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Postharvest Biology and Technology 85 (2013) 77–82

Contents lists available at ScienceDirect

Postharvest Biology and Technology

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

ypobaric treatment stimulates defence-related enzymes in strawberry

ajid S. Hashmi, Andrew R. East ∗, Jon S. Palmer, Julian A. Heyesentre for Postharvest and Refrigeration Research, Massey University, Private Bag 11222, Palmerston North 4474, New Zealand

r t i c l e i n f o

rticle history:eceived 28 February 2013ccepted 5 May 2013

eywords:efence enzymesragaria × ananassaungal decaynduced resistance

a b s t r a c t

Strawberry fruit are very prone to fungal decay. Postharvest hypobaric treatment is a potential new tech-nique to delay fungal decay in strawberries. Hypobaric treated (50 kPa, 4 h) strawberries had reduced rotincidence from natural infection during subsequent storage for 4 days at 20 ◦C and after subsequent inoc-ulation with Botrytis cinerea or Rhizopus stolonifer spores. Biochemical analysis of strawberries suggestedthat activities of defence-related enzymes were increased with the hypobaric treatment; phenylalanineammonia-lyase (PAL, EC: 4.3.1.24) and chitinase (EC: 3.2.1.14) peaked 12 h after treatment, while peroxi-dase (POD, EC: 1.11.1.7) increased immediately. Polyphenol oxidase (PPO, EC: 1.10.3.1) activity remainedunaffected during subsequent storage for 48 h at 20 ◦C. In addition, the effect of low oxygen treatment

ow pressure (10% at 101 kPa, 4 h) was investigated to determine if the lower partial pressure of oxygen generatedduring hypobaric treatment contributed to the observed effect. However the low oxygen treatment didnot influence rot development, suggesting that the treatment effects were pressure rather than oxygenrelated. The results suggest that hypobaric treatment causes reduced decay incidence due to stimulationof defence-related enzymes. Studies of defence-related genes are required to further explore the inducedresistance mechanisms of hypobaric treatments.

. Introduction

Strawberries are a highly perishable fruit with a storage lifef 1–2 days at room temperature (Cao et al., 2010). Apart fromigh physiological activity, postharvest fungal decay by Botrytisinerea and Rhizopus stolonifer are the main causes of reductionn strawberry storage life (Nigro et al., 2000; Perdones et al.,012). Although pre-harvest application of fungicides reduces fun-al decay in strawberries, fungicides should not be applied afterarvest, as high residue levels may remain present (Romanazzit al., 2013). Fungal resistance development to chemicals andesidue contamination from fungicides has led to the investiga-ion of alternative approaches for controlling rots in fresh produceTripathi and Dubey, 2004).

In recent years, many innovative physical techniques have beensed for the potential reduction of fungal decay in fresh pro-uce. These include heat (Wang et al., 2010; Liu et al., 2012),V (Xu and Du, 2012), ultrasound (Cao et al., 2010), and hyper-aric (Goyette et al., 2012) and hypobaric treatments (Romanazzit al., 2001). Research into some of these techniques remains at

n elementary stage and requires further investigation into theundamental mechanisms causing the observed reduction in fun-al decay. Hypobaric treatments have produced some promising

∗ Corresponding author. Tel.: +64 6356 9099; fax: +64 6350 5610.E-mail address: A.R.East@massey.ac.nz (A.R. East).

925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.postharvbio.2013.05.002

© 2013 Elsevier B.V. All rights reserved.

results, with reduced fungal growth of R. stolonifer and B. cinerea insweet cherries, grapes and strawberries (Romanazzi et al., 2001,2003). However, the mechanisms of these effects of hypobarictreatment remain unknown, although the current literature sug-gests that hypobaric treatment is not affecting the in vitro growth offungi (Hashmi et al., 2013). This leads to the hypothesis that hypo-baric treatment may act as a resistance inducer to tackle fungalinfection.

Different approaches have been taken to understand the mech-anism of induced resistance in plants. It is believed that fruit releasedefence related phytochemicals upon biotic and abiotic stress(Ferreira et al., 2007), and therefore creation of stress conditionsbefore infection could also lead to the stimulation of these defence-related compounds (Yao and Tian, 2005). Inoculation of challengefungi after treatment has been used to evaluate the response of fruitto the corresponding treatment (Jin et al., 2009). Similarly, defenceenzymes have been studied to understand the mechanisms of decaycontrol in different treatments (Zeng et al., 2006; Jin et al., 2009;Wang et al., 2010; Pombo et al., 2011). These defence enzymesinclude phenyl alanine ammonia-lyase (PAL), chitinase, peroxidase(POD), polyphenol oxidase (PPO) and �-1,3-glucanase. In addition,molecular studies of these enzymes have also been used to inves-tigate the mechanism of induced resistance (Pombo et al., 2009;

Dotto et al., 2011).

