Effects of different salt sources and salinity levels on emergence and seedling growth of faba bean genotypes

Suitability of poor quality water for irrigation depends on salinity level and solute concentration in the water and selected crop. Salt stress is a major potential constraint for faba bean. The present study aimed to investigate the effects of different Cl- and SO4-containing salt sources in irrigation water with different salinity levels on emergence, early seedling growth and photosynthetic capacity of six faba bean genotypes. The negative effect order of salinity level was high (3 dS/m) > medium (2 dS/m) > low (1 dS/m) > control (0.05 dS/m) for all investigated parameters except dry root weight. The negative effects of Cl-containing salt sources were higher than that of SO4-containing salt sources. The worst and the best performing genotypes were determined as III-28 and III-29 on emergence percentage at 10th DAS, I-29 and III-1 on mean emergence time, III-22 and III-1 on shoot height, III-1 and I-29 on fresh biomass weight, III-22 and III-28 on fresh shoot weight, III-29 and I-29 on fresh root weight, respectively. This study showed that faba bean genotypes have different behaviors in terms of response to the increasing salinity levels artificially makeup by using different salt sources indicating that salt response of faba bean is genotype-specific.


Results
The results of ANOVA for faba bean genotypes (G), salt sources (S) and salinity levels (L) as the main factors, and their interactions including G × S, G × L, S × L and G × S × L were given in Table 1. According to these statistical analyses, faba bean genotypes and salinity level as the main factors and S × L interaction had significant effects on all investigated parameters including emergence percentage at 10th, 17th (P < 0.05 for S × L) and 24th (P < 0.05 for S × L) DAS, mean emergence time; shoot height; fresh and dry biomass, shoot and root weights; and F v /F m ratio. However, emergence percentage at 17th and 24th DAS for salt sources as the main effect; emergence percentage at 17th DAS, mean emergence time, dry shoot weight and F v /F m ratio for G × S interaction; and only F v /F m ratio for G × L interaction were not statistically significant (Table 1). Since a three-way ANOVA did not show significant differences for all parameters (except fresh root weight), the results were interpreted by following two-way interaction of S × L for each faba bean genotype and also by plotting S × G and L × G interactions by ignoring salinity level and salt sources, respectively 19 . Therefore, the performances of each faba bean genotype under S × L interaction, and individual effect of salinity levels averaged over salt sources and salt sources averaged over salinity level for all faba bean genotypes were evaluated.
Performance of I-29 faba bean genotype. Considering S × L interaction, F v /F m ratios showed a significant difference for I-29 genotype, whereas emergence percentage at 10th (ranged between 0.0 and 26.7%), and at 24th (ranged between 63.3 and 100.0%) DAS, mean emergence time (ranged between 11.47 and 15.77 days), shoot height (ranged between 4.51 and 9.23 cm), fresh biomass weight (ranged between 6.73 and 12.40 g), dry biomass weight (ranged between 1.03 and 1.51 g), fresh shoot weight (ranged between 2.35 and 5.40 g), dry shoot weight (ranged between 0.31 and 0.71 g), fresh root weight (ranged between 4.38 and 7.56 g) and dry root Table 1. P values (significance) from analysis of variance for the effects of faba bean genotype, salt source and salinity level on investigated parameters. www.nature.com/scientificreports/ weight (ranged between 0.56 and 0.92 g) parameters were not affected from S × L interaction ( Table 2). None of the emergence percentage values starting from on the 9th DAS until the experiment lasted was not statistically significant. As an indication of photosynthetic capacity, the lowest F v /F m ratio was observed under low, medium and high salinity levels with Na 2 SO 4 salt source, however F v /F m ratios under all salinity levels with other salt sources did not show significant difference from that of the control salinity level (Table 2). www.nature.com/scientificreports/ levels with all salt sources except CaSO 4 and also low salinity level with CaCl 2 cause significant reduction on fresh root weights. In general, marked decreases on dry biomass weights under medium or high salinity levels with all Cl containing salts and dry shoot and root weights under medium and/or high salinity levels with CaCl 2 and NaCl salt sources were observed. In contrast, none of the salinity level with SO 4 containing salt sources have an adverse effect on these dry weight parameters. The F v /F m ratio was ranged between 0.58 and 0.72. High salinity levels with CaSO 4 , and low and medium salinity levels with MgSO 4 resulted in an increase on F v /F m ratios whereas the lowest F v /F m ratio was observed under high salinity level with Na 2 SO 4 salt source (Table 7).    www.nature.com/scientificreports/ III-22 and III28 for dry shoot weight, III-1/III-22/III-29 and I-29/III-28 for fresh root weight, III-1 and I-29/ III-28 for dry root weight, and I-29/III-1/III-28 and USK-1 for F v /F m ratios (Fig. 1).

