Dodo6/Topic_Modelling_using_LDA

1agriculture2Article3 4Influence of Harvest Stage and Rootstock Genotype5on Compositional and Sensory Profile of the6Elongated Tomato cv. “Sir Elyan”7Rosario Paolo Mauro 1, * , Valeria Rizzo 1 , Cherubino Leonardi 1 , Agata Mazzaglia 1 ,8Giuseppe Muratore 1 , Miriam Distefano 1 , Leo Sabatino 2 and Francesco Giuffrida 19110 11212 13*14 15Dipartimento di Agricoltura, Alimentazione e Ambiente (Di3A), University of Catania, via Valdisavoia 5,1695123 Catania, Italy; vrizzo@unict.it (V.R.); cherubino.leonardi@unict.it (C.L.);17agata.mazzaglia@unict.it (A.M.); g.muratore@unict.it (G.M.); miriam.distefano@unict.it (M.D.);18francesco.giuffrida@unict.it (F.G.)19Dipartimento di Scienze Agrarie, Alimentari e Forestali (SAAF), University of Palermo, viale delle Scienze20Ed. 4, 90128 Palermo, Italy; leo.sabatino@unipa.it21Correspondence: rosario.mauro@unict.it; Tel.: +39-095-478331422 23Received: 5 February 2020; Accepted: 12 March 2020; Published: 16 March 202024 25


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27 28Abstract: The present experiment addressed the effects of two harvest stages, namely breaker (S1 )29and turning (S2 ), on compositional and sensory profile of grafted, greenhouse elongated tomatoes30cv. “Sir Elyan”. The rootstocks “He-Man”, “Interpro”, and “Armstrong” were used. The S1 fruits31showed a higher dry matter content, firmness and titratable acidity when compared to the S2 ones32(by 6%, 3%, and 15%, respectively). They showed, also, the highest L-ascorbic acid concentration33and antioxidant activity. Differently, soluble solid content, lycopene and β-carotene increased in the34S2 fruits (by 4%, 92%, and 26%, respectively). Excepting methyl salicylate, all the volatiles detected35peaked in the S2 fruits, which were scored by panelists as sweeter and more flavorful than the S1 ones.36Among the rootstock genotypes, “He-Man” promoted fruits firmness, carotenoids concentration and37antioxidant activity, irrespective of the harvest stage. “Interpro” enhanced the sensory attributes of38the S1 fruits in terms of bitterness, sourness, sweetness and flavor. Differently, sweetness, sourness,39and tomato flavor of the S2 fruits were promoted by “Armstrong”. The results highlight the influence40of rootstock genotype on the nutraceutical and sensorial profile of “Sir Elyan” fruits harvested at41early ripening stages.42Keywords: Solanum lycopersicum L.; fruit ripening; grafting; nutraceutical profile; volatile composition43 441. Introduction45In the Mediterranean Basin, tomato (Solanum lycopersicum L.) represents a resource of primary46economic and dietary importance [1]. Where quality is concerned, organoleptic traits and47health-promoting compounds of fresh vegetables are becoming increasingly important [2,3], and for48tomato these characteristics nowadays play a pivotal role in influencing consumers’ preferences. From49a nutraceutical viewpoint, tomato is a primary source of antioxidant in the human diet, with ascorbic50acid, lycopene, and β-carotene being among the most effective in protecting human health from several51degenerative diseases [4].52Tomato flavor results from the interaction among taste (deriving from sugars and organic acids)53and aroma [5]. Tomato aroma flows from over 400 volatile organic compounds (VOCs), although54only few of them are reputed of primary importance [5,6]. It has been demonstrated that these55traits involve the developmentally-regulated expression of many polygenic systems, making them56strongly influenced by environmental and agronomic factors [6]. Nowadays, greenhouse tomato57Agriculture 2020, 10, 82; doi:10.3390/agriculture1003008258 59www.mdpi.com/journal/agriculture60 61Agriculture 2020, 10, 8262 632 of 1764 65cultivations are strongly dependent on the adoption of grafting, in order to increase their yield and66resistance to biotic and/or abiotic stressors [7–9]. It has been reported that tomato quality variables67such as acidity, sugar, flavor, aroma, color, carotenoid content, and texture can be differently influenced68by the rootstock-scion combinations, also because of their interaction with external factors such as69climate conditions and cultural practices [10]. Tomato fruits are harvested at different ripening stages,70according to its destination. Fruit destined for the fresh market can be harvested from the mature-green71to fully ripe stage [11], according to the consumers’ demand and fruit typology.72In the paste decades, breeding has