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Article

Salt Spray and Surfactants Induced Morphological, Physiological, and Biochemical Responses in Callistemon citrinus (Curtis) Plants

by
Stefania Toscano
,
Giovanni La Fornara
and
Daniela Romano
*
Department of Agriculture, Food and Environment (Di3A), Università degli Studi di Catania, Via Valdisavoia 5, 95123 Catania, Italy
*
Author to whom correspondence should be addressed.
Horticulturae 2022, 8(3), 261; https://doi.org/10.3390/horticulturae8030261
Submission received: 18 February 2022 / Revised: 15 March 2022 / Accepted: 16 March 2022 / Published: 17 March 2022
(This article belongs to the Special Issue Plant Physiology under Abiotic Stresses)
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Abstract

:
The growth and aesthetic value of ornamental plant species used near coastlines are negatively influenced by salt spray. The presence of surfactants could enhance salt damage. To analyze the influences of salt spray and surfactants alone and in combination with each other, individual Callistemon plants were subjected to different treatments for 8 weeks: a solution simulating the composition of seawater (salt spray), a solution containing an anionic surfactant (surfactant), a solution with salt spray and anionic surfactant (salt plus surfactants), and deionized water (control). To study the influence of different climatic conditions, two growing periods, from January to March (I CP) and from May to July (II CP), were established. Salt spray, alone or with surfactant action, influences plants’ growth and aesthetic features in different cycle periods. The percentage of leaf damage significantly increased with salt spray and salt plus surfactants during II CP (~27%). Additionally, the Na+ and Cl− contents were enhanced in the leaves in both CPs, but the contents in the roots were only enhanced in the II CP. The gas exchanges were significantly influenced by the treatments, especially during the II CP, when a reduction in net photosynthesis due to salt spray was observed starting from the second week of stress. At the end of the experiment, in both cycle periods, the leaf proline content increased in the salt spray and salt plus surfactants treatments. In both CPs, PCA revealed that the morphological and physiological parameters were directly associated with the control and surfactants treatments, whereas the mineral contents and biochemical parameters were directly correlated with the salt and salt plus surfactants treatments. The additive effect of surfactant stress, compared to salt stress, did not appear to be significant, with the exception of CP II, and for some parameters, the solubilization action of surfactants was favored by higher temperatures.

