Food Chemistry 159 (2014) 29–37

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

c-Aminobutyric acid induces resistance against Penicillium expansumby priming of defence responses in pear fruit

http://dx.doi.org/10.1016/j.foodchem.2014.03.0110308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +86 571 88982398; fax: +86 571 88982191.E-mail address: yuting@zju.edu.cn (T. Yu).

Chen Yu, Lizhen Zeng, Kuang Sheng, Fangxia Chen, Tao Zhou, Xiaodong Zheng, Ting Yu ⇑College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R&D Center for Food Technologyand Equipment, Zhejiang University, Hangzhou 310058, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 October 2013Received in revised form 9 February 2014Accepted 4 March 2014Available online 13 March 2014

Keywords:c-Aminobutyric acid (GABA)Induced resistancePrimingPenicillium expansumPearPostharvest

The results from this study showed that treatment with c-aminobutyric acid (GABA), at 100–1000 lg/ml,induced strong resistance against blue mould rot caused by Penicillium expansum in pear fruit. Moreover,the activities of five defence-related enzymes (including chitinase, b-1,3-glucanase, phenylalnine ammo-nialyase, peroxidase and polyphenol oxidase) and the expression of these corresponding genes weremarkedly and/or promptly enhanced in the treatment with GABA and inoculation with P. expansumcompared with those that were treated with GABA or inoculated with pathogen alone. In addition, thetreatment of pear with GABA had little adverse effect on the edible quality of the fruit. To the best ofour knowledge, this is the first report that GABA can effectively reduce fungal disease of harvested fruit.Its mechanisms may be closely correlated with the induction of fruit resistance by priming activation andexpression of defence-related enzymes and genes upon challenge with pathogen.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Pathogen infection is an important factor that affects fruit post-harvest physiology and metabolism (Prusky et al., 2010). Penicilliumexpansum, the causal agent of blue mould, is a widespread fungalpathogen and causes considerable pome fruit losses, in apples,cherries, pears and peaches (Cao, Yang, Hua, & Zheng, 2011; Juricket al., 2009; Malandrakis et al., 2013; Quaglia, Ederli, Pasqualini, &Zazzerini, 2011; Venturini, Oria, & Blanco, 2002). Besides the eco-nomic impact, P. expansum is also regarded as the major producerof patulin (4-hydroxy-4H-furo[3,2-c]pyran-2[6H]one), mycotoxinand secondary metabolites toxic to humans (Malandrakis et al.,2013). Moreover, the intense use of synthetic fungicides has led tothe increasing resistance of fungal pathogens. Therefore, there is agrowing need for eco-friendly alternative strategies to inhibit thefungal decay (Droby, Wisniewski, Macarisin, & Wilson, 2009).

Attempts to exploit induced resistance through the applicationof defence response elicitors are being pursued, to control posthar-vest diseases as a safer technology (Shoresh, Harman, & Mastouri,2010). Several chemical elicitors have been reported to induceresistance to pathogens, e.g. salicylic acid (SA) and its functionalanalogue acibenzolar-S-methyl (ASM) and b-aminobutyric acid

(BABA) (Quaglia et al., 2011). A common feature of induced resis-tance caused by these elicitors is called priming (Conrath, Pieterse,& Mauch-Mani, 2002; Wang, Xu, Wang, Jin, & Zheng, 2013), whichcan cause a faster and stronger activation of defence responsemechanisms after pathogen infection (Tonelli, Furlan, Taurian,Castro, & Fabra, 2011).

c-Aminobutyric acid (GABA), a four carbon, non-protein freeamino acid, is widespread in most prokaryotic and eukaryoticorganisms (Bown, MacGregor, & Shelp, 2006). In plants, multiplesignalling roles have been attributed to GABA, including involve-ment in pH regulation, nitrogen storage, plant development anddefence against abiotic stresses, such as drought, oxidative stress,salinity and cold stress (Shelp et al., 2012). Experimental evidenceshows that exogenous GABA can alleviate oxidative damage causedby H+ and Al3+ toxicities in barley seedlings (Song, Xu, Wang,Wang, & Tao, 2010) and reduce chilling injury of peach fruit byinducing proline accumulation, enhancing the enzymatic antioxi-dant system and maintaining energy status (Shang, Cao, Yang,Cai, & Zheng, 2011; Yang, Cao, Yang, Cai, & Zheng, 2011). However,there is little information about the effect of GABA on the posthar-vest fungal diseases of fruit.

The aim of this research was to assess the effect of GABA oninduction of resistance to blue mould rot caused by P. expansumin pear fruit and the possible action mechanisms involved.

30 C. Yu et al. / Food Chemistry 159 (2014) 29–37

2. Materials and methods

2.1. Fruit, pathogen and GABA

Pear fruit (Pyrus pyrifolia Nakai. cultivar ‘‘Shuijing’’) were sortedon the basis of size, colour, maturity and absence of physical injuryor infection. The fruit surfaces were disinfected in 0.1% sodiumhypochlorite solution for 2 min, thoroughly washed in tap waterand air-dried at 25 �C prior to use.

An isolate of P. expansum obtained from rotted pear fruit wasmaintained on potato dextrose agar (PDA) medium (containingthe extract from 200 g of potato, 20 g of glucose and 20 g of agarin 1 l of distilled water) for 7 days at 25 �C. Sterile distilled waterwas used to flood the surface of the PDA culture and conidia werescraped by a sterile loop. The spore concentrations obtained weredetermined using a hemacytometer and adjusted with sterile dis-tilled water as required.

GABA was purchased from Sangon Biotech (Shanghai, China).The stock solution of GABA was diluted to a series (1, 10, 100and 1000 lg/ml).

2.2. Efficacy of GABA for control of blue mould rot caused byP. expansum in pear fruit

2.2.1. Effect of GABA at different concentrations on inhibition of bluemould rot in pear fruit

The fruit were wounded (5 mm wide by 3 mm deep) with thetip of a sterile border at five different sites. 30 ll of the aqueouspreparation which contained GABA at 1, 10, 100 and 1000 lg/mlwere injected into each wound. Wounds treated with the sameamounts of sterile distilled water served as control. After 2 or24 h, each wound was inoculated with 30 ll of a conidial suspen-sion of P. expansum at 1 � 104 spores/ml. The fruit were then air-dried and incubated in the dark at 25 �C to maintain 90% relativehumidity (RH). Each treatment included 3 replicates and each rep-licate consisted of 9 fruit.

