**1. Introduction**

In arid, semi-arid and coastal areas, natural resources for good quality water have decreased. They are often characterized by high contents of total soluble salts due to groundwater overexploitation, seawater intrusion into aquifers and increased demand for freshwater, particularly in densely populated areas [1,2]. Long term degradation of water quality has led to the use of alternative water resources for irrigation derived from water reuse and recycling that is also saline [3,4]. Irrigation with saline water affects the growth and development of many plant species, even at low concentrations [5,6]. In the ground whether in the wild, field or garden, the effect of salinity on plants is determined by various variables such as ion concentration, soil composition, proximity to the sea, altitude, evapotranspiration rate, temperature and rainfall frequency [5–7]. In many parts of the world, salinity affects agricultural production and is predicted to become more intense in future decades [8]. It is considered as one of the most important stress factors in plant growth and yield that could lead to plant death under persisting

saline conditions [6,9]. Plant tolerance to salinity stress depends on the capacity of plants to exclude salt from the shoots or tolerate high leaf salt concentrations [10].

Irrigation with saline water initially creates a water deficit induced by osmotic stress and demonstrated by the reduced ability of plants to absorb water hence reduced plant growth rate [11]; the high saline concentrations cause osmotic and ionic imbalances between soil and plants, and plants exhibit signs of wilt despite the fact they have been irrigated [12,13]. Afterwards, a salt-specific or ion-excess effect of salinity is demonstrated by the salt entrance into the plant transpiration stream, causing eventual injury of transpiring leaf cells and further reduction of plant growth [11]. The high saline concentrations within the plant affect the anatomy, physiology and morphology of plant parts and particularly of leaves [4,6,14,15]. The salts absorbed by the plant are concentrated within the mature leaves, leading to leaf death over an extended time period due to the inability of leaf cells to compartmentalize salts in the vacuole; hence the salts either accumulate in the cytoplasm, inhibiting enzyme function, or accumulate in the cell walls, dehydrating the leaf cells [11]. The level of stress caused by salinity is dependent on the plant species and variety, the growth substrate and the applied method of irrigation. The more tolerant nonhalophytic species avoid the ion-excess effect. However, they may exhibit water deficits affecting cell extension and/or division. Therefore, potential reductions in photosynthesis may represent a secondary effect of reduced growth [16].

In floriculture, the use of saline water for the production of nursery crops requires an understanding of plant response to the effect of salinity through irrigation [17]. Some effects of salinity, on one hand, could be desirable such as decreased length and/or number of internodes [17] and others on the other hand could be undesirable such as chlorosis and marginal leaf necrosis. The effect of saline irrigation on floriculture has received less attention, as ornamental plants are normally irrigated with good-quality water [18,19]. In areas with limited or poor water quality resources, the cultivation of floriculture crops that can tolerate saline water irrigation can be an advantage [20]. Lavender species and varieties are popular floriculture crops. Lavender plants such as *Lavandula angustifolia*, *L. dentata*, and *L. stoechas* and their numerous cultivars are sold as ornamental plants for the garden. These species exhibit a variety of leaves and inflorescences with ornamental value and are highly aromatic due to the essential oils present in glands that cover much of the plant surface. The *Lavandula* genus includes 47 species and many varieties [21]. Some *Lavandula* species such as *Lavandula stoechas* and *Lavandula angustifolia* are found naturally growing in the Mediterranean coniferous coastal dune woodlands, coastal garrigues and sea cliffs, often exposed to sea spray [22,23]. The literature on the effect of saline irrigation on *Lavandula* species for nursery crops is limited. Potted *L. multifida* plants were able to grow in a mixture of sphagnum peat-moss and perlite when irrigated with 60 mM NaCl without significant biomass reduction [24]; however, the total plant dry weight of *L. multifida* decreased when irrigated with 100 mM and 200 mM NaCl [20,24]. Despite this, there are no comparative studies among different lavender species grown under greenhouse conditions and irrigated with different NaCl solutions.

This study examines the pot growth of four *Lavandula* species irrigated with different concentrations of saline water for nursery production to support floriculture in areas with poor water quality, using saline water for irrigation.

