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Postharvest Biology and Technology 68 (2012) 47–53

Contents lists available at SciVerse ScienceDirect

Postharvest Biology and Technology

jou rna l h omepa g e: www.elsev ier .com/ locate /postharvbio

ffect of X-ray irradiation on nutritional and antifungal bioactive compounds ofClemenules’ clementine mandarins

ristina Rojas-Argudoa, Lluís Paloua,∗, Almudena Bermejob, Antonio Canob, Miguel Angel del Ríoa,. Carmen González-Masb,c

Centro de Tecnología Poscosecha, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Valencia, SpainCentro de Citricultura y Producción Vegetal, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Valencia, SpainFundación AGROALIMED, Fundación de la Comunidad Valenciana para la Investigación Agroalimentaria, 46113 Moncada, Valencia, Spain

r t i c l e i n f o

rticle history:eceived 23 November 2011ccepted 12 February 2012

eywords:itrus reticulata Blanco

a b s t r a c t

X-ray irradiation (510 and 875 Gy) of intact unwounded ‘Clemenules’ mandarins increased the rindbiosynthesis of the phytoalexins scoparone and scopoletin after storage at 20 ◦C for up to 14 d, but notat 5 ◦C for up to 60 d. In general, irradiation did not affect the content of polymethoxyflavones in therind and vitamin C and flavanone glycosides in the juice. Clementines that were wounded, irradiated at510 Gy and inoculated with Penicillium digitatum, contained higher scoparone and scopoletin levels than

ostharvestcoparonecopoletinodium carbonatereen moldenicillium digitatum

non-irradiated ones. Sodium carbonate treatment (3% SC) increased scoparone but not scopoletin levelsin inoculated fruit. SC treatment and irradiation at 510 Gy induced highest scoparone and scopoletinaccumulation 3 d post-fungal inoculation at 20 ◦C (125.3 and 15.4 �g/g rind dry weight, respectively),and effectively inhibited green mold. However, phytoalexin levels declined and disease control failed 5 dafter fungal inoculation. Irradiation at 875 Gy appeared to be phytotoxic and did not induce phytoalexinproduction nor prevented green mold in rind wounds.

. Introduction

The incidence of postharvest green and blue molds, caused byhe pathogens Penicillium digitatum (Pers.:Fr.) Sacc. and Penicil-ium italicum Wehmer, respectively, is one of the major economicssues challenging citrus fruit production and commercialization.ifferent phenolic compounds such as coumarins, flavanones, andolymethoxyflavones (PMFs) are bioactive compounds that haveeen found to induce some degree of resistance in citrus fruitgainst the development of pathogenic fungi (Ortuno et al., 2011).he roles of the PMFs sinensetin (5,6,7,3′,4′-pentamethoxyflavone)SIN), nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone) (NOB), tan-eretin (5,6,7,8,4′-pentamethoxyflavone) (TAN), and 3,5,6,7,8,3′,4′-eptamethoxyflavone (HMF) in defense mechanisms of ‘Valencia’ranges have been previously discussed by Del Río et al. (2004).he levels of some flavonoids involved in resistance mecha-isms against the pathogenic fungus P. digitatum in citrus fruitan be maintained or increased by the application of posthar-

est treatments such as heat or ultraviolet light (Ben Yehoshuat al., 1992). Likewise, phytoalexins are frequently produced innfected tissues of plants as a defense mechanism in response

∗ Corresponding author. Tel.: +34 96 342 4000; fax: +34 96 342 4001.E-mail address: palou llu@gva.es (L. Palou).

925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.postharvbio.2012.02.004

© 2012 Elsevier B.V. All rights reserved.

to microbial infection. They can also be biosynthesized asa physiological response to chemical or physical postharvesttreatments (Dubery, 1992; Venditti et al., 2005). It has beendemonstrated that some coumarin-type phytoalexins such asscoparone (6,7-dimethoxycoumarin) and scopoletin (7-hydroxy-6-methoxycoumarin) play an important role in the resistance of citrusfruit against fungal pathogens (Rodov et al., 1994). On the otherhand, the juice of citrus fruit is an important source of vitamin Cas well as bioactive compounds such as polyphenolic compounds,mainly flavonoids, with high antioxidant properties. Similarly todisease resistance inducers, the content of health-promoting com-pounds in citrus fruit may be altered by postharvest treatmentssuch as irradiation. For instance, recent studies showed that irra-diation of citrus fruit reduced significantly the total ascorbic acid(TAA) content when irradiation doses were high (Patil et al., 2004;Girennavar et al., 2008). However, information is still scarce onthe effect of X-ray irradiation on nutritional quality of many citruscultivars.

X-ray irradiation is an effective alternative disinfestationquarantine treatment against the Mediterranean fruit fly, Ceratitiscapitata (Hallman and Loaharanu, 2002). We observed in previous

research that ‘Clemenules’ is a mandarin cultivar highly toler-ant to X-ray irradiation and the commercial quality of the fruitwas not adversely affected by postharvest quarantine applica-tions that effectively controlled the Medfly (Alonso et al., 2007).