2.2.2. Effect of GABA treatment time on induction of disease resistanceagainst blue mould rot in pear fruit

Five wounds were made on each pear fruit as above and eachwound was treated with 30 ll of GABA, at 100 lg/ml, or steriledistilled water as the control. After 0, 6, 12, 24 and 36 h, fruit wereinoculated with 30 ll of the P. expansum suspension at 1 � 104

spores/ml in each wound. The fruits were then air-dried and incu-bated in the dark at 25 �C to retain about 90% RH. Each treatmentincluded 3 replicates and each replicate consisted of 9 fruit.

2.2.3. Effect of GABA on blue mould rot at different sporeconcentrations in pear fruit

Six wounds were made on the surface of each pear fruit as de-scribed above and three of the wounds were treated with 30 ll ofGABA at 100 lg/ml or with 30 ll of sterile water as control. After24 h, 30 ll of 1 � 102, 1 � 104 or 1 � 106 spores/ml suspension ofP. expansum were respectively inoculated into each wound. Thefruits were then air-dried and incubated in the dark at 25 �C to re-tain about 90% RH. Each treatment included 3 replicates and eachreplicate consisted of 9 fruit.

2.2.4. Effect of GABA on blue mould rot development at lowtemperature in pear fruit

Pear fruit were wounded at two sites as above. Aliquots of 30 llof GABA at 100 lg/ml and sterile water as a control, were pipettedonto each wound and 24 h later wounds were then inoculated with30 ll of a 1 � 104 spores/ml suspension of P. expansum. The fruitswere then air-dried and incubated in the dark at 4 �C to maintain

90% RH. Each treatment included 3 replicates and each replicateconsisted of 9 fruit.

In the above four tests, the percentage of wounds showing rotsymptoms was assessed on a daily basis. Disease incidence was de-fined as decayed fruit/total fruit � 100%. Each test was performedat least twice. The discussed data were from one individual exper-iment and representative of the two experiments with similarresults.

2.3. Effect of GABA on germination and survival of P. expansum sporesin vitro

The effect of GABA on spore germination of P. expansum wastested in potato dextrose broth (PDB) with various concentrationsof GABA (0, 1, 10, 100, 1000 lg/ml). Aliquots of 100 ll of pathogensuspensions were put into 10 ml glass tubes containing 2 ml of PDBto obtain a final concentration of 1 � 106 spores/ml. All treatedtubes were placed on a rotary shaker at 200 rpm at 25 �C. After12 h of incubation, approximately 150–200 spores of the pathogenper replicate were measured for germination rate. Spores wereconsidered germinated when germ tube lengths were equal to orgreater than spore lengths. All treatments consisted of three repli-cates and the experiment was repeated twice.

Equal amounts of P. expansum spores were mixed with a solu-tion of GABA in final concentrations of 0, 1, 10, 100, 1000 lg/ml,respectively and kept for 1 min. Then 100 ll of spore suspensionwere plated on PDA. The colonies per plate were counted after72 h of incubation at 25 �C. There are three replicates per treat-ment with three plates per replicate and the experiment was con-ducted twice.

2.4. Effect of GABA on spore germination of P. expansum in pear fruitwounds

Two of the four wounded sites on each fruit were injected with30 ll of sterile distilled water (as the control) and 30 ll of GABA, at100 lg/ml, were placed in the other two wounds. After 2 or 24 h,each wound was inoculated with 30 ll of a conidial suspensionof P. expansum at 1 � 107 spores/ml. After being air-dried, pear fruitwere stored in enclosed plastic trays to maintain a high relativehumidity at 25 �C. After 12 h postinoculation, 150–200 spores perreplicate were examined microscopically to determine germina-tion rate. Each treatment included 3 replicates and each replicateconsisted of nine fruit for each time point.

2.5. Sample treatment for enzyme activity and gene expressionanalysis

The wounds were inoculated with 30 ll of the P. expansum sus-pension at 1 � 104 spores/ml 24 h after the treatment of GABA at100 lg/ml. Tissue samples of pulp from the GABA or water-treatedfruit, with or without inoculation, were collected at the same inter-vals (0, 12, 24, 36 and 48 h after the treatment) and immediatelyfrozen in liquid N2. They were then stored at �80 �C for furtheranalysis of enzyme activity and gene expression.

2.6. Assay of enzyme activities in pear fruit

Frozen tissue samples (0.6 g) were ground in a mortar and pes-tle with different buffers to assay different enzymes: 1.2 ml of so-dium acetate buffer (50 mM, pH 5.0), that contained 1% (w/v)polyvinyl-pyrrolidone (PVP) for chitinase and 5.4 ml for b-1,3-glucanase, 1.2 ml of 200 mM sodium borate buffer (pH 8.8) for PALand 1.2 ml of sodium phosphate buffer (50 mM, pH 7.8) containing1.33 mM EDTA and 1% (w/v) polyvinyl-pyrrolidone (PVP) for PODand PPO. The homogenate was centrifuged at 13,000g for 20 min

C. Yu et al. / Food Chemistry 159 (2014) 29–37 31

at 4 �C. The supernatants were used as the crude enzyme source toassay enzymatic activities and protein contents. Each sample, con-taining three replicates, was obtained from nine fruit of each treat-ment or inoculation and the experiment was repeated twice.

