1. Introduction
Basil (
Ocimum basilicum L.) is an herbaceous species with a short-production cycle, appreciated as leafy green and aromatic plant [
1,
2]. A characterizing feature is that the secretory glandular trichomes of the basil leaves accumulate an essential oil giving the plant its easily recognizable, composite flavor [
3,
4]. Differently from many other spices, basil is not only used as food garnish and it is often consumed fresh. Therefore, this herb is also highly valued in different dietary regimes for its antioxidant capacity and as a source of mineral elements [
1,
5]. Finally, other uses of
Ocimum spp. are in the perfume and cosmetics industry, in food processing or as folk medicine [
2,
6].
As any other complex trait, the nutritional and the functional quality of basil, including flavor, depends on genetic (e.g., the variety) and environmental factors, and their interaction. Especially for green vegetables, the growing conditions and the time of the leaves’ harvest are central to manage crop quality. Among pre-harvest factors, plant nutrition is arguably the most important and easily tractable element that governs growth, yield, and the phytochemical composition of a plant.
In recent years, the modulation of the mineral nutrient solutions in soilless cultivation has been increasingly applied as a tool to study the various adaptive mechanisms to nutrient availability in higher plants [
7]. Moreover, these studies also gained popularity in addressing environmental issues (e.g., soil and groundwater contamination) [
7]. A wealth of knowledge has been gained especially in relation to the availability of essential minerals (e.g., nutrient limitation) or toxic elements (e.g., excessive amounts). It is long established that plant uptake and use of different ions depend on their relations yet, relatively little is known on the morphological and physiological responses (e.g., indexes of growth, leaf gas exchanges, ionic accumulation, production of secondary metabolites, etc.) to the concurrent modulation of ions in nutrient solutions with identical electrical conductivities (“ionic strength”). Moreover, understanding the effect of isosmotic variations in the nutrient solution (NS) provides knowledge to alter the phytochemical profile of a plant, an area of increasing importance also for research dietitians and the food industry [
8,
9].
The selective uptake and mobilization of nutrients by plants is often shaped by counterions and ion antagonisms. For instance, it has been long known that nitrate and chlorine have a competitive anionic interaction that in some cases can lead to nutritional imbalance [
10,
11]. These two anions have similar physical and osmotic properties, and share uptake and transport mechanisms in higher plants [
12,
13,
14]. This anion-anion interaction can be exploited to deliver valuable outcomes. For instance, nitrate uptake and accumulation in vegetables can be limited by high amount of chlorine [
15,
16]. On the other hand, nitrate can be exploited to mitigate chloride toxicity in Cl-sensitive crops [
17,
18]. Chloride is a plant micronutrient and since deficiency in soil rarely occurs, it has traditionally attracted attention as a potential toxic element when present at concentrations that exceed those required by a plant species (e.g., in saline environment). While it is well established that chloride excess limits yield [
19], recent studies also underlined the beneficial functions of this microelement, present also when it accumulates in an amount typical of a macronutrient [
14,
19]. In particular, chloride may positively influence nitrogen use efficiency and water-holding capacity, being also an effective osmoticum [
14,
20]. It has been shown that limited amount of chloride increased biomass and leaf area in
Nicotiana tabacum, although the largest biomass variation was achieved through nitrate modulation [
21]. The potential agronomical relevance of these effects, therefore, encourages new studies for sustainable crop management.
The impact of macro and micro-elements to increase yield and quality was studied in several aspects, and more detailed integrative investigations were recently conducted to determine the effect of the concurrent modulation of ions [
22]. Since chloride may have beneficial effects especially in comparison with nitrate [
14], in this work, we sought to examine the impact on basil of the simultaneous, opposite variation of nitrate and chlorine in nutrient solutions with the same ionic strength. Our hypothesis was to exploit the functional relations of the nitrate-chlorine modulation to sustainably increase basil quality while maintaining yield. To this aim, using a full factorial design, we evaluated changes in morpho-physiological, anatomical, chemical, and quality-related traits in basil in response to four different NO
3−:Cl
− ratios in two consecutive harvests.