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owever, these postharvest applications did not satisfactorilyontrol established infections of P. digitatum or P. italicum on arti-cially inoculated ‘Clemenules’ clementines (Palou et al., 2007).s an oxidative stress, ionizing radiation can directly affect theontent of bioactive compounds, such as coumarins (phytoalexins)nd PMFs (phytoanticipins) present in citrus fruit (Oufedjikh et al.,000), and therefore it could potentially increase fruit diseaseesistance by modulation of flavonoid biosynthesis. Irradiation,owever, can adversely affect fruit quality and even produce

mportant phytotoxicities if it is incorrectly applied.Carbonic acid salts, such as sodium carbonate (SC, Na2CO3, soda

sh), are common food additives allowed with no restrictions forany applications. SC aqueous solutions have been re-examined

uring recent years as a potential alternative to synthetic fungicideso manage citrus postharvest diseases because SC is inexpen-ive, readily available, and can be used with a minimal risk ofnjury to the fruit (Palou et al., 2002). Carbonic acid salts haveeen reported to induce the accumulation of scoparone in rindounds of citrus fruit (Venditti et al., 2005). In general, carbonic

cid salts are considered to be good candidates to be used in com-ination with other chemical, physical, or biological methods foron-polluting integrated control of citrus postharvest diseases. In

act, they have shown additive or synergistic effects when appliedith heat (heated aqueous solutions or fruit curing), antagonisticicroorganisms or other chemicals such as conventional fungicides

t reduced doses (Montesinos-Herrero and Palou, 2010).The objective of this work was to study the effect of X-ray irra-

iation (at doses of 510 and 875 Gy) on scoparone and scopoletinlicitation in ‘Clemenules’ clementine rind after short-term shelf-ife at 20 ◦C (up to 14 d) and long-term cold storage at 5 ◦C (upo 60 d). In addition, the effect of X-ray irradiation on the levelsf citrus health-promoting compounds (vitamin C and flavanonelycosides, FGs) was studied in irradiated ‘Clemenules’ mandarinsfter a 60 d cold storage period at 5 ◦C followed by a 7 d period ofimulated shelf-life at 20 ◦C. Likewise, scoparone and scopoletinontents were studied in mandarins inoculated with P. digitatumfter combining SC dips with X-ray irradiation treatments.

. Materials and methods

.1. Fruit

Mature clementine mandarins (Citrus reticulata Blanco) cv.Clemenules’ from commercial orchards in the Valencia area (Spain)

ere selected from field bins after harvest, randomized, washedith fresh water, air-dried, and used in the experiments before any

ommercial postharvest treatments were applied. Intact healthyruit were separated to determine the initial levels of bioactiveompounds (levels at harvest) as described below.

.2. Irradiation treatment

Mandarins were placed in commercial 40 cm × 29 cm × 27 cmardboard boxes with lids and transported to the irradiation plantBeta Gamma Service, BGM, Bruchsal, Germany). During trans-ortation, the fruit were kept at 20 ± 3 ◦C. About 36 h later, theruit were exposed to X-rays from a source with beam energy of.8 MeV and a conveyor speed of 5 m/min. The theoretical doseselected were 500 and 900 Gy. Each dose was applied to three boxesontaining about 150 mandarins each. The mandarins were intactr previously treated depending on the experiment and poste-

ior analytical procedure. Actual doses were determined by placing

cm2 radiochromic dosimetry films (Gafchromic® HD-810, Inter-ational Specialty Products, Wayne, NJ, USA) at three differenteights within each box. Readings (nine per dose) were made with a

y and Technology 68 (2012) 47–53

spectrophotometer at 560 nm and ranged from 492 to 526, and 843to 921 Gy for the respective theoretical doses. Mean and standarderror values were 510 ± 10, and 875 ± 27 Gy, respectively.

2.3. Effect of X-ray irradiation on bioactive compounds inclementine rind

In this study, intact mandarins were irradiated and stored ateither 20 ◦C for 14 d or 5 ◦C for 60 d. Samples were obtained aftereither 7 and 14 d at 20 ◦C or 30 and 60 d at 5 ◦C. For each treat-ment, the rind of 4 replicates of 5 mandarins each was removedand immediately frozen at −80 ◦C for later high performance liquidchromatography (HPLC) analysis.

2.3.1. Extraction of PMFs and phytoalexinsThe samples of mandarin rind were defrosted at room tem-

perature and dried in an oven until constant weight. They werethen powdered in a laboratory mill (Fritsch 14 Pulverisette GmbH,Germany) and 40 mg of citrus rind powder were mixed with1 mL of dimethylsulfoxide (DMSO) (Scharlau, Scharlab, Sentmenat,Barcelona, Spain) for 2 h. Then, the samples were filtered througha nylon filter (0.45 �m) before being HPLC analyzed.