prioritized yield, shelf-life, and diseases resistance, which may73have contributed to compromise flavor characteristics in fresh market tomatoes. However, the lack of74flavor of retail tomatoes is partly due also to harvesting fruits before their full ripening is achieved. In75fact, the concentration of individual volatiles in tomato fruits depends by the ripening stage at harvest,76and fruit harvested before full ripening usually do not produce the characteristic volatiles associated to77high quality tomatoes [11]. However, the threshold concentrations detected by humans for various78aroma compounds range over many orders of magnitude. Sensory observations could be used to79confirm the contribution of these compounds to odor and aroma [12].80The elongated-type tomato, that represents a significant commercial niche in Italy, could be81consumed at breaker or turning stage. In a survey in Italy, the first ripening stages are preferred by the8220% of fresh tomato consumers [13].83The objective of this work was to investigate the composition and sensorial properties of tomato84harvested at breaker and turning stages of an elongated tomato cultivar, grafted onto three common85rootstocks in Mediterranean greenhouse cultivation.862. Materials and Methods872.1. Experimental Materials and Growth Conditions88The experiment was conducted in a 2400 m2 greenhouse located in Southwest Sicily (36◦ 50’ N,89E; 18 m a.s.l.). Tomato plants cv. “Sir Elyan” F1 , belonging to the medium-sized, elongated90type, were grafted onto 3 rootstocks, characterized by different ability to imprint vegetative vigor to91the scion: “He-Man” F1 (low-vigor) “Interpro” F1 (medium-vigor), and “Armstrong” F1 (high-vigor).92Transplanting was effected at the end of January adopting the following distances: 1.20 m (between93double rows), 0.80 m (between paired rows) and 0.70 m (within single rows) (1.43 plant m−2 ,942 stems plant−1 ). A typical fertilization program was applied, whereas drip irrigation was provided95when accumulated daily evaporation reached 25 mm. The crop was grown up to the half of July. A96two-way randomized blocks design with four replications was adopted, using 6.8 × 3.6 m experimental97plots, each containing 16 plants (net of borders).98 9914◦ 28’100 1012.2. Carpometric Determinations102On 10 May, 32 commercial tomato fruits from the third trusses per replicate were hand-harvested103at 2 different ripening stages. These were breaker stage (16 fruits per replicate), i.e., when the berries104started turning to red by their stylar end (rank 3 of the OECD Tomato Colour Gauge, hereafter S1 );105turning stage (16 fruits per replicate), i.e., when colour change regarded ~30 of the esocarp (rank1064 of OECD Tomato Colour Gauge, hereafter S2 ). Soon after harvest, fruits were transported in the107laboratory and processed for further analysis. Fruits fresh weight was determined, whereas the fruit108shape index was calculated as the ratio among longitudinal and transversal diameters. Fruit firmness109was determined through a Digital Texture Analyser mod. TA-XT2 (Stable Micro Systems, Godalming,110UK) and defined as the force (N) needed to impress a 2 mm fruit deformation along its equatorial axis.111Subsamples of collected fruits were kept in a thermo-ventilated oven at 70 ◦ C (Binder, Milan, Italy)112until constant weight was reached, in order to determine their dry matter content.113 114Agriculture 2020, 10, 82115 1163 of 17117 1182.3. Fruit Quality Determinations119Subsamples of harvested fruit were washed with demineralized water, dried with paper and120blended with a domestic food processor at room temperature. The resulting puree was centrifuged121and an aliquot of the supernatant was used to determine the soluble solids content (SSC) by using a122digital refractometer DBX-55A (Atago Co., Ltd., Tokyo, Japan) provided with an automatic temperature123compensation system. Titratable acidity (TA) was determined using 10 g aliquots of tomato fruits124poured in 50 mL of distilled water and titrated with 0.1N NaOH to an end-point of pH 8.1. TA was125expressed as g L−1 citric acid (CA). The SSC/TA ratio was also calculated. Lycopene and β-carotene126were extracted using the method described by Sharma and Le Maguer [14] and quantified by HPLC127(equipped with a C30 Acclaim column) according to Gregory et al. [15] and Subagio et al. [16].128Ascorbic acid was extracted and quantified by HPLC (with an Ultra AQ C18 column) according129to Nisperos-Carriedo et