Salinity Tolerance of Four Hardy Ferns from the Genus Dryopteris Adans. Grown under Different Light Conditions

: Hardy ferns form a group of attractive garden perennials with an unknown response to abiotic stresses. The aim of this study was to evaluate the tolerance of three species of ferns of Dryopteris genus ( D. affinis , D. atrata and D. filix-mas ) and one cultivar ( D. filix-mas cv. “Linearis-Polydactylon”) to salinity and light stress. The plants were grown in full sun and shade and watered with 50 and 100 mM dm − 3 NaCl solution. All taxa treated with 100 mM NaCl responded with reduced height, leaf greenness index and fresh weight of the above-ground part. In D. affinis and D. atrata salinity caused leaf damage manifested by necrotic spots, which was not observed in the other two taxa. The effect of NaCl depended on light treatments and individual taxon. D. affinis and D. atrata were more tolerant to salinity when growing under shade. Contrary to that, D. filix-mas cv. “Linearis-Polydactylon” seemed to show significantly greater tolerance to this stress under full sun. Salt-treated D. filix-mas cv. “Linearis-Polydactylon” plants accumulated enhanced amounts of K + in the leaves, which might be associated with the taxon’s tolerance to salinity. Among the investigated genotypes, D. filix-mas cv. “Linearis-Polydactylon” seemed the most and D. affinis and D. atrata the least tolerant to salinity and light stress.


Introduction
Ornamental plants are constantly exposed to adverse effects of stress factors that disturb their growth and diminish their decorative value [1]. These factors operate both during production and after planting at the target site. The stress can be caused by an excess or deficiency of any abiotic factor [2]. Very often stresses evoked by unfavorable environmental conditions overlap with each other [3]. Most studies conducted so far have focused on the response of ornamental plants to single stress factors [1,4,5], while their response to multiple stresses experienced simultaneously is less known.
Excessive substrate salinity causing salt stress is one of the most common abiotic stresses that negatively affect the quality of ornamental plants [4]. Exceeding tolerance threshold for salt triggers numerous adverse changes in plant growth and development [6,7]. Too high salinity limits water availability, lowers turgor and leads to inhibition of cell elongation growth, tissue necrosis, yellowing, drying and falling of leaves [8]. The negative effects of salinity are due to toxicity of sodium ions that accumulate in plant tissues and disturb plant ionic balance [9]. The assessment of ion content in plants grown under salt stress is important from a cognitive and practical perspective, as the ion level might be a marker for selecting species and cultivars tolerant to elevated salinity [10,11].
Global climatic changes increase the intensity of extreme weather phenomena, including heat waves and ensuing intense solar radiation that causes light stress in plants [12,13]. Excessive solar radiation results in photoinhibition and photodestruction of pigments, and further consequences include reduced photosynthesis efficiency and extensive tissue damage [14]. Ornamental plants exposed to adverse light conditions respond with growth

Plant Material
The study involved three species of hardy ferns belonging to Dryopteris genus (D. affinis, D. atrata and D. filix-mas) and one cultivar of this genus (D. filix-mas "Linearis-Polydactylon"). The plants were propagated in vitro and acclimatized in pots (0.5 dm 3 capacity) in a greenhouse at a horticultural farm (Rzgów, Poland). Each plant had 5-7 leaves and a very well developed rhizome clump.

Culture Conditions
The plants were planted on 15 July 2019. Each round, a black PVC pot of 1.7 dm 3 capacity harbored a single plant and was filled with peat substrate (pH 6.0) supplemented with PG Mix fertilizer (Yara, Poland) at a dose of 1.0 kg m −3 . The fertilizer contained: 5.5% N-NO 3 , 8.5% N-NH 4 , 16% P 2 O 5 , 18% K 2 O, 0.8% MgO, 19% SO 3 , 0.03% B, 0.12% Cu, 0.09% Fe, 0.16% Mn, 0.20% Mo, and 0.04% Zn. The pots were placed on white nursery mats in an unheated plastic tunnel of the area of 225 m 2 , and covered with a double layer of inflated poly film (lat. 53 • 25 N, long. 14 • 32 E; elevation, 25 m). The plants were watered every three days with tap water of pH 6.4, electrolytic conductivity 0.64 mS cm −1 containing (mg dm −3 ) 6.2 K + , 98 Ca 2+ and 25 Na + . The tunnel's roof ventilation opened when the temperature inside exceeded 18 • C. Air temperature and relative humidity were recorded with a portable USB data logger. The average monthly maximum/minimum air temperature and average maximum/minimum relative humidity (RH) in the plastic house were, respectively: July 28.6 • C/15.