2. Materials and Methods
2.1. Plant Material, Growth Conditions and Experimental Design
At the three true leaves stage, sweet basil plantlets (Ocimum basilicum L. var. basilicum, large leaved Genovese type) were transferred to dark plastic pots with 1.3 L of a 2:1 mixture (v/v) of peat:perlite at the Experimental Station of the Department of Agricultural Sciences (University of Naples Federico II) in Bellizzi (SA), 43°31 N, 14°58 E (60 m a.s.l.). Transplant was carried out on April the 28th, 2016. Pots were in a greenhouse, and the mean air temperature during the experiments was 26 °C (min: 16 °C; max: 33 °C). The relative humidity was set at 57% during day and 80% during night. Plastic pots were on an aluminum bench and each experimental unit (plot) comprised ten plants. The first and the last two were treated as guard plants not to be analyzed. Plants were in single rows with a 23 plants per square meter density. The experimental design was a randomized complete-block design (RCBD), with three replicates per level of factor, for a total of 24 experimental units. As fixed factors we considered the NO3−:Cl− ratio (R) in the nutrient solution (NS) (four levels) and the harvest (CT) of the leaves and stems (two levels). The four levels of factor R were named 80:20, 60:40, 40:60 and 20:80 according to the relative molar ratio of NO3− and Cl− in the nutrient solution. The NS was a modified Hoagland solution with the following composition: 1.5 mM phosphorus, 4.5 mM potassium, 6.5 mM calcium, 2.5 mM sulfur, 2.0 mM magnesium, 20 μM iron, 20 μM boron, 9 μM manganese, 1.6 μM zinc, 0.3 μM cupper, and 0.3 μM molybdenum. The electrical conductivity was 2.0 ± 0.1 dS m−1 and the pH was monitored and, when necessary, adjusted to be within the 6.0 ± 0.2 interval. We obtained the four NO3−:Cl− ratios by using different amounts of Ca(NO3)2 and CaCl2. Specifically, nitrate concentration (expressed as NO3− mM) and salt quantity (expressed as mg L−1 of Ca(NO3)2 and of CaCl2) in the NS were the following. Ratio 80:20: 9.6 mM, 867.1 and 133.2 mg L−1; 60:40: 7.2 mM, 650.3 and 266.4 mg L−1; 40:60: 4.8 mM, 433.5 and 399.6 mg L−1; 20:80: 2.4 mM, 216.8 and 532.8 mg L−1.
2.2. Harvest, Yield, Growth Analysis and Leaf Colorimetry
The sweet basil plants were harvested twice (47 and 75 days after transplanting, hereafter CT1 and CT2, respectively), when the plant height reached approximately 35 cm and before flowering. At each harvest date and for each experimental unit (i.e., replicate) six plants were collected. The above-ground plant was harvested and immediately weighed. Leaves were separated, weighed and the leaf area quantified with a LiCor 3100C area meter (LI-COR Biosciences, Lincoln, NE, USA). A sub-sample of the entire plant was dried in a forced-air oven at 70 °C for three days until constant weight to determine the shoot dry biomass. Dry matter percentage was also calculated using the following formula: DM = 100 × Dry weight/Fresh weight. On the center of the upper basil leaf surface 10 color measurements were performed with a Minolta Chroma Meter (CR-300, Minolta Camera Co. Ltd., Tokyo, Japan). The color space was measured as defined by the International Commission on Illumination, and color was expressed with three values: L* for the lightness from black (0) to white (100), a* from green (−) to red (+), and b* from blue (−) to yellow (+).
2.3. Quality Analysis Sampling
At both harvest dates, basil fresh samples (three replicates per treatment) were harvested, immediately frozen in liquid nitrogen, and stored at −80 °C before being lyophilized in a freeze-dryer Alpha 1-4 LSC Basic (M. Christ, Osterode, Germany) for further analysis (i.e., antioxidant activity, phytochemicals and volatile compounds). For the analysis of the mineral composition, the dry leaf material was milled with a Wiley Laboratory Mill model 4 to pass through an 841 µm sieve mesh.
2.4. Leaf Gas Exchange
Net photosynthetic carbon dioxide assimilation rate (ACO2) and transpiration (E) were determined just before the two harvests whit a portable gas exchange analyzer (LCA-4; ADC BioScientific Ltd., Hoddesdon, UK) equipped with a broadleaf chamber (cuvette window area: 6.25 cm2). The physiological measurements were carried out between 11.00 and 13.00 h on nine replicate plants per treatment. The climatic conditions (e.g., the photosynthetically active radiation, relative humidity, and carbon dioxide concentrations) were set at ambient temperature and the flow air rate was 400 mL s−1. Intrinsic water use efficiency was calculated as the ratio between ACO2 and E.
