Journal of the Science of Food and Agriculture J Sci Food Agric 88:707–713 (2008)

Effect of nitrogen fertilisation levelson melon fruit quality at the harvest timeand during storageAntonio Ferrante,1∗ Anna Spinardi,1 Tommaso Maggiore,1 Armando Testoni2 andPietro M Gallina1

1Dip. Produzione Vegetale, University of Milano, via celoria 2, 20133 Milan, Italy2CRA – Istituto per la Valorizzazione Tecnologica delle Produzioni Agricole, Milan, Italy

Abstract

BACKGROUND: The quality of melon fruit depends upon many factors that can be affected by growing conditionsand post-harvest management. The aim of this work was to investigate the effect of nitrogen fertilisation levels onthe fruit quality at harvest time and during storage. Experiments were performed in an open field using melonplants (Cucumis melo L. Var. Reticulatus cv. Prodigio). The nitrogen (N) was applied through fertigation usingfour fertilisation levels: 0, 55, 110 and 165 kg ha−1. After harvest the fruits were stored at 10 ◦C for 8 days. At harvesttime the yield, flesh firmness, skin and pulp colours, content of carotenoids, total phenols, ascorbic acid (AsA)and ethylene production were measured on fruits differently fertilised.

RESULTS: The total marketable fruit yield and fruit nitrogen content linearly increased with N levels. Antioxidantcompounds decreased after storage but were not affected by N fertilisation levels. However, total carotenoids, totalphenols, and AsA declined during storage.

CONCLUSION: All the quality parameters did not appear to be affected by N level at either harvest time or afterstorage. Therefore it is advisable to reduce nitrogen input for cultivation without compromising quality and yields. 2007 Society of Chemical Industry

Keywords: ascorbic acid; ethylene; firmness; phenols; sugar

INTRODUCTIONMelons are a very important commercial crop inItaly and are available from early spring to latesummer. The production peaks are often concentratedwithin a few weeks and are strictly influenced byweather conditions, mainly temperature. Therefore,good storage conditions may delay fruit availabilityon the market, keeping the melon at a remunerativeprice. Market availability may be expanded usingprotected cultivations or by importation from othercountries.1 However, long distance transport or longstorage periods may reduce fruit quality. Melon qualityis defined by internal and external parameters, whichcan be grouped in terms of rind colour, size, firmness,aroma and taste.2 These variables are importantfor consumer attractiveness. Sweetness is probablythe most important quality parameter commonlyevaluated by consumers.

Fruit quality must be achieved in the field because,after harvest, during the post-harvest stages, thequality can only be preserved. Quality parametersmainly depend on plant genotype; growing conditions,management and the ripening state of the fruits at

harvesting time are also important.3 Soil characteris-tics, water availability, light intensity and temperatureare the most important environmental factors thathave to be considered for obtaining maximum pro-duce quality. The management of fertilisation andirrigation is crucial for assuring the yield with goodfruit quality.4

Melon fruit quality is strictly related to flavour but itis also important to maintain desirable health-relatedattributes such microbial safety and compounds thatcontribute to human well-being (e.g. carotenoids andvitamin C) at harvest time and during distribution orstorage.

The nitrogen fertilisation should be carefullyplanned in order to avoid environmental pollutionand to the maximum benefit to the plants. The mostefficient practice for nitrogen distribution is throughthe irrigation water (fertigation). In this way, nitrogenis applied when the plants really need it.5

Little information is available on the effects ofnitrogen fertilisation on fruit quality during post-harvest storage, although it is known that nitrogen

∗ Correspondence to: Antonio Ferrante, Dip. Produzione Vegetale, University of Milano, via celoria 2, 20133 Milan, ItalyE-mail: antonio.ferrante@unimi.it(Received 25 January 2007; revised version received 19 September 2007; accepted 20 September 2007)Published online 13 December 2007; DOI: 10.1002/jsfa.3139

