Interactive salinity and water stress severely reduced the growth, stress tolerance, and physiological responses of guava (Psidium Guajava L.)

Salinity and water stress are serious environmental issues that reduced crop production worldwide. The current research was initiated (2012) in the wirehouse of the Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan to investigate the growth, stress tolerance, and physiological responses of guava to salinity and water shortage. Guava was grown for one year in pots containing soil with Eight treatments (control, 10 dS m−1, 20 dS m−1, 40 dS m−1, control + water stress (WS), 10 dS m−1 + WS, 20 dS m−1 + WS, 40 dS m−1 + WS) in a completely randomized design. The results indicated that plant growth, stress tolerance, and physiological parameters declined at higher salinity and water stress and could not survive at 40 dS m−1. The 20 dS m−1 + WS caused a > 70% decline in dry weights of shoot and root regarding control. Similarly, the highest decrease in stress tolerance was noticed in 20 dS m−1 + WS followed by the 20 dS m−1 treatment than control. Our findings validated that guava can be cultivated on soils having salinity ≤ 10 dS m−1 but it could not be cultivated on soils having salinity ≥ 20 dS m−1 with limited water supply.


Results
Plant growth. The plants growing under salinity level of 40 dS m −1 and interactive salinity and water stress treatment i.e., 40 dS m −1 + WS (water stress) did not survive in these high saline and drought conditions and died within three weeks. Therefore, their data are not available. Increasing levels of soil salinity significantly affected shoot and root growth parameters such as shoot fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW), and root dry weight (RDW) of guava plants (Fig. 1A-D). However, water stress alone did not appreciably affect these attributes. Among the salinity treatments, the soil salinity level of 20 dS m −1 caused the highest reduction in shoot and root biomass compared with the control. Water stress further worsen the effect of high salinity levels on the shoot growth of guava. The combined treatment of high salinity level and the water stress (20 dS m −1 + WS) showed the most prominent decline in fresh and dry weights of the shoot (67% and 71%, respectively), in contrast to the control treatment. Both the fresh and dry root weights were decreased to a similar extent in the salt treatment of 20 dS m −1 , alone and its combination with water stress (20 dS m −1 + WS). The reduction in root biomass was more than 40% in these treatments in comparison to the control.
The combined treatment of salt and the water stress (20 dS m −1 + water stress) caused the highest decline in fresh and dry weights of the root (68% and 80%, respectively), in comparison to the control. Under 20 dS m −1 soil salinity level, fresh and dry weights of root were respectively decreased by 61% and 64% regarding control treatment. The second combined treatment of salinity and water stress conditions (10 dS m −1 + water stress) led to a 48% and 52% decline in fresh and dry weights of the root in contrast to the control treatment (p < 0.05). Similarly, the highest decrease in stress tolerance (73%) was noticed in 20 dS m −1 + WS followed by the 20 dS m −1 treatment where a 48% decrease in stress tolerance was found compared with control ( Fig. 2B). An exponential decay model was employed between salinity (EC dS m −1 ) and relative total plant biomass (Y r ) based on the best fitting of the data points (Fig. 2C).
Chlorophyll contents and gas exchange attributes. Chlorophyll contents were depreciated only under salt stress while water stress has a non-significant effect on pigment contents in guava (Fig. 3A). Interestingly, the combined treatments of salinity and water stress caused a similar reduction in chlorophyll contents as did the salinity alone treatments.
Increasing levels of salinity had detrimental effects on photosynthetic rate, transpiration rate, and stomatal conductance ( Fig. 3B-D). These attributes also significantly declined under water stress conditions as compared to control. The higher level of salinity alone (20 dS m −1 ) and two combined treatments of salinity and water stress (10 dS m −1 + WS; 20 dS m −1 + WS) had a similar effect on the photosynthetic rate and stomatal conductance of guava. However, the highest decrease in transpiration rate as compared to control (60%) was observed in the combined treatment of salt stress and water stress (20 dS m −1 + WS).
