Salt Tolerance of Forage Kochia, Gardner's Saltbush, and Halogeton: Studies in Hydroponic Culture

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1 Utah State University All Graduate Theses and Dissertations Graduate Studies 2016 Salt Tolerance of Forage Kochia, Gardner's Saltbush, and Halogeton: Studies in Hydroponic Culture Joseph Sagers Follow this and additional works at: Part of the Plant Sciences Commons Recommended Citation Sagers, Joseph, "Salt Tolerance of Forage Kochia, Gardner's Saltbush, and Halogeton: Studies in Hydroponic Culture" (2016). All Graduate Theses and Dissertations This Thesis is brought to you for free and open access by the Graduate Studies at It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of For more information, please contact

2 SALT TOLERANCE OF FORAGE KOCHIA, GARDNER S SALTBUSH, AND HALOGETON: STUDIES IN HYDROPONIC CULTURE by Joseph Sagers A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Plant Science Approved: J. Earl Creech, Ph.D. Blair L. Waldron, Ph.D. Major Professor Committee Member Bruce Bugbee, Ph.D. Committee Member Ivan Mott, Ph.D. Committee Member Mark R. McLellan, Ph.D. Vice President for Research and Dean of the School of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2016

3 ii Copyright Joseph K. Sagers 2016 All Rights Reserved

4 iii ABSTRACT Salt Tolerance of Forage Kochia, Gardner s Saltbush and Halogeton: Studies in Hydroponic Culture by Joseph K. Sagers, Master of Science Utah State University, 2016 Major Professor: Dr. J. Earl Creech Department: Plants, Soils, and Climate Halogeton (Halogeton glomeratus) is a halophytic, invasive species that displaces Gardner s saltbush (Atriplex gardneri) on saline rangelands. Forage kochia (Bassia prostrata) is a potential species to rehabilitate these ecosystems. This study compared the salinity tolerance of these species and tall wheatgrass (Thinopyrum ponticum) and alfalfa (Medicago sativa). Plants were evaluated for 28 days in hydroponics where they were maintained at 0, 150, 200, 300, 400, 600, and 800 mm NaCl. Shoot growth and ion accumulation were determined. Alfalfa and tall wheatgrass were severely affected by salt with both species shoot mass just 32% of control at 150 mm NaCl. Alfalfa did not survive above 300 mm NaCl, while, tall wheatgrass did not survive at salt levels above 400 mm NaCl. In contrast, forage kochia survived to 600 mm, but produced little shoot mass at that level. Halogeton exhibited halophytic shoot growth, reaching maximum mass at 141 mm, and not less mass than the control until salinity reached 400 mm. Gardner s saltbush did not show a dramatic decrease in dry mass produced until it

5 iv reached salt levels of 600 and 800 mm NaCl. Forage kochia yielded high amounts of dry mass in the absence of salt, but also managed to survive up to 600 mm NaCl. Salt tolerance ranking (GR 50 = 50% reduction in shoot mass) was Gardner s saltbush=halogeton>forage kochia> alfalfa>tall wheatgrass. Both halogeton and Gardner s saltbush actively accumulated sodium in shoots, indicating that Na + was the principle ion in osmotic adjustment. In contrast, forage kochia exhibited a linear increase (e.g. passive uptake) in Na + accumulation as salinity increased. This study confirmed that halogeton is a halophytic species and thus well adapted to salt-desert shrubland ecosystems. Gardner s saltbush, also a halophyte, was equally salt tolerant, suggesting other factors are responsible for halogeton displacement of Gardner s saltbush. Forage kochia is a halophytic species that can survive salinity equal to seawater, but is not as salt tolerant as Gardner s saltbush and halogeton. (72 pages)

6 v PUBLIC ABSTRACT Salt Tolerance of Forage Kochia, Gardner s Saltbush and Halogeton: Studies in Hydroponic Culture Joseph Sagers Halogeton (Halogeton glomeratus) is a halophytic, invasive species that displaces Gardner s saltbush (Atriplex gardneri) on saline rangelands. Forage kochia (Bassia prostrata) is a potential species to rehabilitate these ecosystems. This study compared the salinity tolerance of these species and tall wheatgrass (Thinopyrum ponticum) and alfalfa (Medicago sativa). Plants were evaluated for 28 days in hydroponics where they were maintained at 0, 150, 200, 300, 400, 600, and 800 mm NaCl. Shoot growth and ion accumulation were determined. Alfalfa and tall wheatgrass were severely affected by salt with both species shoot mass just 32% of control at 150 mm NaCl. Alfalfa did not survive above 300 mm NaCl, while, tall wheatgrass did not survive at salt levels above 400 mm NaCl. In contrast, forage kochia survived to 600 mm, but produced little shoot mass at that level. Halogeton exhibited halophytic shoot growth, reaching maximum mass at 141 mm, and not less mass than the control until salinity reached 400 mm. Gardner s saltbush did not show a dramatic decrease in dry mass produced until it reached salt levels of 600 and 800 mm NaCl. Forage kochia yielded high amounts of dry mass in the absence of salt, but also managed to survive up to 600 mm NaCl. Salt tolerance ranking (GR 50 = 50% reduction in shoot mass) was Gardner s saltbush=halogeton>forage kochia> alfalfa>tall wheatgrass. Both halogeton and

7 vi Gardner s saltbush actively accumulated sodium in shoots, indicating that Na + was the principle ion in osmotic adjustment. In contrast, forage kochia exhibited a linear increase (e.g. passive uptake) in Na + accumulation as salinity increased. This study confirmed that halogeton is a halophytic species and thus well adapted to salt-desert shrubland ecosystems. Gardner s saltbush, also a halophyte, was equally salt tolerant, suggesting other factors are responsible for halogeton displacement of Gardner s saltbush. Forage kochia is a halophytic species that can survive salinity equal to seawater, but is not as salt tolerant as Gardner s saltbush and halogeton.