2007 Society of Chemical Industry. J Sci Food Agric 0022–5142/2007/$30.00

A Ferrante et al.

fertilisation affects the post-harvest life of manyperishables, such as strawberries and cut flowers.6–8

During post-harvest life, temperature managementis extremely important for preserving fruit quality. Theoptimal storage temperatures depend upon the speciesand cultivar. Melons are classified as highly perishableand their potential storage life in air at near optimumtemperature and relative humidity ranges between2 and 4 weeks.9 The optimal storage temperaturedepends upon the melon, and usually ranges between2.2 and 13 ◦C;10,11 it is 8–10 ◦C for reticulate varieties.

The main goal of this work was to investigate theeffect of different nitrogen fertilisation levels on themarketable yield and fruit quality at both harvest timeand during storage.

EXPERIMENTALPlant material and growing conditionsFruits were derived from an open field trial withfour nitrogen fertilisation levels and three replicationsarranged in a complete randomised block design.Netted melons (Cucumis melo var. Reticulatus) cv.Prodigio were transplanted in the experimental openfield in Viadana (44◦56′08′′N and 10◦32′28′′E),Mantova, Italy, in April–July 2004. Transplantingbeds were covered with transparent plastic film,120 cm wide. Plant density was 0.4 plant m−2. Theexperimental field was chosen after soil analysis toidentify a low N content. Soil analyses were performedon the top layers (0–30 cm) and results are reportedin Table 1.

Phosphorus (P2O5 74 kg ha−1) and potassium(K2O 200 kg ha−1) fertilisers were applied beforetransplanting, considering their content in the soil,and of nitrogen during the cultivation.

Plants were fertilised using four nitrogen (N) levels:0 kg ha−1 (N I), 55 kg ha−1 (N II), 110 kg ha−1 (N

Table 1. Proprieties of 0–30 cm depth soil as measured in March

before the beginning of the experiments

Parameter u.m. Value

pH units 8.2CaCO3 g kg−1 79Sand g kg−1 424Silt USDA g kg−1 358Clay g kg−1 218Soil texture loamOrganic matter g kg−1 20.7N tot. g kg−1 0.9N-NH4

+ mg kg−1 0.2N-NO3

− mg kg−1 3.2P mg kg−1 9.8CEC cmol(+) kg−1 14.5Ca mg kg−1 2549Mg mg kg−1 164K mg kg−1 159

Analysis were performed according to MIPAF methods.31 The valuesare means of eight samples randomly taken from the experimentalfield.

III) and 165 kg ha−1 (N IV). The N was applied asNH4NO3 34.5% soluble fertiliser.

The N was totally applied by fertigation starting25 days after planting until 60 days later. The totalamount of N was split into 17 constant dressingsand two dressings were applied each week. The finalapplication was performed 5 days before harvesting.The N III level was calculated by a simplified nitrogenbalance approach. The N II and N IV levels were,respectively, 50% lower and higher than the calculateddose. Fertigations were performed by drip irrigationsystems, with tubes placed under the plastic film toavoid evaporation. Water nitrogen content was lowerthan 0.25 mg L−1, therefore water was not consideredas a relevant source of this element. Preventivetreatments were performed against insects and diseasesin accordance with local farming practices.

Measurements at the harvesting stageHarvesting was performed when fruits changed colour,at full slip, when the detachment circle became visible.Fruits on which measurements were performed wererandomly taken from harvested batches on the 18 July,which was almost the fruits’ production peak. Twelvefruits for each fertilisation level were used to determinethe harvesting stage.

Flesh firmness, pulp and skin colour, totalsoluble solidsFlesh firmness was determined at two points on a ringtransversally cut at the equator in the middle of themesocarp with an Instron instrument using an 8 mmprobe (Milan, Italy).