PAL plays a key role in the phenylpropanoid pathway (Krugeret al., 2002). Synthesis of phytoalexins and lignin directly involvePAL (Sticher et al., 1997; Baarlen et al., 2007), which is related

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8 M.S. Hashmi et al. / Postharvest Bi

o induced resistance to pathogen invasion (Wang et al., 2011).imilarly the importance of plant chitinases in defence againstathogens is well documented (Van Loon et al., 2006; Grover, 2012).hitin is the main cell wall constituent of fungi and the insect skele-on (Sharma et al., 2011). Pathogenesis-related proteins, chitinasesn combination with �-1,3-glucanases, hydrolyse chitin present inungal cell walls resulting in inhibition of fungal growth (Grover,012). POD is also considered to be one of the anti-fungal proteinsFerreira et al., 2007) and also an important enzyme in the biosyn-hesis of phenolic compounds which can be stimulated by variousnducers.

Previous work by the authors demonstrated that hypobaricreatment (50 kPa, 4 h) can reduce fungal decay in strawberriesHashmi et al., 2013). One possible mechanism of the hypobaricondition could be the modification of oxygen partial pressure (Ant al., 2009). As a result of applying the successful hypobaric treat-ent (50 kPa, 4 h) the oxygen partial pressure also decreases from21 kPa (≈21% at 101 kPa) to ≈10 kPa (or ≈21% at 50 kPa). This

eduction in oxygen partial pressure may be responsible for a mildtress that results in induced resistance and causes the delay inungal decay of strawberries. A study of low oxygen concentrationreatment at atmospheric pressure is needed to determine if lowxygen partial pressure in hypobaric treatment contributes to theotential stress response.

The objective of this study was to (1) determine if low O2 partialressure achieved during hypobaric treatment contributes to theeduced decay development of hypobaric-treated strawberries, (2)nvestigate the response of hypobaric treated strawberries to thenoculation of B. cinerea and R. stolonifer, and (3) assess the effectf hypobaric treatment on the activity of defence-related enzymesn strawberry. As a whole, this study explores the possible modesf action for decay reduction as caused by pre-storage hypobaricreatments.

. Materials and methods

.1. Strawberry fruit

Field grown strawberries (Fragaria × ananassa) cv. Camarosa,ere obtained from a commercial grower in the Whanganui region,ew Zealand. On arrival at Massey University, bruised and dam-ged fruit were removed from the population. Fruit with calyx andedicel were selected for experimental work. In the third experi-ent where fungal spores were inoculated onto strawberries, 400

ydroponically grown ‘Camarosa’ strawberries were obtained fromhe Massey University Plant Growth Unit, Palmerston North, Newealand.

.2. Treatments

Three independent experiments were conducted, each aimed atddressing a single objective. In the first experiment 56 clamshells≈250 g) of 12–15 sound fruit were selected and were divided intowo treatments: 50 kPa for 4 h in a vacuum tank, or atmosphericonditions (≈101.3 kPa) for the same time at 20 ◦C. After hypobaricreatment (50 kPa, 4 h), 24 clamshells were used for collection ofamples for enzyme analysis 0, 6, 12, 18, 24 and 48 h after hypobaricreatment, while 32 clamshells were used for storage.

In a second experiment 56 clamshells of strawberries weresed and divided into two treatments: ≈10% O2 treatment for 4 h,r atmospheric conditions (≈21% O2) for the same time at 20 ◦C.

fter O2 treatments, samples were collected as for the first exper-

ment.In the third experiment, four hundred fully ripe strawberries

ere disinfected by dipping in commercial grade NaClO solution

and Technology 85 (2013) 77–82

(2%) for 5 s. After drying the fruit at room temperature, each fruitwas placed in a cylindrical plastic container with a porous lid toavoid cross-contamination and ensure respiration. Half of the fruitwere treated with hypobaric pressure (50 kPa for 4 h), with theothers used as a control (≈101.3 kPa). Each strawberry fruit fromboth groups was inoculated by spreading 10 �L of spore suspension(104 mL−1) of B. cinerea or R. stolonifer on the surface 0, 6, 12, 18 and24 h after hypobaric treatment. For each treatment, 20 individualfruit were used at each time interval.

