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Research ArticleReceived: 18 January 2009 Revised: 1 April 2009 Accepted: 28 April 2009 Published online in Wiley Interscience: 23 June 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3659

Effects of preharvest applicationof antagonistic yeast combined with chitosanon decay and quality of harvested table grapefruitXianghong Menga,b and Shiping Tiana∗

Abstract

BACKGROUND: Decay caused by fungal pathogens is responsible for significant economic loss of grape fruit in vineyardsworldwide. However, postharvest treatment is not advisable for this fruit owing to its thin waxy pericarp and succulent flesh,which are easily damaged. Therefore preharvest treatment even at 1 day before harvest has been considered as a promisingmethod to control postharvest decay of table grape fruit in storage. Integrative effects of preharvest application of Cryptococcuslaurentii combined with low-concentration chitosan on decay and quality of table grape fruit during storage were investigatedin this study.

RESULTS: Spraying of antagonistic yeast combined with chitosan before harvest significantly reduced natural decay of fruitstored at 0 ◦C. Preharvest treatment stabilised polyphenol oxidase activity, increased peroxidase and phenylalanine ammonia-lyase activities and decreased superoxide dismutase activity compared with control fruit. There was a higher ratio of solublesolid content to titratable acidity in treated fruit than in control fruit during storage. In addition, preharvest treatment affectedthe total phenolic content in fruit during storage.

CONCLUSION: The results suggest that integrative application of C. laurentii and low-concentration chitosan before harvestmay be a promising technology to control decay of table grape fruit in storage.c© 2009 Society of Chemical Industry

Keywords: antagonistic yeast; chitosan; spray; Vitis vinifera L; decay; quality

INTRODUCTIONDecay caused by fungal pathogens is responsible for significanteconomic loss of grape fruit in vineyards worldwide.1 Postharvesttreatment with chitosan,2,3 ethanol,4,5 hot water6 or antagonisticagent7 was confirmed in trials to be an effective method for decaycontrol of grape fruit during storage. However, it is difficult to carryout commercially, because grape fruit has a thin waxy pericarpand succulent flesh, so the fruit is easily damaged and cannot beexposed to water-based treatment.8 In fact, to avoid handling anddeleterious effects in commercial storage, table grape bunches areusually packed directly in the field and stored at 0 ◦C for variousdurations. Therefore postharvest treatment is not feasible exceptfor fumigation with sulfur dioxide (SO2), an effective method fortable grape fruit storage at present.9,10 However, SO2 applicationusually causes injury to table grape fruit;11 moreover, it is harmfulto human health.12 With the increase in public concern on foodsafety, SO2 as a fungicide is limited in its use for grape fruitstorage.12 In view of this, preharvest treatment even at 1 daybefore harvest should be considered as a promising method tocontrol postharvest decay of table grape fruit in storage.8

Biological control, as an alternative to chemical fungicides,has been regarded as a promising method for controllingpostharvest diseases of fruits and vegetables.13 Previous studies

have shown that Cryptococcus laurentii inhibited growth of manyphytopathogens, including Botrytis cinerea, Penicillium expansum,Alternaria alternate and Rhizopus stolonifer, but also controlledcorresponding diseases.14 – 18 Ippolito et al.8 considered thatpreharvest application of biocontrol agents was an appropriatestrategy for decay control of table grape fruit. Tian et al.19 reportedthat C. laurentii sprayed before harvest on the surface of sweetcherry fruit showed relatively high survival at harvest time andeven during controlled atmosphere storage, resulting in effectivecontrol of postharvest decay in storage.19

Chitosan, as a natural polycation compound, could limit fungaldecay of fruits by its direct antifungal activity,20,21 capability forinduction of host resistance to pathogens22 or self-polymerisation

∗ Correspondence to: Shiping Tian, Key laboratory of Photosynthesis and Environ-mental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences,Nanxincun 20, Xiangshan, Beijing 100093, China. E-mail: tsp@ibcas.ac.cn

a Key laboratory of Photosynthesis and Environmental Molecular Physiology,Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Xiangshan,Beijing 100093, China

b College of Marine Life Science, Ocean University of China, Qingdao 266003,China

