ECOPHYSIOLOGY OF SALINITY

TOLERANCE IN THREE HALOPHYTIC

TURFGRASSES

Ghazi Abu Rumman

2011

This thesis is presented for the degree of

Doctor of Philosophy at

The University of Western Australia

School of Plant Biology

2011

I

Summary Growth and physiological mechanisms of salt tolerance in three halophytic turfgrasses

(Distichlis spicata, Sporobolus virginicus and Paspalum vaginatum) and a non-halophyte

(Pennisetum clandestinum) were studied. Field experiments were conducted at a site in

Western Australia with plots irrigated either with saline groundwater (13.5 dS m-1

) or potable

water, to assess changes in soil salinity and responses of the turfgrass species. Glasshouse

experiments further characterised physiological responses to high levels of salinity.

Key questions addressed by this study regarding the use of saline irrigation water were:

(i) Will build up of salts in the soil have adverse effects on growth and quality of turfgrass,

and what irrigation volumes are required to best manage salt accumulation? (ii) Will

halophytic grasses prevent large increases in Na+ and Cl

- concentrations in leaf tissues and

thus retain high leaf colour, as compared to the non-halophyte, as a major criterion for

salinity tolerance? (iii) Will turfgrass water use by the halophytes be maintained under saline

irrigation, whereas declines in water use are expected for the non-halophyte if suffering from

salinity stress? (iv) After salts are leached out of the root-zone by autumn/winter rains, how

well can the halophytic and non-halophytic turfgrasses recover?

In a field experiment, saline water ECw of 13.5 dS m-1

and potable water were used to irrigate

replicated plots of the four species. Changes in soil salinity were evaluated; ECsoil solution was

~6.5 dS m-1

prior to saline irrigation and increased gradually to ~ 40 dS m-1

by mid-summer,

and growth of the non-halophyte was severely reduced. By contrast, growth of two of the

halophytes was not impeded (S. virginicus and D. spicata). Colour remained unchanged for

the three halophytes, but it declined in P. clandestinum. In addition, the species differed in

vigour; P. vaginatum was the most vigorous of the studied species.

Toxic ions (Na+ and Cl

-) increased in concentration in the leaf tissues of the four species

when irrigated with saline water, however, mechanisms of ion exclusion (or excretion)

enabled the halophytes to maintain lower Na+ and Cl

−, and retain higher K

+:Na

+ ratio than the

non-halophyte. S. virginicus and D. spicata contained ~50% less Na+ and Cl

- than P.

clandestinum.

II

Two weeks after ceasing irrigation, the plots had received 38 mm of rain and the minimum

and maximum air temperatures had declined by 3 and 4 oC, respectively. Over this time, the

ECsoil solution decreased by 37% in the topsoil (0-25 cm), and despite the cooler temperatures

growth (clipping DM) doubled for the non-halophyte P. clandestinum compared with its

previous growth during the last two weeks when irrigated with saline water. By contrast, the

clipping DM of the three halophytes after ceasing the irrigation treatments were 82-91% of

those during the final two weeks of saline irrigation. Thus, the non-halophyte showed

recovery whereas the halophytes continued their previous good performance.

A second field experiment evaluated P. vaginatum when irrigated with saline water at four

volumes (40, 60, 80 and 100% replacement of net evaporation). Soil salinity increased as

irrigation volume was decreased; 100% replacement of net evaporation resulted in 30-60%

lower soil salinity compared to the lowest irrigation volume (40% replacement). Turf colour

did not differ across these treatments, but clippings DM was generally reduced when

irrigation volume was decreased. In an outdoors lysimeter experiment, irrigation with saline

water (13.5 dS m-1

) reduced the water use of the non-halophyte greatly (31%), whilst for the

three halophytes the reductions were more modest (7 – 21% of those in the non-saline

irrigation).

In a glasshouse experiment, growth was tested at higher levels of salinity (0, 125, 250, 500

and 750 mM NaCl) and both the aboveground (leaves and stolons) and the belowground

(roots and rhizomes) tissues were evaluated. The declines in growth with saline irrigation

were associated with high Na+ and Cl

- concentrations in the aboveground tissues reaching

inhibitory levels. However, for the belowground tissues, D. spicata increased by 14% at the

treatment of 125 mM NaCl and S. virginicus increased by 60% at 230 mM NaCl. D. spicata

and S. virginicus controlled the Na+ and Cl

- accumulation in leaves better than the higher Na

+

and Cl- in the third halophytic species P. vaginatum. The high toxic ion concentration in P.

vaginatum occurred despite a higher tissue water content. Halophytes maintained adequate

tissue K+ (~0.4 mmol g

-1 DM) in saline treatments, whereas K

+ declined in the tissues of the

non-halophyte.

In conclusion, the ranking for salt-tolerance was: D. spicata, S. virginicus, P. vaginatum and

then P. clandestinum. The three halophytes performed well when irrigated with saline

groundwater (13.5 dS m-1

) at a field site with a hot, dry summer environment. Salt tolerance

III

was also demonstrated under controlled conditions at higher levels of salinity. The species

also differed in vigour. Irrigation volume dose-response trials in the field established that P.

vaginatum can maintain high colour quality even though salt accumulation in the soil can be

considerable, but growth had declined. D. spicata and S. virginicus are also expected to

maintain high growth and colour quality under high irrigation volumes (80 and 100%

replacement of net evaporation) based on the results from the field experiment when irrigated

at 60% replacement of net evaporation and also considering their superior tolerance to

salinity in the controlled environment experiment.