al. [17]. The antioxidant activity was determined using the free radical1302,2-diphenyl-1-picrylhydrazyl (DPPH) assay, according to Brand-Williams et al. [18].1312.4. Volatile Extraction and Analysis132Soon after harvest, fresh tomato sample were chopped and mixed. The aroma compounds133identification was performed using SPME coupled with GC/MS. The fiber was chosen according to134Beltran et al. [19], i.e., a 75 µm Carboxen/PDMS (CAR/PDMS) fiber (Supelco, Bellefonte, PA, USA).135Before use, the fiber was preconditioned in the GC injection port at 300 ◦ C for 1 h, then exposed for 1 h136to the headspace of a 25 mL septum-sealed glass vial containing 20 g aliquot of homogenized fresh137tomato. Each vial was previously immersed in a bath water at 60 ◦ C for 15 min. GC–MS analyses138were performed using an Agilent (Palo Alto, CA, USA) 6890 N GC equipped with a 30 cm length,1390.20 mm i.d., 0.20 µm film thickness, fused silica capillary column (SUPELCOWAX™ 10, Supelco).140During the analysis, the GC injection port temperature was 250 ◦ C, with a split ratio of 5:1. Helium141was used as the carrier gas at a flow rate of 1.1 mL min−1 . The column temperature was held at 40 ◦ C142for 5 min, then programmed to increase by 5 ◦ C min−1 to 220 ◦ C, which was held for 10 min. Mass143spectrometry conditions were as follows: ion source, 230 ◦ C; electron energy, 70 eV; multiplier voltage,1441247 V; GC/MS interface zone, 280 ◦ C; and a scan range of 35–350 mass units. Duplicate analyses were145performed for each sample. Identification of the compounds was carried out by comparison of the146analytes fragmentation patterns with the spectra libraries (NIST 98, US).1472.5. Sensory Analysis148The UNI EN ISO 13299:2016 [20] sensory profile method was used to measure any difference in149sensory characteristics of tomatoes. Twelve trained (ISO 8586:2012) [21] panelists (six females and six150males, 28–40 years old) with a broad expertise in vegetables were trained in 3 sessions, using both151commercial and experimental samples to familiarize with scales and procedures. The panelists, using a152discontinuous scale between 1 (absence of sensation) and 9 (extremely intense), evaluated the intensity153of the sixteen attributes selected on the basis of frequency (≥60%): 1 for appearance (freshness); 1 for154tactile hand feel (firmness); 3 for odor (herbaceous, tomato and off odors), 3 for flavor (herbaceous,155tomato and off-flavors); 4 for taste (salt, sour, sweet and bitter); 4 for rheological properties (crunchy,156juicy, mealy, peel thick) (Table 1). The evaluation sessions were conducted in the sensory laboratory157(UNI EN ISO 8589:2014) [22] of Di3A (University of Catania) from 11:00 a.m. to 12:00 a.m. in individual158booths illuminated with a white light. Tomato samples were served on plates, coded with three-digit159numbers and water was provided to panelists for rinsing between samples. The order presentation160was randomized among panelists and sessions. All data were acquired by a direct computerized161registration system (FIZZ Byosistemes. ver. 2.00 M, Couternon, France).162 163Agriculture 2020, 10, 82164 1654 of 17166 167Table 1. List of evaluated sensory attributes and their definitions.168Attribute169 170Description171 172Freshness173Firmness174Tomato odor175Herbaceous odor176 177Degree of freshness of the product by visual estimation178Strength required to compress a food between the moles179Characteristics odor of tomato perceived with the sense of smell180Characteristics odor of herbaceous perceived with the sense of smell181Unpleasant odor not characteristic of the product concerned, perceived through182the sense of smell183One of the four basic tastes caused by aqueous solutions of salt compounds184perceived on the tongue185One of the four basic tastes caused by aqueous solutions of acid compounds186perceived on the tongue187One of the four basic tastes caused by aqueous solutions of sweet compounds188perceived on the tongue189One of the four basic tastes caused by aqueous solutions of bitter compounds190perceived on the tongue191The sensation of muffled grinding of a foodstuff192The amount of liquid released from the samples during first and second chew193The amount of small particles perceived in the mouth when biting the sample194Resistance of the epicarp to removal195Characteristic flavor of tomato perceived by the sense of smell and mouth with196the swallowing197Characteristic flavor of herbaceous perceived by the sense of smell