Effects of irrigation water salinity on evapotranspiration modified by leaching fractions in hot pepper plants

We investigated whether leaching fraction (LF) is able to modify the effects of irrigation water salinity (ECiw) on evapotranspiration (ET). We conducted an experiment with a completely randomized block design using five levels of ECiw and two LFs. Results showed that the electrical conductivity of drainage water (ECdw) in an LF of 0.29 was considerably higher during the 21–36 days after transplanting (DAT), and considerably lower after 50 DAT than in an LF of 0.17. The hourly, nighttime, daily, cumulative and seasonal ET all decreased considerably as a result of an increase in the ECiw. The daily ET started to be considerably higher in the LF of 0.29 than in the LF of 0.17 from 65 DAT. Compared with the LF of 0.17, the seasonal ET in the LF of 0.29 under various ECiw levels increased by 4.8%–8.7%. The Maas and Hoffman and van Genuchten and Hoffman models both corresponded well with the measured relative seasonal ET and the LF had no marked effects on these model parameters. Collectively, an increase in the level of ECiw always decreased the ET substantially. An increase in the LF increased the ET considerably, but there was a time lag.

SCIEntIFIC REPORts | 7: 7231 | DOI: 10.1038/s41598-017-07743-2 In addition to ET, the level of EC iw also affects the salinity of the root zone. In an experiment using bell peppers 2 , the salinity of the drainage water leaving the root zone (EC dw ) was 1.5-2 times higher than for an EC iw level of 7-9 dS m −1 . Similarly, in the case of two young pomegranates, a considerably higher EC dw was observed in an EC iw level of 8 dS m −1 7 . On the whole, there is almost no uptake of salts from the soil by plant roots. The EC dw was increased as a successive accumulation of salts in the soil.
Hot peppers are one of the most popular and widely grown vegetables in the world, and are considered moderately sensitive to salt stress [14][15][16] . Most studies have been conducted to determine the effect of the EC iw on growth, yield and quality rather than directly determining ET. In addition, a limited number of studies have been conducted to analyze the effects of LFs on drainage water salinity and ET. There is also scant information for nighttime ET under varying EC iw levels and LFs, which accounted for a considerable proportion of the total daily ET and lower crop water productivity. The objective of this study is to combine varying EC iw levels and LFs and assess whether the effect of EC iw on the EC dw and ET can be modified by using LFs.

Results
Variations in the EC dw and EC e . Variations in the EC dw over time are illustrated in Fig. 1. The EC dw became a linear function of EC iw and there were considerable differences among treatments after the second application of saline water (17 days after transplanting (DAT)) for the two LFs. The EC dw reached the EC iw level approximately 25 days after the treatment had been initiated for both LFs. The EC dw of higher salinity levels in the LF of 0.29 reached more or less constant values within 10 weeks. The values of the EC dw for these treatments were 2.0-3.2 times higher than that of the EC iw (Fig. 1). The EC dw of the higher salinity levels in the LF of 0.17 continued to increase throughout the season, however, reaching levels of up to 3.1-4.4 times their corresponding EC iw values.
During 21-36 DAT, the EC dw in the LF of 0.29 was considerably higher than in the LF of 0.17, while the LF had no significant (P > 0.05) effect on the EC dw during 1-20 and 37-50 DAT. After 50 DAT, the EC dw in the LF of 0.29 was considerably lower than in the LF of 0.17. At the end of the experiment, the EC dw was 21.4%-35.2% higher in the LF of 0.17 than in the LF of 0.29, except for the EC iw of 0.9 dS m −1 . There were marked interactions between the EC iw and LF on the EC dw from 54 DAT to the end of the experiment, indicating that the greatest EC dw belonged to the highest EC iw and the LF of 0.17, and the lowest EC dw occurred in the EC iw of 0.9 dS m −1 with the LF of 0.29.
The salinity in the soil accumulated when it was irrigated with saline water. The EC e in the different soil layers increased linearly with an increase EC iw level for the two LFs. A reduction in the LF increased the EC e significantly (P < 0.001) ( Table 1). The average EC e in the LF of 0.17 was 16.0%-33.4% higher than in the LF of 0.29, except for the EC iw of 0.9 dS m −1 . Salinity was mainly concentrated in the top layer of the soil. The EC e in the 10 cm soil layer was significantly (P < 0.001) higher than in the 20 cm soil layer (Table 1). There were significant (P < 0.01) interactions between the EC iw and LF in terms of an average EC e .