IV

Table of Contents

SUMMARY I

TABLE OF CONTENTS IV

ACKNOWLEDGEMENTS VII

DEDICATION IX

STATEMENT OF CANDIDATE CONTRIBUTION X

PUBLICATIONS AND PRESENTATIONS ARISING FROM THIS THESIS XI

ABBREVIATIONS XII

CHAPTER 1- INTRODUCTION 1

CHAPTER 2 – LITERATURE REVIEW 5

2.1 INTRODUCTION 5

2.2 IRRIGATION WITH SALINE WATER 7

2.2.1 SOIL SALINITY AND SODICITY 13

2.3 HALOPHYTIC TURFGRASSES 16

2.3.1 COLOUR, A SELECTION CRITERIA FOR SALT TOLERANCE IN

TURFGRASSES 23

2.4 INTERACTION BETWEEN SALINITY AND ENVIRONMENTAL

FACTORS 26

2.4.1 EVAPORATIVE DEMAND AND TURFGRASS WATER USE 27

2.5 MECHANISMS OF SALT-TOLERANCE IN PLANTS 30

2.5.1 Na+ AND Cl

- EXCLUSION AT DIFFERENT LEVELS IN THE

PLANT BODY 31

2.5.2 ION SECRETION VIA SALT GLANDS 34

2.5.3 OSMOTIC ADJUSTMENT 35

2.5.3.1 GLYCINEBETAINE 37

2.5.3.2 PROLINE 38

2.5.3.3 SUGARS 39

2.6 CONCLUSIONS AND HYPOTHESES FOR THESIS 40

V

CHAPTER 3 43

IRRIGATION OF THREE HALOPHYTIC TURFGRASSES WITH SALINE WATER:

GROWTH, TURF QUALITY AND PHYSIOLOGY OF SALT-TOLERANCE IN FIELD

PLOTS

3.1 ABSTRACT 43

3.2 INTRODUCTION 44

3.3 MATERIALS AND METHODS 46

3.4 RESULTS 51

3.5 DISCUSSION 68

3.6 CONCLUSIONS 73

3.7 APPENDICES 74

CHAPTER 4 77

RESPONSES OF TISSUE IONS AND GROWTH OF FOUR TURFGRASS SPECIES TO

INCREASINGLY SALINE CONDITIONS

4.1 ABSTRACT 77

4.2 INTRODUCTION 78

4.3 MATERIALS AND METHODS 80

4.4 RESULTS 83

4.5 DISCUSSION 95

4.6 CONCLUSIONS 99

4.7 APPENDICES 100

CHAPTER 5 105

VOLUME OF SALINE IRRIGATION CONTROLS SALINITY OF THE SOIL

SOLUTION, GROWTH AND QUALITY OF THE HALOPHYTIC TURFGRASS

PASPALUM VAGINATUM

5.1 ABSTRACT 105

5.2 INTRODUCTION 106

5.3 MATERIALS AND METHODS 108

5.4 RESULTS 112

5.5 DISCUSSION 126

5.6 CONCLUSIONS 131

5.7 APPENDICES 133

VI

CHAPTER 6 137

SALINITY TOLERANCE OF DISTICHLIS SPICATA: ION CONCENTRATIONS WITHIN

SEGMENTS OF RHIZOMES, AND IN ROOTS AND LEAVES

6.1 ABSTRACT 137

6.2 INTRODUCTION 138

6.3 MATERIALS AND METHODS 140

6.4 RESULTS 144

6.5 DISCUSSION 152

6.6 CONCLUSION 156

6.7 APPENDICES 158

CHAPTER 7 163

CONCLUDING COMMENTS

7.1 OVERVIEW 163

7.2 TESTED HYPOTHESES 163

7.3 REFLECTIONS ON EXPERIMENTAL OUTCOMES 166

7.4 CONCLUSIONS 176

LITERATURE CITED 177

VII

Acknowledgements After thanking The Lord who assisted me with my thesis which would not have been possible

without the support and guidance of Prof. Timothy D. Colmer, thank you Tim for your

vigilance in keeping the project focused. Lessons learned went far beyond physiology of salt

tolerance. Thank you A/Prof. Edward G. Barrett-Lennard for co-supervising my project.

There are a number of people that provided help and encouragement throughout the duration

of this project. Firstly I would like to thank Kirsten, Andres, Melissa, Shinpie and Francis

Lopez for their assistance in the field work. Most importantly I would like to thank The Shire

of Wagin in Western Australia for allocating the field site and assisting in the operational

budget of the project. The significant contribution from Allan and his colleagues at Wagin

made it possible to set up the field site and maintain it over the two years. I also thank The

Landcare Programme in Wagin.

This project was funded by The Australian Research Council in collaboration with The Shire

of Wagin, appreciation also extends to the Department of Agriculture and Food WA for the

in-kind contribution in the project. Also, I thank The Centre of Excellence for Ecohydrology

and Faculty of Natural & Agricultural Sciences for the top-up scholarship, and also Grassland

Society of Southern Australia for a travel award.

To the ones very special to me, who opened their hearts before houses to accommodate my

son every fortnight while I was on field trips or out of country, I am grateful to Bandar,

Sultan and Ahmed Wagdy. To all I owe a debt of gratitude, I know of no other way of

repayment than of extending the kindness appreciation to all of you.

I would also like to thank Adrian Pitsikas for his help with commissioning the automated

irrigation system, and Total Eden Irrigation for the in-kind support in system design.

To the School of Plant Biology, The University of Western Australia, grateful

acknowledgement is made for the assistance given during the period of this study. Thanks

extend to my peers for supporting me as the postgraduate representative over two years.

I must give a heartfelt thanks to my parents, my sisters and brothers, for their love and

support throughout my life adventures. Without their help none of this thesis would have

VIII

been possible. I also thank other members of my family for encouraging me throughout my

studies.

And lastly, I am forever thankful for having such a loving and supporting son, to Ahmed,

who has sacrificed so much during my study and whilst I had been away from him several

times.

IX

Dedication

To the loving memory of my late wife “Amal” and late

son “Abdul Razzaq”

X

Statement of candidate contribution I declare that the thesis hereby submitted for the Philosophy of Doctorate (PhD) degree at

The University of Western Australia is my own composition and the results of my own

research and has not been previously submitted by me at another University for any degree.

To the best of my knowledge, all sources are acknowledged. Contributions made by other

individuals have been duly acknowledged.

Ghazi Abu Rumman

2011

XI

Publications Arising From This Thesis

International scientific conference papers

Abu Rumman, G, Barrett-Lennard, E and Colmer, TD. 2011. Enhanced quality of

turfgrasses by irrigating with saline groundwater in a Mediterranean environment.

Proceedings of peer-reviewed ICSC 2010. Abu Dhabi – UAE (in press).

