Plnt Pthol. J. 3(5) : 46-468 (6) http://x.oi.org/.543/ppj.oa.3.6.6 pissn 598-54 eissn 93-98 Reserh Artile Open Aess The Plnt Pthology Journl The Koren Soiety of Plnt Pthology Posthrvest Disese of Colletotrihum gloeosporioies n Peniillium expnsum on Store Apples y Gmm Irrition Comine with Fumigtion Wonsu Cheon, Young Soo Kim, Kotnl Blrju, Bong-Su Kim 3, Byeong-Ho Lee 3, n Yongho Jeon * Deprtment of Bioresoure Sienes, Anong Ntionl University, Anong 3679, Kore Agriulturl Siene n Tehnology Reserh Institute, Anong Ntionl University, Anong 3679, Kore 3 Dongu Agriulturl Reserh Institute, Dongu Frm Hnnong Co., Lt., Nonsn 33, Kore (Reeive on Mrh 5, 6; Revise on June 5, 6; Aepte on June 7, 6) To stuy the ontrol of posthrvest ey use y Colletotrihum gloeosporioies n Peniillium expnsum, gmm irrition lone or in omintion with fumigtion ws evlute to exten the shelf life of pples in South Kore. An irrition ose of. kgy resulte in the mximum inhiition of C. gloeosporioies n P. expnsum spore germintion. The gmm irrition ose require to reue the spore germintion y 9% ws. n.35 kgy for C. gloeosporioies n P. expnsum, respetively. Mirosopi oservtions revele tht when the fungl spores were trete with gmm irrition (4. kgy), oniil germintion ws stoppe ompletely resulting in no germ tue formtion in C. gloeosporioies. Tretment with the eo-frienly fumignt ethneinitrile h greter ntifungl tivity ginst C. gloeosporioies n P. expnsum in omprison with the non-trete ontrol uner in vitro onitions. The in vitro ntifungl effets of the gmm irrition n fumigtion tretments llowe us to further stuy the effets of the omine tretments to ontrol posthrvest ey on store pples. Interestingly, when pples were trete with gmm irrition in omine with fumigtion, isese inhiition inrese more t lower (<.4 kgy) thn t higher oses of irrition, suggesting tht omine tretments reue the neessry irrition ose in phytosnitry irrition *Corresponing uthor. Phone) +8-54-8-557, FAX) +8-54-8-63 E-mil) yong@nu..kr This is n Open Aess rtile istriute uner the terms of the Cretive Commons Attriution Non-Commeril Liense (http:// retiveommons.org/lienses/y-n/4.) whih permits unrestrite nonommeril use, istriution, n reproution in ny meium, provie the originl work is properly ite. Artiles n e freely viewe online t www.ppjonline.org. proessing uner storge onitions. Keywors : Colletotrihum gloeosporioies, omintion, ethneinitrile, gmm irrition, Peniillium expnsum Apple (Mlus pumil Mill.) is one of the most eonomilly importnt fruit rops in South Kore n is wiely ultivte in temperte regions. Kore is onsiere one of the mjor pple-prouing ountries in Asi (Cheon et l., 6; Mostfvi et l., ). Usully, pples re hrveste for limite perio; therefore, it is neessry to store the fruits for long perios until the ompletion of mrketing. The urility of vrious rops, inluing pples, is ffete y numer of ftors, inluing posthrvest iseses tht use losses ue to metoli tivity uring storge (Jeong et l., 5; Kzemi et l., ; Omim n Krim, 7). When fresh fruits n vegetles re exporte, phytosnitry tretment is importnt to preserve qulity uring ol storge in orer to prevent from rotting (Jung et l., 4). Posthrvest pthogens use mjor losses in pple proution n more thn 9 fungl pthogen speies hve een reporte s usl gents in pple ey uring storge (Jones n Alwinkle, 99; Mostfvi et l., ). Colletotrihum gloeosporioies uses nthrnose in mny plnts n is one of the most ommon Colletotrihum fungl plnt pthogens. It lso uses nthrnose in vrious rops, prtiulrly perennils, in tropil regions (Onofre n Antonizzi, 4; Wller, 99). However, pple ultivrs re suseptile to iseses, inluing C. gloeosporioies, whih n use losses fter hrvesting. Peniillium expnsum is posthrvest pthogen tht infets severl host plnts, inluing pples; it uses lue mol on store pples (Sutton et l., 4). This plnt
