Nitrogen and phosphorus levels affected plant growth, essential oil composition and antioxidant status of lavender plant (Lavandula angustifolia Mill.)

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Nitrogen and phosphorus levels affected plant growth, essential oil composition and antioxidant status of lavender plant (Lavandula angustifolia Mill.) Antonios Chrysargyris, Christakis Panayiotou, Nikos Tzortzakis ∗ Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, Limassol 3603, Cyprus

a r t i c l e

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Article history: Received 18 October 2015 Received in revised form 14 December 2015 Accepted 23 December 2015 Available online xxx Keywords: Antioxidants Essential oil Lavandula angustifolia Mineral accumulation Soilless culture

a b s t r a c t Lavandula angustifolia (Mill.) is a multidisciplinary medicinal and aromatic plant of great importance in fragrance and pharmaceutical industries and/or landscaping. Minerals rate affect yield and quality of medicinal plants therefore, this experiment was conducted in order to determine the effects of nitrogen (N: 150–175–200–225–250 mg/L) and phosphorus (P: 30–40–50–60–70 mg/L) levels on the morphological and biochemical characteristics of lavender under hydroponic condition. The results indicated that P levels mainly affected plant growth, while lower N levels (150 mg/L) reduced chlorophylls content. Essential oil yield was remained unaffected under N and P levels. The N levels greater than 200 mg/L as well as 60 mg/L of P, benefited antioxidant status (total phenols, DPPH, FRAP, flavonoids). The main constituents of leaves essential oil (1.8-cineole, borneol, camphor, ␣-terpineol, myrtenal) and mineral accumulation were affected by N and P treatments. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Lavender (Lavandula angustifolia Mill.), a perennial shrub of Lamiacea family, is used medicinally, in balms, salves, perfumes, cosmetics and consisted as model plant for isoprenoid studies (Biswas et al., 2009). Essential oil (EO) of lavender has antiseptic and anti-inflammatory, analgesic, antifungal and bactericidal properties because it is rich in terpenes (Tzortzakis and Economakis, 2007; Biesiada et al., 2008). Several bioactive constituents have been reported for lavender, such as polyphenols, anthocyanins, carotenoids, chemicals that act as antioxidants in the human body, but little information on lavender antioxidant properties are available in literature (Miliauskas et al., 2004). The essential oil of lavender is mainly comprised of monoterpenes (the C10 class of isoprenoids), and is produced and stored in the glandular trichomes (or oil glanda), which cover the surface of the aerial parts (both leaves and inflorescences) of the plant. Lavender main compounds responsible for the typical aroma are linalool, linalyl acetate, 1,8-cineole, ocimene, terpene, and camphor (Biswas et al., 2009; Haddanpouraghdam et al., 2011).

∗ Corresponding author at: Department of Agricultural Science, Biotechnology and Food Science, Faculty of Geotechnical Sciences and Environmental Management, Cyprus University of Technology, 3603 Limassol, Cyprus. Fax: +357 25002838. E-mail address: [email protected] (N. Tzortzakis).

Chemical and biological diversity of medicinal plants are depending on several variables such as cultivation area, microclimate, vegetation stage as well as genetic modifications (Miliauskas et al., 2004). There is lack information on effect of fertilization on antioxidant activity and essential oil yield and constitutes of lavender plants. Due to poor commercial cultivation of aromatic plants in the past years, little is known about cultivation techniques and mineral needs (Biesiada et al., 2008). However, nowadays, aromatic plants are cultivated and commercialized extensively, and appropriate cultural practices are necessary. Thus, hydroponics (soilless culture) controls minerals uptake and combined to the controlled environment of the greenhouses, allows a faster plant growth in relation to soil cultivation, shortening the productive cycle and increasing productivity for vegetables and floriculture (Savvas and Passam, 2002). Plant fertigation and mineral uptake/accumulation are one of the most important factors that increase plant production. It is well known that nitrogen (N), phosphorous (P), and potassium (K) affect the growth and essential oil synthesis in medicinal plants. These components influence the levels of enzymes that are very important in the terpenoides biosynthesis (Sell, 2003). Nitrogen is considered a plant essential mineral contributing on synthesis of many organic compounds such as amino acids, proteins, enzymes and nucleic acids. The high concentration of N increased the leaf cell number and size with an overall increase in leaf production as

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well as biomass yield that attributed to the well-known functions of N in plant life. Indeed, amino acids and enzymes play a key role in the biosynthesis of numerous compounds which are essential oil constituents (Koeduka et al., 2006). Nitrogen fertilization has been reported to reduce EO content in creeping juniper (Juniperus horizontalis) (Robert and Francis, 1986), although it has been reported to increase EO yield in thyme (Thymus vulgaris L.) (Baranauskienne et al., 2003) and in cumin (Cuminum cyminum) (Azizi and Kahrizi, 2008). Phosphorus plays an important role in various metabolic processes, being a constitute of nucleic acid, phospholipids, coenzymes activating the amino acid production used in protein synthesis, DNA, RNA and ATP (Rouached et al., 2010). The P deficiency is related with the reduction of chloroplast carbon fixation as a consequence to the photosynthetic potential. High P rates decreased chamomile (Matricaria chamomilla L.) EO yield (Emonfor et al., 1990) but increased in feverfew (Tanacetum parthenium L.) (Saharkhiz and Omidbaigi, 2008) and in sage (Salvia officinalis L.) (Nell et al., 2009) EO yield. In addition minerals interaction often is stronger than their individual action. At present there is increasing interest both, in industry and in scientific research for medicinal and aromatic plants due to their strong antioxidant and antimicrobial properties, which exceeded many commonly used natural and synthetic antioxidants. These properties are related to the presence of some vitamins, carotenoids, chlorophyll, catechins, phytoestrogens, minerals, etc. and render aromatic/medicinal plants and/or their antioxidant components as food preservatives (Parejo et al., 2002). The objective of the present study was to examine the effects of nitrogen and phosphorus concentration on growth characters, nutrient accumulation, antioxidant activity as well as quality and quantity of essential oil of L. angustifolia plant, just before flowering, as quite often plant early harvested in a mixed vegetative and flowering stage.

and Na = 1.91 mmol/L, respectively; and B = 18.21, Fe = 71.56, Mn = 18.21, Cu = 4.72, Zn = 1.53, and Mo = 0.52 ␮mol/L, respectively. Fertigation was applied during daytime through a timer (8 times with 1 min every time at a flow rate of 30 mL/min, due to low water holding capacity of perlite medium) with a drip irrigation system (via emitters; one emitter/plant) by means of pressure pumps. Nutrient solution target pH and EC were 5.8 and 2.1 mS/cm respectively. 2.2. Plant growth and tissue analysis In this experiment, plants were fertigated with a complete nutrient solution and grown over 1 week for better root establishment; a commercial amino acid solution (1 mL/L v/v) used once for the same reason. Following eight weeks of plant growth under different nutrient solutions, considering five concentrations for N and five concentrations for P, six individual plants for each treatment were considered for detail plant growth analysis. Plant height, leaf number, leaf length, stem thickness, plant fresh and dry weight observed for upper and root part were determined. 2.3. Determination of leaf chlorophyll content and stomatal conductance

2. Material and methods

Lavender leaf tissues (six replications/treatment; each replication consisted of a pool of two plants tissue; leaf disk: 0.1 g) were incubated in heat bath at 65◦C for 30 min, in the dark, with 10 mL dimethyl sulfoxide (DMSO, Sigma Aldrich, Germany) for chlorophyll extraction. Photosynthetic leaf pigments, chlorophyll a (Chl a), chlorophyll b (Chl b) and total chlorophyll (t-Chl) concentrations were calculated using the equations of Arnon (1949) as follows: Chl a = 0.0127 × A663 − 0.00269 × A645 ; Chl b = 0.0229 × A645 –0.00468 × A663 ; and tChl = 0.0202 × A645 + 0.00802 × A663 (Richardson et al., 2002). Stomatal conductance was measured using a T-Porometer AP4 (Delta-T Devices-Cambridge, UK) according to the manufacturer’s instructions.