Abu Rumman, G, Barrett-Lennard, E and Colmer, TD. 2008. Understanding salt and

water dynamics to enhance the quality of turfgrasses irrigated with saline ground water in

a Mediterranean environment. An evaluation of four species. 5th

International Crop

Congress, 13-18 April, 2008, Jeju, Korea.

Abu Rumman, G, Barrett-Lennard, E and Colmer, TD. 2008. Understanding salt and

water dynamics to enhance the quality of turfgrasses irrigated with saline ground water in

a Mediterranean environment. 2nd

International Salinity Forum, 30 March – 3 April, 2008,

Adelaide, Australia.

Abu Rumman, G, Barrett-Lennard, E and Colmer, TD. 2007. Understanding salt and

water dynamics to enhance the quality of turfgrasses irrigated with saline ground water in

a Mediterranean environment. ASA-CSSA-SSSA International Annual Meetings, 4-8

November, 2007, New Orleans, USA.

Industry publications and seminars

Abu Rumman, G, Barrett-Lennard, E and Colmer, TD. 2009. Halophytic turfgrasses and

their potential use on salt-affected areas. Australian Turfgrass Management Journal, 11.1,

pp. 46-47.

Abu Rumman, G, Barrett-Lennard, E and Colmer, TD. 2007. An evaluation of four

turfgrass species when irrigated with saline groundwater. The Fourth UWA Turf

Research Seminar Day, 28 June 2007, UWA.

XII

Abbreviations

FAO: Food and Agriculture Organization

SCARM: The Standing Committee on Agriculture and Resource Management

DU: Distribution uniformity

EU: Emission uniformity

USAIA: United States of America Irrigation Association

MDBC: Murray Darling Basin Commission

FC: Field capacity

PWP: Permanent wilting point

DM: Dry mass

PAR: Photosynthetically active radiation

Kc: Crop coefficient

ECe: Electrical conductivity of a saturated soil extract

EC1:5: Electrical conductivity of 1:5 soil:water extract (one part by weight (g) air dried soil to

five parts by volume (ml) distilled water)

ECw: Electrical conductivity of water

CEC: Cation exchange capacity

ET: Evapotranspiration

ETo: Potential evapotranspiration

OP: Osmotic potential

GB: Glycinebetaine

1

Chapter 1 - Introduction

Demand for scarce fresh water resources is increasing, and in some locations climate change

will decrease rainfall (Barnett et al., 2005), so use of alternative water sources is increasing in

priority for turfgrass management. Quality turfgrass surfaces require supplementary irrigation

during hot and dry summer months in many locations. Lower availability of high-quality

water and the need to use other sources of water (e.g. treated wastewater, brackish water

sources, etc.) requires consideration of the species that are able to perform under these

challenging conditions (Helmuth et al., 2005; Harley et al., 2006; Duncan et al., 2009). In

addition to shortages of fresh water resources, soil and water salinity problems are increasing

in many regions, so the turfgrass industry also needs to manage turfgrasses on saline sites in

addition to further development of saline water resources in irrigation (Brede, 2000).

Demand on fresh water resources will continue to increase, so water for turfgrass irrigation

will probably be assigned a lower priority than some other uses, such as use in households for

drinking, washing, bathing, etc. In recent years in most areas of Australia, watering lawns has

faced restrictions in order to conserve water, and this trend is likely to continue. Use of fresh

water resources to irrigate out-door landscapes in Western Australian homes is estimated to

account for 60 % of the water used by residential customers (cf. Pathan et al., 2007), so the

state water supplier has identified use on gardens as one area for water conservation.

Identifying and developing turfgrass species with low water requirements to face the

shortages in fresh water resources might help, but turfgrass quality will be downgraded if

sufficient water is not applied (Short, 2002; Bushman et al., 2007). These studies showed a

decline in turfgrass colour after reducing irrigation water to less than 60% replacement of pan

evaporation for a range of warm-season species. In addition to quality, survival is also

important. The ability of turfgrasses to recover from drought is related to species, cultivars

and management practices (Fu and Dernoeden, 2009).

In regional Australia, secondary salinity has developed across large areas as a consequence of

clearing the land of native vegetation (removal of perennial shrubs and trees with deep roots)

and planting of shallow-rooting annual crop and pastures - this has caused rising water tables

2

that bring salts up to the soil surface (George et al., 1997). The reduction in

evapotranspiration as a result of clearing the native perennial vegetation has resulted in

excess amounts of water contributing to groundwater recharge and runoff. Discharge of

saline groundwater makes low-lying areas unproductive for conventional plant species. Thus,

saline farming systems aim to introduce perennial halophytes to use saline groundwater, and

to lower water tables, as well as to provide forage for livestock (Barrett-Lennard, 2003).

An alternative might be to pump this shallow saline groundwater to irrigate shallow-rooted

grasses and crops that are salt tolerant, as well as possible use on turfgrass areas in some

situations. The distribution of salt-affected soils in landscapes cleared for agriculture follows

the climatic gradients in Australia, mainly where the average annual rainfall lies within the

250-600 mm range (Northcote and Skene, 1972). Turfgrass areas in these rainfall zones

require irrigation over summer months in the Mediterranean-type environment. Groundwater

is also often saline in salt-affected areas (Williams et al., 1997), so its use for irrigation will

add to the level of salts in the soil that plants must be able to tolerate.

The scale of the problem of saline soils in the world is large Szabolcs (1989) estimated the

total saline areas worldwide at 955 million ha as a result of both primary and secondary

salinisation while FAO (2005) reported 397 million ha of saline soils and 434 million ha of

sodic soils globally. Ghassemi et al. (1995) estimated approximately 77 million ha of

agricultural land is affected by salinity, of which approximately 45 million ha are in irrigated

areas and 30 million ha are in dryland farming areas. Estimates of the extent of salinisation in

Australia suggest about 5.7 million ha and the area will expand to 17 million ha by 2050

(Dolling, 2001).

The aim of the research described in this thesis was to generate knowledge on the

comparative physiology of salt tolerance in four turfgrass species; Distichlis spicata,

Sporobolus virginicus and Paspalum vaginatum (three halophytic species) and a non-

halophytic turfgrass which has been used widely in Western Australia, Pennisetum

clandestinum. Trials were conducted in a field situation within a rural town in south-western

Australia where plots were irrigated with saline groundwater or with potable water. In

addition, glasshouse experiments were also conducted to assess salinity responses of the

grasses. Using saline water, rather than potable water, to irrigate turfgrass areas will help

3

conserve precious high-quality water resources within town sites in the Western Australia

wheat belt.