Posthrvest Dey y Gmm Irrition n Fumigtion 46 pthogen n e isolte from wie rnge of hosts, in ition to pples (Ashizw n L, ; Morles et l., 7). P. expnsum is one of the most ggressive n ommonly reporte Peniillium spp. (Pinzzol et l., 4); it proues myotoxin with neurotoxi funtions on pple fruits (Bruse et l., 996). Thus, it is importnt to ontrol posthrvest iseses to inrese the shelf life of pples. Gmm irrition, prtiulrly from olt-6, is use s n effetive non-hemil tretment to sterilize griulturl ommoities in orer to ontrol posthrvest losses use y vrious iseses (Chu et l., 5; Hllmn, ; Mostfvi et l., ). During irrition, highenergy photons re emitte from n isotope soure (e.g., olt-6) throughout the ojet (Choi n Lim, 5; Mostfvi et l., ); this proess mges the ells t the moleulr level, n the resulting DNA hnges my use eth or the inility to reproue. In ition to the irrition, the fumigtion metho ws use in our present stuy. In generl, the fumignt methyl romie is wiely use to ontrol pests n iseses euse it hs wie spetrum of tivity (Fiels n White, ). However, ue to its negtive impts on humn helth s well s environmentl risks (UNEP, 995), the use of methyl romie hs een nne in the worl y the yer 5 (Klose et l., 6). Therefore, mny reserhers hve fouse on eo-frienly posthrvest tehnologies to ontrol iseses. Numerous vile lterntives, suh s het, ol, n irrition tretments hve een use to reple methyl romie (Diehl, ; Hllmn, ). Ethneinitrile (EDN) is lso known s ynogen n generlly use s n eo-frienly fumignt to isinfet the soil in the nurseries, s n lterntive to methyl romie (Mttner et l., 6). EDN is n environmentlly-sfe fumignt, olorless gs with n lmon-like oor. It is gs t room temperture n esily solule in wter. EDN s min route of eomposition is to erivtives of oxli i (Hooper et l., 3). There is no informtion regring the effetiveness of gmm irrition omine with fumigtion to ontrol posthrvest iseses in pples. Therefore, the ojetive of this stuy ws to investigte the effiy of gmm irrition tretment in omintion with EDN fumigtion to ontrol posthrvest ey in pples, n to etermine the optiml oses of gmm irrition to ontrol storge-relte iseses in pples. Mterils n Methos Rw mteril preprtion. Apple (ultivr Fuji) fruits of uniform shpe n size, with firm texture n t proper mturity, were proure from the Agriulturl Prouts Proessing Center (APPC), Anong, Gyeonguk Provine, South Kore from Otoer to Novemer, 4. Fruits were pre-oole t o C for 4 h in ol storge hmer. The pre-oole fruits were mnully gre in orer to ensure uniformity. The fruits were surfeisinfete in % soium hypohlorite for 3 min n rinse with sterile istille wter (SDW); then they were ir-rie n pke in ror oxes. Eighteen fruits were rrnge on ror trys n ple in ror oxes. One ox ws use for eh tretment n therefore eh tretment onsiste of 8 replites. Fungl strins, ulture onitions, n spore suspension preprtion. The two pthogeni fungi (C. gloeosporioies n P. expnsum) use in this stuy were originlly isolte from Fuji pples in Kore, n isolte fungi were ientifie y PCR (White et l., 99). The fungi were mintine on potto extrose gr (PDA; Difo, Frnklin Lkes, NJ, USA) t 8 o C. To prepre spore suspensions, symptomti tissues were ut from the pple fruits n were sujete to surfe steriliztion using % soium hypohlorite (NOCl) solution for min n 7% ethnol for 3 s, n were then rinse twie in SDW. After steriliztion, the tissues were rie on sterile filter pper, trnsferre to PDA pltes, n inute t 5 o C for 7 ys. Conii suspensions of pthogeni fungi were prepre y suspening myeli with SDW srpe from 7-y-ol ultures onto PDA pltes. The resulting suspensions were