Chitinase (CHI) activity was measured according to the methodof Yalpani and Pantaleone (1994) with chitin azure (Sigma–Aldrich) as the substrate. CHI activity was measured by mixing300 ll of crude enzyme solution with 100 ll of 5 mg/ml chitinazure in 3 M AcOH. After 12 h of shaking at 37 �C, the reaction mix-ture was cooled and centrifuged for 5 min at 9168g and the absor-bance of the supernatant was measured at 550 nm. One unit of CHIactivity was defined as the amount of enzyme that caused an in-crease in absorbance of 0.001 at 550 nm in 1 h under the assay con-ditions. The results were expressed as units per mg protein.

b-1,3-Glucanase (GLU) activity was assayed by measuring theamount of reducing sugar released from the substrate, followingthe method described by Ippolito, El-Ghaouth, Wilson, andWisniewski (2000) with some modification. 250 ll of enzymepreparation were incubated with 250 ll of 50 mM sodium acetatebuffer (pH 5.0) containing 0.5% laminarin (w/v) for 4 h at 40 �C. Theblank was the crude enzyme preparation mixed with laminarin atzero time incubation. The reaction was terminated by adding500 ll of 3,5-dinitrosalicylate and heating the sample in boilingwater for 5 min. The solution was diluted with 4 ml of distilledwater and the amount of reducing sugars was measured at500 nm. The specific activity of GLU was expressed as units permg of protein. One unit (U) was defined as the enzyme activitycatalysing the formation of 1 lmol of glucose equivalents per hour.

Phenylalanine ammonialyase (PAL) activity was assayed, refer-ring to the method of Zhang et al. (2012) with slight modifications.The assay mixture contained 500 ll of crude enzyme and 200 ll of50 mM L-phenylalanine in sodium borate buffer (200 mM, pH 8.8).After incubation at 37 �C for 8 h, the reaction was stopped byaddition of 40 ll of 6 M HCl. One unit of PAL activity was definedas the amount of enzyme that caused an increase in absorbanceof 0.01 at 290 nm in 1 h. The results were expressed as units permg of protein.

Peroxidase (POD) activity was determined, using guaiacol assubstrate (Lurie, Fallik, Handros, & Shapira, 1997). The reactionmixture consisted of 100 ll of crude enzyme extract, 140 ll of0.3% (v/v) guaiacol (in 50 mM sodium phosphate buffer, pH 6.4).The increase in absorbance at 470 nm was measured after 60 llof 0.3% (w/v) H2O2 (in 50 mM sodium phosphate buffer, pH 6.4)were added. One unit of POD activity was defined as the amountof enzyme that caused an increase in absorbance of 0.01 at470 nm per minute and the activity was expressed as units permg of protein.

Polyphenol oxidase (PPO) activity was assayed, following themethod of Mohammadi and Kazemi (2002). The reaction mixtureswere 250 ll 50 mM sodium phosphate buffer (pH 6.4) containing10 mM pyrocatechol and 50 ll of crude enzyme. One unit of PPOactivity was defined as the amount of enzyme extracts producingan increase of A398 nm by 0.01 in 1 min and the activity was ex-pressed as units per mg of protein.

Protein content in the enzyme extracts was determined by theBradford (1976) method, bovine serum albumin (BSA) being usedas a standard.

2.7. Gene expression analysis by real-quantitative PCR

Total RNA was extracted, using a modified CTAB method. First-strand cDNA was synthesised from 500 ng of total DNA-free RNAwith a PrimeScript� RT reagent Kit (TaKaRa, Japan) according tothe manufacturer’s instructions. The cDNA was diluted 20-foldand 2 ll of the diluted cDNA was used as the template for real-timequantitative PCR (Q-PCR) analysis.

The reference housekeeping gene coding for actin and five tar-get genes coding for defence proteins were chosen: PpCHI, PpGLU,PpPAL, PpPOD and PpPPO. The corresponding P. pyrifolia nucleotidesequences were obtained from the GENBANK database and used todesign gene-specific primer pairs as follows, employing Primer5.0software: PpCHI (FJ589786), forward 50-CACAGACGATGCCTACTGC-30 and reverse 50-AACTTGCGTCCGCCTGAT-30; PpGLU (JX127223),forward 50-CCTTACTTCAGCTACAATGACAAC-30 and reverse 50-GTACTGAGCGTCCAGGAGAG-30; PpPAL (JQ749640), forward 50-ATCTTGTCCCGCTGTCCT-30 and reverse 50-TGAGGGTCTGACCGTTTG-30;PpPOD (JX290377), forward 50-CAACATGGACCCAACCAC-30 and re-verse 50-TTGGCCCACCTTCTTACC-30; PpPPO (AY338251), forward50-GACATTCGCTATGCCGTTCT-30 and reverse 50-TCGGTCCCGTTGTAATCG-30; PpACTIN (JN684184), forward 50-CCATCCAGGCTGTTCTCTC-30 and reverse 50-GCAAGGTCCAGACGAAGG-30.

PCR reactions were carried out in a 48-well system (20 ll perwell). The reaction mixture contained 10.0 ll of SYBR� Premix ExTaq™ (TaKaRa, Japan), 0.4 ll of each primer (10 lM), 0.4 ll ROXReference Dye (50�), 2 ll of cDNA and 6.8 ll of RNase-free water.The reactions were performed on a StepOne Real-Time PCR System(Applied Biosystems) at 95 �C for 30 s, followed by 40 cycles at95 �C for 5 s and at 60 �C for 30 s. Relative quantifications werethen calculated, using the 2�DDC(T) method (Livak & Schmittgen,2001). All Q-PCR reactions were normalised, using the Ct value cor-responding to the Pyrus actin gene. Efficiency of primers was ana-lysed by the dilution method. Melting curve analysis wasconducted to confirm the specificity of the primers. Each treatmentincluded 3 biological replicate samples.

2.8. Effect of GABA on quality parameters of pear fruit

The fruit were immersed in solutions of 0, 10, 100, or 1000 lg/ml of GABA for 10 min. and then air-dried. Samples were collectedafter 7, 15 and 30 days of storage at 25 �C for quality evaluation.

Firmness was determined at the equator of the fruit, using ahand-held penetrometer (GY-2, Fruit Firmness Tester, Zhejiang,China). Total soluble solids (TSS) content was assayed by using aportable refractometer (WZ-103, Top instrument Co., Ltd., China)and the results were expressed as percentages. Titratable acidity(TA) content was measured by titrating 10 ml of the filtered pearjuice to pH 8.2 with 0.01 M NaOH. Ascorbic acid (AA) contentwas determined, using the 2,6-dichloro-indophenol method andthe results were expressed as mg/100 g. Each treatment was repli-cated three times with three fruits per replicate.

