Enhancement of biocontrol activity of yeasts by adding sodium bicarbonate or ammonium molybdate to control postharvest disease of jujube fruits

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1 Lttrs in Applid Miroiology 2003, 37, doi: /j x x Enhnmnt of ioontrol tivity of ysts y dding sodium iront or mmonium molydt to ontrol posthrvst diss of juju fruits Y.K. Wn, S.P. Tin nd G.Z. Qin Ky Lortory of Photosynthsis nd Environmntl Molulr Physiology, Institut of Botny, Th Chins Admy of Sins, Bijing, Chin 2003/0109: rivd 10 Frury 2003, rvisd 22 April 2003 nd ptd 1 Jun 2003 ABSTRACT Y. K. W A N, S. P. T I A N A N D G. Z. Q I N Aims: To ssss th potntil of sodium iront nd mmonium molydt s dditivs in nhning th ioontrol ffiy of Rhodotorul glutinis nd Cryptoous lurntii ginst lu mould in juju fruits. Mthods nd Rsults: Two ysts t onntrtion of 10 7 CFU ml )1, in omintion with 238 mmol l )1 sodium iront or 15 mmol l )1 mmonium molydt, showd signifint inhiition fft on lu mould of juju fruits stord t 20 C for 5 dys. Th olonizing ility of th ysts in woundd sits ws signifintly drsd in th prsn of mmonium molydt. Conlusions: Comining R. glutinis or C. lurntii with sodium iront or mmonium molydt providd mor fftiv ontrol of posthrvst diss thn using th ntgonisti ysts or th hmils lon. Signifin nd Impt of th Study: Th ddition of sodium iront or mmonium molydt rdud th numr of ntgonists rquird to ffiintly ontrol diss of posthrvst fruits, whih ould rsult in th rdution of osts. Kywords: ntgonisti ysts, ioontrol ffiy, nutritionl sustns, posthrvst diss. INTRODUCTION Juju (Zizyphus juju v. Dongzo) fruit is kind of fvourl nd profitl fruit, nd is muh dmird for its high nutritionl vlu. Th fruit is vry prishl nd highly susptil to posthrvst dy. Although Altrnri ltrnt is min diss of juju fruit in fild vi ltnt inftion, lu mould usd y Pniillium xpnsum is lso on of th most srious posthrvst disss of th fruit in Chin. This diss usully ours in th nd of storg nd during shlf lif nd us srious losss. Us of iprodion, s ommon fungiid, to ontrol this diss is in prti. Howvr, puli onrn ovr food sfty nd th dvlopmnt of fungiid rsistn y pthogns hs inrsd th Corrspondn to: S.P. Tin, Institut of Botny, Th Chins Admy of Sins, Xingshn Nn xinun 20, Hiding Distrit, Bijing , Chin (-mil: shiping@95777.om). srh for ltrntiv mns potntilly lss hrmful to humn hlth nd th nvironmnt (EI-Ghouth nd Wilson 1995; Holms nd Ekrt 1999). In th lst fw yrs, iologil ontrol of posthrvst disss of fruits hs n dvlopd s promising ltrntiv to hmil ontrol (Wilson nd Wisniwski 1989; Wisniwski nd Wilson 1992). Mny tri nd ysts hv n rportd to fftivly ontrol vrious posthrvst disss of fruits (Fn nd Tin 2001; Nuns t l. 2001). Som ysts r now ommrilly vill nd svrl mor r in dvnd stgs of ommriliztion (Krulut t l. 2001). Som ntgonisti miroorgnisms hv shown to fftiv in ontrolling posthrvst dy in vrity of fruits, howvr, thir ioontrol ffiy undr smi-ommril onditions is oftn lowr omprd with hmil ontrol (Droy t l. 1998; EI-Ghouth t l. 1998). In ordr to nhn ioontrol tivity, high onntrtions of ntgonists r ª 2003 Th Soity for Applid Miroiology

