Light quantity and quality supplies sharply affect growth, morphological, physiological and quality traits of basil

Industrial Crops & Products 122 (2018) 277–289

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Light quantity and quality supplies sharply affect growth, morphological, physiological and quality traits of basil

T

⁎

Fabio Stagnaria, Carla Di Mattiaa, Angelica Galienia,b, , Veronica Santarellia, Sara D'Egidioa, Giancarlo Pagnania, Michele Pisantea a b

Faculty of Bioscience and Technology for Agriculture Food and Environment, University of Teramo, Italy Council for Agricultural Research and Economics – Research Centre for Vegetable and Ornamental Crops, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Shading Light spectra manipulation Colored cover films Morphological traits Single phenolic acids Pigment content

Plants sharply adapt their growth and physiology to light availability. This study aimed at evaluating the effect of light quantity and quality manipulation on growth, morphological traits, pigment and secondary metabolites content in basil as well as comprehending the mechanisms which regulate such responses. Two experiments were carried out under greenhouse in 2014 (spring transplanting, Spr_Tr) and 2015 (summer transplanting, Sum_Tr). On a complete randomized block design, plants of basil were exposed to three modifications of the transmitted solar radiation with colored plastic films: yellow (YF), green (GF) and blue films (BF), plus a control (Control). Leaf pairs, axillary shoots, total fresh and dry biomass, specific leaf area, soil-plant analysis development, reflectance indices (Normalized Different Vegetation Index670, NDVI670, and Optimized Soil-Adjusted Vegetation Index, OSAVI), total chlorophyll, chlorophyll a, chlorophyll b, carotenoids, single and total polyphenol content and radical scavenging activity were recorded and examined. Shading induced stem elongation, a greater leaf area expansion and a lower leaf thickness; moreover, shaded plants increased chlorophyll accumulation (on average +29.4% and +21.6% during Spr_Tr and Sum_Tr, respectively). YF treatment allowed always the highest biomass accumulation (averaged over crop cycle: 2.1 and 3.4 g plant−1 during Spr_Tr and Sum_Tr, respectively). OSAVI and NDVI670 seem the more suitable indicators for chlorophyll accumulation. Light manipulation influenced specific phenolic compounds concentration. The application of colored films lowered rosmarinic and caftaric acids (by 29.8% and 33.2%, respectively, averaged over treatments and crop cycle). Antiradical activity was linearly correlated only with caffeic acid. Light manipulation represents a promising tool for the manipulation of basil morphological, physiological and quality traits.

1. Introduction Plants constantly modify their physiology and morphology to adapt to different environmental conditions. Among the environmental factors, which strongly influence plant growth and development, both quantity and quality of light transmitted to canopy play a major role (Shahak et al., 2004). Indeed, light represents both the primary source

of energy and the most important regulatory factor in plant's cycle: i.e. seed germination, seedling establishment, transition to flowering and morphogenesis (i.e. stem elongation) (Folta and Carvalho, 2015; Galvão and Fankhauser, 2015). Plants respond to light modified environments with physiological (photosynthetic rate, nutrient uptake) and biochemical (pigment and carbohydrate content) adaptations (Chang et al., 2008), which in turn

Abbreviations: ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); ANOVA, analysis of variance; BF, blue film; Car, carotenoids; Chl, chlorophyll; Chla, chlorophyll a; Chlb, chlorophyll b; CIred-edge, chlorophyll index red edge; CRI700, carotenoid concentration index700; DAT, days after transplanting; DMF, N,Ndimethylformamide; DW, total dry weight; FW, total fresh weight; GAE, gallic acid equivalents; GDD, growing degree days; GF, green film; GNDVI, green normalized difference vegetation index; HCl, hydrochloric acid; HPLC, high performance liquid chromatography; I%, percentage of inhibition; LA, leaf area; LED, light-emitting diode; LSD, least significant difference; MCARI, modified chlorophyll absorption ratio index; NDVI670, normalized difference vegetation Index670; NIR, near-infrared; NumAS, axillary shoots; NumLP, leaf pairs on the main stem; OSAVI, optimized soil-adjusted vegetation index; PAR, photosynthetically active radiation; SED, standard error of the difference; SLA, specific leaf area; SPAD, soil-plant analysis development; Spr_Tr, spring transplanting; Sum_Tr, summer transplanting; TEAC, trolox equivalent antioxidant capacity; TPC, total polyphenol content; UV, ultraviolet; UV/vis, ultraviolet/visible; YF, yellow film ⁎ Corresponding author at: Council for Agricultural Research and Economics – Research Centre for Vegetable and Ornamental Crops – Via Salaria, 1, Monsampolo del Tronto (AP), Italy. E-mail address: [email protected] (A. Galieni). https://doi.org/10.1016/j.indcrop.2018.05.073 Received 18 December 2017; Received in revised form 17 May 2018; Accepted 29 May 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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hypotheses that light quantity and quality manipulation – with colored cover films application (i.e. yellow, green and blue cover films) – on basil plants, should significantly affect: (i) growth and morphological traits (i.e. leaf biomass accumulation and expansion), (ii) pigment content, (iii) secondary metabolites content and activity (i.e. total polyphenols, phenolic compounds as well as antiradical activity). The main assumption of this research activity was that light sharply affects plant responses, consequently some agro-technologies can be conveyed to meet the consumers demand for edible herbs with high nutritional values.