4

5

Chapter 2 – Literature Review

2.1 Introduction

Water is essential for maintaining healthy ecosystems. As the human population increases,

pressure increases on the allocation of groundwater and surface water for the domestic,

agriculture and industrial sectors. Water use has been growing at more than twice the rate of

population increase in the last century. By 2025, 1,800 million people will be living in

countries or regions with water scarcity (UN-Water, 2006), and two-thirds of the world

population could be under water limited conditions. The situation will be exacerbated as

rapidly growing urban areas place heavy pressure on neighbouring water resources. Water

scarcity can impact adversely on human health and on economic growth.

Worldwide, the withdrawals of groundwater for irrigation purposes doubled between 1950-

1995 (FAO, 2003), according to Scott et al. (2004) the use of wastewaters is certain to

increase, in terms of volumes and areas irrigated to meet the increase in population and

industrial activities. Alternative sources of water supply can be drawn on, provided this is

done so sustainably. For example, the use of saline groundwater resources, particularly for

the irrigation of salt tolerant pastures/crops and turfgrass areas could be sustainable, provided

that guidelines and standards are adhered to (Brede, 2000; Helmuth et al., 2005; Harley et al.,

2006).

Turfgrass is an important component of the urban environment, providing aesthetic and land

use benefits (e.g. sports ovals) as well as having a role as one environmental solution for ever

increasing urbanisation (Morgan, 1998). The advantages of turfgrass as a component of our

urban environments have been classified into three main categories: (i) individuals, (ii)

environment, and (iii) workplaces and societies (Beard, 1994). Twenty benefits of turfgrasses

have been listed in Beard (1994). Other advantages of turfgrass have also been reported.

Turfgrass areas could be used as sites for disposal of effluent waters (Taylor et al., 2005).

During heavy rainfall, turfgrasses characterised by dense shoots and leaves that cover the soil

surface will prolong the opportunity for water infiltration and reduce surface runoff (Gross,

1990; King, 2000). Grasses can also provide a buffer to intercept transported nutrients in

6

drainage waters or on soil particles by overland flow. Grasses are one of the best traps to

intercept mobile nutrients. Dense ground cover slows and makes uniform overland flow and

increased infiltration under grass compared with under riparian forest (Hairsine, 1996;

Hairsine and Prosser, 1997). Ground covers are important as they influence runoff and help

promote settling of suspended sediments from floodwaters (McIvor et al., 1995). Like other

perennials, turfgrasses can play a role in stopping soil erosion (Hairsine and Prosser, 1997;

Romani et al., 2002). Turfgrasses reduce wind erosion through stabilizing the soil in newly

constructed sites; this has also some safety advantages through preventing accidents

originated from sand particles in industrial plants and airports which create some engine

faults (Joo, 2001). Turfgrasses can also act as a firebreak in retarding the spread of wildfires

(Beard and Green, 1994).

Only a few species within the 7500 in the Poaceae family are adapted to frequent mowing

and traffic, these being characteristics essential for a successful turfgrass. Thus the number of

species selected as potential turfgrasses are relatively few. Key C4 species include:

Zoysiagrasses (Zoysia spp.), Bermudagrass (Cynodon dactylon), Seashore paspalum

(Paspalum vaginatum), Kikuyu (Pennestium clandestinum), Buffalograss (Buchloe

dactyloides), St. Augustinegrass (Stenotaphrum secundatum), Carpetgrass (Axonopus affinis)

and Centipedegrass (Eremochloa ophiuroides) (Turgeon, 1980). These grasses are

characterised with aggressive rhizomes and stolons which made them adaptable to low

mowing regimes and also tolerant to wear (Carrow et al., 2005; Bowman and Turner, 1993).

In addition to the long-standing turfgrass species listed in the preceding paragraph, interest is

increasing is some ‘niche’ species. In some situations, use of native species as turfgrasses

could aid harmonious fits into specific ecological structures that evolved over a long period

of time for particular locations (Hobbs and Huenneke, 1992) and these might also have

economical advantages if native species require less time, money, and energy to maintain

(Klink, 1996). Also of increasing interest are turfgrass species that can tolerate high levels of

salts in irrigation waters and soils (Qadir and Oster, 2004). Some turfgrass species are

halophytes (e.g. Seashore paspalum) and additional turfgrass-quality halophytic species that

can grow well with high salinity need to be identified. Halophytic turfgrasses, however, will

need to be managed and maintained in a modified way to practices used for traditional

species and cultivars in respect to fertilizer application and irrigation scheduling (Carrow et

al., 2001).

7

The scope of this review is to addresses the significance of use of halophytic turfgrass species

in soils or sites with saline irrigation water. The review addresses how halophytes employ

different physiological mechanisms to adjust to the external salinity stress. Current

management practices for turfgrass on saline sites will be summarised and consideration will

be given on how to evaluate and manage the performance and physiology of salt tolerant

turfgrasses, as well as soil water and salt dynamics, under low- and high-salinity irrigation.

Such information is needed to develop best practices for management of turfgrasses on saline

sites and when using saline irrigation water.

2.2 Irrigation with saline water

This section will address the use of saline water in irrigation and its impact on soils. A few

scenarios and successful management practices of saline water will be discussed. The

scenarios include: species selection, impact of saline irrigation on soil, impact of saline

groundwater on agricultural lands, design of irrigation systems, irrigation management,

leaching fraction and the necessity of frequent monitoring of soil salinity.

Irrigation with saline water requires the use of salt-tolerant plants. Nevertheless, use of saline

water for irrigation poses a threat to soil chemical and physical properties, such as increasing

the salinity, sodicity and soil structural stability (Balks et al., 1998), if not associated with

proper management practices (Ayers and Westcot, 1985; Ayars et al., 1994). Management

will also depend on the specific soils and water chemistry. As one example, a study of saline

drainage water from fields of irrigated wheat in California of EC up to 18.8 dS m-1

showed

that subsurface drainage in combination with an appropriate periodic leaching fraction

eliminated the accumulation of toxic elements (Na+, Cl

-, Se and B) in the soil profile (Sharma

and Rao, 1998), but disposal of the drainage water remains unsolved. Lagoons are potential

destinations for high saline drainage water (Sorour et al., 2003). Reducing the drainage water

through applying less leaching fractions will reduce irrigation costs and decrease drainage

water disposal, but plants must be able to tolerate salts that build up in the soil (Pitman and

Lauchli, 2002).

