Industrial Crops & Products 122 (2018) 277–289

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Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

Light quantity and quality supplies sharply affect growth, morphological, T

physiological and quality traits of basil
⁎
Fabio Stagnaria, Carla Di Mattiaa, Angelica Galienia,b, , Veronica Santarellia, Sara D'Egidioa,
Giancarlo Pagnania, Michele Pisantea
a
Faculty of Bioscience and Technology for Agriculture Food and Environment, University of Teramo, Italy
b
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: Plants sharply adapt their growth and physiology to light availability. This study aimed at evaluating the effect
Shading of light quantity and quality manipulation on growth, morphological traits, pigment and secondary metabolites
Light spectra manipulation content in basil as well as comprehending the mechanisms which regulate such responses.
Colored cover films Two experiments were carried out under greenhouse in 2014 (spring transplanting, Spr_Tr) and 2015
Morphological traits
(summer transplanting, Sum_Tr). On a complete randomized block design, plants of basil were exposed to three
Single phenolic acids
Pigment content
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, re-
spectively). 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 of energy and the most important regulatory factor in plant's cycle: i.e.
seed germination, seedling establishment, transition to flowering and
Plants constantly modify their physiology and morphology to adapt morphogenesis (i.e. stem elongation) (Folta and Carvalho, 2015; Galvão
to different environmental conditions. Among the environmental fac- and Fankhauser, 2015).
tors, which strongly influence plant growth and development, both Plants respond to light modified environments with physiological
quantity and quality of light transmitted to canopy play a major role (photosynthetic rate, nutrient uptake) and biochemical (pigment and
(Shahak et al., 2004). Indeed, light represents both the primary source 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.
F. Stagnari et al. Industrial Crops & Products 122 (2018) 277–289

are reflected into modification of plant growth, as well as into altera- hypotheses that light quantity and quality manipulation – with colored
tions of morphological and anatomical traits (Peralta et al., 2002). Low cover films application (i.e. yellow, green and blue cover films) – on
irradiance leads to more biomass allocated in leaves at the expense of basil plants, should significantly affect: (i) growth and morphological
roots optimizing leaf area per unit leaf biomass as well as maximizing traits (i.e. leaf biomass accumulation and expansion), (ii) pigment
light interception (Valladares and Niinemets, 2008). Shaded plants, content, (iii) secondary metabolites content and activity (i.e. total
indeed, have the thickness of mesophyll layer reduced (Terashima polyphenols, phenolic compounds as well as antiradical activity). The
et al., 2005) and pigment density per unit leaf area increased (Xu et al., main assumption of this research activity was that light sharply affects
2009). plant responses, consequently some agro-technologies can be conveyed
In the last decades, studies on light manipulation have become more to meet the consumers demand for edible herbs with high nutritional
interesting due to the relationship between light and physiology of values.
secondary metabolism under different light spectra and/or light in-
tensities (Bantis et al., 2016). As already known in many horticultural 2. Materials and methods
and herbal crops, light quantity and quality plays an important role in
the synthesis of many antioxidants such as phenolic acids, carotenoids, 2.1. Plant material and growing conditions
flavonoids, anthocyanins, and α-tocopherol (Stagnari et al., 2014;
Bantis et al., 2016), mainly due to phytomorphogenic responses of Experiments were carried out at the greenhouse of the Agronomy
phytochromes (Henschel et al., 2017). and Crop Sciences Research and Education Center, University of
Light manipulation is commonly applied using narrow-bandwidth Teramo (altitude 15 m above sea level; 42° 42′ N,13° 54' 10′ E) during
light, such as that produced by light-emitting diode (LED)-based light two different crop growing seasons, from 4 April to 29 May 2014 (early
sources (Samuolienė et al., 2016), coloured shade nets (Ilić and Fallik, spring transplanting, Spr_Tr) and from 28 May to 8 July 2015 (later
2017) and different coloured plastic covers or photoselective plastic spring transplanting, named as summer transplanting, Sum_Tr). The
films (Stagnari et al., 2014; Henschel et al., 2017). In particular, the use greenhouse was covered with a single layer of ethylene-vinyl acetate
of photoselective cover materials (i.e. films or nets) could be a suitable film (PATILUX) provided by P.A.T.I. S.p.A. (San Zenone degli Ezzelini,
agro-technological alternative to improve the quality of the edible TV, Italy) and characterized by a natural ventilation system.
products, of both horticultural and herbal crops. Beside plant protection Seeds of sweet basil (Ocimum basilicum L. cv. Emily, Enza Zaden
from high rainfall, hail and frost (mainly films) as well as reduced Italia S.r.l., Tarquinia, VT, Italy) were sown in a nursery substrate and
physiological disorders and flower abortion (mainly nets), other im- maintained in a growth chamber until transplanting, which occurred 20
portant biochemical modifications could be obtained with the appli- and 16 days after sowing in Spr_Tr and Sum_Tr, respectively. Uniformly
cation of cover materials. Black and yellow nets increased the total sized seedlings (two-leaves stage) were transplanted into 9 cm side
antioxidant activity at harvest in oregano, while pearl nets favoured plastic pots, at a density of 1 plant per pot. Pots were filled with a
flavonoid (quercetin) accumulation in oregano, marjoram and cor- mixture of peat-based compost (Terraplant® 2, COMPO Italia S.r.l.,
iander (Buthelezi et al., 2016). Blue cover film enhanced the levels of Cesano Maderno, MB, Italy), potting soil (HOCHMOOR HORTUS
anthocyanins and ascorbic acid in fruits of strawberry, probably due to ESTIVO, Terflor S.r.l., Capriolo, BS, Italy), vermiculite and perlite at the
the antioxidant capacity of these compounds which act as an ultra- ratio of 1.5:2:1:1 (v/v). The growth substrate was saturated with tap
violet (UV) light filter (Henschel et al., 2017); conversely, UV black water before transplanting of seedlings and each pot was supplemented
plastic films significantly lowered the total phenols and flavonoid gly- with 50 mL of fertilizer solution (Cifoumic 10-10-10, Cifo S.r.l.,
cosides contents in red leaf lettuce (García-Macías et al., 2007). On the Bologna, BO, Italy) five days after transplanting (DAT). No insecticide
other hand, Liu et al. (2015) observed higher biomass and camp- or fungicide treatments were performed.
tothecin yield in Camptotheca acuminate, grown under red plastics. Starting from transplanting, the greenhouse’s environmental con-
Sweet basil (Ocimum basilicum L.) is an aromatic herb belonging to ditions were constantly monitored with sensors of temperature and
Lamiaceae family, which is extensively used fresh or dried to add a humidity connected to a data logger system (EM50 Data Collection
distinctive aroma and flavor to food, due to the strong content of es- System, Decagon Devices Inc., Pullman, WA, USA) (Fig. 1).
sential oils; it contains high concentrations of phenolic compounds that
contribute to its strong antioxidant capacity (Kwee and Niemeyer, 2.2. Treatments and experimental design
2011; Bufalo et al., 2015). Among these, rosmarinic acid represents the
most prevalent basil’s phenolic compound and it is associated with its The experiments were arranged as a completely randomized block
functional properties; other important phenolic compounds are re- design with two replicates. Experimental treatments consisted on three
presented by caffeic acid derivatives, such as chicoric acid (Kwee and modifications of the transmitted solar radiation achieved with the use
Niemeyer, 2011). of three different colored plastic films: a yellow film (named as YF), a
To date, in basil only few investigations on the effects of light ma- green film (GF) and a blue film (BF); an uncovered treatment was in-
nipulation on morphological and physiological traits, as well as on cluded as control (Control). The colored films used in this study are
some biochemical compounds, have been carried out. Growth and yield actually made to be used for other purposes than the agricultural sector,
performances of basil plants were investigated under both artificial LED but have been adapted to cover the basil plants through the support of a
or/and fluorescent lamps (Frąszczak et al., 2014; Bantis et al., 2016) hand-made rigid and removable structure (1.5 m × 1.5 m) placed above
and photoselective nets (Shahak et al., 2008). Regarding quality traits, the vegetation. The structures were covered from the top and sides to
the content of total volatile oils was strongly reduced under heavy ∼1 cm above the bottom of the pots to allow air circulation and secure
shaded conditions (Chang et al., 2008), while the setting of specific proper temperature and humidity; light was not filtered from below. In
spectra has been demonstrated to significantly influence phenolic particular, the colored films were purchased from a local store and
content as well as antioxidant activity. According to Bantis et al. provided by RiPlast S.r.l. (Pogliano Milanese, MI, Italy); yellow and
(2016), LED applications with high blue light portion induce phenolic blue films were 90 μM thick scratched polypropylene films, while green
compounds accumulation as well as red light has a preeminent role in was a 80 μM thick polyvinyl chloride frosted film; thickness was mea-
the regulation of phenolic acids biosynthesis (Taulavuori et al., 2017). sured by a thickness gauge (Vogel S.r.l., Leno, BS, Italy). The Fig. 2
Irradiation (fluorescent lamps) with red and white rather than blue shows a picture detail of the three films. The colored films were applied
light, seems to favor higher rosmarinic acid accumulation (Shiga et al., starting from transplanting and each treatment consisted of 98 pots,
2009). with 49 pots representing one experimental unit; pots were south-north
On these basis, the main objective of this study was to test the (S-N) oriented.

