Introduction
Lilies are one of the most popular floricultural crops and are widely used in the horticultural industry as bulbs, cut flowers, and garden plants. Lilies can be divided into five types: Asiatic-, Oriental-, LA-, LO-, and OT-hybrids (Gill et al. 2006). In Korea, lily is the third most cultivated cut flower, with a cultivated area of 77 ha and a value of 10,104 million won (MAFRA 2022). While lily bulbs are mostly imported into Korea from the Netherlands, there have been recent attempts to grow lilies on reclaimed land to reduce import costs (Kang et al. 2021). Reclaimed land often has a high salinity, which can be a problem for plant growth (García-Caparrós and Lao 2018). Similarly, crops grown in a greenhouse with controlled environments for year-round production can often exhibit saline stress from fertilization (Machado and Serralheiro 2017).
Salinity in irrigation and soil is one of the major abiotic factors, affecting nearly 6% of the world's countries (Parihar et al. 2015). Salinity can limit plant growth and development by causing osmotic stress, reduced water potential, ionic imbalances, and toxicity (Munns and Tester 2008). Initially, when roots are exposed to salinity, changes in osmotic pressure at the cellular level make water uptake difficult, and Na+ becomes more concentrated in the roots and stems than in the aerial parts, resulting in reduced plant growth (Machado and Serralheiro 2017).
When ornamental plants are exposed to saline conditions, they have a negative effect on plant biomass. Reduction in plant biomass and flower numbers by increasing NaCl concentration has been reported in several floricultural crops, including chrysanthemum, daffodil, and garden rose (Bandurska et al. 2022;Cai et al. 2014;Veatch-Blohm et al. 2014). In high salinity soils, ion imbalance inhibits photosynthesis and accelerates the senescence of mature leaves (Munns and Tester 2008). High Na+ concentrations mainly reduce stomatal conductance, limiting the availability of CO2 for carboxylation, while high Cl- concentrations reduce the photosynthetic capacity through non-stomatal effects and chlorophyll degradation (Everard et al. 1994;Tavakkoli et al. 2011).
Photosynthesis is one of the main processes affected by salt stress (Munns et al. 2006). Abiotic stress influences photosynthesis in both the short and long term. In the short term, salinity can affect photosynthesis through stomatal limitation, leading to a decrease in carbon assimilation (Allel et al. 2017). In the long term, salinity stress can also affect the photosynthetic process through NaCl accumulation in young leaves and a reduction in chlorophyll and carotenoid concentrations (Acosta-Motos et al. 2017). In addition, saline conditions can induce a decrease in PSII efficiency and photochemical quenching parameters (Niu et al. 2008).
Salinity can also inhibit plant growth and flower quality. A previous study has reported that Asiatic hybrid, L. longiflorum, Asiatic hybrid, Oriental hybrid, and Oriental Trumpet hybrid lilies show reduced growth and flower development at high salinity (Kang et al. 2021). In this study, we hypothesized that salt stress inhibits the growth and photosynthetic capacity of lily and the consequent bulb growth. Therefore, this study was conducted to determine the effects of salinity stress on the bulb growth and photosynthetic capacity in Lilium LA hybrid lily ‘Serrada’.
Materials and Methods
Plant material and growth conditions
The experiment was conducted at the Seoul National University Farm Greenhouse (37.27°N, 126.99°E, Suwon, Korea). The Lilium LA hybrid lily ‘Serrada’ bulbs, 10/12 cm in diameter (Jan de Wit en Zonen B.V., Enkhuizen, Netherlands), were planted in bulb boxes [60 (L) × 40 (W) × 20 (H) cm] on 15 September 2023. The box was filled with five liters of commercial horticultural substrate (Sunshine Mix #4, Sun Gro Horticulture, Agawam, MA, USA). Five bulbs were planted per box and the planting depth was approximately 10 cm. A 15 N–4.8 P–10.8 K + 2 Mg + TE controlled-release fertilizer (Osmocote Plus; Everris International B.V., Heerlen, Netherlands) was applied at 20 g per box. The plants were watered twice a week until the end of the experiment. The experiment was lasted three months, from 15 September to 22 December 2023. Air temperature and relative humidity were recorded at one-minute intervals using a data logger (Watch Dog Model 2000, Spectrum Technologies Inc., Aurora, USA). During the experimental period, the average air temperature and relative humidity were 16.6°C and 73.8%, respectively.