Saline groundwater threatens some agricultural lands, but extraction and use of this water in

irrigation could assist in lowering the water table, and also create an opportunity to replant

some agricultural lands that have been abandoned (Rogers et al., 2005). Controlling water

8

tables requires knowledge of both temporal changes in the levels at a given location as well

as the spatial variation across the area, based on new methods of monitoring and interpreting

the spatially distributed data (Western et al., 1998). Management practices include drainage

systems and dewatering of the particular location (George et al., 1996), normally, the quality

of the drain water drops over time (Neal and Robson, 2000). Pumping the saline groundwater

out to reduce waterlogging and salinisation of irrigated land decreases the negative

environmental impacts on soil and subsequently plants.

Proper irrigation design results in even distribution of water across the turfgrass surface.

Uniformity in irrigation prevents the formation of brown spots as a result of uneven irrigation

resulting in localised dry patches. Irrigation scheduling should be based on the plant water

use and the evaporative demand (Fereres and Soriano, 2007; Raes et al., 2009).

Understanding the rooting depth across the different turfgrass species is also useful for

establishing irrigation schedules, as it determines the upper amount of water that can be

applied to the soil before water leaches beyond the reach of the roots.

Design of irrigation systems for efficient and uniform distribution of water is a vital practice

for successful turfgrass surfaces (Carrow, 1995). The design of irrigation systems should

achieve a minimum operational distribution uniformity (DU) or emission uniformity (EU)

and the design should also account and mitigate the effects of wind by using low-trajectory

sprinkler nozzles along with the appropriate modified sprinkler head spacing. Designs also

require consideration of the soil type, slope, expected root depth, turfgrass species,

microclimates, weather conditions and water source. Uniform irrigation water application is

important to maintain uniform appearance of turfgrass (Baum et al., 2005).

DU is the ratio of the dry or under watered area to the average applied within the sprinkler

coverage area. The calculation requires ranking the catch can values from highest to lowest,

with the average of the lowest 25% divided by the overall average of the catch cans to

calculate the Low Quarter Distribution Uniformity (DULQ), which is a measure of irrigation

efficiency defined as the ratio of dry areas to wet areas (Zoldoske, 2003). DULQ indicates

how uniform the distribution of sprinkler water delivery is on the ground surface but it can

under estimate the uniformity of irrigation water distribution in the root zone (Li and Rao,

2000). The spatial variability in soil moisture is controlled mainly by irrigation delivery, but

other soil factors such as (organic matter, texture, hydraulic properties, non-wetting soils, and

9

management practices) also influence soil moisture content (Adriano, 1980; Pathan et al.,

2003). The American Irrigation Association (USAIA) states that 70% uniformity (DU) is

what should be expected on golf course irrigation systems that are properly installed and

functioning (USAIA, 2003).

The type of irrigation system influences the quantity of water required for leaching, drip

irrigation localised the accumulated salts within the root zone while sprinkler irrigation

system distributes salts across the irrigated area, the influence of rainfall contribute to the

leaching. For example, the volume of water would be required to reduce the salinity in a golf

course from (ECe 8 dS m-1

- ECe 2 dS m-1

) where the leaching water has an ECw (1.5 dS m-1

).

To leach (~ 44 cm) using an irrigation water with ECw (1.5 dS m-1

), the depth of irrigation

water should replenish the available water supply before reaching the permanent wilting point

and also count for the leaching requirements, it is clear that water quality has a similar

important influence on reclamation leaching and on maintenance of leaching fraction

(Carrow et al., 2005). The use of subsurface drip irrigation system to irrigate turfgrass with

effluent water resources proved that this technique is effective to reduce salt accumulation in

the soil surface beyond salinity threshold levels of the studied species (bermudagrass), so

long as short distances were kept between drippers (Devitt and Miller, 1988).

In case of salt affected lands, or when using saline irrigation water, an extra volume of

irrigation water has to be applied to leach the excess salts or toxic ions in the soil (Harivandi

et al., 2008). The water requirement for leaching can be reduced by intermittent applications

of ponded water, particularly for fine-textured soils. With sprinkler irrigation as it is the most

common irrigation system for turfgrasses, reclamation by leaching can occur under

continuously unsaturated conditions (Oster et al., 1999). In field experiments on a silty clay

soil, the leaching efficiency of intermittent ponding exceeded that of sprinkler applications

(Oster et al., 1972).

Leaching of accumulated salts is of environmental concern. One example is a study in soil

columns that showed 118 mm rain leached 27% of the accumulated salts in 3 months, using

rain simulator and undisturbed heavy soil with 55% clay (Tanton et al., 1995). Similarly, in a

study of permanent grassland soil in England, frequent soil sampling collected from the top

1.2 m, found that after an increase of 11% in ECe in the top soil following summer season,

salts were then leached during winter season with 630 mm rainfall and 540 mm annual

10

average potential evaporation (Armstrong et al., 1996). Thus, the fate of salts needs to be

considered and collected using drainage systems, as a management practice of saline

irrigation systems not only during the irrigation season, but also when leaching occurs

following rains.

Determining the precise leaching requirement and plant response under efficient irrigation

systems require a better understanding and quantification of salt movement within the soil

profile (Mmolawa and Or, 2000). The use of saline water requires close monitoring of the salt

levels in the soil and particularly within the root-zone. However, monitoring water and solute

movement and accumulation associated with turfgrass response is costly and time consuming.

Several methods can be used to assess soil salinity; such as the electrical conductivity (EC) of

a saturated soil extract (ECe) (Richards, 1954), or in some countries the EC of a 1:5 soil:water

extract (EC1:5) (Slavich and Petterson, 1990), or apparent EC can be estimated in situ from

electromagnetic induction survey techniques (e.g. EM38) (De Jong et al., 1979; Williams and

Baker, 1982). EC measurements cover the sum of all ions in the soil (Thomas and Langdale,

1980), not just Na+ and Cl

- which are often thought of as ‘salinity’, and EC values are also

influenced by temperature (2% for each increase of 1° C, Seyfried and Murdock, 2001).