8 vii ACKNOWLEDGMENTS I would like to thank the members of my committee, Dr. J. Earl Creech, Dr. Blair Waldron, Dr. Bruce Bugbee and Dr. Ivan Mott. Dr. Creech encouraged and supported the idea of continuing my education, and introduced me to Blair Waldron who facilitated this study. Dr. Waldron also contributed to and helped with all aspects of writing this thesis. Dr. Ivan Mott was available when I needed help with writing and equations. Dr. Bruce Bugbee provided the greenhouse space, as well as the knowledge and experience in the realm of greenhouse hydroponic research. His office door was always open and he was always willing to give input when I needed it. This study would have been impossible without his expertise. I would like to acknowledge Jason Newhall and Brandon Gilbert for their role in experimental setup, application of treatments, and data collection. Above all, I am most grateful for my wife, Katie, and children, Annabeth and Willard, who have been patient and supported me throughout this whole experience. I am also grateful for my father, Joel Sagers, who taught me the value of hard work and persistence. Joseph K. Sagers

9 viii CONTENTS Page ABSTRACT... iii PUBLIC ABSTRACT... v ACKNOWLEDGMENTS... vii LIST OF TABLES... x LIST OF FIGURES... xi LIST OF PICTURES... xiii CHAPTER 1. INTRODUCTION Salt Tolerance and Halophytes Halogeton, Gardner s Saltbush, and Forage Kochia Objectives METHODS Plant Materials Hydroponics Treatments Plant Growth and Elongation Element Accumulation in Plant Tissues Statistical Analysis RESULTS Plant Growth Sodium, Potassium, Calcium, Na + /K + and Ca 2+ /K + ratios, Magnesium, and Phosphorous Accumulation DISCUSSION Halogeton and Gardner s Saltbush s Comparative Salinity Tolerance Is Bassia prostrata a Halophytic Species? Conclusions about Comparative Salt Tolerance REFERENCES... 23

10 ix TABLES FIGURES PICTURES APPENDICES APPENDIX A - TABLES...

11 x LIST OF TABLES Table Page 1 Parameter estimates of shoot dry mass in response to increasing salinity levels in a hydroponic study. Models used were Sigmoidal Logistic 3 Parameter (SL3), Sigmoidal Sigmoid 3 Parameter (SS3), or Peak Lorentizan 3 Parameter (PL3). Standard error stated in parenthesis Parameter estimates of shoot dry mass as a percent of the control in response to increasing salinity levels in a hydroponic study. Models used were Sigmoidal Logistic 3 Parameter (SL3), Sigmoidal Sigmoid 3 Parameter (SS3), or Peak Lorentizan 3 Parameter (PL3). Standard error stated in parenthesis Parameter estimates of shoot elongation rate in response to increasing salinity levels in a hydroponic study. Model used was Sigmoidal Logistic 3 Parameter (SL3). Standard error stated in parenthesis Parameter estimates of shoot length at harvest in response to increasing salinity levels in a hydroponic study. Models used were Sigmoidal Logistic 3 Parameter (SL3). Standard error stated in parenthesis Parameter estimates of the percent root mass in response to increasing salinity levels in a hydroponic study. Model used was polynomial, linear. Standard error stated in parenthesis...31

12 xi LIST OF FIGURES Figure Page 1 Shoot dry mass(x ± SE;n=6for150, 30, and600mm, and3for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table Shoot dry massas a p ercent ofthecontrol (x ±SE;n=6for150, 30, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table Shoot elongat ionrate ( x ±SE; n = 6 for 150, 30, and 60 mm, an d 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table Shoot lengt h(x ±SE; n=6for 150, 30, and600mm, and3for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table Per cent r otmas ( root/ro t+shot; x ±SE;n=6for150, 30, and600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table Percent Na + (x ±SE; n = 6 for 150, 300, and 60 mm, and 3 for 20, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix table A Percent K + (x ±SE; n = 6 for 150, 300, and 60 mm, and 3 for 20, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix table A Na + /K + rat io (x ±SE; n=6 for 150, 300, and 60 mm, and 3 f or 20, 400, and 800 mm) of plants grown in hydroponics with increasing

13 amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix table A Percent Ca 2+ (me an x ±SE;n=6for 150, 30, and600mm, and3for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix table A Na + /Ca 2+ ratio(x ±SE;n=6for150, 30, and600mm, and3for200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix table A Percent Mg 2+ (x ± SE; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix table A Percent P ( x ±SE; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix table A Per cent ash (x ± SE; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix table A xii

14 xiii LIST OF PICTURES Picture Page 1 Alfalfa (Medicago sativa ssp. Falcata) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm Tall wheatgrass (Thinopyrum ponticum) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm Immigrant forage kochia (Bassia prostrata ssp virescens; cv Immigrant ) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm Snowstorm forage Kochia (Bassia prostrata spp grisea; cv Snowstorm ) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm Gardner s saltbush (Atriplex Gardneri) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm Halogeton (Halogeton glomeratus) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm...50

15 1. INTRODUCTION 1.1 Salt Tolerance and Halophytes Approximately 6% of the earth s land, and 20% of agricultural lands, are affected by salt (Munns and Tester, 2008). While salt exists as many different compounds, sodium chloride is the most common salt in saline soils that negatively impacts plant growth (Flowers et al., 1977b; Glenn et al., 1999). Most salts in soils of arid and semiarid regions was generated from natural causes of weathering of materials containing salt. In addition, agricultural practices have increased the salinity of arid and semiarid lands, particularly the evaporation of irrigation water from saline sources (Flowers et al., 1977a; Munns and Tester, 2008). Plant growth is reduced by salt because of both osmotic and specific ion effects on plant cells (Romo and Haferkamp, 1987; Dodd and Donovan, 1999). The osmotic pressure effect reduces available water at the root zone, which causes a loss of water from the cells and a decrease in turgor pressure. The uptake of sodium and chloride ions interfere with other internal biochemical processes, causing toxicity (Masters et al., 2007; Munns and Tester, 2008). Mechanisms used by plants to tolerate and survive in saline conditions include; excluding salt at the root level, limiting transportation to the shoot, moving sodium and excess chloride into the vacuoles, or excreting excess salt from the leaves. (Glenn et al., 1999; Masters et al., 2007; Munns and Tester, 2008). Accumulation of osmoprotectants (i.e. Proline) is another major mechanism of salt tolerance (Jahantigh et al., 2016). Many plants use a combination of these methods. In addition, salt tolerant species generally have a lower osmotic potential, allowing them to absorb water from the environment and giving them an advantage in arid climates (Zhang et al., 2010).

16 2 Calcium and potassium often interact with sodium within plants. Calcium is an essential element that plants use to preserve the structural and functional integrity of cell membranes and cell walls, and to facilitate ion transport and exchange and enzyme activities within cell walls (Rengel, 1992). Calcium is readily displaced by ions such as sodium (Lynch et al., 1987; Rengel, 1992; Tuna et al., 2007). Consequently, high concentrations of sodium within a plant can result in calcium deficiency (Cambrolle et al., 2011). Adequate calcium in saline soil solutions can reduce the severity of specific ion toxicities, particularly in crops which are susceptible to sodium and chloride injury (Maas, 1993; Grattan and Grieve, 1999; Cambrolle et al., 2011). Potassium is an important element in many biochemical and physiological processes within the plant. The ratio between sodium and potassium within the plant is considered a key factor in determining salt tolerance (Singh and Singh, 1999; Cambrolle et al., 2011). Flowers et al. (Flowers et al., 1986) define a halophyte as a plant that can complete the life cycle in a salt concentration of at least 200 mm NaCl under conditions similar to those that might be encountered in the natural environment, implying that a halophyte can survive the osmotic pressure applied by saline soils. Flowers et al. (Flowers et al., 1977a) further defined halophytes as plants that have optimal growth at the range of 20 mm to 500 mm NaCl, and that all halophytes appear to respond positively to NaCl. Greenway and Munns (Greenway and Munns, 1980) separate their classification of halophytes into two different categories: halophytes that grow rapidly at mm NaCl, versus those that grow very slowly above 200 mm NaCl. Some reviews further categorize halophytes into two groups: monocotyledonous and dicotyledonous (Flowers and Colmer, 2008; Munns and Tester, 2008), where