2.1. Plant material and growing conditions

2.4. Plant ion concentration analysis

The present investigation was carried out at the Hydroponic Infrastructures (fully controlled plastic greenhouse) of the Experimental Farm, Cyprus University of Technology, Limassol, Cyprus, during two spring-summer seasons of 2013 and 2014. The effect of N and P concentration into nutrient solution was examined, resulting in five concentration levels for each mineral. Thus, two sub-set created: (i) N varied in 150–175–200–225–250 mg/L with constant 50 mg/L of P and (ii) P varied in 30-40-50-60–70 mg/L with constant 200 mg/L of N, resulting in nine treatments, considering as reference treatment the intermediate concentrations i.e., 200 mg/L N and 50 mg/L P based on preliminary studies on aromatic plants and/or previous reports (Economakis et al., 2002; Mollafilabi et al., 2010). Each treatment consisted of 5 replications (3 plants in each replication; 15 plants in total for each treatment). Average minimum and maximum air temperatures during this period were 18 and 30 ◦ C, respectively; temperature reached up to 33–35 ◦ C during sunny hours in late spring and summer. Lavender (L. angustifolia Mill.) plants were purchased from the Cypriot National Centre of Aromatic Plants in trays at the stage of 3–4 leaves and 4–5 cm height. Seedlings were transplanted into pots (1 plant per pot) with perlite (5 Lt/pot). Pots arranged in singles row (rows were 0.3 m apart and plants were separated by 0.2 m). The soilless culture system was open with the excess nutrient solution drained away. A solution (1:100 v/v) in water containing the following concentration of nutrients was used: NO3 − N = 14.29, K = 8.31, PO4 -P = 1.61, Ca = 7.48, Mg = 5.76, SO4 −2 -S = 1.56

Leaf (6 replications/treatment) and root (3 replications/treatment) plant tissue samples were dried at 75◦ C for 6 d, weighted, and grounded in a Wiley mill to pass through a 40 mesh screens. Sub samples (0.2–0.3 g) were digested using hydrochloric acid (2 N HCl). Determination of K, P, Ca, Mg, Fe, Cu, Mn, Zn, Na, and B was done by inductively coupled plasma atomic emission spectrometry [ICP-AES; PSFO 2.0 (Leeman Labs Inc., USA) and N by the Kjeldahl (BUCHI, Digest automat K-439 and Distillation Kjelflex K-360) method. 2.5. Essential oil extraction Three samples (pooled of three individual plants/sample) for each treatment, harvested just before flowering, and air-dried lavender leaves (in oven at 42 ◦ C) were chopped and approx. 15–20 g of sample were subjected to hydrodistillation for 3 h using Clevenger apparatus. The essential oil (dried over anhydrous sodium sulphate) yield was measured and calculated as ␮L of oil/g dry tissue. Oils were kept in amber glass bottles at −20 ◦ C until GC/MS analysis. 2.6. Gas chromatography/mass spectrometry analyses of EOs Analytical gas chromatography was carried out on a Shimadzu GC2010 gas chromatograph interfaced Shimadzu GC/MS QP2010 plus mass spectrometer. An aliquot of 2 ␮L was injected in a split

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Table 1 Influence of nitrogen and phosphorous on plant height (cm), leaf length (cm), stem thickness (mm), biomass fresh weight (FW; g/plant), biomass dry matter (DM;%), root fresh weight (FW; g/plant), root dry matter (DM;%), biomass:root ration and root length (cm) of lavender plants grown hydroponically in perlite. N

Plant height

Leaf length

Stem thickness

Biomass FW

Biomass DM

Root FW

Root DM

Biomass: root

Root length

N150 N175 N200 N225 N250

21.00 ± 0.81a Y 20.66 ± 0.55a 20.33 ± 0.71a 20.50 ± 1.41a 21.66 ± 0.61a

5.91 ± 0.14a 6.55 ± 0.26a 6.24 ± 0.10a 6.21 ± 0.26a 6.20 ± 0.21a

5.80 ± 0.35a 5.88 ± 0.34a 5.50 ± 0.29a 5.67 ± 0.22a 5.78 ± 0.19a

18.04 ± 1.18a 19.27 ± 1.90a 15.91 ± 1.05a 18.60 ± 1.46a 16.11 ± 1.31a

28.01 ± 0.72a 27.18 ± 0.36a 28.66 ± 0.61a 28.06 ± 0.95a 29.06 ± 1.06a

18.62 ± 1.67a 12.86 ± 1.94b 19.07 ± 1.39a 17.87 ± 0.70a 7.15 ± 1.00c

9.70 ± 0.29b 10.82 ± 061b 12.02 ± 0.27b 9.90 ± 0.411b 15.38 ± 1.89a

0.98 ± 0.07c 1.63 ± 0.17b 0.80 ± 0.08c 1.05 ± 0.10c 2.40 ± 0.26a

23.41 ± 1.46a 23.66 ± 1.83a 21.91 ± 1.68a 22.83 ± 1.10a 20.66 ± 1.80a

16.44 ± 0.70b 12.69 ± 0.43c 15.91 ± 1.05b 16.49 ± 0.99b 19.47 ± 1.03a

28.28 ± 0.98a 29.09 ± 0.90a 28.66 ± 0.61a 28.84 ± 0.54a 27.54 ± 0.83a

P P30 P40 P50 P60 P70

20.66 ± 0.88ab 19.00 ± 0.51b 20.33 ± 0.71ab 21.06 ± 0.51a 22.16 ± 0.79a

5.97 ± 0.06bc 5.82 ± 0.11c 6.24 ± 0.10abc 6.36 ± 0.16ab 6.62 ± 0.19a

4.85 ± 0.45ab 4.00 ± 0.19b 5.50 ± 0.29a 5.45 ± 0.25a 5.53 ± 0.67a

11.30 ± 0.93b 7.37 ± 0.86c 19.07 ± 1.39a 11.08 ± 1.03b 13.63 ± 1.56b

8.57 ± 0.28b 9.33 ± 0.89b 12.02 ± 0.27a 8.45 ± 0.48b 8.27 ± 0.20b

1.51 ± 0.16a 1.80 ± 0.16a 0.80 ± 0.08b 1.53 ± 0.12a 1.46 ± 0.10a

24.91 ± 1.91a 23.25 ± 0.51a 21.91 ± 1.68a 23.08 ± 1.20a 23.26 ± 1.32a

Y values (n = 6) in columns followed by the same letter are not significantly different, P ≤ 0.05.

mode (split ratio 20:1) into the gas chromatograph fitted with a ZB-5 column (Zebron, Phenomenex, USA) coated with 5% pheny95% dimethylpolysiloxane with film thickness of 0.25 ␮m, length of 30.0 m and a diameter of 0.25 mm. The flow of the carrier gas (Helium) was 1.03 mL/min. The injector temperature was set at 230 ◦ C. Electron impact mass spectra with ionization energy of 70 eV was recorded at the 35–400 m/z. The column temperature was programmed to rise from 60◦ C to 240◦ C at a rate of 5◦ C/min, with a 5 min hold at 240◦ C. The solution of standard alkanes mixtures (C8–C20) was also analyzed using the above conditions. The identity of the oil components was assigned by comparison of their retention indices relative to (C8–C20) n-alkanes with those of literature or with those of authentic compounds available in our laboratory (Fig S1). Further identification was made by matching their recorded mass spectra with those stored in the NIST08 mass spectral library of the GC–MS data system and other published mass spectra (Adams, 2012). The percentage determination was based on peak area normalization without using correction factors. 2.7. Polyphenol extraction and analyses 2.7.1. Preparation of extracts Six leaves samples (pooled by two individual plants/sample) for each treatment, of the freshly cut plants (0.5 g) were milled with 10 mL methanol (50%) and extraction was assisted with ultrasound. The samples were centrifuged for 30 min on 4000 × g at 4 ◦ C (Sigma 3–18 K, Sigma Laboratory Centrifuge, Germany). The supernatant was transferred to a 15 mL falcon tube, stored at 4◦ C until analysis (within 48 h) for evaluation of total phenolic and flavonoids content and total antioxidant activity by the DPPH and FRAP radical scavenging assay.