Ionic contents. The concentration of Na + was significantly higher in root, stem, and leaf when the guava plants were exposed to the combination of salt and water stress conditions (Table 1). The highest increase in Na + concentration in the above-mentioned plant parts was observed under soil salinity treatment of 20 dS m −1 + water stress. The next highest level of Na + was recorded in soil salinity treatment of 20 dS m −1 followed by the combined treatment of salt and water stress (10 dS m −1 + water stress).
Soil salinity alone and in combination with water stress significantly reduced K + concentration in the root, stem, and leaf (Table 1). The concentration of K + in the afore-mentioned plant parts was noticed to be the highest in the control treatment. Contrarily, it was the lowest in the combined treatment of salinity and water stress conditions (20 dS m −1 + water stress), followed by a salinity treatment of 20 dS m −1 and a combined salinity and deficit supply of water (10 dS m −1 + water stress). www.nature.com/scientificreports/ The highest K + /Na + ratios (4.55, 2.85, and 14.33) in stem, leaf, and root were noted in the control, while the least K + /Na + average values were found in 20 dS m −1 + water stress. Following the control, water stress showed K + / Na + ratios of 4.22, 1.50, and 2.11 in the stem, leaf, and root, respectively. Nonetheless, a non-significant (P > 0.05) change in the K + / Na + ratio of the root was observed between 20 dS m −1 , and 10 dS m −1 + water stress (Table 1).   www.nature.com/scientificreports/ Relationships of Na + content in shoot and root with stress tolerance. Figure 4 shows that significant negative relationships are recorded between Na + content in shoot and root, and stress tolerance. For instance, Na + content in the shoot explains a 73% variance in stress tolerance. While increasing Na + content in the root explains 96% variability in salinity stress (Fig. 4B). In contrast, K + content in shoot and root, respectively showed positive correlations (R = 0.974; R = 0.918; Table 1) with stress tolerance (Fig. 4C,D).
Relationships of K + /Na + ratio in leaf, stem, and root with biomass and physiological attributes. An exponential model was observed between the K + /Na + ratio in shoot and SDW (Fig. 5A) while a linear model was recorded between the K + /Na + ratio in root and RDW (Fig. 5B). Similarly, highly significant regressions were observed between the K + /Na + ratio (leaf, stem, and root) and physiological attributes ( Fig. 5C-F). For instance, a clear relationship between K + /Na + ratio and chlorophyll content, and K + /Na + ratio and photosynthetic rate with the slope and regression values (b = 5.11; R 2 = 0.834) in leaf, stem, and root, respectively.  Table 1. Regression between ionic contents (Na + and K + ) in shoot and root of guava plant (Psidium Guajava L.), and stress tolerance against salinity and water stress treatments. Na + and K + denote sodium and potassium, respectively. Results are significant at p ≤ 0.0001 represented by ****. Regression is based on a first-order kinetics equation (y = bx + a) where "a" is the intercept, and "b" is the slope.  Fig. 5E,F the highest slope values of 0.316 and 0.170 were observed in regressions between the K + / Na + ratio and transpiration rate (leaf), and K + /Na + ratio and stomatal conductance (leaf), respectively (Table 2).

Principal component analysis. The first two Principal Components axes (PC1 and PC2) explained 90%
of the total variability. The shoot dry weight, root dry weight, K + in the root, and K + in the shoot correlated positively with pH, and BD, and negatively with Na + in the root and Na + in the shoot (Fig. 6). The PC1 and PC2 axes explained 72.4% and 17.1% of the total variability. We found that ionic contents (Na + in the shoot and root had highly significant negative correlations with K + in the shoot and K + in the root, respectively. Similarly, SOM and BD also had a negative relationship. While EC of the soil had positive associations with the shoot and dry weights ( Fig. 5; Tables S1 and S2 Supplementary materials). Na + contents in shoot and root were negatively correlated with the shoot and root dry weight, and K + concentration in shoot and root, respectively. Similarly, OM was negatively associated with SAR, pH, BD, and EC (Fig. 6A). While shoot and root dry weight and K + concentration of shoot and root were positively correlated with each other. Moreover, no significant relationships were observed among BD, pH, EC, and SAR (Fig. 6A).