Experimental Design and Treatments
Form 5 August 2019 until the end of the trial (23 October 2019), half plants of each fern taxon were grown in a tunnel in full sun, and the other half of the plants were grown in a tunnel under shading screens (a highly reflective aluminized shade fabric). Measurements with a Radiometer-Fotometr RF-100 (Sonopan, Białystok, Poland) determined the photosynthetic photon flux density (PPFD) on a cloudless day of 4 August 2019 at 609.1 µmol m −2 s −1 for full sun and 151 µmol m −2 s −1 , i.e., 24% of this value, in shade.
Starting on 5 August 2019, plants at the two sites (full sun and shade) were watered four times, i.e., every five days with a solution of sodium chloride (NaCl) pure p.a. 99.9% (Chempur, Poland). NaCl concentration was either 50 or 100 mM, and each plant was provided with 100 mL of the solution per watering. The control plants were irrigated with tap water. NaCl concentrations were selected based on a study by Bogdanovic et al. [24]. After the last dose of NaCl was applied, all plants were watered with tap water until the end of the experiment.
The experimental design was a sub-block one, with three repetitions per combination and nine plants per repetition.

Assessment of Greenness Index, Ornamental Value and Morphological Features
On the last day of the experiment we measured leaf greenness index in Soil Plant Analysis Development (SPAD) units with a Chlorophyll Meter SPAD-502 (Minolta, Japan). The measurements were conducted between 10.00 a.m. and noon and included three fully developed leaves without any signs of necrosis, located in the middle of the plant. Three readings were taken per each leaf. Nine plants were assessed per each combination.
To determine the decorative value of the plants, five researchers conducted a bonitation assessment (visual score) by rating all plants according to a five-point scale, where 1 meant low attractiveness, expressed as insufficient foliage, poor growth and unattractive habit, and 5 meant the maximum decorative value manifested in vigorous growth, attractive habit and healthy foliage.
All plants in all combinations were also assessed for their height (from the soil surface to the tip of the tallest leaf) and fresh weight of the above-ground part cut at the substrate level in the pots.

Analysis of Na + , K + and Ca 2+ Content
To determine the content of Na + , K + and Ca 2+ , the collected leaves were rinsed twice with deionized water, blotted dry, placed into brown paper bags and left in an oven at 65 • C for 72 h. Dried material was pulverized into particles of diameter below 1 mm, and wet mineralized in 17 mL of 96-97% H 2 SO 4 per 2.0 g of the material. The ion content was determined by the flame photometry on a flame photometer AFP-100 (Biotech Engineering Management, Nicosia, Cyprus, as described by Ostrowska [27]. Each mineral was determined in three analytical replicates per treatment.

Statistical Analysis
The experimental data were statistically analyzed by means of a variance analysis for two-factor (salinity and light) experiments in Statistica Professional 13.3 package (TIBCO Software, Palo Alto, CA, USA). Date were analyzed separately for each taxon. The multiple comparison procedure based on the Tukey's HSD post-hoc test with the significance level p ≤ 0.05 was used to identify differences between the means.