2.7.2. Total phenolic amounts The total phenolic content of the methanol (50% v/v) extracts was determined by using Folin–Ciocalteu reagent (Merck), according to the procedure described by Tzortzakis et al. (2007). Briefly, 125 ␮L of plant extract was mixed with 125 ␮L of Folin reagent. The mixture was shaken, before addition of 1.25 mL of 7% Na2 CO3 , adjusting with distilled water to a final volume of 3 mL, and mixed thoroughly. After incubation in the dark for 90 min, the absorbance at 755 nm was measured versus the prepared blank. Total phenolic content was expressed as ␮mol of gallic acid equivalents per gram of fresh weight (␮mol GAE/g FW), through a calibration curve with gallic acid. All samples were analysed in triplicate. 2.7.3. DPPH and FRAP radical scavenging assay Radical-scavenging activity was determined according to Goulas and Manganaris (2011). DPPH (2,2-diphenyl-1picrylhydrazyl) radical scavenging activity of the plant extracts was measured from the bleaching of the purple-colored 0.3 mM solution of DPPH. One milliliter of the DPPH solution, 1.98 mL 50% methanol and 0.02 mL of plant extract were mixed. After shaking, the mixture was incubated at room temperature in the dark for 30 min, and then the absorbance was measured at 517 nm. DPPH radical-scavenging activity was expressed as the inhibition percentage (I%) and was calculated using the following formula: DPPH radicalscavenging activityI(%) = [100 − 100 × [

(Abs − Abb ) ] Abc

where Abb is the absorbance of the blank sample, Abs is the absorbance of the test sample and Abc is the absorbance of the control, with DPPH and 50% methanol.

Table 2 Influence of nitrogen and phosphorous on leaf stomata conductivity (cm/s), chlorophylls (Chl a, Chl b, Total Chl) content (mg/g fresh weight) and essential oil (EO) yield (%) of lavender plants grown hydroponically in perlite. N

Stomatal conductivity

Chl a

Chl b

Total Chl

EO

N150 N175 N200 N225 N250

2.59 ± 0.43a Y 1.59 ± 0.58a 1.30 ± 0.21a 1.59 ± 0.41a 2.05 ± 0.88a

0.89 ± 0.02b 1.01 ± 0.03a 1.09 ± 0.01a 1.09 ± 0.02a 1.04 ± 0.04a

0.27 ± 0.00b 0.31 ± 0.01a 0.33 ± 0.00a 0.33 ± 0.00a 0.32 ± 0.01a

1.16 ± 0.03b 1.31 ± 0.05a 1.43 ± 0.02a 1.42 ± 0.02a 1.37 ± 0.06a

1.14 ± 0.00a 0.76 ± 0.13a 0.89 ± 0.16a 0.91 ± 0.01a 0.90 ± 0.09a

P P30 P40 P50 P60 P70

1.19 ± 0.44a 1.48 ± 0.69a 1.30 ± 0.21a 1.88 ± 0.40a 1.23 ± 0.28a

1.05 ± 0.03a 1.14 ± 0.05a 1.09 ± 0.01a 1.05 ± 0.02a 1.11 ± 0.04a

0.33 ± 0.01a 0.34 ± 0.01a 0.33 ± 0.00a 0.31 ± 0.01a 0.34 ± 0.01a

1.38 ± 0.05a 1.49 ± 0.07a 1.43 ± 0.02a 1.36 ± 0.03a 1.44 ± 0.06a

0.89 ± 0.04a 0.96 ± 0.25a 0.89 ± 0.16a 0.76 ± 0.06a 0.71 ± 0.18a

Y values (n = 6) in columns followed by the same letter are not significantly different, P ≤ 0.05.

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Ferric reducing/antioxidant power (FRAP) assay performed as well. A sample of 3 mL of freshly FRAP solution (0.3 mol/L acetate buffer, pH 3.6), containing 10 mmol/L TPTZ (Tripyridil-s-triazine) and 40 mmol/L FeCl3 10H2 O and 20 ␮L of extract (50 mg/mL) were incubated at 37◦ C for 4 min and the absorbance was measured at 593 nm. The absorbance change was converted into a FRAP value, by relating the change of absorbance at 593 nm of the test sample to that of the standard solution of Trolox ((±) - 6- hydroxy2,5,7,8- tetramethylchromane- 2- carboxylic acid), and the results were expressed as mg Trolox/g fresh weight (Pantelidis et al., 2007). 2.7.4. Total flavonoid analysis The total flavonoid content was determined according to aluminium chloride colorimetric method (Meyers et al., 2003). Plant extracts and 0.75 mL of 5% sodium nitrite (NaNO2 ) were incubated for 6 min. After 5 min, 0.15 mL of AlCl3 solution (10%) was added. After an additional 5 min, a 0.5 mL of NaOH (1 M) solution was added and the volume was brought up to 2.5 mL with distilled water. The solution was mixed thoroughly and the absorbance was measured at 510 nm. The total flavonoid concentrations are expressed as Rutin equivalents (mg Rutin/g of fresh tissue). 2.8. Statistical methods The whole experiment was carried out twice, with similar outcomes (EO was analysed only in the 2nd year), and the 2nd year experiment was further analyzed/presented. Data were statistically analysed using analysis of variance (ANOVA) by IBM SPSS v.22 for Windows, and presented as treatment mean ± SE of six biological measurements. Percentage values were log-transformed prior to subjecting data to ANOVA. The chemical treatments structure and the relationship among nutrients concentration were determined by Linear Discriminate analysis (LDA) performed on the percentages of all identified compounds for all treatments using the SPSS program. Duncan’s multiple range tests were calculated for the significant data at P < 0.05. 3. Results and discussion 3.1. Plant growth Data presented in Table 1 indicated that plant growth variables influenced mainly by P than by N levels. The greater root fresh weight was observed in 200 mg/L N application comparing to 175 mg/L and 250 mg/L N. The highest ratio of upper plant biomass: root was in 250 mg/L N due to low root fresh weight of that treatment. There were no differences among N concentrations into nutrient solution for plant height (averaged in 20.8 cm), leaf length (averaged in 6.2 cm), stem thickness (averaged in 5.7 mm), fresh biomass (averaged in 17.6 g/plant) and root length (averaged in 22.5 cm). Leaf dry matter ranged from 27.18% to 29.09% among the different N and P concentrations into the nutrient solution. Barreyro et al. (2005) showed that N application enhanced oregano (Origanum x applii) yield, but had no effect on the final quality, while the highest yield of oregano may be achieved in different N (40 kg N/ha) levels compared with the highest essential oil content that may be achieved at 60 kg N/ha (Ozguven et al., 2006). Data in Table 1 showed that, nitrogen had no a pronounced effect on plant growth related parameters while considerable effects were observed in root development. Jacimovic et al. (2010) found no effect of increasing N doses on shoot and leaf dry mass of sweet basil (Ocimum basilicum L), being in accordance with the present study, while the oppose reported by El Gendy et al. (2015) as a high positive correlation between N doses and chervil (Anthriscus cerefolium L.) plant biomass was observed. In a field study, the most suitable N level for lavender yielding appeared to be the medium N