filtere through oule-lyere heeseloth, n their onentrtions were juste to 5 onii/ml using hemoytometer (Ekert n Brown, 986). Effets of gmm rition on spore germintion of C. gloeosporioies n P. expnsum. For irrition tretments, the spore suspensions were spre onto PDA pltes for ulturing. The pltes were expose to gmm rys t oses of.. kgy using olt-6 soure t ose of.6 kgy/h from the Kore Atomi Energy Reserh Institute, Jeongeup, Kore. A olt-6 gmm irritor (5 TBq pity, ACEL; MDS Norion, Ottw, ON, Cn) ws use for irrition. All sore oses were lirte using n lnine osimeter with imeter of 5 mm (Bruker Instruments, Rheinstetten, Germny); n EMS 4 EPR nlyzer (Bruker Instruments) ws use to etermine the free ril signls. The ose uniformity rtio, or the rtio of the mximl n miniml ose sore in the irrite mteril (D mx /D min ), ws ~. Spore germintion ws reore t, 4, n 48 h fter inuting the pltes t 5 o C, n spore viility ws otine y seril ilution on PDA pltes. Lter, these preprtions were oserve with light mirosope (BX4; Olympus, Tokyo, Jpn) t mgnifition of
46 Cheon et l. 4. The spore germintion perentge ws etermine using hemoytometer for the ifferent oses of irrition fter spore suspensions inute for 3 ys t 5 o C. Germ tue length ws reore using ProgRes Cpture Pro v.8.8 (JENOPTIK, Jen, Germny). Three replites were use for eh tretment. The viility of irrite n non-irrite spores ws etermine using ilution plting metho in three susets (up to 4 or 5 ilution with SDW) for 7 h on PDA pltes (Ley et l., 98). The survivl urves were onstrute y plotting the survivl spores/ml ginst the tul irrition ose. In vitro ntifungl tivity ginst C. gloeosporioies n P. expnsum y fumigtion. The fungi C. gloeosporioies n P. expnsum were ulture on PDA pltes t 5 o C in the rk. EDN ws pplie s fumignt using vuum esitor. For fumigtion, the spore suspensions were ulture y spreing on PDA pltes. The pltes were expose to fumigtion t vrious oses (.,.5, n. g/m 3 ) for h t 4 o C in the vuum esitor. After inution t 5 o C in the rk for 3 ys, myelil growth of C. gloeosporioies n P. expnsum ws mesure s the verge olony rius. The experiment ws repete three times. Evlution of omine gmm irrition n fumigtion to ontrol C. gloeosporioies n P. expnsum in pples uner storge onitions. A -μl oniil suspension ( 4 spores ml ) ws ple on surfesterilize pple fruits tht h een woune y piering to epth of to mm with pin. The pple fruits were fumigte with EDN t oses of. n.5 g/m 3 using vuum esitor uner ommeril onitions. The trete smples n untrete ontrols were ple in storge ontiner pke in ror oxes. Irrition of pple fruits ws performe fter fumigtion. The preoole fumigte fruits were irrite using oses of. n.4 kgy t minimum ose rte of.6 kgy/h with PANBIT irritor (ACEL; MDS Norion, Ottw, ON, Cn) n olt-6 s the gmm irrition soure in 4 n 5. After irrition, fruits were kept t 5 ± o C n 4 o C storge onitions for ys n 3 ys, respetively, fter whih ey ws estimte s perentge. Eh tretment ontine 8 replites (fruits), n the experiment ws repete t lest two times. Sttistil nlysis. The t were sujete to nlysis of vrine using SAS JMP softwre (SAS Institute, 995). Differenes mong tretment mens were ssesse using the lest signifint ifferene test n signifine ws estlishe t P <.5. All the experiments were performe t lest two times. For eh experiment, the t were nlyze seprtely, n the results of one representtive experiment re presente. Results n Disussion Effet of gmm irrition on oniil germintion of C. gloeosporioies n P. expnsum. When the oniil spores of C. gloeosporioies n P. expnsum were expose to irrition t oses rnging from. to. kgy uner in vitro onitions, ifferent egrees of impirment of oniil germintion n germ tue lengths were oserve, n these were onfirme