Effects of the EC iw and LF on ET
Hourly scale. Figure 2 shows the diurnal variation of ET every two hours from 7:00 to 19:00 under various EC iw levels for the two LFs at 34, 38 and 76 DAT. An increase in the EC iw levels always linearly decreased the hourly ET, even when there was a low demand for evaporation -at 7:00 and 19:00, for example. Hourly ET was always a function of the EC iw and the slopes of the regression functions were higher at 11:00-15:00 when the demand for evaporation was high. The difference in terms of hourly ET over the different treatments became more marked as time went by (Fig. 2). There was no significant (P > 0.05) difference between the two LFs at 34 and 38 DAT with respect to the hourly ET, and no interactive effect between the EC iw and LF at 34, 38 and 76 DAT. At 76 DAT, except for at 7:00, the hourly ET in the LF of 0.29 was considerably higher than in the LF of 0.17, especially when the EC iw were high. The hourly ET of the EC iw level of 4.7 and 7 dS m −1 was 20.0%-26.1% higher in the LF of 0.29 than in the LF of 0.17. Nighttime ET. Figure 3 shows the variation in terms of nighttime ET under various EC iw levels for the two LFs at 33, 38 and 76 DAT. Nighttime ET accounted for 1.9%-5.6% of the total daily ET. An increase in the EC iw significantly (P < 0.001) decreased the nighttime ET ( Table 2). The highest nighttime ET was obtained for the EC iw of 0.9 dS m −1 , and the lowest from the EC iw of 7.0 dS m −1 . The LF had no significant (P < 0.05) effect on nighttime ET at 33 and 38 DAT, while the nighttime ET in the LF of 0.29 was significantly (P < 0.001) higher than in the LF of 0.17 at 76 DAT (Table 2). There were no marked interactions between the EC iw and LF in terms of nighttime ET.
Daily scale and cumulative ET. Figures 4 and 5 show the evolution of daily and cumulative ET by aggregating their respective daily ET values under various EC iw levels and LFs. Daily and cumulative ET was nearly identical when all the plants were irrigated using tap water at the beginning of the experiment. A significant (P < 0.05) reduction in daily and cumulative ET under varying EC iw levels was observed once treatment commenced (10 DAT). While daily and cumulative ET started to decrease linearly as the EC iw level increased after the second (16 DAT) and third applications (20 DAT) of saline water, respectively, for the both LFs. The absolute value of the regression function slope between daily ET and the EC iw was higher when the demand for evaporation was also high. Salinity reduced the cumulative ET and the extent of the reduction increased with time. Daily and cumulative ET in the LF of 0.29 was considerably higher than in the LF of 0.17 from 65 DAT and 75 DAT, respectively. The difference in terms of cumulative ET between the two LFs increased over time. There was a considerable difference in terms of daily ET between the two LFs on sunny days, especially when the EC iw was higher, for instance the daily ET in the LF of 0.29 was 23.7%-33.3% higher at 75 DAT (a sunny day) and 7.7%-24.8% higher at 73 DAT (a cloudy day) than in the LF of 0.17. Throughout the experiment, there were no marked interactions between the EC iw and LF in terms of the daily and cumulative ET. Table 3 shows that the seasonal irrigation, amount of drainage water and ET were 18.0-35.9 kg, 3.2-9.7 kg and 15.2-26.8 kg, respectively. An increase in the EC iw level significantly (P < 0.001) decreased the seasonal ET. The application of 7.0 dS m −1 in the EC iw caused ET to be reduced by 39.5% and 38.1% as compared with the EC iw of 0.9 dS m −1 for the two LFs. The seasonal ET increased significantly (P < 0.01) because of an increase in the LF, as well as the seasonal irrigation and amount of drainage water. Under varying EC iw levels, the seasonal ET in the LF of 0.29 was 4.8%-8.7% higher than in the LF of 0.17. There were no significant (P > 0.05) interactions between the EC iw and LF in terms of the amount of irrigation water and seasonal ET.