application (100 kg N/ha; Biesiada et al., 2008), which actually be in accordance with the present study, highlighting the importance of the appropriate mineral ratio (i.e., N:K, N:P) for plant nutrition. In different regions of the world with intensive fertilizer use, considering soil buffering capacity and mineral accumulation, the excessive N use has led the ground water pollution, i.e., nitrate, the most mobile form of N in any ecosystem. High P concentrations generally affected positively the plant growth parameters. Plants grown under the highest P concentration (70 mg/L P) revealed the greatest biomass of 19.47 g/plant whereas plant grown in 40 mg/L P revealed the lowest biomass, averaged in 12.69 g/plant (Table 1). The application of 50 mg/L P supported and increased root fresh weight and dry matter content but decreased upper plant biomass: root ratio. Additionally, plant height, leaf length and stem thickness were not benefited by increased P concentration into the nutrient solution. The leaf biomass of garden sage (S. officinalis L.) and EO content increased with adding P fertilizer (Nell et al., 2009) as reported with the 70 mg/L P in the present study. It was found that high P concentrations in Calendula officinalis (L.) did not increase flower production, but instead produced significantly more leaf biomass (Stewart and Lovett-Doust, 2003), while similar biomass increment was found at the present study with 70 mg/L P. 3.2. Physiological parameters Considering the fact that N is directly participating in chlorophyll molecule structure, it could be expected a positive correlation between leave’s N and chlorophyll content (El Gendy et al., 2015), which actually stimulates vegetative growth. In the present study, low N concentration (150 mg/L N) reduced the content of chlorophylls (Chl a, Chl b and total Chl) while higher N concentration or P application did not affect the Chlorophyll content (Table 2). Similar findings were observed for lavender (Lavender officinalis L.) regarding the N concentrations, in a field study (Biesiada et al., 2008). Thus, nitrogen greater than 175 mg/L did not differ either the chlorophyll content or stomatal conductance, and this may attributed to the fact that potassium concentration was 325 mg/L into the nutrient solution whereas, different nitrogen application affected the nitrogen:potassium ratio ranging from 1:2.16 (N:K) at 150 mg/L N up to 1:1.3 (N:K) at 250 mg/L N. It has been reported that appropriate N:K ratio for several vegetables and flowers crops should varied among 1.5–2.2, and actually include the 150 mg/L, 175 mg/L and 200 mg/L applications (Savvas and Passam, 2002). Potassium is an important element in plant metabolism as well, promoting carbohydrates synthesis. An antagonistic relationship between nitrogen and potassium as related to dry weight of Echinacea was noted (Shalaby et al., 1997). Increasing nitrogen concentration (>200 mg/L N) resulted in increased antioxidative status of the lavender plants. Thus, total phenols, DPPH and FRAP radical scavenging activity increased (up to 52%, 70% and 88% respectively) in N > 200 mg/L comparing with lower concentrations (Fig. 1). Similar, total flavonoids increased up to 79% in lavender plants grown in nitrogen concentration greater than 200 mg/L. Nitrogen applied in the cultivation of herbal plants not only may affect the plant biomass but also may stimulates the synthesis of other biologically active substances. Nguyen and Niemeyer (2008) proved that changes in the level of nitrogen fertilization during the growing period of basil had a significant impact on the production of phenolic compounds, in particular rosmarinic acid. Phosphorus total phenols, DPPH and FRAP (including 30 mg/L P) activity increased with the application of 60 mg/L P into nutrient solution comparing with 70 mg/L P, while no differences observed at lower P levels. Total flavonoids content did not differ regarding the P concentrations applied into nutrient solution (Fig. 1). Plant

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Fig. 1. Effects of nitrogen (N) and phosphorous (P) concentrations on total phenol mg GAE/g fresh weight. antioxidant activity (DPPH, FRAP, in mg Trolox/g fresh weight) and flavonoids (mg Rutin/g fresh weight) of lavender plants grown hydroponically in perlite.

material with higher antioxidant status is more appreciable and acceptable by consumers and markets/industry, providing added value products. 3.3. Nutrient uptake Increasing N concentration affected macronutrients and micronutrient in both of leaves and roots (Table 3). Lavender plants grown under different N concentrations presented the following order of accumulation of macronutrients: N > K> Ca > Mg > P > Na and micronutrients: Fe > Al > Mn > B > Zn > Cu. Regarding lavender roots grown under different N concentrations presented the following order of accumulation of macronutrients: N > K> Ca > Na > Mg > P and micronutrients: Fe > Al > Mn > Cu > B > Zn. Plant tissue mineral content ranged

from 18.62 to 20.94 g/Kg for N; 16.43 to 18.31 g/Kg for K; 1.62 to 1.65 g/Kg for P. The greater accumulation for N (including NO3 and NH4 form, data no presented) and K was observed in 175 mg/L N. Moreover, the greater accumulation for Ca, Mg and Na and the least Al (known for putative toxicidal effects in great concentrations) accumulation observed in 200 mg/L N treatment. Increasing N concentration at 225 mg/L, revealed higher Fe, Zn and Cu accumulation which are involved in several enzymes metabolism. In general, lower (i.e., 175 mg/L N) nitrogen levels into nutrient solution affected strongly N, K and Mn accumulation, medium (200 mg/L N) nitrogen level affected Ca, Mg and Na accumulation while higher (225 mg/L N) nitrogen level affected mainly micronutrients such as Fe, Zn and Cu accumulation. In roots, the application of 200 mg/L N increased the accumulation of Mg, Na, Mn and B, while low (150 mg/L) nitrogen concentration revealed

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Ca (g/kg)

P (g/kg)

Mg (g/kg)

Na (g/kg)

Al (mg/kg)

Fe (mg/kg)

Mn (mg/kg)

Zn (mg/kg)

Cu (mg/kg)

B (mg/kg)

17.12 ± 0.26ab 18.31 ± 0.32a 16.43 ± 0.55b 17.66 ± 0.24ab 16.46 ± 0.55b

7.34 ± 0.13bc 7.23 ± 0.13c 8.05 ± 0.17a 7.45 ± 0.09bc 7.73 ± 0.12ab

1.65 ± 0.04a 1.66 ± 0.03a 1.68 ± 0.08a 1.65 ± 0.01a 1.62 ± 0.06a

2.05 ± 0.05ab 1.87 ± 0.06c 2.18 ± 0.04a 1.96 ± 0.02bc 2.16 ± 0.07a

0.62 ± 0.05ab 0.49 ± 0.03b 0.75 ± 0.06a 0.59 ± 0.01ab 0.73 ± 0.10a

144.50 ± 10.35a 129.65 ± 3.96a 76.65 ± 2.42b 126.83 ± 5.14a 137.00 ± 6.43a

192.33 ± 10.45b 219.20 ± 19.40b 147.00 ± 9.67b 391.50 ± 26.21a 165.50 ± 8.09b

41.28 ± 1.99a 46.04 ± 1.44a 29.51 ± 1.22c 34.78 ± 2.08b 23.52 ± 1.78d

7.00 ± 0.65b 5.57 ± 0.30c 3.83 ± 0.23d 8.70 ± 0.25a 6.65 ± .47bc

4.49 ± 0.24b 4.76 ± 0.27ab 3.85 ± 0.23b 5.65 ± 0.69a 4.27 ± 0.30b

30.03 ± 0.91b 35.56 ± 0.91a 32.60 ± 0.77ab 34.80 ± 0.49a 35.03 ± 1.59a

Roots

N (g/kg)

K (g/kg)

Ca (g/kg)

P (g/kg)

Mg (g/kg)

NA (mg/kg)

Al (mg/kg)

Fe (mg/kg)

Mn (mg/kg)

Zn (mg/kg)

Cu (mg/kg)

B (mg/kg)

N150 N175 N200 N225 N250

24.46 ± 0.61a 24.53 ± 0.99a 25.87 ± 1.55a 27.02 ± 1.20a 21.71 ± 1.07a

21.26 ± 1.06a 21.13 ± 1.35a 22.63 ± 0.68a 21.66 ± 0.82a 19.90 ± 1.28a

8.11 ± 0.46a 7.46 ± 0.08a 9.52 ± 0.52a 8.05 ± 0.52a 10.11 ± 1.73a

3.07 ± 0.34a 2.95 ± 0.09a 2.87 ± 0.37a 3.02 ± 0.07a 2.86 ± 0.27a

3.47 ± .21b 3.65 ± 0.29b 4.42 ± 0.31a 3.46 ± 0.02b 3.62 ± 0.20b

5.68 ± 0.44ab 5.39 ± 0.85ab 7.71 ± 0.80a 5.83 ± 0.11a 4.97 ± 1.08b

146.00 ± 2.64a 151.00 ± 10.69a 151.33 ± 22.82a 132.65 ± 8.08a 107.56 ± 15.06a

331.66 ± 19.78a 284.00 ± 26.68a 324.00 ± 31.21a 294.00 ± 28.00a 275.00 ± 45.21a

151.66 ± 16.16a 139.33 ± 8.95a 144.66 ± 17.74a 122.00 ± 10.69ab 82.76 ± 8.06b

23.26 ± 2.60a 19.83 ± 1.35a 22.26 ± 4.04a 20.30 ± 1.47a 16.20 ± 1.06a

92.83 ± 7.34a 74.50 ± 6.68b 70.03 ± 3.14b 79.16 ± 4.97b 68.83 ± 1.35b

24.46 ± 1.14b 23.40 ± 1.35b 36.66 ± 4.18a 29.60 ± 0.80ab 26.10 ± 2.90b

Y

Y values (n = 6 for leaves and n = 3 for roots) in columns followed by the same letter are not significantly different, P ≤ 0.05.