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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 measure-
ments 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 trans-
planting (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 environ-
ment, 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
Fig. 1. Patterns of air temperature (T, °C; solid line chart) and relative air hu-
calculated as the sum of leaves and stems DW. Before drying, shoot
midity (RH, %; dashed line chart) as recorded under greenhouse during the two
basil cycles, starting from transplanting: (A) spring transplanting 2014, Spr_Tr, length (cm) was measured and leaf area (LA, cm2) was recorded after
and (B) summer transplanting 2015, Sum_Tr. Each point represents the daily acquiring scanned leaves’ images with an image analysis software
mean of 24 h collected values. (ImageJ, National Institutes of Health, Bethesda, MD, USA). The spe-
cific leaf area (SLA, cm2 g−1) was then calculated.
Colored films were characterized for their optical properties. The
photosynthetically active radiation (PAR, range 400–700 nm) trans- 2.4. Physiological traits
mitted to canopy was measured with PAR photon flux sensors (Decagon
Devices, USA) placed above the vegetation and connected to a data The canopy reflectance was measured with a HandHeld 2 Pro
logger (EM50 data collection system, Decagon Devices, USA). Starting Portable FieldSpec Spectroradiometer (ADS Inc., Boulder, CO, USA) in
from transplanting and for each experimental treatment, the measure- eight plants – without cover – per treatment (four from each plot of 49
ments were constantly recorded (every 10 min) during the crop plants). Measurements were carried out starting from 150 and 210 GDD
growing cycles. The percentages of shading with respect to Control, in Spr_Tr and Sum_Tr, respectively, for a total of six sampling dates
averaged over growing cycles, were reported in Fig. 3 (box). In ac- during the whole crop growing cycles (matching with the growth
cordance with Shahak et al. (2004), the amount of scattered light in the analysis determinations). To minimize the effects of the sun’s position,
PAR region was measured at noontime of clear days using an opaque the reflectance measurements were taken within 1 h, near solar noon.
disc, held to 30–40 cm above the PAR sensor. The percentage of scat- Using the reflectance data, some important vegetation indices –
tering was then calculated as the ratio between not-direct light to total Modified Chlorophyll Absorption Ratio Index (MCARI), the Normalized
light (box in Fig. 3). 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.)

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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 char-
acterized 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° Spr_Tr 2014 Sum_Tr 2015

DATa GDDb NumLPc NumASd DAT GDD NumLP NumAS

I 14 150 13 210
Control 2.8 2.8 4.6 7.5
YF 3.0 2.0 4.8 7.8
GF 3.0 4.2 4.8 7.7
BF 3.0 2.8 5.0 8.0
II 20 230 16 264
Control 3.5 5.8 5.2 8.0
YF 4.0 6.7 5.2 8.7
GF 4.7 7.2 5.5 8.0
BF 4.0 7.3 5.0 8.0
III 26 313 18 300
Control 4.2 7.8 5.2 10.0
YF 4.7 8.3 5.3 9.3
GF 5.0 8.7 5.5 10.2
BF 4.5 8.5 5.0 10.0
IV 29 363 20 339
Control 5.0 9.3 5.2 8.7
YF 5.2 7.7 5.5 11.0
GF 5.7 11.7 5.8 12.0
BF 5.0 10.2 5.2 11.0
V 32 412 23 403
Control 5.0 9.3 6.0 13.3
YF 5.5 9.7 6.0 13.7
GF 6.2 12.3 6.0 14.3
BF 5.7 10.3 6.0 13.0
VI 35 457 25 449
Control 5.7 10.0 7.0 14.0
YF 6.3 10.8 7.5 15.0
GF 6.7 12.8 8.0 16.0
BF 6.0 11.5 7.0 14.0

