile pplitions for preventing grpe dey during storge. Mterils nd methods Fungi used in this study were isolted from grpes nd dried vine fruits olleted from ustrli nd Spin: spergillus ronrius (UdLT 3Æ122) ple of OT prodution, C. herrum (UdLT 3Æ129), Peniillium jnthinellum (UdLT 3Æ126), spergillus niger (FRR 5694) OT positive nd lternri lternt (FRR 478). UdLT strins were from the ulture olletion of the Food Tehnology Deprtment, Lleid University, Spin nd FRR strins were from the ulture olletion t CSIRO Food Siene ustrli, North Ryde, NSW ustrli. For spore prodution, eh speies ws inoulted onto Mlt Extrt gr (ME; Rper nd Thom 1949) nd onto Dihlorn Chlormpheniol Mlt gr for lternri lternt (DCM; ndrews 1992) nd inuted for 7 dys t 25 C. Spores were hrvested into queous Tween 8 (Æ5%), quntified using hemoytometer nd diluted to give finl suspension of 1 3 spores ml )1. The growth medium ws Syntheti Nutrient Medium (SNM), hosen euse its omposition is similr to tht of grpes etween verison nd ripeness (Delfini 1982), djusted to Æ97 w (Vlero et l. 25). liquots (Æ1 ml) of eh 1 3 spore suspension were inoulted onto SNM y spreding uniformly on the gr surfe nd fterwrds irrdited with UVC (254 nm), for (ontrol), 1, 2, 3, 6, 3 nd 6 s. Pltes without the lids were set upside t 1 m from the lmp (G2T1 Snkyo Denki, Co., Ltd, Tokyo, Jpn) in losed hmer previously sterilised. Nominl lmp power ws 19 W nd 75Æ8 lwm )2 of irrdine (t 1 m of distne). Eh omintion of speies nd irrdition time hd four replites. Pltes were inuted t 25 C for 7 dys nd olonies ounted dily up to 7 dys. Survivl frtions (S) of the five miroorgnisms were plotted ginst UVC irrdition time nd n eqution desriing the one-stge exponentil dey urve S ¼ expð ktþ where k = dey rte (s )1 ); t = irrdition time, ws generted using GrphPd Prism version 4Æ for Windows (GrphPd Softwre, Sn Diego, Cliforni, US). Covrine nlysis of germintion perentges vs time nd relted Dunn s multiple rnge tests were performed using SS (SS Institute In., Cry, NC, US). Results lternri lternt nd spergillus ronrius were the most resistnt fungi when irrdited with UVC for 1 or 2 s (Fig. 1). Conidil germintion in these speies ws redued y pprox. 25% fter 1 s of exposure, ompred with greter thn 7% redution for the remining speies tested. For UVC exposures longer thn 2 s, lternri lternt ws the only speies ple of growing fter 3 nd 6 s exposure to UVC light. In ontrst, P. jnthinellum spores were the most suseptile t this wvelength, with totl onidil intivtion fter 3 s of exposure. UVC exposure of 6 s redued survivl of ll fungi tested y more thn 9%. The exponentil dey of the survivl frtions ws modelled so s to generte single eqution desriing the UVC resistne for eh isolte ginst exposure time (Fig. 2). More resistnt fungi showed lower dey rtes (k). UVC irrdition t distne of 1 m for 3 s prevented spergillus growth for 3 dys nd UVC exposures of 3 s prevented growth of ll isoltes studied, prt from lternri lternt, for longer thn 7 dys (Tle 1). The pity to repir dmged spores ws ffeted y UVC irrdition in ll speies tested, s longer inution times were required for ll vile spores to germinte when irrdited with higher umultive UVC dosges. In ft ll vile spores tht were not irrdited germinted etween 2 3 dys, while the irrdited spores germinted differently, up to the sixth dy, depending on the strins (Fig. 3). UVC irrdition for 1 s produed signifint redution (P <Æ5) in germintion of ll fungi ssyed, ompred with germintion of non-irrdited spores. Generlly, susequent exposures of 1 s led to suessive signifint redutions. Disussion Integrtion of these findings on resistne to UVC with dt on the effets of temperture nd wter tivity on ª 27 The uthors Journl ompiltion ª 27 The Soiety for pplied Miroiology, Letters in pplied Miroiology 45 (27)