Table 4 Macronutrient and micronutrient leaf and root analysis (g/kg or mg/kg) of lavender plants grown hydroponically in perlite under phosphorus rates. Leaves

N (g/kg)

K (g/kg)

Ca (g/kg)

P (g/kg)

Mg (g/kg)

Na (g/kg)

Al (mg/kg)

Fe (mg/kg)

Mn (mg/kg)

Zn (mg/kg)

Cu (mg/kg)

B (mg/kg)

P30 P40 P50 P60 P70

18.79 ± 0.90a 17.85 ± 0.66a 18.62 ± 0.43a 18.43 ± 0.32a 19.11 ± 0.56a

17.46 ± 0.56a 16.13 ± 0.44a 16.43 ± 0.55a 16.25 ± 0.41a 16.45 ± 0.53a

6.91 ± 0.19c 7.77 ± 0.36bc 8.05 ± 0.17a 7.19 ± 0.19bc 6.96 ± 0.13c

1.43 ± 0.08c 1.42 ± 0.06c 1.68 ± 0.08b 1.92 ± 0.06a 1.93 ± 0.06a

1.81 ± 0.03b 2.23 ± 0.13a 2.18 ± 0.04a 1.89 ± 0.08b 1.830.14b

0.49 ± 0.06b 0.79 ± 0.14a 0.75 ± 0.06a 0.42 ± 0.03b 0.36 ± 0.02b

52.74 ± 2.75b 79.08 ± 3.88a 76.65 ± 2.79a 76.15 ± 4.39a 83.90 ± 4.48a

91.08 ± 3.03b 171.66 ± 12.47a 138.91 ± 11.30ab 136.23 ± 36.09ab 112.01 ± 7.10b

32.13 ± 3.84ab 20.80 ± 2.78c 29.51 ± 1.22b 33.25 ± 1.82ab 39.18 ± 3.25a

4.29 ± 0.53ab 3.13 ± 0.57bc 3.52 ± 0.36abc 2.79 ± 0.49c 4.74 ± 0.21a

4.19 ± 0.24a 4.28 ± 0.41a 3.85 ± 0.23a 3.62 ± 0.23a 4.12 ± 0.27a

27.40 ± 0.62b 26.82 ± 0.70ab 32.60 ± 0.77a 21.12 ± 1.68b 22.24 ± 1.31b

Roots

N (g/kg)

K (g/kg)

Ca (g/kg)

P (g/kg)

Mg (g/kg)

Na (g/kg)

Al (mg/kg)

Fe (mg/kg)

Mn (mg/kg)

Zn (mg/kg)

Cu (mg/kg)

B (mg/kg)

P30 P40 P50 P60 P70

21.28 ± 0.44b 16.33 ± 0.56c 25.87 ± 1.55a 19.29 ± 0.49b 19.38 ± 0.69b

22.76 ± 2.06a 20.06 ± 0.28a 22.63 ± 0.68a 19.63 ± 1.59a 21.43 ± 1.23a

8.45 ± 0.46a 9.03 ± 0.52a 9.52 ± 0.52a 9.02 ± 0.75a 8.79 ± 0.80a

2.17 ± 0.12b 2.15 ± 0.06b 2.87 ± 0.37ab 3.07 ± 0.34a 3.60 ± 0.09a

3.35 ± 0.28b 3.98 ± 0.17b 4.42 ± 0.31a 3.49 ± 0.02b 3.37 ± 0.23b

4.91 ± 0.91b 7.31 ± 0.58ab 7.70 ± 0.80a 5.15 ± 0.25b 5.58 ± 0.82ab

148.00 ± 24.33a 117.60 ± 15.66a 151.33 ± 22.82a 143.00 ± 7.37a 122.00 ± 9.45a

324.33 ± 42.72a 258.33 ± 28.81a 324.00 ± 31.21a 278.66 ± 13.32a 291.66 ± 13.61a

203.66 ± 18.22a 101.60 ± 14.50c 144.66 ± 17.74bc 161.33 ± 21.67ab 187.00 ± 5.50ab

23.43 ± 4.38a 18.66 ± 0.81a 22.26 ± 4.04a 21.26 ± 2.96a 23.86 ± 0.77a

80.76 ± 10.02a 52.33 ± 3.18b 70.03 ± 3.14ab 63.76 ± 4.36ab 64.73 ± 2.13ab

25.43 ± 3.44b 27.86 ± 2.03ab 36.66 ± 4.18a 21.83 ± 0.97b 23.86 ± 3.06b

Y values (n = 6 for leaves and n = 3 for roots) in columns followed by the same letter are not significantly different, P ≤ 0.05.

ARTICLE IN PRESS

K (g/kg)

19.85 ± 0.85abcY 20.94 ± 0.24a 18.62 ± 0.43c 20.37 ± 0.47ab 19.39 ± 0.63bc

G Model

N (g/kg)

N150 N175 N200 N225 N250

A. Chrysargyris et al. / Industrial Crops and Products xxx (2015) xxx–xxx

Leaves

INDCRO-8635; No. of Pages 10

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Please cite this article in press as: Chrysargyris, A., et al., Nitrogen and phosphorus levels affected plant growth, essential oil composition and antioxidant status of lavender plant (Lavandula angustifolia Mill.). Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.12.067

Table 3 Macronutrient and micronutrient leaf and root tissue analysis (g/kg or mg/kg) of lavender plants grown hydroponically in perlite under nitrogen rates.

G Model INDCRO-8635; No. of Pages 10

ARTICLE IN PRESS A. Chrysargyris et al. / Industrial Crops and Products xxx (2015) xxx–xxx

Cu accumulation in roots. No chances observed in N, K, Ca, P, Al, Fe and Zn accumulation under different nitrogen concentrations into nutrient solution. Nitrogen application may resulted in increased K level which actually depressed Mg (Table 3), and this was also evidence in wheat shoots but not in roots, although K did not affect the rate of Mg influx (Wilkinson, 1994). Examining the effects of phosphorus application into nutrient solution, the results obtained showed that there was an influence by phosphorus concentration on mineral accumulation in both plant and root tissues (Table 4). Lavender plants grown under different P concentrations presented the following order of accumulation of macronutrients: N > K > Ca > Mg > P > Na and micronutrients: Fe > Al > Mn > B > Zn ≈nCu. Regarding lavender roots grown under different P concentrations the following order observed for accumulation of macronutrients: K ≥ N> Ca > Na > Mg ≥ P and micronutrients: Fe > Al ≈ Mn > Cu > B > Zn. Plant tissue mineral content ranged from 17.85 to 19.11 g/Kg for N; 16.13 to 17.46 g/Kg for K; 1.42 to 1.93 g/Kg for P. The greater accumulation for Mn, Na and Fe was observed in 40 mg/L P, for Ca and B was observed in 50 mg/L P whereas for P, Al, Mn and Zn in 70 mg/L P. In general, medium (40-50–60 mg/L) P levels supported better the mineral accumulation compared with low or high P levels. Different P levels did not affect the N and K accumulation in lavender plants. In roots, the application of 50 mg/L phosphorus increased the accumulation of N, Mg, Na and B, while low (30 mg/L) phosphorus concentration revealed Mn and Cu accumulation in roots. No chances observed in K, Ca, Al, Fe and Zn accumulation under different phosphorus concentrations into nutrient solution. Obviously, increasing phosphorus concentration into nutrient solution resulted in greater P accumulation in both leaves and roots. As mentioned, phosphorus at 50 mg/L has a favorable effect on root growth (Table 1), an action which resulted in better absorption and uptake of other nutrients (see Table 4). Enhancement in rate of metabolic processes with higher dose of N and P may also result in increased demand and utilization of other plant nutrients, among which K is of prime importance. A synergistic role of K with both either N or P already has been reported (Mandal et al., 2002). 3.4. Essential oil yield and constitutes Production of essential oil in aromatic plants may be affected positively or negatively by type and amount of fertilizers (Fonseca et al., 2006; Ramezani et al., 2009). In the present study, essential oil yield as well as leaf stomatal conductance did not chance either by increasing N or P concentration (Table 2). Nitrogen application presents conflicting results in regard to growth, essential oil yield and contents of medicinal plants. Economakis et al. (1999) and Baranauskienne et al. (2003) showed that N fertilization had no effect on essential oil content of Origanum dictamnus and T. vulgaris, respectively, being in accordance with the present study. Similar, Arabaci and Bayram (2004) reported that N fertilization increased the herb yield but had no effect on essential oil content of basil. It was reported that P (5, 30, and 60 mg/L) concentrations in the nutrient solution affected the essential oil yield and constitutes in O. dictamnus (Economakis et al., 2002) which differ compared to our findings and this may related to the really low (i.e., 5 mg/L) P concentrations used in the past study, whereas their high concentration (i.e., 60 mg/L) was similar to the high P levels used in the current work. Phosphorus levels and interaction with nitrogen were not significant on sage oil content (Verma et al., 2010). Phosphorus application significantly increased basil essential oil content, but fresh and dry weight of herbage remained unaffected (Ramezani et al., 2009), being in accordance with the present findings regarding the plant development status. The effect of different N and P levels into nutrient solution on chemical composition of the essential oil of L. angustifolia and reten-

