Efecto Agua Caliente Antracnosis Papaya

Postharvest Biology and Technology 86 (2013) 437446

Contents lists available at ScienceDirect

Postharvest Biology and Technology

jou rn al h om epage: www.elsev ier .com/ l

Effects diand its

Xueping WeState Key Labo ovinciTechnology of F ngzhou

a r t i c l

Article history:Received 11 MAccepted 22 Ju

Keywords:Hot water treaFruit ripeningEpicuticular wDisease resistaPapaya

due (HWontroe resiolletohracnoringn the

ata barri

CpPGIP and promptly induced the expression of CpNPR1, and then regulated the expression of the CpPR1gene, which may enhance the resistance of the fruit to anthracnose disease and reduce the decay rate.Together, these results conrm that HWT could reduce disease incidence and induce resistance, and thusmaintain postharvest quality during storage and prolong the shelf-life of papaya fruit.

2013 Elsevier B.V. All rights reserved.

1. Introdu

Papaya ishable aftesusceptibilifactor that extension ois one of thet al., 2007vest diseasfruit and rehard to prereduce quaand transpof postharvHowever, dcern over ththe environstrategies fet al., 2009)

CorresponE-mail add

1 These auth

0925-5214/$ http://dx.doi.oction

(Carica papaya L.) is a climacteric fruit and very per-r harvesting, with rapid ripening and softening, andty to biotic or abiotic stresses (Kader, 2002). An importmay affect papaya postharvest quality and limit thef storage life is the occurrence of rots. Anthracnosee most important postharvest diseases in papaya (Cia), as well as other fruit (Shi et al., 2011). Posthar-es, especially anthracnose, can infect young papayamain latent during fruit growth in the eld Thus it isvent disease development, and this may signicantlylity and commercial value of the fruit during storageort. Anthracnose is usually controlled by applicationest fungicides such as prochloraz or propiconazole.evelopment of fungicide resistance, and public con-e potential impact of fungicides on human health andment have created interest in seeking new alternativeor disease management (Gamagae et al., 2004; Droby. Extensive research is imperative to develop advanced

ding author. Tel.: +86 20 38294892; fax: +86 20 85288280.ress: [email protected] (W. Chen).ors contributed equally to this work.

postharvest treatments in environmentally friendly ways tomaintain high commercial quality during the storage and mar-keting period. Different postharvest diseases of various fruit havebeen effectively controlled using modied atmosphere packag-ing (Karabulut and Baykal, 2004), sodium chloride (Malakou andNanos, 2005), biocontrol (Shi et al., 2010), antagonistic microor-ganisms (Calvo et al., 2010), salicylic acid (Chan et al., 2008), borateapplication (Shi et al., 2011, 2012), etc.

Among various non-chemical approaches, hot water treatment(HWT) appears to be one of the most effective and promising meth-ods, especially for organically grown crops, to control relativelyhigh rates of postharvest decay in environmentally friendly ways(Mari et al., 2007; Jemric et al., 2011; Fruk et al., 2012; Liu et al.,2012). HWT has been known for a relatively long time as an effec-tive and economic method for controlling plant pathogens (Rodovet al., 1995, 2000; Schirra and DHallewin, 1997; Hong et al., 2007;Liu et al., 2012), improving fruit resistance to chilling (Rodov et al.,1995; Schirra et al., 2004; Lu et al., 2010a; Rui et al., 2010), inhibitingripening of many fruit and vegetables, and alleviating some physio-logical storage disorders (Fallik, 2004; Ciou et al., 2011; Jemric et al.,2011), thus, maintaining fruit quality and prolonging storage (Kleinand Lurie, 1991; Malakou and Nanos, 2005; Fruk et al., 2012; Liuet al., 2012). There has therefore, been increasing interest in usingpostharvest heat treatments for fruit. Ripening of most climac-teric fruit is characterized by softening of the esh, enhanced color

see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.postharvbio.2013.07.037 of hot water treatment on anthracnose possible mechanism