NaCl treatments
The plants were divided into three treatment groups: 0, 200, and 400 mM NaCl. Saline solutions were prepared by adding sodium chloride (NaCl; Duchefa Biochemie B.V. Inc., Haarlem, Netherlands) to tap water. Sodium chloride solutions, one liter per box, were applied manually by drenching every two weeks for 14 weeks. The same amount of water without added NaCl was used as a control. Plants were irrigated twice a week with one liter per box. The experiment was designed as a completely randomized design with nine replicates, each containing five plants, per treatment.
The analysis of pH and electrical conductivity (EC)
Substrate samples with three replications in each treatment were collected at the end of the experiment. The substrate was saturated to the maximum water-holding capacity, and then the substrate solution was extracted using a vacuum pump. The extract was measured for pH and EC using a portable pH/EC/TDS meter (Hanna Instruments Inc., Woonsocket, RI, USA).
Plant growth parameters
Plant height and SPAD value were measured weekly. Plant height was measured from the substrate surface to the top of the stem, and the relative chlorophyll content of mature leaves was measured using a chlorophyll meter (SPAD-502, Konica Minolta Inc., Tokyo, Japan). Days to flowering, number of flowers, and flower width were measured at the flowering stage. The days to flowering were calculated from the planting date to the first flowering. At the end of experiment, leaf area was measured using a leaf-area meter (LI-3100C, LI-COR Inc., Lincoln, NE, USA) and bulb size was measured with a tape measure, centered on the widest part of the bulb. After 14 weeks, fresh weight (FW) and dry weight (DW) were measured for each part of the three plants including root, bulb, stem, and leaf per treatment. The DW of the plants was obtained by drying the samples in an oven at 60°C for one week.
Photosynthetic capacity
At the flowering stage, leaf gas exchange was measured on a sunny day using a portable photosynthesis system (LI-6400XT, LI-COR Inc., Lincoln, NE, USA). Four fully mature healthy leaves from each treatment were selected for the measurement. The mature leaf, attached to the fifth node from the top, was clamped to a 6400-02B LED light source chamber. Light response of photosynthesis was measured at PPFD levels between 0 and 1500 μmol·m–2·s–1. The starting level of PPFD was 1500 μmol·m–2·s–1, followed by 1200, 1000, 900, 600, 300, 200, 100, 50, and 0 μmol·m–2·s–1 PPFD. Net assimilation rate (An) was measured under controlled conditions with 300 μmol·s-1 of air flow, 400 μmol·mol–1 of CO2 concentration, and 60% of relative humidity, and a block temperature was set at 25°C.
Three plants from each treatment were randomly chosen for measuring chlorophyll fluorescence. Stomatal conductance (gs) was measured at midday using a chlorophyll fluorometer (LI-600PF, LI-COR Inc., Lincoln, NE, USA). During the measurement, the average light intensities for 0, 200, and 400 mM NaCl treatments were 92.7, 93.8, 94.3 μmol·m–2·s–1, respectively. And, Light-adapted leaves from the fifth node from the top were induced with a saturating light pulse to obtain maximum fluorescence (Fm’). Fq’, the difference between Fm’ and steady-state level of fluorescence (F’), was calculated to determine photosystem II (PSII) operating efficiency (ΦPSII). The maximum quantum yield of photosystem II (Fv/Fm) was measured after 20 min of dark adaptation. Dark-adapted leaves were exposed to light less than 0.1 μmol·m–2·s–1 to obtain the minimum fluorescence in the dark-adapted state (Fo). A saturating light pulse was irradiated to induce maximum fluorescence in the dark-adapted state (Fm). The yield of variable fluorescence (Fv) was calculated from the equation Fv = Fm – Fo.