7

tion indices are given in Table 5. Thirty eight components were identified in the essential oils of lavender plants underwent at different treatments that represented 98.67–99.67% of the oils. It can be noticed that, hydrocarbon compounds ranged from 9.17 to 10.59% while oxygenated (monoterpenes and sesquiterpenes) compounds ranged from 84.70 to 87.76% and 2.05 to 3.57%, respectively. major components were 1.8-cineole (alcohol; The 51.67–62.28%), borneol (alcohol; 8.45–12.34%), camphor (ketone; 7.43–11.26%), ␤-pinene (monoterpene hydrocarbon;3.02–3.67%), (alcohol; 2.16–3.41%), myrtenal (aldehyde; ␣-terpineol 2.04–2.76%), ␣-pinene (monoterpene hydrocarbon;1.66–2.21%), limonene (monoterpene hydrocarbon; 1.61–1.94%), ␣-bisabolol (alcohol; 1.05–1.79%) and trans-pinocarveol (alcohol; 0.98–1.51). Other components were present in amounts less than 1% in most treatments. It has been reported in L. officinalis that 1.8-cineole, borneol and camphor were the predominant components of leaf volatile oil while linalool, 1.8-cineole, borneol and camphor were the major component of inflorescence oil (Hassanpouraghdam et al., 2011), being in agreement with current leaf oil constitutes, indicating the lavender chemotype (CT) of CT-1,8 cineole. Examining the N effects on oil constitutes, 1.8-cineole reached to its maximal percentage (58.55%) as a result of N 200 mg/L application. Camphor and borneol, reached the greatest (11.26% and 11.55%) value at 150 mg/L N and the lowest (8.02% and 9.10%) value at 200 mg/L N, respectively. Oil quality decreases with increasing camphor ratios (Biswas et al., 2009), consisting the 200 mg/L N treatment as the most appropriate one. Myrtenal greatest percentage reached at 150 mg/L N, while the lowest value was obtained at the 250 mg/L N. Other components which showed significance were trans-Pinocarveol (1.15–1.51%), ␣-Terpineol (2.16–3.32%) and ␣-Bisabolol (1.08–1.79%). Considering P effects on oil constitutes, 1.8-cineole reached to its maximal percentage of 62.28% as a result of P 60 mg/L application, while pinocarvone reached to its greatest percentage (1.01%) at 50 mg/L P. Increasing P levels (>50 mg/L) into the nutrient solution, resulted in increased ␣-Terpineol percentage. No differences on camphor and borneol presentences were observed among P treatments. Other components which showed significance were trans-Pinocarveol (0.98–1.23%) and ␣-Terpineol (2.16–3.01%). To identify possible relationships between volatile compounds and nutrient concentrations, linear discriminate analysis (LDA) was applied (Fig. 2). The LDA, performed on average contents of all compounds for each nutrient concentration, showed that the first two principal axes represented 91.6% of the total variation. The first axis (82.3% of the total variation) was mainly correlated with thuja-2,4(10)-diene, ␤-Pinene, cineole, cis-Sabinenehydrate, p-mentha-2,4(8)-diene, camphene and fenchol. The second axis represented 9.3% of the total variation, and ␣-Pinene, sabinene, ␤-Myrcene, o-Cymene, ␥-Terpinene, ␣-Terpinene and limonene were the main compounds contributing to its definition. The plot of the projection of the average values of all the compounds onto the first two principal axes, revealed a high chemical dispersion among nutrient concentrations (Fig. 2). Therefore, according to the linear discriminate analysis, three concentration groups in relation to the nutrient solution could be distinguished. The first group represented by concentrations 150 mg/L N, situated at the periphery of the plot (inferior semiarid zone), situated at the positive side of axis 1 and at the negative side of axis 2. The second group represented by 175 mg/L N; 200 mg/L N/50 mg/L P; 225 mg/L N; 250 mg/L N and 40 mg/L P situated in the center of axis 1 and 2. The third group represented by 30 mg/L P; 60 mg/L P and 70 mg/L P situated at the periphery of the plot (inferior semiarid zone), situated at the positive side of axis 2 (30 mg/L P) and at the negative side of axis 1 (60–70 mg/L P).

Please cite this article in press as: Chrysargyris, A., et al., Nitrogen and phosphorus levels affected plant growth, essential oil composition and antioxidant status of lavender plant (Lavandula angustifolia Mill.). Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.12.067

N175 1.67 ± 0.38 a 0.37 ± 0.05 ab 0.08 ± 0.01 a 0.91 ± 0.13 a 3.22 ± 0.32 a 0.53 ± 0.09 ab 0.20 ± 0.01 a 0.18 ± 0.02 b 1.90 ± 0.00 a 54.86 ± 2.12 ab 0.41 ± 0.00 a 0.63 ± 0.03 a 0.14 ± 0.00 a 0.28 ± 0.00 a 0.08 ± 0.00 a 0.32 ± 0.01 a 1.26 ± 0.06 bc 0.11 ± 0.00 a 9.64 ± 0.27 b 0.99 ± 0.04 a 9.81 ± 0.27 ab 0.72 ± 0.04 ab 0.12 ± 0.01 ab 0.25 ± 0.03 a 2.92 ± 0.39 ab 2.54 ± 0.02 abc 0.04 ± 0.03 a 0.27 ± 0.02 a 0.31 ± 0.02 a 0.27 ± 0.00 a 0.16 ± 0.00 a 0.11 ± 0.01 a 0.11 ± 0.03 a 0.71 ± 0.04 a 0.78 ± 0.01 a 0.34 ± 0.01 a 1.59 ± 0.07 ab 0.44 ± 0.04 a

N200 2.10 ± 0.02 a 0.50 ± 0.00 a 0.10 ± 0.01 a 0.98 ± 0.03 a 3.55 ± 0.00 a 0.44 ± 0.00 b 0.20 ± 0.02 a 0.21 ± 0.00 a 1.69 ± 0.07 a 58.55 ± 0.59 a 0.40 ± 0.03 a 0.67 ± 0.12 a 0.13 ± 0.01 a 0.24 ± 0.02 a 0.09 ± 0.01 a 0.34 ± 0.02 a 1.17 ± 0.01 c 0.11 ± 0.00 a 8.02 ± 0.11 d 1.01 ± 0.04 a 9.10 ± 0.37 b 0.66 ± 0.02 b 0.11 ± 0.00 b 0.23 ± 0.02 a 2.16 ± 0.02 c 2.38 ± 0.03 bc 0.05 ± 0.02 a 0.27 ± 0.01 a 0.29 ± 0.02 a 0.27 ± 0.05 a 0.18 ± 0.00 a 0.11 ± 0.00 a 0.09 ± 0.02 a 0.49 ± 0.02 b 0.57 ± 0.03 b 0.27 ± 0.01 a 1.08 ± 0.04 c 0.40 ± 0.02 a