are reflected into modification of plant growth, as well as into alterations of morphological and anatomical traits (Peralta et al., 2002). Low irradiance leads to more biomass allocated in leaves at the expense of roots optimizing leaf area per unit leaf biomass as well as maximizing light interception (Valladares and Niinemets, 2008). Shaded plants, indeed, have the thickness of mesophyll layer reduced (Terashima et al., 2005) and pigment density per unit leaf area increased (Xu et al., 2009). In the last decades, studies on light manipulation have become more interesting due to the relationship between light and physiology of secondary metabolism under different light spectra and/or light intensities (Bantis et al., 2016). As already known in many horticultural and herbal crops, light quantity and quality plays an important role in the synthesis of many antioxidants such as phenolic acids, carotenoids, flavonoids, anthocyanins, and α-tocopherol (Stagnari et al., 2014; Bantis et al., 2016), mainly due to phytomorphogenic responses of phytochromes (Henschel et al., 2017). Light manipulation is commonly applied using narrow-bandwidth light, such as that produced by light-emitting diode (LED)-based light sources (Samuolienė et al., 2016), coloured shade nets (Ilić and Fallik, 2017) and different coloured plastic covers or photoselective plastic films (Stagnari et al., 2014; Henschel et al., 2017). In particular, the use of photoselective cover materials (i.e. films or nets) could be a suitable agro-technological alternative to improve the quality of the edible products, of both horticultural and herbal crops. Beside plant protection from high rainfall, hail and frost (mainly films) as well as reduced physiological disorders and flower abortion (mainly nets), other important biochemical modifications could be obtained with the application of cover materials. Black and yellow nets increased the total antioxidant activity at harvest in oregano, while pearl nets favoured flavonoid (quercetin) accumulation in oregano, marjoram and coriander (Buthelezi et al., 2016). Blue cover film enhanced the levels of anthocyanins and ascorbic acid in fruits of strawberry, probably due to the antioxidant capacity of these compounds which act as an ultraviolet (UV) light filter (Henschel et al., 2017); conversely, UV black plastic films significantly lowered the total phenols and flavonoid glycosides contents in red leaf lettuce (García-Macías et al., 2007). On the other hand, Liu et al. (2015) observed higher biomass and camptothecin yield in Camptotheca acuminate, grown under red plastics. Sweet basil (Ocimum basilicum L.) is an aromatic herb belonging to Lamiaceae family, which is extensively used fresh or dried to add a distinctive aroma and flavor to food, due to the strong content of essential oils; it contains high concentrations of phenolic compounds that contribute to its strong antioxidant capacity (Kwee and Niemeyer, 2011; Bufalo et al., 2015). Among these, rosmarinic acid represents the most prevalent basil’s phenolic compound and it is associated with its functional properties; other important phenolic compounds are represented by caffeic acid derivatives, such as chicoric acid (Kwee and Niemeyer, 2011). To date, in basil only few investigations on the effects of light manipulation on morphological and physiological traits, as well as on some biochemical compounds, have been carried out. Growth and yield performances of basil plants were investigated under both artificial LED or/and fluorescent lamps (Frąszczak et al., 2014; Bantis et al., 2016) and photoselective nets (Shahak et al., 2008). Regarding quality traits, the content of total volatile oils was strongly reduced under heavy shaded conditions (Chang et al., 2008), while the setting of specific spectra has been demonstrated to significantly influence phenolic content as well as antioxidant activity. According to Bantis et al. (2016), LED applications with high blue light portion induce phenolic compounds accumulation as well as red light has a preeminent role in the regulation of phenolic acids biosynthesis (Taulavuori et al., 2017). Irradiation (fluorescent lamps) with red and white rather than blue light, seems to favor higher rosmarinic acid accumulation (Shiga et al., 2009). On these basis, the main objective of this study was to test the

2. Materials and methods 2.1. Plant material and growing conditions Experiments were carried out at the greenhouse of the Agronomy and Crop Sciences Research and Education Center, University of Teramo (altitude 15 m above sea level; 42° 42′ N,13° 54' 10′ E) during two different crop growing seasons, from 4 April to 29 May 2014 (early spring transplanting, Spr_Tr) and from 28 May to 8 July 2015 (later spring transplanting, named as summer transplanting, Sum_Tr). The greenhouse was covered with a single layer of ethylene-vinyl acetate film (PATILUX) provided by P.A.T.I. S.p.A. (San Zenone degli Ezzelini, TV, Italy) and characterized by a natural ventilation system. Seeds of sweet basil (Ocimum basilicum L. cv. Emily, Enza Zaden Italia S.r.l., Tarquinia, VT, Italy) were sown in a nursery substrate and maintained in a growth chamber until transplanting, which occurred 20 and 16 days after sowing in Spr_Tr and Sum_Tr, respectively. Uniformly sized seedlings (two-leaves stage) were transplanted into 9 cm side plastic pots, at a density of 1 plant per pot. Pots were filled with a mixture of peat-based compost (Terraplant® 2, COMPO Italia S.r.l., Cesano Maderno, MB, Italy), potting soil (HOCHMOOR HORTUS ESTIVO, Terflor S.r.l., Capriolo, BS, Italy), vermiculite and perlite at the ratio of 1.5:2:1:1 (v/v). The growth substrate was saturated with tap water before transplanting of seedlings and each pot was supplemented with 50 mL of fertilizer solution (Cifoumic 10-10-10, Cifo S.r.l., Bologna, BO, Italy) five days after transplanting (DAT). No insecticide or fungicide treatments were performed. Starting from transplanting, the greenhouse’s environmental conditions were constantly monitored with sensors of temperature and humidity connected to a data logger system (EM50 Data Collection System, Decagon Devices Inc., Pullman, WA, USA) (Fig. 1). 2.2. Treatments and experimental design The experiments were arranged as a completely randomized block design with two replicates. Experimental treatments consisted on three modifications of the transmitted solar radiation achieved with the use of three different colored plastic films: a yellow film (named as YF), a green film (GF) and a blue film (BF); an uncovered treatment was included as control (Control). The colored films used in this study are actually made to be used for other purposes than the agricultural sector, but have been adapted to cover the basil plants through the support of a hand-made rigid and removable structure (1.5 m × 1.5 m) placed above the vegetation. The structures were covered from the top and sides to ∼1 cm above the bottom of the pots to allow air circulation and secure proper temperature and humidity; light was not filtered from below. In particular, the colored films were purchased from a local store and provided by RiPlast S.r.l. (Pogliano Milanese, MI, Italy); yellow and blue films were 90 μM thick scratched polypropylene films, while green was a 80 μM thick polyvinyl chloride frosted film; thickness was measured by a thickness gauge (Vogel S.r.l., Leno, BS, Italy). The Fig. 2 shows a picture detail of the three films. The colored films were applied starting from transplanting and each treatment consisted of 98 pots, with 49 pots representing one experimental unit; pots were south-north (S-N) oriented. 278

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Reflectance under colored films was measured with the HandHeld 2 Pro Portable Spectroradiometer (FieldSpec, ADS Inc., Boulder, CO, USA), which measures radiation in the visible and near-infrared (NIR) wavelengths from 325 to 1075 nm ( ± 2 nm), using a reflective surface characterized by a certificated reflectivity of 99%. The instrument was placed to a distance of 9 cm from the reflective surface and measurements were taken, after calibration, with (covered treatments) and without (Control) the rigid structures that supported the colored film. In this way it was possible to have information about the absorbed radiation by the plastic films and of the radiation transmitted below the plastic films, in order to compare the treatments with the Control and between each other. Measurements were taken at noontime and within a few minutes of sunny days during the middle phases of the crop cycles (Fig. 3). 2.3. Growth analysis and morphological traits In both Spr_Tr and Sum_Tr, three plants per experimental unit (each plot of 49 plants) were sampled at almost regular intervals until the final harvest (visible inflorescence on the vegetative apex). Indications of sampling data, in terms of both DAT and thermal time after transplanting (growing degree days, GDD), as well as of plants phenology (number of leaf pairs on the main stem and number of axillary shoots), are reported in Table 1. Growing degree days (°C) were calculated as the accumulation of mean air temperature, of the uncovered environment, exceeding the base temperature of 6.11 °C (Fallahi et al., 2015). The leaf pairs on the main stem (NumLP) were counted considering the effectively formed leaf (i.e. of a length of 1.5 cm) and the axillary shoots (NumAS) were considered independently from the length they reached. Sampled plants were separated into leaves and stems for organs and total fresh (FW) and dry weights (DW) determinations, after drying in an oven at 80 °C, until constant weight; total aerial biomass was then calculated as the sum of leaves and stems DW. Before drying, shoot length (cm) was measured and leaf area (LA, cm2) was recorded after acquiring scanned leaves’ images with an image analysis software (ImageJ, National Institutes of Health, Bethesda, MD, USA). The specific leaf area (SLA, cm2 g−1) was then calculated.