2.9. Statistical analysis

Experiments were performed by a completely randomized de-sign and conducted at least twice. The data are from one individualexperiment and are representative of two independent experi-ments with similar results. Data were subjected to one-way analy-sis of variance (ANOVA univariate, SPSS 17.0; SPSS Inc., Chicago IL,USA). When the number of means in each group is two, the inde-pendent samples t test is applied for means separation. The Dun-can’s multiple range test was used for separation of means.

3. Results

3.1. Effect of GABA on induction of resistance to P. expansum in pearfruit wounds

Exogenous GABA had no direct fungitoxic activity against P.expansum in pear fruit wounds when the time interval betweenGABA treatment and P. expansum inoculation was within 6 h(Fig. 1A and B). In contrast, the resistance to blue mould in pear

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Fig. 1. Efficacy of c-aminobutyric acid (GABA) for control of blue mould rot caused by Penicillium expansum in pear fruit. (A) Effect of GABA at different concentrations oninhibition of blue mould rot in pear fruit wounds at 25 �C. Surface wounds were treated with 30 ll of various concentrations of GABA and then were inoculated with P.expansum at 1 � 104 spores/ml after 2 or 24 h, respcetively. (B) Effect of the time interval between treatment with GABA and pathogen inoculation on induction of diseaseresistance against blue mould rot in pear fruit wounds at 25 �C. Fruit wounds were treated with 30 ll of GABA at 100 lg/ml and then were inoculated with 30 ll P. expansumat 1 � 104 spores/ml after 0, 6, 12, 24 and 36 h, respectively. (C) Effect of GABA on blue mould rot caused by P. expansum at different spore concentrations in pear fruit woundsat 25 �C. Fruit wounds were treated with 30 ll of GABA at 100 lg/ml and then were inoculated with P. expansum at 1 � 102, 1 � 104 and 1 � 106 spores/ml after 24 h,respectively. Disease incidence was evaluated after 4 days of incubation. Different letters in Fig. 1(A–C) indicate significant differences (P = 0.05) according to Duncan’smultiple range test. (D) Effect of GABA on blue mould rot development in pear fruit wounds at 4 �C. Fruit wounds were treated with 30 ll of GABA at 100 lg/ml and then wereinoculated with P. expansum (1 � 104 spores/ml) after 24 h at 4 �C. The rot symptoms were observed from 16 days of inoculation. The independent samples t test is applied formeans separation for each time point. Standard errors of three replications are given as the short bars in Fig. 1(A–D).

32 C. Yu et al. / Food Chemistry 159 (2014) 29–37

fruit pre-treated with GABA was gradually enhanced when thetime interval was increased from 12 to 36 h (P < 0.05, Fig. 1A andB). Moreover, the inhibition effect was constantly increased whenthe concentration of GABA increased from 10 to 1000 lg/ml atthe internal of 24 h (Fig. 1A). The GABA treatment of 100 lg/mlinhibited the incidence of the blue mould rot by more than 50%with respect to control at 24 h which was not statistically differentfrom the GABA at 1000 lg/ml or the treatment of GABA at 100 lg/ml for 36 h (P < 0.05, Fig. 1A and B).

At the optimal time interval of 24 h, the treatment with GABA at100 lg/ml was highly effective against the pathogen and reducedthe level of infection caused by P. expansum at 1 � 102 or 1 � 104

spores/ml compared with the control. As regards P. expansum at1 � 106 spores/ml, the effect of GABA proved to be not obvious insuppressing the blue mould rot (P < 0.05, Fig. 1C).

The results in Fig. 1D indicated that the application of GABAcompletely inhibited the pathogen infection after storage at 4 �Cfor 16 days. Although the decay symptoms developed in the con-trol and GABA treatment after 20–28 days of storage, the diseaseincidence in GABA-treated pear fruit was still significantly lowerthan that in the control (P < 0.05).

3.2. Effect of GABA on germination and growth of P. expansum sporesin vitro

In general, GABA, at 1–1000 lg/ml, did not significantly influ-ence the spore germination of P. expansum in PDB after 12 h ofincubation. Similarly, there was no effect on the survival of P.

expansum on PDA between the treatments with GABA at differentconcentrations and the control (Table 1).

3.3. Effect of GABA on spore germination of P. expansum in pear fruitwounds

Approximately 80% of spores germinated in pear wounds with-in 12 h of inoculation, regardless of the presence or absence ofGABA at 100 lg/ml when the time interval between the treatmentwith GABA and P. expansum inoculation was 2 h. Conversely, ger-mination rate was effectively inhibited in wounds inoculated withP. expansum 24 h after treatment with GABA, by 57.6% comparedwith that in the control fruit (P < 0.01, Fig. 2).

3.4. Effect of GABA and P. expansum on defence-related enzymeactivities in pear fruit wounds

CHI activity was increased gradually by GABA as compared tothe control and then sharply decreased after 36 h. Pear fruit thatwere treated with GABA and followed by inoculation with P. expan-sum showed 28.8% higher activity of CHI than with the inoculatedpathogen alone after 24 h of inoculation (Fig. 3A).

GLU activity increased immediately, within 12 h after inocula-tion with P. expansum alone. The activity reached its highest valueat 24 h in GABA-treated and inoculated fruit and then decreasedgradually afterwards whereas it still maintained a relatively higherlevel than after other treatments (Fig. 3B). Similarly, the change

Table 1Effect of c-aminobutyric acid (GABA) at different concentrations on Penicilliumexpansum spore germination in PDB and survival on PDA.

GABA (lg/ml) Spore germination in PDB (%) Survival on PDA (CFU per plate)

0 83.7 ± 1.5 a,b 27.2 ± 3.2 a1 77.6 ± 3.0 b 27.2 ± 0.3 a

10 79.5 ± 3.6 a,b 24.3 ± 1.3 a100 85.8 ± 1.6 a 26.6 ± 1.7 a

1000 83.5 ± 1.0 a,b 22.4 ± 1.6 a

Each value is the mean of three separate determinations and the standard errors.Different letters indicate significant differences (P = 0.05) according to Duncan’smultiple range test.