y omprison with non-trete ontrol. In n nlysis of survivl with respet to irrition ose, the gmm irrition oses require to reue the oniil popultion y 9%, the osimetry vlues were. n.35 kgy for C. gloeosporioies (Fig. A) n P. expnsum (Fig. B), respetively. Irrition oses rnging from.4 to. kgy resulte in n inrese perentge of oniil germintion inhiition for C. gloeosporioies to greter level ompre with the non-trete ontrol. However, the oses. kgy use 37.6% n 9.6% inhiition for C. gloeosporioies (Fig. C) n P. expnsum (Fig. D). The mirosopi oservtions of C. gloeosporioies t mgnifition of 4 showe tht the germ tue length erese grully when spores were trete with inresing oses of rition. An irrition ose t 5. kgy use no germ tue formtion in C. gloeosporioies t 7 h fter inution t 5 o C, while germ tue formtion ws oserve t.5 kgy ose (Fig. A, B). In the spore germintion nlysis using hemoytometer, the gretest perentge of spore inhiition ws oserve t 5. kgy ose, n spore germintion ws rreste ompletely t 4. kgy (Fig. C). However, lower level of germintion ws oserve for ose of. kgy in omprison with the non-trete ontrol. Doses of >. kgy re neessry to ontrol some posthrvest pthogens, ut Fuji pples n Niitk pers irrite t oses greter thn.8 kgy show ltertions in the physiologil progress of firmness (Jung et l., 4). A previous stuy y Ci et l. (7) reporte tht posthrvest nthrnose isese use y C. gloeosporioies in ppy ws ontrolle y the omine pplition of ultrviolet-c n gmm irrition. Similrly, prestorge het tretment improves the qulity of mngo fruits y reuing posthrvest ey use y C. gloeosporioies following ol storge t 4 o C (Kest et l., ). In the se of P. expnsum, there ws no germ tue formtion or spore germintion t ny ose of irrition, even 3 ys fter inution (Fig. 3). Previous stuies hve shown tht irrition tretment inhiits myelil growth in P. expnsum t oses > 3. kgy (Mostfvi et l., ). A ose of pproximtely 6 Gy frequently
Posthrvest Dey y Gmm Irrition n Fumigtion 463 A Colletotrihum gloeosporioies B Peniillium expnsum 5 6 y= 4.4837x +4.38 4 R =.8868 5 y=.7839x +5.36 D =.3 4 R =.9786 3 D =.359 Log (spores/ml)..4.6.8. Log (spores/ml) 3..4.6.8...4.6.8. C D Growth inhiition rte (%) 8 6 4 Growth inhiition rte (%) 8 6 4..3.4.5.. e..3.4.5.. Fig.. Effet of ifferent oses of gmm irrition on the oniil germintion of Colletotrihum gloeosporioies n Peniillium expnsum. Spores per milliliter of C. gloeosporioies (A) n P. expnsum (B) n inhiition rte (%) of oniil germintion of C. gloeosporioies (C) n P. expnsum (D) t ifferent oses of irrition tretment ompre with the non-trete ontrol. Surviving vile miroorgnisms were plotte ginst irrition ose. The experiment ws repete t lest two times with three replites per tretment. Brs with the sme letters inite no sttistil ifferene etween the trete n non-trete ontrol, oring to the lest signifint ifferene test (P <.5). kille P. expnsum spores. Our results re onsistent with those of Jitreert et l. (5), who showe tht gmm irrition ontrols spore germintion n myelil growth in P. expnsum. In ition, the omplete inhiition of fungl growth hs een reporte s result of rition-inue DNA mge, whih results in nonfuntioning ells n ell wouning (Monk et l., 995; Smith n Pilli, 4). Gmm irrition hs een shown to suessfully intivte fungi from vrious griulturl ommoities (De Silv et l., 6). Interestingly, reent stuy y Jeong et l. (5) hs shown tht three fungl pthogens hve greter sensitivity to the e-em tretment thn to gmm or X-ry irritions. However, few previous stuies hve shown tht plnt pthogeni fungi iffer with respet to riosensitivity to irrition (Sleh et l., 988; Tiryki, 99). Aoringly, gmm irrition oul e n lterntive metho to reue fungiie use (Ci et l., 7). In vitro ntifungl