1. Introduction

In coastal gardens and ornamental green areas, one of the main problems observed is related to salt stress originating from sea spray, which damages plants [1,2]. In this landscape, salt spray may limit plant growth and survival [3], altering coastal species’ composition [4] and thus influencing the ornamental and ecological value of plant communities [5,6].
The negative effects of salts on ornamental plants are observed above all on their aesthetic appearance, which is an essential trait [7]. The visual aspect, although a subjective parameter, is of fundamental importance when evaluating the salt tolerance of landscape plants [8]. Among the different species used in urban landscaping, salt tolerance differs considerably [9] according to the morphological and physiological characteristics of different genotypes. Most ornamental plant species are non-halophytes; therefore, the assessment of salt tolerance is necessary [10]. The species selected for inclusion in urban green areas must not only survive but must also maintain suitable aesthetic characteristics [11].
Although plant species typical of coastal areas are resistant to the presence of salt on their leaves [12], plant growth and reproduction can be negatively affected when they are exposed to salt spray, and when salt water penetrates into the groundwater [13]. When salt is applied to roots, the damage is less significant than that seen from salt sprays [14]. Salt spray can reduce plant growth because it determines water stress; leads to the disruption of membranes and enzyme systems; causes the inhibition of nutrient uptake, necrosis, and the loss of leaves, and can lead to mortality [15].
In modern societies, surfactants are often used in the form of detergents and pesticides. Given their wide usage, these substances cause pollution in aquatic environments. The foliar absorption of sea salt through stomatal and cuticular penetration can be increased by the presence of surfactants in seawater [16].
The wide uses of surfactants in chemical industries, such as household products, industrial cleaning, ink, pharmaceuticals, and personal care, can affect the environment and human health. In 2014, 15.93 million tons of surfactants were used, and in 2022 this figure is expected to be over 1/3 more (24.19 million tons of surfactants) [17]. The phenomenon is worrying for the coastal ecosystem because about 10% of this amount is released into the sea [18].
The damage to coastal flora is mainly caused by the synergic effects of marine salt and surfactants, but also by the direct actions of the surfactants [19] that are responsible for the solubilization of cell membranes [20], the increase in the permeability of cuticles [21], and the dissolution of epicuticular waxes [22], all of which are processes that facilitate the foliar absorption of salt, and its phytotoxic effects. The effect of surfactants is to degenerate epicuticular waxes and modify the photosynthetic processes by causing the leaves to absorb greater quantities of sea salt [23].
Bussotti et al. [24] observed that resistance to salt spray depends on different behaviors determined by leaf structure (sclerophyllia, cuticle thickness). Reductions in gas exchanges and photosystem II (PSII) efficiency have been observed in the presence of salt spray because, although the cuticle of the leaves protects against external agents, it cannot prevent the penetration of ions and the consequent osmotic and ionic stress [25]. Modifications in the chlorophyll content and consequently reductions in photosynthesis activity occur in the presence of saline stress [26]. Stomatal limitation reduces the diffusion of CO2 in the mesophyll and can contribute to the generation of additional reactive oxygen species (ROS), which may cause irreversible damage [27].
Photosynthetic pigments can be damaged by ROS, altering the metabolism because they are highly reactive in the absence of protective mechanisms [28]. In cellular compartments, to counteract the effects of reactive oxygen species, plants have developed different mechanisms. Generally, plants activate various antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX)) [29]. In many species, a close relationship is clear between stress tolerance and antioxidant activity [30,31,32]. Catalase, guaiacol peroxidase, and L-ascorbate peroxidase are all involved in the plant’s responses to biotic and abiotic stresses. In particular, these enzymes are involved in the scavenging of reactive oxygen species (ROS), partially reduced forms of atmospheric oxygen, that are highly reactive and capable of causing oxidative damage to the cell.
Among the ROS, the most dangerous is the superoxide anion; this is scavenged in plants by superoxide dismutase, which converts the superoxide anion to hydrogen peroxide [33]. Furthermore, in the context of the detoxification of toxic ROS, the superoxide anion is a fundamental component of the ascorbate-glutathione cycle [34].
In the presence of abiotic stress, plants accumulate metabolic components, which act as plant osmotic regulators, such as proline (Pro) and malondialdehyde (MDA) [35,36,37].
Few studies have been conducted on the response of plants to interactions between natural stresses in coastal areas; research has generally focused on single stress, leading to a suitable but incomplete understanding of the response mechanism of plants [38]. The effect of stress depends on both the intensity of the stress factor and on its duration and therefore makes it more difficult to understand the response mechanisms and interactions between other stresses [39]. In coastal areas, salt spray is often associated with pollutants, and the role of surfactants is critical in some cases of environmental damage to coastal vegetation [40]. Interactions with environmental conditions are often important as well; surfactant experiments carried out during winter produced damage lower down in the plants than experiments in warmer seasons [41].
To better understand the influence of some of the stronger factors affecting the ornamental plants used in the coastal landscape, we selected Callistemon of the Myrtaceae family; this entire genus is endemic of Australia and occurs in the form of shrubs or small trees. The Callistemon genus is characterized by a suitable tolerance to drought, salt, and root restriction stresses [42,43,44]. One of the most interesting ornamental species is Callistemon citrinus (Curtis) Skeels, characterized by fast growth and abundant blooming with bright colors and various shapes and volumes [45].
The research related to the effects of salt spray and its interactions with surfactants in ornamental shrubs is sparse. The aims of this study were to define (i) the influence of salt spray and surfactants and their interactions on Callistemon plants during two cycle periods with different climatic conditions; (ii) the morphological, physiological, and biochemical mechanisms involved in responding to salt spray and surfactants; (iii) the relationships among different parameters, in order to better understand the action mechanisms.

2. Materials and Methods

2.1. Plant Materials and Treatments

Both cycle periods were operated in an unheated greenhouse located near Catania, Italy (37°41′ N 15°11′ E 89 m a.s.l.). In 3 L pots (16 cm), filled with peat and perlite (2:1 v/v), rooted cutting (two months old) of Callistemon (Callistemon citrinus (Curtis) Skeels) were transplanted; 2 g L−1 of Osmocote Plus (14:13:13 N, P, K plus microelements) was added to each pot. A drip system with one 2 L h−1 emitter into each pot was used, and the plants were irrigated daily. The plants were subjected for 8 weeks to different treatments: (a) a solution simulating the composition of seawater-salt spray [46] with a concentration of 401.8 mM (salt spray); (b) a solution containing an anionic surfactant (sodium dodecylbenzene-sulfonate 82.52%, 50 mg L−1) (surfactants) [3]; (c) a solution containing salt spray and anionic surfactant (salt plus surfactants); (d) control plants treated only with deionized water (control). The plants were sprayed twice a week with different aqueous solutions. To avoid salt deposition on the soil, plastic discs were placed over the soil surfaces of the potted plants. To investigate the effects of different climatic conditions, two growing periods were considered: January to March (I CP), with transplant in November, and May to July (II CP), with transplant in February.

2.2. Plant Materials and Treatments

At the ends of both cycle periods, three pots per replication were randomly chosen for the determination of shoots’ and roots’ fresh and dry biomass, total leaf area, leaf number, chlorophyll content, and leaf damage. The dry biomass (DW) of the shoots (leaves plus stems) and the whole plant (leaves, stems, and roots) was determined by drying the weighed samples in a thermo-ventilated oven at 70 °C until constant weights were obtained. A leaf area meter (Delta-T Devices Ltd., Cambridge, U.K.) was used for determining the total leaf area and percentage of leaf damage (necrotic area on total area). The protocol of Moran and Porath [47] was adopted to measure the chlorophyll content. Dry leaves and roots were ground in a Wiley Mill and then passed through a 20-mesh screen for the mineral analysis. Na+ and Cl− contents were determined by chromatography on a Dionex IC 25 (Dionex Corp., Sunnyvale, CA, USA). Ion concentrations were expressed in g kg–1 DW.