Li1, Xiaoyang Zhu1, Nan Zhao, Danwen Fu, Jun Li, ratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Prruits and Vegetables, College of Horticulture, South China Agricultural University, Gua

e i n f o

ay 2013ly 2013

tment

axnce

a b s t r a c t

Harvested papaya fruit are perishableabiotic stresses. Hot water treatmentstudy investigated effects of HWT on canism by which HWT induced diseaseffect on reducing the carrier rate of Cicantly inhibited the incidence of antslightly promoted the rate of fruit colcertain extent and induced changes iwax to melt. The cracks and most stomwax, thereby providing a mechanicalocate /postharvbio

sease in papaya fruit

n Chen, Weixin Chen

al Key Laboratory for Postharvest Science and 510642, PR China

to rapid ripening and softening and susceptibility to biotic orT) can preserve fruit quality by reducing decay. The presentlling fungal pathogens of papaya fruit and the possible mech-stance. HWT (54 C, 4 min) of papaya fruit had a pronouncedtrichum gloeosporioides (C. gloeosporioides) in fruit peel, signif-ose and stem-end rot, effectively delayed fruit softening, but. HWT reduced the anthracnose index and fruit ripeness to a

wax arrangement on the surface of treated fruit, causing theappeared to be partially or completely plugged by the melteder against wound pathogens. HWT induced the expression of

438 X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446

development, increased sugar/acid ratio, respiratory activity andethylene production (Prasanna et al., 2007). Exposing fruit to hightemperatures retards some of these processes while enhancingothers. The physiological and biochemical changes in heat-treatedfruit are motreated fruiduring stor

Postharvon both plaet al., 2012nounced efmaintainingHowever, ia negative ihave reportand growth2012). Withdecay by Hinduce a dpreventing Yehoshua ea few studihot water, tapplicationet al., 1998;atic studiesunderlying

Expansinplant growtand cell expcell walls foripening, pacell wall prorole in cell relationshiphave been s2008). Fruiassociated (Barritt, 198

In the prfor papayamolecular bstudied. Thphysiologicanisms of s

2. Materia

2.1. Plant m

Papaya frity indicatharvested fSouth Chinaand maturiease or bledipped in ainvestigatiomost effecting of papawater (54

25 C. The cature watertimes and eplaced into

25 C. Samples were collected at 0, 12, 24 and 36 h and 2, 3, 4, 5,6, 7, 8, 9 and 10 days after treatment. For all samples, peel aroundthe equatorial fruit, about 2 mm thick, and 2 cm thick esh, werecollected, then frozen in liquid nitrogen and stored at 80 C until

lor in

it peus st

tering tdiam

=

X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446 439

and made up to volume with distilled water for total pectin exam-ination.

A mixture of 80 mg of AIS and 20 mL of distilled water wasstirred in a centrifuge tube for 5 min at room temperature and thecentrifugedextracted tnatants werup to volumnation.

In an ice6 mL of 12.5and then hement followincubation added instetotal pectinturonic acid

2.5. Scannin

SEM anaet al. (Hu ettissue (2 mmserved in a and 5 mL ofthe slices wdried at a crwere mounsilver glue ried out witNetherland

2.6. RNA ex

Total RNby Wan andusing an RNtial DNA coevaluated. (i.e. OD260analyses. Threverse tranfor RT-PCR.PCR ampliand CpPR1 NCBI and Cpprimers weto one cyclefor 45 s andPCR producpGEM-Teasby Beijing G

2.7. Northe

Total RNby electrop(morpholinpositively-cCo., Sarasoting for 2 h aan ultraviolThe membrized formamDiagnostics(pH 7.0) an

Hybridization was then performed overnight in the same buffercontaining the gene-specic digoxin (DIG)-labeled probes at 45 C.Probes were prepared with a DIG probe synthesis kit (Roche Diag-nostics) by following the manufacturers instructions. Following

ization, membranes were washed twice for 10 min with 2ntaining 0.1% SDS at 37 C, followed by washing twice in 0.1ntaining 0.1% SDS for 30 min at 63 C. The chemilumines-ignals were detected using CDP-Star TM (Roche Diagnostics,

Mannheim, Germany) as described by the manufacturer.ch treatment (membrane), three repeats were performed.ecic primers used for synthesis of DIG-labeled probes wereal to those used for RT-PCR listed in Supplementary Table 1.

