Growth Responses and Nitrogen Uptake by Saltgrass ( Distichlis spicata L . ) , a Halophytic Plant Species , under Salt Stress , Using the 15 N Technique

Various saltgrass clones were studied hydroponically, using Hoagland solution, in a greenhouse to evaluate their DM weights and nitrogen uptake under control and salt stress conditions. Treatments included control (no added salt) and plants grown under NaCl salinity. Twelve clones were grown with 4 replications of each treatment in a RCB design trial. Ammonium sulfate, 5.3%15N was used to enrich the plants by adding 5 mg 15N as 22.931 mg (NH4)2SO4, per liter of the culture solution per day. Plant shoots were harvested weekly, oven-dried at 65◦C, and DM weights were recorded. At the last harvest, plant roots were also harvested, oven-dried at 65◦C, and DM weights were determined. Harvested plant materials were analyzed for total-N and 15N contents. The results showed non-significant differences in shoot DM weights and total-N and 15N concentrations and contents in salinized plants compared with the controls. Total-N and 15N concentrations of the roots were higher than that of the shoots under either control or saline condition. Overall, due to the high degree of salt tolerance of saltgrass, the results showed generally no difference in nitrogen uptake by most of the clones under salt stress compared with the control plants.

Except for one publication of these authors on only one accession of this grass [8], to our knowledge, there is not any other work reported in the literature regarding the nitrogen (particularly, 15 N) nutrition of saltgrass.The previous reports and those of Sigua and Hudnall [10], Sowa and Towill [11], Enberg and Wu [12], Miyamoto et al. [13], Rossi et al. [14], and Miller et al. [15] are concerned only with the growth of this species either under normal or stressful conditions apart from nitrogen absorption.Since saltgrass is a potential turfgrass and landscaping species with a low maintenance/cultural practices, it would be a substantial savings in using this grass as a turf species.However, due to the lack of the adequate information on its water and nutrient requirements, more research should be conducted on this plant before being used as a turfgrass species and a landscaping plant in large scale.Therefore, the objectives of this study were to gather more information on this grass and to compare growth responses in terms of dry-matter (DM) yields and nitrogen requirements (i.e., total-N and 15 N absorptions) of various clones of saltgrass grown under control (nonsaline) and salt stress conditions.