and mouth198with the swallowing199Unpleasant flavor not characteristic of the product concerned, perceived by the200sense of smell and mouth with the swallowing201 202Off-odor203Salt204Sour205Sweet206Bitter207Crunchy208Juicy209Mealy210Peel thick211Tomato flavor212Herbaceous flavor213Off-flavor214 2152.6. Statistical Procedures216Collected and calculated data attributable to ratio scales were firstly subjected to Shapiro–Wilk217and Levene’s test, in order to check for normal distribution and homoscedasticity, respectively, then218to a factorial “rootstock × ripening stage” (R × S) analysis of variance (ANOVA), according to the219experimental layout adopted in the greenhouse. Percentage data were Bliss’ transformed before the220ANOVA (untransformed data are reported and discussed), whereas multiple mean comparisons were221performed through Fisher’s protected LSD test (p = 0.05). Sensory data were subjected to a two-way222non-parametric ANOVA using Friedman’s test followed by the calculation of Kendall’s coefficient of223concordance, in order to check the independence of observations. Means separation was performed in224all pairwise comparisons by using the Mann-Whitney’s U-test, with an associated P-level calculated225according to the Bonferroni’s correction. A correlation analysis was also performed, in order to226define possible relationships among volatiles concentration and sensory scores. All calculations were227performed using Excel version 2016 (Microsoft Corporation, Redmond, WA, USA) and Minitab version22816.1.1 (Minitab Inc., State College, PA, USA).2293. Results2303.1. Carpometric Traits231Average fruit weight and fruit shape index were both affected by R × S interaction. Passing from S1232to S2 , the former variable significantly increased only in “Sir Elyan” grafted onto “Interpro” (+13.6%),233whereas shape index decreased only in “Sir Elyan” grafted onto “Armstrong” (−5.3%) (Table 2). Fruit234dry matter showed a similar trend in all the grafting combinations, decreasing from 7.4 (S1 ) to 7.0%235(S2 ), whereas fruit firmness proved to be higher in “Sir Elyan” grafted onto “He-Man” than onto the236other rootstocks, and in S1 than S2 stage (Table 2).237 238Agriculture 2020, 10, 82239 2405 of 17241 242Table 2. Carpometric traits of tomatoes “Sir Elyan” as affected by rootstock and ripening stage243(mean ± standard error). Different letters among factor means (bold numbers) indicate significance at244Fisher’s LSD test (p = 0.05). NS: not significant.245 246Average fruit247weight (g)248Shape index249(adimensional)250Fruit dry matter251(%)252Fruit firmness253(N)254 255Rootstock256 257Ripening Stage258 259Variable260 261S1262S2263Rootstock mean264S1265S2266Rootstock mean267S1268S2269Rootstock mean270S1271S2272Rootstock mean273 274“He-Man”275 276“Interpro”277 278“Armstrong”279 28088.1 ± 1.728183.7 ± 2.128285.9 ± 2.4 a2831.67 ± 0.022841.65 ± 0.012851.66 ± 0.02 a2867.3 ± 0.12877.2 ± 0.12887.2 ± 0.1 a28913.91 ± 0.3529013.64 ± 0.2829113.78 ± 0.32 a292 29379.5 ± 2.329490.3 ± 1.929584.9 ± 3.6 a2961.66 ± 0.022971.68 ± 0.012981.67 ± 0.02 a2997.5 ± 0.23006.9 ± 0.13017.2 ± 0.2 a30212.74 ± 0.4730312.23 ± 0.4630412.49 ± 0.49 b305 30678.7 ± 1.930782.3 ± 1.930880.5 ± 2.0 a3091.70 ± 0.023101.61 ± 0.013111.66 ± 0.03 a3127.3 ± 0.13136.9 ± 0.13147.1 ± 0.2 a31511.81 ± 0.4231611.77 ± 0.3131711.79 ± 0.47 b318 319Ripening320Stage Mean321 322LSDinteraction323(p = 0.05)324 32582.1 ± 3.0 a32685.4 ± 2.8 a327 3287.0329 3301.68 ± 0.02 a3311.65 ± 0.02 a332 3330.06334 3357.4 ± 0.2 a3367.0 ± 0.1 b337 338NS339 34012.82 ± 0.70 a34112.55 ± 0.56 b342 343NS344 3453.2. Taste Variables346Soluble solid content (SSC) proved to be significantly higher in S2 than in S1 fruits, and when347“Sir Elyan” was grafted onto “Interpro” (Table 3). Differently, a significant R × S interaction was348recorded for titratable acidity (TA) since, passing from S1 to S2 , it significantly decreased in “Sir Elyan”349grafted onto “Interpro” and “Armstrong” (−19.7% and −12.8%, respectively) and did not change using350“He-Man” as rootstock (Table 3). Both main factors significantly affected the SSC/TA ratio, as in S2351fruits it was 18.7% higher than the S1 ones, showing also a higher value in “Sir Elyan” grafted onto352“He-Man” (Table 3).353Table 3. Soluble solid content (SSC), titratable acidity (TA) and their ratio in tomatoes “Sir Elyan” as354affected by rootstock and ripening stage (mean ± standard error). Different letters among factor means355(bold numbers) indicate significance