Fig. 1. Patterns of air temperature (T, °C; solid line chart) and relative air humidity (RH, %; dashed line chart) as recorded under greenhouse during the two basil cycles, starting from transplanting: (A) spring transplanting 2014, Spr_Tr, and (B) summer transplanting 2015, Sum_Tr. Each point represents the daily mean of 24 h collected values.

Colored films were characterized for their optical properties. The photosynthetically active radiation (PAR, range 400–700 nm) transmitted to canopy was measured with PAR photon flux sensors (Decagon Devices, USA) placed above the vegetation and connected to a data logger (EM50 data collection system, Decagon Devices, USA). Starting from transplanting and for each experimental treatment, the measurements were constantly recorded (every 10 min) during the crop growing cycles. The percentages of shading with respect to Control, averaged over growing cycles, were reported in Fig. 3 (box). In accordance with Shahak et al. (2004), the amount of scattered light in the PAR region was measured at noontime of clear days using an opaque disc, held to 30–40 cm above the PAR sensor. The percentage of scattering was then calculated as the ratio between not-direct light to total light (box in Fig. 3).

2.4. Physiological traits The canopy reflectance was measured with a HandHeld 2 Pro Portable FieldSpec Spectroradiometer (ADS Inc., Boulder, CO, USA) in eight plants – without cover – per treatment (four from each plot of 49 plants). Measurements were carried out starting from 150 and 210 GDD in Spr_Tr and Sum_Tr, respectively, for a total of six sampling dates during the whole crop growing cycles (matching with the growth analysis determinations). To minimize the effects of the sun’s position, the reflectance measurements were taken within 1 h, near solar noon. Using the reflectance data, some important vegetation indices – Modified Chlorophyll Absorption Ratio Index (MCARI), the Normalized Difference Vegetation Index670 (NDVI670), the Optimized Soil-Adjusted Vegetation Index (OSAVI) and the Green Normalized Difference

Fig. 2. Details of colored films. From left to right: yellow film (YF), blue film (BF) and green film (GF). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 279

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Fig. 3. Reflectance as recorded for the colored films (yellow film, YF; green film, GF; blue film, BF) and Control (greenhouse) using a reflective surface characterized by a 99% certificated reflectivity. In box: shading percentage (%) with respect to Control (greenhouse conditions) in the photosyntetically active radiation (PAR) range; scattering percentage (%) with respect to Control (greenhouse conditions) in the photosyntetically active radiation (PAR) range. All these characteristics are obtained as a mean of values measured and calculated during the two different transplanting times (spring transplanting, Spr_Tr, and summer transplanting, Sum_Tr). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1 Sampling details of basil plants grown under different colored plastic films (yellow film, YF, green film, GF and blue film, BF) plus an uncovered Control, during the whole crop cycles (spring transplanting – Spr_Tr – 2014 and summer transplanting – Sum_Tr – 2015). For each sampling date and growing condition (i.e. treatments) were reported: the time expressed in terms of days after transplanting (DAT) and the thermal time, corresponding to cumulative average daily air temperature exceeding 6.11 °C starting from transplanting (growing degree days, GDD, °C), and the phenological stage indicated as number of leaf pairs on the main stem (NumLP) and number of axillary shoots (NumAS). Sampling N°

I Control YF GF BF II Control YF GF BF III Control YF GF BF IV Control YF GF BF V Control YF GF BF VI Control YF GF BF a b c d

Spr_Tr 2014

Sum_Tr 2015

DATa

GDDb

14

150

20

26

29

32

35

NumLPc

NumASd

2.8 3.0 3.0 3.0

2.8 2.0 4.2 2.8

3.5 4.0 4.7 4.0

5.8 6.7 7.2 7.3

4.2 4.7 5.0 4.5

7.8 8.3 8.7 8.5

5.0 5.2 5.7 5.0

9.3 7.7 11.7 10.2

5.0 5.5 6.2 5.7

9.3 9.7 12.3 10.3

5.7 6.3 6.7 6.0

10.0 10.8 12.8 11.5

230

DAT

GDD

13

210

16

313

18

363

20

412

23

457

25

DAT: days after transplanting. GDD: growing degree days. The leaf pairs were counted considering the effectively formed leaf (i.e. of a length of 1.5 cm). The axillary shoots were considered independently from the length they reached. 280

NumLP

NumAS

4.6 4.8 4.8 5.0

7.5 7.8 7.7 8.0

5.2 5.2 5.5 5.0

8.0 8.7 8.0 8.0

5.2 5.3 5.5 5.0

10.0 9.3 10.2 10.0

5.2 5.5 5.8 5.2

8.7 11.0 12.0 11.0

6.0 6.0 6.0 6.0

13.3 13.7 14.3 13.0

7.0 7.5 8.0 7.0

14.0 15.0 16.0 14.0

264

300

339

403

449

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(0.1–1.0 μL) was diluted with deionized water to a volume of 5 mL and then 0.5 mL of Folin-Ciocalteau reagent were added; after 3 min 1.5 mL of a 25% Na2CO3 solution was added and then deionised water up to 10 mL final volume. Solutions were maintained at room temperature under dark conditions for 60 min and the total polyphenols content was determined at 765 nm using a Perkin Elmer Lambda Bio 20 spectrophotometer. Gallic acid standard (Fluka, Buchs, CH) solutions were used to calibrate the method.

Vegetation Index (GNDVI) – were calculated in order to estimate chlorophyll (Chl), as follows: MCARI = [(R700-R670) − 0.2 × (R700R550)] × (R700/R670) NDVI670 = (R800 − R670)/(R800 + R670)

(Daughtry et al., 2000) (Rouse et al., 1974) OSAVI = (1 + 0.16) × (R800 − R670)/ (Rondeaux et al., (R800 + R670 + 0.16) 1996) GNDVI = (R800 − R500)/(R800 + R500) (Gitelson et al., 1996) The literatures have proposed many spectral indices also for carotenoids (Car) estimation using diverse ratios of wavelengths in the visible and visible/NIR. Following the results of Yi et al. (2014) in cotton, the Carotenoid Concentration Index700 (CRI700) and the Chlorophyll Index Red Edge (CIred-edge) were calculated as follows:

2.8. Identification and quantification of phenolic compounds Identification and quantification of the phenolic compounds were carried out according to the method described by Llorach et al. (2008) with slight modifications. Chromatographic analyses were performed on a 1200 Agilent Series HPLC (Agilent Technologies, Milano, Italy) equipped with a G1322 degasser, a G1311A quaternary pump, a G136A Column thermostat, an autosampler injection system and a diode array detector. The system was controlled with Agilent ChemStation for Windows (Agilent Technologies). Separations were achieved on a Kinetex C18 column 5 μm 100A 250 × 4.6 mm (Phenomenex, Castel Maggiore, Italy). The mobile phases were water with 5% formic acid (A) and methanol (B) with a solvent flow rate of 1 mL min−1 in a gradient program starting with 5% B in A, reaching 40% B at 25 min, and then isocratic for 5 min. UV chromatograms were recorded at 330 nm. Caffeic acid, caftaric acid, rosmarinic, and cichoric acid were quantified by means of calibration curves obtained in the range 1.25–100 mg L−1.

