Impaginato 353 Adv. Hort. Sci., 2018 32(3): 353-362 DOI: 10.13128/ahs-21927 Influence of soil and soilless agricultural growing system on postharvest quality of three ready-to-use multi-leaf lettuce cultivars B. Pace 1, 2, I. Capotorto 1, 2, M. Gonnella1, F. Baruzzi 1, M. Cefola 1, 2 (*) 1 Istituto di Scienze delle Produzioni Alimentari, Consiglio Nazionale delle Ricerche (CNR), Via G. Amendola, 122/O, 70126 Bari, Italy. 2 Istituto di Scienze delle Produzioni Alimentari, Consiglio Nazionale delle Ricerche (CNR), URT c/o CS-DAT, Via M. Protano, 71121 Foggia, Italy. Key words: ammonium, Lactuca sativa L., nitrate, microbial quality, postharvest storage, soilless cultivation. Abstract: In this study the influence of soil and soilless growing systems (sub- strate 3:1 v/v perlite:peat) on quality and microbial traits of three multi-leaf lettuce cultivars (two green, ‘Eztoril’ and ‘Ezabel’, and one red, ‘Ezra’) was eval- uated at harvest and after 7 and 13 days of storage at 8°C. At harvest, ‘Ezra’ showed a respiration activity and a total phenol content respectively 2-fold and 25% significantly higher than the green cultivars. Soil lettuces resulted more stressed than those grown in soilless, as indicated by their initial content in antioxidants. As for nitrate content, soilless grown lettuces at harvest showed an average concentration higher than soil-grown ones, although values are gen- erally lower than limits imposed by the EU Regulation (No. 1258/2011). During storage, soilless lettuces showed no ammonium accumulation, differently from those cultivated in soil. In addition, lettuce cultivars grown in soilless condition showed unchanged content in the antioxidant activity and total phenols, and lower microbial counts than soil lettuces. Results of the present study showed that soilless growing system can positively affect qualitative and microbiologi- cal parameter of lettuces studied, and it can be considered a good soilless growing technique in order to obtain high quality multi-leaf lettuces for ready- to-use industry. 1. Introduction The consumer’s demand of ready-to-use fruits and vegetables, and in particular that for minimally processed leafy vegetables, is continuously growing. Although iceberg lettuce is still the main lettuce used in the ready-to-use industry, consumers are requesting other types of lettuce with attractive colours and shapes combining the best quality characteris- tics from all varieties (Rijk Zwaan, 2009). The new baby-sized leaves, (*) Corresponding author: maria.cefola@ispa.cnr.it Citation: PACE B., CAPOTORTO I., GONNELLA M., BARUZZI F., CEFOLA M., 2018 - Influence of soil and soilless agricultural growing system on postharvest qua- lity of three ready-to-use multi-leaf lettuce culti- vars. - Adv. Hort. Sci., 32(3): 353-362 Copyright: © 2018 Pace B., Capotorto I., Gonnella M., Baruzzi F., Cefola M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 10 October 2017 Accepted for publication 12 January 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(3): 353-362 354 baby- and multi-leaf have been developed recently as high quality lettuce varieties for the ready-to-use market. Some benefits of baby-sized lettuce, when compared with whole-head lettuce, include: i) greater efficiency with higher percentage of usable product; ii) easier and faster processing; iii) more attractive colour and shapes, and iv) minimal oxida- tion due to smaller stem diameter (Martínez-Sánchez et al., 2012). Moreover, for both multi- and baby-leaf lettuces, no physical wounding was undertaken, except that of the harvesting, avoiding the physical damage that occurs during preparation of fresh-cut lettuce that causes an increase in respiration activity, biochemical changes and microbial spoilage, which may result in degradation of colour, texture and flavour of the ready-to-use produce (Cantwell, 2004). Likewise, cultivar selection is of great importance in the ready-to-use industry since quality characteristics (such as leaf colour, shape, freshness, texture and