2.3.2. PMF analysisFiltrated samples were analyzed with a HPLC system equipped

with an Alliance 2695 separation module coupled to photodiodearray (PDA) detector 2996 and ZQ mass spectrometer (Waters,Milford, MA, USA). Samples and column oven temperature was30 ◦C. A reverse phase Gemini C18 150 mm × 3 mm (5 �m) col-umn (Phenomenex®, Torrance, CA, USA) was used with a gradientmobile phase tetrahydrofuran (A):water (B):acetonitrile (C) witha flow rate of 1 mL/min. The initial conditions were 16% A and84% B, reaching 12% A, 68% B and 20% C in 20 min. After that, thephase reached 12% A, 18% B and 70% C in the following 20 min, held5 min and then back to the initial conditions in 1 min and held for14 min (total run time was 60 min). All solvents used were of HPLC-grade and ultrapure water (Milli-Q, Millipore, Billerica, MA, USA)was used. The injection volume for each sample was 20 �L. Massspectra were performed working in electrospray ion positive con-ditions. The capillary voltage was 3.3 kV, cone voltage 20 V, sourcetemperature 100 ◦C, desolvation temperature 225 ◦C, cone gas flow70 L/h and desolvation gas flow 500 L/h. Full data acquisition wasperformed scanning 200–900 uma in centroid mode.

The identification and quantification of PMFs were per-formed using commercially available standards except hep-tamethoxyflavone (HMF), which was quantified using the responsefactor standard peak of nobiletin. The quantification of PMFs wasdone selecting � at 340 nm. Concentration in �g/g rind dry weight(DW) of HMF was expressed as the response factor standard peakof NOB.

The retention times for the analyzed PMFs were 7.55 min forSIN ([MH]+ 373 m/z; �max (nm) = 240/265/334), 11.22 min for NOB([MH]+ 403 m/z; �max (nm) = 250/270/337), 12.88 min for HMF([MH]+ 433 m/z; �max (nm) = 254/270/337), and 18.38 min for TAN([MH]+ 373 m/z; �max (nm) = 270/326).

2.3.3. Phytoalexin (scoparone and scopoletin) analysisFiltrated samples were analyzed with a Waters HPLC system

equipped with a Alliance 2695 separation module coupled toMulti� Fluorescence Detector 2475 (Waters). Samples in the auto-matic injector and column oven temperatures were 25 ◦C and 50 ◦C,

respectively. A reverse phase Gemini C18 5 �m 150 mm × 3 mmcolumn (Phenomenex®) was used with a gradient mobile phasemethanol (A): 0.05 M (pH 4.25) ammonium acetate tampon (B) at aflow rate of 0.5 mL/min with an initial condition of 20% A for 10 min,

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eaching 80% A for the following 5 min, then back to initial condi-ion in 5 min and held for 5 min previously to inject the next sampletotal run time was 25 min). The injection volume for each sampleas 2 �L.

The identification and quantification of phytoalexins were per-ormed using known concentrations of commercially availablecoparone and scopoletin (Sigma Aldrich, Madrid, Spain) selectinghe �ex at 300 nm and the �em at 430 nm. The retention times forhe compounds above were 7.15 and 9.34 min for scopoletin andcoparone, respectively.

.4. Effect of X-ray irradiation on health-promoting compoundsn mandarin juice

At the end of a storage period of 60 d at 5 ◦C followed by 7 d at0 ◦C, 3 replicates of 10 mandarins each were squeezed to deter-ine the total content of vitamin C and the content of the main FGs

n the mandarin juice of irradiated and non-irradiated clementineandarins.

.4.1. Total vitamin C analysisOne milliliter of filtered juice was mixed with 1 mL of 5%

etaphosphoric acid. The samples were centrifuged at 12,000 rpmor 15 min at 4 ◦C, and the aqueous phase was filtered through a.45 �m filter. Total vitamin C was determined after the reductionf 1 mL aliquot of aqueous phase with 200 �L of 1,4-dithio-dl-hreitol (DTT, 200 mg/mL) (Dhuique-Mayer et al., 2005), allowedo react for 2 h in the dark, then filtered as described above, andsed for total ascorbic acid determination by HPLC–DAD–MS (HPLCaters Alliance-2996 PDA-ZQ mass spectrometer), using a reverse-

hase column C18 Tracer Excel 5 �m 120 OSDB (250 mm × 4.6 mm)Teknokroma, Barcelona, Spain) and a binary isocratic phase con-isting in methanol:0.6% acetic acid (5:95, v/v). The total run timeas 10 min at a flow rate of 1 mL/min. Quantification of ascorbic

cid was performed at 245 nm by external standard calibration.-Ascorbic acid and DTT were obtained from Sigma Aldrich.

.4.2. FG analysisOne milliliter of filtered juice was mixed and homogenized with

mL of DMSO:methanol (1:1, v/v), and the sample was then cen-rifuged at 12,000 rpm for 15 min at 4 ◦C. The supernatant wasltered through a 0.45 �m nylon filter and analyzed by HPLC–DADnd HPLC–MS, using a reverse-phase column C18 Tracer Excel 5 �m20 OSDB (250 mm × 4.6 mm) (Teknokroma) and a gradient mobilehase consisting in acetonitrile (A) and 0.6% acetic acid (B). Theonditions were as follows: initial conditions 10% A for 2 min;hen reach 75% A in the following 28 min; and then back to initialonditions and held for 5 min, total run time 35 min at 1 mL/minMata-Bilbao et al., 2007). HPLC–MS analysis was performed work-ng in electrospray ion positive conditions. The capillary voltage

as 3.50 kV, cone voltage 20 V, source temperature 100 ◦C, desol-ation temperature 225 ◦C, cone gas flow 70 L/h and desolvationas flow 500 L/h. Full data acquisition was performed scanning00–800 uma in centroid mode. Compounds were identified onhe basis of comparison their retention times, UV–Vis spectrand mass spectrum data with corresponding authentic standards.oncentrations were determined using an external calibrationurve with narirutin (NAR: rT = 14.2 min; [MH]+ 581 m/z; �max

nm) = 227.0/283.6/331.2), hesperidin (HESP: rT = 14.8 min; [MH]+

11 m/z; �max (nm) = 227.0/283.6) and didymin (DID: rT = 17.6 min;