17 3 dicotyledonous halophytes generally show optimal growth in concentrations of mm NaCl, while monocotyledonous halophytes generally grow optimally in the absence of salt (Flowers and Colmer, 2008). In addition, halophytes can be generally categorized as salt accumulators or salt excluders (Greenway and Munns, 1980). A typical growth curve for salt accumulating halophytes shows increased growth as sodium chloride increases, followed by a decrease in growth as salinity approaches toxic levels, with optimum growth at salinity levels ranging from 20 to 500 mm NaCl (Flowers and Colmer, 2008). In contrast, salt excluders, such as many monocot species, have optimum growth in the absence of salt (Flowers et al., 1977b; Flowers and Colmer, 2008). 1.2 Halogeton, Gardner s Saltbush, and Forage Kochia Halogeton (Halogeton glomeratus) is a fleshy annual weed, native to Eurasia that was discovered in the United States in 1935 in Nevada (Dayton, 1951; Holmgren and Anderson, 1971; Young, 2002). Halogeton is a halophyte that alters the environment in which it lives to obtain a competitive advantage over other plant species (Eckert and Kinsinger, 1960). Soil salts, primarily sodium chloride, are taken up by halogeton roots and transported to the foliage, which then falls to the soil surface as plant litter to decompose at the end of the growing season. This process, known as salt pumping, increases ph, salinity, and exchangeable sodium on the soil surface. Salt persists at the soil surface in arid landscapes where halogeton prevails because there is not enough precipitation to move the salt out of the root zone (Smith et al., 2016). Halogeton has shown optimal growth in the presence of sodium chloride (Cronin and Williams, 1966), which enables it to survive in these altered soils while the competition cannot (Harper et

18 4 al., 1996; Duda et al., 2003). For livestock producers, halogeton is of concern because it develops oxalates which are toxic to livestock (Tisdale and Zappetini, 1953; Cronin and Williams, 1966). Gardner s saltbush (Atriplex gardneri) is a native shrub that is a valuable source of feed for livestock and wildlife in the salt desert shrub ecosystems of the western USA, typically characterized by saline, clay, or poorly developed soils (Smith et al., 2016). Gardner s saltbush shows a low growth habit that reaches about 8 to 20 inches tall. The twigs and stems are slender and usually soft and herbaceous, making it a good candidate for livestock fodder. A weakness of Gardner s saltbush is its vulnerability to invasion from halogeton. One study showed how an ecosystem that began with 26% Gardner s saltbush groundcover was completely dominated by halogeton within 16 years (Goodrich and Zobell, 2011). Another study showed that the establishment of Gardner s Saltbush proved to be difficult even in its native habitat when a monoculture of halogeton persisted (Smith et al., 2016). Forage kochia [Bassia prostrata (L.) A.J. Scott], a perennial chenopod shrub, is an important forage in its native environment of Eurasia, where it is utilized by sheep, goats, camels, and horses (Waldron et al., 2010b). Waldron et al. (Waldron et al., 2011) recommended the use of forage kochia on western U.S.A. rangelands as it increases nutritional value, carrying capacity, and livestock performance on semiarid rangelands, especially for fall/winter grazing. Forage kochia is well adapted to these semiarid and arid rangelands of the western U.S. where it been successfully grown in soils approaching salinity EC values of 20 ds m -1 (Francois, 1976; McFarland et al., 1990; Waldron et al.,

19 5 2010b). Because of forage kochia s high salt and drought tolerance, forage kochia has been shown to have potential to rehabilitate disturbed rangeland areas where frequent fire occurs and invasive annuals such as halogeton are displacing native perennials (Monaco et al., 2003; Newhall et al., 2004; Bailey et al., 2010; Smith et al., 2016). 1.3 Objectives The objectives of this study were to (1) document the comparative salinity tolerance of halogeton, Gardner s saltbush, and forage kochia, and (2) to determine and/or verify if these species are halophytes by defining their growth and ion accumulation response to increasing levels of salinity. By conducting this trial in a hydroponic environment, comparisons of response to salinity can be made between species, without the confounding effect of drought tolerance or limited nutrients. 2. METHODS 2.1 Plant Materials The study was conducted in a greenhouse on the campus of Utah State University. The temperature was maintained at 25 to 27 ºC during the daytime and 20 to 25 ºC at night. Entries included in study were: halogeton (Halogeton glomeratus; wildland collection), Gardner s saltbush (Atriplex gardneri; commercial source, variety not stated), alfalfa (Medicago sativa ssp. falcata; USDA experimental population HS-B, selected for salt tolerance), tall wheatgrass (Thinopyrum ponticum; USDA experimental population originated from accession PI255149), gray-type forage kochia (Bassia prostrata ssp grisea; cv Snowstorm ), and green-type forage kochia (Bassia prostrata ssp virescens; cv Immigrant ). Entries were started from seed in containers filled with

20 silica sand and grown for 12 weeks until the juvenile plants reached cm in ht. During establishment, plants were watered 2x per week by submersing for a brief time into a nutrient solution (Hoagland Solution (Hoagland and Arnon, 1938)). 2.2 Hydroponics Following establishment, roots of the juvenile plants were washed and the plants were placed in a hydroponic system. Hydroponic tanks were made of high density polyethylene and covered with closed cell foam insulation boards. Plant roots were submersed in the hydroponic solution through holes drilled into the foam insulation and soft closed cell foam plugs were used to hold the plants securely in place. The system was aerated by forcing an air supply through PVC pipe with small holes that lay across the bottom of each tank. The hydroponic solution consisted of mixing 175 g of nutrient mix (Scotts stock no /53 Hydro-Sol), 87.5 g of calcium nitrate, 25.7 g calcium chloride (dehydrate), and 525 ml of 0.1 M potassium silicate with 175 L municipal tap water. Calcium nitrate was the main source of nitrogen for the plants, while calcium chloride (dehydrate) was added to ensure that ample calcium was available. Inasmuch as the purpose of this study was to test the plants ability to monitor osmotic potential, and not necessarily to investigate salinity toxicity, the calcium helped keep sodium levels at a lower level of toxicity (Greenway and Munns, 1980; Munns, 2002). Silica is not an essential element, but has shown to be beneficial for plant growth in hydroponics (Samuels et al., 1993; Cocker et al., 1996; Suriyaprabha et al., 2012); therefore, potassium silicate was added to provide the plants with sufficient silica. The solution ph was maintained at a ph of 5.0