The advantage of using the saturated paste extract (ECe) is that the soil water content of the

saturated extract is typically twice that of the soil at field capacity and four times more than at

permanent wilting point (PWP) (Richards, 1954), so it is easy to gain an indication of the

salinity that plant roots might experience by use of these simple conversion factors. ECe

values also enables use of databases of relationships between growth and ECe developed for

over 100 plant species (Maas and Hoffman, 1977; Maas, 1986; Maas and Grattan, 1999;

Steppuhn et al., 2005). ECe can, however, be time-consuming, so that some researchers use

EC1:5 and then convert those data to estimated ECe (Slavich and Petterson, 1990) conditional

to knowing the soil type. The conversion factor varies according to soil texture; heavy soils

with high clay content have high water holding capacity and saturation percentage of about 2-

fold more than that of light sandy soils, so the conversion factors are 8, 11 and 14 for clay,

loam and sand, respectively (Slavich and Petterson, 1993).

The spatial variability in both soil physical and chemical properties leads to a spatial salt

distribution in soils, so that large numbers of sampling points are needed to assess salinity

11

across paddocks. Thus, quick and reliable measuring devices have been developed to enable

survey of target areas (Rhoades et al., 1989), such as EM38 used to estimate the electrical

conductivity of the bulk soil profile. A strong correlation of the EM38 readings and the ECe

was found in various soils (Slavich, 1990). Given the ease of using the EM38 to measure soil

salinity, it is recommended for frequent monitoring subject to on-site calibrations.

The use of automated irrigation controllers and soil moisture monitoring systems can be a

useful tool as these can save up to 25% of water resources used on turfgrass areas (Snyder et

al., 1984). Automation of irrigation systems based on soil moisture sensors may improve

water use efficiency by maintaining soil moisture at optimum levels, rather than having

cycles of very wet to very dry as a result of larger manual irrigation events. The technique

allows control of the volume of irrigation water applied, according to soil water status

(Pathan et al., 2003; Jones, 2004). Significant advances have been made in the application of

electromagnetic methods to measure soil water content based on the use of high relative

permittivity (dielectric constant) of the water in soil (Jones, 1990; Barrett et al., 2003).

Closely monitoring and controlling the soil moisture content by adjusting the irrigation

system (sprinklers) is vital in turfgrass management to avoid brown spots in turfgrass (Jiang

and Carrow, 2007).

Adequate irrigation is critical to maintain healthy turfgrasses, decreasing irrigation frequency

resulted in reduction in water use by 6–18% for some warm-season turfgrasses (Fry and

Huang, 2004; Short and Colmer, 2007). Over irrigation can cause turfgrass to decline in

tolerance to some environmental stresses (i.e. heat and water stresses). For example, in

creeping bentgrass, excessive irrigation resulted in a shallow root system mainly within the

top 7.5 cm, whereas a 4-day interval in irrigation during the growing season improved the

shoot and root density and improved turfgrass quality when compared with daily irrigation

(Jordan et al., 2003).

Infrequent irrigation can increase root growth of turfgrass. As one example, roots of ‘Meyer’

zoysiagrass irrigated at first sign of leaf roll had the ability to extract moisture from deeper in

the soil profile (below 55-75 cm) than if watered daily to replace ET (extracted only to 27

cm) (Madison and Hagan, 1962; Qian et al., 1996). As the majority of turfgrass roots were

found in the upper 10 cm of soil (Liu and Huang, 2002), infrequent irrigation can be used as a

turfgrass management practice so as to enhance deeper rooting to access water at depth.

12

Deeper rooting would reduce the pressure on delivering water through the irrigation network

since turfgrass root systems can typically have 90% of the roots in the top 10 cm (Short and

Colmer, 1999a,b), which can dry down relatively quickly in hot environments.

Different irrigation strategies can be employed to reduce the negative impact on turfgrasses

when using saline water for irrigation, these practices include irrigation scheduling (Carrow

et al., 2005). Cyclic irrigation refers to applying water in several cycles rather than one long

irrigation application per zone, such scheduling can reduce leachate volumes. Cyclic

irrigation has different forms including alternating between irrigation volumes (Baldwin et

al., 2006) or use of water sources of different quality (Schaan et al., 2003). The later has

many advantages such as dilution of soil salinity to control the salt level in the root zone and

can be targeted to critical growth stages of plants as salinity tolerance varies between growth

stages (Grattan and Rhoades, 1990; Minhas and Gupta, 1992). Cyclic irrigation should be

associated with irrigation scheduling (irrigation time and volume) to meet plant water

requirements and control salt accumulation (Rhoades, 1972).

It is crucial to prevent the development of excessive soil salinisation within the root zone,

some irrigation management practices can be used to achieve this goal such as irrigation

scheduling (volume and interval); leaching scheduling (volume and timing) and irrigation

method (Shalhevet, 1984) and cyclic irrigation. Irrigation by sprinklers allows close control

of the volume and distribution of irrigation water. Leaching scheduling is the key factor by

which soil salinity can be maintained at acceptable levels without undue decline in the quality

of turfgrass areas therefore, installing drainage and disposal systems is essential. Blending or

diluting excessively saline water with good quality water supplies assists in maintaining soil

salinity within acceptable limits and also reduces the salinity of the drainage water. Uniform

irrigation systems, and approaches such as soil water monitoring, can aid in production of

high-quality turfgrass surfaces. The key to the effective use of saline irrigation waters and

salinity control is to monitor soil and plant status continuously and provide the proper amount

of water to the plant at the appropriate time. The ideal irrigation scheme should provide water

as needed to keep the soil water content in the root zone within optimum safe limits.