2.2.1. Hypobaric treatmentHypobaric pressure was generated in a hermetically sealed tank

with a vacuum pump. Clamshells/containers of strawberries wereplaced inside the tank at 20 ◦C as described by Hashmi et al. (2013).The vacuum generated during treatment was measured with acalibrated pressure gauge (EN 837-1, Nuova Fima, Invorio, Italy).Clamshells/containers placed inside the same room at atmosphericpressure (101.3 kPa) were used as controls. Hypobaric pressure,temperature and relative humidity (RH) inside the chamber weremonitored as previously described (Hashmi et al., 2013).

2.2.2. Oxygen treatmentFor experiment 2, Strawberries in clamshells were kept in 7 L air-

tight Perspex boxes (6 clamshells in each box) at 20 ◦C and exposedto 10% O2 (at 101 kPa) or control (21% O2) by supplying the desiredhumidified gas mixture in a flow-through system. The 10% O2 treat-ment was achieved by reducing the oxygen content of air by mixingwith nitrogen (BOC, Auckland, New Zealand). The flow rate of bothtreatments was maintained (0.5 L min−1) for 4 h, to ensure no mod-ification of the gas environments due to strawberry respiration.

2.2.3. Inoculation of strawberry with B. cinerea and R. stoloniferIn order to investigate the possibility of induced resistance

by hypobaric treatment against fungi, an inoculum was requiredfor experiment 3. B. cinerea and R. stolonifer were isolated fromdiseased strawberries, grown on potato dextrose agar (PDA) andincubated at 20 ◦C until conidia developed. Spores from 7-day-oldcultures were centre seeded on another set of Petri dishes to obtainpure cultures. After 7 days the conidia from each pure culture wereharvested by suspending in sterile water containing 0.03% (v/v)Tween20. This conidial suspension was then filtered through a ster-ile cheese cloth. The concentration of the filtrate was adjusted toapproximately 104 spores mL−1 with the addition of sterile waterby estimating fungal spore concentration through the use of ahaemocytometer.

2.3. Storage and decay development

Samples were subsequently stored after pressure treatment at20 ◦C and 80–90% RH. Observations of decay development wererecorded daily. For natural rot incidence (experiments 1 and 2) 4independent clamshells per treatment were assessed destructivelyat each sampling day, while rot incidence was visually assessed byexamining each berry. Fruit with visible decay were counted andthe results recorded as percentage of rot incidence per clamshell.

For the inoculation third experiment, for each inoculation time,20 individual strawberries were repeatedly assessed daily for rotseverity. The extent of macroscopic fungal development of indi-

vidual strawberry fruit was externally analysed by estimating theaffected area percentage of each fruit to nearest 10%. From thesefigures, mean percentage severity of infected strawberry fruit wascalculated.

ology and Technology 85 (2013) 77–82 79

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10% O2

B

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Fig. 1. Effect of hypobaric treatment (A) and 10% O2 (B) on rot incidence of straw-berries stored at 20 ◦C. Each data point represents the mean value of four replicate

not be due to low oxygen partial pressure but to some other mech-

M.S. Hashmi et al. / Postharvest Bi

.4. Enzyme assays

Fruit (5 per replicate) with no sign of decay were cut into smallieces with a sharp blade, immediately frozen with liquid nitrogennd stored at −30 ◦C for later analysis.

The phenylalanine ammonia-lyase (PAL) activity was measuredy a method modified from Civello et al. (1997) and Pombo et al.2011). Frozen strawberries (5 g) were homogenized in a high-erformance dispersing instrument (Ultra-Turrax® T25 Basic) for

min with 20 mL of extraction buffer (0.1 mol L−1 Na2B4O7·10H2O, mmol L−1 2-mercaptoethanol, 2 mmol L−1 EDTA, 3% (w/v)olyvinylpolypyrrolidone (PVPP), pH 8.8). The mixture was left totir for 1 h at 4 ◦C and centrifuged at 10,000 × g for 20 min. Theupernatant was used for phenylalanine ammonia-lyase assay. Theollowing reaction mixture was employed: 2550 �L 0.03 mol L−1

a2B4O7·10H2O pH 8.8, 450 �L 0.01 mol L−1 l-phenylalanine in.03 mol L−1 Na2B4O7·10H2O (pH 8.8), and 1500 �L of extract.he optical density (OD) of reaction mixture was measured at90 nm after 0, 0.5, 2, 3 h while incubating at 37 ◦C in a waterath. The rate was linear; therefore specific enzymatic activ-

ty was calculated as increase in �OD kg−1 s−1 between 0 and h.