MH]+ 595 m/z; �max (nm) = 228.2/282.5/328.8). NAR was pur-hased from Extrasynthese (Genay, France), HESP was obtainedrom Sigma Aldrich, and DID was purchased from ChromaDexIrvine, CA, USA).

y and Technology 68 (2012) 47–53 49

2.5. Effect of the combination of sodium carbonate and X-rayirradiation on phytoalexin content in clementine rind

2.5.1. Sodium carbonate treatmentPrior to dip treatments, a total number of 240 mandarins were

wounded in four points along the equatorial zone. Wounded man-darins were placed in stainless steel grid baskets and submergedfor 150 s in 22.5-L stainless steel buckets containing aqueous 3%(wt/vol) SC (Na2CO3; Guinama S.L., Alboraia, Valencia, Spain) solu-tions at 20 ◦C. Control fruit (half of the fruit, 120 mandarins) weredipped in water at 20 ◦C for the same time. SC-treated fruit wererinsed with fresh water at low pressure in a spray 20 cm above thefruit for about 5 s, placed on plastic cavity trays on cardboard trays,and let to air-dry at room temperature.

2.5.2. X-ray irradiation treatmentWater- and SC-treated mandarins were divided into three

groups and, about 36 h after dip treatments, irradiated at 0 (non-irradiated control), 510 or 875 Gy as previously described. Therewere, therefore, six different treatments with 40 fruit per treat-ment.

2.5.3. Fungal inoculation of treated mandarinsTwo days after X-ray irradiation treatment, mandarins were

inoculated with P. digitatum by immersing a stainless steel rod witha probe tip 1 mm wide and 2 mm in length into a suspension of105 spores/mL and wounding each fruit at the same four equato-rial points where the rind had been wounded before dipping. Afterinoculation, mandarins were incubated at 20 ◦C for 3 or 5 d (20 fruitper each incubation time). After incubation, disks of rind tissueincluding the inoculated wounds were removed with a cork borer(0.8 cm diameter, 0.4 cm thick) and immediately stored at −80 ◦Cuntil phytoalexin analysis. Eighty rind disks were excised from 20fruit per treatment and incubation time (4 disks per fruit). Sco-parone and scopoletin quantification were performed as describedabove on 4 replicates of 5 fruit (20 disks) per treatment and incu-bation time.

2.6. Statistical analysis

Analyses of variance (ANOVA) were performed using STAT-GRAPHICS Plus 4.1 (Manugistics Inc., Rockville, MD, USA). Specificdifferences among means were determined by Fisher’s pro-tected least significant difference (LSD) test (P = 0.05). To avoidheteroscedasticity, data on green mold incidence on artificiallyinoculated mandarins were transformed prior to ANOVA to thearcsine of the square root of the proportion of infected rind wounds.

3. Results and discussion

3.1. Effect of X-ray irradiation on bioactive compounds inclementine rind

3.1.1. PMFsTable 1 shows the content of the main PMFs detected in the rind

of ‘Clemenules’ mandarins: TAN, NOB, SIN and HMF. The PMFs rindcontents were lower than those previously found by other authorsin oranges and grapefruit (Del Río et al., 2004; Ortuno et al., 2006).In accordance with our results, these authors reported that NOBand HMF were the most abundant PMFs detected. They found NOBlevels from 110 �g/g DW in the rind of ‘Valencia’ oranges to 17 �g/g

DW in the rind of ‘Shambar’ grapefruit. In contrast, TAN levels inthe flavedo of grapefruit were around 6 �g/g DW while SIN levelswere negligible. These authors also showed the antifungal activityof these PMFs against P. digitatum. Furthermore, Del Río et al. (1998)

50 C. Rojas-Argudo et al. / Postharvest Biology and Technology 68 (2012) 47–53

Table 1Contents of polymethoxyflavones at harvest and after X-ray irradiation at 0 (control), 510 or 875 Gy in the flavedo of ‘Clemenules’ mandarins stored at either 20 ◦C for 7 or14 d or 5 ◦C for 30 or 60 d.