CRI700 = (1/R515 − 1/R700) (Gitelson et al., 2006) CIred-edge = ((R800/R750) − 1) (Gitelson et al., 2005) The reflectance results were also compared with that obtained by soil-plant analysis development (SPAD) readings. Measurements were performed at the same sampling dates using a 502 plus portable chlorophyll meter (Konica Minolta, Inc., Tokyo, Japan). The Chl content was estimated in the mid-section of three fully expanded and same sun-oriented leaves per experimental unit. 2.5. Chlorophylls and carotenoids analysis The concentrations of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Car) – as well as the related Total Chl (Chla + Chlb) – were determined at 35 and 25 DAT (corresponding to 457 and 449 GDD) in Spr_Tr and Sum_Tr, respectively. Two leaf round sub-samples of 78.5 mm2 (from both sides of the central vein) were obtained by cutting with a metal tube and weighted for FW determinations. The sub-samples were collected from two plants per experimental unit. In particular, for each plant leaves were distinguished on the basis of: (i) their relative sun position, as upper, sunny exposed leaves and lower, shaded leaves; (ii) their relative plant position, as leaves of main shoot and leaves of axillary shoot. In summary for each plant, four analytical determinations were performed on: main shoot-upper leaf, main shootlower leaf, axillary shoot-upper leaf, and axillary shoot-lower leaf. Pigments were extracted by placing the leaf discs in 5 mL of N,Ndimethylformamide (DMF) for 36 h in dark conditions at 5 °C (Moran and Porath, 1980). Absorbance of the extracts was measured at wavelengths of 664, 647 and 480 nm for Chla, Chlb and Car, respectively, with a UV/vis spectrophotometer (PerkinElmer, Waltham, MA, USA) and 1.00-cm quartz cuvettes. Pigment concentrations were then calculated with the equations proposed by Wellburn (1994).

2.9. Radical scavenging activity The radical scavenging activity of the extracts was measured according to the method described by Re et al. (1999) with some modifications. ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) – Fluka, Buchs, Switzerland) was dissolved in water to a 7 mM concentration; the ABTS radical was formed by reacting ABTS stock solution with 2.45 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. The ABTS radical solution was diluted with deionised water to reach an absorbance of 0.70 ± 0.02 at 734 nm and at 30 °C. The absorbance at 734 nm was evaluated by a Perkin Elmer (Boston, MA, USA) Lambda Bio 20 spectrophotometer. The reaction was started by the addition of 30 μL of extract to 2970 μL of ABTS%+. The time of analysis, 5 min, was chosen after preliminary tests and represented the time necessary to reach at least 80% of the overall inhibition of the ABTS radical at a given concentration. For each sample, the percentage of inhibition (I%) was plotted as a function of concentration and the TEAC (Trolox Equivalent Antioxidant Capacity) calculated as the ratio of the linear regression coefficient of the sample to that of the Trolox standard (Fluka). Results were expressed as micromoles of Trolox equivalents per gram of fresh weight. The coefficient of variation of the method was below 5%.

2.6. Extraction of phenolic compounds For the extraction of polyphenols, basil was freeze-dried immediately after collection and the powder was stored at −40 °C until extraction and analysis. One aliquot (0.5 g) of freeze-dried powder was added to 5 mL of methanol/acetone/HCl (70:29:1 v/v/v) and stirring for 3 h at 200 rpm by an orbital shaker, then the solution was centrifuge for 5 min at 4000 rpm and the supernatant was filtered with cellulose filters. The extracts were used to evaluate total polyphenolic content, phenolic pattern, and antiradical activity. All the extracts were stored at −40 °C until analysis. Quality traits were assessed only during Sum_Tr.

2.10. Statistical analysis A one-way analysis of variance (ANOVA) was applied to test (F-test) the effects of treatment (YF, BF, GF and Control). When significant differences were detected, means separation was conducted by applying the Fisher’ LSD (Least Significant Difference) at the 5% (p < 0.05) level of significance; the standard error of the difference (s.e.d.) between means was also reported in Tables. ANOVA assumptions were tested through graphical methods. The statistical analyses were performed using R (R Core Team, 2017).

2.7. Total polyphenols content The total polyphenol content (TPC) of the free and bound phenolic fractions was evaluated using the Folin-Ciocalteau reagent and following a method adapted from Singleton and Rossi (1965). The sample 281

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Control (199.4, 191.1, 167.3, and 148.4 cm2 g−1, averaged over GDD and transplanting time) (Figs. 5E and F).

3. Results 3.1. Optical characteristics of coloured films

3.3. Photosynthetic pigments The three films altered the quantity and quality of the radiation transmitted to canopy (Fig. 3). Blue film showed the highest PAR reduction (51.8% of shading), followed by GF (42.7%) and YF (28.3%), as well as the highest relative amounts of scattered PAR (48.7% vs. 42.0% and 42.1% for BF, YF and GF, respectively). With respect to the single wavelengths of the transmitted radiation, as expected, YF absorbed in the blue-violet region (400–500 nm), with reflectance values (on the certified reflective surface) lower than the two other covered treatments, while transmitted more than GF and BF, starting from around 540 nm to 850 nm (Fig. 3). Conversely, GF and BF behaved similarly in the reflectance spectra, showing a valley from about 550–750 nm. Blue film treatment exhibited higher transmittance values with respect to GF only around 400–450 nm (blue-violet region) and around 760–820 nm (near-infrared region). Green film also showed the highest reflectance values in the NIR region (from around 850 nm) (Fig. 3).

Table 3 shows the Chla, Chlb, Total Chl, and Car contents in basil leaves, differently positioned on the plant, hence characterized by different age and relative sun exposition. Although significant differences restricted only for some variables, few clear trends were observed. In the leaves of the main shoot, Green colored film gave higher Chla, Chlb, and Total Chl contents, while Control the lowest, regardless of leaf position (i.e. upper or lower leaf) and transplanting time (Total Chl: 1339 and 860 μg g−1 FW for GF and Control, respectively, averaged over leaf position and transplanting time). In general, lower leaves showed higher pigment content (10% higher than upper ones) (Table 3). In Sum_Tr, in the leaves collected from the axillary shoots, the lowest Chl values were always found in Control treatment and GF confirmed to exhibit the highest values (Total Chl: 1547 and 1038 μg g−1 FW for GF and Control, respectively, averaged over leaf position); conversely, in Spr_Tr BF registered the highest values (Table 3). Differences in Car content were significant only in lower leaves of axillary shoots during Spr_Tr, with GF confirming to stimulate pigment accumulations (101 μg g−1 FW) (Table 3). The indices used to estimate the Chl and Car content in basil leaves, are reported in Table 4. Differences between SPAD values were not always significant among treatments: GF gave higher values, regardless of transplanting time (on average, 39.9 and 39.3 in Spr_Tr and Sum_Tr, respectively). However, no correlation was observed between SPAD and Chl content in basil leaves of both ages and position. The values of MCARI, NDVI670, GNDVI, and OSAVI remained fairly stable among the two transplanting times; anyway, the discriminatory capacity of such indices was higher during Spr_Tr (Table 4), when the values of some indices (at 457 GDD) were correlated with the Total Chl content in leaves (see the correlations in Table 5). Note that NDVI670 and OSAVI gave the highest and significant correlations ((i) NDVI670 vs. Total Chl (main shoot) = 0.638; (ii) NDVI670 vs. Total Chl (axillary shoot) = 0.748; (iii) OSAVI vs. Total Chl (main shoot) = 0.722; (iv) OSAVI vs. Total Chl (axillary shoot) = 0.819). In general, BF enhanced the CIred-edge values, followed by GF and YF, in both Spr_Tr and Sum_Tr; on the other hand, the behaviour of CRI700 resulted pretty unclear (Table 4). Also for CIred-edge and CRI700 − which were selected because their potential as Car indicators − the higher correlations with the analytically measured Car contents, were observed during Spr_Tr (CIred-edge vs. Car − axillary shoot: 0.692) (data not shown).