2.5. Nitrate and Mineral Analysis
One gram of basil ground material was used for the determination of total nitrogen (N) using the Kjeldahl method [
23]. Ground material (0.25 g) was analyzed by ion chromatography (ICS-3000, Dionex, Sunnyvale, CA, USA) to determine the mineral content (P, K, S and Cl) as described [
24]. The results were expressed as g kg
−1 dry weight (dw), whereas nitrate was expressed as mg kg
−1 fresh weight (fw), according to each sample dry matter content.
2.6. Total Chlorophyll and Total Ascorbic Acid Measurement
Total chlorophyll and total ascorbic acid (TAA) were analyzed on the fresh basil material. Total chlorophyll content (chlorophyll a and b) and TAA were determined as reported [
25,
26] using a Hach DR 4000 (Hach Co., Loveland, CO, USA) spectrophotometer. Total chlorophyll and TAA were expressed in µg g
−1 and in mg AA 100 g
−1 fw, respectively.
2.7. Antioxidant Activity and Total Phenols Analyses
The lipophilic antioxidant activity (LAA) and the total phenols (TP) content were analyzed on freeze-dried leaf samples using UV–Vis spectrophotometric approached [
27,
28]. Absorbance was measured at 505 nm for LAA and 525 nm for TP. LAA and TP were expressed in mmol eq. trolox 100 g
−1 and mg eq. gallic acid g
−1, respectively.
2.8. Analysis of Volatile Compounds
Flavor compounds (e.g., volatiles and semi-volatiles, C3–C20) were analyzed using the solid-phase microextraction (SMPE) sampling technique coupled with gas chromatography/mass spectrometry (GC/MS). Compounds were analyzed using three biological replicates. An SPME fiber assembly coated with Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS; film thickness: 50/30 µm) (Supelco, Bellefonte, PA, USA) was employed for the extraction of the analytes. Freeze-dried powdered leaves (2 g) were introduced into a 20 mL vial of headspace. The SPME fiber was exposed in the headspace for 20 min at 45 °C and then, introduced into the GC injector. Analytes desorption was carried out at 250 °C for 5 min. GC/MS was performed in a6890N Network Gas Chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a 5973 Mass Detector System (MDS) (Agilent Technologies). Separation was carried out on a 30 m × 0.250 mm capillary column coated with 0.25 µm of 5% diphenyl −95% dimethylpolysiloxane (HP5MS J&W Scientific, Folsom, CA, USA), directly inserted into the ion source of the MDS. After column conditioning (50 °C for 2 min), oven temperature was ramped with a 10 °C per minute speed up to 150 °C and then, with a 15 °C per minute speed up to 300 °C, which was held for 10 min. The spitless injection and ion source temperatures were 250 and 230 °C, respectively. Helium (99.999%) was used as the carrier gas at a flow rate of 1 mL per minute. The electron ionization energy was 70 eV. The scanned mass range was 40–450 atomic mass units (in full-scan mode). Volatile compounds were identified considering their mass spectra and retention indices using the NIST Atomic Spectra Database version 1.6 libraries, with a range of similarity values of 85–100%. Relative quantification was performed on the basis of the peak area.
2.9. Leaf Anatomical Traits
Leaf sampling for the anatomical analyses was performed at the second harvest stage (CT2) on six replicate plants per treatment. The lamina of fully expanded leaves was fixed in a solution made of 38% formaldehyde, glacial acetic acid, 50% ethanol (5:5:90 by volume). For the analysis of epidermal traits, epidermal strips were peeled off from the abaxial and the adaxial lamina surfaces, flattened, and mounted with distilled water on microscope slides. Peels were observed under an epi-fluorescence microscope (BX60; Olympus, Hamburg, Germany) set to detect, with different emissions, stomata and glandular trichomes (mercury lamp, band-pass filter of 330–385 nm, dichromatic mirror of 400 nm and above, and a barrier filter of 420 nm and above). Collected images (Camedia C4040; Olympus) were analyzed through the software program AnalySIS 3.2 (Olympus) to quantify stomata frequency (SF) and size (area of each couple of guard cells – GCA, guard cells area). SF was calculated in three regions per peel, while GCA was measured in 20 stomata per peel. For the analyses of mesophyll traits, leaf lamina samples were divided into 5 × 5 mm pieces, which were dehydrated in a graded ethanol series (up to 95%), infiltrated and embedded in the acrylic resin JB4 (Polysciences, Hirschberg, Germany). Cross-sections (5-µm thick) were cut through a rotary microtome, stained with 0.5% Toludine blue in water [
29], mounted with Canadian balsam and observed under a light microscope (BX60, Olympus). Digital images were captured and analyzed as reported above to measure: the thickness and area of palisade and spongy cells, and the density of spongy parenchyma in terms of quantity of intercellular spaces (IS, expressed as the percentage of tissue occupied by air spaces over a given surface) [
30]. All these traits were measured in six positions along the lamina, avoiding veins.