Evapotranspiration response functions. The effect of salinity on ET is further demonstrated by exam-
ining ET as a function of EC iw , EC e and EC dw . Figure 6 shows the relative seasonal ET measured and estimated by the Maas and Hoffman model and van Genuchten and Hoffman model. The LFs had no significant (P < 0.05) effect on the parameters of either model. In both models, the relative seasonal ET estimated across the LFs bears a close resemblance to the measured data, with R 2 ranging from 0.98 to 0.99 (n = 5, P < 0.01 or 0.001) (Fig. 6). The estimated values for the EC iw , EC e and EC dw threshold given in the Maas and Hoffman model were 0.92-1.02, 0.79-1.70 and 0.94-2.52 dS m −1 , respectively, for the two LFs, indicating that seasonal ET starts to decrease when the EC iw , EC e and EC dw are higher than these values. The slope parameters of the model were 0.0626-0.0669, 0.0332-0.0373 and 0.0146-0.0177 m dS −1 respectively for the two LFs (Fig. 6). The curves represented in Fig. 6  Leaf area, root dry weight and K + and Na + concentrations. Leaf area and root dry weight values under various EC iw and LFs are shown in Fig. 7. The leaf area and root dry weight showed a pronounced reduction with an increase in the EC iw level, especially when the EC iw level was high. Compared with the EC iw of 0.9 dS m −1 , the leaf area and root dry weight in the EC iw of 7.0 dS m −1 decreased by 61.6%-65.5% and 43.3%-62.8% respectively for the two LFs. The high LF significantly (P < 0.01) increased the leaf area (Table 2). Neither LF had any effect on root dry weight, however. There were no marked interactions between the EC iw and LF on the leaf area and root dry weight. An increase in the EC iw led to an increase in the K + and Na + concentrations in the roots (Fig. 7). The Na + concentration in the roots decreased significantly (P < 0.01) owing to an increase in the LF, while the LF did not affect the K + concentration ( Table 2).

Discussion
As we have shown, the EC iw and LFs have a strong effect on the EC dw and EC e . Non-saline soil was used in this study. After being irrigated with saline water several times, the salt accumulated rapidly in the soil in the high LF as a result of the introduction of more saline water; this is reflected by the considerably higher EC dw in the LF of 0.29 than in the LF of 0.17 during 21-36 DAT. When salt began to accumulate in the soil, more drained water in the higher LF resulted in more salt being leached from the root zone, so that the salt that had accumulated in the higher LF became lower than in the lower LF, as is reflected by the difference in the EC dw after 50 DAT (Fig. 1). At the end of the experiment, the EC dw and average EC e in the LF of 0.17 were 21.4%-35.2% and 16.0%-33.4% higher than in the LF of 0.29 respectively, except for the EC iw of 0.9 dS m −1 (Table 1). In an experiment using    Table 2. Output of the two-way analysis of variance (ANOVA) for nighttime evapotranspiration (ET) at 33, 38 and 76 days after transplanting (DAT), leaf area, root dry weight and K + and Na + concentrations of hot pepper as affected by various irrigation water salinity levels (EC iw ) and leaching fractions (LF). *, ** and *** significant differences between means at 0.05, 0.01 and 0.001 level of probability, respectively; NS, non-significant.    Table 3. Effects of irrigation water salinity (EC iw ) and leaching fraction (LF) on seasonal irrigation, amount of drainage water, evapotranspiration (ET) and actual LF using two-way analysis of variance (ANOVA) ** and *** represent significant differences between means at 0.01 and 0.001 level of probability, respectively; NS, nonsignificant. Each value is mean ± S.D. (n = 4).