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

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Vegetation Index (GNDVI) – were calculated in order to estimate (0.1–1.0 μL) was diluted with deionized water to a volume of 5 mL and
chlorophyll (Chl), as follows: 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
MCARI = [(R700-R670) − 0.2 × (R700- (Daughtry et al., 10 mL final volume. Solutions were maintained at room temperature
R550)] × (R700/R670) 2000) under dark conditions for 60 min and the total polyphenols content was
NDVI670 = (R800 − R670)/(R800 + R670) (Rouse et al., determined at 765 nm using a Perkin Elmer Lambda Bio 20 spectro-
1974) photometer. Gallic acid standard (Fluka, Buchs, CH) solutions were
OSAVI = (1 + 0.16) × (R800 − R670)/ (Rondeaux et al., used to calibrate the method.
(R800 + R670 + 0.16) 1996)
GNDVI = (R800 − R500)/(R800 + R500) (Gitelson et al.,
1996) 2.8. Identification and quantification of phenolic compounds
The literatures have proposed many spectral indices also for car-
otenoids (Car) estimation using diverse ratios of wavelengths in the Identification and quantification of the phenolic compounds were
visible and visible/NIR. Following the results of Yi et al. (2014) in carried out according to the method described by Llorach et al. (2008)
cotton, the Carotenoid Concentration Index700 (CRI700) and the Chlor- with slight modifications. Chromatographic analyses were performed
ophyll Index Red Edge (CIred-edge) were calculated as follows: on a 1200 Agilent Series HPLC (Agilent Technologies, Milano, Italy)
equipped with a G1322 degasser, a G1311A quaternary pump, a G136A
CRI700 = (1/R515 − 1/R700) (Gitelson et al., 2006) Column thermostat, an autosampler injection system and a diode array
CIred-edge = ((R800/R750) − 1) (Gitelson et al., 2005) detector. The system was controlled with Agilent ChemStation for
The reflectance results were also compared with that obtained by Windows (Agilent Technologies). Separations were achieved on a Ki-
soil-plant analysis development (SPAD) readings. Measurements were netex C18 column 5 μm 100A 250 × 4.6 mm (Phenomenex, Castel
performed at the same sampling dates using a 502 plus portable Maggiore, Italy).
chlorophyll meter (Konica Minolta, Inc., Tokyo, Japan). The Chl con- The mobile phases were water with 5% formic acid (A) and me-
tent was estimated in the mid-section of three fully expanded and same thanol (B) with a solvent flow rate of 1 mL min−1 in a gradient program
sun-oriented leaves per experimental unit. 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,
2.5. Chlorophylls and carotenoids analysis caftaric acid, rosmarinic, and cichoric acid were quantified by means of
calibration curves obtained in the range 1.25–100 mg L−1.
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 2.9. Radical scavenging activity
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 The radical scavenging activity of the extracts was measured ac-
cutting with a metal tube and weighted for FW determinations. The cording to the method described by Re et al. (1999) with some mod-
sub-samples were collected from two plants per experimental unit. In ifications. ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic
particular, for each plant leaves were distinguished on the basis of: (i) acid) – Fluka, Buchs, Switzerland) was dissolved in water to a 7 mM
their relative sun position, as upper, sunny exposed leaves and lower, concentration; the ABTS radical was formed by reacting ABTS stock
shaded leaves; (ii) their relative plant position, as leaves of main shoot solution with 2.45 mM potassium persulphate and allowing the mixture
and leaves of axillary shoot. In summary for each plant, four analytical to stand in the dark at room temperature for 12–16 h before use. The
determinations were performed on: main shoot-upper leaf, main shoot- ABTS radical solution was diluted with deionised water to reach an
lower leaf, axillary shoot-upper leaf, and axillary shoot-lower leaf. absorbance of 0.70 ± 0.02 at 734 nm and at 30 °C. The absorbance at
Pigments were extracted by placing the leaf discs in 5 mL of 734 nm was evaluated by a Perkin Elmer (Boston, MA, USA) Lambda
N,Ndimethylformamide (DMF) for 36 h in dark conditions at 5 °C Bio 20 spectrophotometer. The reaction was started by the addition of
(Moran and Porath, 1980). Absorbance of the extracts was measured at 30 μL of extract to 2970 μL of ABTS%+. The time of analysis, 5 min, was
wavelengths of 664, 647 and 480 nm for Chla, Chlb and Car, respec- chosen after preliminary tests and represented the time necessary to
tively, with a UV/vis spectrophotometer (PerkinElmer, Waltham, MA, reach at least 80% of the overall inhibition of the ABTS radical at a
USA) and 1.00-cm quartz cuvettes. Pigment concentrations were then given concentration. For each sample, the percentage of inhibition (I%)
calculated with the equations proposed by Wellburn (1994). was plotted as a function of concentration and the TEAC (Trolox
Equivalent Antioxidant Capacity) calculated as the ratio of the linear
2.6. Extraction of phenolic compounds regression coefficient of the sample to that of the Trolox standard
(Fluka). Results were expressed as micromoles of Trolox equivalents per
For the extraction of polyphenols, basil was freeze-dried im- gram of fresh weight. The coefficient of variation of the method was
mediately after collection and the powder was stored at −40 °C until below 5%.
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 2.10. Statistical analysis
for 5 min at 4000 rpm and the supernatant was filtered with cellulose
filters. The extracts were used to evaluate total polyphenolic content, A one-way analysis of variance (ANOVA) was applied to test (F-test)
phenolic pattern, and antiradical activity. All the extracts were stored at the effects of treatment (YF, BF, GF and Control). When significant
−40 °C until analysis. Quality traits were assessed only during Sum_Tr. differences were detected, means separation was conducted by applying
the Fisher’ LSD (Least Significant Difference) at the 5% (p < 0.05)
2.7. Total polyphenols content level of significance; the standard error of the difference (s.e.d.) be-
tween means was also reported in Tables. ANOVA assumptions were
The total polyphenol content (TPC) of the free and bound phenolic tested through graphical methods.
fractions was evaluated using the Folin-Ciocalteau reagent and fol- The statistical analyses were performed using R (R Core Team,
lowing a method adapted from Singleton and Rossi (1965). The sample 2017).