2.3. Chlorophyll Content, Gas Exchanges, Chlorophyll a Fluorescence and RWC Measurements

The gas exchanges were registered during both cycle periods, every two weeks (10:00 am and 2:00 pm). Net photosynthetic rate (AN) and stomatal conductance (gs) were registered on fully expanded leaves through a CO2/H2O IRGA (LCi, ADC Bioscientific Ltd., Hoddesdon, UK). Simultaneously, the efficiency of PSII was registered using a modulated chlorophyll fluorimeter OS1-FL (Opti-Sciences Corporation, Tyngsboro, MA, USA). Using cuvettes, the leaves were darkened for 20 min before measurement. The chlorophyll a fluorescence was reported as the Fv/Fm ratio, which shows the maximal quantum yield of PSII photochemistry, where Fm = the maximal fluorescence and Fv = the variable fluorescence.

2.4. Estimation of Proline Content

The protocol of Bates et al. [48] was used for determining the proline content, using L-proline as the standard. In total, 1 g of fresh leaf samples was homogenized in 5 mL of 3% aqueous sulfosalicylic acid and centrifuged at 14,000× g for 15 min (Neya 10R, REMI, Mumbai, India). The supernatant (2 mL) was blended with an equal volume of acetic acid and acid ninhydrin, vortexed, and incubated for 1 h at 100 °C. An ice bath was used to stop the reaction, and the solution was extracted with 4 mL of toluene. The sample was read spectrophotometrically at 525 nm using toluene as the blank (7315 Spectrophotometer, Jenway, Staffordshire, UK).

2.5. Extraction and Assay of Antioxidant Enzymes

In total, 0.5 g fresh leaf sample was extracted with 4 mL of buffer (50 mM potassium phosphate, 1 mM EDTA, 1% PVP, 1 mM DTT and 1 mM PMSF, pH 7.8). The samples were centrifuged at 15,000× g for 30 min at 4 °C [49]. The supernatant was picked up and stored at −80 °C for the determination of enzyme activity. The catalase activity (CAT) was measured via the protocol of Aebi [50]. Then, 20 µL of supernatant was blended with 830 µL of potassium phosphate buffer (50 mM, pH 7). The reaction was initiated by the addition of 150 µL of H2O2, and we registered the decrease in absorbance (240 nm) for 2 min. The GPX activity was determined using the protocol described by Ruley et al. [34]. A reaction mixture consisting of suitable quantities of enzymatic extracts was employed, with equal amounts of 17 mM H2O2 and 2% guaiacol. The reaction was registered for 3 min at 510 nm. The Giannopolitis and Ries [51] protocol was used for determining the SOD activity (560 nm).

2.6. Climate Conditions

The climate conditions (air temperatures (°C) and relative humidity (RH %)) in both cycle periods were registered using a CR 1000 data logger (Campbell Scientific Ltd., Loughborough, UK). The mean air temperatures during I CP and II CP were 13.4 °C and 21.4 °C, respectively, and the mean relative humidity values were 86% and 76.0%, respectively (Figure S1).

2.7. Statistical Analysis

Both experimental trials were conducted via a randomized complete design; three replicates in each cycle period (CP) and for each treatment (T) were used. Statistical analysis of the data was performed using CoStat release 6.311 (CoHort Software, Monterey, CA, USA). The data were subjected to two- and one-way analysis of variance (ANOVA), and the means were compared via a Tukey’s test at p < 0.05. The data presented in the figures are the means ± standard error (SE).