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to cover the fruit surface.23 Spraying with chitosan before harvestreduced decay incidence of table grape fruit during storage.2,3

Moreover, chitosan did not influence the population growth ofC. laurentii when applied in vivo at concentrations ranging from0.01 to 1 g L−1, and combination of chitosan with C. laurentiiresulted in synergistic inhibition of blue mould rot.24 Thereforecombining chitosan with an antagonist makes it possible to exploitnot only the antifungal and eliciting properties of chitosan butalso the biological activity of the antagonist. The objective of thisstudy was to investigate the effects of spraying C. laurentii mixedwith chitosan at 10 days before harvest on natural decay controland quality attributes of table grape fruit during storage at 0 ◦C.

MATERIALS AND METHODSFruit and treatmentTable grapes (Vitis vinifera L., cv. Jingxiu) were grown in an organicorchard of the Institute of Botany, Chinese Academy of Sciences,Beijing, China. No fungicide was applied prior to harvest. Chitosan(5 g, deacetylation degree 90%, viscosity 15 cP) was dissolved in100 mL of distilled water containing 5 g L−1 acetic acid to prepared50 g L−1 chitosan solution. The mixed solution for spraying beforeharvest, containing chitosan at a final concentration of 1 g L−1 (bydilution of 50 g L−1 chitosan solution) and C. laurentii at a finalconcentration of 1×108 cells mL−1 as well as 0.5 mL L−1 Tween-80as a surfactant to increase affinity with the fruit, was prepared justbefore use. At 10 days before harvest, more than 150 grape clusterswere immediately sprayed once with the mixed solution until allfruits were wet to run-off. Additional clusters were sprayed withdeionised water containing 0.5 mL L−1 Tween-80 as the control.At harvest time, all treated fruits were randomly distributed intogroups of 15 clusters of about 10 kg each. One group as a replicateand three replicates per treatment were used. The treated andcontrol fruits were packaged in plastic boxes, then overwrappedwith plastic bags to maintain the relative humidity (RH) at 90–95%and finally stored at 0 ◦C. Fruits were sampled at 17 and 42 daysimmediately following removal from 0 ◦C and, in the case of thosestored for 42 days, after a further 3 days of incubation at 20 ◦C. Allfruit samples were evaluated by the methods described below.The experiment was repeated twice in this study.

Decay determinationDuring storage, natural decay incidence was evaluated in termsof a decay index. The disease severity of each grape berry in eachbunch was assessed according to the following empirical scale:0 = healthy berry; 1 = one lesion less than 2 mm in diameter;2 = one lesion less than 5 mm in diameter; 3 = several lesionsor less than 25% of berry surface infected; 4 = more than 25% ofberry surface infected, sporulation present. The decay index (DI)was calculated by the formula DI = ∑

(d × f )/N/D, where d isthe degree of rot severity scored on a berry and f is its respectivequantity, N is the total number of berries examined and D is thehighest degree of disease severity occurring on the scale.

Enzyme activitiesFruit flesh samples (10 g) taken randomly from ten grapes of onereplicate were homogenised in 20 mL of ice-cold extraction buffercontaining 0.5 g of polyvinyl polypyrrolidone. The extraction bufferwas 100 mmol L−1 sodium phosphate (pH 6.4) for peroxidase(POD) and polyphenol oxidase (PPO) assays, 50 mmol L−1 sodiumborate (pH 8.8) containing 5 mol L−1 β-mercaptoethanol for

phenylalanine ammonia-lyase (PAL) assay and 100 mmol L−1

potassium phosphate (pH 7.8) for superoxide dismutase (SOD)assay. The homogenates were centrifuged at 15 000×g for 30 minat 4 ◦C and the supernatants, as crude enzyme extracts, were usedfor the assays.