olor inater trnt rep at 10,000 rpm for 10 min at 4 C. The residues werewice with 2 20 mL of distilled water. All the super-e transferred into a 100 mL volumetric ask and madee with distilled water for water soluble pectin exami-

bath, 1 mL of the above sample solution was added to mM sodium tetraborate (in concentrated sulfuric acid)ated for 5 min in a boiling water bath. Color develop-ed addition of 0.1 mL of 0.15% m-hydroxydiphenyl andfor 20 min at room temperature. NaOH (0.1 mL) wasad of 0.15% m-hydroxydiphenyl to the control. Both

and water soluble contents were expressed as galac- equivalents.

g electron microscopy (SEM) analysis

lysis was conducted by the method described by Hu al., 2012), with minor modications. A small piece of

2 mm 1 mm) was torn with forceps, xed and pre-xative solution (90 mL of 50% ethanol, 5 mL of formalin,

acetic acid) until further processing. Before scanning,ere dehydrated in a series of ethanol solutions anditical point of liquid CO2 in a desiccator. The specimensted onto aluminum pecimen stubs using conductiveand sputter-coated with gold. SEM scanning was car-h an XL-30 scanning electron microscope (Philips, Thes) at 20 kV.

traction and genes clone

A was extracted using the hot borate method described Wilkins (1994) and then treated with DNAse I digestionAse-free kit (TaKaRa, Japan) to eliminate the poten-

ntamination. The RNA concentration and purity wereThose RNA samples that had fully lled the criteria/280 > 1.8 and OD260/230 > 2.0) were used for furthere extracted total RNA was reversed transcribed withscription kit into cDNA, which were used as templates

The product (the rst-strand cDNA) was subjected tocation. The primer pairs of CpEXP1, CpPGIP, CpNPR1,were designed based on the sequences obtained fromEXP2 was cloned by using degenerate primers. All the

re listed in Supplementary Table 1. PCR were subjected of 94 C for 3 min, 35 cycles each at 94 C for 1 min, 52 C

72 C for 2 min, and then one cycle of 72 C for 10 min.ts of the predicted size were puried and cloned intoy vector (Promega, Madison, WI, USA) and sequencedenomics Institute (BGI, China).

rn blot analysis

A (10 g per lane) from each sample was separatedhoresis on 1.0% agarose-formadehyde gel in 1 MOPSopropanesulfonic acid) buffer and capillary blotted ontoharged nylon membrane (Biodyne B, 0.45 m, PALLa, FL). The RNA was xed to the membrane by bak-t 80 C and then cross-linked to the membranes usinget cross linker (Amersham Biosciences, Piscataway, NJ).anes were pre-hybridized in SDS buffer [50% deion-ide (v/v), 5 SSC, 7% SDS, 2% blocking reagent (Roche

, Mannheim, Germany), 50 mM sodium-phosphated 0.1% N-lauroylsarcosine (w/v)] for more than 3 h.

hybridSSC coSSC cocence sGmbh,For eaThe spidentic

Fig. 1. Cof hot wdata poidex (A), water soluble pectin content (B) and total pectin content (C)eated fruit or untreated fruit (control) during ripening at 25 C. Eachresents a mean standard error (n = 3).


2.8. Statistical analysis

All experiments were arranged in completely randomizeddesign, and each treatment comprised of three replicates. Datawere tested by the analysis of variance using SPSS version 16.0.The Duncans multiple range tests (P < 0.05) were used to comparedifferences among mean values.

3. Results

3.1. Fruit evaluation during storage

Changes in visual inspection of the peel color, respiration, andethylene for at least thirty fruit per treatment group were period-ically monitored. As shown in Fig. 1A, fruit color index graduallyincreased as a function of storage time and almost turned yellow at6 days in the HWT-group, but the control fruit fully turned yellowat 8 days. Clearly the HWT accelerated peel de-greening of thefruit (Fig. 1A).

Correlation analysis showed that ethylene production, colorchange and respiration rate had positive correlations. Signicantcorrelation was observed between the ethylene production andthe respiration rate (R2 = 0.8151) (Supplementary Fig. 1).