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Fig. 2. In vivo detection of spore germination of Penicillium expansum in pear fruitwounds treated with c-aminobutyric acid at 100 lg/ml. Each column representsthe mean of triplicate samples. Standard errors of three replications are given as theshort bars. Different letters indicate significant differences (P = 0.01) according toDuncan’s multiple range test.

C. Yu et al. / Food Chemistry 159 (2014) 29–37 33

tendency of GLU activity was also obtained in GABA-treated alonefruit.

The treatment with GABA, or with pathogen inoculation alone,caused only a slight elevation in PAL activity levels compared tothe respective water control within 12 h. However, PAL activitywas activated quickly in GABA-treated and P. expansum-inoculatedfruit and reached a maximal level at the same time, which wasapproximately 2.5-fold that in pathogen-inoculated fruit. After36 h, PAL activity in pathogen-inoculated fruit was also markedlyincreased (Fig. 3C).

The treatment with both GABA and P. expansum resulted in themost evident inducible effect on the activities of POD and PPO inpear fruit. The two enzyme activities both reached a peak value,at 12 and 36 h, respectively (Fig. 3D and E).

3.5. Effect of GABA and P. expansum on defence-related genesexpression in pear fruit wounds

Expression of the five defence-related genes was retained atvery low level in fruit treated with GABA alone, while pathogenchallenge triggered accelerated gene expression at different timepoints with the exception of PpPOD. Transcript levels of all fivegenes in fruit treated with GABA and inoculated with P. expansumwere enhanced faster and to greater extents than after the otherthree treatments at 12 h (Fig. 4). A priming effect, of 19.4-,2.9-, 2.8-, 3.4- and 4.0-fold, was observed in GABA-treated andP. expansum inoculated fruit for an up-regulated expression of PpCHI,PpGLU, PpPAL, PpPOD and PpPPO, respectively, in comparison withthat in the control at 12 h. At 24 h, GABA induced stronger

expression of PpPOD and PpPPO in pear fruit when challenged withP. expansum. Moreover, the change in expression level of PpCHI,PpGLU and PpPAL exhibited a similar tendency in pathogen-inoculated fruit that increased from 24 h and reached a higher levelthan after other treatments at 48 h.

3.6. Effect of GABA treatment on quality parameters of pear fruit

There were no significant differences of postharvest qualityparameters, including firmness, TSS, TA and AA of pear fruit, amongall treatments during 1 month of storage at 25 �C (Table 2).

4. Discussion

Most of the postharvest pathogens are incapable of penetratingdirectly through the fruit cuticle, depending on the wound for theirinvasion (Janisiewicz & Korsten, 2002). The active pathogenic pro-cess can start immediately after spores land on the wounded tis-sue, which results in the full spectrum of conditions conductiveto pathogen development (Prusky et al., 2010). The results fromthis study, for the first time, showed that GABA effectively inhib-ited the blue mould rot caused by P. expansum in pear fruit withoutadverse influence on its edible quality. The use of GABA in all foodcommodities has been approved if it meets the statutory require-ment of reasonable certainty of no harm when applied in accor-dance with good agricultural practises (US-EPA, 2004) and GABAmay have a potential postharvest use on organic produce (Prange,Ramin, Daniels-Lake, DeLong, & Braun, 2006). The application ofGABA, therefore, as a new anti-fungal biopesticide, might be devel-oped, promisingly, into an economically feasible biocontrol agentin control of postharvest fungal rots.

To further understand the mechanisms by which GABA con-trolled the blue mould rot of pear fruit, we investigated the effectof GABA on P. expansum in vitro and in vivo. Our results indicatedthat GABA, at a concentration ranging from 1 to 1000 lg/ml, didnot significantly inhibit the spore germination in PDB and the sur-vival of P. expansum on PDA. Similarly, GABA (at 100 lg/ml) had nodirect antifungal activity against P. expansum in vivo when the timeinterval between GABA treatment and pathogen inoculation waswithin 2 h. By contrast, GABA at 100 lg/ml showed clear and astrong inhibitory effect on the spore germination of P. expansumin fruit wounds when the time internal was increased to 24 h. The-ses results imply that the induction of host-mediated resistancemay be an important mechanism for GABA in reducing postharvestrot of pear fruit. It is well known that plants respond almost imme-diately to an inducing agent but, nevertheless require a lag period(Yu, Chen, Lu, & Zheng, 2009). For instance, some chemical elici-tors, such as chitosan, harpin, indole-3-acetic acid, or SA, wereeffective in developing disease resistance against postharvest path-ogen attacks only after they were applied one or several days be-fore pathogen inoculation (de Capdeville et al., 2003; Ma, Yang,Yan, Kennedy, & Meng, 2013; Yu et al., 2009).

The protection of fruit from invasion by fungal pathogens is lar-gely due to activation of a highly coordinated defensive systemthat helps ward off the spread of pathogens (Tian, Wan, Qin, &Xu, 2006; Wang et al., 2013). Generally, CHI, GLU and PAL playsan important role in induced resistance of fruits (Tian et al.,2006; Yu, Shen, Zhang, & Sheng, 2011). CHI has been proved to de-grade chitin, which is the major component of pathogen cell walls(Tian et al., 2007). GLU, one of the most fully characterisedpathogenesis-related (PR) proteins, can also act indirectly byreleasing oligosaccharide and eliciting defence reactions and thenact synergistically with CHI to inhibit fungal growth (Tian et al.,2007). PAL, as the first enzyme in the general pathway of phenyl-propanoid metabolism, is directly involved in the synthesis of

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120

140

160

0 12 24 36 48

Hours after treatment

PAL

activ

ity (U

/mg

prot

ein)

Control GABA P.exp GABA+P.exp

D

0

100

200

300

400

500

600

700

0 12 24 36 48Hours after treatment

POD

act

ivity

(U/m

g pr

otei

n)

Control GABA P.exp GABA+P.exp E

0

100

200

300

400

500

600

700

0 12 24 36 48Hours after treatment

PPO

act

ivity

(U/m

g pr

otei

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Control GABA P.exp GABA+P.exp

B

50

100

150

200

250

300

0 12 24 36 48Hours after treatment

GLU

act

ivity

(U/m

g pr

otei

n)

Control GABA P.exp GABA+P.exp

Fig. 3. Effect of c-aminobutyric acid (GABA) and Penicillium expansum on the activities of chitinase (CHI; A), b-1,3-glucanase (GLU; B), phenylalanine amonialyase (PAL; C),peroxidase (POD; D), and polyphenol oxidase (PPO; E) in pear fruit wounds. The wounds were inoculated with P. expansum 24 h after the treatment with GABA at 100 lg/ml.After the various intervals, tissue samples from GABA or water-treated fruit, with or without inoculation, were taken for enzyme extractions. Standard errors of threereplications are given as the short bars.