3.2. Basil growth In both Spr_Tr and Sum_Tr the number of leaf pairs on the main stem increased as the growing cycle went on (Table 1). In Spr_Tr GF showed always the higher NumLP while Control the lowest; in Sum_Tr such trend was confirmed, but with generally higher values at the same GDD. In general, also for NumAS the highest values were registered by GF while the lowest by Control; nevertheless Sum_Tr allowed higher values, although such differences among treatments emerged starting from 300 GDD (Table 1). Light manipulation significantly influenced main shoot length (Fig. 4). Also with this trait, GF induced the highest values than the other treatments while Control the lowest. Differences between YF and BF were inconsistent, probably related to sampling time. Such trend was observed in both growing seasons (Fig. 4). Total aerial biomass (DW) resulted significantly influenced by plastic films application (Table 2). In Spr_Tr, YF induced higher values already starting from 150 GDD, reaching 4.03 g plant−1 at 457 GDD; GF and Control gave the lowest biomass accumulation reaching 2.77 and 2.70 g plant−1 at 457 GDD, respectively (Fig. 5A). In general, Sum_Tr allowed higher biomass DW accumulation than Spr_Tr (1.66 and 3.06 g plant−1 respectively, averaged over treatments and GDD) (Fig. 5A and 5B). Significantly differences among treatments emerged only from 339 GDD, i.e. the middle phase of the crop cycle (Table 2). Yellow film confirmed to give highest values (6.47 g plant−1 at 449 GDD), while GF the lowest (4.92 g plant−1 at 449 GDD); conversely, Control plants dry weight reached interesting values (5.80 g plant−1 at 449 GDD) (Fig. 5B). The trend of LA over thermal time showed similar patterns in all theses and in both growing seasons (Figs. 5C and D). During Spr_Tr, plants under YF film reached the highest LA values at all sampling dates (see also Table 2), followed by BF and GF (on average: 318.4, 272.7 and 259.2 cm2 plant−1, respectively); Control plants exhibited always the lowest values (202.5 cm2 plant−1 averaged over GDD) (Fig. 5C). Differently, in Sum_Tr, GF showed significantly higher values than BF during the middle phases of the crop cycle (averaged over 339 and 403 GDD: 462.9 vs. 411.3 cm2 plant−1 for GF and BF, respectively); nevertheless, YF maintained high values, especially from 339 GDD, and Control confirmed the lowest LA values (253.2 cm2 plant−1 averaged over GDD) (Fig. 5D). Specific leaf area gave the same responses to the different covered treatments regardless of transplanting times, despite a greater influence it would seem observable during Spr_Tr (Figs. 5E and F). Green film allowed significantly higher SLA values, followed by BF, YF, and

3.4. Quality traits The total phenolic content and the antiradical activity in basil, as affected by colored films application, at different GDD are presented in Fig. 6. The TPC ranged from 37 to 70 mg GAE g−1 DW, 90–150 mg GAE g−1 DW and from 70 to 130 mg GAE g−1 DW for samples at 264, 339 and 449 GDD respectively. At the first sampling time, the application of yellow, blue, and green films lowered phenolic compounds accumulation, with respect to Control. As the crop growing cycle went on, TPC increased, although at 339 GGD significant differences among treatments were not detected. At 449 GDD, while YF, GF, and BF confirmed their values, Control reduced significantly its values to 41.4 mg GAE g−1 DW. The utilization of colored films did not enhance TEAC values; on average, the TEAC values were 131, 113 and 106 μmol TE g−1 DW at 264, 339 and 449 GGD, respectively. Regarding the phenolic pattern of the extracts, the content of caftaric, caffeic, chicoric and rosmarinic acids are reported in Table 6. The application of colored films always reduced the concentration of rosmarinic and caftaric acids in basil leaves; in the case of rosmarinic acid, the reduction was pretty severe when GF was applied. Caftaric and rosmarinic acids showed an 282

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Fig. 4. Dynamics of main shoot lenght (cm plant−1) as recorded for basil plants grown under different colored plastic films (grey chart: yellow film, YF; diagonal crossed chart: green film, GF; forward slashed chart: blue film, BF) plus an uncovered Control (white chart), during the whole crop cycles (A: spring transplanting – Spr_Tr – 2014; B: summer transplanting – Sum_Tr – 2015). Reported data represent averages ± standard error of the mean (degrees of freedom: Blocks 1; Treatment 3; Residual 3); in box, for each sampling date: p-values (treatment effect) from analysis of variance (ANOVA). Different letters stand for statistically significant differences at p < 0.05 (Fisher’s LSD test). Time is expressed in thermal time after transplanting (growing degree days, GDD; base temperature of 6.11 °C).

4. Discussion

increased accumulation over time whilst a decreasing trend was observed for caffeic acid. Interestingly, GF significantly enhanced caffeic acid content at harvesting (449 GDD), while YF induced a higher accumulation of chicoric acid in the last two samplings although differences were not significant.

The manipulation of solar irradiance through the application of photo-selective colored plastic films significantly affected some morphological and biochemical traits of basil plants; anyway, a discrimination between the contribution of spectra modification and the total amount of the transmitted radiation is not reliable. Both basil growth and plant morphology were sharply influenced by

Table 2 Summary of p-values (treatment effect) from analysis of variance (ANOVAa) on total aerial biomass, leaf area (LA), specific leaf area (SLA), leaf temperature (TIR) during the whole crop cycles (spring transplanting – Spr_Tr – 2014 and summer transplanting – Sum_Tr – 2015). Variables Spr_Tr 2014 Total biomass (g plant−1) LA (cm2 plant−1) SLA (cm2 g−1) TIR (°C) Sum_Tr 2015 Total biomass (g plant−1) LA (cm2 plant−1) SLA (cm2 g−1) TIR (°C)