browning potential) can change largely depending on the genotype (Nicola et al., 2009). The quality and shelf-life of ready-to-use leaves depend on genotypic traits of raw material and on several aspects from preharvest to postharvest processing (Clarkson et al., 2003; Cantwell, 2004). Some physical and chemical indicators can be used for objective assessment of visual quality (Barrett et al., 2010; Salinas-Hernández et al., 2015). Among these, ammonium (NH4+), pro- duced during storage as a consequence of senes- cence in various vegetables (Cefola et al., 2010; Pace et al., 2014), might be used as predictors of shelf-life (Cefola et al., 2017). In general, preharvest factors should be aimed to optimize their impact on posthar- vest quality (Crisosto and Mitchel, 2002). From this point of view, soilless system is becoming of high interest since it can improve both, preharvest and postharvest quality of vegetables (Rodríguez-Hidalgo et al., 2010). In particular, soilless agricultural grow- ing system allows to set optimal conditions and nutri- ent concentration for plant growth (Silberbush and Ben-Asher, 2001; Selma et al., 2012) with the follow- ing advantages: higher yields (Lopez-Medina et al., 2004; Recamales et al., 2007), better quality vegeta- bles (Recamales et al., 2007; Cefola et al., 2011) and higher earliness (Recamales et al., 2007; Valenzano et al., 2008), compared to soil cultivation. The success of lettuce production depends to a great extent on the maintenance of a continuous growth rate by the optimal management of nutrients (Luna et al., 2013). In addition, especially for leafy vegetables, the use of soilless system can avoid soil contaminants and improve the sanitary quality respect to traditional soil cultivation, leading benefits on raw materials for postharvest industry (Selma et al., 2012). Starting from these findings, the aim of this work was to eval- uate the influence of two growing system (soil and soilless) on postharvest quality of three multi-leaf let- tuce genotypes, including two green and one red, stored under refrigeration for 13 days. 2. Materials and Methods Reagents Extraction solvents (MeOH, EtOH), 2,2-diphenyl-1- picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetram- ethylchroman-2-carboxylic acid (Trolox) and all stan- dards used in the experiments were obtained from Sigma-Aldrich (St. Louis, Mo., USA). Folin-Ciocalteu’s p h e n o l r e a g e n t w a s p u r c h a s e d f r o m M e r c k (Germany). Plant material and growing system Three types of Dutch multi-leaf lettuces (Lactuca sativa L.), two green (cv. Eztoril and Ezabel) and one red (cv. Ezra) (Enza Zaden, Enkhuizen, Netherland) were cultivated in an unheated plastic tunnel under soil (S) or soilless (SL) agricultural growing system in the same greenhouse at the experimental farm “La Noria” of the Institute of Sciences of Food Production (CNR-ISPA) located in the South of Italy (Mola di Bari). A split-plot design with three replications was applied, randomizing the growing systems (GS) in the main plots and cultivars in the subplots. Main plots were of 3.6 m2 (0.9 m wide and 4 m long). The SL sys- tem consisted of three single benches (4 m long x 0.3 m wide x 0.1 m high, with a slope of 2%) each plot containing a 3:1 (v:v) perlite:peat mixture as sub- strate. The nutrient solution was supplied to the SL system without recirculation and had the elemental composition given in Table 1, where the soil charac- teristics are reported too. The irrigation water had the following composition (expressed in mmol L-1): 0.3 N-NO3, 0.23 K, 0 P, 1.73 Mg, 1.82 Ca, 7.39 Cl, 4.05 Na. Nutrient solution and water were supplied to SL and S units based on a timer controlled schedule, using minimum substrate water content values, mon- itored by tensiometers. Furthermore, as additional reference control taking into account two different threshold levels of -5 and -25 kPa to start the irriga- tion supply in SL and S, respectively. For SL system a nutrient supply level criterion was additionally