34 C. Yu et al. / Food Chemistry 159 (2014) 29–37

active metabolites, including phenols, phytoalexins and lignin, thatare associated with the localised resistance processes (Cao et al.,2008). In addition, POD and PPO are both linked to lignificationof host plant cells and they participate in cell wall reinforcement.They are considered key enzymes in host defence reactions againstpathogen infections (Cao et al., 2008; Ma et al., 2013; Zhang, Wang,Zhang, Hou, & Wang, 2011). Various chemical elicitors have beenreported to induce resistance against fungal infection in harvestedfruits by the stimulation of pathogenesis- or defence-related en-zyme activities and gene expression. For example, BABA treatmentinduced a significant increase in the activities of CHI, GLU and POD,thereby promoting resistance against blue mould in apple fruit(Zhang et al., 2011). The induction of resistance to Penicillium

digitatum by BABA was accompanied by activation of CHI geneexpression and an increase in PAL activity in grapefruit peel tissue(Porat et al., 2003). SA, oxalic acid and calcium chloride signifi-cantly enhanced PAL and PPO activities and reduced the diseaseincidence caused by Alternaria alternata in pear fruit (Tian et al.,2006). Benzothiadiazole-induced disease resistance againstanthracnose in harvested mango fruits was related to up-regulatedexpression of PPO and POD genes (Lin, Gong, Zhu, Zhang, & Zhang,2011). Ma et al. (2013) reported that chitosan and oligochitosan in-duced the expression of GLU and POD and elevated the resistanceof peach fruit against brown rot caused by Monilinia fructicola.Thus, the results from this present study demonstrate that GABAstrongly enhanced the activities or gene expression of CHI, GLU

PpCHI

0

5

10

15

20

25

12 24 48Hours after treatment

Fold

cha

nge

ControlGABAP.expGABA+P.exp

APpGLU

0

1

2

3

4

5

12 24 48Hours after treatment

Fold

cha

nge

ControlGABAP.expGABA+P.exp

B

PpPAL

0

2

4

6

8

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14

16

12 24 48Hours after treatment

Fold

cha

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ControlGABAP.expGABA+P.exp

C

PpPPO

0

1

2

3

4

5

12 24 48Hours after treatment

Fold

cha

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ControlGABAP.expGABA+P.exp

EPpPOD

0

1

2

3

4

12 24 48Hours after treatment

Fold

cha

nge

ControlGABAP.expGABA+P.exp

D

Fig. 4. Expression analysis of chitinase (PpCHI; A), b-1,3-glucanase (PpGLU; B), phenylalnine ammonialyase (PpPAL; C), peroxidase (PpPOD; D) and polyphenol oxidase (PpPPO;E) genes in wound tissue of pear fruit by qRT-PCR. Fruit wounds were inoculated with Penicillium expansum 24 h after the treatment of c-aminobutyric acid (GABA) at 100 lg/ml. After various intervals, tissue samples from GABA- or water-treated fruit, with or without inoculation, were removed. Values were normalised to control at each timepoint, arbitrarily set to 1. PpActin was used as endogenous reference gene. ⁄Indicates up-regulated expression (fold change P2) for each time point. The vertical bars representthe standard error of three replicates.

C. Yu et al. / Food Chemistry 159 (2014) 29–37 35

and PAL as well as the levels of POD and PPO in pear fruit, espe-cially when inoculated with P. expansum, which might contributeto the fruit resistance against P. expansum by the treatment withGABA.

Moreover, in view of the pronounced and rapid increase in theactivities of enzymes and the expression levels of defence-relatedgenes after treatment with GABA followed by P. expansum in con-trast to the treatment with the pathogen alone, we infer that themode of the action for the induced resistance by GABA might beassociated with mechanisms of priming. The benefit of applyingGABA is, at least, partially due to the triggering of defence re-sponses, at enzymatic and transcript level, related to induced resis-tance mechanisms, which make the primed fruit less prone todisease development after infection. It is well known that the

mechanisms of induced resistance appear to involve the primingof plant defences that display a more rapid response followinga challenge inoculation with a virulent strain of a pathogen(Hammerschmidt, 2009). The enhanced defensive capacity bypriming is correlated with a potentiated expression of defencegenes and de novo synthesis of antimicrobial compounds, such aspathogenesis-related proteins (Solano, Maicas, de la Iglesia,Domenech, & Mañero, 2008). Priming leads, not only to an alarmedstate of defence, but also offers low energy cost protection underconditions of relatively high disease pressure (van der Ent et al.,2009). This phenomenon, described as ‘‘priming’’, occurs in somechemical compound (BABA, BTH and ASM) – or beneficial microor-ganism (rhizobacterium, Bacillus cereus AR156) – mediated resistance(Conrath et al., 2002; van der Ent et al., 2009; Wang et al., 2013).

Table 2Effect of c-aminobutyric acid (GABA) on quality parameters of pear fruit during 1 month of storage at 25 �C.