2 250 Y.K. WAN ET AL. usully ndd (Pusy nd Wilson 1984; Jnisiwiz 1987; Fn nd Tin 2001). Grt intrst hs rntly n fousd on omining ntgonists with som hmil sustns suh s lium hlorid, 2-doxy-D-gluos nd glyolhitosn to nhn thir ffiy ginst fungl pthogns (EI-Ghouth t l. 2000,; Tin t l. 2001). Compounds lik food dditivs or non-hmil fungiids r prfrrd s thy pos miniml risk to hlth nd n usful in th mngmnt of fungiid-rsistnt strins (Nuns t l. 2002). Among thm, sodium iront nd mmonium molydt xhiit rod-sptrum ntifungl tivity (Corrl t l. 1988; Nuns t l. 2002). Th ddition of mmonium molydt to th yst Cndid sk, signifintly nhnd its ioontrol ffiy in ontrolling posthrvst dy in pr fruits (Nuns t l. 2002). Howvr, thr ws littl rportd us of sodium iront s n dditiv to improv th ffiy of ysts ginst fungl disss in fruits. Th ims of th prsnt study wr (i) to ssss th potntil of sodium iront or mmonium molydt s n dditiv to nhn th ffiy of ysts Rhodotorul glutinis nd Cryptoous lurntii ginst lu mould of juju fruits usd y P. xpnsum, nd (ii) to dtrmin th fft of sodium iront nd mmonium molydt on th growth of th ysts in th juju wounds. MATERIALS AND METHODS Plnt mtril, miroil ulturs nd hmils Juju (Z. juju v. Dongzo) fruits wr hrvstd t ommril mturity from n orhrd in Bingzhou (Shndong Provin, Chin). Th fruits wr stord t 1 C for 1 wk prior to trtmnt. Fruits wr dippd in th solution of 2% sodium hypohlorit for 2 min, rinsd with tp wtr, nd ir-drid for wounding. Pniillium xpnsum ws isoltd from inftd juju fruits nd ulturd on potto dxtros gr t 25 C for 2 wks. Conidil suspnsions wr prprd y flooding th ulturs with stril distilld wtr ontining 0Æ05% (v/v) Twn 80. Th onidil onntrtion of th pthogn ws dtrmind with th id of hmoytomtr nd djustd to spors ml )1. Th ysts R. glutinis nd C. lurntii wr ulturd in 250 ml Erlnmyr flsks with 50 ml of nutrint yst dxtros roth (NYDB: nutrint roth 8 g l )1 ; yst xtrt 5gl )1 ; dxtros 10 g l )1 ) on rotry shkr t 200 rv min )1 for 24 h t 28 C. Th mdium ws ntrifugd t 2500 g for 10 min. Th lls of th ysts wr rsuspndd in stril distilld wtr. Th ll suspnsions wr djustd to onntrtions of 10 7 or 10 8 CFU ml )1, rsptivly, y mns of hmoytomtr. Th sodium iront (NHCO 3 ) (hrftr SBC) nd mmonium molydt [(NH 4 ) 6 Mo7O 24 4H 2 O] (hrftr NH 4 -Mo) wr purhsd from Bijing Chmil Rgnt Compny, Chin. Invstigtion of SBC nd NH 4 -Mo on th growth of ysts First, th onntrtions of SBC nd NH 4 -Mo wr sltd in th prliminry xprimnt. Th rsults inditd tht th ysts omind with 238 nd 15 mmol l )1 NH 4 -Mo showd th st inhiition fft on lu mould of juju fruits (dt not shown). Th fft of 238 mmol l )1 SBC nd 15 mmol l )1 NH 4 -Mo on th popultion of two ysts ws dtrmind in th wounds of juju fruits. Wounds wr md s dsrid prviously (Tin t l. 2001), nd 20 ll 10 7 CFU ml )1 ll suspnsion of h ntgonist lon or in omintion with 238 mmol l )1 SBC or 15 mmol l )1 NH 4 -Mo ws ddd to th wounds. Yst ntgonists wr rovrd from wounds t diffrnt points of tim (1, 24, 48, 72 nd 96 h) ftr trtmnt. Th four rsulting ylindrs (h of 1 m in dimtr nd 1 m dp) from four juju fruits wr pld in mortr nd ground with 10 ml of stril distilld wtr. Thn 50 ll of th sril 10-fold dilutions ws pltd on NYDA mdium. Colonis wr ountd ftr inution t 28 C for 48 h. Dt wr xprssd s log CFU pr wound. Thr wr thr rplits for h trtmnt, nd th xprimnt ws prformd two tims. Invstigtion of SBC nd NH 4 -Mo on ioontrol tivity of th ysts A uniform wound (3 mm dp nd 3 mm wid) ws md t th qutor of h fruit (put on its sid) using stril dissting ndl. Aliquots of 20 ll of 238 mmol l )1 SBC or 15 mmol l )1 NH 4 -Mo lon or in omintion with R. glutinis or C. lurntii t 10 7 CFU ml )1, rsptivly, R. glutinis or C. lurntii t 10 7 nd 10 8 CFU ml )1 nd stril distilld wtr (ontrol) ws pipttd into h wound sit. Aftr 4 h, 20 ll ofp. xpnsum t spor ml )1 ws hllngd. All trtd fruits wr pld in plsti gs to mintin rltiv humidity of out 95% nd kpt t 20 C. Diss inidn ws osrvd ftr 5 dys of inution. Thr wr 10 fruits in h trtmnt nd th trtmnts wr rplitd thr tims. Th xprimnt ws rptd two tims. Sttistil nlysis In this study, ll xprimnts wr rptd two tims nd th rsults of two xprimnts wr ordnt. Th dt from on of th xprimnts wr prsntd in this ppr. All sttistil ª 2003 Th Soity for Applid Miroiology, Lttrs in Applid Miroiology, 37, , doi: /j x x