150 GDD

230 GDD

313 GDD

363 GDD

412 GDD

457 GDD

0.021 0.034 0.007 0.036 210 GDD

0.007 0.005 0.006 0.125 264 GDD

0.030 0.006 < 0.001 < 0.001 300 GDD

0.007 0.003 0.013 0.003 339 GDD

< 0.001 0.006 0.002 0.077 403 GDD

< 0.001 0.001 0.058 0.019 449 GDD

0.137 0.027 0.004 0.035

0.068 0.021 0.005 0.008

0.060 0.012 0.007 < 0.001

0.017 0.002 0.033 0.002

0.032 0.003 < 0.001 0.008

0.047 0.009 < 0.001 0.010

GDD, growing degree days. a One-way ANOVA: degrees of freedom: Blocks 1; Treatment 3; Residual 3. 283

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Fig. 5. Dynamics of total aerial biomass dry weight (DW, g plant−1), leaf area (LA, cm2 plant−1), and specific leaf area (SLA, cm2 g−1) as recorded for basil plants grown under different colored plastic films (white rhombus: yellow film, YF; white triangle: green film, GF; white circle: blue film, BF) plus an uncovered Control (black star), during the whole crop cycles (A, C, and E: spring transplanting – Spr_Tr – 2014; B, D, and F: summer transplanting – Sum_Tr – 2015). Reported data represent averages ± standard error of the mean (degrees of freedom: Blocks 1; Treatment 3; Residual 3); for each sampling date, p-values (treatment effect) from analysis of variance (ANOVA) are reported in Table 2. Time is expressed in thermal time after transplanting (growing degree days, GDD; base temperature of 6.11 °C) and in days after transplanting (DAT) (below and above x-axis, respectively).

covered plastic films, as an adaptive response to the modified environment (Bantis et al., 2016; Ye et al., 2017). All the covered treatments enhanced plant height (as recorded by the higher values of the main shoot length), independently from the transplanting times, probably due to the shading effect which in turn influence the red to far red ratio (R:FR). It is notorious that shading lowers R:FR, inducing photomorphogenic responses of plants, such as stem elongation (DemotesMainard et al., 2016; Han et al., 2017): basil plants supplied with different LED light treatments (Bantis et al., 2016) as well as chrysanthemum and bell pepper plants grown under photoselective plastic films (Li et al., 2000) exhibited higher plant heights. It has to be highlighted that in this work higher heights were given by GF and not by the most shaded film (i.e. blue film); however, GF and BF are

characterized by very similar patterns in terms of transmitted radiation to canopy, even though BF has higher scatter properties. The latter characteristic has a heavy impact on plant growth, independently from the alteration of light spectrum, due to the improved deeper light penetration, which induces a compact habitus in plants (Nissim-Levi et al., 2008; Ilić and Fallik, 2017). In any case, the relation between shading level and scattering, as observed under BF, should not be neglected. Despite low R:FR mainly stimulates internode elongation rather than the total number of internodes (Franklin and Quail, 2010), the application of colored plastic films enhanced the NumLP as well as NumAS with respect to Control plants. Nevertheless, low-light or dark conditions generally induce a faster plant growth than bright light, in order to complete plant’s life cycle and improve biological yield (Ye 284

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Table 3 Pigment content (chlorophyll a, Chla, chlorophyll b, Chlb, Total Chl (Chla + Chlb), carotenoids, Car) as determined in leaves of basil plants grown under different colored plastic films (yellow film, YF, green film, GF and blue film, BF) plus an uncovered Control, at 35 (spring transplanting – Spr_Tr – 2014) and 25 (summer transplanting – Sum_Tr – 2015) days after transplanting (DAT), corresponding to 457 and 449 growing degree days (GDD), respectively. Leaves were sampled from both main and axillary shoots and subsequently separated into “upper” and “lower” leaves, depending on their position on the main stem (i.e. upper half or lower half) or on the position of the axillary shoot on the main stem (i.e. upper half or lower half). For each column, means labeled with the same letter did not differ significantly at p < 0.05 (Fisher’s LSD test). Treatments

Spr_Tr 2014 Chl a (μg g

Main shoot Upper leaves Control YF GF BF p-value s.e.d.

−1

Sum_Tr 2015 Chl b

Total Chl

Car

a

FW )

Chl a (μg g

−1

Chl b

Total Chl

Car

FW)

427 533 615 577 0.408

258 347 415 376 0.218

685 881 1030 952 0.322

126 126 124 124 0.999

563 b 567 b 791 a 590 b 0.012 30.410

381 385 861 468 0.179

944 952 1652 1057 0.066

113 121 82 101 0.757

595 736 800 784 0.349

387 462 530 454 0.476

982 1199 1330 1239 0.367

141 152 158 157 0.823

473 559 758 662 0.064

355 450 587 475 0.123

827 1009 1346 1137 0.081

104 95 110 100 0.426

Axillary shoot Upper leaves Control YF GF BF p-value s.e.d.

328 c 463 bc 507 ab 650 a 0.034 53.572

214 285 331 381 0.067

542 c 749 bc 838 ab 1031 a 0.042 88.409

106 119 115 146 0.136

540 603 727 658 0.741

361 518 580 497 0.054

901 1121 1307 1154 0.414

106 113 117 113 0.994

Lower leaves Control YF GF BF p-value s.e.d.

325 b 430 ab 524 a 547 a 0.048 46.261

224 273 329 364 0.288

549 703 853 910 0.120

72 b 89 ab 101 a 96 a 0.049 5.918

683 522 991 756 0.330

492 366 797 567 0.426

1175 889 1788 1323 0.373

157 117 155 172 0.779

Lower leaves Control YF GF BF p-value s.e.d.

s.e.d., standard error of differences between means. One-way ANOVA: degrees of freedom: Blocks 1; Treatment 3; Residual 3. a FW: fresh weight.

Such morphological adaptations were associated also to physiological changes such as an increase in chlorophyll content in basil leaves. Shaded grown leaves receive lower light amounts, and consequently contain more chlorophyll than those exposed to high irradiance levels (see also Chl content in lower leaves) (Ilić and Fallik, 2017; Yang et al., 2018). Also for this trait, the scatter properties of BF emerged, inducing a reduction in Chl content. The analytical measured Chl amount was properly by the estimation through the reflectance indices. Apart SPAD, some indices already validated and calibrated at canopy scale have been selected (MCARI, OSAVI, NDVI700 and GNDVI) (Haboudane et al., 2002). The Optimized Soil-Adjusted Vegetation Index and NDVI700 seem the more suitable indicators for such trait, despite other spectral indices, based on other NIR and red wavelengths (i.e. 875–900 and 720–725) (Wang et al., 2015) or originated by specific combinations (i.e. OSAVIxSIPI or OSAVIxCIred-edge) (Jin et al., 2013), could better perform. Filtering the transmitted solar radiation through photoselective films or shade nets is known to sharply affect quality and yield of crops (Nissim-Levi et al., 2008). The effect of light quantity and quality manipulation on some important molecules for human health, such as phenolic acids, has been assessed. In basil leaves the amount of total phenolic compounds resulted higher than the values reported in Kwee and Niemeyer (2011) but comparable to Ghasemzadeh et al. (2016). Interestingly, as already observed, the treatments did not induce any