2.10. Statistics
The statistical analyses were carried out using a two-way between-subject analysis of variance (ANOVA), followed by post-hoc analysis with the Duncan’s multiple range-test (α < 0.05). The fixed factors were the NO
3−:Cl
− ratio (R) in the nutrient solution, and the harvest (CT) of the leaves and stems. Differences between cuts were evaluated with an independent Student’s t-test. For anatomical traits, a one-way ANOVA was performed followed by post-hoc analysis with the Duncan’s multiple range-test (α < 0.05). The statistical analysis was carried out with the Statistical Package for Social Sciences (SPSS) software version 20 (IBM Corp., Armonk, NY, USA). Principal Component Analysis and correlation analysis were performed and visualized in R [
31].
4. Discussion
In this work, we demonstrated the broad range of alterations in basil caused by the variation of two antagonist anions (nitrate and chloride) in four nutritional formulations that have the same anion concentration. We also compared leaves from two harvests, considering that basil is harvested at least twice in professional horticulture.
Crops are typically supplied with ample inorganic nutrients and it is well demonstrated that nitrogen fertilization is a key determinant of plant growth. Accordingly, the highest yield was obtained at the highest nitrate concentration (i.e., the 80:20). Moreover, the overall yield was higher at the first cut [
32,
33]. Nonetheless, it is interesting that yield did not significantly decrease with the 60:40 ratio. A large yield reduction was evident for the 20:80 and only at this ratio, the yield at CT2 was not statistically lower than at CT1. Yield differences can be explained by the concomitant, specific action of the two anions. As expected for nitrogen fertilization, yield and dry biomass correlated well and likewise, plants with higher yield had larger leaves. The dry biomass did not show differences between cuts, while the percentage of dry matter was always higher at CT2, indicating a predominant role of the water content in yield differences. Water content was visibly reduced at high chlorine concentration and the strong yield reduction at 20:80 associated with a strong increase in the percentage of dry matter. The reduction of leaf lamina according to the decrease in the nitrogen/chloride ratio in the nutrient solution, was accompanied by an increase in the leaf lamina thickness up to 40:60. The trend of increasing thickness was inverted at 20:80, where the mesophyll showed signs of stress (e.g., severe cell shrinkage) that are consistent with the reduced water content. It is worth adding that in our experimental system, different amounts of chlorine were supplied with solutions with the same osmotic potential, therefore not affecting water movement. For instance, the constant amount of sulfur in leaves is consistent with the lack antagonism with the sulfate anion in the NS [
34]. Similarly, a gradual antagonist effect of chlorine on phosphate accumulation was not evident, and phosphate content in leaves was reduced only at the 20:80 ratio [
16,
18]. In addition, the reduction of nitrate in the NS (and in leaves) did not cause a significant decrease in chlorophyll, pointing towards a compensatory effect of lower concentrations of chlorine, which may be present also for gas exchange parameters. For instance, chlorophyll content was significantly reduced at the second cut and net photosynthetic carbon dioxide assimilation rate only mildly decreased with lower nitrogen supplies. Being present in plants with a smaller leaf surface, the lower assimilation rate per leaf area should result in a large effect on the whole plant photosynthetic activity. Moreover, the reduction in A
CO2, along with an unchanged or increase in evapotranspiration, according to the increase in higher chlorine concentration can be explained by a coordination of leaf structural traits [
35,
36]. At 40:60, the occurrence of thicker leaves (due to larger cells) coupled with reduced intercellular spaces explains the higher evapotranspiration due to the maintenance of high the leaf hydraulic conductance (Kleaf), but reduced photosynthesis due to lower volume available for CO
2 exchange in comparison with the other conditions. Many studies indicated a negative effect of low nitrogen supply on intrinsic water use efficiency [
37]. iWUE, in both cuts, reduced only from 80:20 to 60:40, suggesting that Cl allows maintaining the same iWUE regardless of a mild increase of transpiration and a reduced biomass. In addition, the adjustment of stomata traits at the adaxial lamina surface supports the induction of a leaf response towards the quicker control of gas exchange thanks to a reduction in the frequency and size of stomata at 20:80 and 40:60. For instance, smaller stomata allow a better and faster stomatal response [
38].