Pace et al. - Postharvest quality of three ready-to-use multi-leaf lettuce cultivars 355 applied. At transplant soil plots were fertilized with ammonium nitrate and monopotassium phosphate giving the equivalent of 50, 80, 50 kg ha-1 of N, P2O5 and K2O, respectively, and after a month a 30 kg ha-1 i n t e g r a t i o n o f N f r o m a m m o n i u m n i t r a t e w a s applied. Seedlings were produced in greenhouse in polystyrene trays on peat and were transplanted 25 days after sowing on February 22. Harvest was per- formed 55 days after transplanting (on April 18) for the SL and after 73 days (on May 6) for the S system. Greenhouse ventilation temperature was 20°C. In fig- ure 1 the climatic parameters measured in the green- house are reported. Daily air temperature was on average 21°C, and minimum and maximum air tem- perature ranged from - 0.2 to 17.5 and from 17.5 to 46.0°C, respectively (Fig. 1A). Air relative humidity was on average 50.5%; daily minimum and maximum relative humidity ranged from 6 to 56% and from 45 to 85% (Fig. 1B). The average photosynthetically active radiation was 282 µmol m-2 s-1; its mean and maximum values changed from 80 to 420 and from 400 to 2,080 µmol m-2 s-1, respectively (Fig. 1C). After harvest, lettuces were immediately transported under refrigerated condition in polystyrene boxes to the CNR ISPA- postharvest laboratory. Processing and storage After harvest, for each multi-leaf lettuce cultivar (‘Ezra’, ‘Eztoril’ and ‘Ezabel’), and for each agricultur- al growing system (S or SL), leafs were selected in order to avoid damaged samples, and no washing or pre-treatment were applied. For each cultivar and GS about 600 g of leaves were used for quality evalua- tion at harvest, whereas about 1.2 Kg were used for the quality evaluations during storage. Thus, leaves were put in open polyethylene bags (about 200 g each bag), and stored at 8°C in dark conditions. For each cultivar, 12 bags (3 replicates ´ 2 GS, S or SL, ´ 2 storage periods, 7 and 13 days) were prepared. Fig. 1 - Climatic parameters (A= Temperature; B= air Relative humidity; C= Photosynthetically Active Radiation) measu- red in the greenhouse during the experiment, from seed- ling transplantation to harvest days. The blue and red perpendicular lines indicate the harvest day for lettuce cultivated in soilless and soil condition, respectively. Mineral composition Soil composition Soilless nutrient solution Sand 24.30% - Silt 31.90% - Clay 43.80% - pH 7.6 6.5 EC (dS m-1) 2.5 2.3 Cl- - 7.39 Mg2+ - 1.73 Na+ - 4.05 K+ - 5.12 Ca2+ - 4.74 NH 4 + - 0.5 NO 3 - - 9.43 P-H2PO4- - 1.61 S-SO42- - 0.81 CEC (cmol kg-1 dw) 31.8 - Organic matter (g kg-1 dw) 14 - Total N (g kg-1 dw) 0.95 - Available P (g kg-1 dw) 110 - Available K (g kg-1 dw) 244 - CaCO 3 (g kg-1 dw) 0.11 - Table 1 - Soil and soilless nutrient solution composition. Soil classified as clay soil (USDA textural soil classification, 1987). Values of nutrient solution are expressed in mmol L-1. Micronutrients were supplied according to Johnson et al. (1957) Adv. Hort. Sci., 2018 32(3): 353-362 356 Respiration activity and the analysis of nitrate con- tent were performed at harvest. In addition, antioxi- dant activity, total phenols, ammonium content and microbiological analysis were evaluated at harvest and after 7 and 13 days of storage. Respiration activity The respiration activity of each cultivar was mea- sured using a closed system as reported by Kader (2002). About 100 g of leaves for each replicate were put into 6 L sealed plastic jars (one jar for replicate) where CO2 was allowed to accumulate until the value of 0.1%. The time needed to reach this value was cal- culated, making CO2 measurement at regular time intervals. For the CO2 analysis, 1 mL gas sample was taken from the head space of the plastic jars through a rubber septum and injected into the gas chromato- graph (p200 micro GC, Agilent, Santa Clara, CA) equipped with dual columns and thermal conductivi- ty detector. CO2 was analyzed with a retention time of 16 s and total