21 7 with doses of 0.1 M of nitric acid. In addition, 1 ml of a fungicide (Ridomil Gold EC, active ingredient: Mefenoxam) was added to each tank as a preventative measure. As evapotranspiration occurred, the tank was refilled every 7 days with a slightly modified hydroponic solution. The refill solution consisted of 100 L municipal tap water mixed with 30 g of nutrient mix, 50 g of calcium nitrate, and 300 ml 0.1 M potassium silicate. These measurements are similar to the original refill solution, however the nutrient mix was reduced and calcium chloride was not added because previous experience had indicated that nutrients and calcium are not taken up by the plants at the same rate as evapotranspiration occurred (B. Bugbee, personal communication). 2.3 Treatments Treatments consisted of four levels of salinity and arranged in an RCB design with three replications. The experiment was repeated three times (runs) with start dates of 15 July 2015, 30 September 2015, and 9 March Salinity levels in the first run were 0 mm, 200 mm, 400 mm and 800 mm of NaCl, and, thereafter, changed to 0 mm, 150 mm, 300 mm and 600 mm for runs 2 and 3, due to death of most entries at the 800 mm level. Salinity levels were gradually increased over a period of 10 days until the full molarity was reached in order to minimize plant shock. This was accomplished by, each day, dissolving in nutrient solution one-tenth of the total NaCl needed in 175 L and adding it to the respective tanks (153.4, 204.6, 306.8, 409.2, 613.0, and g NaCl each day for the 150, 200, 300, 400, 600, and 800 mm treatments, respectively). At the end of 10 days, the solution EC was checked and was always close to the expected ECs of 15, 20, 30, 40, 60, and 80 ds/m for the 150, 200, 300, 400, 600, and 800 mm

22 treatments, respectively. Once final solution molarity was reached, the plants were grown for an additional 28 days in the hydroponic solution Plant Growth and Elongation Plant Shoot elongation rate was calculated using the method of Maguire (Maguire, 1962) by measuring the longest shoot length weekly and calculated as: Elongation rate (ER) = ([cm 7 - cm 0 ]/7) + ([cm 14 - cm 0 ]/14) + ([cm 21 - cm 0 ]/21) + ([cm 28 - cm 0 ]/28), where cm 0 is the shoot length at the time that solution salinity reached full molarity, and cm 7, cm 14, cm 21, and cm 28 are the shoot length at 7, 14, 21, and 28 days thereafter. Following 28 days of growth in hydroponics at full salinity levels, plant shoots and roots were harvested separately. Shoot and root length were measured following the harvest from the base of the plant to the furthest point on the shoots and the roots. Shoot and root mass were determined by weighing shoots and roots at harvest to determine fresh weight and then were dried in an oven at 65 ºC for 72 hours and weighed again to determine dry weight. The percent of total plant mass comprised of shoot mass was calculated and used as an estimate of differential carbon partitioning by these species. 2.5 Element Accumulation in Plant Tissues Ground shoot samples were sent to the Utah State University Analytical Laboratory (Logan, Utah) for analysis of ion content using a Thermo Electron icap ICP (Inductively-Coupled Plasma Spectrophotometer) following their standard operating procedure. Root samples were not evaluated. In addition, ground shoot samples were ashed to determine total inorganic content. Ground samples were placed in a microwave ashing oven (Milestone Pyro), and the temperature was raised to 550 ºC and maintained

23 for 120 minutes. Following ashing, percent ash on dry matter (DM) basis was calculated. The equation used to calculate total ash was: 9 % ASH (DM basis) = (W3 W1) x 100 / (W2 W1) where W1 is equal to the tare weight of crucible (in grams), W2 is the weight of the crucible and the added sample weight after being dried at 105 ºC for 12 hours and W3 is the weight of the crucible and the sample after being ashed at 550 ºC. 2.6 Statistical Analysis All data were analyzed with the mixed procedure of SAS to test main effects and get estimates of the Entry Salinity Level lsmeans and standard errors. Response curves across salinity levels were then fit using Sigmaplot. Shoot and root growth responses were fit to standard dose responses as three-parameter sigmoidal logistic (Equation 1) or sigmoid (Equation 2) models as shown: Y = a/(1 +!!!! ) [Equation 1] Y = a/(1 + e (!!!!!! ) ) [Equation 2] where a indicates the upper limit, x 0 represents the 50% biomass or growth reduction (e.g. GR 50 ) value, and b is the slope of the line around the GR 50 values. The resulting GR 50 values provide an objective comparison of salinity tolerance among species. In the case of halogeton, response of shoot mass also required fitting a Lorentzian threeparameter peak model as shown: Y = a/(1 +!!!!!! ) [Equation 3]

24 10 where a indicates the height of the peak, x 0 represents the location (e.g. salt level) of the peak, and b is the scaling parameter which specifies the half-width at half-maximum (interquartile range). In contrast, ion content response to increasing salinity was fit using the best available model. In many cases, the best fit for the ion data was sigmoidal such as the three-parameter logistic model. However, some species at the higher salinity levels lacked sufficient plant growth for ion analysis, and those responses were mostly fit to a polynomial (linear, quadratic, or cubic) model, while a few required hyperbola and exponential decay models. Growth response models and parameters are listed in tables 1-5. All response fitting analyses were performed on individual plant data. 3. RESULTS 3.1 Plant Growth Species varied in growth response to increasing salt level (Fig. 1), and, in general, could be categorized into three distinct groups: low salt tolerance (alfalfa and tall wheatgrass), medium salt tolerance (forage kochia), and highly salt tolerant with halophytic characteristics (Gardner s saltbush and halogeton) (Fig. 2). Plant growth in the absence of salt (control) had an inverse pattern, favoring growth of low and medium salt tolerant species (Fig. 1). Alfalfa and tall wheatgrass were severely affected by increasing salt with both species shoot mass reduced to just 32% of the control plants at their lowest salt level (150 mm) (Fig. 2). Interestingly, alfalfa produced greater (P = ) shoot mass (g) than tall wheatgrass at the 150 mm level (Fig. 1), validating that salt tolerance had been improved in this experimental population of alfalfa. However, tall wheatgrass exhibited