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3. Results Control (199.4, 191.1, 167.3, and 148.4 cm2 g−1, averaged over GDD
and transplanting time) (Figs. 5E and F).
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 re- Table 3 shows the Chla, Chlb, Total Chl, and Car contents in basil
duction (51.8% of shading), followed by GF (42.7%) and YF (28.3%), as leaves, differently positioned on the plant, hence characterized by dif-
well as the highest relative amounts of scattered PAR (48.7% vs. 42.0% ferent age and relative sun exposition. Although significant differences
and 42.1% for BF, YF and GF, respectively). restricted only for some variables, few clear trends were observed. In
With respect to the single wavelengths of the transmitted radiation, the leaves of the main shoot, Green colored film gave higher Chla, Chlb,
as expected, YF absorbed in the blue-violet region (400–500 nm), with and Total Chl contents, while Control the lowest, regardless of leaf
reflectance values (on the certified reflective surface) lower than the position (i.e. upper or lower leaf) and transplanting time (Total Chl:
two other covered treatments, while transmitted more than GF and BF, 1339 and 860 μg g−1 FW for GF and Control, respectively, averaged
starting from around 540 nm to 850 nm (Fig. 3). Conversely, GF and BF over leaf position and transplanting time). In general, lower leaves
behaved similarly in the reflectance spectra, showing a valley from showed higher pigment content (10% higher than upper ones)
about 550–750 nm. Blue film treatment exhibited higher transmittance (Table 3). In Sum_Tr, in the leaves collected from the axillary shoots,
values with respect to GF only around 400–450 nm (blue-violet region) the lowest Chl values were always found in Control treatment and GF
and around 760–820 nm (near-infrared region). Green film also showed confirmed to exhibit the highest values (Total Chl: 1547 and
the highest reflectance values in the NIR region (from around 850 nm) 1038 μg g−1 FW for GF and Control, respectively, averaged over leaf
(Fig. 3). position); conversely, in Spr_Tr BF registered the highest values
(Table 3). Differences in Car content were significant only in lower
3.2. Basil growth leaves of axillary shoots during Spr_Tr, with GF confirming to stimulate
pigment accumulations (101 μg g−1 FW) (Table 3).
In both Spr_Tr and Sum_Tr the number of leaf pairs on the main The indices used to estimate the Chl and Car content in basil leaves,
stem increased as the growing cycle went on (Table 1). In Spr_Tr GF are reported in Table 4. Differences between SPAD values were not
showed always the higher NumLP while Control the lowest; in Sum_Tr always significant among treatments: GF gave higher values, regardless
such trend was confirmed, but with generally higher values at the same of transplanting time (on average, 39.9 and 39.3 in Spr_Tr and Sum_Tr,
GDD. respectively). However, no correlation was observed between SPAD and
In general, also for NumAS the highest values were registered by GF Chl content in basil leaves of both ages and position.
while the lowest by Control; nevertheless Sum_Tr allowed higher va- The values of MCARI, NDVI670, GNDVI, and OSAVI remained fairly
lues, although such differences among treatments emerged starting stable among the two transplanting times; anyway, the discriminatory
from 300 GDD (Table 1). capacity of such indices was higher during Spr_Tr (Table 4), when the
Light manipulation significantly influenced main shoot length values of some indices (at 457 GDD) were correlated with the Total Chl
(Fig. 4). Also with this trait, GF induced the highest values than the content in leaves (see the correlations in Table 5). Note that NDVI670
other treatments while Control the lowest. Differences between YF and and OSAVI gave the highest and significant correlations ((i) NDVI670 vs.
BF were inconsistent, probably related to sampling time. Such trend Total Chl (main shoot) = 0.638; (ii) NDVI670 vs. Total Chl (axillary
was observed in both growing seasons (Fig. 4). shoot) = 0.748; (iii) OSAVI vs. Total Chl (main shoot) = 0.722; (iv)
Total aerial biomass (DW) resulted significantly influenced by OSAVI vs. Total Chl (axillary shoot) = 0.819).
plastic films application (Table 2). In Spr_Tr, YF induced higher values In general, BF enhanced the CIred-edge values, followed by GF and
already starting from 150 GDD, reaching 4.03 g plant−1 at 457 GDD; GF YF, in both Spr_Tr and Sum_Tr; on the other hand, the behaviour of
and Control gave the lowest biomass accumulation reaching 2.77 and CRI700 resulted pretty unclear (Table 4). Also for CIred-edge and CRI700 −
2.70 g plant−1 at 457 GDD, respectively (Fig. 5A). In general, Sum_Tr which were selected because their potential as Car indicators − the
allowed higher biomass DW accumulation than Spr_Tr (1.66 and 3.06 g higher correlations with the analytically measured Car contents, were
plant−1 respectively, averaged over treatments and GDD) (Fig. 5A and observed during Spr_Tr (CIred-edge vs. Car − axillary shoot: 0.692) (data
5B). Significantly differences among treatments emerged only from 339 not shown).
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 3.4. Quality traits
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) The total phenolic content and the antiradical activity in basil, as
(Fig. 5B). affected by colored films application, at different GDD are presented in
The trend of LA over thermal time showed similar patterns in all Fig. 6. The TPC ranged from 37 to 70 mg GAE g−1 DW, 90–150 mg GAE
theses and in both growing seasons (Figs. 5C and D). During Spr_Tr, g−1 DW and from 70 to 130 mg GAE g−1 DW for samples at 264, 339
plants under YF film reached the highest LA values at all sampling dates and 449 GDD respectively. At the first sampling time, the application of
(see also Table 2), followed by BF and GF (on average: 318.4, 272.7 and yellow, blue, and green films lowered phenolic compounds accumula-
259.2 cm2 plant−1, respectively); Control plants exhibited always the tion, with respect to Control. As the crop growing cycle went on, TPC
lowest values (202.5 cm2 plant−1 averaged over GDD) (Fig. 5C). Dif- increased, although at 339 GGD significant differences among treat-
ferently, in Sum_Tr, GF showed significantly higher values than BF ments were not detected. At 449 GDD, while YF, GF, and BF confirmed
during the middle phases of the crop cycle (averaged over 339 and 403 their values, Control reduced significantly its values to 41.4 mg GAE
GDD: 462.9 vs. 411.3 cm2 plant−1 for GF and BF, respectively); g−1 DW. The utilization of colored films did not enhance TEAC values;
nevertheless, YF maintained high values, especially from 339 GDD, and on average, the TEAC values were 131, 113 and 106 μmol TE g−1 DW at
Control confirmed the lowest LA values (253.2 cm2 plant−1 averaged 264, 339 and 449 GGD, respectively. Regarding the phenolic pattern of
over GDD) (Fig. 5D). the extracts, the content of caftaric, caffeic, chicoric and rosmarinic
Specific leaf area gave the same responses to the different covered acids are reported in Table 6. The application of colored films always
treatments regardless of transplanting times, despite a greater influence reduced the concentration of rosmarinic and caftaric acids in basil
it would seem observable during Spr_Tr (Figs. 5E and F). Green film leaves; in the case of rosmarinic acid, the reduction was pretty severe
allowed significantly higher SLA values, followed by BF, YF, and when GF was applied. Caftaric and rosmarinic acids showed an

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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).

increased accumulation over time whilst a decreasing trend was ob- 4. Discussion
served for caffeic acid. Interestingly, GF significantly enhanced caffeic
acid content at harvesting (449 GDD), while YF induced a higher ac- The manipulation of solar irradiance through the application of
cumulation of chicoric acid in the last two samplings although differ- photo-selective colored plastic films significantly affected some mor-
ences were not significant. phological and biochemical traits of basil plants; anyway, a dis-
crimination 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 150 GDD 230 GDD 313 GDD 363 GDD 412 GDD 457 GDD

Spr_Tr 2014
Total biomass (g plant−1) 0.021 0.007 0.030 0.007 < 0.001 < 0.001
LA (cm2 plant−1) 0.034 0.005 0.006 0.003 0.006 0.001
SLA (cm2 g−1) 0.007 0.006 < 0.001 0.013 0.002 0.058
TIR (°C) 0.036 0.125 < 0.001 0.003 0.077 0.019
210 GDD 264 GDD 300 GDD 339 GDD 403 GDD 449 GDD

Sum_Tr 2015
Total biomass (g plant−1) 0.137 0.068 0.060 0.017 0.032 0.047
LA (cm2 plant−1) 0.027 0.021 0.012 0.002 0.003 0.009
SLA (cm2 g−1) 0.004 0.005 0.007 0.033 < 0.001 < 0.001
TIR (°C) 0.035 0.008 < 0.001 0.002 0.008 0.010

GDD, growing degree days.

a
One-way ANOVA: degrees of freedom: Blocks 1; Treatment 3; Residual 3.

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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 dif-
ferent 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 trans-
planting – Sum_Tr – 2015). Reported data re-
present 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 ex-
pressed 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 en- characterized by very similar patterns in terms of transmitted radiation
vironment (Bantis et al., 2016; Ye et al., 2017). All the covered treat- to canopy, even though BF has higher scatter properties. The latter
ments enhanced plant height (as recorded by the higher values of the characteristic has a heavy impact on plant growth, independently from
main shoot length), independently from the transplanting times, prob- the alteration of light spectrum, due to the improved deeper light pe-
ably due to the shading effect which in turn influence the red to far red netration, which induces a compact habitus in plants (Nissim-Levi et al.,
ratio (R:FR). It is notorious that shading lowers R:FR, inducing photo- 2008; Ilić and Fallik, 2017). In any case, the relation between shading
morphogenic responses of plants, such as stem elongation (Demotes- level and scattering, as observed under BF, should not be neglected.
Mainard et al., 2016; Han et al., 2017): basil plants supplied with dif- Despite low R:FR mainly stimulates internode elongation rather
ferent LED light treatments (Bantis et al., 2016) as well as chry- than the total number of internodes (Franklin and Quail, 2010), the
santhemum and bell pepper plants grown under photoselective plastic application of colored plastic films enhanced the NumLP as well as
films (Li et al., 2000) exhibited higher plant heights. It has to be NumAS with respect to Control plants. Nevertheless, low-light or dark
highlighted that in this work higher heights were given by GF and not conditions generally induce a faster plant growth than bright light, in
by the most shaded film (i.e. blue film); however, GF and BF are order to complete plant’s life cycle and improve biological yield (Ye

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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 Sum_Tr 2015

Chl a Chl b Total Chl Car Chl a Chl b Total Chl Car

−1 a −1
(μg g FW ) (μg g FW)

Main shoot
Upper leaves
Control 427 258 685 126 563 b 381 944 113
YF 533 347 881 126 567 b 385 952 121
GF 615 415 1030 124 791 a 861 1652 82
BF 577 376 952 124 590 b 468 1057 101
p-value 0.408 0.218 0.322 0.999 0.012 0.179 0.066 0.757
s.e.d. 30.410

Lower leaves
Control 595 387 982 141 473 355 827 104
YF 736 462 1199 152 559 450 1009 95
GF 800 530 1330 158 758 587 1346 110
BF 784 454 1239 157 662 475 1137 100
p-value 0.349 0.476 0.367 0.823 0.064 0.123 0.081 0.426
s.e.d.