LF EC iw (dS m −1 ) Irrigation (kg) Drainage (kg) ET (kg) ET (mm)
wheat and barley, the EC e in an LF of 0.2 decreased by 49.7%-65.2% when compared with that in an LF of 0.5 17 . The salinity changes in the soil and drainage water for different LFs demonstrate that the addition of water, in excess of that required by hot peppers, could be applied to ensure leaching, thereby controling soil salinity. The EC dw and EC e increased linearly as the EC iw increased and the extent of the increment increased with time. EC dw that was 1.5 to 2-fold higher than the EC iw was reported for an EC iw of 7-9 dS m −1 in an experiment using bell peppers 2 . Bhantana and Lazarovitch 7 found that EC dw was more than 5 times higher than the EC iw for an EC iw of 8 dS m −1 during the peak season. At the end of the experiment covered in this paper, the EC dw was 3.2-4.4 times higher than the EC iw when the EC iw was at 7.0 dS m −1 for both LFs. The higher EC dw in the LF of 0.17 did not reach its steady state with a target value of 42 dS m −1 according to the concept of LF for steady state conditions, with no precipitation or dissolution and good drainage, i.e., LF = V d / V i = EC iw / EC dw 18 , where V d and V i represent the drainage and amount of irrigation water.
The EC e in the 10 cm soil layer was approximately 1.25 times higher than in the 20 cm soil layer in this study. This is because salts always move with water when it evaporates, indicating that salts tend to accumulate in the upper part of the root zone 19,20 .
The ET of the hot peppers decreased considerably as a result of an increase in the EC iw . The hourly ET during the daytime linearly decreased even in the morning when solar radiation was lower (Fig. 2). Similar results were also recorded at night (Fig. 3; Table 2). This means that salinity always affects ET. Root water uptake is mainly driven by the soil's osmotic and matric potential, which controls their respective symplastic and apoplastic pathways independently 7,21 . The effect of salinity on ET has generally been assumed to reduce water availability by reducing the osmotic potential 18,22,23 . The osmotic stress reduces the free energy of water and causes a plant to spend more biological energy in taking up water from the soil solution, thus causing a reduction in transpiration and ET [24][25][26][27] . In addition, the excessive absorption of Na + by the roots in the high EC iw is another reason limiting ET ( Fig. 7; Table 2). Salinity also has an adverse effect on the leaf area and root of the plant (Fig. 7; Table 2), limiting the root water uptake rate, which in turn decreases transpiration and ET 6,28 .
High LF can control soil salinity and in turn increase ET. In this study, the seasonal ET increased significantly (P < 0.01) by 4.8%-8.7% in the high LF (Table 3). The possible reasons for this result are as follows: (1) a low LF increases soil salinity (Table 1), thus reducing water availability and causing a reduction in transpiration and ET; Figure 7. Leaf area (a), root dry weight (b) and K + (c) and Na + (d) concentrations of roots under various irrigation water salinity levels (EC iw ) and leaching fractions (LF). The error bars indicate standard deviation.
(2) when the LF is low, the reduced leaf area (Fig. 7; Table 2) contributes to a reduction in transpiration and ET; (3) a low LF causes the root to absorb more Na + (Table 2) which limites transpiration and ET; and (4) the roots have no effect on the reduction of ET because the LF has no effect on root dry weight (Table 2).
However, the LF did not have an effect on ET once treatment commenced. There were no significant (P > 0.05) differences between the two LFs at 34 and 38 DAT in terms of hourly ET (Fig. 2). There was a considerable difference between the two LFs with respect to the daily and cumulative ET from 65 and 75 DAT, respectively. Interestingly, the effect of the LF on ET and EC dw was not synchronous, as described above. The response time of ET to LF was delayed by 15-25 days when compared with that of the EC dw . The possible reason for this is that osmotic stress needs time to affect plant growth (e.g. leaf area), which in turn affects plant transpiration and ET.