3 UVC on fungi from grpes nd risins. Vlero et l. 1 9 Survivl frtion (dy 7) C D E UVC Irrdition time (s) 3 6. lternt. ronrius. niger C. herrum P. jnthinellum Figure 1 Survivl frtion of the five isoltes (lternri lternt, spergillus ronrius, spergillus niger, Cldosporium herrum nd Peniillium jnthinellum) fter inution for 7 dys (verge of four replites). For omprison of speies within eh time point, rs with different letters re signifintly different (P < Æ5). Survivl frtion e d,,. ronrius. lternt. niger C. herrum P. jnthinellum. lternt. ronrius. niger C. herrum P. jnthinellum UVC time (s) lternri spergillus spergillus Cldosporium Penillium lternt ronrius niger herrum jnthinellum k SE R Figure 2 Models for single stge exponentil dey urves of five isoltes s influened y UVC irrdition. Exponentil dey rtes for eh urve re detiled in tle: Survivl frtion = exp () kt ). k, dey onstnt (s )1 ); t, UVC irrdition time (s). SE: stndrd error, R 2 : regression oeffiient. Isoltes: (n), Peniillium jnthinellum; (m), spergillus niger ; (s), Cldosporium herrum ; d (r), spergillus ronrius ; e( ), lternri lternt. fungl growth nd survivl provides good explntion of the inidene of fungi found on oth fresh nd sun-dried grpes. lternri is the most ommon fungl genus found on grpes, followed y Peniillium, spergillus, Epioum nd Cldosporium (Sge et l. 22; ellí et l. 24; Vlero et l. 25). Leong et l. (26) reported derese (1 5 ) in spergillus ronrius spore viility on grpes fter n exposure to UV irrdition equivlent to 1 week of high UV intensity with loudless skies in Sydney, NSW ustrli, umultive energy estimted in 3Æ6 1 3 joules (1 mwh, milliwtt hour). When grpes re dried in intense sunlight, the most ommonly found fungi re spergillus, Peniillium nd lternri (Romero et l. 25; Vlero et l. 25). Likewise, Ulevičius et l. (24) isolted fungl propgules from ir on the Lithunin ost t different hours during the dy nd found tht spergillus niger, letrnri lternt, Cldosporium spp., rthrinium pherosporum nd demtieous sterile myelium were prevlent fter exposure to solr rdition. Our dt demonstrting the resistne of lternri lternt to UVC gree with the findings of Rotem nd ust (1991), who noted tht propgules of lternri sp. were the most resistnt to ultrviolet rdition or sunlight, followed y Myosphell sp., spergillus niger nd otrytis inere. lternri lternt hs multi-elled spores with ª 27 The uthors 24 Journl ompiltion ª 27 The Soiety for pplied Miroiology, Letters in pplied Miroiology 45 (27)

4 . Vlero et l. UVC on fungi from grpes nd risins Tle 1 Lg phse (dys) of five isoltes of fungi s influened y durtion of UVC irrdition Durtion of UVC irrdition (s) lternri lternt spergillus ronrius n.g. n.g. spergillus niger n.g. n.g. Cldosporium herrum n.g. 5 n.g. n.g. Peniillium jnthinellum n.g. n.g. n.g. n.g. n.g., no growth d,d,d for the survivl nd longevity of fungl spores (ell nd Wheeler 1986), wheres non-melnin ompounds re poor protetnts ginst ultrviolet rdition (Durrell nd Shields 196). Grishkn et l. (23) found signifint orreltion etween res reeiving high solr irrdition nd the inidene of melnin-ontining fungl speies mong soil mirofungi isolted in Isrel. spergillus ronrius nd spergillus niger produe single-elled onidi with melnin nd spergilline in their ell wlls (Duguy nd Klironomos 2; itsky nd Shher 22), ut differ in their UVC resistne d,d,d,d d, ,, , Figure 3 Germintion urves following rdition of different fungi gener. 1, lternri lternt; 2, spergillus ronrius; 3, spergillus niger; 4, Cldosporium herrum; 5, Peniillilum jnthinellum. UVC irrdition times: +, s; d, 1s;, 2s; m, 3s;, 6s; h, 3 s; n, 6 s. Lower se letters denote finl germintion perentges tht re signifintly different (P < Æ5). thik, melnised wlls tht re elieved to onfer tolerne to sunlight (Crznig et l. 22). The photoprotetive properties of melnin re elieved to e importnt nd their inidene on grpes. spergillus ronrius spores re thought to possess thiker wlls thn the 9 16 nm thik wll of spergillus niger (Tiedt 1993). The ª 27 The uthors Journl ompiltion ª 27 The Soiety for pplied Miroiology, Letters in pplied Miroiology 45 (27)