The internal and external colour was measuredusing a Minolta Chromameter (Nieuwegein, Nether-lands) based on light reflectance. The colour wasexpressed using the Commission Internationale del’Eclairage (CIE) system where the L∗, a∗ and b∗ valuesrepresent the lightness, green–red and blue–yellow,respectively. Four equidistant and independent read-ings were collected in the equatorial zone of each fruit.Soluble solids were measured using a bench refrac-tometer (Model RFM 81; Bellingham Stanley Ltd,Kent, UK) on juice obtained from 1–2 g of pulp takenfrom two opposite sides of the fruit equatorial zones.The values were expressed in ◦Brix.

Determination of total N in fruitsTotal N was determined with the Dumas method per-formed by using an elemental analyser (ThermoQuestNA 1500 N; Thermo Electron, Milan, Italy). Analysiswere carried out with whole fruits (n = 3 per plot).

Measurements after storageSix melons for each fertilisation level were stored at10 ◦C in darkness, with 80% RH. Three fruits per Nlevel were used for post-harvest determinations after8 days. The aim of this post-harvest storage trial wasto investigate the changes in fruit quality during the

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distribution chain from the production zones to theend users.

Carotenoids were extracted using methanol (99.9%)as solvent. Samples were kept in a dark room at4 ◦C for 24 h. Absorbance readings were performedat 470 nm and carotenoid concentrations were calcu-lated by Lichtenthaler’s formula12 and expressed asβ-carotene.

For extraction of total phenols, 1-g sampleswere placed in 3 mL of 1.2 mol L−1 HCl in 80%methanol/water. The samples were vortexed for 1 min,and subsequently heated to 60 ◦C for 3 h. Duringincubation the samples were vortexed every 30 min.After incubation the samples were cooled at roomtemperature and centrifuged at 3000 × g for 1–2 minto remove solids.

Quantification of total phenols was performed usingFolin–Ciocalteu reagent diluted 10-fold before use.13

Absorbance readings of reactions containing 200 µLextract and 0.5 mL Folin–Ciocalteu reagent plus waterup to 10 mL were performed at 760 nm after 2 hincubation at room temperature.

For determining ascorbic acid (vitamin C), samplesof flesh melon were homogenised in cold 5% (w/v)trichloroacetic acid, using a cold mortar, and cen-trifuged at 16000 × g for 15 min. Total ascorbate wasdetermined in the supernatant following the methodof Wang et al.14 This assay is based on the reductionof ferric to ferrous ion by ascorbic acid in acidic solu-tion followed by formation of a red chelate betweenferrous ion and 4,7-diphenyl-1,10-phenanthrolin(bathophenantroline) which absorbs at 534 nm. Totalascorbate was determined by reduction of DHA toAsA using 3.89 mmol L−1 dithiothreitol, and DHAlevels were estimated on the basis of the differencebetween total ascorbate and AsA values. A standardcurve covering the range 0–10 nmol AsA was used.

Estimation of lipid peroxidationThe lipid oxidation of pulp was determined using1 g of pulp that was homogenised in 3 mL 0.1% (w/v)trichloroacetic acid (TCA) using a mortar. The extractwas centrifuged at 3000 × g for 5 min. The thiobar-bituric acid–malondialdehyde (TBARS–MDA) assaywas performed heating the reaction mixture containing1 mL crude extract, 4 mL 20% TCA and 25 µL 0.5%thiobarbituric acid at 95 ◦C for 10 min.15 Absorbance

readings of the reaction mixture were performed at532 and 600 nm.

Ethylene biosynthesisEthylene production was measured by enclosing onemelon fruit in airtight containers (5 L). Gas samples(1 mL each) were taken from the headspace ofthe containers with a hypodermic syringe after 1 hincubation at 20 ◦C. The ethylene concentration ineach sample was measured by gas chromatographyusing a flame ionisation detector (FID), a stainlesssteel column (150 × 0.4 cm diameter, packed withHysep T), column and detector temperatures of 70◦and 350 ◦C, respectively, and nitrogen carrier gas at aflow rate of 30 mL min−1.