Axillary shoot
Upper leaves
Control 328 c 214 542 c 106 540 361 901 106
YF 463 bc 285 749 bc 119 603 518 1121 113
GF 507 ab 331 838 ab 115 727 580 1307 117
BF 650 a 381 1031 a 146 658 497 1154 113
p-value 0.034 0.067 0.042 0.136 0.741 0.054 0.414 0.994
s.e.d. 53.572 88.409

Lower leaves
Control 325 b 224 549 72 b 683 492 1175 157
YF 430 ab 273 703 89 ab 522 366 889 117
GF 524 a 329 853 101 a 991 797 1788 155
BF 547 a 364 910 96 a 756 567 1323 172
p-value 0.048 0.288 0.120 0.049 0.330 0.426 0.373 0.779
s.e.d. 46.261 5.918

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.

et al., 2017). Differences among crop growing seasons (i.e. Spr_Tr and Such morphological adaptations were associated also to physiolo-
Sum_Tr) are probably related to air temperatures and day-length con- gical changes such as an increase in chlorophyll content in basil leaves.
ditions, since basil is a warm season crop, mainly grown at relative high Shaded grown leaves receive lower light amounts, and consequently
temperatures (Fallahi et al., 2015). It was confirmed by the different contain more chlorophyll than those exposed to high irradiance levels
behavior of Control plants: the highest Sum_Tr temperatures (on (see also Chl content in lower leaves) (Ilić and Fallik, 2017; Yang et al.,
average, 24.2 °C vs. 19.2 °C in Spr_Tr) guaranteed optimal plant growth 2018). Also for this trait, the scatter properties of BF emerged, inducing
(biomass accumulation in leaves), while during Spr_Tr, plants grown a reduction in Chl content. The analytical measured Chl amount was
under the colored plastic films received slightly higher temperatures properly by the estimation through the reflectance indices. Apart SPAD,
than greenhouse conditions (data not shown). some indices already validated and calibrated at canopy scale have
Differences in biomass accumulation among covered treatments been selected (MCARI, OSAVI, NDVI700 and GNDVI) (Haboudane et al.,
were probably related to both shade levels and light spectrum. Yellow 2002). The Optimized Soil-Adjusted Vegetation Index and NDVI700
film treatment allowed the highest biomass accumulation while GF the seem the more suitable indicators for such trait, despite other spectral
lowest, regardless of the crop growing season. Such results are related indices, based on other NIR and red wavelengths (i.e. 875–900 and
to a lower shade percentage in combination with the highest trans- 720–725) (Wang et al., 2015) or originated by specific combinations
mission in the 540–700 nm PAR range, which guarantees a high pho- (i.e. OSAVIxSIPI or OSAVIxCIred-edge) (Jin et al., 2013), could better
tosynthetic activity. Also scattering properties play an important role in perform.
biomass accumulation as demonstrated by the higher values of plants Filtering the transmitted solar radiation through photoselective
grown under BF (Shahak et al., 2008; Ilić and Fallik, 2017). films or shade nets is known to sharply affect quality and yield of crops
The effect of shading emerged also in terms of a greater leaf area (Nissim-Levi et al., 2008). The effect of light quantity and quality ma-
expansion confirming previous results in bell pepper (Díaz-Pérez, 2013; nipulation on some important molecules for human health, such as
Ilić et al., 2017a), tomato (Ilić et al., 2015), and lettuce (Ilić et al., phenolic acids, has been assessed. In basil leaves the amount of total
2017b), as well as of a lower leaf thickness (higher SLA values) as an phenolic compounds resulted higher than the values reported in Kwee
adaptive response to capture more light (Ballaré and Pierik, 2017; and Niemeyer (2011) but comparable to Ghasemzadeh et al. (2016).
Bertel et al., 2017; Han et al., 2017). Interestingly, as already observed, the treatments did not induce any

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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 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

SPAD
Control 39.6 40.7 bc 41.4 40.5 35.3 34.4 38.3 39.4 bc 39.6 41.2 b 34.9 33.6
YF 39.3 39.2 c 38.0 39.5 35.9 33.1 38.3 38.0 c 39.6 42.8 a 34.2 34.4
GF 42.3 42.8 a 41.3 40.5 36.7 35.8 41.9 41.4 ab 41.9 39.1 c 36.5 35.1
BF 41.2 41.6 ab 41.0 39.2 36.5 35.5 40.7 41.7 a 37.3 41.6 b 35.8 36.3
p-value 0.546 0.033 0.126 0.649 0.418 0.543 0.198 0.029 0.234 0.007 0.730 0.449
s.e.d. 0.604 0.665 0.356

MCARI
Control 0.498 a 0.121 ab 0.180 0.361 0.333 0.599 0.299 0.475 a 0.291 0.595 0.407 0.540
YF 0.438 a 0.151 a 0.195 0.323 0.407 0.540 0.275 0.507 a 0.238 0.539 0.308 0.476
GF 0.453 a 0.122 ab 0.196 0.303 0.367 0.631 0.246 0.346 b 0.218 0.502 0.317 0.480
BF 0.399 b 0.091 b 0.166 0.278 0.320 0.521 0.201 0.315 b 0.242 0.420 0.296 0.463
p-value 0.049 0.046 0.127 0.526 0.211 0.188 0.051 0.032 0.195 0.179 0.215 0.620
s.e.d. 0.006 0.011 3.719

NDVI670
Control 0.832 0.697 0.823 b 0.859 b 0.859 b 0.840 c 0.761 c 0.820 0.835 0.817 b 0.814 0.809
YF 0.825 0.789 0.880 a 0.878 a 0.869 a 0.863 ab 0.821 bc 0.820 0.840 0.856 a 0.807 0.808
GF 0.826 0.741 0.877 a 0.859 b 0.876 a 0.855 bc 0.876 ab 0.847 0.839 0.812 b 0.813 0.834
BF 0.823 0.768 0.884 a 0.865 b 0.877 a 0.878 a 0.899 a 0.839 0.831 0.806 b 0.816 0.805
p-value 0.843 0.223 0.025 0.014 0.021 0.029 0.024 0.050 0.975 0.044 0.977 0.499
s.e.d. 0.011 0.003 0.003 0.006 0.022 0.010

GNDVI
Control 0.603 0.507 0.656 0.658 b 0.669 0.627 b 0.516 c 0.514 b 0.534 0.508 0.484 0.464
YF 0.594 0.529 0.699 0.704 a 0.692 0.617 b 0.582 bc 0.524 b 0.569 0.558 0.493 0.481
GF 0.588 0.493 0.693 0.669 b 0.678 0.627 b 0.653 ab 0.568 a 0.550 0.509 0.468 0.484
BF 0.602 0.526 0.721 0.687 ab 0.704 0.673 a 0.697 a 0.571 a 0.568 0.534 0.472 0.509
p-value 0.657 0.553 0.148 0.049 0.059 0.012 0.016 0.025 0.412 0.121 0.286 0.248
s.e.d. 0.009 0.007 0.024 0.011