Discussion
Plant biomass. The current study was designed to evaluate the influence of water stress on the salt tolerance Our findings revealed that water stress alone was not as much detrimental to the plant growth of guava (Fig. 1). However, the combined application of water stress with the higher salinity level (20 dS m −1 + WS) was more damaging than salinity alone treatment (20 dS m −1 ), for fresh and dry weight of shoot. The effect of the combination of both these treatments was not additive in the case of   2016) reported that the plant biomass of two acacia species was significantly decreased when plants were grown under salinity and water stress. In the present study, we employed stress tolerance (%) as indices of plant biomass, which were considerably depreciated in the combined salinity and water stress conditions ( Fig. 2A). Salinity-induced growth reduction can be attributed to ion toxicity, osmotic effect, and nutrient www.nature.com/scientificreports/ deficiency 3,8,11 . Similarly, limited water supply to plants may cause osmotic problems 11 , and deficiency of nutrients in most plants 16 . The above-mentioned adverse effects operate at the cellular and whole plant level, thereby decreasing plant growth and biomass production. Osmotic effects and perhaps nutrient deficiency effects caused by water stress augmented the harmful effects of salt stress ( Fig. 3 and Table 3). As a result of additive effects, guava plants grown at the same salinity level but having a shortage of water produced less biomass (Fig. 1).
Chlorophyll contents and gas exchange attributes. Leaf chlorophyll contents were decreased under salinity stress in guava. In line with our results, pigment contents of Acacia 30 , and soybean 31 were decreased under salinity stress conditions. Salt stress conditions trigger the formation of the reactive oxygen species, which are known to have deleterious effects on chloroplast 20,32,33 . It has been reported that under limited water supplies, stomata are closed to conserve moisture in the leaves; resultantly, stomatal conductance is decreased 16,34 . Reduced stomatal conductance under salinity or drought leads to a corresponding decrease in photosynthesis 35 , and transpiration rates 36 . Adequate stomatal conductance is crucial for maintaining water balance in the cell. In severe conditions of water deficiency, the activities of some essential enzymes including ribulose-1,5-bisphosphate carboxylase/oxygenase, and rubisco activase are inhibited, leading to a reduction in CO 2 fixation 37 . According to Munns 38 , salinity stress may cause a decrease in the water uptake potential of plants, leading to a decrease in transpiration rate. We found that when guava was grown at the salt treatment of 10 dS m −1 with or without water stress conditions, these attributes were mostly less affected, again indicating the suitability of this tree plant to grow on saline soils (from slightly to moderately) successfully.
Relationships of Na + content and K + /Na + ratio with stress tolerance, and morpho-physiological attributes. The Na + concentration was increased under salinity stress, whereas the K + concentration was decreased in the root and shoot of guava. Further, an increase in the concentration of Na + , on the contrary, a decrease in the concentration of K + was recorded upon the combined application of salinity and water stress conditions. Through PCA, we also noticed negative associations between Na + content in shoot and root and K + content in shoot and root (Fig. 6A). Moreover, an increase in salinity in the soil matrix or within plant tends to decrease the K + content in plant organs such as shoot or root (Fig. 6A). This ionic imbalance is well documented in plants when grown under salinity stress 8,12 and a blend of salt and water stress conditions 3,11,12 . In this research work, results showed that guava leaves had the highest Na + concentration, followed by stem and roots. Different plant species/genotypes have different Na + uptake and sequestration potential. Some plant species exclude Na + at the root level, while others accumulate and sequester excessive Na + in their leaf vacuoles 6,12,38 . The Na + uptake Table 2. Regression between ionic contents (Na + and K + ) in shoot and root of guava plant (Psidium Guajava L.), and stress tolerance against salinity and water stress treatments. Na + and K + denote sodium and potassium, respectively. Results are significant at p ≤ 0.0001 represented by ****y. All regressions are based on first order kinetics equation (y = bx + a) where "a" is the intercept, and "b" is the slope value for the regression except regression between K + /Na + (shoot) and shoot dry weight is based on exponential model (y = a −exp(bx) ). www.nature.com/scientificreports/ behavior of guava indicated that the prominent salt tolerance mechanism in this plant is the vacuolar sequestration of Na + . In contrast to Na + ions, the uptake of K + ions was reduced when plants were grown under combined treatments of salinity and water stress 3,8,11,39 . Potassium has a vital role both in the salt and drought resistance of plants. Under the combined application of salt and water stress, the uptake of Na + ions in the shoot was greater as compared to a decrease in plant essential K + ions, which might have contributed to the lower salt tolerance potential of guava at a higher salinity level 12 . This greater accumulation of Na might be due to higher concentrations of Na ions in the saline soil.