Principle component analysis (PCA).
According to PCA, three eigenvalues higher than 1 were found, but the first two components are presented in Fig. 3 www.nature.com/scientificreports/ positioned far from the origin in the biplot indicates that it largely affected the genotypes. The genotypes I-29 and III-28 had a specific respond to salt sources CaSO 4 and MgSO 4 , while the genotypes III-1 and USK-1 had a specific respond to salt sources CaCl 2 , MgCl 2 and NaCl. This can be explained by the fact that genotypes react as distinct from to different salt sources (Fig. 3). Also, it was determined that the parameter that best explains the salinity tolerance is F v /F m . F v /F m ratios showed a significant difference for I-29 and III-28 genotypes. Fresh root weight, dry shoot weight, fresh biomass weight, fresh shoot weight and shoot height were found to be positively and significantly correlated with each other and they were negatively correlated with mean emergence time. As www.nature.com/scientificreports/ expected, these parameters decrease as mean emergence time increases. Dry root weight, dry biomass weight and F v /F m parameters were positively correlated with each other. It was determined that I-29 and III-28 genotypes were better than other genotypes, especially in terms of dry root weight and dry biomass weight. While the www.nature.com/scientificreports/ salt sources that most negatively affected the genotypes were NaCl and Na 2 SO 4 , it was determined that the salt source with less effect was determined for CaSO 4 (Fig. 3).  Table 8. There were significantly important (P < 0.01) positive correlations between parameters of shoot height, fresh biomass weight, dry biomass weight, fresh shoot weight, dry shoot weight, fresh root weight and dry root weight, although, most of the R values seems to be low which indicates a non-linear correlation. Mean emergence time has a negative correlation with all other investigated parameters except dry biomass weight and F v /F m ratio (not significant), and dry root weight (a positive correlation). The substantial positive linear correlation was obtained between fresh biomass weight versus fresh root weight (96%) and fresh shoot weight (91%); dry biomass weight versus dry root weight (89%) and dry shoot weight (82%); shoot height versus fresh shoot weight (80%) and fresh shoot weight versus dry shoot weight (80%). The prominent negative relationship was observed between emergence percentage at 10   www.nature.com/scientificreports/ DAS versus mean emergence time (-75%). However, the relationship between emergence percentage at 10th DAS versus dry shoot weight, fresh root weight and F v /F m ratio; emergence percentage at 24th DAS versus dry biomass weight, dry root weight and F v /F m ratio; and mean emergence time versus dry biomass weight and F v / F m ratio was not statistically significant (Table 8).