run time of 120 s on a 10 m porous polymer (PPU) column at a constant temperature of 70°C. Respiration activity was expressed as mL CO2·kg-1·h-1. Antioxidant activity, total phenol, ammonium and nitrate content To determine both antioxidant activity and total phenols content, the extraction procedure reported by Cefola et al. (2012), was followed. In detail, 5 g samples were homogenized (Ultraturrax T-25, IKA Staufen, Germany) in a MeOH: water (80:20) solution for 1 min, and then centrifuged at 5°C at 6440 x g for 5 min. The supernatant was therefore used for the assays. The antioxidant activity assay was performed following the procedure described by Brand-Williams et al. (1995) with minor modifications. Briefly, the supernatant, proper diluted, was pipetted into 0.95 mL of DPPH solution to start the reaction. The absorbance was read after about 30 min at 515 nm. Trolox was used as a standard and the antioxidant activity was expressed in g of Trolox equivalents per kg of fresh weight (g TEAC kg-1 fw). The total phenol content was determined according to the method of Singleton and Rossi (1965). Each extract (100 μL), proper diluted, was mixed with 1.58 mL water, 100 μL of Folin-Ciocalteu reagent and 300 μL of sodium carbonate solution (200 g L-1). The absorbance was read after 2 h at 765 nm. Total phenol content was calculated on the basis of the calibration curve of gal- lic acid and expressed as g of gallic acid per kg of fresh weight (g GA kg-1 fw). For ammonium content the method reported by Weatherburn (1967) was used. In detail, 5 grams of chopped sample were homogenized (Ultraturrax T- 25, IKA Staufen, Germany) with 20 mL distilled water for 2 min, centrifuged at 6440 x g for 5 min, and 0.5 mL extract was used for the analysis. Color develop- ment, caused by the reaction with a phenol nitro- prusside reagent and alkaline hypochlorite solution, was determined after an incubation of 20 min at 37°C, by reading the absorbance at 635 nm (UV- 1800, Shimadzu, Kyoto, Japan). Ammonium content was expressed as µmole NH4+ per kg of fresh weight (µmole NH4+ kg-1 fw). As for nitrate content, samples (about 100 g for replicates) were dried in the oven (65°C until con- stant weight) and were ground to fine powder. The powder (0.5 g for each replicate) were extracted on orbital shaker for 20 minutes with 50 mL of a solution containing 3.5 mmol L-1 of sodium carbonate and 1 mmol L-1 sodium bicarbonate. Analysis were carried out using a ion exchange chromatography (Dionex DX 200, Dionex Corp, Sunyvale, CA, USA) with a conduc- tivity detector, using an IonPac AG14 precolumn and a n I o n P a c A S 4 A s e p a r a t i o n c o l u m n ( D i o n e x Corporation). Results were expressed in mg of nitrate per kg of fresh weight (mg NO3- kg-1 fw). Microbiological analysis Samples (30 g for replicates) were homogenized for 1 min in 0.1% sterile buffered peptone water (Difco Laboratories, Detroit, MI, USA) (1:5 dilution) using a stomacher (Seward, London, UK). Total aero- bic mesophilic bacteria count was evaluated using plate count agar (Difco) incubated at 30°C for 48 h. Yeasts and moulds were counted on Sabouraud Dextrose Agar (Difco) supplemented with chloram- phenicol and chlortetracycline (both 0.05 g L-1) and incubated at 25°C for 5-7 days. Total counts of Enterobacteriaceae were obtained by pour-plating dilutions (1 mL) in Violet Red Bile Glucose agar (Difco) and plates were incubated at 37°C for 24 h. Microbiological counts were expressed as log CFU g-1 of fresh weight (log CFU g-1 fw). Statistical analysis In order to study the effect of GS (S or SL), culti- vars, CV (‘Ezra’, ‘Eztoril’ and ‘Ezabel’) and their inter- action (GS x CV) on quality parameters at harvest, and the effect of GS, CV, storage (0-7-13 days) and their interaction (GS x CV x storage) on quality para- meters, two multifactor ANOVA were performed (Statistica Software). When significant effect of fac- tors were detected, the Student Newman Keuls (SNK) test was applied to separate means. For a visual Pace et