Enzyme activities were assayed according to the methods ofMeng et al.3 Specific activities of enzymes were expressed asU mg−1 protein, where one unit (1 U) was defined as 1�OD398

min−1 mg−1 protein for PPO, as 0.01 �OD460 min−1 mg−1 proteinfor POD, as a 50% decrease in SOD-inhibited nitroblue tetrazolium(NBT) reduction for SOD and as 1 �OD290 min−1 mg−1 proteinfor PAL (�OD, change in optical density). Protein content wasmeasured according to the method of Bradford25 using bovineserum albumin (BSA) as standard. All measurements above wereperformed in triplicate.

Total phenolic contentTotal phenolics were determined according to Zhang andQuantick.26 Fruit flesh samples (10 g) were homogenised in 10 mLof 1% (v/v) HCl/methanol and then centrifuged at 15 000 × gfor 50 min at 4 ◦C. The supernatants were collected and theirabsorbance (A) was measured at 280 nm. Total phenolic contentwas expressed as A280 g−1 fresh weight (FW). All assays wereperformed in triplicate.

Soluble solid content and titratable acidityFruit flesh samples (10 g) taken randomly from ten grapes weresuspended in 25 mL of distilled water, homogenised in a grinderand centrifuged at 15 000 × g for 30 min at 4 ◦C. The supernatantswere collected for assay. Soluble solid content (SSC) was measuredusing an AO MRK II refractometer (AO Scientific Instruments, NY,USA) at 20 ◦C and expressed as ◦Brix. Titratable acidity (TA) wasdetermined by titration with 0.01 mol L−1 NaOH to pH 8.2 usingphenolphthalein as indicator and expressed as mmol H+ per 100 gFW. All assays were performed in triplicate.

Statistical analysisAll data were subjected to one-way analysis of variance (ANOVA).Separation of means was performed by Duncan’s multiple rangetest. Differences were considered significant at P ≤ 0.05.

RESULTSEffect of preharvest treatment on decay indexAs shown in Fig. 1, preharvest spraying with the mixed solutionof antagonist and chitosan significantly reduced natural decayof table grape fruit compared with control fruit after 42 days ofstorage at 0 ◦C and also after a further 3 days of shelf life at 20 ◦C(P ≤ 0.05).

Effect of preharvest treatment on enzyme activitiesSpraying of the mixed solution at 10 days prior to harvestsignificantly enhanced PAL activity but had no noticeable effecton PPO, POD and SOD activities in table grape fruit comparedwith control fruit at harvest (Fig. 2). PPO, POD and SOD activitiesincreased and PAL activity decreased in control fruit stored at 0 ◦C,whereas preharvest spraying significantly reduced SOD activityand enhanced PAL activity (Fig. 2). In addition, POD and PALactivities were higher in treated fruit than in control fruit after3 days of shelf life at 20 ◦C following 42 days of storage at 0 ◦C(Fig. 2).

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Figure 1. Decay index of table grape fruit stored for 42 days at 0 ◦C andfollowing shelf life of 3 days at 20 ◦C. CK, control; Pre(A+C), preharvestspray of Cryptococcus laurentii combined with chitosan. Bars representstandard deviations of means.

Effect of preharvest treatment on total phenolic contentThe content of total phenolics in table grape fruit at harvestwas higher than that at 10 days before harvest, and there wasno significant difference between preharvest-treated fruit andcontrol fruit (Fig. 3). Preharvest spraying reduced the total phenoliccontent of table grape fruit after 17 days of storage at 0 ◦C butenhanced it during further storage at 0 ◦C and subsequent shelflife at 20 ◦C compared with control fruit (Fig. 3).

Effect of preharvest treatment on SSC and TASpray treatment before harvest had no significant effect onSSC (Fig. 4(a)) and TA (Fig. 4(b)) of fruit at harvest. However, it

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Figure 3. Effects of preharvest spray of Cryptococcus laurentii combinedwith chitosan on total phenolic content of table grape fruit stored at 0 ◦C.Bars represent standard deviations of means.

significantly enhanced the SSC/TA ratio (Fig. 4(c)) by increasingSSC and decreasing TA in table grape fruit compared with controlfruit during storage at 0 ◦C and subsequent shelf life at 20 ◦C.