Materials and Methods
Various saltgrass (Distichlis spicata) clones (Table 1) collected from several southwestern states of the United States [2] were studied in a greenhouse to evaluate their nitrogen uptake under normal (control) [no added salt, but, EC 0.95 dSm −1 (deci Siemens per meter) equal to 608 mg L −1 TDS (total dissolved solids) due to the half strength Hoagland solution reagents] and salt stress condition [NaCl at EC 20 dSm −1 equal to 12,800 mg L −1 TDS (total dissolved solids)] using 15 N in a hydroponics system.Salt stress is the stress caused by increasing the osmotic pressure (decreasing osmotic potential) of the growth medium (i.e., the culture solution) by adding any salt or solids (i.e., sodium chloride, NaCl) to the growth medium (i.e., culture solution).Saltgrass, a true halophytic plant species, has a very high level of salt tolerance.Several studies by the authors of this paper have shown that, at EC 6 dSm −1 (salt concentrations of 3,840 mg L −1 TDS), this grass performed better than the control plants.The EC 6, 20, 34, and 48 dSm −1 have been considered, normal, low, medium, and high, respectively, for the degree of salt tolerance of saltgrass.These EC values are equal to 3,840, 12,800, 21,760, and 30,720 mg L −1 TDS, respectively.Nitrogen-15 was used as a marker to find exactly how much nitrogen was uptaken and partitioned between the roots and the shoots of the grasses under stress and control conditions.
The plants were grown vegetatively in cups, 9 cm diameter and 7 cm height, following the procedures of Marcum et al. [3], Pessarakli et al. [8], Pessarakli [5], and Pessarakli and Kopec [6].Briefly, silica sand was used as the plant anchor medium.Each cup was fitted into one of the 9 cm diameter holes cut in a rectangular plywood sheet measuring 46 cm × 37 cm × 2 cm.The plywood sheets served as the lids for the hydroponics tubs and supported the cups above the solution to allow for root growth.
The sheets were placed on 42 cm × 34 cm × 12 cm Carb-X polyethylene tubs (total of 8 tubs, 4 replications of 2 treatments) containing half strength Hoagland solution number 1 ([16], Table 2), modified with Fe-sodium ferric diethylenetriamine pentaacetate (DTPA) chelate to provide 3 mg L −1 elemental Fe.Plants were grown in a randomized complete block (RCB) design with four replications of each treatment.The experiments were conducted in two different seasons (started on March 15, 2008 and repeated on March 15, 2009) for more validity of the data and to take the possible seasonal variations on the studied parameter into consideration.The averages of the data for the two experiments (two growing seasons) are reported here.The day/night temperatures of the greenhouse were 30 • C/20 • C, with maximum photosynthetically active radiation (PAR) of 1200 µmol m −2 s −1 .Light levels were supplemented for 2 h during early morning (after sunrise) and late afternoon (before sunset) with high-pressure sodium lamps (1000 W; Energy Technics, York, Pa).In each experiment, the plants were allowed to grow in this nutrient solution for 6 months.During this period, the plant shoots (clippings, all above ground biomass) were harvested weekly in order to allow the grass to reach full maturity and develop uniform and equal plant size.The harvested plant materials were discarded.At the last harvest, all roots and shoots were cut to have uniform roots and shoots prior to the initiation of the salt stress phase of the study.
The salt treatments were initiated by adding sodium chloride (NaCl) to the culture solutions to raise the electrical conductivity (EC) of the solutions 5 dSm −1 (3,200 mg L −1 TDS) every other day until the final EC 20 dSm −1 (12,800 mg L −1 TDS) was reached.Two treatments were used, including control (no salt addition) and salinized (EC = 20 dSm −1 (12,800 mg L −1 TDS).The culture solution levels in the tubs were maintained at 10 liter volume, and solution conductivity was monitored and adjusted to maintain prescribed treatment salinity levels.After the final salinity level was reached, the shoots were harvested and the harvested plant materials were discarded prior to beginning the 15 N treatment.
The 15 N treatment was started by adding 5 mg 15 N as 22.931 mg ammonium sulfate (( 15 NH 4 ) 2 SO 4 ), 5.3% 15 N (atom percent 15 N) per liter of the culture solution per day (following procedures used by Pessarakli and Tucker [17,18], Al-Rawahy et al. [19], Pessarakli [20], and Pessarakli et al. [8]).Briefly, 22.931 mg ammonium sulfate (( 15 NH 4 ) 2 SO 4 ) with 5.3% 15 N (Atom percent 15 N) enrichment to provide exactly 5 mg 15 N was added per liter of the culture solution per day.After the 15 N addition, plant shoots were harvested weekly for the determination of the 15 N absorption.The harvested plant materials were oven dried at 65 • C and dry weights (DM) were measured and recorded.Six harvests were made in each of these experiments.Plant samples were analyzed for total N and 15 N concentrations using Automated 15 N Analysis by the Rittenberg Technique (the complete system is referred to as an ANCA-MS for automated N/C analyzer-mass spectrometer and CF-IRMS for continuous flow-isotope ratio mass spectrometer (Spectrumedix Corporation, State College, PA)), following procedures reported Pessarakli [20], and Pessarakli et al. [8].Briefly, 0.5 g ground dry plant samples were digested using concentrated sulfuric acid (H 2 SO 4 ) on heated block under the hood, followed by distillation and titration, and then were analyzed for total-N and 15 N content using the aforementioned apparatus.Nitrogen isotope ( 15 N) analyses were performed by the Rittenberg technique, in which alkaline hypobromite was used to oxidize NH 4 + -N to N 2 in the absence of air.For the 15 N analysis, conversion of sample N to NH 4 + was done by the Kjeldahl method, which involves digestion with concentrated sulfuric acid (H 2 SO 4 ) to convert plant organic forms of N to NH 4 + -N, followed by steam distillation of the digest with alkali substance.Then, the following steps were performed.
(1) The distillate was placed on microplate that is moved with an x-y plotter to position a well-containing the NH 4 + -N sample beneath a pneumatically actuated reaction head.
(2) The head drops, and air is purged from the well with nitrous oxide (N 2 O).
(3) A small amount of lithium bromide (LiOBr) is introduced by a peristaltic pump to oxidize NH 4 + -N to N 2 .
(4) A valve opens briefly to admit a small amount of the gaseous headspace to a vacuum manifold.Nitrous oxide is frozen out in a cold trap immersed in liquid N 2 .
(5) The pressure of residual gas (N 2 ) is measured with a pressure transducer and is regulated as required.
(6) The N 2 is admitted to the mass spectrometer for isotope-ratio analysis.
(7) When data collection is complete, the N 2 is evacuated, and the cold trap is heated to remove N 2 O.
(8) Reference N 2 (air without O 2 ) is analyzed for calibration of the mass spectrometer.
The combined data of the two experiments were subjected to analysis of variance (ANOVA), using SAS statistical package [21].The combined means of the two experiments were separated, using Duncan Multiple Range test at the 0.05 probability level.