3. Results

Table 1 shows the main effects and the interaction effects (over cycle periods and treatments) of morphometric parameters and mineral contents. The two-way ANOVA showed that the parameters were influenced by cycle and treatments (Table 1). The total and shoot dry biomass were statistically different in different cycle periods. Significant differences were found in the root/shoot ratio depending on the two factors and their interactions. Leaf number and total leaf area were only statistically significant for the cycle periods. There were no significant differences in chlorophyll content for any factors. The mineral contents in the leaves and in the roots were significantly different between cycle periods and treatments, and their interactions had an effect too. Leaf damage was only significantly different between treatments. To better understand the effects of the cycle periods on the treatments, the two cycle periods were separately analyzed.
Salt spray, alone or in combination with the surfactant, significantly influenced the biomass accumulation in both growing periods (Table 2). The total dry biomass was reduced in I CP under all treatments by 30% in relation to the control plants (Figure 1a). During II CP, the total dry biomass was only reduced in the treatment with salt by 33% (Table 2). Shoot dry biomass showed a similar trend in both cycle periods (Table 2).
All treatments slightly modified root dry biomass (data not shown), and in I CP, enhanced the root/shoot ratio (Table 2).
The effects of treatments on leaf numbers and total leaf area were observed in both cycle periods (Table 2). Leaf number and total leaf area were significantly reduced during I CP in all treatments, with a reduction of 64% in relation to the control plants (Table 2). A similar trend was registered during II CP, but with only a minor reduction for both parameters (by 20% and 14% for leaf number and total leaf area, respectively).
The chlorophyll contents in both cycle periods did not change according to the treatments (Table 2).
At the end of I CP, a seven-fold higher leaf Na+ content was registered in plants treated with salt spray and salt spray plus surfactants in relation to the control plants and those treated with surfactants only. In II CP, this increase was amplified; 8- and 11-fold increases were noted in plants treated with salt spray and salt spray plus surfactants, respectively (Table 2).
A similar trend was registered for the leaf Cl− content; at the end of the I CP, a five-fold higher Cl− content was noted in the plants treated with salt spray and with salt spray plus surfactants in relation to the control plants and those treated with surfactants only. In CP II, we noticed a six-fold increase in plants treated with salt spray and salt spray plus surfactants (Table 2).
No significant differences were observed in the root mineral content during I CP. In II CP, on the other hand, an increase in the Na+ content by 33% was observed in plants treated with salt spray and by 44% in those treated with salt plus surfactants. The Cl− content increased by 50% in plants treated with salt spray and salt spray plus surfactants (Table 2).
The leaf damage percentages were significantly different among treatments. During I CP, the average leaf damage percentage was 18% in plants treated with salt spray and salt spray plus surfactants. During II CP, the maximum value (~27%) was observed in salt spray plus surfactants plants; no leaf damage was observed in plants treated with only surfactants in either cycle period (Table 2).
Values are the means ± SE of three replicate samples. n.s. = not significant; *, **, *** significant at p < 0.05, 0.01 and p < 0.001, respectively. The values in the same column followed by the same letter are not significantly different at p ≤ 0.05 (Tukey’s test). The gas exchange in both cycle periods was significantly modified as a result of the treatments (Figure 1). From the first measurement (2 weeks), Callistemon plants in I CP treated with salt spray alone or in combination with surfactants showed reductions of 33% and 50% in AN, compared with the control plants; at the end of I CP, a reduction of 23% in this activity in salt spray and salt spray and surfactant plants was observed (Figure 1a). The stomatal conductance values during I CP showed no significant differences (Figure 1c).
During II CP, the reduction in photosynthesis activity was significant immediately after the second week, and this trend was maintained until the end of the experimental period. Under control conditions, the net photosynthesis was about 5 µmol CO2 m−2 s−1. Salt spray alone or in combination caused a progressive decrease in net photosynthesis assimilation; at the end of the cycle period, a reduction of ~60% was reached (Figure 1b). A similar response was registered in the stomatal conductance. During the warmer season, stomatal conductance showed higher values throughout the growth cycle, but a more intense reduction was seen at the end of the trial in the salt spray plus surfactants group (~60%) (Figure 1d), compared with the control plants.
The different values of chlorophyll a fluorescence (Fv/Fm ratio) after exposure to different treatments in different growth periods are presented in Figure 2a,b. The plants treated with salt spray and salt spray plus surfactant showed reductions in both cycle periods, compared with the control plants; the Fv/Fm achieved respective values of 0.67 and 0.71 in I CP, and 0.67 and 0.65 during II CP.
In the final part of the experiment, in both cycle periods, the leaf proline content increased in the salt spray and salt plus surfactants treatment groups, compared with the control and surfactants plants, in both cycle periods, by 65% and 37% in I CP, and 75% and 63% in II CP (Figure 3).
The enzyme activities, SOD, CAT, and GPX, exhibited significant differences in the cycle periods, depending on treatments (Figure 4).
The CAT activity at the end of I CP showed no significant differences. In II CP, however, the salt spray and the salt spray plus surfactants treatment groups showed significant increases in this enzyme by 47% compared with the control and surfactants treatments (Figure 4a).
The GPX activity during I CP was lower in the surfactants compared with the other treatments (Figure 4b). In the final part of II CP, however, the salt treatments increased the activity of this enzyme by 42% and 12% in the salt and salt plus surfactants treatments, respectively (Figure 4b).
In I CP, the SOD activity was much lower and showed its highest values in the salt treatments, with an increase of 20%. In the second cycle period, the observed values were much higher and were again even higher in the salt treatment groups, with increases of 21% and of 42% in the salt spray and salt plus surfactants groups, respectively (Figure 4c).
To visualize the associations among the different treatments in terms of the plants’ morphological, physiological, and biochemical parameters in the two cycle periods, a principal component analysis was performed for all analyzed parameters (PCA; Figure 5a,b; Tables S1 and S2). The first two principal components (PC1 and PC2) accounted for 59% and 28% and 73% and 19% of the total variance during I CP and II CP, respectively, and these components were associated with eigenvalues higher than one.
During I CP, PCA revealed that the morphological and physiological parameters were directly affected by the control and surfactants treatments, whereas the mineral contents and the biochemical parameters were directly correlated with the salt and salt plus surfactants treatments (Figure 5a).
A similar trend was found at the end of II CP (Figure 5b).