Chitinase activity was determined by the method of Pombo et al.2011) with some modifications. Frozen strawberries (5 g) wereomogenized (Ultra-Turrax® T25 Basic) with 15 mL of 10 mmol L−1

H3COONa. 3H2O buffer, 2% (w/v) Polyvinylpyrrolidone (PVP), pH. The homogenate was stirred for 3 h at 4 ◦C and centrifuged at2,000 × g for 30 min. The pellet was discarded and chitinase activ-

ty was assayed from supernatant using the following reactionixtures as employed by Pombo et al. (2011): 739 �L of 2 g L−1

hitin azure (Sigma) and 2210 �L of enzyme extract. The reactionixture was kept at 37 ◦C while stirring, 710 �L of extract was taken

t 0, 2, 4 and 6 h and the reaction was stopped by adding 178 �Lf 2 M HCl at each time interval. The samples were kept on ice for

min and increase in absorbance was measured at 575 nm between and 6 h.

The method for determination of POD activity was modi-ed from Civello et al. (1995). The extraction buffer consisted of.02 mol L−1 Na2HPO4, 0.08 mol L−1 NaH2PO4, 0.1% (v/v) Triton X-00, 1 mol L−1 NaCl, 30 g L−1 PVPP, at pH 6. Frozen strawberries5 g) were mixed with extraction buffer (1:4) and homogenizedith a high-performance dispersing instrument (Ultra-Turrax®

25 Basic) for 2 minutes. The mixture was left to stir for 1 hnd then centrifuged at 12,000 × g for 20 min. The reaction mix-ure consisted of: 500 �L of 0.02 mol L−1 Na2HPO4/0.08 mol L−1

aH2PO4 buffer at pH 6, 200 �L of 2 mmol L−1 pyrogallol,00 �L 4 mmol L−1 H2O2 and 100 �L enzyme extract. The mix-ure was kept at 20 ◦C and the enzymatic activity determinedy calculating the increase in OD at 470 nm between 0 and5 min.

For measuring polyphenoloxidase (PPO) activity, the enzymextract was prepared according to Pombo et al. (2011) with someinor modifications at 4 ◦C. Enzyme extracts were prepared as

escribed for POD. The resulting supernatant was used in thenzyme assay. The following reaction mixture was used: 950 �L.02 mol L−1 Na2HPO4/0.08 mol L−1 NaH2PO4 buffer, pH 6, 400 �Lf strawberry extract and 150 �L of 20 mmol L−1 pyrocatechol. Theixture was kept at 20 ◦C and increase in optical density (OD) at

10 nm was measured as the enzyme activity over a 90 min period.he enzyme activity was determined by calculating the increase inbsorbance between 0 and 90 min.

In all cases, control reactions were carried out in buffers withoutnzyme and no increase in OD was observed. The enzyme activityas expressed as optical density change per kilogram of fruit per

econd (�OD kg−1 s−1). Optical density was measured with Shi-adzu UV 160A UV-VIS spectrophotometer.

clamshells. Overall significant difference (P < 0.05) is represented by * as determinedby Tukey’s test. Vertical bars represent the standard error of mean values.

2.5. Data analysis

All the data were subjected to analysis of variance (ANOVA) withMinitab Version 16 (Minitab Inc., State College, PA, USA). Meanswere compared by a Tukey test at a significance level of 0.05 and0.01. The enzyme activities of PAL and chitinase represent the meanof 4 independent samples while POD and PPO value is the mean of3 independent replicates.

3. Results and discussion

3.1. Rot incidence

Hypobaric-treated (50 kPa, 4 h) strawberries showed significant(P < 0.05) reduction in natural rot incidence during subsequent stor-age for 4 days at 20 ◦C as compared to the control (Fig. 1A). Visiblefungal rots were observed on day 2 in both treated and controlsamples. This data set is additional to that presented previously(Hashmi et al., 2013) and hence represents the 4th independent setof data collected that demonstrates the delay in development ofdecay for hypobaric-treated strawberries. Similar results have pre-viously been reported for hypobaric-treated sweet cherries, grapes,strawberries (Romanazzi et al., 2001) and blueberries (Hashmiet al., 2012).

Meanwhile, strawberries treated with 10% O2 demonstrated nodifference from the control during subsequent storage (Fig. 1B).This indicates that reduction in rots by hypobaric treatment may

anism that triggers the response of the fruit to the low pressurestress.