Discussion
Final emergence percentages were always high (80-100%) for III-22, III-29 and USK-1 faba bean genotypes. Mean emergence time values of I-29, III-1, III-29 and USK-1 faba bean genotypes did not show significant difference under S × L interaction, indicated that the emergence time of these faba bean genotypes was not affected from increasing salinity levels with all salt sources. However, seed emergence was significantly retarded; under high salinity level with CaCl 2 and under medium and high salinity levels with NaCl salt source for both III-22 and III-28 faba bean genotypes. Similar results have been reported for common bean by Cavalcanti et al. 20 who studied the effect of water salinity (from 0.7 to 4.7 dS/m), the ionic composition of Na + Mg, and their interaction. They did not observe a significant influence on the percent emergence and emergence speed index, which shows that different genotypes from the same species can have different behaviors. In general, increasing salinity level regardless of the salt source resulted in decreases in shoot heights of these genotypes. Similarly, it was reported that the emergence and growth of castor bean were more affected by the salinity level than by the cationic composition of the irrigation water 21 . Also, it was stated that shoot height values of common bean genotypes were affected by salinity (NaCl) only at 200 mM 13 whereas shoot height of pinto bean 22 and emergence rate, shoot height and leaf area of castor bean 23 were reduced by increased salinity (NaCl) level. The latter researchers also concluded that the reduction in emergence, plant height, leaf area and chlorophyll a and b was much greater for an increase in salinity from 50 to 100 mM NaCl than that from 0 to 50 mM NaCl.
Significant decreases on these parameters were observed especially under high salinity level with majority of the salt sources. The decreases were more pronuonced with Cl containing salt sources. Dry biomass weight values of III-1, III-22, III-29 and USK-1; dry shoot weight values of III-1, III-22 and USK-1; and dry root weight values of III-29 and USK-1 faba bean genotypes were significantly different under S × L interaction. The dry biomass and shoot weight values, in general, significantly decreased under high salinity level with especially some Cl containing and/or Na 2 SO 4 salt sources whereas, no harmfull effect was observed at any salinity level with CaSO 4 and MgSO 4 salt sources. Del Pilar Cordovilla et al. 24 reported that shoot and dry root weights of faba been at 25th days after onset of salt treatment were 0.76 and 0.35 g under control and 0.69 g under 100 mM NaCl applications, repectively. Similarly, Helal and Mengel 15 mentioned that NaCl salinity depresses faba been seedling growth and restricts protein formation, CO 2 assimilation, and especially the incorporation of photosynthates into the lipid fraction.
It is explained that the photosynthetic performances of higher plants can vary widely according to growth forms, species, organs and stages concerning diverse eco-physiological demands 25 . Similarly, it was reported that exposure to salt did not have any immediate effect on F v /F m in the Glycophyte Arabidopsis and the Halophyte Thellungiella species. However, during the development of salt stress over a 14 d period, this parameter fell in Arabidopsis exposed to either 100 or 150 mm NaCl. In Thellungiella, no change in F v /F m occurred, even at the highest salt concentration 26 . In general, F v /F m ratios under medium and high salinity levels with Na 2 SO 4 salt source were the lowest almost for all faba bean genotypes.
Averaged over all faba bean genotypes, the order of salinity level in terms of negative effect was high  21 and Nobre et al. 29 observed that increasing water NaCl salinity (only with NaCl) level caused significant decreases in percent emergence, emergence speed index and plant height values of caster bean. Similarly, for common bean, it was claimed that seedling growth at 200, 250 and 300 mM NaCl were drastically affected with regard to control 30 . In general, growth reduction due to increased salinity illustrates the negative effect of salinity on plants, activated by altered osmotic potentials of salt in the root system that limits the gain of the required amount of water 31 ≥ MgSO 4 on dry root weight; and Na 2 SO 4 > NaCl ≥ MgCl 2 ≥ CaCl 2 > MgSO 4 ≥ CaSO 4 on F v /F m ratios. These orders show that, in general, the negative effects of Cl containing salt sources were higher than that of SO 4 containing salt sources. In a study to investigate the effect of Na-, Cl-and NaCl-treated soil on faba bean, it was concluded that both high Na and high Cl reduced growth of faba bean but plants were more sensitive to Cl than to Na 33 . The researchers also mentioned that reductions in growth and photosynthesis were greater under NaCl stress and the effect was mainly additive. Also, they claim that salinity caused by high concentrations of NaCl can reduce growth by the accumulation of high concentrations of both Na and Cl simultaneously, but the effects of the two ions may differ. It was reported that the emergence speed index and the emergence percentage of castor bean seedlings were significantly affected by the studied types of water salinity 21 . They claimed that the order of the cations in the irrigation water, in terms of negative effects, was Na > Na + Ca > Ca > Na + Ca + Mg > K. In another study, it was concluded that common bean seedling growth decreases in different type of salinity, due to the seedlings being unable to adjust osmotically or due to the toxic effects of Cl, SO 4 , and/or Na 34 . They observed that inhibiting effects of Na 2 SO 4 on shoot height, fresh biomass and root weight parameters were by 20% stronger than those of NaCl. However, in our experiment negative effects of NaCl on these parameters were somewhat higher than those of Na 2 SO 4 salt source.