4. Discussion

The negative influence of salinity on ornamental plants has been studied. Nevertheless, only a limited number of papers have provided information on the effects of salt spray alone or in combination with surfactants. The identification of plants tolerant to salt spray is particularly important in the selection of shrubs and trees to be used in green areas [9].
A large number of studies on salt stress have used NaCl as the salinizing agent [52,53,54]. However, the compositions of salts in seawater are rather different. Saline solutions containing a single salt may give rise to misleading and erroneous interpretations of plants’ responses to salinity [55,56]. In our studies, we have added to deionized water a solution reproducing the composition of seawater [46] so as to simulate the effects of salt spray along coastal areas.
Salt spray can hinder plant growth because it causes drought stress, disrupts membranes and enzyme systems, reduces nutrient uptake, causes necrosis or leaf loss, and can lead to plant mortality [15].
In both cycle periods, the total and shoot dry biomass were reduced in the presence of salt spray, alone or in combination with surfactants. Fragmented cuticles, disaggregated chloroplasts, and nuclei disrupted stomata, collapsed cell walls, coarsely granulated cytoplasm, and disorganized phloem leading to reduced biomass were observed after the application of spray NaCl [57].
Salt treatments reduced the epigeous dry biomass, reduced the leaf number and total leaf area, and did so more extensively during II CP. This behavior confirms that the plants reduce their leaf area to overcome stress [58]. Plants implement different mechanisms to overcome abiotic stress (drought, saline stress), including a reduction in the total leaf area, which leads to a reduction in water losses through stomata closure, which is the principal defense strategy of species subject to osmotic stress [12].
Reductions in leaf area can therefore be a strategy of adaptation. The reduction in this parameter is generally related to a decrease in the total leaf number and size. In the presence of abiotic stress (drought and salt stress), plants reduce their leaf size so as to reduce the surface available for both the depositing of salt and the loss of water through transpiration [59]. Reduction in leaf number was observed in plants of Scaevola sericea [60] and Crambe maritime [61] sprayed with seawater. This agrees with the results of our study. The plants grown during II CP and treated with salt spray showed a greater reduction in leaf number. This result agrees with the experiments of Bussotti et al. [41], who found that the damage of surfactants was greater in the warmer season.
Aesthetic quality is the most important trait in ornamental plant species [62]. Visual quality is a practical tool used to evaluate the salt response of ornamental species [63]. Common responses in the presence of salt stress include leaf necrosis and reductions in chlorophyll [64]; greater sensitivity has been observed in older leaves [65]. In our study, leaf necrosis as a symptom of leaf damage was registered, and this ranged from minimal (surfactant) to moderate (salt spray and salt spray plus surfactants). Leaf damage could lead to a reduction in the photosynthetic rate, which might manifest in diminished growth [66].
A potential biomarker of salinity tolerance is indicated by the absorption and accumulation of ions in the leaves [2]. The Na+ contents significantly increased during salt treatments in both cycle periods, although this was greater during the summer cycle, and the presence of surfactants intensified this effect further still. In this case, the presence of surfactants increased the phytotoxic effects, as reported by Sànchez-Blanco et al. [67]. High-rated absorptions of Na+ and Cl− in the shoots reduce growth rate due to ion toxicity. Excessive Na and Cl accumulation leads to molecular damage-causing growth arrest and plant death because these ions induce cytoplasmic toxicity [68].
Different studies suggest ornamental plants exposed to salt treatments show a reduction in net photosynthesis and stomatal conductance. In our work, salt treatments, alone or in combination with surfactants, caused a reduction in net photosynthesis as a consequence of the reduced CO2 assimilation following stomatal closure, which particularly occurred during II CP.
The reduction in Fv/Fm is a clear signal that the PSII was affected by the presence of salt and that photoinhibition occurred. Generally, plants under normal conditions presented Fv/Fm ratios close to 0.8 [7], but in our case, the reduction in Fv/Fm was correlated with salt spray. The plants showed a reduction in this index in the presence of salt, and this reduction was greater when the salt was combined with a surfactant. For different plant species, regardless of whether the PSII damages occur in the leaves, this index is considered one of the most effective tools for the measurement of plant stress response, particularly before any morphological changes manifest [69]. The reduction in photosynthetic capacity is correlated with reductions in chlorophyll fluorescence in ornamental species; these cause physiological modifications that will lead to losses in sales for the growers [70].
One of the mechanisms adopted to defend against salt stress is proline accumulation, which can be used as a selection criterion for salt tolerance because an increase in this amino acid may be positively correlated with the level of salt tolerance [71]. In fact, modifications in soluble protein and proline content could regulate cellular redox potential [72] and help scavenge free radicals [73].
Increased ROS production, which damages biomolecules (e.g., lipids, proteins, and nucleic acids) and harms redox homeostasis, was observed in the presence of salt stress [74]. The increase in ROS activity causes an increase in oxidative stress due to ionic toxicity and osmotic imbalance and has negative effects on photosynthetic pigments [75]. In the presence of saline stress, plants produce reactive oxygen species (ROS), such as hydroxyl radical (OH•), singlet oxygen (1O2), superoxide radical (O2•−), and hydrogen peroxide (H2O2), which have a toxic effect on plants [76]. Our results show that the enzyme activity was similar between the control and surfactant treatments but was significantly increased under the salt spray treatment. These results showed that Callistemon plants have significant salt tolerance, and as reported by Sklodowska et al. [77], a greater increase in SOD activity could help preserve membrane integrity and osmoregulation by helping regulate the level of superoxide radical in the presence of high saline levels.
To better understand the tolerance mechanisms related to salt stress and surfactants, it will be relevant to investigate and understand the morphological, physiological, and biochemical parameters of plants grown under stressed and not-stressed conditions (Supplementary Tables S1 and S2). To evaluate stress tolerance and the relationships among the different parameters analyzed in our study, an effective approach is the analysis of principal components (PCA) [12]. Our PCA results demonstrate that the variations in stress tolerance between Callistemon during I CP and II CP are linked to variations in biochemical and mineral contents.