at Fisher’s LSD test (p = 0.05). NS: not significant.356Variable357 358SSC359(◦ Brix)360TA361(g CA L−1 )362SSC/TA363 364Rootstock365 366Ripening Stage367S1368S2369Rootstock mean370S1371S2372Rootstock mean373S1374S2375Rootstock mean376 377“He-Man”378 379“Interpro”380 381“Armstrong”382 3835.66 ± 0.093846.04 ± 0.083855.85 ± 0.15 b3862.88 ± 0.083872.73 ± 0.113882.81 ± 0.10 c3891.97 ± 0.013902.22 ± 0.033912.09 ± 0.08 a392 3936.06 ± 0.073946.32 ± 0.103956.19 ± 0.16 a3963.61 ± 0.053972.90 ± 0.093983.25 ± 0.22 a3991.68 ± 0.024002.18 ± 0.034011.92 ± 0.11 b402 4035.80 ± 0.054045.84 ± 0.044055.82 ± 0.03 b4063.20 ± 0.074072.79 ± 0.054083.00 ± 0.13 b4091.81 ± 0.024102.09 ± 0.044111.95 ± 0.09 b412 413Ripening414Stage Mean415 416LSDinteraction417(p = 0.05)418 4195.84 ± 0.19 b4206.07 ± 0.13 a421 422NS423 4243.23 ± 0.18 a4252.81 ± 0.10 b426 4270.30428 4291.82 ± 0.06 b4302.16 ± 0.06 a431 432NS433 4343.3. Fruit Nutraceutical Profile435As regards the L-ascorbic acid content, the S2 fruits showed a significant decrease when “Sir436Elyan” was grafted onto “He-Man” (−21.4%) while for the other rootstocks no statistical variations437among ripening stages were recorded (Table 4). On the average of grafting combinations, lycopene438content increased by 91.5% passing from S1 to S2 , whereas showed significantly lower values in “Sir439Elyan” grafted onto “Armstrong”, and the highest content in “Sir Elyan” grafted onto “He-Man”440(Table 4). The β-carotene content was affected by R × S interaction since, passing from S1 to S2 , the441higher increase was noticed in the grafting combination “Sir Elyan”/“Armstrong” than in the other442ones (Table 4). DPPH significantly decreased passing from S1 to S2 using “Interpro” and “Armstrong”443rootstocks (Table 4).444 445Agriculture 2020, 10, 82446 4476 of 17448 449Table 4. L—ascorbic acid, main carotenoids content and antioxidant activity in tomatoes “Sir Elyan” as450affected by rootstock and ripening stage (mean ± standard error). Different letters among factor means451(bold numbers) indicate significance at Fisher’s LSD test (p = 0.05). NS: not significant.452Variable453L – ascorbic acid454(µg g−1 FW)455Lycopene456(µg g−1 FW)457β – carotene458(µg g−1 FW)459DPPH460(µmol TEAC g−1461FW)462 463Rootstock464 465Ripening Stage466S1467S2468Rootstock mean469S1470S2471Rootstock mean472S1473S2474Rootstock mean475S1476S2477Rootstock mean478 479“He-Man”480 481“Interpro”482 483“Armstrong”484 485126 ± 448699 ± 3487113 ± 9 a4888.3 ± 0.348915.3 ± 0.549011.8 ± 2.3 a4919.5 ± 0.349211.4 ± 0.549310.5 ± 0.6 a4941.90 ± 0.084951.82 ± 0.064961.86 ± 0.12 a497 498103 ± 3499100 ± 5500102 ± 4 b5017.1 ± 0.350213.0 ± 0.650310.1 ± 1.6 b5046.6 ± 0.45058.5 ± 0.65067.6 ± 0.8 c5071.84 ± 0.065081.44 ± 0.045091.64 ± 0.16 b510 51197 ± 451291 ± 351394 ± 4 b5145.9 ± 0.251512.4 ± 0.35169.2 ± 1.9 c5177.9 ± 0.251810.1 ± 0.35199.0 ± 1.0 b5201.67 ± 0.065211.05 ± 0.045221.36 ± 0.2 c523 524Ripening525Stage Mean526 527LSDinteraction528(p = 0.05)529 530109 ± 8 a53197 ± 5 b532 53314534 5357.1 ± 0.6 b53613.6 ± 0.9 a537 538NS539 5408.0 ± 0.6 b54110.1 ± 0.9 a542 5431.6544 5451.80 ± 0.18 a5461.44 ± 0.13 b547 5480.13549 5503.4. Fruit Volatile Profile551Twelve volatile compounds, which were suggested to be key tomato aroma contributors [23],552were identified in our study, including 4 alcohols (3-methyl-1-butanol, 1-pentanol, 1-hexanol, and5533-henex-1-ol), 5 aldehydes (3-methylbutanal, hexanal, E-2-hexenal, E-2-heptenal and octanal), plus554the apocarotenoids β-ionone and 6-methyl-5-hepten-2-one and the ester methyl salicylate (Table 5).555Excepting methyl salicylate, the average concentration of all detected volatiles was higher in the S2556fruits (Table 5), but all these differences were rootstock-dependent. Among the alcohols volatiles,5573-methyl-1-butanol concentration increased in “Sir Elyan” grafted onto “Interpro” and “Armstrong”558(+2.3 and 24.2-fold, respectively, passing from S1 to S2 ) and decreased when grafted onto “He-Man”.559“He-Man” and “Armstrong” determined a higher rise in 1-pentanol (+2.6 and 1.5-fold, respectively)560than “Interpro”. “He-Man” caused also the highest rise in 1-hexanol (+1.6-fold) and in 3-hexen-1-ol561concentration (+1.2-fold) (Figure 1). Considering the aldehydes volatiles, “Armstrong” determined, in562the S2 fruits, the strongest increase in 3-methylbutanal (+1.1-fold), “Interpro” in hexanal (+16.7-fold)563and “He-Man” proved the most marked increase in E-2-hexenal (+4.3-fold), E-2-heptenal (+37.7-fold)564and octanal (+5.3-fold) (Figures 2 and 3). Octanal significantly decreased in fruit harvested at S2565when grafted onto “Interpro” (Figure 3). Among the remaining compounds, β-ionone displayed566the highest increase in the S2 fruits in “Sir Elyan” grafted onto “He-Man” (+2.1-fold), whereas5676-methyl-5-hepten-2-one proved the highest rise on “He-Man” and “Interpro” (+116.5 and 49-fold,568respectively) (Figure 3). Methyl salicylate concentration peaked in the S1 fruits that, compared to569the S2 ones, proved the highest concentration in the grafting combination “Sir Elyan”/“Armstrong”570(+0.4-fold) (Figure 3).571 572Agriculture 2020, 10, 82573 5747 of 17575 576Table 5. Peak area (×106 ) of volatile organic compounds detected in tomatoes “Sir Elyan”, as affected by rootstock and ripening stage (main effects) (mean ± standard577error). Different letters among factor’s means indicate significance at Fisher’s LSD test (p = 0.05).578Rootstock579 580Compound581Alcohols5823-methyl-1-butanol5831-pentanol5841-hexanol5853-hexen-1-ol586Aldehydes5873-methylbutanal588Hexanal589E-2-hexenal590E-2-heptenal591Octanal592Others593β-ionone5946-methyl-5-hepten-2-one595Methyl salicylate596 597Ripening Stage598 599Overall Mean600 601Odor Description602[24,25]603 604“He-Man”605 606“Interpro”607 608“Armstrong”609 610S1611 612S2613 614118.6 ± 24.5 c61524.8 ± 6.3 b61629.9 ± 5.9 b61722.3 ± 4.8 b618 619336.1 ± 81.1 b62014.2 ± 2.9 c6218.0 ± 1.0 c62249.7 ± 2.8 a623 624559.7 ± 118.5 a62526.6 ± 5.2 a62636.3 ± 2.9 a62715.9 ± 2.7 c628 629124.0 ± 25.4 b63012.0 ± 1.5 b63117.7 ± 4.4 b63224.3 ± 6.9 b633 634552.3 ± 80.3 a63531.8 ± 4.2 a63631.8 ± 6.7 a63734.3 ± 6.1 a638 639338.1 ± 154.064021.9 ± 5.164124.7 ± 6.264229.3 ± 6.6643 644Whiskey, malt, burnt645Green646Flower, green647Herbal, green648 6499.9 ± 1.5 c650635.3 ± 69.0 a651600.8 ± 84.2 a652106.6 ± 15.5 a653395.0 ± 49.4 a654 65519.6 ± 2.7 b656527.2 ± 91.5 b657227.7 ± 55.2 b65823.3 ± 2.9 c659236.0 ± 60.0 b660 66150.0 ± 8.1 a662556.3 ± 47.9 ab663111.0 ± 19.9 c66489.5 ± 5.2 b665131.2 ± 19.2 c666 66720.6 ± 4.2 b668379.5 ± 74.2 b669144.2 ± 24.2 b67037.3 ± 13.5 b671196.3 ± 53.3 b672 67332.4 ± 5.0 a674766.4 ± 74.3 a675482.1 ± 64.1 a676108.9 ± 23.6 a677311.8 ± 64.4 a678 67926.5 ± 8.4680572.9 ± 119.6681331.2 ± 134.168273.1 ± 29.1683254.1 ± 89.9684 685Malt686Grass, tallow, fat687Green, apple688Soap, fat, almond689Soap, lemon, green, fat690 69122.6 ± 5.3 a692377.0 ± 86.5 a69331.4 ± 3.7 b694 69519.1 ± 3.9 b69690.9 ± 39.2 c69715.6 ± 1.4 c698 69917.6 ± 4.2 c700145.4 ± 28.8 b70172.2 ± 5.1 a702 7039.9 ± 0.6 b70430.6 ± 15.8 b70548.8 ± 12.2 a706 70729.6 ± 1.7 a708378.3 ± 94.0 a70932.7 ± 8.6 b710 71119.7 ± 4.3712204.4 ± 107.571339.7 ± 10.7714 715Ripe tomato716Sweet, nutty, raspberry717Wintergreen718 719Agriculture 2020, 10, x FOR PEER REVIEW720Agriculture 2020, 10, 82721 7222 of 237238 of 17724 725A726 727B728 729C730 731D732 733Figure 1. Peak area (×106 ) of 3-methyl-1-butanol (A), 1-pentanol (B), 1-hexanol (C), and 3-hexen-1-ol (D) in tomatoes “Sir Elyan” as affected by ‘rootstock × ripening734interaction.735bars: S1 . Dark bars:736S21-pentanol737.738Figurestage’7391. Peak740area (×106Light741) of 3-methyl-1-butanol742(A),743(B), 1-hexanol (C), and 3-hexen-1-ol (D) in tomatoes “Sir Elyan” as affected by ‘rootstock x ripening stage’744 745interaction. Light bars: S1. Dark bars: S2.746 747Agriculture 2020, 10, x FOR PEER REVIEW748Agriculture 2020, 10, 82749 7503 of 237519 of 17752 753A754 755B756 757C758 759D760 761Figure 2. Peak area (×106 ) of 3-methylbutanal (A), hexanal (B), E-2-hexenal (C), and E-2-heptenal (D) in tomatoes “Sir Elyan” as affected by ‘rootstock × ripening762Figure 2. Peak area (×106) of 3-methylbutanal (A), hexanal (B), E-2-hexenal (C), and E-2-heptenal (D) in tomatoes “Sir Elyan” as affected by ‘rootstock x ripening763stage’ interaction. Light bars: S1 . Dark bars: S2 .764stage’ interaction. Light bars: S1. Dark bars: S2.765 7661767 768Agriculture 2020, 10, 82769Agriculture 2020, 10, x FOR PEER REVIEW770 77110 of 17772 7731 of 23774 775A776 777B778 779C780 781D782 783Figure 3. Peak area (×106 ) of octanal (A), β-ionone (B), 6-methyl-5-hepten-2-one (C), and methyl salicylate (D) in tomatoes “Sir Elyan” as affected by ‘rootstock × ripening784Figure 3. Peak area (×106) of octanal (A), β-ionone (B), 6-methyl-5-hepten-2-one (C), and methyl salicylate (D) in tomatoes “Sir Elyan” as affected by ‘rootstock x785stage’ interaction. Light bars: S1 . Dark bars: S2 .786ripening stage’ interaction. Light bars: S1. Dark bars: S2.787 788Agriculture 