Pectin degradation plays an important role in fruit softening.As shown in Fig. 1B, total pectin showed a moderate increase inthe earlier period but decreased rapidly in later stages. During thelater storage times, the level of total pectin in the HWT-fruit washigher than that in the control fruit. HWT signicantly delayedand reduced papaya fruit total pectin decrease during the laterstorage period. In contrast, water soluble pectin (WSP) contentgradually increased during the entire storage time (Fig. 1C), andWSP in HWT-fruit was obviously lower than in the control fruit.

Correlation analysis showed that fruit rmness with watersoluble pectin, PME activities and PG activities displayed negativecorrelations. Fruit rmness was signicantly and negatively corre-lated with WSP context (R2 = 0.9407) (Supplementary Fig. 2A) andPME activities (R2 = 0.9958) (Supplementary Fig. 2D). However, noobviously close relationship was observed between fruit rmnessand total soluble pectin (Supplementary Fig. 2B).

3.2. The effect on carrier rate of C. gloeosporioides in fruit peel

As shown in Fig. 2A, HWT had a strong impact on carrier rateof C. gloeosporioides in fruit peel. After 4 days storage, the carrierrate of C. gloeosporioides in control fruit was about 48%, comparedto 20% in the HWT fruit, HWT effectively reduced the carrier rateof C. gloeosporioides in peel by 58.3%.

3.3. Effect of hot water treatment on disease inhibition

HWT signicantly reduced the incidence of decay. As shown inFig. 2B, HWT signicantly prevented anthracnose. The DI was 13.12in the control fruit at 6 d but was only 4.37 in the HWT-fruit, witha maximum inhibition of 66.6%. At 7 d after treatment, the con-trol fruit were nearly decayed with a DI of 23.35, whereas the DIfor the HWT-fruit was 10.37. The controlling effect reached a levelof 55.4%. At 8 d after treatment, the control fruit were over-ripenand anthracnose expanded rapidly, with a DI of 43.34. However,anthracnose expanded at a relatively low rate in the treated fruit,with a DI of 27.12. The controlling effect reached a level of 37.42%.

HWT signicantly controlled stem-end rot as well. As shown inFig. 2C, HWT signicantly reduced stem-end rot incidence and DI(P < 0.05). At 7 d after treatment, the disease incidence was 35.3%that of the control fruit, compare with a disease incidence of 11.8%

Fig. 2. The eff er rateon control of a t. Eachdifferent at P ects of HWT on the carrier rate of Colletotrichum and disease control. (A) The carrinthracnose. (C and D) The effect of hot water treatment on control of stem end ro

0.05. of Colletotrichum gloeosporioides in fruit peel. (B) The effect of HWT data point represents a mean standard error (n = 3). *Signicantly


Fig. 3. Relative correlation coefcient of fruit ripeness with disease index.

in HWT-fruit, which was reduced by almost 3-fold as compared tothat of the control fruit. The DI in HWT-fruit was 9.88 at 7 d, whichwas 50% that of the control (Fig. 2D).

3.4. Correlation of anthracnose index and fruit ripeness

The relationship between anthracnose index and fruit ripenessis shown inlated with fwas highly0.9796. Signnose indexa relativelythe correlaripeness to cantly delay

3.5. The effeepicuticular

As showregularly a

wax melted to oil drop dimensions and unevenly covered the fruitsurface in the treated fruit (Fig. 4B1). There were several naturalopenings on the untreated fruit surface (Fig. 4A2 and A3), but theopenings or stomas were sealed with molten wax material causedby HWT (Fig. 4B2 and B3). It is supposed that the molten wax causedby HWT partially or entirely sealed natural openings in the epider-mis, and this could physically prevent the invasion of the pathogen,and thus reduce disease incidence.