5. Conclusions

Our trial has demonstrated the combined effects of salt spray and surfactant on the ornamental shrub species largely used in Mediterranean green areas near the sea. Salt spray, alone or in combination with surfactant action, influences plants’ growth and aesthetic features, especially during warmer seasons when transpiration is more intense and causes stomatal opening. To overcome stressful conditions, plants undergo several changes, such as the activation of enzymes and the accumulation of proline and minerals (Na+ and Cl−). Interaction with surfactants amplifies these adverse effects.
The results of this trial help us to better understand the responses of plant species used in green areas near the coastline and will thus guide the choice of plant species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8030261/s1, Figure S1: Air temperature (°C) and relative humidity (%) during the two cycle periods, Table S1: Pearson’s correlation coefficients among morphometric, physiological, and biochemical parameters from Callistemon potted plants exposed to different treatments during I CP, Table S2: Pearson’s correlation coefficients among morphometric, physiological, and biochemical parameters from Callistemon potted plants exposed to different treatments during II CP.

Author Contributions

Conceptualization, D.R. and S.T.; methodology, D.R. and S.T.; software, S.T.; validation, D.R.; formal analysis, S.T. and G.L.F.; investigation, S.T. and G.L.F.; resources, D.R.; data curation, S.T.; writing—original draft preparation, D.R. and S.T.; writing—review and editing, D.R. and S.T.; supervision, D.R. 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.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data for this study has been included in the tables, figures, and Supplemental Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Horticulturae 08 00261 g001 550
Figure 1. Net photosynthesis (AN) (a,b) and leaf stomatal conductance (gs) (c,d) in Callistemon plants during I CP and II CP. Plants were subjected twice a week to nebulization treatments for 8 weeks, depending on their treatments: Control (deionized water), salt spray (solution simulating the composition of seawater), surfactants (sodium dodecylbenzene-sulfonate), and a solution with seawater and anionic surfactant. Mean values ± SE (n = 4).
Figure 1. Net photosynthesis (AN) (a,b) and leaf stomatal conductance (gs) (c,d) in Callistemon plants during I CP and II CP. Plants were subjected twice a week to nebulization treatments for 8 weeks, depending on their treatments: Control (deionized water), salt spray (solution simulating the composition of seawater), surfactants (sodium dodecylbenzene-sulfonate), and a solution with seawater and anionic surfactant. Mean values ± SE (n = 4).
Horticulturae 08 00261 g001
Horticulturae 08 00261 g002 550
Figure 2. Maximum quantum efficiency of the PSII (Fv/Fm) of Callistemon plants during I CP (a) and II CP (b) treated with distilled water (control), salt spray, surfactants, and salt plus surfactants. Values are means ± SE (n = 3).
Figure 2. Maximum quantum efficiency of the PSII (Fv/Fm) of Callistemon plants during I CP (a) and II CP (b) treated with distilled water (control), salt spray, surfactants, and salt plus surfactants. Values are means ± SE (n = 3).
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Horticulturae 08 00261 g003 550
Figure 3. Proline contents in leaves of Callistemon during I CP ( Horticulturae 08 00261 i001) and II CP ( Horticulturae 08 00261 i002) treated with distilled water (control, Horticulturae 08 00261 i003 and Horticulturae 08 00261 i004), salt spray ( Horticulturae 08 00261 i005 and Horticulturae 08 00261 i006), surfactants ( Horticulturae 08 00261 i007 and Horticulturae 08 00261 i008), and salt plus surfactants ( Horticulturae 08 00261 i009 and Horticulturae 08 00261 i010). Values are means ± SE (n = 3). Values with the same letter are not significantly different as determined by Tukey’s test (p < 0.05).