2020, 10, 82789 79011 of 17791 7923.5. Sensory Analysis793Tomato samples significantly differed for 11 out the 16 sensory attributes (Table 6). In particular, the794bitter perception decreased in the S2 fruits only in the grafting combinations “Sir Elyan”/“Armstrong”795(−21.3%); the crunchy decreased in the S2 fruits of “Sir Elyan” grafted onto “He-Man” and “Interpro”796with a trend particularly evident in the latter (−32.6%). Both firmness and freshness in general dropped797in the S2 fruits, particularly in “Sir Elyan” grafted onto “Interpro” (−19.2% and −6.0%, respectively).798Similarly, herbaceous flavor and odor showed a general decrease in the S2 fruits with the strongest799reduction in tomato grafted onto “He-Man” (−15.6% and −18.2%, respectively). Overall, salt and800sour perceptions were higher in the S1 fruits as compared to the S2 ones, with the highest differences801recorded in “Sir Elyan” grafted onto “Interpro” (+34.4%) and “Armstrong” (+13.8%), respectively.802Differently, the sweet perception showed a different trend passing from S1 to S2 stage in the rootstock803treatments. This sensory attribute increased using “He-Man” (+13.1%) and ‘Armstrong (+12.3%)804rootstocks and decreased in “Interpro”. Tomato flavor and odor generally peaked in the S2 fruits,805with a gradient recorded in the grafting combinations “Sir Elyan”/“Armstrong” (+18.8%) and “Sir806Elyan”/“Interpro” (+24.5%).807Table 6. Mean scores of 16 sensory attributes of tomatoes “Sir Elyan” differing for rootstock and808ripening stage. Different letters within each row indicate significance at Mann–Whitney’s U-test809(p = 0.04885).810Attribute811 812“He-Man”813 814“Interpro”815 816“Armstrong”817 818“He-Man”819 820S1821Freshness822Firmness823Tomato odor824Herbaceous odor825Off-odor826Salt827Sour828Sweet829Bitter830Crunchy831Juicy832Mealy833Peel thick834Tomato flavor835Herbaceous flavor836Off-flavor837 8387.36 ab8397.54 ab8405.18 d8415.55 a8421.54 a8434.54 b8443.45 ab8454.90 c8461.81 b8476.18 a8487.09 a8493.09 a8505.63 a8516.00 c8525.27 a8531.18 a854 8557.54 a8568.09 a8575.54 cd8585.63 a8591.90 a8605.00 a8613.18 b8625.36 a8632.09 b8647.00 a8657.45 a8662.81 a8675.81 a8686.27 b8695.18 a8701.63 a871 872“Interpro”873 874“Armstrong”875 876S28777.63 a8787.90 a8795.81 c8805.36 a8811.36 a8824.90 a8833.72 a8844.45 d8852.54 a8866.36 ab8877.27 a8883.18 a8895.90 a8905.81 d8915.18 a8921.18 a893 8947.00 b8956.36 c8966.27 b8974.54 b8981.54 a8993.72 c9003.36 b9015.54 a9022.09 b9036.09 b9047.27 a9052.81 a9065.90 a9076.81 a9084.45 b9091.36 a910 9117.09 b9126.54 bc9136.90 a9144.81 b9151.09 a9163.72 c9173.00 c9185.19 b9192.09 b9204.72 c9217.54 a9223.09 a9235.36 a9246.27 b9254.45 b9261.90 a927 9287.27 b9297.00 b9306.54 b9314.54 b9321.72 a9334.90 a9343.27 b9355.00 c9362.00 b9375.54 bc9387.18 a9392.63 a9405.63 a9416.90 a9424.63 b9431.27 a944 9453.6. Correlation among Volatiles Concentration and Sensory Scores946Globally, 132 correlations were analyzed, of which 60 (45% of total) showed significance, revealing94737 negative and 23 positive relationships (Table 7). In the case of the alcohol volatiles, 17 out of 44948correlations (39% of total) were significant, whereas they were 26 out of 55 (47%) for the aldehydes949and 22 out 33 (67%) for the remaining volatiles. Among the negative correlations, the lowest r-values950were recorded among β-ionone concentration, herbaceous flavor (−0.961 ***), firmness (−0.946 ***) and951herbaceous odor (−0.932 ***), followed by that between hexanal concentration and crunchy (−0.929 ***)952(Table 7). Differently, the strongest relationship in the data frame of positive correlations was found953between 6-methyl-5-hepten-2-one concentration and tomato flavor (0.873 ***), β-ionone and tomato954flavor (0.834 ***), methyl salicylate and sour (0.813 ***) and among β-ionone and sweet (0.798 ***)955(Table 7).956 957Agriculture 2020, 10, 82958 95912 of 17960 961Table 7. Pearson’s product-moment correlation coefficients (r) among volatiles concentration and sensory attributes. *, ** and *** indicate significance at p ≤ 0.05, 0.01962and 0.001, respectively. NS: not significant.963Attribute964Bitter965Crunchy966Firmness967Freshness968Herbaceous969flavor970Herbaceous odor971Salt972Sour973Sweet974Tomato flavor975Tomato odor976 9773-methyl1-butanol978 9791-pentanol980 9811-hexanol982 9833-hexen-1-ol984 9853-methylbutanal986 987Hexanal988 989E-2-hexenal E-2-heptenal Octanal990 991β-ionone992 9936-methyl-5hepten-2-one994 995Methyl996Salicylate997 998NS999−0.583 *1000NS1001NS1002 1003NS1004NS1005−0.716 ***1006−0.644 **1007 1008NS1009NS1010NS1011NS1012 1013NS1014NS1015NS1016NS1017 1018NS1019NS1020NS1021NS1022 1023NS1024−0.929 ***1025−0.788 ***1026−0.709 ***1027 1028NS1029NS1030−0.695 **1031−0.742 ***1032 1033−0.478 *1034 1035−0.760 ***1036 1037NS1038 1039NS1040 1041NS1042 1043−0.663 **1044 1045−0.556 *1046NS1047−0.484 *1048NS10490.522 *10500.586 *1051 1052−0.916 ***1053NS1054NS10550.470 *10560.875 ***10570.605 **1058 1059−0.683 **1060NS1061NS1062NS10630.558 *1064NS1065 1066NS1067NS1068−0.854 ***10690.656 **1070NS1071NS1072 1073NS10740.511 *1075NS1076NS1077NS1078NS1079 1080−0.627 **1081−0.706 **1082NS1083NS1084NS10850.659 **1086 1087NS1088NS1089−0.524 *1090NS1091 1092NS1093NS1094NS1095NS1096 1097NS1098−0.623 **1099−0.946 ***1100−0.922 ***1101 1102NS1103NS1104−0.768 ***1105−0.747 ***1106 11070.579 *1108NS1109NS11100.551 *1111 1112−0.600 **1113 1114−0.539 *1115 1116NS1117 1118−0.961 ***1119 1120−0.727 ***1121 1122NS1123 1124−0.553 *1125−0.724 ***1126NS11270.752 ***11280.607 **1129NS1130 1131−0.672 **1132NS1133NS1134NS11350.656 **1136NS1137 1138NS1139NS1140NS11410.628 **11420.580 *1143NS1144 1145−0.932 ***1146−0.723 ***1147−0.490 *11480.798 ***11490.834 ***11500.788 ***1151 1152−0.749 ***1153−0.668 **1154NS11550.662 **11560.703 **1157NS1158 1159NS11600.568 *11610.813 ***1162−0.827 ***1163NS1164NS1165 1166Agriculture 2020, 10, 821167 116813 of 171169 11704. Discussion1171Under the specific conditions of our experiment, the S1 fruits showed a higher dry matter content,1172consistent with their higher firmness, this last feature indicating less advanced metabolic processes1173when the reference ripening stage was achieved. Indeed, the decline in fruit firmness coincides with1174the up-regulation of several cell wall degrading enzymes, as well as with the dissolution of the middle1175lamella, leading to the reduction of the intercellular adhesion and cell wall depolymerization [26]. The1176grafting combination “Sir Elyan”/“He-Man” yielded the fruits with the highest firmness in both harvest1177stages, indicating the possibility to influence this trait by selecting the most suitable rootstock. This is1178an important commercial modification brought by grafting, since textural properties are implicated in1179fruits’ shelf life and transportability, as well as on the perception of their flavor profile [10].1180Soluble sugars (mainly glucose, fructose and sucrose) and organic acids (mainly citric and malic)1181are primary compounds of tomato fruits, whose amount are commonly measured through the soluble1182solid content (SSC) and titratable acidity (TA), respectively. From a sensorial viewpoint, their measure1183is linked to the perceived sweetness (SCC) and sourness (TA) of tomatoes, whereas the SCC/TA ratio1184describes the overall balance among them in the perceived taste [27]. All these variables are reputed1185primary contributors to the perceived flavor of tomato fruits [27]. In our experiment, the S1 fruits were1186characterized by a decreased SSC and an increased TA, overall indicating their less sweet, more acidic1187taste. So our results confirm the lower sugar content characterizing tomato fruits harvested at earlier1188ripening stages [28]. Moreover, our results agree with previous reports of a TA increase up to breaker1189stage, and its subsequent decline with further ripening [29].1190It is generally accepted that vigorous rootstocks show a higher sink strength, competing with the1191fruits for photosynthates accumulation [30]. Accordingly, we recorded a higher SCC/TA ratio when1192“Sir Elyan” was grafted onto “He-Man” (i.e., the least vigorous rootstock) than onto “Armstrong” (the1193most vigorous one), indicating a modified tendency of the fruits to accumulate sugars on the basis of1194the rootstock vigor.1195When compared to other fruits, tomato shows only moderate ascorbic acid (AsA) content, but its1196dietary importance implies that even small variations in this micronutrient can have relevant effects1197for consumers [31]. The ascorbic acid concentration we recorded showed a tendency to peak in S11198fruits. This is consistent with previous findings about the higher AsA biosynthetic capacity of younger1199fruits (up to 1.4-fold higher), probably to support their higher rates of cell division and expansion [31].1200Indeed, it has been reported that AsA plays an important role in plants, related to cell division and cell

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