Plants prefer to keep a higher K + concentration compared with Na + in stem and root. Various studies reported that plants try to acquire enough K + concentrations that could actively ameliorate the adverse impacts of Na + in tissues [40][41][42] . In contrast, the increased leaf Na + content related to the decreased leaf K + content in salinity treatments depreciates the K + /Na + ratios. Our findings revealed that the K + /Na + ratio declined with an increment in salt content (Table 1). Furthermore, potassium is an essential element for activating more than 50 enzymes 43 including those enzymes responsible for biologically synthesizing the chlorophyll. Likewise Na + , the K + ion has an identical ionic radius thus in higher salinity, Na + ions via K + channels enter the cell 44 . The higher Na + www.nature.com/scientificreports/ concentration in cytoplasm results in a lowering K + /Na + ratio, which consequently limits plant metabolism. Therefore, the plants' ability to minimize K + loss and maintain a higher cytoplasmic K + /Na + ratio is an expression of their salt tolerance potential 45 . In this study, we observed a significantly elevated K + /Na + ratio in the control, in comparison to the other treatments (Table 1). The least K + /Na + ratio under 20 dSm −1 + WS also limited the physiological attributes such as photosynthetic and transpiration rates, chlorophyll contents, and stomatal conductance by 68%, 60%, and 61%, 92%, respectively, compared with control ( Fig. 3 and Table 1). Linear regressions between the K + /Na + ratio and physiological parameters also revealed that all the physiological parameters increased with increasing K + /Na + ratio (Fig. 5). For instance, in agreement with our findings, Al-Karaki 46 reported that retention of higher amounts of K + /Na + ratio in leaf enhances salt tolerance in tomato. Moreover, Giri et al. 47 concluded that enhanced tolerance to salinity may partially be attributed to the increased K + /Na + ratios in shoot and root.

Conclusions
It can be concluded that higher salinity stress (20 dS m −1 ) drastically reduced plant growth, chlorophyll contents, and leaf gas exchange attributes of guava plants. Plants could not survive at a salinity level of 40 dS m −1 due to severe salt stress. The effect of water stress was non-additive except for shoot biomass. Increased accumulation of Na + and limited uptake of K + concentration greatly contributed to lowering the salt tolerance of guava plants.
The results demonstrated that guava is a moderately salt-tolerant plant and can be cultivated on saline soils having salinity levels up to 10 dS m −1 under sufficient water supply. However, the cultivation of this fruit tree is not recommended on soils having higher salinity level (20 dS m −1 ) accompanied by limited water supply Nevertheless, these findings need further validation on saline soils prone to drought stress under actual field conditions.