#### **2. Materials and Methods**

#### *2.1. Experimental Site and Growth Conditions*

Uniform in size, young (5 months old) and fully developed potted lavender plants were supplied by the nursery Kalantzis Plants (Marathonas, Greece). The pot size was 2.5 L (dimensions: 17 cm top diameter, 12.3 cm base diameter and 15 cm height). The growth substrate of the supplied potted lavender plants contained pure sphagnum peat (Base Substrate 2 medium, Klausmann-Deilmann Europe GmbH, Germany) and perlite (Perloflor, ISOCON S.A., Piraeus, Greece) in a 96:4 ratio (v/v) with pH 5.5–6.0 and EC 0.8 mS m−<sup>1</sup> . Plants were placed on metal benches (dimensions: 2.5 m length, 0.85 m width and 0.80 m height) in an automated glass greenhouse of the Laboratory of Floriculture & Landscape Architecture of the Agricultural University of Athens (lat. 37◦58057"N and long. 23◦42017"E), with average daily and night temperatures of 21.4 ± 0.311 ◦C and 14.3 ± 0.065 ◦C, respectively, and average humidity during daytime of 57.6 ± 0.705% and night-time average humidity of 84.6 ± 0.309%. Plants were acclimatized to the new growth conditions for a month and the experiment took place in late winter-early spring over 56 days (from 3 February; day 1 to 30 March 2018; day 56). All plants received the same cultivation practices (i.e., applications of fertilizer, fungicide, etc.) throughout the duration of the experiment that included the application of 2 g L−<sup>1</sup> H2O fertilizer 20-20-20 (Fast-Grow, Humofert S.A., Metamorfosi, Greece) and pesticide (Decis 25 EC, Bayer AG, Leverkusen, Germany) at monthly intervals.

#### *2.2. Experimental Design and Irrigation Treatments*

Four lavender species were studied: *Lavandula angustifolia*, *Lavandula dentata* var. *dentata*, *Lavandula dentata* var*. candicans* and *Lavandula stoechas*. The effect of salinity was investigated using different concentrations of NaCl solutions through irrigation that included 0 (control), 25, 50, 100 and 200 mM of NaCl. The corresponding EC levels for the irrigation water were 0.3, 3.0, 5.8, 10.6 and 20.7 dSm−<sup>1</sup> and pH values were in the range 8.0–8.2 (at 25 ◦C).

Plants were arranged in a randomized complete block that consisted of 4 lavender species, 5 NaCl solution irrigation treatments and 6 replicates (plants) arranged in 3 blocks (metal benches) i.e., 2 plants per species and NaCl solution irrigation treatment per metal bench. The number of plants totaled 120 and the experimental surface area occupied approximately 6.5 m<sup>2</sup> (Figure 1).

At the start of the experiment (day 1), all plants were irrigated with the corresponding NaCl solutions to saturation and weighed half an hour later to determine the water container capacity of the substrate. Substrate water content was monitored using a handheld TDR moisture sensor (HH2, Delta-T Devise, Cambridge, UK) set at the 'organic soil' setting, appropriate for use with peat-based substrates and calibrated to the used substrate. The probes were fully inserted into the substrate with the central rod positioned 5 cm away from the plant center. Irrigation was performed manually when the TDR sensor showed a water content value of approximately 0.46 cm<sup>3</sup> cm−<sup>3</sup> , which was determined from the substrate water retention curve at corresponding a pressure head of −50 cm (Figure 2), and with an amount of water ensuring substrate water availability within the easily available water area (Table 1). This amount of irrigation water of plants was determined with the mean accumulated daily difference in weight of six potted plants from each NaCl treatment between two consecutive irrigations that corresponded to the amount of water lost from evapotranspiration.