25 11 overall greater (P = ) salt tolerance than alfalfa, producing low amounts of shoot mass up to the 400 mm level (Fig. 1, Picture 2). Alfalfa plants only survived up to the 300 mm level (Fig. 1, Picture 1), at which point shoot mass amounted to only 3.71% of the control (Fig. 2). Shoot elongation rate and shoot length of alfalfa and tall wheatgrass followed a similar pattern, dropping off sharply between the control and the lowest level of salt (Fig. 3 and 4). In contrast, the forage kochia entries exhibited greater (P = to ) salt tolerance than alfalfa and tall wheatgrass, surviving up to the 600 mm, although very little shoot growth was produced at that level (Fig. 1 and 2; Pictures 3 and 4). Forage kochia shoot mass was reduced (P = ) compared to the control even at low salt levels, and thus did not exhibit a typical halophytic response of increased growth at low amounts of salts (Fig. 2). Overall, Immigrant was more (P = ) salt tolerant than Snowstorm with greater shoot mass up to the 400 mm level (Fig. 1 and 2). This difference was most pronounced at the 200 mm level (P = 0.001), where Immigrant shoot growth was 61% of the control as compared to 34% of the control for Snowstorm (Fig. 2). However, the two forage kochia entries did not differ in shoot elongation rate or total shoot length (Fig. 3 and 4), perhaps suggesting that the difference was Immigrant s ability to produce new shoots in saline conditions. Gardner s saltbush and halogeton produced the least overall shoot mass (Fig. 1), but their shoot growth indicated that they were the most salt tolerant entries with halophytic-type growth responses to increasing salinity (Fig. 1 and 2). Both species had either increasing or stable shoot mass through the lowest salinity levels (Fig. 2) and were

26 12 still producing 15 and 9% of the control, respectively, at the highest 800 mm level (Fig. 2; Pictures 5 and 6). Shoot elongation rate and total length verified their salt tolerance with a relatively small decrease as salinity levels increased (Fig. 3 and 4). The percent of total biomass comprised of root mass generally increased as salinity increased, indicating that carbon continued to be allocated to root growth even after shoot growth was restricted (Fig. 5; Pictures 1-6). The notable exception was Immigrant forage kochia, in which % root mass did not change in response to increased salinity (Fig. 5). In contrast, alfalfa exhibited a rapid increase in % root mass from the control to the 300 mm salt level (Fig. 5). Rapid death or no additional shoot or root growth occurred at salinities greater than 300 mm in alfalfa, 400 mm in tall wheatgrass, and 800 mm salt level in forage kochia; therefore, % root masses are not shown for those species at those levels (Fig. 5). With the exception of Immigrant forage kochia, the total biomass comprised of root mass was inversely related to salinity tolerance (shoot mass as a % of control), with halogeton and Gardner s saltbush partitioning the least amount of carbon to roots, forage kochia exhibiting an intermediate response, and tall wheatgrass and alfalfa having the greatest % root mass (Fig. 5). 3.2 Sodium, Potassium, Calcium, Na+/K+ and Ca2+/K+ ratios, Magnesium, and Phosphorous Accumulation Similar to the growth response, Na + accumulation in shoot tissues followed three distinct patterns. Gardner s saltbush and halogeton followed a typical 3-parameter logistic pattern, rapidly accumulating Na + with an average of 9% at the 150 mm level, and then gradually leveling off across the higher salinity levels to achieve a maximum accumulation of 12% of Na + at the 800 mm level (Fig. 6). In contrast, the forage kochia

27 13 subspecies exhibited a linear increase in Na + accumulation as salinity levels increased, reaching a maximum of 8% at the 600 mm salt level (Fig. 6). The 300 mm level was the highest salinity dose where alfalfa and tall wheatgrass produced adequate shoot mass to allow for ion analyses. Up to 300 mm, Na + accumulation in alfalfa was the lowest of all species (approximately 2%) and was linearly increasing with greater salinity levels; whereas, tall wheatgrass Na + accumulation was almost exactly that of Immigrant forage kochia, with an approximate maximum of 4% Na + at 300 mm salt level (Fig. 6). Potassium content of shoots rapidly decreased in all species as salinity increased and Na + accumulated (Fig. 7). The decrease in K + was most pronounced in those species that accumulated the greatest amount of Na +, reaching their lowest % K + levels at the low to medium salinity doses (Fig. 7). Decline in K + in tall wheatgrass and alfalfa was gradual with a liner trend. In comparison, the sodium to potassium ratio increased linearly with greater salinity in alfalfa, tall wheatgrass, and forage kochia, and, as expected, alfalfa had the lowest Na + /K + ratio of all species (Fig. 8). Gardner s saltbush and halogeton exhibited a typical logistic dose-response for the Na + /K + ratio, and as expected based upon their rate of Na + accumulation, reached maximum Na + /K + ratios at medium salinity doses of 300 and 400 mm, respectively (Fig. 8). In general, Ca 2+ accumulation in shoot tissues decreased with increasing salinity, with the greatest Na + accumulator (halogeton) exhibiting the lowest Ca 2+ accumulation (Fig. 9). Interestingly, Ca 2+ accumulation in halogeton reached its lowest level at 300 mm, then slightly increasing thereafter. In the addition, the most distinguishable Na + /Ca 2+ ratio response was exhibited by halogeton, with a rapid increase in Na + /Ca 2+ up

28 14 to the 200 mm level followed by a sharp decrease as salinity continued to increase (Fig. 10). The dose responses for Mg 2+ and P accumulation are shown (Fig. 11 and 12). The greatest Mg 2+ accumulation occurred in Gardner s saltbush across all salt levels, while halogeton had the high levels of P in its shoot tissues in the presence of salinity. Ash content has implications to forage nutritive value and is an indicator of inorganic material in tissues. Halogeton and Gardner s saltbush shoots were comprised of large amounts of ash, exceeding 30%, at all salinity levels (Fig. 13). This further indicated that these species rapidly accumulate salt in shoot tissues when grown in saline conditions. The forage kochia entries and tall wheatgrass exhibited intermediate ash content in comparison to other species, and alfalfa had low levels of ash validating that it did not accumulate salt in its shoots. 4. DISCUSSION 4.1 Halogeton and Gardner s Saltbush s Comparative Salinity Tolerance Halogeton and Gardner s saltbush have been reported to be salt-tolerant species, especially in the salt desert shrublands where they commonly grow (Cronin and Williams, 1966; Masters et al., 2010). However, we believe this is the first time the salt tolerance of these two species have been compared side by side, in a controlled hydroponic setting that eliminates the confounding effect of drought and limited nutrients. These two salt accumulators were both slow growing, but tolerated salt as high as 800 mm of NaCl when grown in a hydroponic system. Halogeton exhibited a typical halophytic increase in shoot growth at the lower salinity levels reaching its maximum shoot mass at 141 mm NaCl (Fig. 1; Table 1; x0 of the Lorentzian model is NaCl level