al. - Postharvest quality of three ready-to-use multi-leaf lettuce cultivars 357 analysis of the data, principal component analysis (PCA) (PRINCOMP procedure, SAS software, Cary, NC, USA; biplot by XLStat, Addinsoft, Paris, France) was performed on mean centered and standardized (unit variance scaled) data prior to analysis. The data matrix submitted to PCA was made up of 18 observa- tions - 3 cultivars (‘Ezra’, ‘Eztoril’ and ‘Ezabel’) x 2 growing system (S and SL) x 3 storage times (0-7-13 days) and 6 quality parameters (antioxidant activity, total phenols, ammonium, mesophilic bacteria, yeasts and moulds, Enterobacteriaceae). 3. Results Effect of growing systems and cultivars on lettuces quality traits at harvest Yield response was influenced by genotypes more than GS (S or SL), since a lower fresh weight was pro- duced by the red lettuce compared to the other two cultivars (2.7 vs 3.6 kg m-2) and only in cv. Eztoril there was a higher yield in S compared to SL system (4.3 vs 3.3 kg m-2). The effect of GS, multi-leaf lettuce CV (‘Ezra’, ‘Eztoril’ and ‘Ezabel’) and their interaction on the quality parameters measured at harvest was investi- gated (Table 2). Ammonium content, antioxidant activity, total phenols and nitrate content were sig- nificantly affected by GS and CV, while respiration activity was affected only by CV. The interaction GS x CV was statistically significant only for nitrate content (Table 2). The respiration activity of cv. Ezra was two- fold higher than the green cultivars (‘Eztoril’ and ‘Ezabel’). Regarding ammonium content, the values found for lettuces cultivated in SL were statistically higher respect to S and, between cultivars, red multi- leaf lettuce had mean values statistically higher than green cultivars (Table 2). Growing system affects sig- nificantly the antioxidant activity and total phenols: plants cultivated in S showed significantly higher mean content than SL samples (Table 2). Regarding CV, there were no differences between green multi- leaf lettuces in antioxidant activity and total phenols, while the red cultivar Ezra had lower values of antiox- idants and higher values of total phenols respect to the green cultivars (Table 2). As for nitrate content was almost double in SL lettuces than S grown ones (838 vs 432 mg NO3- kg-1 fw). The red multi-leaf let- tuce (cv. Ezra) had mean values of nitrate statistically higher than cv. Eztoril but not different from cv. Ezabel (Table 2). Effect of growing systems and cultivars on lettuces’ quality traits and microbial parameters during cold storage The results of Multifactor Anova on antioxidant activity, total phenols and ammonium content as affected by GS, CV, storage time (0, 7 and 13 days) and their interaction were reported in Table 3. Growing system affected antioxidant activity and total phenols, CV affected total phenols and ammoni- um, while storage time affected antioxidant activity Respiration activity (mL CO 2 kg-1 h-1) Ammonium content (µmole NH 4 + kg-1fw) Antioxidant activity (g TEAC kg-1 fw) Total phenols (g GA kg-1 fw) Nitrate content (mg NO 3 - kg-1 fw) Growing system (GS) Soilless 47.44 66.7 2.33 1.32 838.12 Soil 45.73 49.6 4.82 1.84 431.96 Cultivar Ezra 72.67 a 72.80 a 3.07 b 1.89 a 765.71 a Eztoril 35.82 b 48.10 b 3.82 a 1.41 b 524.32 b Ezabel 31.26 b 53.60 b 3.84 a 1.44 b 615.09 ab GS NS ** *** ** *** Cultivar *** ** ** * * GS x cultivar NS NS NS NS * Table 2 - Effect of growing system (soil and soilless) and cultivar (Ezra, Eztoril and Ezabel) on quality parameters measured at harvest When interaction among factors was not significant, the results of the mean separation test (SNK test) are reported. Different letters indicate statistical difference within cultivars, respectively, for P≤0.05. NS, not significant; * P≤0.05; ** P≤0.01; *** P≤0.001. 