Results and Discussion
The results for the average weekly shoot and final root dry matter (DM) weights and nitrogen (total-N and 15 N) contents and concentrations of various saltgrass clones are presented in Tables 3-7.

Shoot Dry Matter (DM)
Weight.Except for the A136 and A138 clones, there was statistically no difference between dry matter (DM) weights of the other clones under salt stress compared with the control plants (Table 3).Three entries, clones 72, A136, and A138, produced the highest shoot DM weights and were statistically the same under the control (nonsaline) condition.Only clone A86 produced significantly lower DM weight than the previous group under the control condition.The rest of the clones produced statistically the same DM weights under the control condition, and the values were statistically the same as those of A86 clone but significantly lower than those of A136 and A138 clones.While there were statistically some differences in shoot DM weights of some of the clones under the control condition, their DM weights were statistically the same under salt stress condition (Table 3).Therefore, there was a wider range among the clones in regards to shoot DM weights under the control compared to that under salt stress condition.

Root Dry Matter (DM) Weight.
As was reported for the shoot DM weights, there was a wider range in root DM weights of the various clones under the control compared to that under salt stress condition.Clone 72 produced the highest root DM weight under either the control or salt stress condition, and the root DM weights of this clone were statistically the same under salt stress compared with that under the control condition (Table 3).Three entries, clones A49, A126, and 239, produced numerically the lowest root DM weights under the control condition which were statistically the same as root DM weights of the clones A50 and A107.Under the control condition, the root DM weights of the rest of the clones were statistically the same and were between the highest and the lowest groups.Under salt stress condition, clone 72 produced numerically the highest root DM weight which was statistically the same as that of clone 240.Two entries, clones A126 and 239, produced the lowest root DM weights, but statistically the same as the root DM weights of the rest of the clones, except clone 72 (Table 3).
Salt stress had more severe effects on shoots DM weights than that of the roots.Sagi et al. [22], Pessarakli [5,20], Marcum et al. [3], Pessarakli et al. [4,8,9], and Pessarakli and Kopec [6,7] also found that the adverse effect of salinity stress was more pronounced on the shoot than the root growth and DM weight, which is in agreement with the results of the present study.The major differences of the  Values are total N concentrations and atom % 15 N of the shoots, averages of 4 replications and 6 harvests for both experiments.
Values for Total-N concentration or % 15 N for control and salt stress treatment columns compared together followed by the same letters are not statistically different at the 0.05 probability level.
present study with the ones conducted with these authors and listed previously are as follows.First, saltgrass, a true halophytic plant species, with a very high degree of salt tolerance was much less affected by salt stress compared with the other plants used in the aforementioned studies.Second, to the best of our knowledge the nitrogen nutrition, particularly using 15 N, has not been conducted on this plant and not been reported in the literature, except for the only one study carried out by the senior author of the present study that was done only on one accession of this plant species. 15N Percentage of Saltgrass Shoots.The average values of the total-N concentration Values for Total-N or 15 N content for control and salt stress treatment columns compared together followed by the same letters are not statistically different at the 0.05 probability level.