OSAVI
Control 0.783 a 0.524 0.688 0.779 0.784 b 0.836 c 0.751 0.793 0.765 0.806 b 0.747 0.759
YF 0.759 b 0.580 0.723 0.822 0.842 a 0.853 bc 0.794 0.810 0.776 0.828 a 0.722 0.766
GF 0.766 ab 0.538 0.723 0.795 0.828 a 0.862 ab 0.838 0.800 0.765 0.800 b 0.710 0.760
BF 0.717 c 0.527 0.731 0.793 0.834 a 0.880 a 0.823 0.800 0.776 0.792 b 0.708 0.775
p-value 0.001 0.290 0.288 0.054 0.009 0.017 0.092 0.504 0.259 0.019 0.318 0.828
s.e.d. 0.007 0.007 0.006 0.005

CIred-edge
Control 0.043 0.063 b 0.038 0.027 0.035 c 0.027 b 0.025 0.015 b 0.048 0.047 0.037 c 0.027
YF 0.050 0.044 c 0.057 0.032 0.055 b 0.058 b 0.033 0.017 b 0.048 0.057 0.042 bc 0.041
GF 0.042 0.068 b 0.059 0.031 0.054 b 0.031 b 0.034 0.032 ab 0.068 0.045 0.056 ab 0.040
BF 0.048 0.093 a 0.110 0.040 0.089 a 0.117 a 0.068 0.044 a 0.053 0.053 0.066 a 0.050
p-value 0.645 0.002 0.202 0.092 0.009 0.012 0.155 0.036 0.396 0.321 0.049 0.145
s.e.d. 0.003 0.006 0.012 0.006 0.006

CRI700
Control 5.300 4.368 9.022 7.459 a 6.343 3.960 a 2.617 c 4.073 4.308 3.508 b 3.841 4.489
YF 5.412 5.446 9.617 6.588 b 4.490 2.848 b 3.704 bc 3.486 3.756 3.166 bc 4.220 4.264
GF 5.202 3.812 9.758 5.529 c 4.557 3.283 ab 5.469 b 4.575 2.638 2.769 c 3.015 3.603
BF 7.473 4.706 9.386 6.103 bc 4.493 3.007 b 8.563 a 3.663 3.699 4.235 a 3.749 4.741
p-value 0.092 0.358 0.854 0.011 0.071 0.049 0.007 0.251 0.103 0.007 0.058 0.067
s.e.d. 0.223 0.226 0.585 0.145

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

significant difference in the radical scavenging activity at 264 and 449 agreement also with previous data in literature where rosmarinic acid
GDD, regardless the differences in the TPC values. Probably, the was found to be the most abundant phenolic compound (Jayasinghe
treatments influenced also the accumulation of specific phenolic com- et al., 2003; Kim et al., 2006; Lee and Scagel, 2009; Nguyen et al.,
pounds with different antiradical activity. Thus, the phenolic pattern of 2010). Rosmarinic and chicoric acids contents were comparable to the
the extracts was also investigated. In the conditions adopted, four main ones found by Taulavuori et al. (2017).
peaks were detected and identified as caftaric, caffeic, chicoric and The application of colored films always reduced the concentration
rosmarinic acids. Rosmarinic acid was the main phenolic compound, of rosmarinic and caftaric acids in basil leaves; for the former, the re-
followed by chicoric, caftaric and caffeic acids; this same order was duction was more severe when GF were applied. No significant differ-
reported also by Lee and Scagel (2009), with the exception of caffeic ences were observed for chicoric acid content, in contrast with
acid, which was not found by these authors. These results are in Taulavuori et al. (2017) who found an increased accumulation of

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Table 5 chicoric acid under the application of an enhanced blue light. Inter-
Correlation coefficients between Total Chlorophyll content in main and axillary estingly, at harvesting (449 GDD), caffeic acid was positively influenced
shoots (averaged over upper or lower positions) of basil plants − grown under by the application of colored films and in particular of GF.
different colored plastic films − and seven indices used to estimate pigment When the relationship among the total phenolic content, or the
content in leaves (soil plant analysis development (SPAD), Modified
amount of each phenolic acid, and the antiradical activity of the sam-
Chlorophyll Absorption Ratio Index (MCARI), Normalized Difference
ples was explored, it was interesting to observe that a linear correlation
Vegetation Index670 (NDVI670), Optimized Soil-Adjusted Vegetation Index
was found only with the content of caffeic acid.
(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 5. Conclusions
degree days (GDD), respectively.
Indices Total Chla(μg g−1 FW) Shading of basil plants, and thus lower R:FR, generally induced an
higher stem elongation, greater leaf area and lower leaf thickness.
Spring transplanting – Spr_Tr Summer transplanting – Sum_Tr Among the colored films, GF, with very similar transmitted radiation
spectra to BF, showed the highest effect on plant height, whilst YF al-
Main shoot Axillary shoot Main shoot Axillary shoot
ways allowed the highest biomass accumulation. The alteration of the
SPAD 0.343 n.s. 0.331 n.s. 0.513 n.s. 0.682* transmitted radiation also significantly influenced Chl biosynthesis as a
MCARI 0.118 n.s. -0.110 n.s. -0.101 n.s. -0.024 n.s. strategy amelioration of photosynthetic activity under unfavorable
NDVI670 0.638* 0.748** 0.399 n.s. 0.473 n.s. environments (i.e., shading conditions). Besides, analytical determina-
GNDVI 0.272 n.s. 0.621 n.s. 0.350 n.s. 0.466 n.s.
tions, Chl content in basil leaves could be proven estimated using re-
OSAVI 0.722** 0.819** 0.301 n.s. 0.445 n.s.
flectance data and vegetation indices (i.e. NDVI700 or OSAVI). Shading
n.s. = not-significant. showed a positive impact also on phenolic compounds accumulation;
a
Total Chlorophyll content in basil leaves. the application of GF generally decreased the content of rosmarinic and
* significant effect at the 0.05 probability level. caftaric acids, whilst at harvest increased caffeic acid accumulation;
** significant effect at the 0.01 probability level. 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

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).