Quantification was performed against an externalstandard and results were expressed on a fresh weight(FW) basis (µL kg−1 h−1 FW).

Statistical analysisAccording to the field experimental design, resultswere subjected to one-way or two-way ANOVA, andthe differences among means were determined byTukey’s post-test.

RESULTSYield, total nitrogen content, firmness and fruitcolourThe first ripe fruits were harvested 72 days aftertransplanting (DAT) and the last harvesting wasperformed 82 DAT. The yield of marketable fruitsincreased statistically by increasing the nitrogen levels.The cumulative yield at the 28 July (82 days aftertransplanting) was on average 6.2a, 7.8b, 9.6c and10c kg plant−1, respectively, in the N I, N II, N III,and N IV fertilisation levels (letters indicate statisticaldifferences for P < 0.05). Also, the total N uptakesignificantly increased by increasing the N fertilisationrates, although no difference was found between thefirst two fertilisation levels (Table 2).

The flesh firmness of fruits was measured within24 h after harvest and the highest firmness value wasfound in the N I dose (14.7 N), even if no differencewas observed among treatments (Table 2).

The skin colour was not influenced by the nitrogenapplications and no statistically differences were found

Table 2. Total nitrogen, flesh firmness and pulp colour of melon fruits harvested from melon plant fertilised with 0 (N I), 55 (N II), 110 (N III) and

165 kg ha−1 (N IV) nitrogen

Pulp colour

Nitrogen (kg ha−1) Total nitrogen (% DW) Firmness (newtons) L∗ a∗ b∗

0 1.32 ± 0.069c 14.99 ± 1.775 61.63 ± 0.243ab 15.12 ± 0.161 37.32 ± 0.09655 1.47 ± 0.107bc 12.20 ± 0.146 60.76 ± 0.464bc 14.76 ± 0.196 38.06 ± 0.104110 1.64 ± 0.058b 11.19 ± 0.467 62.79 ± 0.554a 14.29 ± 0.242 37.80 ± 0.272165 1.87 ± 0.108a 12.01 ± 0.605 63.09 ± 0.387a 14.88 ± 0.247 38.94 ± 0.448

Values are means with SE. Data were subjected to one-way ANOVA and differences among means were determined by Tukey’s post-test. Differentletters mean that values are statistically different at least for P < 0.05.

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Figure 2. Total phenols (expressed as gallic acid equivalent µmol g−1

fresh weight) in fruits harvested from melon plant fertilised with 0 (N I),55 (N II), 110 (N III) and 165 kg ha−1 (N IV) nitrogen and stored at 10 ◦Cfor 8 days. Values are means with SE (n = 3). Data were subjected totwo-way ANOVA analysis. No significant differences were foundamong treatments, while the reduction after storage was significant(P < 0.0001).

among the colour components L∗, a∗ and b∗ (data notshown). On the contrary, the L∗ values of the pulpslightly, but significantly, increased by increasing Nfertilisation level (Table 2).

Antioxidant compoundsThe antioxidants (carotenoids, total phenols andascorbic acid) measured in the flesh immediatelyafter harvest were not affected by the fertilisationlevels. Total carotenoids ranged between 105 and238 µg mg−1 FW. However, the lowest value wasobserved in the highest N fertilisation level (Fig. 1).Total phenols ranged between 1.91 and 2.72 µmol g−1

FW; the lowest value was found in the fertilisationN III level (Fig. 2). Total ascorbic acid (AsA+DHA)ranged from 748 to 1573 nmol g−1 FW (Fig. 3).

Figure 1. Total carotenoids (expressed as µg g−1 fresh weight) infruits harvested from melon plant fertilised with 0 (N I), 55 (N II), 110 (NIII) and 165 kg ha−1 (N IV) nitrogen and stored at 10 ◦C for 8 days.Values are means with SE (n = 3). Data were subjected to two-wayANOVA analysis. No significant differences were found amongtreatments, while the reduction after storage was significant(P < 0.0001).