N225 1.89 ± 0.10 a 0.35 ± 0.02 b 0.09 ± 0.00 a 0.91 ± 0.06 a 3.25 ± 0.21 a 0.62 ± 0.01 a 0.19 ± 0.01 a 0.15 ± 0.01 b 1.94 ± 0.155 a 52.18 ± 1.50 ab 0.39 ± 0.03 a 0.61 ± 0.02 a 0.13 ± 0.01 a 0.28 ± 0.01 a 0.08 ± 0.00 a 0.28 ± 0.01 a 1.34 ± 0.02 b 0.12 ± 0.00 a 10.00 ± 0.15 b 0.92 ± 0.04 a 10.86 ± 0.14 ab 0.79 ± 0.00 a 0.14 ± 0.00 a 0.28 ± 0.00 a 3.41 ± 0.06 a 2.61 ± 0.12 ab 0.07 ± 0.01 a 0.29 ± 0.01 a 0.27 ± 0.01 a 0.25 ± 0.01 a 0.16 ± 0.00 a 0.12 ± 0.00 a 0.08 ± 0.01 a 0.62 ± 0.09 ab 0.82 ± 0.08 a 0.34 ± 0.05 a 1.79 ± 0.17 a 0.48 ± 0.06 a

N250 2.13 ± 0.17 a 0.44 ± 0.02 ab 0.07 ± 0.00 a 1.00 ± 0.07 a 3.63 ± 0.09 a 0.55 ± 0.06 ab 0.18 ± 0.01 a 0.17 ± 0.00 b 1.94 ± 0.09 a 56.35 ± 1.89 ab 0.36 ± 0.02 a 0.59 ± 0.06 a 0.13 ± 0.00 a 0.24 ± 0.02 a 0.07 ± 0.00 a 0.29 ± 0.01 a 1.15 ± 0.05 c 0.09 ± 0.00 a 8.94 ± 0.20 c 0.91 ± 0.04 a 9.72 ± 0.94 ab 0.67 ± 0.04 ab 0.11 ± 0.00 b 0.25 ± 0.02 a 2.63 ± 0.19 bc 2.31 ± 0.10 c 0.04 ± 0.02 a 0.23 ± 0.02 a 0.26 ± 0.00 a 0.23 ± 0.00 a 0.13 ± 0.01 a 0.10 ± 0.00 ab 0.10 ± 0.02 a 0.54 ± 0.06 ab 0.66 ± 0.07 ab 0.28 ± 0.03 a 1.39 ± 0.15 bc 0.39 ± 0.02 a

P30 2.21 ± 0.06 A 0.48 ± 0.01 A 0.06 ± 0.00 B 0.87 ± 0.06 AB 3.58 ± 0.17 A 0.46 ± 0.05 A 0.16 ± 0.00 A 0.16 ± 0.00 B 1.66 ± 0.13 A 60.70 ± 2.14 AB 0.34 ± 0.01 A 0.45 ± 0.03 A 0.09 ± 0.01 B 0.17 ± 0.00 B 0.05 ± 0.01 B 0.23 ± 0.00 B 0.98 ± 0.09C 0.07 ± 0.02 A 7.71 ± 0.86 A 0.84 ± 0.00 B 9.36 ± 0.40 A 0.61 ± 0.05 B 0.12 ± 0.02 A 0.26 ± 0.04 A 2.17 ± 0.14 B 2.04 ± 0.13 A 0.07 ± 0.03 A 0.20 ± 0.04 B 0.24 ± 0.00 A 0.22 ± 0.00 B 0.12 ± 0.03 A 0.08 ± 0.03 AB 0.03 ± 0.03 BC 0.45 ± 0.12 AB 0.57 ± 0.19 A 0.25 ± 0.09 A 1.19 ± 0.42 A 0.38 ± 0.14 A

P40 1.96 ± 0.01 A 0.40 ± 0.00 B 0.05 ± 0.00 B 0.80 ± 0.00 B 3.25 ± 0.01 A 0.48 ± 0.00 A 0.14 ± 0.00 A 0.16 ± 0.00 B 1.64 ± 0.01 A 62.28 ± 0.23 A 0.30 ± 0.00 A 0.56 ± 0.00 A 0.08 ± 0.00 B 0.17 ± 0.00 B 0.01 ± 0.01C 0.23 ± 0.00 B 1.00 ± 0.00 BC 0.08 ± 0.00 A 8.15 ± 0.04 A 0.87 ± 0.01 B 8.45 ± 0.04 A 0.64 ± 0.00 AB 0.12 ± 0.00 A 0.27 ± 0.00 A 2.28 ± 0.00 B 2.07 ± 0.01 A 0.04 ± 0.02 A 0.22 ± 0.00 AB 0.24 ± 0.00 A 0.24 ± 0.00 B 0.12 ± 0.00 A 0.00 ± 0.00 B 0.00 ± 0.00C 0.36 ± 0.00 B 0.45 ± 0.00 A 0.18 ± 0.00 A 1.05 ± 0.02 A 0.35 ± 0.00 A

P50 2.10 ± 0.02 A 0.50 ± 0.00 A 0.10 ± 0.01 A 0.98 ± 0.03 A 3.55 ± 0.00 A 0.44 ± 0.00 A 0.20 ± 0.02 A 0.21 ± 0.00 A 1.69 ± 0.07 A 58.55 ± 0.59 AB 0.40 ± 0.03 A 0.67 ± 0.12 A 0.13 ± 0.01 A 0.24 ± 0.02 A 0.09 ± 0.01 A 0.34 ± 0.02 A 1.17 ± 0.01 AB 0.11 ± 0.00 A 8.02 ± 0.11 A 1.01 ± 0.04 A 9.10 ± 0.37 A 0.66 ± 0.02 AB 0.11 ± 0.00 A 0.23 ± 0.02 A 2.16 ± 0.02 B 2.38 ± 0.03 A 0.05 ± 0.02 A 0.27 ± 0.01 AB 0.29 ± 0.02 A 0.27 ± 0.00 A 0.18 ± 0.00 A 0.11 ± 0.00 A 0.09 ± 0.02 AB 0.49 ± 0.02 AB 0.57 ± 0.03 A 0.27 ± 0.01 A 1.08 ± 0.04 A 0.40 ± 0.02 A

P60 2.02 ± 0.12 A 0.42 ± 0.01 B 0.07 ± 0.00 AB 0.81 ± 0.03 B 3.35 ± 0.16 A 0.52 ± 0.02 A 0.19 ± 0.01 A 0.17 ± 0.01 B 1.61 ± 0.09 A 55.12 ± 1.15 B 0.40 ± 0.02 A 0.45 ± 0.02 A 0.10 ± 0.00 AB 0.22 ± 0.00 A 0.07 ± 0.00 AB 0.25 ± 0.01 B 1.23 ± 0.03 A 0.11 ± 0.00 A 7.73 ± 0.81 A 0.89 ± 0.03 B 12.34 ± 1.44 A 0.75 ± 0.03 A 0.16 ± 0.01 A 0.30 ± 0.03 A 3.01 ± 0.02 A 2.44 ± 0.10 A 0.11 ± 0.03 A 0.31 ± 0.01 A 0.25 ± 0.01 A 0.24 ± 0.00 B 0.19 ± 0.01 A 0.11 ± 0.00 A 0.08 ± 0.01 AB 0.58 ± 0.06 AB 0.65 ± 0.05 A 0.31 ± 0.03 A 1.43 ± 0.10 A 0.43 ± 0.04 A