DISCUSSIONAlthough the postharvest application of biocontrol agents and/orfungicides was considered to be an effective method for decaycontrol of fruits during storage, it was not a suitable technology forsome berries, e.g. table grape, sweet berry and strawberry, owing

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Figure 2. Effects of preharvest spray of Cryptococcus laurentii combined with chitosan on (a) PPO, (b) SOD, (c) POD and (d) PAL activities in table grapefruit stored at 0 ◦C. Bars represent standard deviations of means.

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Figure 4. Effects of preharvest spray C. laurentii combination with chitosanon SSC (A), TA (B) contents and rate of SSC/TA (C) and of fruits stored at0 ◦C. Bars represented standard deviations of the means.

to their brittle pericarp and succulent flesh.8 Spraying with naturalor synergic compounds before harvest to reduce storage decayof table grapes seems to be the best method, since exposure topostharvest liquid-based treatments is not advisable for this com-modity, as it could damage the bloom. Preharvest spraying withchitosan alone reduced natural decay of table grapes during stor-age, perhaps owing to its direct antifungal activity and induction ofdisease resistance.2,3 The direct antifungal activity of chitosan hasbeen well documented in both in vitro and in situ studies.20 Prehar-vest chitosan treatments reduced the propagules of filamentousfungi such as B. cinerea, Penicillium sp., Aspergillus sp. and Cladospo-rium sp. occurring naturally on table grape berries but lacked asignificantly negative impact on the naturally occurring microflora

of yeasts and yeast-like fungi, among which antagonistic micro-organisms are frequent.2 Pre- or postharvest chitosan treatmentsenhanced disease resistance of the host by regulating defence-related enzymes such as PAL, chitinase, β-1,3-glucanase, POD,PPO and SOD in table grapes,2,3 litchi,27 mango,28 tomato29,30

and potato.31 The antagonistic activity of C. laurentii againstpathogenic fungi has been widely investigated.16,32,33 Tian et al.34

proved that C. laurentii could stimulate activity and expression ofβ-1,3-glucanase genes. Spraying with antagonistic micro-organismsbefore harvest was regarded as a promising technology for appli-cation in fruit storage owing to its significant effect on reducingincidence of rot.8,19 Attachment to the fruit surface is probably animportant trait for antagonists to possess for successful prehar-vest applications, since persistent attachment would contributeto better colonisation and avoid dislodgement due to wind, rainor water level fluctuations.35 Production of slime, mainly compris-ing extracellular polysaccharides, on the phylloplane could play animportant role in the adhesion of yeast-like fungi,36 but the mecha-nism is still unclear. Chitosan has a particular adhesiveness towardsbiological surfaces owing to its positive charge and filmogenicproperty.37 Additionally, chitosan or chitin at low concentrationnot only had no significantly negative effect on the populationgrowth of C. laurentii24 but also induced antagonistic activity.33 Asynergic effect on the control of blue rot in pear by postharvest in-oculation of C. laurentii combined with chitosan or chitin has beenreported.24,33 Therefore addition of low-concentration chitosan tothe solution of C. laurentii may be beneficial in adhesion, coloni-sation of yeast on the fruit surface and biocontrol efficacy. In thisstudy the results showed that preharvest spraying of C. laurentiimixed with chitosan effectively decreased natural disease inci-dence of table grape fruit during storage at 0 ◦C and subsequentshelf life at 20 ◦C, and PAL activity was higher in preharvest-treatedfruit than in control fruit (Fig. 2(d)). Therefore the effectiveness ofpreharvest treatment by antagonists and chitosan on decay was re-lated to the combination of its antifungal and adhesion propertiesas well as its ability to stimulate defence responses in the host.

CONCLUSIONIntegrative application of C. laurentii and chitosan at lowconcentration before harvest had a beneficial effect on decaycontrol and quality. The results suggested that it should be apromising technology for decay control of table grape fruit.

ACKNOWLEDGEMENTSThis research was supported by the National Natural ScienceFoundation of China (30600481) and the Ministry of Science andTechnology of China (2006BAD22B03).

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