13

2.2.1 Soil salinity and sodicity

Soils are divided into three categories in regards to salt contents; saline soils, sodic soils and

saline-sodic soils. Saline soils are those having an ECe of 4 dS m-1

or above (Miller and

Donahue, 1990; Brady and Weil, 1999) and contains soluble salts of Na+, Ca

2+ and Mg

2+

(Szalbolcs, 1989), usually the pH will be less than 8.5, whereas, sodic soils have a pH more

than 8.5, and contain exchangeable Na+. Saline-sodic soils contain both the soluble salts and

exchangeable Na+ and the pH of this soil type is typically < 8.5 (Bresler et al., 1982; Carrow

and Duncan, 1998). Both salinity and sodicity are limiting factors in regards to the

sustainability of plant productivity on such soils, owing to plant and soil constraints

(Surapaneni and Olsson, 2002; Tillman and Surapaneni, 2002; Miller and Donahue, 1990;

Brady and Weil, 1999; Barrett-Lennard, 2002).

Soil sodicity is caused by high levels of exchangeable Na+ adsorbed on the surface of clay

particles, due to the negative charges attracting the positive charge of Na+. The Australian

definition of sodic soil is if Exchangeable Sodium Percentage (ESP) exceeds 6% (Isbell,

1996). The dominance of rainfed agriculture in Australia and the low total electrolyte levels

in the soil solution in Australia, led to consider the low percentage of ESP (6%) to classify a

particular soil as a sodic soil (ASPAC) whereas the American standards classify a soil with

(15% ESP) or more as a sodic soil (Richards, 1954).

Cation exchange capacity (CEC) is defined as the sum total of the exchangeable cations that a

soil can absorb (Brady and Weil, 2000), another definition of sodicity is: CEC = Mx+

excess

+Ax-

excess where Mx+

and Ax-

are the cations and anions in the system. CEC is a measure of

the quantity of sites on soil surfaces that can retain positively charged ions (cations) by

electrostatic forces. Cations retained electrostatically are easily exchangeable with other

cations in the soil solution and are thus readily available for plant uptake. However, the

magnitude of the anion deficit is small and is often ignored (Sumner and Miller, 1996).

Cation exchange capacity is a function of the amount and type of clay minerals and the

amount of organic matter present in the soil, expanding clay minerals tend to have a higher

CEC than non-expanding clay minerals. The amount of organic matter has a positive

influence on the CEC.

14

Increases in CEC can increase the soil’s capability to retain cationic nutrients, such as

NH4+, K

+, Ca

2+, and Mg

2+. Organic soils can have CEC values of 50 to 100 cmol kg

-1, while

values for sands can be as low as 3 to 5 cmol kg-1

(Tisdale et al., 1985). The CEC of golf

greens are usually quite low (0 – 6 cmol kg-1

) due to the textural properties of the

infrastructure and materials used in their construction (USGA Green Section staff, 1993).

Oxidative loss of organic matter causes reductions in CEC of soils during the warm, moist

conditions in some locations (Carrow et al., 2001).

A major problem in sodic soils is a loss of structure causing poor infiltration of water.

Minimising the effect of sodicity on poorly structured soils can be achieved by incorporation

of organic matter to the soil surface layers. Organic matter promotes development of larger

soil aggregates with higher porosity (Landschoot and McNitt, 2000). A potential of

increasing soil organic matter in turfgrass areas is by returning clippings after mowing

(Waddington et al., 1976). Organic matter binds with soil particles and increases soil porosity

(Dawson, 1993) as well as keeps higher level of soil K+ when compared to removing

clippings off turfgrass, since clippings contain 1 to 2.5% K+ and higher on a dry mass basis

(Nus, 1993).

Poor water infiltration in sodic or sodic-saline soils also reduces capacity to leach salts out of

the root zone. Drainage and percolation rates assist in leaching the salts out of the root zone,

and plant roots provide root channels to increase the flow of water through soil profile; this is

known as preferential flow. There is not enough data in the literature about whether turfgrass

roots and rhizomes aid to leach the salts out of the root zone. The below ground growth of

turfgrasses may create channels for water infiltration and increase the preferential flow as

some of the potential turfgrasses have strong rhizomes (Yensen, 2002a,b). This is quite

important in particular with heavy soil types. By contrast with the relatively little knowledge

of leaching of salts, deep root and rhizome depth was reported to enhance leaching of N

(Miltner, 1996; Barton et al., 2005) and pesticides leaching (McLeod et al., 2001; Starett et

al., 2000).

In the case of turfgrass culture, the range of required physical (bulk density and porosity) and

chemical properties of soils to achieve high quality turfgrass areas in silt loam/clay loam soils

are summarised in Table 2.1.

15

Table 2.1. Favourable soil physical and chemical properties to achieve high quality

turfgrasses in silt loam/clay loam soils.

Soil properties Minimum Maximum Reference

Physical properties

Bulk density (g cm-3

) 1.30 1.70 Waltz et al. (2000)

Porosity (%) 36 51 Waltz et al. (2000)

Aggregate Stability (MWD, mm) 1.0 6.4 Kemper and Rosenau (1984)

Chemical properties

Total Carbon (g C kg-1

) 10.0 50.0 Carrow et al. (2001)

Total Nitrogen (g C kg-1

) 0.5 4.5 Carrow et al. (2001)

Electrical Conductivity (ECw)

(mmho cm-1

)

0 4 Carrow (1995)

CEC (cmol kg-1

) 15 30 Carrow (2001 )

pH 4.5 7 Duncan and Carrow (1993);

Musser (1950)

Mg - Mg/CEC (%) 10 20 Skorulski (2001); Christians (1993)

K- K/CEC (%) 2 7 Skorulski (2001); Christians (1993)

Ca - Ca/CEC (%) 60 85 Skorulski (2001); Christians (1993)

Free CaCO3 0 2 Carrow (2001 )

Na - Na/CEC (%) 0 15 Carrow (1995); Mitra (2001)

In conclusion for section 2.2 (and 2.2.1), use of irrigation water containing relatively high

Na+ concentrations can be managed by blending or cyclic irrigation. Leaching by periodic

larger applications of irrigation water can flush salts further down the soil profile, but

requires both drainage and disposal systems for on-going use as a management practice.

Applying amendments such as gypsum can also be useful in terms of reducing the water

sodicity and the bicarbonate concentrations; gypsum is also a useful soil amendment for sodic

soils. Using cyclic irrigation could provide appropriate management to turfgrass culture in

16

saline-sodic soils.