Principal Component Analysis was performed to examine how the genotypes differ in terms of salt sources and salinity levels. It was determined that III-28 and I-29 genotypes were more tolerant to MgSO 4 and CaSO 4 salt sources and were better explained by dry root weight, dry biomass weight and F v /F m parameters. The III-22 genotype had a late mean emergence time than the others. III-29, III-1 and USK-1 genotypes have more sensitivity to salinity than I-29 and III-28 genotypes with all salt sources. In studies, investigating salinity stress in faba bean, principal component analysis was carried out in limited numbers. However, there are a few studies evaluating common parameters with this study. Rajhi et al. 28 (2020) tested salinity (NaCl) resistance in six different bean genotypes. It was reported that mass of the fresh root, mass of fresh shoot, mass of total fresh plant, mass of the dry root, length of the root and length of the shoot parameters explained the phenotypic variance according to PCA 28 . In another study using NaCl salt source in faba bean, it was reported that the parameters evaluated by PCA, root dry weight, shoot fresh weight, shoot dry weight, root fresh weight, dry seed weight and fresh seed weight were collected in one group 35 . The distribution of parameters is similar to our study.
Salinity is an ever-present major constraint and a major threat to most crops, particularly in areas with irrigated agriculture. It is quite evident that salt stress also significantly affects legume crops. Suitability of poor quality water as a supplemental source for irrigation depends on the level of salinity and solute concentration in the water and the selected crop. Even genotypes of the same species may demonstrate distinct responses to the amount and kind of salts present in irrigation and/or soil, especially during the emergence and early seedling growth. There is an immense need about the knowledge of genotype response to both salt source and salinity level for higher yield across saline environments. This study showed that faba bean genotypes have different behaviors in terms of response to the increasing salinity levels artificially makeup by using different salt sources indicating that salt response of faba bean is genotype-specific. These results provide support for further studies on the effect of the salt sources with different salinity level on emergence and early seedling growth, but these still need to be tied into a general response of the genotypes during vegetative and reproductive phases under these conditions.

Methods
Experimental site and plant material. The experiment was carried out in plastic seed trays at Akdeniz University Experimental Research Area of Agricultural Faculty, Antalya, located in the south-west region of Turkey, latitude of 36° 53′ 15″ North and longitude of 30° 38′ 53″ East and 58 m above the sea level. Seeds of six faba bean genotypes including I-29, III-1, III-22, III-28, III-29 and USK-1 which are widely extended varieties in Turkey were used in this study. Some traits of plant materials were given in Table 9.
Plastic trays, of dimension 50 × 30 × 5 cm (length × width × height) each containing 45 cells (4.5 cm deep, 5.0 and 4.0 cm upper and bottom diameter per cell), were used for seed emergence and seedling development. Soils used in the experiment as a sowing substrate were sieved with a 4 mm screen in order to remove large particles and filled to the cells of each seed tray. The experimental soil used in the experiment had sandy-loam with 54.8% sand, 16.9% silt and 28.3% clay particles with a bulk density of 1.43 g/cm 3 . The original electrical conductivity and pH of the saturated soil paste extract were 0.37 dS/m and 7.63, respectively. Soil water contents at field capacity and permanent wilting point were measured as 23.8 and 9.4%, respectively.