358 Adv. Hort. Sci., 2018 32(3): 353-362 and ammonium (Table 3). Considering the interaction among factors, antioxidant activity was affected by GS x Storage and CV x Storage; total phenols were influenced by GS x CV and by GS x CV x Storage, and ammonium was affected only by GS x Storage (Table 3). In figure 2, changes in antioxidant activity (A), total phenols (B) and ammonium content (C) during storage of the three multi-leaf lettuce cultivars, culti- vated in S and SL conditions, are reported. At har- vest, lettuces cultivated in S showed values of antiox- idant activity significantly higher than SL lettuce. However, during storage, antioxidant activity of let- tuces cultivated in S decreased rapidly, reaching approximately the same values of samples cultivated in SL after 7 day of storage at 8°C; after it remained unchanged for cv. Ezabel (about 2 g TEAC kg-1 fw) and slightly increased for cv. Eztoril (about 3 g TEAC kg-1 fw). The cultivar Ezra cultivated in S showed a con- tent in antioxidant activity almost constant during storage, with a 30% reduction at the end of storage (Fig. 2A). Whereas, lettuce cultivated in SL showed unchanged values of antioxidant activity during time, with a slight reduction at the end of storage for cv. Ezabel (about 1.3 g TEAC kg-1 fw) (Fig. 2A). Regarding the content of total phenols (Fig. 2B), green multi- leaf lettuces cultivated in S showed a slight decrease during the first week of storage, after then values rise again until the end of storage, reaching values of about 1.6 and 1.7 g GA kg-1 fw for cv. Eztoril and Ezabel, respectively (Fig. 2B). A specular trend for green multi-leaf lettuce cultivated in SL was observed (Fig. 2B). The cv. Ezra cultivated in SL showed the same behavior of green lettuces; whereas ‘Ezra’ culti- vated in S showed an initial total phenol content of about 2.3 g GA kg-1 fw, which increased during the first week of storage, reducing to initial values until the end of the storage (Fig. 2B). As regard data of ammonium content (Fig. 2C) let- tuces cultivated in SL showed unchanged values dur- ing postharvest storage, starting from the initial m e a n v a l u e s o f a b o u t 7 9 . 9 ± 7 . 5 , 5 7 . 0 ± 9 . 3 a n d 63.2±9.6 mmole NH4+ kg-1 fw in cv. Ezra, Eztoril and Ezabel, respectively. Whereas, lettuces cultivated in S showed an increase in ammonium content during storage, which doubled for all cultivars, starting from initial mean values of 65.5±15.7, 39.2±3.5 and 44.0±7.0 mmole NH4+ kg-1 in cv. Ezra, Eztoril and Ezabel, respectively. Fig. 2 - Changes in antioxidant activity (A), total phenols (B) and ammonium content (C) of three multi-leaf lettuce culti- vars (‘Ezra’, ‘Eztoril’ and ‘Ezabel’), cultivated in soil or soilless condition, during storage at 8°C. Mean ± SD. Table 3 - Multifactor Anova of antioxidant activity total phenols and ammonium content as affected by growing system (soil or soilless), cultivar (‘Ezra’, ‘Eztoril’ and ‘Ezabel’) and storage time (0, 7 and 13 days) Antioxidant activity (g TEAC kg-1 fw) Total phenols (g GA kg-1 fw) Ammonium content (µmole NH 4 + kg-1fw) Growing system (GS) *** ** NS Cultivar (CV) NS ** ** Storage time *** NS * GS x CV NS ** NS GS x Storage * NS * CV x Storage *** NS NS GS x CV x Storage NS * NS NS= not significant; * P≤0.05; ** P≤0.01; *** P≤0.001. 359 Pace et al. - Postharvest quality of three ready-to-use multi-leaf lettuce cultivars As concerns microbial populations evaluated dur- ing the trial, results of multifactor Anova statistical analysis of mesophilic bacteria, yeasts and moulds and Enterobacteriaceae as affected by growing sys- tem (GS), cultivars (CV) and storage time are report- ed in Table 4. All factors and their interactions affect- ed significantly the microbial populations evaluated, with the exception of GS x CV interaction for yeasts and moulds. As concerns total mesophilic bacteria, lettuces cultivated in SL showed an increase of about 1.5 log unit during storage, starting from initial mean values of 3.7±0.05 log CFU g-1 (cv. Eztoril), 4.9±0.22 log CFU g-1 (cv. Ezabel) and 6.1±0.29 log CFU g-1 (cv. Ezra), whereas lettuces cultivated in S growing condi- t i o n s h o w e d a n h i g h e r s i g n i f i c a n t i n c r e a s e i n mesophilic population during storage, of