*Water* **2020**, *12*, x FOR PEER REVIEW 4 of 18

**Figure 1.** The layout of the experiment studying the effect of different NaCl solution irrigation treatments on the growth of 4 lavender species. Plants were arranged in a randomized complete block that consisted of 4 lavender species, 5 NaCl solution irrigation treatments (0, 25, 50, 100, 200 mM NaCl) and 6 replicates (plants) arranged in 3 blocks (metal benches; dimensions 2.50 cm length and 0.85 m width). La: *Lavandula angustifolia*, Ld: *Lavandula dentata* var. *dentata*, Lc: *Lavandula dentata* var. *candicans*, Ls: *Lavandula stoechas*, and subscripts denote applied **Figure 1.** The layout of the experiment studying the effect of different NaCl solution irrigation treatments on the growth of 4 lavender species. Plants were arranged in a randomized complete block that consisted of 4 lavender species, 5 NaCl solution irrigation treatments (0, 25, 50, 100, 200 mM NaCl) and 6 replicates (plants) arranged in 3 blocks (metal benches; dimensions 2.50 cm length and 0.85 m width). La: *Lavandula angustifolia*, Ld: *Lavandula dentata* var. *dentata*, Lc: *Lavandula dentata* var. *candicans*, Ls: *Lavandula stoechas*, and subscripts denote applied NaCl solution irrigation treatments.

#### NaCl solution irrigation treatments. *2.3. Plant Growth Variables*

*2.3. Plant Growth Variables*  Measurements started one week (day 7) after irrigation with the NaCl solutions for the first time and ended 56 days later. Plant height (determined from the pot rim of the substrate surface), shoot canopy diameter (mean value of the widest width and perpendicular width), and growth index ((height + widest width + perpendicular width)/3) were measured at weekly intervals. Additionally, during flowering, for each plant, the number and length of all inflorescences that were fully open >60% as well as the corresponding peduncle length, were recorded at weekly intervals. The maximum efficiency of PSII photochemistry (ΦPSIIo) of mature leaves (3 leaves per plant) was determined fortnightly (day 14, 35, 56) using a MINI-PAM Photosynthesis Yield Analyzer (Heinz Walz GmbH, Effeltrich, Germany). All measurements were performed in the morning after dark acclimation of the Measurements started one week (day 7) after irrigation with the NaCl solutions for the first time and ended 56 days later. Plant height (determined from the pot rim of the substrate surface), shoot canopy diameter (mean value of the widest width and perpendicular width), and growth index ((height + widest width + perpendicular width)/3) were measured at weekly intervals. Additionally, during flowering, for each plant, the number and length of all inflorescences that were fully open >60% as well as the corresponding peduncle length, were recorded at weekly intervals. The maximum efficiency of PSII photochemistry (ΦPSIIo) of mature leaves (3 leaves per plant) was determined fortnightly (day 14, 35, 56) using a MINI-PAM Photosynthesis Yield Analyzer (Heinz Walz GmbH, Effeltrich, Germany). All measurements were performed in the morning after dark acclimation of the samples for 30 min using the saturation pulse technique. Saturation pulse (intensity circa 12,000 µmol quanta m−<sup>2</sup> s −1 ) lasted 0.8 s.

samples for 30 min using the saturation pulse technique. Saturation pulse (intensity circa 12,000 μmol quanta m−2 s−1) lasted 0.8 s. At the end of the experiment (day 56) the leaf thickness of mature leaves was determined with cross sections taken at a distance of 3 cm from the leaf base (3 leaves per plant) under a Zeiss Axiolab microscope (Carl Zeiss, Jena, Germany) using the x100 lens. Plants were harvested at the end of the experiment (day 56) and divided at soil level into shoot and root. The substrate was carefully washed At the end of the experiment (day 56) the leaf thickness of mature leaves was determined with cross sections taken at a distance of 3 cm from the leaf base (3 leaves per plant) under a Zeiss Axiolab microscope (Carl Zeiss, Jena, Germany) using the x100 lens. Plants were harvested at the end of the experiment (day 56) and divided at soil level into shoot and root. The substrate was carefully washed off the harvested root. Following both harvested shoots and roots were separately dried in an oven at

off the harvested root. Following both harvested shoots and roots were separately dried in an oven

70 ◦C until a constant weight was reached, and their dry weights were determined. Weekly recordings of observations for signs of salinity stress were undertaken, throughout the duration of the experiment. In plants, the onset of visual symptoms induced by salinity (leaf and stem chlorosis and necrosis) was recorded during the experiment and assessed at the end of the experiment (day 56). Visual symptoms induced by salinity were assessed on a 6 point scale from 0–5, where 0: plant mortality, 1: no leaf injury, 2: mild leaf chlorosis, 3: moderate leaf chlorosis 25–50% approximately, 4: leaf necrosis 50–75% approximately, 5: leaf necrosis >75%.