3.6. Differential expression of CpEXP1 and CpEXP2

Two expansin genes, CpEXP1 and CpEXP2, were cloned and theirexpression patterns were investigated in papaya fruit peel and pulp.As shown in Figs. 5 and 6, the stable levels of CpEXP1 and CpEXP2mRNA gradually increased throughout the entire storage period,both in peel and pulp with fruit ripening. CpEXP1 expression in theHWH-fruit of peel was signicantly lower than that in the controlfruit (Fig. 5). However, the CpEXP2 mRNA levels in HWT-fruit wereslightly higher than those in the control fruit during the rst 2 days,but no noticeable differences were observed thereafter (Fig. 5). Theresults showed that HWT inhibited CpEXP1 expression but slightlyinduced CpEXP2 expression during the early period in the peel. Sim-ilar changes were observed in the pulp (Fig. 6), but the inhibition ofCpEXP1 expression by HWT in pulp was more severe than in peel.

3.7. Regulation of expression of CpPGIP, CpNPR1, CpPR1 genes byter treatment

GIP eith sntly contpapan pesion

andh CpN. 7 an. In p

uncut th1 wa Fig. 3. The DI was signicantly and positively corre-ruit ripeness (Fig. 3). In the control fruit, the correlation

signicant (P < 0.01), with a correlation coefcient oficant correlation was also observed between anthrac-

and fruit ripeness in the treated fruit (P < 0.05), with low correlation coefcient of 0.8535. HWT reducedtion coefcient between anthracnose index and fruitsome extent. These results indicated that HWT signi-ed the development of anthracnose.

cts of hot water treatment on structural changes of wax

n in Fig. 4, the wax material on the untreated fruit wasrrayed as ovuliferous scales (Fig. 4A1). However, the

hot wa

CpPand wtransiein the in the lower iexpres(Figs. 7

Botin Figsin pulpalmostfruit, bCpNPRFig. 4. Structural changes of epicuticular wax induced bxpression decreased during storage, as the fruit ripenedevere levels of disease. However, CpPGIP expressionincreased at 36 h in HWT-fruit and was higher than thatrol fruit in peel (Fig. 7). A similar scenario was shownya fruit pulp (Fig. 8), but the expression of CpPGIP wasel later than in pulp. These results indicated that CpPGIPin both the peel and pulp were enhanced by HWT

8).PR1 and CpPR1 were cloned in papaya fruit. As shownd 8, CpNPR1 was expressed more strongly in peel thaneel, the steady state levels of CpNPR1 mRNA remainedhanged throughout the rst 4 d period in the controley gradually decreased from 5 d to 9 d (Fig. 7). However,s profoundly induced by HWT, increasing signicantly

y hot water treatment.


Fig. 5. Differential expression patterns of CpEXP1 and CpEXP2 in the pericarp during ripening at 25 C. Total RNA (10 g per lane) was separated by electrophoresis on1.0% agarose-formadehyde gel and hybridized with DIG-labeled probes. The top section showed the RNA gel blot analysis while the bottom section indicated the ethidiumbromide-stained rRNA bands as a loading control of the gel. The graphics below indicated the ratio between the signal detected in each lane and the corresponding upperband of the ethidium bromide stained gel (integrating optical density: IOD) using GelPro Analyzer 4.0 software.

Fig. 6. Differential expression patterns of CpEXP1 and CpEXP2 in the peel and pulp during ripening at 25 C. Total RNA (10 g per lane) was separated by electrophoresis on1.0% agarose-formadehyde gel and hybridized with DIG-labeled probes. The top section showed the RNA gel blot analysis while the bottom section indicated the ethidiumbromide-stained rRNA bands as a loading control of the gel. The graphics below indicated the ratio between the signal detected in each lane and the corresponding upperband of the ethidium bromide stained gel (IOD) using GelPro Analyzer 4.0 software.


Fig. 7. Regulahybridized wieach lane and

from 12 h toa low level.control fruiexpressed wno signicaat 2 d (Fig. expression

The exprpathogen inonly be detto 9 d withpeel in conwas delayeincreased. Hcontrol fruiundetectab(Fig. 8). Thfoundly ind

4. Discussi

Papaya irespiration attention dtal pollutiotion of CpPGIP, CpNPR1 and CpPR1 in HWT-fruit in pericarp. Total RNA (10 g per lane) wath DIG-labeled probes. Ethidium bromide-stained rRNA was used as the loading control. the corresponding upper band of the ethidium bromide stained gel (IOD) using GelPro A