29 15 where peak is maximum), and shoot mass was not less than that of the control until salinity reached 400 mm and greater levels (Fig. 2). In a potted plant study, Wang et al. (Wang et al., 2015) reported that halogeton reached maximum growth when irrigated with a 100 mm NaCl solution, and declined thereafter with growth at 200 mm significantly less than the control. Wang et al. (Wang et al., 2015) irrigated plants daily, but still the differences are probably due to the confounding effect of the variable matrix and osmotic potentials. As water is removed in transpiration, the osmotic potential increases rapidly. This effect is particularly significant in containers because of the reduced root-zone volume. Studies in hydroponic culture minimize this confounding interaction. They also reported that halogeton growth was reduced by 64% at the 500 mm salt level, whereas we found that growth was reduced 50% at a similar salinity (Table 1, 463 mm NaCl is the GR 50 value). However, even with these slight differences, both studies further document the high salt tolerance of halogeton. In comparison to halogeton, Gardner s saltbush growth response was stable and not affected by salinity up to the 300 mm level (Fig. 2). Based upon overall average shoot growth (% of control), halogeton had greater (P = ) salt tolerance than Gardner s saltbush, supporting the hypothesis that halogeton is displacing Gardner s saltbush on rangelands by salt pumping to increase soil salinity (Goodrich and Zobell, 2011; Smith, 2015). However, examining salinity levels where growth is reduced by 50% (GR 50 ) allows us to directly compare the salinity tolerance of these species. In our study, the GR 50 values clearly indicate that these two species are much more salt tolerant than all other species (e.g., 250% greater tolerance than Immigrant forage kochia), and that Gardner s saltbush (GR 50 = 489 ± 104 mm NaCl) and halogeton (GR 50 = 463 ± 95 mm

30 16 NaCl) have nearly identical salt tolerance (Table 1). Therefore, this study suggests that factors other than salt tolerance, including drought or rhizosphere alteration by halogeton (Duda et al., 2003; Smith et al., 2016) may be responsible for the displacement of Gardner s saltbush by halogeton. Both halogeton and Gardner s saltbush accumulated sodium in shoot tissues. Even at the lowest salinity dose of 150 mm NaCl, both species had accumulated Na + in shoot tissues that was over 40X greater than salt concentrations considered toxic to plants (0.2%, (Bernstein, 1975)) (Fig. 6). In addition, the Na + /K + ratios were at minimum 5X greater than optimum for non-halophytic plant growth (Greenway and Munns, 1980). These results suggest that the tolerance mechanism of these halophytic species is primarily osmotic adjustment, associated with the compartmentalization of Na + (Munns and Tester, 2008). This is in agreement with Wang et al. (Wang et al., 2015) who reported that halogeton salt tolerance came from osmotic adjustment associated with transport and compartmentalization of sodium in vacuoles. They reported a Na + content of 17% of dry weight in halogeton leaves at the 500 mm NaCl level, while in our study, sodium content at 600 mm NaCl was 12% of dry weight for both halogeton and Gardner s saltbush (Fig. 6). The difference may be because we measured the sodium content of the entire shoot, which suggests that the stems are also compartmentalizing Na + but not to the same level as the leaves. Our data clearly show that Na + was the principle ion involved in osmotic adjustment in both of these species, with Na + accumulation (Fig. 6) resembling that observed for active uptake of essential nutrients resulting in concentrations higher in the plant than that in the external environment (White, 2012). In addition, their ability to transport Na + into the shoot appeared to be

31 17 saturated at relatively low external salinity, similar to that observed for Suaeda maritima (Yeo and Flowers, 1986), a succulent halophyte like halogeton, and Atriplex canescens, another common Atriplex shrub species found on salt desert shrublands of North America (Glenn et al., 1994). In further comparison to reports for four-wing saltbush (A. canescens ssp. canescens), Gardner s saltbush accumulated greater amounts of Na + and had greater Na + /K + ratios in high saline environments (Glenn et al., 1992; Glenn et al., 1994; Glenn et al., 1996). However, it should be noted that Glenn et al. (Glenn et al., 1992) concluded that high salt tolerance wasn t dependent upon high levels of Na + accumulation in A. canescens. Ash content, as a measure of inorganic material in the shoots, was further evidence of the high sodium uptake and accumulation in halogeton and Gardner s saltbush. In this hydroponic study, halogeton and Gardner s saltbush had ash contents ranging from 37 to 42% and 34 to 44%, respectively, for salinity levels ranging from 150 to 600 mm (Fig. 13). These extreme values exceed those previously reported for Gardner s saltbush (25% ash) when sampled from plants growing in its natural salt-desert shrub rangeland environment (Welch, 1978). Most other nutrient and ion concentration trends in halogeton and Gardner s saltbush were as expected with sodium accumulators. In general, as these species increased uptake of sodium there was an associated decrease in uptake of K +, Ca 2+, and Mg 2+ (Fig. 7, 9, 11). The response was rapid, occurring mostly by 200 mm NaCl except in the case of Mg 2+, where a gradual decrease was observed in Gardner s saltbush and no decrease was exhibited by halogeton as salinity increased. The phosphorous uptake by halogeton was also noteworthy (Fig. 12). Halogeton plants at all salinity levels accumulated phosphorous such that shoot concentrations exceeded 10X

32 18 that considered adequate for a growing plant (0.3 to 0.4%). It is not clear what the physiological advantage would be in this type of P uptake trend and we are not aware of other reports with similar findings. 4.2 Is Bassia prostrata a Halophytic Species? Forage kochia is considered a drought and salt tolerant species (Waldron et al., 2010a), and in preliminary studies by these authors, exhibited high salt tolerance including active growth and LD 50 values at salinity levels exceeding that of seawater (600 mm NaCl) (unpublished data), thus suggesting halophytic growth patterns. However, in those studies, older forage kochia seedlings and/or potted plant experiments were used, and they were not compared to a documented halophyte such as halogeton. This is the first known report of forage kochia s salinity tolerance without the confounding effect of drought tolerance. Unlike that observed for halogeton and Gardner s saltbush, shoot mass of forage kochia decreased at even the lowest salt level of 150 mm (Fig. 1). Karimi et al (Karimi et al., 2005) reported that forage kochia growth was not decreased at salinity levels between 50 and 150 mm, and then exhibited a 52% shoot reduction at 200 mm NaCl. Their study was similar to ours with the same initial size of forage kochia seedlings, same rate of incremental increase to reach full salinity, and same duration of the study, but the primary differences were that they used plants potted in sand and looked at salinity levels below 150 mm. Normally, due to evapotranspiration, we would assume potted plants would have higher root zone salinity than the actual solution salinity, but their manuscript does not clearly define how saline treatments were applied (e.g., irrigation or hydroponics) and