Statistical analysis
Statistical analysis was performed using ANOVA in IBM SPSS Statistics 29.0 software (SPSS Inc., Chicago, IL., USA). Mean separations among treatment groups were performed by Tukey’s HSD test at α=0.05. The graphs were created using SigmaPlot 12.5 (Systat Software Inc., Chicago, IL, USA).
Results
The NaCl treatments did not affect any aspect of plant height for 14 days. After 28 days, there was a clear trend showing a significant reduction (p < 0.001) in plant height (Fig. 1A). Specifically, plant height in the 400 mM NaCl significantly decreased compared to the control and 200 mM NaCl. Significant differences in SPAD values were observed from day 42 onwards, particularly between the control and the 400 mM NaCl treatment groups (p < 0.01) (Fig. 1B). The SPAD values decreased by 4.9% at 200 mM and by 8.6% at 400 mM NaCl, respectively (Fig. 1B).
With increasing salinity, the substrate showed a decrease in pH and an increase in EC, which were significantly affected (p < 0.001) by the salinity stress caused by NaCl accumulation in the substrate (Table 1). Regardless of salinity levels, the days to flowering and the number of flowers were similar among treatments, while the flower width decreased with increasing NaCl concentrations by 14.0% at 200 mM and 22.0% at 400 mM NaCl (Fig. 2A and Table 2). Kang et al. (2021) showed that plant height in Asiatic lily and LA hybrids decreased with 8 dS·m-1, but there were no significant changes in the days to flowering and flower width.
Growth characteristics, including bulb size, bulb root length, stem diameter, leaf area, and number of leaves, were significantly affected by the 400 mM NaCl treatment after 14 weeks (Table 3). The effects of increasing NaCl concentrations on the bulbs were clearly detrimental, as shown by the significant reduction in bulb size, bulb root length, and leaf area. At 200 mM NaCl, bulb size decreased by 9.2%, and the reduction was more severe by 20.0% at 400 mM compared to the control group. Similarly, bulb root length decreased by 16.7% at 200 mM NaCl and further decreased by 29.2% at 400 mM NaCl. Leaf area also showed a significant reduction (p < 0.001), decreasing by 23.0% at 200 mM and by 36.4% at 400 mM NaCl, respectively, indicating a severe effect of salt stress on plant growth.
Although ΦPSII and Fv/Fm were not affected by increased salinity (data not shown), An was reduced by 24.7% and 25.4% at 200 mM and 400 mM NaCl, respectively (Fig. 3A). The light saturation point decreased from approximately 900 μmol·m–2·s–1 in the control to 600 μmol·m–2·s–1 in the 400 mM NaCl treatment. Stomatal conductance was also significantly reduced (p < 0.001), decreasing by 46.7 % at 200 mM and 66.7% at 400 mM NaCl (Fig. 3B).
At the end of the experiment, the measured fresh and dry biomasses (root, bulb, stem, and leaf) showed significant reductions due to salinity (p < 0.001) (Fig. 2B and Table 4). In particular, the bulb growth was inhibited with increasing NaCl concentrations. Plants showed the highest biomass production in the control condition, but the biomasses were reduced in the 400 mM NaCl treatment, ranging from 38% to 81% reduction.