tivity ginst C. gloeosporioies n P. expnsum in response to fumigtion. The egree of ntifungl tivity is presente s the rte of inhiition in omprison with the non-trete ontrol. The EDN fumigtion tretment t.5 g n. g oses use % growth inhiition rte in oth posthrvest pthogens C. gloeosporioies n P. expnsum; while fumigtion tretment t oses elow.5 g resulte in 77.5% n 85.7% inhiition of fungl growth for C. gloeosporioies n P. expnsum, respetively (Fig. 4). An EDN ose >.5 g use omplete growth inhiition for the two pthogens. Previous reports (Sholerg n Rnll, 7; Song et l., 996) hve suggeste tht the vpors of hexnl, C-6 ron lehye, re effetive for the ontrol of oth lue n gry mols on mture pples. Mny previous
464 Cheon et l. A 4 h 7 h B.5 kgy.5 kgy. kgy. kgy 4. kgy 5. kgy. Germ tue length ( m) C Coniil germintion (%).5..5 5 5 5.5.. 4. 5. e f.5.. 4. 5. Fig.. (A) Mirosopi oservtions of the fungl spore germintion fter gmm rition tretment on Colletotrihum gloeosporioies uring 4 h n 7 h. Sle r = µm. (B) Germ tue length ws rstilly reue upon the inresing of irrition ose. (C) The perent oniil germintion rte ws reue t higher oses of irrition. The germintion ounting ws one using hemoytometer. The experiment ws repete three times with three replites per tretment. Brs with the sme letters inite no sttistil ifferene etween the trete n non-trete ontrol, oring to the lest signifint ifferene test (P <.5). kgy.5 kgy. kgy. kgy 4. kgy 5. kgy Fig. 3. Mirosopi oservtions of the spore germintion of Peniillium expnsum t 7 h fter gmm irrition tretment in omprison with the non-trete ontrol. There ws no formtion of germ tue. The spore mge ourre y the irrition tretment t the oses >. kgy. stuies support our results. Lee et l. (9) reporte tht the voltile orgni ompouns (VOCs) from Oxyporus ltemrgintus EF69 inhiit severl pthogeni fungi, inluing C. gloeosporioies. The purifie VOC 5-pentyl- -furlehye inhiits the myelil growth of Rhizotoni solni in ose-epenent mnner. The pplition of higher onentrtions of hemils to ontrol nthrnose isese inreses the risk of high levels of toxi resiues
Posthrvest Dey y Gmm Irrition n Fumigtion 465 Colletotrihum gloeosporioies Peniillium expnsum Growth inhiition rte (%) 8 6 4 Growth inhiition rte (%) 8 6 4 EDN. g EDN.5 g EDN. g EDN. g EDN.5 g Tretments Tretments EDN. g Fig. 4. Antifungl tivity of n eo-frienly fumignt ethneinitrile (EDN) ginst Colletotrihum gloeosporioies n Peniillium expnsum in omprison with the non-trete ontrol uner in vitro onitions. The experiment ws repete t lest one with three replites per tretment. Brs with the sme letters inite no sttistil ifferene etween the trete n non-trete ontrol, oring to the lest signifint ifferene test (P <.5). The photogrphx were tken 7 ys fter inution t 5 o C. in ppy (Hernnez-Aliter et l., 7). The myelil growth of C. gloeosporioies n C. uttum ws inhiite y VOCs from yest, n the exposure of pthogeni fungi to VOCs inues n inrese in peroxition levels, initing the ourrene of oxitive stress (Rezene et l., 5). A previous report revele tht the voltile oil of Illiium verum strongly inhiits the myelil growth of the posthrvest pthogens Botrytis inere n C. gloeosporioies y > 9% s nturl fumignt (Lee et l., 7), suggesting tht nturl eo-frienly fumignts re highly effetive for the suppression of fungl growth. Effet of omine gmm irrition n fumigtion to ontrol posthrvest iseses use y C. gloeosporioies n P. expnsum in pples uner storge onitions. The omine tretment of gmm irrition n EDN resulte in greter ontrol of oth iseses uner oth mient (5 ± o C) n ol (4 o C) tempertures s ompre with either tretment lone. The egree of isese reution ws ssesse s the inhiition perentge. The omine tretment with.5 g EDN n.4 kgy of irrition