5 UVC on fungi from grpes nd risins. Vlero et l. greter UVC resistne displyed y spergillus ronrius spores thn spergillus niger spores provides logil explntion for the high numers of spergillus ronrius on grpes sujeted to prolonged sun exposure. In study of fungl ontmintion of grpes fter sun drying, spergillus setion Nigri spp. were found in more thn 8% of dried grpes (Vlero et l. 25). spergillus ronrius ourred in inresed frequeny ompred with spergillus niger on risins nd dried vine fruits (Leong et l. 24; Romero et l. 25; Vlero et l. 25; Gómez et l. 26). This is somewht surprising, s spergillus niger is more tolernt thn spergillus ronrius to low wter tivities (growth limits of Æ77 w nd Æ87 w, respetively; Vlero et l. 27) nd high tempertures (optimum growth t 35 4 C nd 3 C, respetively; Pnsenko 1967; Plios-Crer et l. 25; Vlero et l. 27). Resistne to UVC provides ompetitive dvntge for spergillus ronrius during grpe drying tht overrides the superior growth of spergillus niger in hot nd dry onditions. Cldosporium herrum produes onidi with melnin-like pigment (Mrglith 1992). The thikness of its onidil wll is unknown, ut the pigmented onidi llow Cldosporium to survive under high solr irrdition, whih explins their ommonly ourrene on grpes nd risins (Romero et l. 25; Vlero et l. 25), even though its mximum growth temperture is pproximtely 32 C (Domsh et l. 198; Vlero et l. 27). Some Peniillium speies re known to produe melnin-like ompounds; however, there is little informtion regrding P. jnthinellum (Younghim et l. 24). This speies produes single-elled slightly pigmented onidi nd hyline myelium hene, hs few protetive mehnisms ginst UV rdition. oth the sptil distriution of pigments nd highly effiient DN-repir mehnisms hve umultive effet of eologil signifine, onferring on those miroorgnisms n dvntge in surviving on grpe surfes (Wynn-Willims et l. 22; Ulevičius et l. 24). Our results show tht UVC irrdition plys mjor role in seleting for prtiulr fungi tht dominte the myoiot of drying grpes. Temperture nd wter tivity re lso importnt. In ddition, the UVC irrdition of hrvested grpes ould prevent germintion of ontminnt fungi during storge or further dehydrtion tht my led to prodution of undesired sustnes suh s myotoxins. knowledgements The uthors re grteful to the Spnish Government (CI- CYT, Comisión Interministeril de Cieni y Tenologí, projet GL , grnt ES nd Rmón y Cjl progrm) nd to the Ctlonin Government (CeRT strtegi projet Seguritt iòti y iòti dels liments, Generlitt de Ctluny) for their finnil support. We lso thnk the Myology nd Myotoxins Setion of Food Siene ustrli (CSIRO) for their finnil support, ollortion nd hospitlity. Referenes r, M.L., ensi, F., rgult, M.R., Cstell, G. nd Cñes, F. (23) Contmintion in dried vine fruits from the Spnish mrket. J Food Prot 66, ndrews, S. (1992) Differentition of lternri speies isolted from erels on dihlorn mlt extrt gr. In Modern Methods in Food Myology ed. Smson, R.., Hoking,.D., Pitt, J.I. nd King,.D. pp msterdm: Elsevier. itsky, V.G. nd Shher, V.V. (22) The nture of melnin pigments of severl miro- nd mromyetes. ppl iohem Miroiol 38, ell,.. nd Wheeler, M.H. (1986) iosynthesis nd funtion of fungl melnins. nn Rev Phytopthol 24, ellí, N., Prdo, E., Mrín, S., Frré, G., Rmos,.J. nd Snhis, V. (24) Ourrene of ohrtoxin nd toxigeni potentil of fungl isoltes from Spnish grpes. J Si Food gri 84, Crznig, R., Fioo, D., owyer, P. nd O Connell, R.J. (22) Loliztion of melnin in onidi of lternri lternt using phge disply ntiodies. Mol Plnt-Miroe Intert 15, Delfini, C. (1982) Teni di Miroiologi Enologi. Rome: Luigi Slpi. Domsh, K.H., Gms, W. nd