Materials and methods
Site description, treatments, and plant establishment. The impact of salt and water stress on the growth and physiological parameters of guava was investigated. The pot experiment was conducted in a rainprotected wirehouse of the Institute of Soil and Environmental Sciences (ISES), University of Agriculture Faisalabad (UAF), Pakistan. The protection from the rain was achieved by a polycarbonated sheet used as the roof of the wirehouse. Non-saline (ECe = 2.30 dS m −1 ) sandy clay loam soil was taken from the field of ISES. The collected soil was dried in the open air and subsequently sieved through a 2 mm sieve. Then, 12 kg of this soil was filled per plastic pot. Different salinity levels, i.e., 10, 20, and 40 dS m −1 were developed in the soil of each pot by properly mixing NaCl salt. The NaCl was added based on the difference between the initial EC and the required EC of the soil. The required TTS were multiplied by the equivalent weight of NaCl and the given amount of NaCl was applied to the soil. The soil was kept for two months for equilibrium before transplanting the seedlings. These salinity levels were calculated based on saturated soil extract ECe. Pots without salt addition served as control. Eight treatments of salinity and water stress (control, 10 dS m −1 , 20 dS m −1 , 40 dS m −1 , control + water stress, 10 dS m −1 + water stress, 20 dS m −1 + water stress, 40 dS m −1 + water stress) were used in the study. In water stress treatments, plants remained under water stress for 15 days (no irrigation), and 15 days of normal watering (irrigation twice a week). The plants that did not receive water stress treatments were regularly irrigated twice a week to maintain the moisture level (70%, water holding capacity). One liter of water was applied for reirrigation. Initially, they were acclimatized for one month, and water stress treatments were used afterward. These treatments were continued until the harvesting of the plants. Each treatment was replicated four times, and the pots were randomly arranged in the wirehouse. Uniformly growing guava plants (cultivar 'Safeda') at the age of three months (since seed germination; with about 20 cm plant height) were shifted to pots (one plant per pot). The plants were grown and acclimatized for three months before transplanting. On the off chance, when a plant died during the first month, it was replaced with a new plant having the same size and age, just to let it establish in stress conditions. The soil used for the pot experiment was analyzed for different physico-chemical soil properties (Table 3) according to the method proposed by Ryan et al. 48 .
Plant growth. The plants were harvested after one year, and subsequently, the data related to growth parameters such as fresh weight (shoot and root), and dry weight (shoot and root) were recorded. The shoot and roots Table 3. Concentrations of Na + , K + , and K + /Na + ratio in Psidium guajava exposed to the salt (NaCl) and drought stress (low water supply). Data refer to mean values (n = 3). The same letters along with columns are not significantly different according to the Tukey HSD post hoc test after ANOVA (p ≤ 0.05). D = Water stress; dw = dry weight; dSm −1 = desiSiemens per meter.

Salinity tolerance index.
Total root and shoot dry masses were used (in terms of yield) to model the response functions of guava to applied salt and drought treatments 23 . The total dry mass (Y) was first calculated as relative dry mass (Y r ) to compare the obtained data by using a scaling divisor (Y m ) based on the highest amount of total biomass under non-treated conditions 51 . Each Y r value has been calculated by the Eq. (1): To check the response of dry mass to salinity, next to the data transformation according to Eq. (1), an exponential model was used as a function of the best-fit line and the highest R 2 : Here EC i represents the irrigation water electrical conductivity; "a" reflects the curve shape and is a constant, and "b" defines the model intensity and is negative always. The salt tolerance index (ST-index) shows the inherited tolerance of crops to salinity in the root zone.
If Yr = 0.5, the irrigation water electrical conductivity for dry mass reduction of 50% of the maximum dry mass (EC i50 ) is gained from Eq. (3). The ST-index can be calculated by the following equation adapted from Steppuhn et al. 52 : Statistical analysis. Each treatment was repeated thrice. The data were analyzed following standard procedure in a completely randomized design (CRD) with one-way ANOVA at a significance level of p < 0.05. The least significant differences between mean values were determined by Tukey Honest Significant Difference (HSD) posthoc test. All of these statistical analyses were performed by using IBM-SPSS 23.0 software (SPSS Inc. Chicago, IL., USA). Principal component analysis (PCA) was employed to evaluate the relationships among soil physico-chemical properties, plant growth attributes such as shoot and root dry weights, ionic contents e.g., Na + content in shoot and root, and K + content in shoot and root using the princomp function of the factoextra and factoMineR packages of R software (Version 4.1.0) (R Core Team, Vienna, Austria). The PCA biplot, contribution plot and eigenvalues corresponding to the variation explained by each principal component were performed using the fviz function of factoextra.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. (1)