Figure 3. Effect of fertilisation levels on the ASA+DHA contents ofmelon fruits at the harvest time and after storage. Values are meanswith SE (n = 3). Data were subjected to one-way ANOVA andsignificant differences among treatments were determined by Tukey’stest. Different letters mean that values are statistically different atP < 0.05. The reduction after storage (time) was significant(P < 0.0001).

During storage the antioxidant content significantlydecreased, but no differences were observed amongtreatments, except for AsA+DHA in the case of NII treatments. At end of the storage period totalcarotenoids, total phenols and AsA+DHA were, onaverage, 53%, 65% and 63% of the initial valuesrespectively (Figs 1, 2 and 3). Briefly, no linear relationbetween the different antioxidant compounds and thenitrogen levels was found.

Weight loss and total soluble solidsThe weight loss significantly increased during storagein all treatments, but the highest fertilised fruitsshowed the lowest weight reduction (Fig. 4).

The total soluble solids were not affected by nitrogenlevels at harvest time. After 8 days of storage a general

Figure 4. Relative weight change (RWC) in fruits harvested frommelon plant fertilised with 0 (N I), 55 (N II), 110 (N III) and 165 kg ha−1

(N IV) nitrogen and stored at 10 ◦C after 8 days. Values are means withSE (n = 3). Data were subjected to one-way ANOVA analysis anddifferences among means were determined by Tukey’s test. Differentletters mean that values are statistically different at least for P < 0.05.

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decrease of total soluble solids seemed occur, evenif higher fertilised fruits showed higher values of thisparameter (Fig. 5).

Lipid peroxidation and ethylene production ofstored fruitsThe malondialdehyde measured as thiobarbituricacid reactive substances, a marker of membranelipid degradation, increased in all treatments duringstorage, but no significant differences were foundamong treatments (Fig. 6). However, the highest valuewas observed in fruits harvested in plots fertilised withthe third nitrogen fertilisation level.

Ethylene production was not statistically differentamong the fruits harvested in the different nitrogenfertilisation rates. Immediately after harvest ethylenebiosynthesis ranged from 30 to 50 µL kg−1 h−1, whileafter 8 days of storage, it significantly decreased to5–8 µL kg−1 h−1 (Fig. 7).

Figure 5. Total solid soluble (expressed as ◦Brix) in fruits harvestedfrom melon plants fertilised with 0 (N I), 55 (N II), 110 (N III) and 165 kgha−1 (N IV) nitrogen and stored at 10 ◦C for 8 days. Values are meanswith SE (n = 3). Data were subjected to two-way ANOVA analysis.Different letters mean that values are statistically different at least forP < 0.05. The reduction after storage (time) was significant(P < 0.0001).

Figure 6. Lipid peroxidation (expressed as TBARS equivalent nmolg−1 fresh weight) in fruits harvested from melon plants fertilised with 0(N I), 55 (N II), 110 (N III) and 165 kg ha−1 (N IV) nitrogen and stored at10 ◦C for 8 days. Values are means with SE (n = 3). Data weresubjected to two-way ANOVA. No significant differences were foundamong treatments. The increase after storage (time) was significant(P < 0.0001).

Figure 7. Ethylene production (expressed as µL kg−1 h−1 freshweight) measured after recovery for 3 h at 20 ◦C (B) from melon fruitsharvested from plants fertilised with 0 (N I), 55 (N II), 110 (N III) and165 kg ha−1 (N IV) nitrogen and stored at 10 ◦C for 8 days. Values aremeans with SE (n = 3). Data were subjected to two-way ANOVA. Nosignificant differences were found among treatments. The reductionafter storage (time) was significant (P < 0.0001).