48 h in treated fruit and then gradually decreasing to The level in HWT fruit was still higher than that in thet during the entire storage period (Fig. 7). CpNPR1 waseakly in the pulp both in control and treated fruit and

nt difference was found between both groups except8). These results indicated that HWT could induce theof CpNPR1 in peel of papaya fruit.ession of CpPR1 gene showed a close relationship withfection. As shown in Fig. 7, CpPR1 accumulation could

ected after 3 d and was dramatically increased from 5 d the occurrence of pathogen, which severely infectedtrol fruit (Fig. 7). In HWT-fruit, CpPR1 accumulationd and was only detected after 5 d, then dramaticallyowever, its expression was much lower than that in

t (Fig. 7). In contrast to expression in peel, CpPR1 wasle in the pulp tissues during the whole storage periodese results showed that CpPR1 expression was pro-uced by the pathogen and inhibited by HWT.

on

s a typical climacteric fruit. Ethylene production andrate increase as fruit ripen. HWT has drawn increasingue to the growing demand of reducing environmen-n caused by postharvest use of chemical fungicides.

Recently, wendogenoupostharvestobserved thdisease (Figmediated breported thsynthesis wet al., 1988the decreasthe delayedreduced eththe reducedsynthase (AHowever, thHWT needsdisease incto many pabiosynthesihas revealetion and rescontrollingchange.

Many stdelayed frus separated by electrophoresis on 1.0% agarose-formadehyde gel andThe graphics below indicate the ratio between the signal detected innalyzer 4.0 software.

e observed that HWT delayed ripening and reduceds ethylene production and the respiration rate in

papaya fruit (Zhao et al., 2008). In present study, weat HWT also controlled the incidence of postharvest. 2BD). The inhibition of ripening by HWT may bey its effect on ethylene (Luo et al., 2010). It has beenat hot air treatments of 3540 C inhibit ethyleneithin hours in mangoes, apples and tomatoes (Biggs

; Klein, 1989; Ketsa et al., 1999). In the present study,ed ethylene production caused by HWT contributed to

ripening of the fruit. Some studies have reported thatylene production in HWT-fruit might be attributed to

activities of 1-aminocyclopropane-1-carboxylic acidCS) and ACC oxidase (Atta-Aly, 1992; Ketsa et al., 1999).e mechanism of the inhibition of ethylene synthesis by

further investigation. Ethylene is also associated withidence, as change in ethylene production is a responsethogen infections in plants. Pathogens induce ethylenes and produce ethylene themselves as well. Our workd that HWT played a role in inhibiting ethylene produc-piration rate, which may be responsible for its effect on

fruit pathogens, as well on inducing fruit physiological

udies have reported that appropriate heat treatmentit softening, and extended postharvest life of fruit such


Fig. 8. Regula ) washybridized wi ntrol. each lane and lPro A

as nectarin1997), wheet al., 20104 min) dela(Zhao et al.inhibition oPG and PMEtant role in and the hemobserved thin associatiHWT-fruit ening. Studand more inin untreate(Shalom et

Expansinplay an impNorthern bferently affein the earlicaused by Hpulp than into delay of sno signicastage, indicfruit ripeni(Dotto et al

Anthracin papaya. Pand results tion of CpPGIP, CpNPR1 and CpPR1 in HWT-fruit in pulp. Total RNA (10 g per laneth DIG-labeled probes. Ethidium bromide-stained rRNA was used as the loading cothe corresponding upper band of the ethidium bromide stained gel (IOD) using Gee (Fruk et al., 2012) and tomatoes (Lurie and Sabehat,reas some reduced rmness of treated tomatoes (Lua,b). Our previous study has shown that HWT (54 C,yed fruit softening and maintained postharvest quality, 2008). The decrease in softening rate may be due tof the synthesis of cell wall hydrolytic enzymes such as

(Zhao et al., 2008). Pectin degradation plays an impor-fruit softening, which leads to disassembly of celluloseicellulose network (Liu et al., 2009). In this work, weat an increase of water soluble pectin (WSP) contenton with decreased total pectin (TP) were delayed in(Fig. 1), which may account for inhibition of fruit soft-ies on cell walls of apple fruit found less soluble pectinsoluble pectin after exposure to hot-air treatment thand fruit, indicating inhibition of uronic acid degradational., 1996).s, cell wall-associated proteins, have been proposed toortant role in fruit softening (Brummell et al., 1999).