Treatment Firmness (�105 Pa) TSS (%) TA (%) AA (mg/100 g)

0 day 12.83 ± 0.13 10.10 ± 0.46 0.09 ± 0.03 0.45 ± 0.067 day control 12.17 ± 0.32 a 10.7 ± 0.41 a 0.11 ± 0.01 a 1.22 ± 0.04 a10 lg/ml GABA 12.90 ± 0.69 a 9.97 ± 0.07 a 0.07 ± 0.01 a 0.76 ± 0.15 a100 lg/ml GABA 12.62 ± 0.20 a 10.6 ± 0.27 a 0.07 ± 0.01 a 0.74 ± 0.19 a1000 lg/ml GABA 12.62 ± 0.40 a 9.97 ± 0.38 a 0.08 ± 0.02 a 0.75 ± 0.15 a15 day Control 12.30 ± 1.30 a 9.80 ± 0.40 a 0.09 ± 0.00 a,b 1.62 ± 0.11 a10 lg/ml GABA 11.78 ± 0.18 a 9.85 ± 0.05 a 0.06 ± 0.02 b 1.44 ± 0.37 a100 lg/ml GABA 12.45 ± 0.55 a 9.70 ± 0.30 a 0.08 ± 0.00 a,b 1.55 ± 0.25 a1000 lg/ml GABA 12.15 ± 1.00 a 10.4 ± 0.40 a 0.11 ± 0.02 a 1.49 ± 0.83 a30 day control 11.75 ± 0.35 a 9.48 ± 0.30 a 0.05 ± 0.01 a 1.57 ± 0.44 a10 lg/ml GABA 12.08 ± 0.44 a 9.93 ± 0.47 a 0.05 ± 0.01 a 0.93 ± 0.36 a100 lg/ml GABA 11.90 ± 0.18 a 9.23 ± 0.47 a 0.06 ± 0.01 a 0.98 ± 0.38 a1000 lg/ml GABA 11.97 ± 0.07 a 9.10 ± 0.06 a 0.06 ± 0.01 a 0.60 ± 0.02 a

The fruit were immersed in solutions of 10, 100 or 1000 lg/ml of c-aminobutyric acid for 10 min. and then air-dried. Samples were collected after 7, 15 and 30 days of storageat 25 �C for quality evaluation. Each value is the mean of three separate determinations and the standard errors. Different letters indicate significant differences (P = 0.05)according to Duncan’s multiple range test for each time point.

36 C. Yu et al. / Food Chemistry 159 (2014) 29–37

Nevertheless, more research into the mode of action of GABA incontrolling postharvest diseases of fruit is needed, particularly tofurther elucidate molecular mechanisms underlying induced resis-tance and priming.

Acknowledgements

This research was partially supported by the Foundation for theAuthor of National Excellent Doctoral Dissertation of PR China(201061) and the Program for Key Innovative Research Team ofZhejiang Province (2009R50036).

References

Bown, A. W., MacGregor, K. B., & Shelp, B. J. (2006). Gamma-aminobutyrate: Defenseagainst invertebrate pests? Trends in Plant Science, 11, 424–427.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein dye binding.Analytical Biochemistry, 72, 248–254.

Cao, S. F., Yang, Z. F., Hua, Z. C., & Zheng, Y. H. (2011). The effects of the combinationof Pichia membranefaciens and BTH on controlling of blue mould decay causedby Penicillium expansum in peach fruit. Food Chemistry, 124, 991–996.

Cao,S.F.,Zheng,Y.H.,Yang,Z.F.,Tang,S.S.,Jin,P.,Wang,K.T.,etal.(2008).EffectofmethyljasmonateontheinhibitionofColletotrichumacutatum infectioninloquatfruitandthepossiblemechanisms.PostharvestBiologyandTechnology,49,301–307.

Conrath, U., Pieterse, C. M. J., & Mauch-Mani, B. (2002). Priming in plant–pathogeninteractions. Trends Plant Science, 7, 210–216.

de Capdeville, G., Beer, S. V., Watkins, C. B., Wilson, C. L., Tedeschi, L. O., & Aist, J. R.(2003). Pre- and postharvest harpin treatments of apples induce resistance toblue mould. Plant Disease, 87, 39–44.

Droby, S., Wisniewski, M., Macarisin, D., & Wilson, C. (2009). Twenty years ofpostharvest biocontrol research: Is it time for a new paradigm? PostharvestBiology and Technology, 52, 137–145.

Hammerschmidt, R. (2009). Challenge inoculation reveals the benefits of resistancepriming. Physiological and Molecular Plant Pathology, 73, 59–60.

Ippolito, A., El-Ghaouth, A., Wilson, C. L., & Wisniewski, M. (2000). Control ofpostharvest decay of apple fruit by Aureobasidium pullulans and induction ofdefense responses. Postharvest Biology and Technology, 19, 265–272.

Janisiewicz, W. J., & Korsten, L. (2002). Biological control of postharvest diseases offruits. Annual Review of Phytopathology, 40, 411–441.

Jurick, W. M., II, Vico, I., Gaskins, V. L., Garrett, W. M., Whitaker, B. D., Janisiewicz, W.J., et al. (2009). Purification and biochemical characterization ofpolygalacturonase produced by Penicillium expansum during postharvestdecay of ‘Anjou’ pear. Phytopathology, 100, 42–48.

Lin, J. H., Gong, D. Q., Zhu, S. J., Zhang, L. J., & Zhang, L. B. (2011). Expression of PPOand POD genes and contents of polyphenolic compounds in harvested mangofruits in relation to benzothiadiazole-induced defense against anthracnose.Scientia Horticulturae, 130, 85–89.

Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression datausing real-time quantitative PCR and the 2�DDC(T) method. Methods, 25,402–408.

Lurie, S., Fallik, E., Handros, A., & Shapira, R. (1997). The possible involvement ofperoxidase in resistance to Botrytis cinerea in heat treated tomato fruit.Physiological and Molecular Plant Pathology, 50, 141–149.

Ma, Z. X., Yang, L. Y., Yan, H. X., Kennedy, J. F., & Meng, X. H. (2013). Chitosan andoligochitosan enhance the resistance of peach fruit to brown rot. CarbohydratePolymers, 94, 272–277.

Malandrakis, A. A., Markoglou, A. N., Konstantinou, S., Doukas, E. G., Kalampokis, J.F., & Karaoglanidis, G. S. (2013). Molecular characterization, fitness andmycotoxin production of benzimidazole-resistant isolates of Penicilliumexpansum. International Journal of Food Microbiology, 162, 237–244.