DISCUSSIONLiterature concerning the effect of nitrogen fertilisationon fruit quality is discussed. Results obtained showedthat the total fruit yield increased linearly up to theN III fertilisation level, in terms of the number ofmarketable fruits. These results agree with previousexperiments performed on melon yield and nitrogenfertilisation16,17 and other vegetables and fruits.18

However, the total nitrogen content, expressed as% of dry weight, continued to increase in melonfruits without a significant increase in the yield. Theseresults suggest that at the higher fertilisation levelsthe nitrogen was not a growth limiting factor and anaccumulation dynamic took place.

Firmness of fruits has not been found to be affectedby nitrogen fertilisation, although the untreated fruitshowed the highest flesh firmness according to thework of Hernandez-Fuentes et al.19 Flesh firmnesswas inversely related to nitrogen fertilisation in manyfruits.19

The colour of the flesh is positively affected bythe highest N dose showing an increase in lightness(L) and yellowness (b). This colour change shouldbe correlated with pigment pulp variations, such ascarotenoids and chlorophylls.20

The sugar content measured as total solids solublewas not affected by nitrogen dose. Contrary resultswere found in analogous experiments performed onmelons grown under different nitrogen fertilisationrates (0, 80, 120 and 160 kg ha−1). The increment ofnitrogen from the 0 to 80 kg ha−1 gave a significantincrease of ◦Brix in melon fruits.21 Total soluble solidsis a reliable marker of the eating quality of melon, witha minimum of 10 ◦Brix for commercialisation.

In our experiment the nitrogen doses used (0, 55,110 and 165 kg ha−1) neither affected the antioxidantcompounds content in fruit at harvest time norprevented their catabolism during storage. However,

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many studies have shown a negative correlationbetween the amount of nitrogen applied and vitaminC content in fruits.22,23 In our experiments, althoughthe results among treatments were not statisticallydifferent. The absence of significant differencesmight be due to (1) a level of nitrogen that wasnot really excessive; (2) no limiting of light; and(3) the management of irrigation was oriented toavoid an excess of water and leaching. Duringstorage the vitamin C significantly decreased after8 days. The reduction might be used by thefruits for preventing oxidative stresses. However, forconfirming this hypothesis all the components ofascorbate–glutathione cycle should be monitored.

During storage the weight loss of the fruit increasedaccording to fertilisation rates. TBARS determina-tions were used as markers of membrane disruptionduring fruit senescence. The amount of TBARS isused to estimate the peroxidation of lipids in bio-logical membranes. The increase after storage in alltreatments indicates the degradation of polyunsatu-rated fatty acids. The TBARS content increases duringfruit ripening and dramatically increases during fruitsenescence.24 Therefore, TBARS in our experimentswere determined for monitoring the melon fruitssenescence process. Results indicate that N levelsapplied in pre-harvest do not affect the polyunsatu-rated fatty acids degradation.

In literature it is reported that lipid peroxidationusually increases after ethylene exposure.25 However,the relationship between TBARS and ethylene isoften controversial. Studies performed on spinachleaves showed that ethylene and TBARS may haveopposite trends during senescence, especially ifethylene production is inhibited.26 Ethylene is animportant hormone which regulates fruit ripening andsoftening. Melon are considered moderately sensitiveto exogenous ethylene.27 The effect of ethyleneon melon ripening has been investigated in severalexperiments using ethylene inhibitors such as amino-ethoxyvinylglycine or 1-methylcyclopropene.28,29

The amount of ethylene produced by fruits atharvest time was in accord with data reportedin other experiments.20,30 During ripening, melonsare climacteric fruits, which means that they showa sharp increase (peak) of respiration rate andethylene production during ripening. Therefore lowtemperatures (above chilling sensitive threshold)and ethylene inhibitors can be used efficiently forpreserving fruit quality.

In conclusion, considering that high levels ofnitrogen fertilisation do not significantly increase fruityield and do not improve fruit quality either at harvesttime or during storage, it is advisable to reducenitrogen input for cultivation to the minimal levelneeded to produce the expected yields.

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