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Table 6 Frąszczak, B., Golcz, A., Zawirska-Wojtasiak, R., Janowska, B., 2014. Growth rate of
Caftaric acid, caffeic acid, chicoric acid and rosmarinic acid as determined in sweet basil and lemon balm plants grown under fluorescent lamps and led modules.
leaves of basil plants grown under different colored plastic films (yellow film, Acta Sci. Pol. Hortorum Cultus 13 (2), 3–13.
Franklin, K.A., Quail, P.H., 2010. Phytochrome functions in Arabidopsis development.
YF, green film, GF and blue film, BF) plus an uncovered Control, at 16, 20 and Environ. Exp Bot. 61 (1), 11–24.
25 days after transplanting, corresponding to 264, 339 and 449 growing degree Galvão, V., Fankhauser, C., 2015. Sensing the light environment in plants: photo receptors
days (GDD), respectively. For each column, means labeled with the same letter and early signaling steps. Curr. Opin. Neurobiol. 34, 46–53.
did not differ significantly at p < 0.05 (Fisher’s LSD test). García-Macías, P., Ordidge, M., Vysini, E., Waroonphan, S., Battey, N.H., Gordon, M.H.,
Wagstaffe, A., 2007. Changes in the flavonoid and phenolic acid contents and anti-
Treatments Phenolic acids (mg g−1 DW) oxidant activity of red leaf lettuce (Lollo Rosso) due to cultivation under plastic films
varying in ultraviolet transparency. J. Agric. Food Chem. 55 (25), 10168–10172.
Caftaric acid Caffeic acid Chicoric acid Rosmarinic acid Ghasemzadeh, A., Ashkani, S., Baghdadi, A., Pazoki, A., Jaafar, H.Z., Rahmat, A., 2016.
Improvement in flavonoids and phenolic acids production and pharmaceutical
264 GDD quality of sweet basil (Ocimum basilicum L.) by ultraviolet-B irradiation. Molecules 21
Control 3.70 a 0.310 9.69 12.30 a (9), 1203.
YF 2.52 b 0.380 9.61 8.02 b Gitelson, A.A., Kaufman, Y.J., Merzlyak, M.N., 1996. Use of a green channel in remote
sensing of global vegetation from EOS-MODIS. Remote Sens. Environ. 58, 289–298.
GF 2.69 b 0.358 8.62 7.83 b
Gitelson, A.A., Vina, A., Ciganda, V., Rundquist, D.C., Arkebauer, T.J., 2005. Remote
BF 2.51 b 0.340 9.67 9.05 b
estimation of canopy chlorophyll content in crops. Geophys. Res. Lett. 32 (8).
p-value 0.029 0.476 0.504 0.004
Gitelson, A.A., Keydan, G.P., Merzlyak, M.N., 2006. Three-band model for noninvasive
s.e.d. 0.216 0.400 estimation of chlorophyll, carotenoids, and anthocyanin contents in higher plant
339 GDD leaves. Geophys. Res. Lett. 33 (11).
Control 3.74 a 0.273 8.84 12.97 Haboudane, D., Miller, J.R., Tremblay, N., Zarco-Tejada, P.J., Dextraze, L., 2002.
Integrated narrow-band vegetation indices for prediction of crop chlorophyll content
YF 3.25 b 0.254 9.77 11.46
for application to precision agriculture. Remote Sens. Environ. 81 (2), 416–426.
GF 2.42 c 0.261 7.57 9.67
Han, S., Chen, S.M., Song, A.P., Liu, R.X., Li, H.Y., Jiang, J.F., Chen, F.D., 2017.
BF 2.71 c 0.278 8.95 10.37
Photosynthetic responses of Chrysanthemum morifolium to growth irradiance:
p-value 0.006 0.823 0.465 0.418 morphology, anatomy and chloroplast ultrastructure. Photosynthetica 55 (1),
s.e.d. 0.131 184–192.
449 GDD Henschel, J.M., Resende, J.T., Giloni-Lima, P.C., Zeist, A.R., Lima Filho, R.B., Santos,
Control 4.97 a 0.124 b 9.76 16.73 M.H., 2017. Production and quality of strawberry cultivated under different colors of
low tunnel cover. Hortic. Bras. 35 (3), 364–370.
YF 4.78 a 0.163 ab 10.14 15.10
Ilić, Z.S., Fallik, E., 2017. Light quality manipulation improves vegetable quality at har-
GF 3.61 b 0.208 a 8.89 12.18
vest and postharvest: a review. Environ. Exp. Bot. 139, 69–90.
BF 3.46 b 0.190 a 9.29 13.38 Ilić, Z.S., Milenković, L., Šunić, L., Fallik, E., 2015. Effect of coloured shade-nets on plant
p-value 0.026 0.034 0.375 0.060 leaf parameters and tomato fruit quality. J. Sci. Food Agric. 95, 2660–2667.
s.e.d. 0.285 1.483 Ilić, Z.S., Milenković, L., Šunić, L., Barać, S., Mastilović, J., Kevrešan, Ž., Fallik, E., 2017a.
Effect of shading by coloured nets on yield and fruit quality of sweet pepper.
s.e.d., standard error of differences between means. One-way ANOVA: degrees Žemdirbystė Agric. 104 (1), 53–62.
of freedom: Blocks 1; Treatment 3; Residual 3. Ilić, S.Z., Milenković, L., Dimitrijević, A., Stanojević, L., Cvetković, D., Kevrešan, Ž.,
Fallik, E., Mastilović, J., 2017b. Light modification by color nets improve quality of
lettuce from summer production. Sci. Hortic. Amsterdam 226, 389–397.
affect growth and morphological traits as well as the accumulation of Jayasinghe, C., Gotoh, N., Aoki, T., Wada, S., 2003. Phenolics composition and anti-
secondary metabolites; light manipulation can thus represent a valid oxidant activity of sweet basil (Ocimum basilicum L.). J. Agric. Food Chem. 51 (15),
4442–4449.
tool for the enhancement of basil quality traits as long as a more
Jin, X.L., Diao, W.Y., Xiao, C.H., Wang, F.Y., Chen, B., Wang, K.R., Li, S.K., 2013.
comprehensive understanding of the mechanisms underpinning bioac- Estimation of wheat agronomic parameters using new spectral indices. PLoS One 8
tive accumulation is provided. (8), e72736.
Kim, H.J., Chen, F., Wang, X., Rajapakse, N.C., 2006. Effect of methyl jasmonate on
secondary metabolites of sweet basil (Ocimum basilicum L.). J. Agric. Food Chem. 54
References (6), 2327–2332.
Kwee, E.M., Niemeyer, E.D., 2011. Variations in phenolic composition and antioxidant
Ballaré, C.L., Pierik, R., 2017. The shade-avoidance syndrome: multiple signals and properties among 15 basil (Ocimum basilicum L.) cultivars. Food Chem. 128 (4),
ecological consequences. Plant Cell Environ. 40 (11), 2530–2543. 1044–1050.
Bantis, F., Ouzounis, T., Radoglou, K., 2016. Artificial LED lighting enhances growth Lee, J., Scagel, C.F., 2009. Chicoric acid found in basil (Ocimum basilicum L.) leaves. Food
characteristics and total phenolic content of Ocimum basilicum, but variably affects Chem. 115 (2), 650–656.
transplant success. Sci. Hortic. Amsterdam 198, 277–283. Li, S., Rajapakse, N.C., Young, R.E., Oi, R., 2000. Growth responses of chrysanthemum
Bertel, C., Schönswetter, P., Frajman, B., Holzinger, A., Neuner, G., 2017. Leaf anatomy of and bell pepper transplants to photoselective plastic films. Sci. Hortic. Amsterdam 84
two reciprocally non-monophyletic mountain plants (Heliosperma spp.): does heri- (3), 215–225.
table adaptation to divergent growing sites accompany the onset of speciation? Liu, Y., Song, L., Yu, W., Hu, Y., Ma, X., Wu, J., Ying, Y., 2015. Light quality modifies
Protoplasma 254 (3), 1411–1420. camptothecin production and gene expression of biosynthesis in Camptotheca acu-
Bufalo, J., Cantrell, C.L., Astatkie, T., Zheljazkov, V.D., Gawde, A., Boaro, C.S.F., 2015. minata Decne seedlings. Ind. Crop. Prod. 66, 137–143.
Organic versus conventional fertilization effects on sweet basil (Ocimum basilicum L.) Llorach, R., Martínez-Sánchez, A., Tomás-Barberán, F.A., Gil, M.I., Ferreres, F., 2008.
growth in a greenhouse system. Ind. Crop. Prod. 74, 249–254. Characterisation of polyphenols and antioxidant properties of five lettuce varieties
Buthelezi, M.N.D., Soundy, P., Jifon, J., Sivakumar, D., 2016. Spectral quality of photo- and escarole. Food Chem. 108 (3), 1028–1038.
selective nets improves phytochemicals and aroma volatiles in coriander leaves Moran, R., Porath, D., 1980. Chlorophyll determination in intact tissues using N, N-di-
(Coriandrum sativum L.) after postharvest storage. J. Photochem. Photobiol. B 161, methylformamide. Plant Physiol. 65 (3), 478–479.
328–334. Nguyen, P.M., Kwee, E.M., Niemeyer, E.D., 2010. Potassium rate alters the antioxidant
Chang, X., Alderson, P.G., Wright, C.J., 2008. Solar irradiance level alters the growth of capacity and phenolic concentration of basil (Ocimum basilicum L.) leaves. Food
basil (Ocimum basilicum L.) and its content of volatile oils. Environ. Exp. Bot. 63, Chem. 123 (4), 1235–1241.
216–223. Nissim-Levi, A., Farkash, L., Hamburger, D., Ovadia, R., Forrer, I., Kagan, S., Oren-
Díaz-Pérez, J.C., 2013. Bell pepper (Capsicum annum L.) crop as affected by shade level: Shamir, M., 2008. Light-scattering shade net increases branching and flowering in
microenvironment, plant growth, leaf gas exchange, and leaf mineral nutrient con- ornamental pot plants. J. Hortic. Sci. Biotech. 83, 9–14.
centration. HortScience 48 (2), 175–182. Peralta, G., Pérez-Lloréns, J.L., Hernández, I., Vergara, J.J., 2002. Effects of light avail-
Daughtry, C.S.T., Walthall, C.L., Kim, M.S., De Colstoun, E.B., McMurtrey, J.E., 2000. ability on growth, architecture and nutrient content of the seagrass zostera noltii
Estimating corn leaf chlorophyll concentration from leaf and canopy reflectance. hornem. J. Exp. Mar. Biol. Ecol. 269 (1), 9–26.
Remote Sens. Environ. 74 (2), 229–239. R Core Team, 2017. R: A language and environment for statistical computing. R
Demotes-Mainard, S., Péron, T., Corot, A., Bertheloot, J., Le Gourrierec, J., Pelleschi- Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/.
Travier, S., Vian, A., 2016. Plant responses to red and far-red lights: applications in Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999.
horticulture. Environ. Exp. Bot. 121, 4–21. Antioxidant activity applying an improved ABTS radical cation decolorization assay.
Fallahi, H.R., Mohammadi, M., Aghhavani-Shajari, M., Ranjbar, F., 2015. Determination Free Radic. Biol. Med. 26 (9), 1231–1237.
of germination cardinal temperatures in two basil (Ocimum basilicum L.) cultivars Rondeaux, G., Steven, M., Baret, F., 1996. Optimization of soil-adjusted vegetation in-
using non-linear regression models. J. Appl. Res. Med. Aromat. Plants 2 (4), 140–145. dices. Remote Sens. Environ. 55 (2), 95–107.
Folta, K., Carvalho, S., 2015. Photoreceptors and control of horticultural plant traits. Rouse, J.W., Hass, R.H., Schell, J.A., Deering, D.W., 1974. In. In: Monitoring vegetation
HortScience 50, 1274–1280. systems in the Great Plains with ERTS. The 3rd Earth Resources Technology Satellite-
1 Symposium, Greenbelt, MD. pp. 309–317.