80 M.S. Hashmi et al. / Postharvest Biology and Technology 85 (2013) 77–82

Time of inoculation after treatment (h)

0 6 12 18 24

Ro

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ty (

%)

0

20

40

60

80

100

0 6 12 18 24

B

**

A

Control (101.3 kPa ) 50 kPa

*

*

* *

Fig. 2. Strawberry rot severity after inoculation of Botrytis cinerea (A) and Rhizopus stolonifer (B) and subsequent storage for 5 and 8 days at 20 ◦C respectively after treatment.Each data point represents the mean value of 20 individual fruit. Significant differences (P < 0.05) and (P < 0.01) at each measurement time are represented by * and **r

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espectively as determined by Tukey’s test.

.2. Response of hypobaric-treated strawberries to fungalnoculation

Control and treated strawberries showed visible B. cinerea and R.tolonifer infection 2 and 3 days after hypobaric treatments respec-ively (data not shown). Hypobaric-treated strawberries inoculatedmmediately and 12 h after treatment demonstrated significantP < 0.05 and P < 0.01) reduction in rot severity of B. cinerea and R.tolonifer after 5 days and 8 days respectively as compared to con-rols, while the treated strawberry samples inoculated after 6, 18nd 24 h were comparable with the control (Fig. 2A and B). Straw-erries inoculated 12 h after hypobaric treatment had a twofoldot severity reduction for both pathogens. As hypobaric treatmentccurred before inoculation, this decrease in rots must be attributedo an effect of treatment on the fruit rather than a direct fungicidalffect. The reduced decay in samples inoculated immediately and2 h after treatment (Fig. 2A and B) suggests a possible inductionf specific fungicidal or fungistatic activity after treatment. Pombot al. (2011) observed a similar twofold reduction in rot sever-ty of UV-C treated strawberries artificially inoculated after 8 h.igro et al. (2000) demonstrated that time of artificial inoculationfter UV-C treatment significantly affected the development of rotsith strawberries inoculated 12 h after UV-C treatment (0.5 kJ m−2)

esulting in minimal rots in comparison to other inoculation times.ur results show a similar dependency of rot development on inoc-lation time. The rot severity in samples inoculated 18 and 24 hfter treatment may be influenced by the reduced resistance of fruitue to over-ripening (Guidarelli et al., 2011).

.3. PAL and chitinase activity

Hypobaric treatment significantly (P < 0.05) increased PAL activ-ty of strawberries as compared to controls in subsequent storageor 48 h. Enzyme activity of treated and control strawberriesemained similar up to 6 h after treatment, but treated sampleshowed a peak value 12 h after treatment (Fig. 3A).

The activities of PAL and chitinase are associated with inducedesistance (Giannakis et al., 1998; Ngadze et al., 2012). Differenttudies have suggested different time ranges of induction of resis-

ance for different treatments (Pombo et al., 2011; Yu et al., 2012;hao et al., 2013). Nigro et al. (2000) observed a twofold increasen PAL activity 12 h after mild UV-C treatment while Pombo et al.2011) reported a threefold rise in PAL activity immediately after

higher doses of UV-C treatment (4.1 kJ m−2). Our results demon-strate a 40% increase in PAL activity 12 h after hypobaric treatment.This suggests that hypobaric treatment (50 kPa, 4 h) may inducemild stress in strawberry as compared to UV-C treatments. Inanother study, Shao et al. (2013) found that treatment of tea treeoil (TTO) vapour stimulated PAL activity of inoculated strawberrywith a 50% rise after 24 h, which also resulted in rot reduction.

Contrary to our results, Civello et al. (1997) observed reductionin PAL activity of strawberry fruit after heat treatment (42 or 48 ◦Cfor 3 h) and this decrease was correlated with delayed anthocyaninaccumulation and surface colour. Our previous work suggested thathypobaric treatment did not affect firmness and respiration rateduring storage (Hashmi et al., 2013), therefore the response of fruitto hypobaric treatment may be different to heat stress.

Chitinase activity remained higher (P < 0.05) in treated samplesas compared to controls during subsequent storage for 48 h. Thepeak value was observed 12 h after hypobaric treatment (Fig. 3B).