Experimental design and treatments. The experimental factors were six salt sources (CaCl 2 , MgCl 2 , NaCl, CaSO 4 , MgSO 4 , and Na 2 SO 4 ), three irrigation water salinity levels with an electrical conductivity (EC w ) of 1.0 (low), 2.0 (medium) and 3.0 dS/m (high) in addition to distilled water with an electrical conductivity of 0.05 dS/m without any salt source as a control and six faba bean genotypes (I-29, III-29, III-1, III-22, III-28 and USK-1).
At the beginning of the experiment, the amount of salt required for each salt source to generate the desired electrical conductivity values in irrigation waters was determined in the laboratory. Considering the determined www.nature.com/scientificreports/ amounts, irrigation waters with targeted three different salinity levels for each salt source were made separately ready for use in the experimental area by using 18 plastic water tanks having 100-l volume capacity. There were three replications with 30 seeds for each salt source and salinity level combinations of each faba bean genotype. Therefore, a total of 570 ([3 salinity levels × 6 salt sources + 1 control] × 3 replication × 30 seed per replication) seeds were used for each faba bean genotype. On the day of sowing, the seeds of uniform size were selected and sown in growth substrate with one seed per cell at a depth of 10 mm depth. Starting from the sowing, each seed tray was equally watered every day considering the salt source and salinity level treatment combination.

Analyses and measurements.
Starting on the 9th day after sowing (DAS) until the experiment lasted at 24th DAS, the number of emerged seeds at each replication was recorded every 24-h. Emergence percentage (EP) was calculated at daily periods as the percent ratio of the number of emerged seeds to the number of total seeds for each replication. Similarly, mean emergence time (MET) was calculated at the end of the experiment as the time (day) average value of all emerged seeds for each replication 36 .
Before terminating the experiment, four replicate fluorescence measurements for each seedling were realized in the middle part of intact undetached leaf blades by using a fluorometer (PAR-FluorPen FP 110/D). The potential quantum efficiency of photosystem II (PSII) was determined using expressions F v /F m = (F m − F 0 )/F m ), in which F v , F 0 and F m are variable, minimal and maximal levels of chlorophyll fluorescence. This is a relative measurement of the PSII, and can, therefore, be used to measure the performance of photosynthesis within a leaf where an F v /F m value near to 0.8 is regarded as healthy in most plants 37 . Reduction in the F v /F m ratio indicates that the plant is subjected to biotic and/or abiotic stresses 38 . After these measurements, all seedlings were removed from seed tray holes and cleaned with water to remove soils from their roots. The shoots and roots of seedlings were separated by cutting tissues with a razor blade on connecting lines that could be easily seen with a different color of the stem and root tissues. Then, the distance between the cutting point and tip of the longest leaves was measured as shoot heights and fresh and oven-dried (at 70 °C until a constant value) shoot and root weight of the seedlings were obtained. Statement on guidelines. All experimental procedures and field studies on plants/seeds comply with relevant institutional, national, and international guidelines and legislation. Data analysis. Data on the emergence, seedling growth variables and F v /F m ratio were statistically analyzed using Analysis of Variance (ANOVA) with SPSS 13.0 (SPSS Inc., Chicago, IL). The main effects of the interactions between salt source and irrigation water salinity level on investigated parameters were analyzed by univariate regression using SPSS 13.0. Homogeneity and normality were checked before subjecting data to ANOVA. Unless otherwise noted, all statistical tests were performed at the 0.01 level of significance. If ANOVA reported significant differences, treatment mean differences were separated using by Duncan's multiple range tests at P ≤ 0.05. Principal Component Analysis was performed on JMP Pro 16.0.0 (SAS Institute Inc.). Considering correlation coefficient (R) values, the strengths of the linear relationships between investigated parameters were evaluated as strong (R ≥ 0.8), moderate (0.5 < R < 0.8) and weak (R ≤ 0.5) 39 .