Discussion
In this study, plant biomass decreased as substrate pH decreased and EC increased with increasing NaCl concentrations. The pH values ranged from 5.7 to 6.2 (Table 1), which is within the optimal pH range for lily, which is 5.5 to 6.5 (Singh and Kumar 2024). These results were consistent with previous research. For example, the garden rose (Rosa× hybrida L.) cultivars 'Caldwell Pink', 'Marie Pavie' and 'The Fairy' exhibited reduced shoot growth and number of flowers at a salinity of 4.0 dS·m−1 (Cai et al. 2014). Increased salinity levels in Lilium ‘EL Divov’ led to a decrease in stem diameter, plant height, and leaf area (Ayad et al. 2019). Considering that soil EC is the concentration of ions in the soil solution (Corwin and Lesch 2005), it was subsequently found that the ion content in the NaCl solution increased in this study. A study by Ouhadi and Goodarzi (2007) reported that acidification can occur when hydrogen ions adsorbed on soil colloids are exchanged, resulting in the displacement of H+ ions from the sorption complex, which in turn increases the soil acidity.
Soil salinity inhibits plant growth through osmotic stress, which immediately reduces growth by disrupting water uptake when roots are exposed to salt and ionic toxicity, which accompanies the salt uptake stress (Munns and Tester 2008). Roots are mainly affected by osmotic and ionic stress, which directly affect plant growth, mainly inhibiting root growth (Bañón et al. 2012;Sánchez-Blanco et al. 2014). Similarly, a significant decrease in root length was observed (Table 3).
Chlorophyll fluorescence has been shown to be useful for detecting salinity stress by assessing PSII functionality (Sánchez-Blanco et al. 2014). Fv/Fm values of non-photoinhibited leaves generally range from 0.79 to 0.84 in various species (Maxwell and Johnson 2000). Despite the detrimental effects of salinity on plant growth and photosynthetic rates, several studies have reported that Fv/Fm value remains stable under salinity stress (Belkhodja et al. 1999). For example, in barley, although plant growth and photosynthetic rate were reduced by salinity, Fv/Fm values were not affected (Allel et al. 2018). In this study, the Fv/Fm values remained within the normal range (data not shown), indicating that photosystem II activity and the functionality of the photosynthetic apparatus were not damaged by the soil salinity.
However, high soil salinity can lead to photo-oxidation of chlorophyll, a decomposition process caused by excessive salt accumulation in the soil, which reduces the chlorophyll content in plant leaves (Hnilickova et al. 2021). This study also showed a significant decrease in SPAD values (Fig. 1B). Salinity stress negatively affects the photosynthetic efficiency per unit of chlorophyll, inhibiting the ability of plants to efficiently convert light energy into chemical energy (Munns and Tester 2008). As a result, lower chlorophyll content significantly reduces both photosynthetic efficiency and photosynthetic rate.
Salinity has also been shown to negatively affect photosynthesis primarily through its effect on stomatal limitation, resulting in reduced carbon assimilation (Hnilickova et al. 2021). Salt stress can partially close the stomata, limiting photosynthetic rates and stomatal conductance, thereby reducing carbon dioxide accessibility (Abbas et al. 2024). Several studies have reported that salinity inhibits enzymes of the Calvin cycle (Li et al. 2011). Under salinity stress, an increase in Na+ interferes with the carboxylase activity of Rubisco, affecting the normal function of this enzyme (Antolín and Sánchez-Díaz 1993). As a result, the efficiency of CO2 fixation could be reduced, leading to a decrease in the net CO2 assimilation rate (Hameed et al. 2021). Similarly, we found that the net CO2 assimilation rate (An) and stomatal conductance (gs) were limited by salinity (Fig. 3).
The plant growth is dependent on photosynthesis, so when plants are grown under saline conditions, a reduction in growth and productivity occurs due to a decrease in photosynthetic rates (Munns and Tester 2008). Salinity stress significantly reduces the fresh and dry weights in plants at early growth stage (Kafi and Rahimi 2011). In this study, despite of reductions in growth and photosynthetic rates, Fv/Fm values remained stable, suggesting that the photosystem II was largely unaffected by salinity. However, stomatal closure significantly reduced net assimilation rates (An) and stomatal conductance (gs) as substrate EC increased. This reduction led to reduced plant growth, such as bulb size and weight, at higher salinity. Therefore, this finding suggests that salinity mitigation is necessary to maintain plant growth and photosynthetic capacity in lily cultivation on salt-affected soils.