Figure 3. Proline contents in leaves of Callistemon during I CP ( Horticulturae 08 00261 i001) and II CP ( Horticulturae 08 00261 i002) treated with distilled water (control, Horticulturae 08 00261 i003 and Horticulturae 08 00261 i004), salt spray ( Horticulturae 08 00261 i005 and Horticulturae 08 00261 i006), surfactants ( Horticulturae 08 00261 i007 and Horticulturae 08 00261 i008), and salt plus surfactants ( Horticulturae 08 00261 i009 and Horticulturae 08 00261 i010). Values are means ± SE (n = 3). Values with the same letter are not significantly different as determined by Tukey’s test (p < 0.05).
Horticulturae 08 00261 g003
Horticulturae 08 00261 g004 550
Figure 4. Effects of different treatments on catalase (CAT (a)), peroxidase (GPX (b)), and superoxide dismutase (SOD (c)) activity in Callistemon plants during I CP ( Horticulturae 08 00261 i011) and II CP ( Horticulturae 08 00261 i012) treated with distilled water (control, Horticulturae 08 00261 i013 and Horticulturae 08 00261 i014), salt spray ( Horticulturae 08 00261 i015 and Horticulturae 08 00261 i016), surfactants ( Horticulturae 08 00261 i017 and Horticulturae 08 00261 i018), and salt plus surfactants ( Horticulturae 08 00261 i019 and Horticulturae 08 00261 i020). Values are means ± SE (n = 3). Values with the same letter are not significantly different by Tukey’s test (p < 0.05).
Figure 4. Effects of different treatments on catalase (CAT (a)), peroxidase (GPX (b)), and superoxide dismutase (SOD (c)) activity in Callistemon plants during I CP ( Horticulturae 08 00261 i011) and II CP ( Horticulturae 08 00261 i012) treated with distilled water (control, Horticulturae 08 00261 i013 and Horticulturae 08 00261 i014), salt spray ( Horticulturae 08 00261 i015 and Horticulturae 08 00261 i016), surfactants ( Horticulturae 08 00261 i017 and Horticulturae 08 00261 i018), and salt plus surfactants ( Horticulturae 08 00261 i019 and Horticulturae 08 00261 i020). Values are means ± SE (n = 3). Values with the same letter are not significantly different by Tukey’s test (p < 0.05).
Horticulturae 08 00261 g004
Horticulturae 08 00261 g005a 550Horticulturae 08 00261 g005b 550
Figure 5. Principal component loading plot and scores of the PCA on the total and shoot dry biomass, R/S, leaf number, total leaf area, chlorophyll content (Chl), Na+ leaf and root content, Cl− leaf and root content, leaf damage, gas exchange (AN and gs), Fv/Fm, proline (Pro), and enzyme activity (GPX, CAT, and SOD), for Callistemon plants undergoing different salt treatments (control, salt spray, surfactants, and salt plus surfactants) according to the first two principal components in I CP (a) and II CP (b).
Figure 5. Principal component loading plot and scores of the PCA on the total and shoot dry biomass, R/S, leaf number, total leaf area, chlorophyll content (Chl), Na+ leaf and root content, Cl− leaf and root content, leaf damage, gas exchange (AN and gs), Fv/Fm, proline (Pro), and enzyme activity (GPX, CAT, and SOD), for Callistemon plants undergoing different salt treatments (control, salt spray, surfactants, and salt plus surfactants) according to the first two principal components in I CP (a) and II CP (b).
Horticulturae 08 00261 g005aHorticulturae 08 00261 g005b
Table
Table 1. Summary of the main effects and interaction effects of cycle periods (I CP and II CP) and treatments (control, salt spray, surfactants, and salt plus surfactants) on total and epigeous dry biomass, root/shoot ratio, leaf number, total leaf area, chlorophyll content, Na+ and Cl− in leaves and roots, and leaf damage of Callistemon plants with the corresponding significance of the F-values.
Table 1. Summary of the main effects and interaction effects of cycle periods (I CP and II CP) and treatments (control, salt spray, surfactants, and salt plus surfactants) on total and epigeous dry biomass, root/shoot ratio, leaf number, total leaf area, chlorophyll content, Na+ and Cl− in leaves and roots, and leaf damage of Callistemon plants with the corresponding significance of the F-values.
Total