3 ENHANCED BIOCONTROL EFFICACY OF YEASTS 251 nlyss wr prformd with SPSS (SPSS In., Chigo, IL, USA). To tst th fft of th trtmnts, th dt wr nlysd y nlysis of vrin (ANOVA). Mn sprtions wr prformd using Dunn s multipl rng tst. RESULTS Efft of SBC nd NH 4 -Mo on growth of th ysts SBC nd NH 4 -Mo showd diffrnt inhiitory ffts on th growth of R. glutinis nd C. lurntii (Fig. 1,). Both ysts multiplid similrly in th prsn or sn of SBC, whil thir growth rts wr signifintly rdud y NH 4 -Mo (P ¼ 0Æ05). Within 24 h in th sn of NH 4 - Mo, th popultions of R. glutinis nd C. lurntii inrsd to 37- nd 14-fold, rsptivly. Whil in th prsn of NH 4 -Mo, only 4- nd 8-fold inrss ould osrvd. Log CFU wound () () Hours of inoultion t 20 C Fig. 1 Popultion dynmis of Rhodotorul glutinis () nd Cryptoous lurntii () in wounds of juju fruits inutd t 20 C. Trtmnts inludd: yst suspnsion lon (10 7 CFU ml )1 )(j), yst suspnsion (10 7 CFU ml )1 ) omind with 238 mmol l )1 SBC (), nd yst suspnsion (10 7 CFU ml )1 ) omind with 15 mmol l )1 NH 4 -Mo (m). Vrtil rs rprsnt S.E.M. At h point of tim, dt inditd with diffrnt lttrs r sttistilly diffrnt ording to Dunn s multipl rng tst t P ¼ 0Æ05 Bioontrol tivity of th ysts omind with SBC or NH 4 -Mo ginst posthrvst dy Juju fruits trtd with 15 mmol l )1 NH 4 -Mo lon showd n ovious rdution in dy inidn whn omprd with th ontrol (P ¼ 0Æ05). Th ddition of 238 mmol l )1 SBC or 15 mmol l )1 NH 4 -Mo to R. glutinis mrkdly nhnd th ioontrol ffiy ginst lu mould of juju fruits (Fig. 2) (P ¼ 0Æ05). Th omintion of R. glutinis with SBC nd NH 4 -Mo showd ttr ioontrol ffiy thn th ntgonist lon t 10 8 CFU ml )1. A similr fft ws osrvd with C. lurntii omind with SBC or NH 4 -Mo for th ontrol of lu mould of juju fruits (Fig. 2). Fruits trtd with C. lurntii nd 15 mmol l )1 NH 4 -Mo hd th lowst inftion rt (3Æ3%) of ll trtmnts. DISCUSSION Th rsults of this study indit tht SBC or NH 4 -Mo hv grt potntil to nhn th ioontrol ffiy of R. glutinis nd C. lurntii ginst lu mould on juju fruits (Fig. 2). Th ioontrol ffiy of oth ysts t 10 7 CFU ml )1 omind with 238 mmol l )1 SBC or 15 mmol l )1 NH 4 -Mo ws ttr thn in th s whr th ntgonists wr usd lon t 10 8 CFU ml )1. Th sm fft ws osrvd in th s of omining NH 4 -Mo with C. sk ginst posthrvst dy usd y P. xpnsum (Nuns t l. 2002), who found tht NH 4 -Mo mrkdly rdud diss inidn nd svrity of lu mould in Inftd wounds (%) d A B C D E F G H I J K Trtmnts Fig. 2 Inhiition of lu mould in juju fruits s fftd y vrious trtmnts. (A) Control; (B) 238 mmol l )1 SBC; (C) 15 mmol l )1 NH 4 -Mo; (D) Rhodotorul glutinis (10 8 CFU ml )1 ); (E) R. glutinis (10 7 CFU ml )1 ); (F) R. glutinis (10 7 CFU ml )1 ) omind with SBC; (G) R. glutinis (10 7 CFU ml )1 ) omind with NH 4 -Mo; (H) Cryptoous lurntii (10 8 CFU ml )1 ); (I) C. lurntii (10 7 CFU ml )1 ); (J) C. lurntii (10 7 CFU ml )1 ) omind with SBC; (K) C. lurntii (10 7 CFU ml )1 ) omind with NH 4 -Mo. Dt in olumns with diffrnt lttrs r sttistilly diffrnt ording to Dunn s multipl rng tst t P ¼ 0Æ05 d f ª 2003 Th Soity for Applid Miroiology, Lttrs in Applid Miroiology, 37, , doi: /j x x