Overall Effects of Salinity Treatments
Salinity stress strongly affected plant height, leaf greenness index (SPAD), fresh weight of the above-ground parts (Figure 1a-c) and visual score (Table 1) of all investigated fern taxa. Plants of all combinations survived the salt stress. NaCl at 50 and 100 mM caused a clear plant height reduction in D. atrata, D. affinis, and D. filix-mas whereas D. filix-mas cv. "Linearis-Polydactylon" responded this way only to 100 mM NaCl. SPAD index in D. atrata, D. affinis and D. filix mas dropped with growing salt concentration, and it was also reduced in D. filix-mas cv. "Linearis-Polydactylon" but did not depend on NaCl levels. In A. atrata and A. affinis the drop in fresh weight of the above-ground parts was more intense at higher NaCl concentration. Fresh weight of D. filix-mas plants was also lower under salt stress but no significant differences were spotted for 50 and 100 mM NaCl. In D. filix-mas cv. "Linearis-Polydactylon" plants the decrease in fresh weight was only visible at 100 mM NaCl. Salinity considerably affected the visual score of A. atrata and D. affinis in a concentration dependent way. In D. filix species and its cultivar "Linearis-Polydactylon" the visual score was also lower in the presence of salt but there was no difference between NaCl concentrations.
Software, Palo Alto, CA, USA). Date were analyzed separately for each taxon. The multiple comparison procedure based on the Tukey's HSD post-hoc test with the significance level p ≤ 0.05 was used to identify differences between the means.

Overall Effects of Salinity Treatments
Salinity stress strongly affected plant height, leaf greenness index (SPAD), fresh weight of the above-ground parts (Figure 1a-c) and visual score (Table 1) of all investigated fern taxa. Plants of all combinations survived the salt stress. NaCl at 50 and 100 mM caused a clear plant height reduction in D. atrata, D. affinis, and D. filix-mas whereas D. filix-mas cv. "Linearis-Polydactylon" responded this way only to 100 mM NaCl. SPAD index in D. atrata, D. affinis and D. filix mas dropped with growing salt concentration, and it was also reduced in D. filix-mas cv. "Linearis-Polydactylon" but did not depend on NaCl levels. In A. atrata and A. affinis the drop in fresh weight of the above-ground parts was more intense at higher NaCl concentration. Fresh weight of D. filix-mas plants was also lower under salt stress but no significant differences were spotted for 50 and 100 mM NaCl. In D. filix-mas cv. "Linearis-Polydactylon" plants the decrease in fresh weight was only visible at 100 mM NaCl. Salinity considerably affected the visual score of A. atrata and D. affinis in a concentration dependent way. In D. filix species and its cultivar "Linearis-Polydactylon" the visual score was also lower in the presence of salt but there was no difference between NaCl concentrations.  In all fern taxa, salinity significantly increased the leaf content of Na + with increasing rates of NaCl (Table 2). Salt treatment resulted in a drop of K + levels in D. atrata and its surge in D. filix-mas cv. "Linearis-Polydactylon" at both NaCl levels. Plants of both taxa exposed to the higher NaCl dose (100 mM) accumulated lower content of Ca 2+ .   In all fern taxa, salinity significantly increased the leaf content of Na + with increasing rates of NaCl (Table 2). Salt treatment resulted in a drop of K + levels in D. atrata and its surge in D. filix-mas cv. "Linearis-Polydactylon" at both NaCl levels. Plants of both taxa exposed to the higher NaCl dose (100 mM) accumulated lower content of Ca 2+ .

Overall Effects of Light Treatments
The effects of light conditions on plant height, leaf greenness index (SPAD), fresh weight of the above-ground parts (Figure 2a-c) and visual score (Table 3) was variable and taxon-dependent. D. atrata plants growing in full sun were lower, had smaller fresh weight of the above-ground parts and a lower SPAD index and visual score than shaded plants. Similarly, D. affinis plants grown under full sun had lower fresh weight and reduced SPAD index and visual score. In D. filix-mas, light conditions did not affect fresh weight or SPAD index but resulted in differences in plant height and visual score. D. filix-mas plants growing in full sun were higher but those growing in the shade had higher visual score. D. filix-mas cv. "Linearis-Polydactylon" plants in high light reached greater SPAD index and higher height than their shaded counterparts. We detected no effects of light availability on the content of Na + , K + or Ca 2+ in all tested ferns (p > 0.05, results not shown).
plants. Similarly, D. affinis plants grown under full sun had lower fresh weight and reduced SPAD index and visual score. In D. filix-mas, light conditions did not affect fresh weight or SPAD index but resulted in differences in plant height and visual score. D. filixmas plants growing in full sun were higher but those growing in the shade had higher visual score. D. filix-mas cv. "Linearis-Polydactylon" plants in high light reached greater SPAD index and higher height than their shaded counterparts. We detected no effects of light availability on the content of Na + , K + or Ca 2+ in all tested ferns (p > 0.05, results not shown).