Total Nitrogen Concentration and
Table 6: Total nitrogen concentration (mg g −1 ) and atom percent 15 N of various clones of saltgrass root tissues under the control and salt (NaCl) stress conditions.

Grass clones ID
Root total N concentration Root atom percent 15  and 15 N percentage of the shoots are presented in Table 4.
At this relatively high level of salt stress (EC 20 dSm −1 (12,800 mg L −1 TDS)), there was not a statistically significant difference found between the shoot total-N concentration (except, for A37 clone) and 15 N percentage (except, for clones 72 and A136) of the salinized plants compared with the controls.Among all the clones, 239 and 240 had numerically the highest total-N concentrations under both the control and salt stress conditions.Under the control condition, the total-N concentrations of clones A37 and A86 were statistically the same as the above group (clones 239 and 240).Except for clones 72, A107, and A126 which had numerically the lowest total-N concentrations under control condition, the rest of the clones had statistically similar total-N concentrations as clones A37, A86, and 239.Under salt stress condition, clones A37, A107, and A126 Values for Total-N or 15 N content for control and salt stress treatment columns compared together followed by the same letters are not statistically different at the 0.05 probability level.
had numerically the lowest total-N concentrations.However, there was statistically no difference between the total-N concentrations of these clones with the rest of the clones, except 239 and 240.Clones A107 and A126 had numerically the lowest 15 N percent under both control and salt stress conditions and their values were statistically the same for the control and the salinized plants.Under salt stress condition, the 15 N percent of clone 239 was among the lowest group.Under the control condition, clones A49 and 239 which had statistically the same 15 N percent as the lowest group, but numerically higher values, had statistically the same 15 N percent as the rest of the clones, except A37, A60, 72, and A136.Under salt stress condition, the range in the 15 N percent of the various clones was narrower.Except for the three entries (clones A107, A126, and 239) which were numerically in the lowest group of the 15 N percent and statistically the same as A49, 72, and A86, the rest of the clones (including the latter 3; A49, 72, and A86) had statistically the same 15 N percent.
For both the total-N concentration and the 15 N percent of the shoots, there were statistically significant differences found among the various clones under the control and salt stress conditions.Clones A107 and A126 had numerically the lowest shoot total-N concentration and 15 N percent under the control and salt stress and their values were statistically the same under both conditions (Table 4).
Some clones (i.e., 240) that were more drought and salt stress tolerant in our previous studies [7,9] had higher shoot total-N concentrations in the present study.This is supported by Khalil et al. [23], Pessarakli and Tucker [17,18], Al-Rawahy et al. [19], Pessarakli and Fardad [24], Pessarakli [20], and Pessarakli et al. [8] that showed various salt and drought tolerant plants had a higher total-N concentrations in shoots and roots under salt stress conditions compared with the control plants.The difference of the present study with the ones conducted with these authors on saltgrass and listed previously is that in the previous studies on saltgrass, except for only one study that was done on only one accession of this plant, nitrogen nutrition, particularly using 15 N, has not been conducted on this plant species, rather only growth responses of saltgrass under salt or drought stress conditions were evaluated.
Since saltgrass is a true halophyte, it is expected to perform better or as good as other salt tolerant plants, thereby, accumulate nitrogen under salt stress conditions, and use the accumulated nitrogen for its continuous growth and development under harsh environmental stress (i.e., salt and drought) conditions. 15N Contents of Saltgrass Shoots.The average values of the total-N and 15 N contents of the shoots are presented in Table 5.Except for 3 entries (clones A37, A136, and A138), the rest of the clones showed statistically no difference in their total-N contents under salt stress condition as compared with the control plants.Nevertheless, all the clones, except A86, had numerically lower total-N contents under salt stress condition as compared with the control (nonsalinized) plants.Under the control condition, clone A138 statistically had the highest and clone A86 numerically the lowest total-N content.However, under salt stress condition, clone 240 had numerically the highest and clone A37 the lowest total-N content.