P70 2.10 ± 0.17 A 0.42 ± 0.02 B 0.07 ± 0.02 AB 0.91 ± 0.00 AB 3.67 ± 0.16 A 0.49 ± 0.05 A 0.19 ± 0.04 A 0.18 ± 0.01 B 1.91 ± 0.04 A 55.08 ± 5.16 B 0.41 ± 0.07 A 0.48 ± 0.08 A 0.09 ± 0.00 B 0.20 ± 0.01 AB 0.06 ± 0.01 AB 0.27 ± 0.01 B 1.10 ± 0.08 AB 0.09 ± 0.01 A 7.43 ± 1.11 A 0.91 ± 0.00 B 12.23 ± 3.39 A 0.67 ± 0.04 AB 0.13 ± 0.01 A 0.24 ± 0.01 A 2.81 ± 0.33 A 2.42 ± 0.27 A 0.05 ± 0.00 A 0.24 ± 0.07 AB 0.28 ± 0.03 A 0.23 ± 0.02 B 0.14 ± 0.05 A 0.06 ± 0.05 B 0.12 ± 0.02 A 0.70 ± 0.21 A 0.76 ± 0.21 A 0.34 ± 0.13 A 1.62 ± 0.38 A 0.43 ± 0.16 A

9.17 ± 0.43 a 0.00 86.59 ± 0.84 a 2.87 ± 0.15 ab 0.72 ± 0.11 a

9.59 ± 0.99 a 0.00 85.36 ± 1.08 a 3.41 ± 0.00 a 0.80 ± 0.04 a

10.31 ± 0.03 a 0.00 85.78 ± 0.31 a 2.41 ± 0.11 b 0.71 ± 0.06 a

9.89 ± 0.63 a 0.00 84.70 ± 1.24 a 3.57 ± 0.39 a 0.82 ± 0.07 a

10.59 ± 0.62 a 0.00 85.05 ± 0.91 a 2.87 ± 0.32 ab 0.74 ± 0.00 a

10.06 ± 0.42 A 0.00 86.44 ± 1.41 A 2.45 ± 0.84 A 0.67 ± 0.19 A

9.25 ± 0.05 A 0.00 87.76 ± 0.14 A 2.05 ± 0.04 A 0.62 ± 0.01 A

10.31 ± 0.03 A 0.00 85.78 ± 0.31 A 2.41 ± 0.11A 0.71 ± 0.06 A

9.67 ± 0.48 A 0.00 85.99 ± 0.70 A 2.96 ± 0.25 A 0.82 ± 0.02 A

10.43 ± 0.58 A 0.00 84.81 ± 1.93 A 3.42 ± 0.93 A 0.79 ± 0.15 A

99.36 ± 0.18a

99.14 ± 0.12a

99.20 ± 0.18a

98.97 ± 0.14a

99.25 ± 0.14a

99.63 ± 0.13A

99.67 ± 0.06 A

99.20 ± 0.18 A

99.43 ± 0.06 A

99.43 ± 0.25 A

ARTICLE IN PRESS

Monoterpenes hydrocarbons Sesquiterpenes hydrocarbons Oxygenated monoterpenes Oxygenated sesquiterpenes Others – Total

P

N150 1.66 ± 0.18 a 0.30 ± 0.06 b 0.10 ± 0.01 a 0.92 ± 0.05 a 3.02 ± 0.16 a 0.53 ± 0.02 ab 0.18 ± 0.01 a 0.15 ± 0.00 b 1.84 ± 0.14 a 51.67 ± 0.76 b 0.37 ± 0.01 a 0.84 ± 0.05 a 0.11 ± 0.02 a 0.32 ± 0.03 a 0.08 ± 0.00 a 0.30 ± 0.02 a 1.51 ± 0.04 a 0.10 ± 0.02 a 11.26 ± 0.17 a 0.99 ± 0.03 a 11.55 ± 0.55 a 0.76 ± 0.02 ab 0.13 ± 0.00 ab 0.27 ± 0.03 a 3.32 ± 0.10 a 2.76 ± 0.04 a 0.05 ± 0.02 a 0.28 ± 0.04 a 0.25 ± 0.02 a 0.24 ± 0.02 a 0.13 ± 0.02 a 0.05 ± 0.02 b 0.06 ± 0.03 a 0.62 ± 0.05 ab 0.69 ± 0.02 ab 0.27 ± 0.05 a 1.29 ± 0.04 bc 0.39 ± 0.03 a

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RI 933 948 954 973 977 991 1017 1024 1028 1031 1058 1067 1089 1100 1114 1127 1139 1141 1145 1163 1166 1178 1185 1187 1191 1197 1211 1219 1241 1244 1291 1329 1443 1587 1642 1656 1685 1689

G Model

N Compound ␣-Pinene Camphene Thuja-2.4 (10)- diene Sabinene ␤-Pinene ␤-Myrcene ␣-Terpinene o-Cymene Limonene 1.8-Cineole ␥-Terpinene cis-Sabinene hydrate p-Mentha-2.4(8)-diene Linalool Fenchol ␣-Campholenal trans-Pinocarveol cis-Verbenol Camphor Pinocarvone Borneol Terpinen-4-ol p-Cymen-8-ol Cryptone ␣-Terpineol Myrtenal Verbenone trans-Carveol Cumin aldehyde Carvone p-Cymen-7-ol p-Mentha-1.4-dien-7-ol Coumarin Caryophyllene oxide tau-Cadinol Bisabolol oxide II ␣-Bisabolol Muurol-5-en-4-one

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Table 5 Chemical composition (%) of essential oils of lavender plants grown hydroponically in perlite under nitrogen and phosphorus rates.

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Fig. 2. Linear discriminant analysis (LDA) for the lavender essential oil compounds under different nitrogen (N) and phosphorous (P) concentrations. Projection of the average contents of the essential oil compounds onto the first two principal axes (+ and – indicate positive and negative correlations with axes, respectively). Coding numbers referred to N and P concentrations.

It is worthwhile to mention that high yielding lavender varieties (e.g., Lavandin; Lavandula intermedia) typically produce low-grade oils. As a consequence, plant biomass and oil yield are not the only factors that should be considered, while the EO constitute is of great interest as well. Metabolic engineering in biosynthetic pathways is an important attempt to overcome this difficulty (Biswas et al., 2009). It’s evidence, that several aromatic species, including Lamiacea family, are being harvested essentially before or during the flowering period, before seed set, resulting in low regeneration and progressive population decline (Abbad et al., 2011). Thus, species cultivation could be a promising solution to ensure their conservation under sustainable scheme of utilization. Nevertheless, the cultivation of wild medicinal species is not so simple, as it requires a good understanding domestication impacts on their biological activities associated with their chemical composition. It has been noted that the lack of reproducibility of bioactivity can represent a major restriction for the successful cultivation and commercialization of medicinal and aromatic plants (Lubbe and Verpoorte, 2011), while hydroponically grown medicinal plants provide stable and controlled-manner bioactivity. Appropriate agricultural practices are necessary while controlled mineral uptake either in soils or through hydroponics is one efficient way for successful plant growth and EO biosynthesis control (Karamanos and Sotiropoulou, 2013).

development. The effects of nitrogen and phosphorus rates on hydroponically grown lavender plants, under controlled nutrition status were examined for plant growth, mineral uptake, antioxidant activity and essential oil constitute changes. P levels mainly affected plant growth, while lower N levels (150 mg/L) reduced chlorophylls content. The N levels greater than 200 mg/L benefited antioxidant status of lavender, while middle P concentrations supported better the antioxidant activity. Considering greater accumulation for Ca, Mg and Na, lower Al accumulation and lower camphor percentage in 200 mg/L N treatment, supporting better N:K balance, the recommended N and P levels are 200 mg/L and 50 mg/L respectively. Acknowledgement This research has been co-financed by the Start-up grant for the research project SALTAROMA in Cyprus University of Technology. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop.2015.12. 067. References

4. Conclusion The present findings highlight that aromatic plants development and essential oil production may be affected positively or negatively by the ratio and amount of minerals. Nitrogen and phosphorus are both important nutrients needed for plant growth and

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Please cite this article in press as: Chrysargyris, A., et al., Nitrogen and phosphorus levels affected plant growth, essential oil composition and antioxidant status of lavender plant (Lavandula angustifolia Mill.). Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.12.067