lot analysis in the present work showed that HWT dif-cted expression of two expansin genes. This difference

er postharvest period may be due to a stress responseWT. In addition, CpEXP1 was expressed more strongly in

peel. These results indicate that CpEXP1 may contributeoftening after HWT. With regard to CpEXP2, there werent differences during fruit softening at the later storageating that CpEXP2 has a less signicant relationship withng. Similar results have been obtained in strawberry., 2011) and Creeping Bentgrass (Zhu et al., 2011).nose is one of the most important postharvest diseasesapaya anthracnose has a latent stage in its developmentin considerable loss in papaya production. The infection

progress ofbation and early stageescent untiemerge as Alvarez, 19that HWT ein peel and anthracnosin the posththat gammanthracnosthe occurreing (Cia et arole in redurot (Fig. 2C

In the pthe surfaceto melt, anplugged bybarrier agadisappearafruit and met al., 2000stomata areearly-germmolten waxThe occlusithe encapshave been against dec separated by electrophoresis on 1.0% agarose-formadehyde gel andThe graphics below indicate the ratio between the signal detected innalyzer 4.0 software. latent pathogens includes preinfection, infection, incu-pathogenic periods. Fruit are infected by the fungus ats of maturity in the eld and the fungus remains qui-l the fruit reach the climacteric phase and symptomsanthracnose or chocolate spot lesions (Dickman and83; Gamagae et al., 2003). The present study showedffectively reduced the carrier rate of C. gloeosporioideshad a pronounced effect on decreasing decay caused bye (Fig. 2B). Gamma and UV-C irradiation have been usedarvest control of papaya anthracnose. It has been founda irradiation could reduce postharvest losses caused bye whereas UV-C irradiation was ineffective in reducingnce of C. gloeosporioides lesions, but caused fruit brown-l., 2007). Similar to gamma irradiation, HWT also play acing another important postharvest disease in stem-endand D).resent work, we found that the wax arrangement on

of HWT-fruit had been changed. HWT caused the waxd most stomata appeared to be partially or completely

melted wax (Fig. 4), thereby providing a mechanicalinst wound pathogens. Heat treatment can cause thence of wax platelets normally present in untreatedake the fruit surface relatively homogeneous (Schirra). Thus, cuticular fractures, microwounds, and most

partially or completely lled by the melted wax, andinated spores may be encapsulated and inactivated by

as well (Fallik et al., 1999, 2000; Schirra et al., 2000).on of possible gaps for wound pathogens as well asulation and inactivation of early-germinated spores,considered as additional factors in protection of fruitay. In apple, it has been found that the epicuticular wax


of non-heated fruit showed a number of deep surface cracks thatformed an interconnected network on the peel surface and thecracks became wider and deeper during long-term storage (Royet al., 1999). The circular cracks disappeared after heat treatment,probably ahad occurreGerminatedand mummpears (Schirinhibitory eof Monilinia

Polygalathat can inhan importa(Toubart et ing and wasstudy (Figs(SAR) is estaregulated eet al., 2005)expression et al., 2009)in Arabidopstudy, CpNPpathogens iby HWT in indicating tthe peel of pin plants whSeveral sturesult in acever, other disease devCpPR1 in thincreased athat HWT indefense-relplayed an iof red sweecinerea andto induce thactivities ofany impairmies also havof postharv

The resurevealed a pfruit, i.e.: (1rates of papripening; (2activation otained a relHWT causenatural opepathogen aand then resion of CpPGwhich mayand enhancpossible mefurther wor

In summcould mainresistance tas an effectistorage and

Acknowledgments

The research work was supported by the National Key Technol-ogy R&D Program of China (2011BAD24B02-4) and National Natural

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BiologY., Linphenong heae, D.J.hemis, M.Brichums-Deehe conR ProInform., 200552..C., P

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bioconolog, 2004harve., Ahareblumort ho., Grinwater sicum, Grinb