Total Nitrogen and
The 15 N content of the shoots did not follow the same pattern as the total-N content.For clones A37, 72, A136, A138, 239, and 240, there were statistically significant differences found in the 15 N contents of their shoots under salt stress condition as compared with the control plants.The differences in the 15 N contents of the shoots of the rest of the clones were not significant between the salinized plants as compared with their corresponding controls.Under control condition, clones A136 and A138 had statistically the highest and clone A107 numerically the lowest 15 N content.However, under salt stress condition, clones A138 and 240 had statistically the highest and clone A107 numerically the lowest 15 N content.As was observed for the total-N content of the shoots, all the clones, except A86, had numerically lower 15 N content under salt stress condition compared with the control plants.
For both total-N and 15 N contents of the shoots, there were statistically significant differences found among the various clones under either the control or salt stress condition.Some clones (particularly, A138 and 240) that were more drought and salt stress tolerant in our previous studies [7,9] had higher shoot total-N and 15 N contents in the present study.As previously mentioned, the difference of the present study with the ones conducted with these authors on saltgrass and listed previously is that in the previous studies on saltgrass, except for only one study that was done on only one accession of this plant, nitrogen nutrition, particularly using 15 N, has not been conducted on this plant species, rather only growth responses of saltgrass under salt or drought stress conditions were evaluated.Again, this is probably because the more tolerant plants perform better and accumulate nitrogen under stress conditions and use it for their continuous growth and development under harsh environmental stress (i.e., salt and/or drought) conditions. 15N Percentage of Saltgrass Roots.Total-N and 15 N concentrations of the roots are presented in Table 6.Total-N and 15 N concentration of the roots essentially followed the same pattern as these values for the shoots, but with substantially higher magnitudes.This is because saltgrass is a true halophytic plant species, continued to absorb nitrogen (both total-N and 15 N) under salt stress condition, and accumulated the excess absorbed nitrogen in its roots before being transported to its shoots for utilization and metabolism.Also, probably some nitrogen in the form of ammonium (NH 4 + cation) was absorbed to the root surfaces of the plants as was suggested by Pessarakli and Tucker [17,18] and Pessarakli et al. [8].There was not statistically any significant difference found between total-N concentrations (except, for A37 clone) and 15 N percentage (except, for clones 72 and A136) of the roots of the salinized or the control plants.Among all the clones, clone 240 had significantly higher root total-N concentrations under both control and salt stress conditions.This phenomenon is probably due to the higher drought and salt stress tolerance of this clone compared to the other clones observed in our previous studies [7,9] as mentioned previously.Again, as previously mentioned, the difference of the present study with the ones conducted with these authors on saltgrass and listed previously is that in the previous studies on saltgrass, except for only one study that was done on only one accession of this plant, nitrogen nutrition, particularly using 15 N, has not been conducted on this plant species, rather only growth responses of saltgrass under salt or drought stress conditions were evaluated.Clone 239 had statistically the same root total-N concentration as that of clone 240 under both the control and salt stress conditions.These two clones have similar growth characters, collected from the same location (Fresno, California), and have been reported as turf-type saltgrass clones [2].Under the control (non-saline) condition, clones A37 and A86 also had statistically the same root total-N concentrations as the previous two clones (239 and 240) but significantly lower total-N concentrations under salt stress condition compared to these two clones.Numerically, the lowest root total-N concentrations and 15 N percents were found in clones A107 and A126 under either control (nonsaline) or salt stress condition.The values of the roots total-N concentrations and 15 N percents of all the other clones were between the aforementioned two groups (Table 6).