Ippolito et al. (2000) reported that inoculation of Aureobasidiumpullulans in apple wounds caused a threefold transient increase inchitinase and glucanase activity after 96 h, which reduced fungalrot development. Similarly Wang et al. (2010) observed that hot airtreatment (48 ◦C, 3 h) elevated chitinase activity of bayberry fruitby 60% after 48 h of inoculation with Leptographium abietinum. Inthe current work, we observed a 38% increase in chitinase activ-ity 12 h after hypobaric treatment of un-inoculated strawberries(Fig. 3B), while the individual strawberries inoculated 0 and 12 hafter treatment also showed significant reduction in rots (Fig. 2Aand B). The magnitude of rise of chitinase activity observed waslower than in previously reported data, which may be a resultof measuring un-inoculated fruit. Earlier studies suggested thatenhancement of chitinase activity in different plant species due tobiotic and abiotic stress ranges from 1 to 28 days, which signifiesthat accumulation of chitinase in diseased tissues can only delay thegrowth of necrotrophic pathogens and is not engaged in early host-pathogen interaction (Punja and Zhang, 1993). However, recentlyGrover (2012) reviewed that accumulation of chitinases and glu-canases after fungal attack were the outcome of induced resistanceand thus play an important role in plant resistance to pathogeninvasion. In this study chitinase activity peaked 12 h after hypobaric

treatment, therefore the possibility of involvement of chitinase inearly stage of fungal attack cannot be ruled out.

These results suggest that hypobaric treatment might have stim-ulated PAL and chitinase activities (Fig. 3A and B), this could be the

M.S. Hashmi et al. / Postharvest Biology and Technology 85 (2013) 77–82 81

F and Pi erence

pl

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oibitdiaYtc(bOsniga

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ig. 3. Effect of hypobaric treatment (50 kPa, 4 h) on PAL (A), chitinase (B), POD (C)ndependent measurements. Vertical bars represent LSD at = 0.05. Significant diff

ossible reason for the reduction in decay of strawberries inocu-ated 12 h post-hypobaric treatment (Fig. 2).

.4. POD and PPO activity

An increase in POD activity of hypobaric treated samples wasbserved immediately after treatment, reverting to the same activ-ty as control from 6 h after treatment (Fig. 3C). PPO activity ofoth control and hypobaric treated strawberries was similar dur-

ng subsequent storage for 48 h at 20 ◦C (Fig. 3D). Given thathe experiment on post-hypobaric treatment fungal inoculationemonstrated a reduction in decay for strawberries inoculated

mmediately after treatment (Fig. 2), the observed increase in PODctually would suggest a role of this enzyme in reducing decay.u et al. (2012) previously demonstrated that yeast saccharidereatment induced the activities of POD, PAL, �-1,3-glucanase andhitinase causing reduction in decay of peach fruit. Wang et al.2010) observed that hot air treatment of inoculated Chinese bay-erry fruit increased POD activity by 38%, reducing rots by 70%.ur findings show an 80% increase in POD activity in un-inoculated

trawberries immediately after treatment, (Fig 3c) resulted in a sig-ificant decrease in rots (Fig 2a and b). Comparable to our results

n strawberry, Liu et al. (2010) and Shao et al. (2010) have also sug-ested that PPO may not be involved in disease resistance in loquatnd apple respectively.

Together, our results suggest that the observed post-hypobaricreatment transient increase of defence-related enzymes (PAL,hitinase and POD) (Fig. 3) might be collectively or individually

ssociated with stimulation of induced resistance limiting fungalevelopment in strawberry. Further study is needed to explore theffect of hypobaric treatment on the expression of defence-relatedenes.

PO (D) activity of strawberries. Each data point represents the mean value of fours (P < 0.05) at each measurement time are represented by *.

4. Conclusion

Our results indicate that hypobaric treatment (50 kPa, 4 h)reduced decay in strawberries subsequently stored at 20 ◦C, andwas not attributable to the low oxygen partial pressure createdduring treatment. Furthermore, hypobaric-treated strawberriesinoculated immediately and 12 h after treatment resulted in subse-quent rot reduction, demonstrating response of fruit to the fungalattack. Similarly, increase in the activity of defence related enzymes(PAL, chitinase after 12 h and POD immediately) indicated stim-ulation of induced resistance in un-inoculated hypobaric treatedstrawberries. To our knowledge this is the first report providing theevidence for a possible induced resistance response of hypobarictreatment in delaying decay development.

Acknowledgements

This research work was part of PhD studies of MajidSuhail Hashmi supported by University of Agriculture Peshawar,Pakistan and Higher Education Commission (HEC) of Pakistan.The authors are obliged to Dr Nick Roskruge for generouslyproviding hydroponic strawberries. Thanks to Abdul Jabbar andSrikanth Rupavatharam for assisting in sourcing strawberries forthe work.

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