Storage period (d) X-ray irradiation (Gy) Tangeretin (�g/g DWa) Nobiletin (�g/g DW) Sinensetin (�g/g DW) Heptamethoxy-flavoneb (�g/g DW)

At harvest 2.63 ± 0.51 4.60 ± 1.32 1.71 ± 0.12 5.20 ± 0.93

7 at 20 ◦C Control 2.41 ± 0.29 a 4.51 ± 0.90 a 1.62 ± 0.29 a 4.41 ± 0.09 b510 2.40 ± 0.47 a 4.11 ± 0.82 a 1.49 ± 0.19 a 4.02 ± 0.08 a875 2.39 ± 0.23 a 4.39 ± 1.21 a 1.62 ± 0.27 a 4.94 ± 0.17 c

14 at 20 ◦C Control 2.31 ± 0.51 a 4.21 ± 1.00 a 1.73 ± 0.41 a 4.29 ± 0.78 a510 2.43 ± 0.42 a 4.13 ± 0.79 a 1.52 ± 0.32 a 4.28 ± 0.27 a875 2.40 ± 0.32 a 4.31 ± 0.71 a 1.62 ± 0.19 a 4.35 ± 0.20 a

30 at 5 ◦C Control 2.31 ± 0.49 a 3.93 ± 0.42 a 1.62 ± 0.43 a 4.11 ± 0.62 a510 2.32 ± 0.58 a 4.34 ± 0.81 a 2.01 ± 0.38 a 4.50 ± 0.69 a875 2.34 ± 0.40 a 4.35 ± 0.37 a 1.83 ± 0.40 a 4.62 ± 0.83 a

60 at 5 ◦C Control 2.31 ± 0.29 a 4.81 ± 0.49 a 2.03 ± 0.18 a 4.92 ± 0.91 a510 2.20 ± 0.78 a 4.40 ± 0.29 a 1.93 ± 0.29 a 5.13 ± 0.32 a875 2.29 ± 0.58 a 5.21 ± 0.58 a 2.11 ± 0.21 a 5.11 ± 0.12 a

The data represent mean values ± standard deviation (n = 4).a DW = dry weight.b

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eported that PMF contents in clementines were lower than thosen sour and sweet oranges.

With the exception of HMF, which increased slightly in 875 Gy-rradiated mandarins after 7 d of storage at 20 ◦C, X-ray irradiationid not affect the PMF contents (TAN, NOB, SIN). Oufedjikh et al.2000) found an increase in the PMF contents of clementine man-arins irradiated at 300 Gy after 14 d of storage at 3–4 ◦C; NOBnd HMF were the most affected compounds by the irradiationreatment. The enhancement of PMF levels was correlated withn increase of phenylalanine ammonialyase (PAL) activity, whichs a key enzyme in flavonoid metabolism. Moussaid et al. (2004)bserved an increase in the SIN and TAN contents of oranges �-rradiated at 1 and 2 kGy. If compared with our results, X-rayrradiation could exert lower effects on flavonoid metabolism ofitrus fruit than �-irradiation because of the higher penetrationower of �-rays.

The biosynthesis and/or accumulation of constitutive bioactiveompounds as a response to different postharvest treatments isighly dependent on fruit age or ripening stage (Ben Yehoshua et al.,992; Patil, 2004). Likewise, Riov et al. (1968) reported that levelsf phenolic compounds in irradiated grapefruit were dependent onaturity.

.1.2. PhytoalexinsScoparone induction has been repeatedly associated by many

orkers to a decrease in the susceptibility of citrus fruit to infectiony P. digitatum or P. italicum (Kim et al., 1991; Ben Yehoshua et al.,992; Rodov et al., 1992; Venditti et al., 2005; Ortuno et al., 2011). Atarvest, neither scoparone nor scopoletin were detected in clemen-ine rind (Table 2), which is in accordance with recent findings byrtuno et al. (2011). These authors only found scoparone in man-arins inoculated with P. digitatum. In these tests, despite that theruit were not inoculated with the pathogen, X-ray irradiation effec-ively induced the biosynthesis of scoparone and scopoletin in theind of clementine mandarins during storage at 20 ◦C (Table 2).he level of scoparone in the rind of 875 Gy-irradiated mandarinstored for 14 d at 20 ◦C reached 24 �g/g flavedo DW (equivalent to.46 �g/g fresh flavedo), an amount 25 times higher than that inhe flavedo of non-irradiated clementines kept at the same storage

onditions. Similarly, Dubery (1992) found that the application of a-radiation dose of 2 kGy significantly increased the total coumarinontent in citrus flavedo. According to early studies in Israel (Kimt al., 1991; Ben Yehoshua et al., 1992), different physiological

cording to Fisher’s protected LSD test (P = 0.05).

responses have been observed after different postharvest treat-ments referred to the induction of phytoalexins in the rind of citrusfruit. For instance, while UV-C illumination induced production ofscoparone in lemons without fungal inoculation, heat treatments(curing at 36 ◦C for 3 d) only induced scoparone when the lemonswere inoculated in rind wounds with P. digitatum. It is clear from thepresent results that in the case of X-ray irradiation the treatmentis able to induce phytoalexins, although at relatively low levels, inthe absence of fungal challenge. We observed in complementaryprevious work (Palou et al., 2007) that non-wounded ‘Clemenules’clementines that had been exposed to X-ray irradiation at dosesfrom 195 to 875 Gy always developed decay when they were arti-ficially wounded and inoculated with P. digitatum either 2, 3, or 6 dafter irradiation. We can conclude now that under those experi-mental conditions, the lack of effective resistance to green moldwas not due to a lack of phytoalexin induction by X-ray irradiation,but to induction of insufficient amounts.