Mohammadi, M., & Kazemi, H. (2002). Changes in peroxidase and polyphenoloxidase activities in susceptible and resistant wheat heads inoculated withFusarium graminearum and induced resistance. Plant Science, 16, 2491–2498.

Porat, R., Vinokur, V., Weiss, B., Cohen, L., Daus, A., Goldschmidt, E. E., et al. (2003).Induction of resistance to Penicillium digitatum in grapefruit by b-aminobutyricacid. European Journal of Plant Pathology, 109, 901–907.

Prange, R. K., Ramin, A. A., Daniels-Lake, B. J., DeLong, J. M., & Braun, P. G. (2006).Perspectives on postharvest biopesticides and storage technologies for organicproduce. HortScience, 41, 301–303.

Prusky, D., Alkan, N., Miyara, I., Barad, S., Davidzon, M., Kobiler, I., et al. (2010).Mechanisms modulating postharvest pathogen colonization of decaying fruits.Postharvest Pathology, Netherlands: Springer (Chapter 4).

Quaglia, M., Ederli, L., Pasqualini, S., & Zazzerini, A. (2011). Biological control agentsand chemical inducers of resistance for postharvest control of Penicilliumexpansum link. On apple fruit. Postharvest Biology and Technology, 59, 307–315.

Shang, H. T., Cao, S. F., Yang, Z. F., Cai, Y. T., & Zheng, Y. H. (2011). Effect of exogenousc-aminobutyric acid treatment on proline accumulation and chilling injury inpeach fruit after long-term cold storage. Journal of Agriculture and FoodChemistry, 59, 1264–1268.

Shelp, B. J., Bozzo, G. G., Trobacher, C. P., Zarei, A., Deyman, K. L., & Brikis, C. J. (2012).Hypothesis/review: Contribution of putrescine to 4-aminobutyrate (GABA)production in response to abiotic stress. Plant Science, 193, 130–135.

Shoresh, M., Harman, G. E., & Mastouri, F. (2010). Induced systemic resistance andplant responses to fungal biocontrol agents. Annual Review of Phytopathology,48, 21–43.

Solano, B. R., Maicas, J. B., de la Iglesia, M. T. P., Domenech, J., & Mañero, F. J. G.(2008). Systemic disease protection elicited by plant growth promotingrhizobacteria strains: Relationship between metabolic responses, systemicdisease protection, and biotic elicitors. Phytopathology, 4, 451–457.

Song, H. M., Xu, X. B., Wang, H., Wang, H. Z., & Tao, Y. Z. (2010). Exogenous c-aminobutyric acid alleviates oxidative damage caused by aluminium andproton stresses on barley seedlings. Journal of Science Food Agriculture, 90,1410–1416.

Tian, S. P., Wan, Y. K., Qin, G. Z., & Xu, Y. (2006). Induction of defense responsesagainst Alternaria rot by different elicitors in harvested pear fruit. AppliedMicrobial and Cell Physiology, 70, 729–734.

Tian, S. P., Yao, H. J., Deng, X., Xu, X. B., Qin, G. Z., & Chan, Z. L. (2007).Characterization and expression of b-1,3-glucanase genes in jujube fruitinduced by the microbial biocontrol agent Cryptococcus laurentii.Phytopathology, 97, 260–268.

Tonelli, M. L., Furlan, A., Taurian, T., Castro, S., & Fabra, A. (2011). Peanut priminginduced by biocontrol agents. Physiological and Molecular Plant Pathology, 75,100–105.

US Environmental Protection Agency. (2004). Pesticide tolerances: Gammaaminobutyric acid. Federal Register 69(207): 62596–62602. <http://www.epa.gov/fedrgstr/EPA-PEST/2004/October/Day-27/p24041.htm>.

van der Ent, S., van Hulten, M., Pozo, M. J., Czechowski, T., Udvardi, M. K., Pieterse, C.M. J., et al. (2009). Priming of plant innate immunity by rhizobacteria and b-aminobutyric acid: Differences and similarities in regulation. New Phytologist,183, 419–431.

Venturini, M. E., Oria, R., & Blanco, D. (2002). Microflora of two varieties of sweetcherries: Burlat and Sweetheart. Food Microbiology, 19, 15–21.

Wang, X. L., Xu, F., Wang, J., Jin, P., & Zheng, Y. H. (2013). Bacillus cereus AR156induces resistance against Rhizopus rot through priming of defense responses inpeach fruit. Food Chemistry, 136, 400–406.

Yalpani, M., & Pantaleone, D. (1994). An examination of the unusual susceptibilitiesof aminoglycans to enzymatic hydrolysis. Carbohydrate Research, 256,159–175.

C. Yu et al. / Food Chemistry 159 (2014) 29–37 37

Yang, A. P., Cao, S. F., Yang, Z. F., Cai, Y. T., & Zheng, Y. H. (2011). C-Aminobutyricacid treatment reduces chilling injury and activates the defense response ofpeach fruit. Food Chemistry, 129, 1619–1622.

Yu, T., Chen, J., Lu, H., & Zheng, X. D. (2009). Indole-3-acetic acid improvespostharvest biocontrol of blue mould rot of apple by Cryptococcus laurentii.Phytopathology, 99, 258–264.

Yu, M. M., Shen, L., Zhang, A. J., & Sheng, J. P. (2011). Methyl jasmonate-induceddefense responses are associated with elevation of 1-aminocyclopropane-1-carboxylate oxidase in Lycopersicon esculentum fruit. Journal of Plant Physiology,168, 1820–1827.

Zhang, C. F., Wang, J. M., Zhang, J. G., Hou, C. J., & Wang, G. L. (2011). Effects of b-aminobutyric acid on control of postharvest blue mould of apple fruit and itspossible mechanisms of action. Postharvest Biology and Technology, 61, 145–151.

Zhang, D., Yu, B., Bai, J. H., Qian, M. J., Shu, Q., Su, J., et al. (2012). Effects of hightemperatures on UV-B/visible irradiation induced postharvest anthocyaninaccumulation in ‘Yunhongli No. 1’ (Pyrus pyrifolia Nakai) pears. ScientiaHorticulturae, 134, 53–59.