Conclusions
In summary, the present study demonstrates that the EC iw always decreases ET considerably in pot-grown hot peppers, even when there is a lower evaporation demand. The Maas and Hoffman and van Genuchten and Hoffman models fitted the measured relative seasonal ET of our EC iw treatments and the LFs had no effect on model parameters. The EC dw and EC e increased linearly with an increase in the EC iw , with soil salinity mainly being concentrated in the 0-10 cm soil layer. The effect of the EC iw on the ET, EC dw and EC e was modified by the LF. The EC dw in the LF of 0.29 was considerably higher during 21-36 DAT and considerably lower after 50 DAT, than in the LF of 0.17. The LF had a marked effect on the daily ET from DAT 65. We can therefore conclude that the effect of the LF on the ET and EC dw was not synchronous. Overall, the EC dw and EC e markedly increased, while the seasonal ET decreased because of an increase in the EC iw and a decrease in the LF. The outcome of this study, together with available information on plant responses to constant salinity and LF, should provide valuable information for agricultural water management when saline water irrigation is used. Evapotranspiration (ET, g) was calculated by using the following water balance method: where W n and W n+1 are the weights of pot, plant and soil before the n th and (n + 1) th irrigation (g). I n and D n are the amounts of applied irrigation and drainage water (L) in the n th irrigation, respectively and ρ is the water bulk density (1000 g L −1 ). The amount of applied irrigation water (AW) was 120% and 140% of the ET, which resulted in an LF of 0.17 and 0.29 in accordance with the equation proposed by Letey et al. 8 :

Materials and Methods
Each pot was weighed just before each irrigation event. Throughout the experiment, the plants were irrigated at 2-5 day intervals at 16:00-17:00. A glass bottle was placed underneath each pot in order to collect the drainage water. The volume and salinity of the collected drainage water were measured after each irrigation event and the actual LF and crop ET were calculated. The application of an LF of 0.17 and 0.29 resulted in an average actual LF of 0.17 and 0.27 (Table 3). The hourly ET was measured every two hours at 34, 38, 76 DAT from 7:00 to 19:00 by weighing. The nighttime ET was measured between sunset (at 19:00) and sunrise (at 5:00) at 33, 38 and 76 DAT. The EC dw was measured after each irrigation event, and the EC e in the 10 and 20 cm soil layers was measured at the end of the experiment by a dual channel pH/mV/Ion/Conductivity benchtop meter (MP522, Shanghai San-Xin Instrumentation Inc., China). The leaf length and maximum leaf width were also measured at the end of the experiment. The leaf area was calculated by summing the lamina length × maximum width of each leaf and multiplied by a factor of 0.54 (our measurement). The roots of each plant were washed in fresh water and dried in an oven at 70 °C to obtain a constant dry weight. The dried roots were then ground into powder. The powdered plant samples were digested by concentrated HNO 3 heated using a heating block and finally dissolved in 5% (v/v) high-purity HNO 3 . The concentrations of Na + and K + were determined by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES, Perkin Elmer Optima 8000).
Evapotranspiration response functions. In this study, the relative seasonal ET (ET / ET m ) data were fitted to the yield reduction model because a reduction in yield as a result of salinity is associated with an equivalent reduction in ET 3, 12 . One is a two-piece linear response function proposed by Maas and Hoffman 10 : where ET m is the maximum ET, which appeared mainly in an EC iw of 0.9, and 1.6 dS m −1 , EC t (dS m −1 ) is the threshold electrical conductivity, and b (m dS −1 ) is the slope parameter, indicating the percentage of ET loss per unit increase in the EC e beyond the threshold value, and EC o is the root zone salinity beyond which the yield is zero.
There is another non-linear yield reduction model that is more accurate in terms of describing the sigmoidal growth response of plants to salinity 11 . It is an initial plateau and subsequent decreasing section that better accounts for higher salinity: where EC e50 represents the EC e when ET/ET m = 0.5, and b is an empirical, presumably crop, soil and climate-specific dimensionless parameter. We applied these two models to assess the effect of salinity on ET. We also used EC iw and EC dw instead of EC e in equations (3) and (4) to assess the EC iw and EC dw on relative seasonal ET.