Combined Effects of Salinity and Light Treatment
The effects of salt stress on plant height, leaf greenness index (SPAD), fresh weight of the above-ground parts (Figure 3a-c) and visual score (Table 4) depended on light condition and taxon. In D. atrata and D. affinis salinity reduced plant height considerably stronger in plants growing in full-sun than in shade. In D. filix-mas and cv. "Linearis-Polydactylon" NaCl only slightly diminished plant height under both light conditions. Exposure to both concentrations of salt resulted in a decrease of fresh weight of D. atrata, D. affinis and D. filix-mas in both light treatments, whereas D. filix-mas cv. "Linearis-Polydactylon" responded with a drop in fresh weight, both in the sun and in the shade, only to 100 mM NaCl. SPAD greenness index decreased in D. atrata and D. affinis with increasing concentration of NaCl both under full sun and shade treatments. In D. filix-mas, its salinity-triggered reduction was only perceived in low light intensity. In salt-exposed D. filixmas cv. "Linearis-Polydactylon" plants SPAD value declined in the shade but grew in the sun. Control (no salt) and shaded plants of D. atrata, D. affinis and D. filix achieved the highest visual score. In D. filix-mas cv. "Linearis-Polydactylon" the most decorative plants were those growing without NaCl pressure in the full sun. Interestingly, we found no leaf discoloration or necrosis in D. filix and D. filix-mas cv. "Linearis-Polydactylon" exposed to salt under both light conditions. Salt-exposed plants of D. atrata and D. affinis responded with leaf margin chlorosis and necrosis, particularly at 100 NaCl mM and under full sun (Figure 4).

Combined Effects of Salinity and Light Treatment
The effects of salt stress on plant height, leaf greenness index (SPAD), fresh weight of the above-ground parts (Figure 3a-c) and visual score (Table 4) depended on light condition and taxon. In D. atrata and D. affinis salinity reduced plant height considerably stronger in plants growing in full-sun than in shade. In D. filix-mas and cv. "Linearis-Polydactylon" NaCl only slightly diminished plant height under both light conditions. Exposure to both concentrations of salt resulted in a decrease of fresh weight of D. atrata, D. affinis and D. filixmas in both light treatments, whereas D. filix-mas cv. "Linearis-Polydactylon" responded with a drop in fresh weight, both in the sun and in the shade, only to 100 mM NaCl. SPAD greenness index decreased in D. atrata and D. affinis with increasing concentration of NaCl both under full sun and shade treatments. In D. filix-mas, its salinity-triggered reduction was only perceived in low light intensity. In salt-exposed D. filix-mas cv. "Linearis-Polydactylon" plants SPAD value declined in the shade but grew in the sun. Control (no salt) and shaded plants of D. atrata, D. affinis and D. filix achieved the highest visual score. In D. filix-mas cv. "Linearis-Polydactylon" the most decorative plants were those growing without NaCl pressure in the full sun. Interestingly, we found no leaf discoloration or necrosis in D. filix and D. filix-mas cv. "Linearis-Polydactylon" exposed to salt under both light conditions. Salt-exposed plants of D. atrata and D. affinis responded with leaf margin chlorosis and necrosis, particularly at 100 NaCl mM and under full sun (Figure 4).   Full sun 0 3.9 ± 0.1b 1 4.8 ± 0.1a 3.9 ± 0.1b 5.00 ± 0.00a 50 2.0 ± 0.0d 2.8 ± 0.1c 3.3 ± 0.6bc 4.55 ± 0.19bc 100 1.0 ± 0.0e 2.6 ± 0.2c 3.1 ± 0.1c 3.96 ± 0.07d Shade 0 5.0 ± 0.0a 5.0 ± 0.0a 5.0 ± 0.0a 4.29 ± 0.25cd 50 3.1 ± 0.2c 4.9 ± 0.1a 4.0 ± 0.0b 4.07 ± 0.13d 100 3.1 ± 0.1c 3.8 ± 0.1b 3.9 ± 0.1b 4.85 ± 0.13ab 1 Means not marked with the same letter are significantly different at p ≤ 0.05. The greatest content of Na + was found in all ferns treated with 100 mM NaCl, irrespective of light conditions. In D. atrata plants cultivated under full sun and shade, salinity at 50 and 100 mM NaCl resulted in lowering K + content. In D. filix-mas cv. "Linearis-Polydactylon" NaCl at 50 and 100 mM boosted K + levels irrespective of light intensity. D. atrata treated with 100 mM NaCl and D. filix-mas cv. "Linearis-Polydactylon" treated with 50 mM NaCl exposed to the shade accumulated smaller amounts of Ca 2+ (Table 5). Table 5. Effect of light conditions and salinity (50 and 100 mM NaCl) on Na + , K + and Ca 2+ content (expressed in % dry weight) in leaves of D. affinis, D. atrata, D. filix-mas and D. filix-mas cv. Linearis-Polydactylon (D. filix-mas cv.). Data are mean ± SD.