Total Nitrogen Concentration and
Compared to the 15 N percent of the roots of the plants under the control with that under salt stress condition, the differences between the values for the clones under salt stress were narrower.Clones A37, A50, A60, A138, and 240, and 3 clones (72, A86, and A136, only under control) had the highest and statistically the same root 15 N percents under the control and salt stress conditions (except for noted previously).Clones A107 and A126 had numerically the lowest root total-N concentrations and 15 N percents under the control and salt stress conditions, and their values were statistically the same under both conditions (Table 6).The values of the root total-N concentrations and 15 N percents of the rest of the clones were between the highest and the lowest groups. 15N Contents of Saltgrass Roots.Table 7 presents total-N and 15 N contents of the roots.As shown in this table (Table 7), total-N and 15 N contents of the roots essentially followed the same pattern as these values for the shoots, but with substantially lower magnitudes.This is mainly due to the substantially lower root DM weights of the plants compared to their corresponding values for the shoots.Among all the clones, clone 72 had significantly higher total-N and 15 N contents under both control and salt stress conditions.As was mentioned before, this phenomenon is probably due to the higher drought and salt stress tolerance of this clone compared to the other clones observed in our previous studies [7,9].Again, as previously mentioned, the difference of the present study with the ones conducted with these authors on saltgrass and listed previously is that in the previous studies on saltgrass, except for only one study that was done on only one accession of this plant, nitrogen nutrition, particularly using 15 N, has not been conducted on this plant species, rather only growth responses of saltgrass under salt or drought stress conditions were evaluated.Clone 240 had the second highest total-N and 15 N contents under both the control and salt stress conditions.As mentioned for clone 72, clone 240 was always more tolerant to stress than the other studied clones in our previous salt and drought stress studies.Therefore, the same reasoning applies for the higher total-N and 15 N contents of this clone.The values of the total-N and 15 N contents of the roots of these clones (72 and 240) and most of the other clones (except for clone A37 for total-N and clone A86 for 15 N content) were markedly higher under salt stress condition compared with those of the control plants.

Conclusions
The results showed that while all the 12 clones produced numerically different amounts of shoot DM weights under the control, they were statistically the same under salt stress condition.Therefore, there was a wider range among the clones in regards to shoot DM weights under the control as compared with those under salt stress condition.Shoots DM weights and nitrogen (total-N and 15 N) concentrations were more severely affected than those of the roots under salt stress condition.
Most of the clones showed statistically no difference in their total-N or 15 N contents or concentrations under salt stress condition as compared with the control plants.Except for clone A86, the rest of the clones had only numerically lower total-N and 15 N contents under salt stress condition as compared with the control (nonsalinized) plants.These findings are significant, indicating that at this relatively high level of salt stress, EC 20 dS m −1 (salt concentration of 12,800 mg L −1 TDS), there was not a statistically significant difference between total-N or 15 N contents of the shoots or roots of the salinized and the control plants.For both total-N and 15 N contents of the shoots and the roots, there were statistically significant differences found among the various clones under either the control or salt stress condition.Some clones (particularly, clones A138 and 240) that were more tolerant to drought and salt stress in our previous studies had higher shoot total-N and 15 N contents and concentrations in the present study.
Overall, although the results showed that due to the high degree of salt tolerance of saltgrass, generally no significant adverse effects of salt stress were found on growth, DM production, and nitrogen (total-N and 15 N) uptake by the various saltgrass clones under this relatively high level of salt stress (EC 20 dS m −1 , salt concentration of 12,800 mg L −1 TDS), there were some differences found among the various clones under both control and salt stress conditions.

Table 1 :
Various saltgrass clones and their locations, where they have been collected.

Table 2 :
Nutrient elements content of Hoagland solution.

Table 3 :
Shoot and root dry matter (DM) weights of various saltgrass clones under the control and salt (NaCl) stress conditions.
Shoot DM values are averages of 4 replications and 6 harvests for both experiments.Root DM values are averages of 4 replications at the final harvest for both experiments.Values for shoot or root under control and salt stress treatment columns compared together followed by the same letters are not statistically different at the 0.05 probability level.

Table 4 :
Total nitrogen concentration (mg g −1 ) and atom percent15N of various clones of saltgrass shoot tissues under the control and salt (NaCl) stress conditions.

Table 5 :
Total nitrogen and15N contents (mg) of various clones of saltgrass shoot tissues under the control and salt (NaCl) stress conditions.
Values are total N and 15 N contents of shoots, averages of 4 replications and 6 harvests for both experiments.

Table 7 :
Total nitrogen and15N contents (mg) of various clones of saltgrass root tissues under the control and salt (NaCl) stress conditions.Values are total N and15N contents of the roots, averages of 4 replications at the final harvest for both experiments.