33 19 perhaps in their study this was not a factor. Our study did not look at salinity below 150 mm so we cannot directly compare to their results at 50 and 100 mm, and it is possible that we would have seen similar results or even increased growth at those lower levels. It is also possible that genetic variation is responsible for the differences between these two studies. Their plants originated from wildland collected seed in Iran (Karimi et al., 2005) that were quite likely growing in a saline environment; whereas, Immigrant germplasm originates from an unknown location in Russia (Stevens et al., 1985) and Snowstorm originates from germplasm sources in Uzbekistan (Waldron et al., 2013). While this species is noted for its salt tolerance, neither of these cultivars was purposely selected for salt tolerance and are both many generations removed from their original habitat. However, even so, our calculated GR 50 of Immigrant (189 mm) is in the same general range of that observed for the Iranian biotype (between 150 and 200 mm). Halophytes often accumulate sodium in shoot tissues as a mechanism for osmotic potential adjustment (Flowers and Colmer, 2008). In contrast to the active uptake observed for halogeton and Gardner s saltbush, forage kochia exhibited passive uptake of Na + as evidenced by a linear increase in sodium content of shoots as salinity increased (White, 2012) (Fig. 6). Karimi et al. (Karimi et al., 2005) also observed a linear increase in shoot sodium content in forage kochia as salinity increased from 0 to 200 mm. However, their sodium accumulation was double (5.5% of shoot dry matter) of that we observed (2.7%) at the 150 mm salt level. The fact that their control plants contained 1.2% sodium in the shoots as opposed to our range of 0.1 to 0.3% in forage kochia control plants, suggests the possibility of their control solution containing higher sodium than ours and may be one reason some results differ. In addition, Karimi et al. (Karimi et

34 20 al., 2005) reported 50% less K + accumulation and nearly triple Na + /K + of that we observed, further indicating that there were likely differences in experimental solutions and overall conditions. They conclude that Bassia prostrata is a halophytic species maintaining osmotic potential by NaCl accumulation in vacuoles, with optimum growth at 150 mm NaCl. Even though we observed a substantial growth decrease at the 150 mm salinity level, our findings support their conclusion that forage kochia is a halophyte as many other indicators were in common including sodium accumulation in the shoot tissues. In addition, our study examined much higher salt levels than they and we found that even though growth was severely reduced, Bassia prostrata plants survived up to the 600 mm salt level (Pictures 3-4), further supporting its classification as a halophytic species. It is interesting to note the lesser salt tolerance of Snowstorm forage kochia compared to Immigrant (GR 50 values of 130 and 189, respectively). Smith et al. (Smith et al., 2016) reported that Immigrant performed better than Snowstorm in a halogetoninvaded Gardner s saltbush ecosystem. They were surprised by this finding inasmuch as they had surmised that Snowstorm and the subspecies grisea had greater salt tolerance than Immigrant and the subspecies virescens. Our results do not support their hypothesis on this relative salt tolerance between these two forage kochia species, and provide evidence that Immigrant may have been better adapted to that particular saline environment.

35 Conclusions about Comparative Salt Tolerance Based upon GR 50 values for shoot mass (Table 1 and 2), the species in this study would be ranked for salt tolerance in this order: Gardner s saltbush = halogeton > forage kochia (Immigrant > Snowstorm) > alfalfa > tall wheatgrass. It is remarkable that alfalfa would be reported to have greater salt tolerance than tall wheatgrass, and based upon these measurements was also equal in salt tolerance to Snowstorm forage kochia. In this study we used a salt-tolerant experimental alfalfa population (HS-B) that in an earlier study exhibited greater salt tolerance than the parent population at the 90 mm salinity level (Rokebul Anower et al., 2013). However, our salt levels were unrealistic for a nonhalophyte like alfalfa, and in our study HS-B had the least shoot biomass at all salt levels above 150 mm. It is probable that a comparison of these entries at salt levels ranging between 0 and 150 mm would give a more accurate estimate of GR 50 and change the salt tolerance ranking between alfalfa, tall wheatgrass, and Snowstorm forage kochia. Nevertheless, it does appear that this alfalfa germplasm has improved salt tolerance and in agreement with their findings we showed that HS-B s salt tolerance comes in part from excluding sodium transport to the shoots. However, it does appear that at salinity levels greater than what they evaluated (e.g., 90 mm NaCl), some sodium does make it into the shoots of this alfalfa population (Fig. 6). Tall wheatgrass has been characterized as both a salt tolerant and a halophytic grass (Shannon, 1978). In our study it was the least salt tolerant species (based upon GR 50 values), but accumulated sodium in a similar pattern and rate (passive accumulation) as forage kochia until Na + levels apparently reached toxicity at salinity of 400 mm and greater (Fig. 6). Further evidence of halophytic growth in tall wheatgrass was a Na + /K + ratio that was intermediate between forage kochia and

36 22 alfalfa and above that expected for a non-halophyte (< 0.6; (Greenway and Munns, 1980)) at salinity levels ranging from 150 to 300 mm. This study evaluated the comparative salt tolerance of several putative halophytic plant species. In conclusion, this study confirmed that halogeton is a halophytic species and thus has an adaptive advantage on the salt-desert shrublands of North America. The salt tolerance of the Atriplex genus (saltbushes) has been widely examined and our study indicates that Gardner s saltbush is yet another Atriplex species with halophytic properties. We have documented that Gardner s saltbush is equally salt tolerant as halogeton, suggesting that other growth and competitive factors are responsible for the displacement of Gardner s saltbush in ecosystems invaded by halogeton. Furthermore, we have confirmed that Bassia prostrata (forage kochia) is a halophytic species that can survive at salinity levels equal to seawater, but does not have as great of salt tolerance as Gardner s saltbush and halogeton. Inasmuch as researchers have reported the potential for forage kochia to rehabilitate halogeton-invaded Gardner s saltbush ecosystems, this further indicates other traits, such as drought tolerance, are important for plant survival and competition on saline rangelands. Additional hydroponic studies examining salinity levels below 150 mm, and possible using older plants and a broader range of genotypes could further elucidate salinity tolerance of forage kochia.