Plants treated with the highest amount of nitrate (resp. chloride) showed the highest leaf content of total nitrogen and nitrate (resp. chlorine). Only chlorine accumulated in higher amount at the second cut, mostly because nitrate is continuously metabolized to sustain the growth after the first harvest, unlike chlorine. The maximum chlorine accumulation in leaves was 1.6 mmol per g dw, a value in the range of a macronutrient. At the 20:80 ratio, the Cl concentration in leaves exceeded the maximum of the conventional range in glycophytes (20 g kg
−1 dw) [
19]. This amount is insufficient to cause visible symptoms of toxicity [
21] (we detected only at microscopic level evidence of anatomical perturbation). For instance, in sweet basil salt stress-induced morphological effects are usually visible starting from a 25 mM NaCl concentration [
39]. A linear response of the mineral composition to the nutrient solution was not evident for all R. The sharp decrease in nitrate and total nitrogen at the 20:80 R, along with the increased efficiency in nitrogen uptake and use at increasing Cl concentration in the NS, suggest that nutrient availability or anion-anion competition in uptake and mobilization are not the only factors that can explain mineral composition of the leaves. In leaves, nitrogen is mainly present in photosynthetic enzymes [
40,
41], and it is well established that total nitrogen content strongly correlates with nitrate and the net photosynthetic rate. In our system, the latter correlation was mild and present mainly for leaves of the second cut. As also recorded for iWUE, this can be explained considering a compensatory effect of chlorine that allows Cl to sustain CO
2 assimilation rate only when present in leaves in the range of a macronutrient (mmol) but not in high amount (roughly, below 5 mmol g dw) [
21,
42].
The nutrient solution also affected basil nutritional properties. As expected, the concentration of total ascorbic acid was inversely correlated with nitrate fertilization [
43]. For leafy products, this is typically justified considering the strong positive correlation between nitrogen and plant biomass. Moreover, also a mild supplement of NaCl is reported to increase the ascorbic acid concentration [
8]. For instance, the addition of 40 mM NaCl in the NS increased the TAA and TP content in soilless cultivated basil [
44]. Similarly, higher polyphenolic contents were observed with reducing nitrates in the NS, consistent with the C/N balance theory [
45]. The lipid-soluble antioxidants content was the quality-related component that little correlated with other variables, including chlorophyll and biomass, and was only affected by the CT factor. Variation of carotenoids content in leaves is usually described in relation to stress, and in sweet basil NaCl salinity induced an increase in carotenoids concentrations of the leaf tissue [
39].
The effect on the volatile compounds was complex. In comparison with the other variables, the aromatic profile was the least affected by the two factors (CT: two compounds out of eight; R: four), and their interaction (two). The frequency of secretory glandular trichomes was reduced according to the decrease in nitrogen supply already at 60:20. NS composition likely affected not only the differentiation of such trichomes at leaf surface, but also their capacity to synthesize and accumulate phenylpropenes. It was previously demonstrated that the reduction of the frequency of trichomes that actively produce volatile oils was not necessarily accompanied by the decrease in the major aroma volatiles analyzed [
4]. Among the major components that are significant for basil aroma and for its antioxidant properties, eugenol was affected by the two factors and their interaction. However, a linear correlation with the variation of the NS ratios was not present. Nitrogen application contributed to a decrease in the contents of volatiles such as linalool, the major component of the European-basil oil [
46]. On the other hand, it has also been proposed that nitrogen application can increase volatile emission through an expansion of the leaf surface [
47]. In our study, the analyzed chemical compounds little correlated with the morphological parameters and in a few cases, such as for hexanal, negatively correlated with yield and leaf area, as well as the photosynthetic rate. A compensatory effect of the two anions cannot be excluded however, very little is known on the effect of chlorine on the aromatic compounds, besides a positive effect of the NaCl stress on essential oil production [
39].