use 7.8% inhiition of C. gloeosporioies, whih ws the highest isese ontrol rte mong ll the tretments with respet to pple nthrnose (Fig. 5A). There ws no ifferene in the perentge of isese ontrol of C. gloeosporioies etween temperture onitions of 5 ± o C n 4 o C for storge perio of ys n 3 ys, respetively. The mximum inhiition of pple nthrnose use y C. gloeosporioies ws oserve for the omine tretment with EDN (.5 g) + irrition (.4 kgy), followe y the omine tretment with EDN (.5 g) + irrition (. kgy) uner oth temperture onitions. Our results re lso onsistent with those of Jung et l. (4), who lso esrie the signifine of the omine tretment with irrition. In the se of lue mol isese ontrol use y P. expnsum, the mximum levels of isese ontrol were 88.4% n 87.7% for the omine tretments of EDN (.5 g) + irrition (.4 kgy) n EDN (.5 g) + irrition (. kgy), respetively, for storge urtion of ys t n mient temperture (5 ± o C). However, interestingly, the fumignt tretment of.5 g of EDN lone ws effetive for isese ontrol n the effets were nerly equl to those oserve in the omine tretment of EDN (. g) + irrition (.4 kgy) (Fig. 5B). The omine tretment EDN (.5 g) + irrition (.4 kgy) showe the mximum isese ontrol of 83.4% for the storge perio of 3 ys t 4 o C, n the next highest isese ontrol ws 7.5% for the omine tretment of EDN (.5 g) + irrition (. kgy). Our results suggest tht gmm irrition n ontriute to reution in posthrvest iseses y reuing the use of fungiies. Fig. 6 shows isese ontrol uring posthrvest ey y C. gloeosporioies n P. expnsum for the omine tretment of EDN (.5 g) + irrition (.4 kgy) in omprison with the untrete ontrol uner two ifferent
466 Cheon et l. A Disese ontrol (%) 8 6 4 Colletotrihum gloeosporioies 5 +C o 5+C o B Disese ontrol (%) 8 6 4 Peniillium expnsum e EDN. g + IR.4 kgy EDN. g + IR. kgy EDN.5 g + IR. kgy EDN.5 g + IR.4 kgy Tretments EDN. g EDN.5 g IR. kgy IR.4 kgy EDN. g + IR.4 kgy EDN. g + IR. kgy EDN.5 g + IR.4 kgy EDN.5 g + IR. kgy Tretments EDN. g EDN.5 g IR. kgy IR.4 kgy 4C o 4C o Disese ontrol (%) 8 6 4 e ef f ef Disese ontrol (%) 8 6 4 f e EDN. g + IR.4 kgy EDN. g + IR. kgy EDN.5 g + IR. kgy EDN.5 g + IR.4 kgy Tretments EDN. g EDN.5 g IR. kgy IR.4 kgy EDN. g + IR.4 kgy EDN. g + IR. kgy EDN.5 g + IR. kgy EDN.5 g + IR.4 kgy Tretments EDN. g EDN.5 g IR. kgy IR.4 kgy Fig. 5. Evlution of omine pplition of irrition with fumigtion t vrious oses in ontrol of nthrnose n lue mol use y Colletotrihum gloeosporioies (A) n Peniillium expnsum (B), respetively, t two ifferent storge tempertures (5 ± o C n 4 o C) on posthrvest pple fruits. The experiment ws repete t lest one with 8 replites (fruits) per tretment. Brs with the sme letters inite no sttistil ifferene etween the trete n non-trete ontrol, oring to the lest signifint ifferene test (P <.5). EDN, ethneinitrile; IR, irrition. temperture onitions 5 ± o C n 4 o C for ys n 3 ys, respetively. Among the evelopment of ionizing rition tehnologies, gmm ry irrition hs een wiely investigte for vrious griulturl ommoities to protet ginst posthrvest fungl pthogens, inluing C. gloeosporioies (Ci et l., 7) n P. expnsum (Mostfvi et l., ). These stuies generlly show posthrvest isese ontrol t low oses thn t high oses, suggesting tht gmm irrition t <.75 kgy inhiits oniil germintion when omine with fumigtion (Ci et l., 7). These results re in greement with our results. In onlusion, low oses of gmm irrition proue stimultory effet on isese ontrol in omintion with fumigtion. The lethl oses reore were. kgy n.4 kgy for riosensitive speies, n these results were ttriute the high suppression of isese evelopment when the tretments were omine with fumigtion.
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