nderson, T.H. (198) Compendium of Soil Fungi. London: demi Press. Duguy, K.J. nd Klironomos, J.N. (2) Diret nd indiret effets of enhned UV- rdition on the deomposing nd ompetitive ilities of sproi fungi. ppl Soil Eol 14, Durrell, L.D. nd Shields, L.M. (196) Fungi isolted in ulture from soil of the Nevd test site. Myologi 52, Gómez, C., rgult, M.R., r, M.L., Mínguez, S. nd Cñes, F.J. (26) Ohrtoxin- produing fungi from grpes intended for liqueur wine prodution. Food Miroiol 23, Green, C.F., Srpino, V.V., Jensen, P., Jensen, N.J. nd Gis, S.G. (24) Disinfetion of seleted spergillus spp. using ultrviolet germiidl irrdition. Cn J Miroiol 5, Grishkn, I., Nevo, E., Wsser, S.P. nd ehrv,. (23) dpttive sptiotemporl distriution of soil mirofungi in Evolution Cnyon II, Lower Nhl Keziv, western Upper Glilee, Isrel. iol J Linnen So 78, Jun, S., Irudyrj, J., Demiri,. nd Geiser, D. (23) Pulsed UV-light tretment of orn mel for intivtion of spergillus niger spores. Int J Food Si Tehnol 38, ª 27 The uthors 242 Journl ompiltion ª 27 The Soiety for pplied Miroiology, Letters in pplied Miroiology 45 (27)

6 . Vlero et l. UVC on fungi from grpes nd risins Leong, S., Hoking,.D. nd Pitt, J.I. (24) Ourrene of fruit rot fungi (spergillus setion Nigri) on some drying vrieties of irrigted grpes. ust J Grpe Wine Res 1, Leong, S., Hoking,.D. nd Sott, E.S. (26) Survivl nd growth of spergillus ronrius on wine grpes efore hrvest. Int J Food Miroiol 111, S83 S87. Levetin, E., Shughnessy, R., Rogers, C.. nd Sheir, R. (21) Effetiveness of germiidl UV rdition for reduing fungl ontmintion within ir-hndling units. ppl Environ Miroiol 67, Mgnoli, C., Violnte, M., Comin, M., Plio, G. nd Dlero,. (23) Myoflor nd ohrtoxin-produing strins of spergillus setion Nigri in wine grpes in rgentin. Lett ppl Miroiol 37, Mrglith, P.Z. (1992) Pigmented Miroiology. London: Chpmn nd Hll. Nigro, F., Ippolito,. nd Lim, G. (1998) Use of UV-C light to redue otrytis storge rot of tle grpes. Posthrvest iol Tehnol 13, Plios-Crer, H., Tniwki, M.H., Hshimoto, J.M. nd Menezes, H.C. (25) Growth of spergillus ohreus,. ronrius nd. niger on ulture medi t different wter tivities nd tempertures. rz J Miroiol 36, Pnsenko, V.T. (1967) Eology of mirofungi. otnil Review 33, Rper, K.. nd Thom, C. (1949) The Genus spergillus. ltimore, Mrylnd: Willims nd Wilkins. Romero, S.M., Comerio, R.M., Lrume, G., Ritieni,., Vmonde, G. nd Fernández Pinto, V. (25) Toxigeni fungi isolted from dried vine fruits in rgentin. Int J Food Miroiol 14, Rotem, J. nd ust, H.J. (1991) The effet of ultrviolet nd solr rdition nd temperture on survivl of fungl propgules. J Phytopthol 133, Sge, L., Krivook, S., Delost, E., Seigle-Murndi, F. nd Creppy, E.E. (22) Fungl flor nd ohrtoxin prodution in grpes nd musts from Frne. J gr Food Chem 5, Tiedt, L.R. (1993) n eletron mirosope study of onidiogenesis nd wll formtion of onidi of spergillus niger. Myol Res 97, Ulevičius, V., Pečiulyt_e, D., Lugusks,. nd ndriejuskien_e, J. (24) Field study on hnges in viility of irorne fungl propgules exposed to UV rdition. Environ Toxiol 19, Vlero,., Mrín, S., Rmos,.J. nd Snhis, V. (25) Ohrtoxin produing speies in grpes nd sun dried grpes nd their reltion to eophysiologil ftors. Lett ppl Miroiol 41, Vlero,., Snhis, V., Rmos,.J. nd Mrín, S. (27) Studies on the intertion etween grpe-ssoited filmentous fungi on syntheti medium. Int J Food Miroiol 113, Wynn-Willims, D.D., Edvrds, H.G.M., Newton, E.M. nd Holder, J.M. (22) Pigmenttion s survivl strtegy for nient nd modern photosyntheti miroes under high ultrviolet stress on plnetry surfes. Int J eroiol 1, Younghim, S., Morris-Jones, R., Hy, R.J. nd Hmilton,.J. (24) Prodution of melnin y spergillus fumigtus. J Med Miroiol 53, ª 27 The uthors Journl ompiltion ª 27 The Soiety for pplied Miroiology, Letters in pplied Miroiology 45 (27)