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41 TABLES Table 1 Parameter estimates of shoot dry mass in response to increasing salinity levels in a hydroponic study. Models used were Sigmoidal Logistic 3 Parameter (SL3), Sigmoidal Sigmoid 3 Parameter (SS3), or Peak Lorentizan 3 Parameter (PL3). Standard error stated in parenthesis. Entry Model a b x 0 Rsqr Alfalfa SS (4.38) (26.36) (21.90) 0.78 Gardner s saltbush SL (1.93) 4.27 (3.11) (103.90) 0.34 Halogeton PL (3.29) (59.83) (35.82) 0.37 Halogeton 2 SL (3.16) 3.53 (2.17) (94.76) 0.33 Immigrant SL (2.77) 2.29 (0.40) (16.21) 0.85 Snowstorm SL (2.67) 2.18 (0.58) (18.67) 0.79 Tall Wheatgrass SL (2.49) 1.54 (1.22) (58.98) x 0 is the salt level that growth is reduced by 50% (GR 50 ) for the logistic model, whereas it is the salt level with highest shoot growth in the Lorentizan peak model. 2 Because halogeton had increased growth at low salt levels, the Lorentizan peak model is a better fit for the data, but we also forced the logistic model in order to obtain the GR 50 value. 27

42 Table 2 Parameter estimates of shoot dry mass as a percent of the control in response to increasing salinity levels in a hydroponic study. Models used were Sigmoidal Logistic 3 Parameter (SL3), Sigmoidal Sigmoid 3 Parameter (SS3), or Peak Lorentizan 3 Parameter (PL3). Standard error stated in parenthesis. Entry Model a b 1 x 0 Rsqr Alfalfa SS (6.13) (18.53) (19.19) 0.91 Gardner s saltbush SL (9.41) 4.44 (2.70) (79.77) 0.39 Halogeton PL (12.05) (39.38) (22.88) 0.52 Halogeton 2 SL (11.57) 4.10 (2.18) (75.16) 0.34 Immigrant SL (2.89) 2.34 (0.23) (9.58) 0.94 Snowstorm SL (4.90) 2.17 (0.48) (15.52) 0.84 Tall Wheatgrass SL (4.08) 2.04 (0.60) (19.44) x 0 is the salt level that growth is reduced by 50% (GR 50 ) for the logistic model, whereas it is the salt level with highest shoot growth in the Lorentizan peak model. 2 Because halogeton had increased growth at low salt levels, the Lorentizan peak model is a better fit for the data, but we also forced the logistic model in order to obtain the GR 50 value. 28

43 Table 3 Parameter estimates of shoot elongation rate in response to increasing salinity levels in a hydroponic study. Model used was Sigmoidal Logistic 3 Parameter (SL3). Standard error stated in parenthesis. Entry Model a b 1 x 0 Rsqr Alfalfa SL (0.69) 8.78 (6.78) (6.81) 0.79 Gardner s saltbush SL (0.23) 1.61 (0.64) (85.38) 0.46 Halogeton SL (0.38) 1.33 (0.61) (144.68) 0.32 Immigrant SL (0.23) 3.00 (0.95) (53.16) 0.55 Snowstorm SL (0.50) 1.90 (0.78) (78.38) 0.37 Tall Wheatgrass SL (0.42) 0.32 (0.96) 1.52 (23.94) x 0 is the salt level that shoot elongation rate is reduced by 50% (GR 50 ) for the logistic model. 29

44 Table 4 Parameter estimates of shoot length at harvest in response to increasing salinity levels in a hydroponic study. Models used were Sigmoidal Logistic 3 Parameter (SL3). Standard error stated in parenthesis. Entry Model a b 1 x 0 Rsqr Alfalfa SL (4.77) 2.17 (0.50) (18.71) 0.83 Gardner s saltbush SL (2.18) 1.11 (0.36) (146.63) 0.53 Halogeton SL (2.00) 2.31 (0.74) (66.61) 0.54 Immigrant SL (1.61) 2.13 (0.42) (48.51) 0.72 Snowstorm SL (2.71) 2.02 (0.45) (52.01) 0.60 Tall Wheatgrass SL (3.46) 0.67 (0.27) (91.99) x 0 is the salt level that shoot length is reduced by 50% (GR 50 ) for the logistic model. 30

45 Table 5 Parameter estimates of the percent root mass in response to increasing salinity levels in a hydroponic study. Model used was polynomial, linear. Standard error stated in parenthesis. Entry a y0 Rsqr Alfalfa 0.07 (0.02) (3.20) 0.40 Gardner s saltbush 0.02 (0.02) (2.59) 0.10 Halogeton 0.01 (0.02) (1.71) 0.06 Immigrant 0.01 (0.02) 7.29 (2.03) 0.18 Snowstorm 0.00 (0.02) (4.39) 0.01 Tall Wheatgrass 0.02 (0.02) (2.52)

46 32 FIGURES 60 Shoot Dry Mass (g) Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm NaCl (mm) Fig 1. Shoot dry mass(x ± SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table 1.

47 33 Shoot Dry Mass (% control) Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm NaCl (mm) Fig. 2. Shoot dry massas a p ercent ofthecontrol (x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table 2.

48 34 10 Elongation Rate (cm/day) Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm mm NaCl Fig. 3. Shoot elongat ionrate ( x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table 3.

49 35 Shoot Length (cm) Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm NaCl (mm) Fig. 4. Shoot lengt h(x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table 4.

50 Percent root mass (%) NaCl (mm) Alfalfa Gardner Saltbush Halogeton Immigrant Snowstorm Tall Wheatgrass Fig 5. Percent rotmas ( root/ro t+shot; x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in table 5.

51 Sodium (%) Alfalfa Gardner Saltbush Halogeton Immigrant Snowstorm Tall Wheatgrass NaCl (mm) Fig. 6. Percent Na + (x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix Table A1.

52 Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm K+ (%) NaCl (mm) Fig. 7. Percent K + (x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix Table A1.

53 39 12 Sodium to Potassium Ratio Na/K Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm NaCl (mm) Fig. 8. Na + /K + rat io (x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix Table A3.

54 Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm Ca2+ (%) NaCl (mm) Fig. 9. Percent Ca 2+ (me an x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix Table A4.

55 Sodium to Calcium Ratio Na/Ca Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm NaCl (mm) Fig. 10. Na + /Ca 2+ rat io (x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix Table A5.

56 Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm Mg2+ (%) NaCl (mm) Fig. 2. Percent Mg 2+ (x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix Table A6.

57 Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm P (%) NaCl (mm) Fig. 3. Percent P ( x ±SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix Table A7.

58 Ash (%) Gardner Saltbush Halogeton Alfalfa Tall Wheat Grass Immigrant Snowstorm NaCl (mm) Fig. 4. Percent ash (x ± SE ; n = 6 for 150, 300, and 600 mm, and 3 for 200, 400, and 800 mm) of plants grown in hydroponics with increasing amounts of NaCl. Best fit dose response lines were drawn using parameter estimates shown in Appendix Table A8.

59 45 PICTURES A B Picture 1. Alfalfa (Medicago sativa ssp. Falcata) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm.

60 46 A B Picture 2. Tall wheatgrass (Thinopyrum ponticum) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm.

61 47 A B Picture 3. Immigrant forage kochia (Bassia prostrata ssp virescens; cv Immigrant ) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm.

62 48 A B Picture 4. Snowstorm forage Kochia (Bassia prostrata spp grisea; cv Snowstorm ) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm.

63 49 A B Picture 5. Gardner s saltbush (Atriplex Gardneri) harvested after 28 days of growth in hydroponics with increasing amounts of NaCl. (A) Shoot length. (B) Root length. Horizontal lines are spaced at 10 cm.