et al., 2017). Differences among crop growing seasons (i.e. Spr_Tr and Sum_Tr) are probably related to air temperatures and day-length conditions, since basil is a warm season crop, mainly grown at relative high temperatures (Fallahi et al., 2015). It was confirmed by the different behavior of Control plants: the highest Sum_Tr temperatures (on average, 24.2 °C vs. 19.2 °C in Spr_Tr) guaranteed optimal plant growth (biomass accumulation in leaves), while during Spr_Tr, plants grown under the colored plastic films received slightly higher temperatures than greenhouse conditions (data not shown). Differences in biomass accumulation among covered treatments were probably related to both shade levels and light spectrum. Yellow film treatment allowed the highest biomass accumulation while GF the lowest, regardless of the crop growing season. Such results are related to a lower shade percentage in combination with the highest transmission in the 540–700 nm PAR range, which guarantees a high photosynthetic activity. Also scattering properties play an important role in biomass accumulation as demonstrated by the higher values of plants grown under BF (Shahak et al., 2008; Ilić and Fallik, 2017). The effect of shading emerged also in terms of a greater leaf area expansion confirming previous results in bell pepper (Díaz-Pérez, 2013; Ilić et al., 2017a), tomato (Ilić et al., 2015), and lettuce (Ilić et al., 2017b), as well as of a lower leaf thickness (higher SLA values) as an adaptive response to capture more light (Ballaré and Pierik, 2017; Bertel et al., 2017; Han et al., 2017). 285

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Table 4 Soil Plant Analysis Development (SPAD) and vegetation indices (Modified Chlorophyll Absorption Ratio Index, MCARI; Normalized Difference Vegetation Index670, NDVI670; Optimized Soil-Adjusted Vegetation Index, OSAVI; Green Normalized Difference Vegetation Index, GNDVI; Carotenoid Concentration Index700, CRI700; Chlorophyll Index Red Edge, CIred-edge) as measured from reflectance data of leaves of basil plants grown under different colored plastic films (yellow film, YF, green film, GF and blue film, BF) plus an uncovered Control. Measures were carried out at 150, 230, 313, 363, 412 and 457 growing degree days (GDD) after transplanting (spring transplanting – Spr_Tr – 2014) and at 210, 264, 300, 339, 403 and 449 GDD (summer transplanting – Sum_Tr – 2015). For each column, means labeled with the same letter did not differ significantly at p < 0.05 (Fisher’s LSD test). Treatments

SPAD Control YF GF BF p-value s.e.d. MCARI Control YF GF BF p-value s.e.d.

Spr_Tr 2014

Sum_Tr 2015

150 GDD

230 GDD

313 GDD

363 GDD

412 GDD

457 GDD

210 GDD

264 GDD

300 GDD

339 GDD

403 GDD

449 GDD

39.6 39.3 42.3 41.2 0.546

40.7 bc 39.2 c 42.8 a 41.6 ab 0.033 0.604

41.4 38.0 41.3 41.0 0.126

40.5 39.5 40.5 39.2 0.649

35.3 35.9 36.7 36.5 0.418

34.4 33.1 35.8 35.5 0.543

38.3 38.3 41.9 40.7 0.198

39.4 bc 38.0 c 41.4 ab 41.7 a 0.029 0.665

39.6 39.6 41.9 37.3 0.234

41.2 b 42.8 a 39.1 c 41.6 b 0.007 0.356

34.9 34.2 36.5 35.8 0.730

33.6 34.4 35.1 36.3 0.449

0.121 0.151 0.122 0.091 0.046 0.011

0.180 0.195 0.196 0.166 0.127

0.361 0.323 0.303 0.278 0.526

0.333 0.407 0.367 0.320 0.211

0.599 0.540 0.631 0.521 0.188

0.299 0.275 0.246 0.201 0.051

0.475 0.507 0.346 0.315 0.032 3.719

0.291 0.238 0.218 0.242 0.195

0.595 0.539 0.502 0.420 0.179

0.407 0.308 0.317 0.296 0.215

0.540 0.476 0.480 0.463 0.620

0.835 0.840 0.839 0.831 0.975

0.817 0.856 0.812 0.806 0.044 0.010

0.814 0.807 0.813 0.816 0.977

0.809 0.808 0.834 0.805 0.499

0.534 0.569 0.550 0.568 0.412

0.508 0.558 0.509 0.534 0.121

0.484 0.493 0.468 0.472 0.286

0.464 0.481 0.484 0.509 0.248

0.765 0.776 0.765 0.776 0.259

0.806 0.828 0.800 0.792 0.019 0.005

0.747 0.722 0.710 0.708 0.318

0.759 0.766 0.760 0.775 0.828

0.048 0.048 0.068 0.053 0.396

0.047 0.057 0.045 0.053 0.321

4.308 3.756 2.638 3.699 0.103

3.508 3.166 2.769 4.235 0.007 0.145

0.498 0.438 0.453 0.399 0.049 0.006

a a a b

ab a ab b

NDVI670 Control YF GF BF p-value s.e.d.

0.832 0.825 0.826 0.823 0.843

0.697 0.789 0.741 0.768 0.223

0.823 0.880 0.877 0.884 0.025 0.011

GNDVI Control YF GF BF p-value s.e.d.

0.603 0.594 0.588 0.602 0.657

0.507 0.529 0.493 0.526 0.553

0.524 0.580 0.538 0.527 0.290

OSAVI Control YF GF BF p-value s.e.d.

0.783 0.759 0.766 0.717 0.001 0.007

a b ab c

CIred-edge Control YF GF BF p-value s.e.d.

0.043 0.050 0.042 0.048 0.645

0.063 0.044 0.068 0.093 0.002 0.003

CRI700 Control YF GF BF p-value s.e.d.

5.300 5.412 5.202 7.473 0.092

4.368 5.446 3.812 4.706 0.358

b c b a

b a a a

0.859 0.878 0.859 0.865 0.014 0.003

b a b b

0.859 0.869 0.876 0.877 0.021 0.003

0.656 0.699 0.693 0.721 0.148

0.658 0.704 0.669 0.687 0.049 0.009

b a b ab

0.669 0.692 0.678 0.704 0.059

0.688 0.723 0.723 0.731 0.288

0.779 0.822 0.795 0.793 0.054

0.784 0.842 0.828 0.834 0.009 0.007

0.038 0.057 0.059 0.110 0.202

0.027 0.032 0.031 0.040 0.092

0.035 0.055 0.054 0.089 0.009 0.006

9.022 9.617 9.758 9.386 0.854

7.459 6.588 5.529 6.103 0.011 0.223

a b c bc

6.343 4.490 4.557 4.493 0.071

b a a a

0.840 0.863 0.855 0.878 0.029 0.006

c ab bc a

0.761 0.821 0.876 0.899 0.024 0.022

c bc ab a

0.820 0.820 0.847 0.839 0.050

0.627 0.617 0.627 0.673 0.012 0.007

b b b a

0.516 0.582 0.653 0.697 0.016 0.024

c bc ab a

0.514 0.524 0.568 0.571 0.025 0.011

b a a a

0.836 0.853 0.862 0.880 0.017 0.006

c bc ab a

0.751 0.794 0.838 0.823 0.092

0.793 0.810 0.800 0.800 0.504

c b b a

0.027 0.058 0.031 0.117 0.012 0.012

b b b a

0.025 0.033 0.034 0.068 0.155

0.015 0.017 0.032 0.044 0.036 0.006

3.960 2.848 3.283 3.007 0.049 0.226

a b ab b

2.617 3.704 5.469 8.563 0.007 0.585

c bc b a

4.073 3.486 4.575 3.663 0.251

a a b b

b b a a

b b ab a

b a b b

b a b b

0.037 0.042 0.056 0.066 0.049 0.006 b bc c a

3.841 4.220 3.015 3.749 0.058

c bc ab a

0.027 0.041 0.040 0.050 0.145

4.489 4.264 3.603 4.741 0.067

s.e.d., standard error of differences between means. One-way ANOVA: degrees of freedom: Blocks 1; Treatment 3; Residual 3.