4 252 Y.K. WAN ET AL. ph fruits. Th ddition of NH 4 -Mo to C. sk nhnd its ioontrol ffiy ginst posthrvst disss of ph fruits. Although Krulut t l. (2001) rportd tht 2% SBC ould signifintly rdu posthrvst lu mould dy of swt hrris, thr is littl informtion out its pplition s n dditiv to improv th ioontrol ffiy of yst ntgonists. This study suggsts nw pproh sd on th omintion of SBC with ntgonists for th ontrol of posthrvst diss in stord fruits. In gnrl, rpid oloniztion nd omptition for sp nd nutrints in wounds ply mjor rol in th ioontrol mhnism of yst ntgonists (Wilson nd Wisniwski 1989; Wilson t l. 1993). Thr is dirt rltionship twn th popultion of th ntgonist nd th ioontrol ffiy (Jnisiwiz 1988; Fn nd Tin 2000). Our rsults showd tht th growth of R. glutinis nd C. lurntii ws grtly fftd y th prsn of NH 4 -Mo in th woundd sits (Fig. 2). Howvr, th rdution in th popultion of th ysts in woundd sits did not fft th ioontrol ffiy of th ntgonists. This rsult ws in ordn with prvious rports of Nuns t l. (2002,), who found tht th ility of C. sk to oloniz wounds of ppl nd pr fruits ws fftd in th prsn of NH 4 -Mo, ut th drs in popultion did not sm to influn th ontrol of dy. NH 4 -Mo hs n rportd to strong inhiitor of id phosphts tivity (PTPs) (Glw t l. 1988), nd th pross of phosphoryltion/dphosphoryltion is of mjor importn in rgultion of mny llulr prosss (Huntr 1995). Nuns t l. (2002) onludd tht in ddition to th dirt improvmnt of ioontrol fftivnss, PTPs tivity ould involvd. Furthr rsrh is ndd to luidt th pris mhnisms y whih SBC nd NH 4 -Mo improv th ioontrol tivity of ysts. SBC nd NH 4 -Mo r nvironmntlly sf nd hp rgnts. SBC is widly usd for ontrolling ph, tst nd txtur in food industry. Toxiologil xprimnts showd tht NH 4 -Mo did not ld to mortlity or ltrtions in tstd nimls (Nuns t l. 2002). Thrfor, in onlusion, this study dmonstrtd tht omining yst ntgonists R. glutinis nd C. lurntii with 238 mmol l )1 SBC or 15 mmol l )1 NH 4 -Mo ould xptd to provid mor fftiv ontrol thn using th ysts or th hmils lon. Mnwhil, using SBC or NH 4 -Mo s n dditiv signifintly dlin th numr of ntgonists rquird to ompltly ontrol diss of posthrvst fruits, s wll s filitt th ntgonists prtil implmnttion. ACKNOWLEDGEMENTS This study ws supportd y th Ntionl Sin Fund for Distinguishd Young Sholrs of Chin ( ) nd th Ntionl Nturl Sin Foundtion of Chin ( ). REFERENCES Corrl, L.G., Post, L.S. nd Montvill, T.J. (1988) Antimiroil tivity of sodium iront. Journl of Food Sin 53, Droy, S., Cohn, L., Dus, A., Wiss, B., Horv, B., Chlutz, E., Ktz, H., Krn-Tzur, M. t l. (1998) Commril tsting of Aspir: yst prprtion for th iologil ontrol of posthrvst dy of itrus. Biologil Control 12, EI-Ghouth, A. nd Wilson, C.L. (1995) Biologilly-sd thnologis for th ontrol of posthrvst disss. 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5 ENHANCED BIOCONTROL EFFICACY OF YEASTS 253 Tin, S.P., Fn, Q., Xu, Y. nd Wng, Y. (2001) Effts of Trihosporon sp. in omintion with lium nd fungiid on ioontrol of posthrvst disss in ppl fruits. At Botni Sini 43, Wilson, C.L. nd Wisniwski, M.E. (1989) Biologil ontrol of posthrvst disss of fruits nd vgtls: n mrging thnology. Annul Rviw of Phytopthology 27, Wilson, C.L, Wisniwski, M.E., Droy, S. nd Chlutz, E. (1993) A sltion strtgy for miroil ntgonists to ontrol posthrvst disss of fruits nd vgtls. Sinti Hortiultur 53, Wisniwski, M.E. nd Wilson, C.L. (1992) Biologil ontrol of posthrvst disss of fruits nd vgtls: rnt dvns. Hortsin 27, ª 2003 Th Soity for Applid Miroiology, Lttrs in Applid Miroiology, 37, , doi: /j x x