E., 199et pep., Grintmentology, Niseharvetainin) 77, 2s a result of the melting of the wax platelets thatd in the cracks (Roy et al., 1994; Cajuste et al., 2010).

spores, conidia and hyphae appeared to be coveredied by molten wax after heat treatment in cactusra et al., 1999). Hot treatment was also found to haveffects on spore germination and germ tube elongation

fructicola in peach fruit (Liu et al., 2012).cturonase-inhibiting protein (PGIP), a cell wall proteinibit fungal endopolygalacturonases, is considered to bent factor for plant resistance to phytopathogenic fungial., 1992). CpPGIP expression was low during fruit ripen-

induced by HWT both in peel and pulp in the present. 7 and 8). In plants, systemically acquired resistanceblished as a result of non-expressor of PR gene (NPR1)-xpression of pathogenesis-related (PR) genes (Wang. NPR1 gene has been shown to be a key regulator of geneduring the onset of a plant SAR (Dong, 2004; Mukhtar. Expression of NPR1 is induced by pathogen infectionsis (Zhang et al., 1999; Yu et al., 2001). In the presentR1 expression decreased along with the occurrence ofn the later storage time, and its expression was inducedpeel. CpNPR1 was mainly expressed in the peel tissue,hat disease resistance might be mainly maintained inapaya. Plant PR proteins are induced and accumulateden they are infected by microbial pathogens or insects.

dies have suggested that the induced resistance couldcumulation of pathogenesis related protein (PR). How-reports have revealed that PR was accumulated as fruiteloped. Therefore, we investigated the expression ofe present work and found that the transcripts of CpPR1s the disease increased. All these results demonstratedduced disease resistance by inducing the expression ofated genes in papaya fruit and that the papaya fruit peelmportant defense role rather than the fruit pulp. HWTt pepper also signicantly reduced decay caused by B.

A. alternata (Fallik et al., 1996a). HWT has been knowne expression of defense-related genes and increase the

their corresponding enzymes in peach fruit, withoutent of fruit quality (Liu et al., 2012). Several other stud-

e shown that HWT had a favorable effect on the controlest disease (Jemric et al., 2011; Fruk et al., 2012).lts of present work and those of other studies haveossible mechanism of HWT on preservation of papaya) HWT decreased ethylene production and respirationaya fruit, and thus reduced the effect of ethylene on fruit) HWT repressed the expression of CpEXP1, delayed thef PG and PME, and so delayed fruit softening, main-ative high rmness and reduced pathogen damage; (3)d the melting of wax, which partially or entirely sealednings in the epidermis, prevented the invasion of thend inactivated quiescent/latent infections of pathogen,duced disease incidence; (4) HWT induced the expres-IP and CpNPR1, and regulated the expression of CpPR1,

result in the gain of systemically acquired resistanceed disease resistance. However, while this could be thechanism involved in HWT-induced disease resistance,k is required for a better understanding.ary, the results showed that HWT at 54 C for 4 min

tain the quality of climacteric papaya fruit and induceo postharvest disease. HWT could be applied to papayave pretreatment to maintain postharvest quality during

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Effects of hot water treatment on anthracnose disease in papaya fruit and its possible mechanism1 Introduction2 Materials and methods2.1 Plant materials and hot water treatment2.2 Color index and disease index evaluation2.3 The determination of carrier rate of Colletotrichum gloeosporioides in fruit peel2.4 Determination of pectin content2.5 Scanning electron microscopy (SEM) analysis2.6 RNA extraction and genes clone2.7 Northern blot analysis2.8 Statistical analysis

3 Results3.1 Fruit evaluation during storage3.2 The effect on carrier rate of C. gloeosporioides in fruit peel3.3 Effect of hot water treatment on disease inhibition3.4 Correlation of anthracnose index and fruit ripeness3.5 The effects of hot water treatment on structural changes of epicuticular wax3.6 Differential expression of CpEXP1 and CpEXP23.7 Regulation of expression of CpPGIP, CpNPR1, CpPR1 genes by hot water treatment

4 DiscussionAcknowledgmentsAppendix A Supplementary dataAppendix A Supplementary data

Efecto Agua Caliente Antracnosis Papaya

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