about 3 log unit, starting from a mean initial count of about 4.67±0.68 log CFU g-1 (Fig. 3A). Yeast and mould loads from S and SL were not found to be significantly dif- ferent. However, during 13 days of cold storage their amount increased significanly for all cultivars, result- ing statistically higher in multi-leaf lettuces cultivated in S respect to the SL ones (Fig. 3B). Also in the case of Enterobacteriaceae, lettuces cultivated in S showed a significant increase of about 4 log unit at the end of the storage, starting from initial values of 3.4±0.9, 2.4±0.26 and 4.10±0.10 log CFU g-1 for cv. Ezra, Eztoril and Ezabel, respectively. In lettuces culti- vated in SL conditions, the increase of about one magnitude order was found for this microbial popula- tion (Fig. 3C). Principal component analysis Principal component analysis revealed that almost 84% of the total variability of data was explained by the first two principal components. PC1 resulted mainly and positively correlated to ammonium and the counts of the three microorganisms groups. Each of them contributed to PC1 for 20-25% of the total variability (Fig. 4). On the other hand, antioxidant activity and total phenols contributed to PC2 for more than 45% each. Among observations, all S growing system samples after 13d storage showed a stronger and positive correlations with PC1, ammoni- um and the microbiological counts. At a proximate position collocated samples of the red cultivar Ezra collected from SL and stored for 7 and 13 days. On the contrary, all cultivars grown on the S system, at Fig. 3 - Changes in total mesophilic bacteria (A), yeasts and moulds (B) and Enterobacteriaceae (C) of three multi- leaf lettuce cultivars (‘Ezra’, ‘Eztoril’ and ‘Ezabel’), culti- vated in soil or soilless condition, during storage at 8°C. Mean ± SD. Mesophilic bacteria Yeasts and moulds (log CFU g-1 fw) Entero- bacteriaceae Growing system (GS) ** * * Cultivar (CV) *** * *** Storage time *** *** *** GS x CV *** NS *** GS x Storage *** *** *** CV x Storage *** ** *** GS x CV x Storage *** * *** Table 4 - Multifactor Anova of mesophilic bacteria, yeasts and moulds and Enterobacteriaceae as affected by growing system (soil or soilless), cultivar (‘Ezra’, ‘Eztoril’ and ‘Ezabel’) and storage time (0, 7 and 13 days) NS= not significant; * P≤0.05; ** P≤0.01; *** P≤0.001. 360 Adv. Hort. Sci., 2018 32(3): 353-362 harvest, were negatively correlated to PC1, on the opposite site of ammonium and microbial parame- ters. It seems that no observations were strongly cor- related to antioxidant activity and total phenols, with the exception of soil-grown Ezra leaves sampled at harvest and at 7 days storage, followed by the other two cultivars coming from S at harvest and soil- grown Ezra at 13 days storage (Fig. 4). Among obser- vations negatively correlated to PC2, one result note- worthy, the SL-grown ‘Ezabel’ sampled at 13 days after storage, since it showed a sharp decrease in the antioxidant activity at the end of the storage, as described in figure 2A. 4. Discussion and Conclusions The three multi-leaves lettuces studied in this research showed different qualities at harvest. In particular, the green cultivars resulted very similar and suitable for postharvest processing, on the basis of respiration activity and ammonium content. At contrary, the red cultivar was considered more per- ishable than the green ones, due to the high respira- tion activity (Kader, 2002) even though it showed an higher content in polyphenols than the green culti- v a r s , a s p r e v i o u s l y r e p o r t e d b y o t h e r a u t h o r s (Martínez-Sánchez et al., 2012; Selma et al., 2012). As regards antioxidant activity and total phenols, the higher contents measured in S cultivated lettuces could be a plant response to applied stress treat- ments (Oh et al., 2009). Compared to the SL growing system, S irrigation management implies necessarily different water conditions, keeping S plants in not optimal and constant water availability all-day. As a