After 60 d of storage at 5 ◦C, both scoparone and scopoletin con-tents in irradiated mandarins did not significantly differ from thosein non-irradiated ones (Table 2). Similar results were obtained byArimoto and Homma (1988), who found that the largest quan-tity of scoparone was produced in citrus rind scars at 25 ◦C whileprogressively lower levels were detected in fruit stored at lowertemperatures; no scoparone was detected in fruit held at either 5or 10 ◦C.

3.2. Effect of X-ray irradiation on health-promoting compoundsin mandarin juice

X-ray irradiation at the doses applied in this work did not signif-icantly modify the levels of total vitamin C or FGs in ‘Clemenules’mandarin juice (Table 3). Results in the literature from work withdifferent citrus fruit show that irradiation effects on vitamin C con-tent depend on irradiation dose, fruit cultivar and maturity stage.In accordance with our results, �-irradiation at doses up to 1000 Gydid not modify the vitamin C content on grapefruit, but it decreasedafter the application of a dose of 1500 Gy or higher (Moshonasand Shaw, 1984; Girennavar et al., 2008). Similarly, we observedin previous work no effect of X-ray irradiation at doses of 164 Gy or

lower on vitamin C content of ‘Clemenules’ mandarins (Contreras-Oliva et al., 2011). In contrast, Hu et al. (2004) reported a vitaminC decrease in oranges irradiated at 500–600 Gy compared to non-irradiated fruit, but they found no significant effect when the doses

C. Rojas-Argudo et al. / Postharvest Biology and Technology 68 (2012) 47–53 51

Table 2Scoparone and scopoletin contents at harvest and after X-ray irradiation at 0 (control), 510 or 875 Gy in the flavedo of ‘Clemenules’ mandarins stored at either 20 ◦C for 7 or14 d or 5 ◦C for 30 or 60 d.

Storage period (d) X-ray irradiation (Gy) Scoparone (�g/g DWa) Scopoletin (�g/g DW)

At harvest – b b

7 at 20 ◦C Control 0.67 ± 0.05 a 1.37 ± 0.12 a510 18.61 ± 1.57 c 9.07 ± 2.03 b875 8.91 ± 1.26 b 7.71 ± 0.53 b

14 at 20 ◦C Control 0.94 ± 0.36 a 1.18 ± 0.24 a510 9.91 ± 3.96 b 7.16 ± 1.91 b875 24.1 ± 4.61 c 10.45 ± 3.43 c

30 at 5 ◦C Control 2.13 ± 1.47 a 1.25 ± 0.29 a510 6.20 ± 2.30 b 6.16 ± 3.15 c875 2.02 ± 0.80 a 3.93 ± 0.77 b

60 at 5 ◦C Control 0.86 ± 1.18 a 2.92 ± 2.41 a510 1.76 ± 0.39 a 4.52 ± 2.13 a875 1.55 ± 0.53 a 3.78 ± 0.49 a

The data represent mean values ± standard deviation (n = 4).a DW = dry weight.b Not detected.

For each storage period, values within columns followed by unlike letters are different according to Fisher’s protected LSD test (P = 0.05).

Table 3Total vitamin C and flavanone glycoside contents at harvest and after X-ray irradiation at 0 (control), 510 or 875 Gy in the juice of ‘Clemenules’ mandarins stored at 5 ◦C for60 d followed by 7 d of shelf life at 20 ◦C.

X-ray irradiation (Gy) Total vitamin C (mg/L) Flavanone glycosides (mg/L)

Narirutin Hesperidin Didymin

At harvest 333.0 ± 17.6 51.1 ± 3.4 229.0 ± 15.9 5.53 ± 0.51Control 309.1 ± 38.5 a 58.7 ± 0.4 a 270.2 ± 40.1 a 5.49 ± 0.61 a510 338.7 ± 16.6 a 60.5 ± 5.8 a 286.1 ± 30.2 a 6.50 ± 0.43 a875 327.9 ± 33.4 a 58.7 ± 3.1 a 286.3 ± 18.1 a 5.82 ± 0.80 a

TV prote

d5a(

F5fis(st

TS(3

BT

V

he data represent means ± standard deviation (n = 3).alues within columns followed by unlike letters are different according to Fisher’s

ecreased to 300–400 Gy. Clementine fruit irradiated at 300 and00 Gy doses along with hot water treatment and stored for 3 weekst 17 ◦C contained higher vitamin C levels than control samplesAbdellaoui et al., 1995).

Irradiation at the applied doses did not modify the levels ofGs in citrus juice (Table 3). After prolonged cold storage (60 d at◦C), the amount of hesperidin (the most frequent FG) increased

or every treatment (Table 3). In agreement with our results, anncrease in the contents of FGs and other phenolic compounds after

torage of different citrus cultivars was reported by other workersDel Caro et al., 2004; Contreras-Oliva et al., 2011). These authorsuggested that such an increase could be due to the stimulation ofhe PAL activity during cold storage.

able 4coparone and scopoletin contents in the flavedo of wounded ‘Clemenules’ mandarins dicontrol), 510, or 875 Gy, artificially inoculated with Penicillium digitatum after 2 d of stora

or 5 d.