agreement also with previous data in literature where rosmarinic acid was found to be the most abundant phenolic compound (Jayasinghe et al., 2003; Kim et al., 2006; Lee and Scagel, 2009; Nguyen et al., 2010). Rosmarinic and chicoric acids contents were comparable to the ones found by Taulavuori et al. (2017). The application of colored films always reduced the concentration of rosmarinic and caftaric acids in basil leaves; for the former, the reduction was more severe when GF were applied. No significant differences were observed for chicoric acid content, in contrast with Taulavuori et al. (2017) who found an increased accumulation of

significant difference in the radical scavenging activity at 264 and 449 GDD, regardless the differences in the TPC values. Probably, the treatments influenced also the accumulation of specific phenolic compounds with different antiradical activity. Thus, the phenolic pattern of the extracts was also investigated. In the conditions adopted, four main peaks were detected and identified as caftaric, caffeic, chicoric and rosmarinic acids. Rosmarinic acid was the main phenolic compound, followed by chicoric, caftaric and caffeic acids; this same order was reported also by Lee and Scagel (2009), with the exception of caffeic acid, which was not found by these authors. These results are in 286

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chicoric acid under the application of an enhanced blue light. Interestingly, at harvesting (449 GDD), caffeic acid was positively influenced by the application of colored films and in particular of GF. When the relationship among the total phenolic content, or the amount of each phenolic acid, and the antiradical activity of the samples was explored, it was interesting to observe that a linear correlation was found only with the content of caffeic acid.

Table 5 Correlation coefficients between Total Chlorophyll content in main and axillary shoots (averaged over upper or lower positions) of basil plants − grown under different colored plastic films − and seven indices used to estimate pigment content in leaves (soil plant analysis development (SPAD), Modified Chlorophyll Absorption Ratio Index (MCARI), Normalized Difference Vegetation Index670 (NDVI670), Optimized Soil-Adjusted Vegetation Index (OSAVI), Green Normalized Difference Vegetation Index (GNDVI)) at 35 (spring transplanting – Spr_Tr – 2014) and 25 (summer transplanting − Sum_Tr − 2015) days after transplanting (DAT), corresponding to 457 and 449 growing degree days (GDD), respectively. Indices

SPAD MCARI NDVI670 GNDVI OSAVI

5. Conclusions Shading of basil plants, and thus lower R:FR, generally induced an higher stem elongation, greater leaf area and lower leaf thickness. Among the colored films, GF, with very similar transmitted radiation spectra to BF, showed the highest effect on plant height, whilst YF always allowed the highest biomass accumulation. The alteration of the transmitted radiation also significantly influenced Chl biosynthesis as a strategy amelioration of photosynthetic activity under unfavorable environments (i.e., shading conditions). Besides, analytical determinations, Chl content in basil leaves could be proven estimated using reflectance data and vegetation indices (i.e. NDVI700 or OSAVI). Shading showed a positive impact also on phenolic compounds accumulation; the application of GF generally decreased the content of rosmarinic and caftaric acids, whilst at harvest increased caffeic acid accumulation; however, the effect on the accumulation of some specific phenolic compounds was not reflected in the antiradical activity which was not affected by the application of the colored films. This work confirms earlier studies according to which light quantity and quality can deeply

Total Chla(μg g−1 FW) Spring transplanting – Spr_Tr

Summer transplanting – Sum_Tr

Main shoot

Axillary shoot

Main shoot

Axillary shoot

0.343 n.s. 0.118 n.s. 0.638* 0.272 n.s. 0.722**

0.331 n.s. -0.110 n.s. 0.748** 0.621 n.s. 0.819**

0.513 n.s. -0.101 n.s. 0.399 n.s. 0.350 n.s. 0.301 n.s.

0.682* -0.024 n.s. 0.473 n.s. 0.466 n.s. 0.445 n.s.

n.s. = not-significant. a Total Chlorophyll content in basil leaves. * significant effect at the 0.05 probability level. ** significant effect at the 0.01 probability level.

Fig. 6. (A) Total polyphenols content (TPC, mg GAE g−1 DW) and (B) antiradical activity (TEAC, μmol TE g−1 DW) as recorded for basil plants grown under different colored plastic films (grey chart: yellow film, YF; diagonal crossed chart: green film, GF; forward slashed chart: blue film, BF) plus an uncovered Control (white chart), during 2015 (summer transplanting – Sum_Tr – 2015). Plants were sampled at 16, 20 and 25 days after transplanting, corresponding to 264, 339 and 449 growing degree days (GDD), respectively. Reported data represent averages ± standard error of the mean (degrees of freedom: Blocks 1; Treatment 3; Residual 3); in box, for each sampling date: p-values (treatment effect) from analysis of variance (ANOVA). Different letters stand for statistically significant differences at p < 0.05 (Fisher’s LSD test). 287

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Table 6 Caftaric acid, caffeic acid, chicoric acid and rosmarinic acid as determined in leaves of basil plants grown under different colored plastic films (yellow film, YF, green film, GF and blue film, BF) plus an uncovered Control, at 16, 20 and 25 days after transplanting, corresponding to 264, 339 and 449 growing degree days (GDD), respectively. For each column, means labeled with the same letter did not differ significantly at p < 0.05 (Fisher’s LSD test). Treatments

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Phenolic acids (mg g−1 DW) Caftaric acid

Caffeic acid

Chicoric acid

Rosmarinic acid

264 GDD Control YF GF BF p-value s.e.d.

3.70 a 2.52 b 2.69 b 2.51 b 0.029 0.216

0.310 0.380 0.358 0.340 0.476

9.69 9.61 8.62 9.67 0.504

12.30 a 8.02 b 7.83 b 9.05 b 0.004 0.400

339 GDD Control YF GF BF p-value s.e.d.

3.74 a 3.25 b 2.42 c 2.71 c 0.006 0.131

0.273 0.254 0.261 0.278 0.823

8.84 9.77 7.57 8.95 0.465

12.97 11.46 9.67 10.37 0.418

449 GDD Control YF GF BF p-value s.e.d.

4.97 a 4.78 a 3.61 b 3.46 b 0.026 0.285

0.124 0.163 0.208 0.190 0.034 1.483

9.76 10.14 8.89 9.29 0.375

16.73 15.10 12.18 13.38 0.060

b ab a a

s.e.d., standard error of differences between means. One-way ANOVA: degrees of freedom: Blocks 1; Treatment 3; Residual 3.

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