Discussion
During their growth and development plants are exposed to different environmental stresses, the effects of which are often synergistic, and their combined outcome is considerably more powerful than that of individual stress factors [28,29]. Understanding the response of individual genotypes to adverse environmental conditions allows for proper selection of tolerant and resistant plants [7,10,30]. Most studies on the effects of stressful conditions have been carried out on flower ornamentals, while the group of leaf ornamental plants has so far received very little attention. The aim of this work was to investigate the response of four ferns of Dryopteris genus, generally considered as shade plants, to multi-stress in the form of salinity and high light intensity. Most plants exposed to excessive salinity limit the elongation growth of cells, which results in reduced growth and biomass production [31,32]. Salt stress often diminishes visual quality of plants by evoking brownish necrosis of leaves [4,8]. In our study, salinity also inhibited growth, reduced fresh weight of the above-ground part and lowered the bonitation score of the investigated ferns, and intensity of these effects depended on the taxon and light conditions ( Figure 3, Table 4). The species of D. affinis and D. atrata turned out the most sensitive to salt and they demonstrated leaf margin browning and drying ( Figure 4). Negative effects of salinity on the growth and quality of D. affinis and D. atrata were particularly visible under full sun. In the shade, the stress affected growth and ornamental value of D. affinis and D. atrata to a lesser degree. Our results confirmed shade affinity of D. affinis and D. atrata, and what is more, shade mitigated negative effects of salt in these species. Similarly, Medina et al. [33] showed that a halophytic fern Acrostichum aureum was much more tolerant to salt stress when growing in the shade than in the sun. In Hibiscus tiliaceus Hau, cultivated under different light conditions, salinity caused stronger total biomass reduction in plants growing in 90% shade than in full sun and 50% shade [34]. In a heliophilous species Vicia faba, the toxic effects of salinity were more considerably alleviated by higher than lower light intensity [35]. In our study, the same relationship was demonstrated in D. filix-mas cv. "Linearis-Polydactylon", as salinity experienced by plants growing under full sun did not reduce fresh weight of their above-ground parts. D. filix-mas cv. "Linearis-Polydactylon" plants cultivated in shade were the smallest and had the lowest fresh weight. We noticed no clear effects of light conditions on fresh weight of D. filix-mas but plants growing under full sun demonstrated lower ornamental value than those under low light intensities. As shown by Ure [36], D. filix-mas tolerates a wide range of light/shade levels.
In sensitive species salt stress reduces chlorophyll content, while in tolerant ones the pigment level remains unchanged or may even rise [37,38]. Our study assessed leaf greenness index that correlates with chlorophyll content [39]. We found a negative effect of salt stress on leaf greenness in all shaded ferns (Figure 3b). In full sun SPAD index was clearly lowered in all ferns exposed to salinity, except for D. filix-mas cv. "Linearis-Polydactylon", where NaCl slightly enhanced SPAD value. Bogdanovic et al. [24] tested the response of Asplenium viride Britton, Ceterach officinarum DC and Phyllitis scolopendrium (L.) Newmann to salt stress (0-500 mM NaCl) in vitro and found that high concentrations of NaCl (250 mM and above) drastically lowered total chlorophyll content in all species, while low concentrations (50 and 100 mM NaCl) enhanced the pigment content in A. viride and C. officinarum. Experimentally demonstrated stimulating effect of salinity on the greenness index of D. filix-mas cv. "Linearis-Polydactylon" may indicate that this cultivar grown under full sun is tolerant to increased salinity.