288
F. Stagnari et al. Industrial Crops & Products 122 (2018) 277–289

Samuolienė, G., Brazaitytė, A., Viršilė, A., Jankauskienė, J., Sakalauskienė, S., comparative eco-development of sun and shade leaves in relation to photosynthetic
Duchovskis, P., 2016. Red light-dose or wavelength-dependent photoresponse of CO2 diffusion. J. Exp. Bot. 57 (2), 343–354.
antioxidants in herb microgreens. PLoS One 11 (9), e0163405. Valladares, F., Niinemets, Ü., 2008. Shade tolerance, a key plant feature of complex
Shahak, Y., Gussakovsky, E.E., Cohen, Y., Lurie, S., Stern, R., Kfir, S., Naor, A., Atzmon, I., nature and consequences. Annu. Rev. Ecol. Evol. Syst. 39.
Doron, I., Greenblat-Avron, Y., 2004. Color nets: a new approach for light manip- Wang, L., Yang, R., Tian, Q., Yang, Y., Zhou, Y., Sun, Y., Mi, X., 2015. Comparative
ulation in fruit trees. Acta Hortic. 636, 609–616. analysis of GF-1 WFV, ZY-3 MUX, and HJ-1 CCD sensor data for grassland monitoring
Shahak, Y., Gal, E., Offir, Y., Ben-Yakir, D., 2008. Photoselective shade netting integrated applications. Remote Sens. 7 (2), 2089–2108.
with greenhouse technologies for improved performance of vegetable and orna- Xu, F., Guo, W., Wang, R., Xu, W., Du, N., Wang, Y., 2009. Leaf movement and photo-
mental crops. In International Workshop on Greenhouse Environmental Control and synthetic plasticity of black locust (Robinia pseudoacacia) alleviate stress under dif-
Crop Production in Semi-Arid Regions 797, 75–80. ferent light and water conditions. Acta Physiol. Plant. 31 (3), 553–563.
Shiga, T., Shoji, K., Shimada, H., Hashida, S.N., Goto, F., Yoshihara, T., 2009. Effect of Yang, F., Feng, L., Liu, Q., Wu, X., Fan, Y., Raza, M.A., Cheng, Y., Chen, J., Wang, X.,
light quality on rosmarinic acid content and antioxidant activity of sweet basil, Yong, T., Liu, W., Liu, L., Du, J., Shu, K., Yang, W., 2018. Effect of interactions be-
Ocimum basilicum L. Plant Biotechnol. 26 (2), 255–259. tween light intensity and red-to-far-red ratio on the photosynthesis of soybean leaves
Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic- under shade condition. Environ. Exp. Bot. 150, 79–87.
phosphotungstic acid reagents. J. Enol. Vitic. 16 (3), 144–158. Ye, S., Shao, Q., Xu, M., Li, S., Wu, M., Tan, X., Su, L., 2017. Effects of light quality on
Stagnari, F., Galieni, A., Speca, S., Pisante, M., 2014. Water stress effects on growth, yield morphology, enzyme activities, and bioactive compound contents in anoectochilus
and quality traits of red beet. Sci. Hortic. 165, 13–22. roxburghii. Front. Plant Sci. 8.
Taulavuori, E., Taulavuori, K., Holopainen, J.K., Julkunen‐Tiitto, R., Acar, C., Dincer, I., Yi, Q., Jiapaer, G., Chen, J., Bao, A., Wang, F., 2014. Different units of measurement of
2017. Targeted use of LEDs in improvement of production efficiency through phy- carotenoids estimation in cotton using hyperspectral indices and partial least square
tochemical enrichment. J. Sci. Food Agric. regression. ISPRS J. Photogram. Rem. Sens. 91, 72–84.
Terashima, I., Hanba, Y.T., Tazoe, Y., Vyas, P., Yano, S., 2005. Irradiance and phenotype:

289