Treatment 3 d post-fungal inoculation

Dip – irradiation Scoparone (�g/g DWa) Scopoletin (�g/g

Water – 0 Gy 28.40 ± 0.05 a 3.10 ± 0.40 a

Water – 510 Gy 46.20 ± 1.57 b 11.74 ± 2.98 c

Water – 875 Gy 22.20 ± 1.26 a 4.20 ± 0.43 a

3% SC – 0 Gy 117.40 ± 0.36 c 3.17 ± 0.62 a

3% SC – 510 Gy 125.30 ± 3.96 c 15.41 ± 3.06 d

3% SC – 875 Gy 45.40 ± 4.61 b 7.23 ± 0.80 b

oth scoparone and scopoletin levels were undetectable at harvest.he data represent means ± standard deviation (n = 4, 4 replicates of 5 fruit per treatmenta DW = dry weight.alues within columns followed by unlike letters are different according to Fisher’s prote

cted LSD test (P = 0.05).

3.3. Effect of the combination of sodium carbonate and X-rayirradiation treatments on phytoalexin content in clementine rind

Table 4 shows the scoparone and scopoletin contents onwounded ‘Clemenules’ clementine mandarins dipped in water or3% SC, irradiated after 36 h at 0, 510 or 875 Gy, artificially inoculatedwith P. digitatum after 2 d of storage at 20 ◦C following irradia-tion, and incubated after fungal inoculation at 20 ◦C for 3 or 5 d.Phytoalexin contents before the application of the treatments (at

harvest) were undetectable.

As expected, antifungal bioactive compounds were synthesizedin wounded, X-ray irradiated and inoculated clementine mandarinsat higher levels than in non-wounded clementines. For instance,

pped in water or 3% sodium carbonate (SC) at 20 ◦C for 150 s, X-ray irradiated at 0ge at 20 ◦C following irradiation, and incubated after fungal inoculation at 20 ◦C for

5 d post-fungal inoculation

DW) Scoparone (�g/g DW) Scopoletin (�g/g DW)

5.96 ± 1.71 a 1.41 ± 0.18 a32.60 ± 1.50 b 3.67 ± 0.34 b

3.44 ± 0.75 a 2.55 ± 0.58 ab71.42 ± 19.1 c 2.87 ± 0.35 b41.21 ± 7.35 b 10.48 ± 1.48 d32.63 ± 10.4 b 5.84 ± 1.59 c

and incubation time, 4 rind disks per fruit).

cted LSD test (P = 0.05).

52 C. Rojas-Argudo et al. / Postharvest Biolog

Table 5Percentage of infected wounds on wounded ‘Clemenules’ mandarins dipped in wateror 3% sodium carbonate (SC) at 20 ◦C for 150 s, X-ray irradiated at 0 (control), 510, or875 Gy, artificially inoculated with Penicillium digitatum after 2 d of storage at 20 ◦Cfollowing irradiation, and incubated after fungal inoculation at 20 ◦C for 3 or 5 d.

Treatment Infected wounds (%)

Dip – irradiation 3 d post-fungal inoculation 5 d post-fungal inoculation

Water – 0 Gy 100 a 97.5 aWater – 510 Gy 91.2 a 100 aWater – 875 Gy 97.5 a 100 a3% SC – 0 Gy 26.2 b 86.2 a3% SC – 510 Gy 18.7 b 81.2 a3% SC – 875 Gy 86.2 a 90 a

Vpt

s3re8ttpiIsa

iSlawoAp2eidStaicidopeifd8e

sawfSmS

s

and scopoletin by the combination of X-ray irradiation and SC

alues within columns followed by unlike letters are different according to Fisher’srotected LSD test (P = 0.05) applied to an ANOVA to arcsine-transformed data. Non-ransformed means are shown.

coparone content in mandarins irradiated at 510 Gy was 18 and2 �g/g DW on unwounded and wounded and water-treated fruit,espectively, about 7 d at 20 ◦C after irradiation (Tables 2–4). Inter-stingly, irradiation at the dose of 510 Gy, but not at the dose of75 Gy, significantly increased scoparone and scopoletin levels inhe rind of inoculated clementine mandarins (Table 4). However,he levels attained were not high enough to prevent decay andractically all water-treated and inoculated wounds (>90%) were

nfected after 3 d at 20 ◦C following fungal inoculation (Table 5).nfection symptoms of green mold at this time were small water-oaked areas surrounding the inoculation point with none or fewerial mycelial growth and absence of spores.

The 3% SC treatment markedly increased the level of scoparonen the rind of non-irradiated and 510 Gy-irradiated clementines.coparone concentration in the peel of these mandarins reachedevels of 117 (31 �g/g fresh weight) and 125 �g/g DW, respectively,fter 3 d of incubation at 20 ◦C (Table 4). Apparently, these dosesere high enough to inhibit the development of P. digitatum since

nly 26 and 19% of inoculated wounds resulted infected (Table 5).lthough the figures and experimental procedures are not com-arable, Kim et al. (1991) reported average scoparone doses of9 and 46 �g/mL, as needed for effective inhibition of germ tubelongation and spore germination of P. digitatum, respectively. Ast was observed after 7 d at 20 ◦C on unwounded irradiated man-arins (Table 2), scoparone induction was significantly lower onC-treated fruit irradiated at 875 Gy (Table 4). Therefore, irradia-ion at this elevated dose showed an inhibitory effect on scoparoneccumulation on SC-treated mandarins in response to Penicilliumnoculation. As a consequence, the resistance to fungal growth asso-iated with scoparone accumulation decreased and green moldncidence was considerably higher (Table 5). This might be due to aetrimental effect of X-rays at this dose on the physical and physi-logical condition of the fruit rind (Palou et al., 2007). Although inrevious work no evident rind pitting was observed to the nakedye on 875 Gy-irradiated mandarins, phytotoxicity leading to incip-ent rind injury could alter the flavedo and albedo cells and facilitateungal mycelial growth. This fact might justify why green moldevelopment was slightly favored on unwounded fruit treated at75 Gy when P. digitatum was inoculated 6 d after irradiation (Palout al., 2007).