NaCl evoked salinity may disturb ion homeostasis and, consequently, disrupt the physiological processes [40]. Usually, excessive content of Na + results in deficiency of K + and Ca + [37,41]. There are, however, also contradictory data suggesting that salinity causes increased accumulation of K + [31] and Ca 2+ [42]. Potassium and calcium ions regulate activity of numerous enzymes [43,44], and their deficiency decreases plant stress tolerance [45]. In our experiment, the content of Na + rose in all taxa exposed to salinity ( Table 2) due to using NaCl solution as a stress factor. Enhanced content of Na + , as a major solute responsible for increased osmotic pressure of the cell sap, was also observed in salttreated fern A. aureum [33]. A particularly interesting outcome of this study was a boost in K + content in D. filix-mas cv. "Linearis-Polydactylon". Similarly, Vogelien et al. [46] showed that a mutant of Ceratopteris richardii stl2, relatively tolerant to NaCl, accumulated greater amounts of K + when grown on NaCl-supplemented medium than other fern genotypes. As mentioned earlier, despite NaCl treatment D. filix-mas cv. "Linearis-Polydactylon" maintained its high bonitation score and greenness index, which may indicate its tolerance to the applied NaCl doses. Furthermore, an increased content of K + may suggest a role of these ions in plant adaptation to salt stress. A precise marker of salt stress in A. aureum was the content of cyclitol d-1-O-methyl-muco-inositol, a cytoplasmic compatible solute [33], while other ferns, i.e., A. viride, C. officinarum and P. scolopendrium responded to NaCl with a shift in total leaf phenolic content [24]. The mechanisms of plant tolerance to stress are highly complex and multidirectional. Therefore, to better understand fern tolerance to salinity, we need further studies, particularly on the level of oxidative stress and compatible solutes that protect protein structure and biological membranes against negative effects of excessive salt concentrations.

Conclusions
From among four investigated fern taxa, D. filix-mas cv. "Linearis-Polydactylon" showed the greatest tolerance to salt stress. Despite salinity, plants of this cultivar maintained intense, green coloration of leaves assessed by SPAD greenness index, high visual score and demonstrated increased accumulation of K + in the leaves. D. affinis and D. atrata turned out sensitive to salinity, as manifested in leaf necrosis. The effects of salt stress on plant growth depended on light condition and taxon; D. filix-mas cv. "Linearis-Polydactylon" plants were more tolerant to salinity when growing under full sun, and D. affinis and D. atrata showed better tolerance to NaCl under shade. Our knowledge on the impact of abiotic stresses on the growth of ornamental garden plants from the fern group is scarce, which is why these findings seem important and may serve as practical recommendations for the selection of fern species intended for areas exposed to environmental stresses.
Author Contributions: Conceptualization, methodology, formal analysis, writing and visualization, P.S.; investigation and data curation, R.P.; All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.