consequence, S plants may have occasionally experi- enced a water stress combined to heat stress during the highest temperature hours under greenhouse, in the few days preceding harvest (Fig. 1). Even a time- limited stress can activate the antioxidant synthesis in the plant metabolism (Oh et al., 2009). On the other hand, the more constant availability of water and nutrients in SL grown plants allowed a higher nitrogen uptake, partially accumulated as nitrate in the vacuoles at higher rate than in S cultivated let- tuces. However, the nitrate content measured at har- vest was generally low in both GS compared to the limits imposed by the EU Regulation No. 1258/2011. In compliance with the current regulation, nitrate accumulation in lettuce grown in the spring-summer period under greenhouse should not exceed 4,000 mg kg-1 fw. This limit is in agreement with the poten- tially high nitrate accumulating capacity of lettuce. However, at our latitude, the optimal light conditions found by plants during the spring months allow an efficient and fast assimilation of the up-taken nitrate. During cold storage, the SL growing system resulted able to preserve the quality of lettuces since no increase in ammonium content (senescence indica- tor) was registered whereas multi-leaf lettuces culti- vated in S that resulted more senescence-prone. Ammonium accumulates in leafy vegetables during storage, as consequence of protein catabolism. Thus, ammonium was used as indicator of quality and shelf-life of green vegetable. (Chandra et al., 2006; Pace et al., 2014; Cefola et al., 2015; Cefola and Pace, 2015; Cefola et al., 2017). Data from ammonium con- firms that SL could be considered a suitable growing system to preserve postharvest quality of the culti- vars analysed, although genotyping characteristics of each cultivar need also to be taken into account (Selma et al., 2012). During storage nitrate measure- ments were not carried out, since in preliminary trials performed on the same lettuce genotype (cv. Ezra) no nitrate changes after storage at 8°C for 10 days w e r e d e t e c t e d . I n p a r t i c u l a r n i t r a t e r e m a i n e d unchanged at 1550 and 1800 mg kg-1 fw, in soilless and soil lettuce, respectively (data not shown). This was supported by several contributes in literature, referring about no modification of nitrate concentra- tion in lettuce and other species during storage at temperature in a range from 1 to 10°C (Siomos et al., 2002; Chung et al., 2004; Konstantopoulou et al., 2010). The SL cultivation showed a positive effect Fig. 4 - PCA biplot (PC1 vs PC2) describing the spatial distribu- tion of quality and microbiological parameters of three multi-leaf lettuce cultivars (‘Ezra’, ‘Eztoril’ and ‘Ezabel’) grown in soil (S) and soilless (SL) system during storage; A= Ezra; B= Eztoril; C= Ezabel. AA= antioxidant activity; Phenols= total phenols; 0d – 7d – 13d: 0-7-13 days of storage. 361 Pace et al. - Postharvest quality of three ready-to-use multi-leaf lettuce cultivars also on microbiological quality of the green cultivars during storage. Similarly, results were reported by other authors on table grape (Cefola et al., 2011) and on soilless growing systems (Scuderi et al., 2011; Selma et al., 2012). In conclusion, the three multi-leaves lettuces studied in this research showed different qualities at harvest. In particular, green cultivars resulted very similar and suitable to postharvest processing, whereas, the red one was considered more perish- able. At harvest, lettuces grown in soil showed the higher content in antioxidant activity and total phe- nols and the lower in nitrate than soilless samples. However, the nitrate content measured at harvest was generally low in both growing systems compared to the limits imposed by the EU Regulation No. 1258/2011. Regarding the postharvest storage, ready-to-use lettuces cultivated in soilless showed microbiological and qualitative performance better than those grown in soil. 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