Dry Biomass		Shoot
Dry Biomass		R/S
Ratio		Leaf NumberTotal
Leaf Area		Chlorophyll
Content		Na+ LeavesCl− LeavesNa+ RootCl− RootLeaf Damage
Main Effects
Cycle periods (CP)F 12.21
**		F 24.42
***		F 178.26
***		F 21.50
***		F 25.59
***		F 0.68
ns		F 19.74
***		F 852.36
***		F 674.85
***		F 98.51
***		F 0.84
ns
Treatments (T)F 1.06
ns		F 1.21
ns		F 3.60
*		F 1.33
ns		F 1.60
ns		F 0.12
ns		F 123.46
***		F 522.64
***		F 346.21
***		F 12.27
***		F 440.64
***
Interaction
CP × TF 0.50
ns		F 0.66
ns		F 3.50
*		F 0.67
ns		F 0.57
ns		F 0.11
ns		F 7.37
**		F 184.15
***		F 164.62
***		F 13.94
***		F 9.23
ns
Significance of differences in parameters: ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 with the corresponding significance of the F-values.
Table
Table 2. Effects of the treatments on Callistemon grown in winter (I CP) and summer (II CP) on total and epigeous dry biomass, root/shoot ratio, leaf number, total leaf area, chlorophyll content, Na+ and Cl− in leaves and roots, and leaf damage.
Table 2. Effects of the treatments on Callistemon grown in winter (I CP) and summer (II CP) on total and epigeous dry biomass, root/shoot ratio, leaf number, total leaf area, chlorophyll content, Na+ and Cl− in leaves and roots, and leaf damage.
TreatmentsTotal
Dry Weight
(g Plant−1)		Shoot
Dry Weight
(g Plant−1)		R/S
Ratio
(g g−1)		Leaf
(n·Plant−1)		Total
Leaf Area
(cm2 Plant−1)		Chlorophyll
Content
(µg cm−2)		Na+ Leaves
(g kg−1 DW)		Cl− Leaves
(g kg−1 DW)		Na+ Root
(g kg−1 DW)		Cl− Root
(g kg−1 DW)		Leaf Damage
(%)
Winter (I CP)Control11.3 ± 0.5a7.6 ± 0.4a0.5 ± 0.0b146.0 ± 6.3a478.3 ± 31.7a39.1 ± 2.82.6 ± 0.3b2.9 ± 0.4b3.4 ± 0.12.9 ± 0.60.0 ± 0.0b
Salt spray8.5 ± 0.8b4.8 ± 0.6b0.8 ± 0.1a56.5 ± 7.9b181.2 ± 29.7b27.8 ± 1.917.9 ± 1.1a14.8 ± 1.3a4.0 ± 0.53.3 ± 0.416.5 ± 1.2a
Surfactant7.7 ± 0.5b4.1 ± 0.2b0.9 ± 0.1a41.2 ± 2.8b137.0 ± 11.2b31.5 ± 3.61.2 ± 0.1b2.0 ± 0.5b3.3 ± 0.22.7 ± 0.70.14 ± 0.0b
Salt spray plus surfactant8.0 ± 0.3b4.7 ± 0.3b0.7 ± 0.1a56.2 ± 7.8b197.2 ± 34.5b35.8 ± 1.616.8 ± 1.2a14.8 ± 1.2a3.9 ± 0.53.4 ± 0.520.3 ± 1.2a
Significance ***********ns******nsns**
Summer (II CP)Control21.6 ± 1.1a18.5 ± 1.0a0.2 ± 0.0220.3 ± 12.5a860.0 ± 45.0a35.2 ± 2.03.2 ± 0.5c9.2 ± 0.5c4.6 ± 0.2c4.3 ± 0.2b0.0 ± 0.0c
Salt spray14.1 ± 0.6b12.0 ± 0.7b0.2 ± 0.0167.5 ± 12.8b624.5 ± 32.1b25.6 ± 2.521.2 ± 2.6b55.0 ± 0.6a7.4 ± 0.8b8.0 ± 0.4a17.1 ± 1.2b
Surfactant20.0 ± 0.9a17.3 ± 0.8a0.2 ± 0.0188.2 ± 8.4b814.0 ± 24.7a30.9 ± 3.51.5 ± 0.1c4.5 ± 0.7d3.2 ± 0.5d4.2 ± 0.1b0.0 ± 0.0c
Salt spray plus surfactant14.3 ± 0.3b11.2 ± 0.8b0.2 ± 0.0179.0 ± 8.5b691.9 ± 19.5b33.2 ± 1.428.9 ± 2.6a51.8 ± 0.8b9.9 ± 0.5a7.3 ± 0.6a27.7 ± 1.2a
Significance ******ns****ns***************
Significance of differences in parameters: ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Data followed by a different letter were significantly different according to Tukey’s test.
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Toscano, S.; La Fornara, G.; Romano, D. Salt Spray and Surfactants Induced Morphological, Physiological, and Biochemical Responses in Callistemon citrinus (Curtis) Plants. Horticulturae 2022, 8, 261. https://doi.org/10.3390/horticulturae8030261

AMA Style

Toscano S, La Fornara G, Romano D. Salt Spray and Surfactants Induced Morphological, Physiological, and Biochemical Responses in Callistemon citrinus (Curtis) Plants. Horticulturae. 2022; 8(3):261. https://doi.org/10.3390/horticulturae8030261

Chicago/Turabian Style

Toscano, Stefania, Giovanni La Fornara, and Daniela Romano. 2022. "Salt Spray and Surfactants Induced Morphological, Physiological, and Biochemical Responses in Callistemon citrinus (Curtis) Plants" Horticulturae 8, no. 3: 261. https://doi.org/10.3390/horticulturae8030261

APA Style

Toscano, S., La Fornara, G., & Romano, D. (2022). Salt Spray and Surfactants Induced Morphological, Physiological, and Biochemical Responses in Callistemon citrinus (Curtis) Plants. Horticulturae, 8(3), 261. https://doi.org/10.3390/horticulturae8030261

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