SC-treated and non-irradiated mandarins did not accumulatecopoletin compared to non-treated mandarins. This result is ingreement with those from the tests by Venditti et al. (2005)ho found an accumulation of scoparone, but not of scopoletin,

ollowing SC treatment of ‘Fairchild’ mandarin fruit. However,C treatment increased scopoletin contents on X-ray irradiatedandarins. The highest content of 15 �g/g DW was observed for

C-treated mandarins irradiated at 510 Gy (Table 4).Irrespective of the postharvest treatments, accumulation of both

coparone and scopoletin in ‘Clemenules’ mandarin rind declined

y and Technology 68 (2012) 47–53

from 3 to 5 d after fungal inoculation (Table 4). Extensive previ-ous research has revealed that the pattern of accumulation anddegradation of phytoalexins deeply varies among citrus speciesand cultivars, elicitating postharvest treatments and fungal inoc-ulation sequences. Kim et al. (1991) found that on heated-lemonfruit previously inoculated with P. digitatum, scoparone concen-tration reached a peak on the sixth day after heat treatment anddeclined later. Venditti et al. (2005) observed an increase in sco-parone accumulation over 5 d on SC-treated ‘Fairchild’ mandarinsafter inoculation with P. digitatum. Kuniga and Matsumoto (2003)studied scoparone accumulation on 30 citrus cultivars, includingmandarins, after inoculation with Botrytis cinerea, the causal agentof citrus gray mold. These workers found important differencesin the period of time needed to reach the peak concentration ofscoparone. In 19 out of 30 citrus cultivars, the peak occurred 7 dafter fungal inoculation while the maximum was reached about4 d after inoculation on the rest of species. ‘Ponkan’ mandarinswere among the species that showed the scoparone peak after4 d while the hybrids ‘Ellendale’ and ‘Murcott’ (C. reticulata × Citrussinensis) presented the maximum scoparone level after 7 d. In ourtests, phytoalexin decline after 5 d of incubation at 20 ◦C was clearlyassociated with an increase of disease incidence on artificially inoc-ulated mandarins and the percentage of infected wounds was notsignificantly different among treatments (Table 5). At this time(5 d post-inoculation), infected lesions were larger with abundantwhite aerial mycelium and, in some cases, green spores beginningto form from the center of the lesion. It is interesting to note thatgreen mold development in infected wounds from SC-treated man-darins irradiated at 0 or 510 Gy was delayed in comparison withthe rest of the treatments (symptoms were mostly only water-soaked areas surrounding the inoculation site), indicating that thespore germination of P. digitatum was delayed and/or its mycelialgrowth was slower. These effects appear to be a consequence ofthe higher phytoalexin levels that were detected in wounds fromthese mandarins 3 d post-inoculation. The important scoparoneand scopoletin decline during the next 2 d at 20 ◦C would haveallowed the infectious process to begin. On the other hand, dataconfirm previous observations (Kim et al., 1991; Ben Yehoshuaet al., 1992) that suggested that the presence of high levels of phy-toalexins within rind wounds inhibits decay by an indirect modeof action preventing the spores to germinate or the germ tubeor mycelium to growth, but do not directly damage the fungalstructures or adversely affect their viability. For this reason, dis-ease can initiate in wounds when phytoalexin levels decrease andthere is a change in the biochemical environment surrounding thefungus.

It has been verified through this study that in ‘Clemenules’clementines inoculated with P. digitatum, the rind phytoalexin lev-els significantly increased 3 d after fungal inoculation when the fruithad been either irradiated at 510 Gy or dipped in a 3% SC solution(this treatment only increased scoparone levels). The combinationof both treatments also increased significantly the scopoletin level.Such increments were associated with temporary effective inhi-bition of green mold in treated wounds. After 2 d more at 20 ◦C,however, phytoalexin levels considerably decreased and diseasecontrol failed.

Provided that irradiation treatment plants become more com-mon in the future for insect quarantine applications, the resultsobtained here showing beneficial effects (or in some cases lack ofinfluence) from X-ray irradiation on the induction of nutritionaland antifungal bioactive compounds on mandarins may be signifi-cant for the citrus industry. The effective induction of scoparone

treatments provides satisfactory preventive activity against citruspenicillium molds that may be useful to complement postharvestdecay control programs.

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cknowledgments

This work was partially funded by the Spanish ‘Ministerio deiencia e Innovación’ (MICINN; project AGL2004-05271/AGR) andhe European Union (FEDER Program). Miquel Alonso is acknowl-dged for his kind collaboration in irradiation treatments.

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