2.3 Halophytic turfgrasses

Halophytes constitute about 1% of the world’s flora (Flowers and Colmer, 2008). Halophytes

are plants that can survive in saline conditions, such as marine estuaries and salt marshes.

Around 2500 halophytic species are reported worldwide and spread across some 500 genera

of flowering plants (Khan and Ansari, 2008). Halophytes are physiologically adapted to

withstand high salinity of the soil water. Some halophytes also possess structural

modifications of xerophytes; for example, Salicornia spp. lack true leaves but have

articulated, fused stem segments and accumulate salts intracellularly, and water storage is

thought to function as a mechanism to reduce salt concentrations within cells (Eggli and

Nyffeler, 2009). Halophytes are a group of plants that grow (survive and produce) in saline

conditions, and some even require salty soils (Flowers and Colmer, 2008).

A range of salt-tolerant plants can be considered to make profitable use of saline water and

soil resources rather than neglecting these resources; halophytic species that have proposed

uses are listed in Table 2.2. In addition, potential environmental gains can be made from

vegetating ‘barren saline land’, although if using saline irrigation water care must be taken to

avoid negative effects of salt on soils and any underlying aquifers. Growing halophytes in

soils affected by ‘dryland salinity’ assists in minimising groundwater recharge, which is an

effective tool to reduce or even prevent further rising of a saline water table (Bell et al., 1990;

Morris and Thompson, 1983).

17

Table 2.2. List of halophytes with potential for cultivation, and their uses (modified from

Khan and Qaiser, 2006 and updated with additional references).

Turfgrasses

Sporobolus virginicus and Distichlis spicata

Loch et al.,

2005; Depew

and Tillman,

2006

Distichlis stricta and Paspalum vaginatum Carrow and

Duncan, 1998

Ornamental

(ground covers,

flowers and

shrubs)

Aeluropus lagopoides, Cenchrus ciliaris,

Clerodendrum inerme, Dalbergia sissoo, Euphorbia

thymifolia, Ficus microcarpa, Knorringia sibirica,

Kochia indica, Limonium axillare,

Mesembryanthemum crystillinum, Nitraria retusa,

Phyla nodiflora, Polypogon monspeliensis,

Psylliostachys spicata, Raphanus raphanistrum,

Sessuvium sessuvioides, Tamarix mascatensis,

Trianthema portulacastrum

Khan and

Qaiser, 2006

Medicinal

halophytes

Artemesia scoparia, Achillea mellifolium, Ammi

visnaga, Artimesia scoparia, Caesalpinea bonduc,

Capparis decidua, Centella asiatica, Ceriops tagal,

Cocos nucifera, Corchorus depressus, Evolvulous

alsinoides, Juncus rigidus, Kochia indica,

Microcephala lamellate, Portulaca quadrifida, Phylla

nodiflora, Plantago lanceolata, Plantago major,

Rumex vesicarius, Salsola imbricate, Salvadora

persica, Seriphidium brevifolium, Solanum incanum,

Solanum surratense, Thespesia populnea, Tribulus

terrestris, Verbena officinalis, Withania sominifera,

Zaleya pentandra, Zygophyllum propinqum

Khan and

Qaiser, 2006

Lycium barbarum Wei et al.,

2006

Salicornia brachiata Stanley, 2008

Fuel wood

Acacia spp., Avicennia marina, Capparis spp., Kochia

spp., Prosopis spp., Salsola spp., Salvadora persica,

Suaeda spp., Tamarix spp.

Dagar, 1995

Aegiceras spp., Ceriops spp., Dalbergia sisso,

Pongamia pinnata, Populus euphratica, Rhizophora

spp., Tamarix aphylla

Khan and

Qaiser, 2006

Food and

derivatives

Apium graveolens, Arundo donax, Atriplex hortensis,

Ceriops tagal, Chenopodium album, Haloxylon

stocksii, Hibiscus tiliaceus, Oxystelma esculentum,

Portulaca oleracea, Rhizophora mucronata, Rumex

vesicarius, Salicornia bigellovi, Salvadora oleoides,

Sesuvium portulacastrum, Suaeda maritima, Thespesia

populnea, Trianthema portulacastrum, Triglochin

maritima and Zizyphus nummularia

Khan and

Qaiser, 2006

Suaeda salsa, Limonium bicolour Liu et al., 2006

18

Forage and

fodder species

Acacia spp., Aegiceras corniculata, Aeluropus

lagopoides, Alhagi pseudoalhagi, Atriplex spp.,

Avicennia marina, Ceriops tagal, Chloris gayana,

Cynodon dactylon, Dactyloctenium sindicum,

Echinochloa turnerana, Paspalum vaginatum, Prosopis

spp., Puccinellia distans, Rhizophora mucronata,

Salicornia spp., Salsola spp., Salvadora persica,

Sporobolus marginatus, Suaeda martima and Zizyphus

spp.

Khan and

Qaiser, 2006

Kochia indica Dagar, 1995

Leptochloa fusca Malik et al.,

1986

Atrilpex canescens Glenn and

Brown, 1998

Atrilpex undulata, Atriplex lentiformis, Atriplex

amnicola, Atriplex nummularia, Atriplex semibaccata

Malcolm and

Swaan, 1989;

Malcolm,

1994; Warren

and Casson,

1994; Barrett-

Lennard and

Malcolm, 1995

Distichlis spicata, Sporobolus virginicus, Paspalum

vaginatum Burvill, 1956

Halosarcia pergranulata Malcolm and

Cooper, 1988

Maireana brevifolia Malcolm, 1963

Prosopis tamarugo Squella, 1986

Puccinellia ciliate

Rogers and

Bailey, 1963;

Barrett-

Lennard and

Holt, 1999

Thinopyrum elongatum

Rogers and

Bailey, 1963;

Carland et al.,

1998

Sporobolus virginicus, Spartina patens, Leptochloa

fusca

Ashour et al.,

2007

Sporobolus virginicus Napper, 1965

Acacia ampliceps

Spartina spp., Distichlis palmeri, Paspalum vaginatum,

Juncus roemerianus, Salicornia bigelovii, Batis

maritima

Noaman and

El-Haddad,

2000

Oil seeds

Arthrocnemum macrostachyum, Halogeton glomeratus,

Kochia scoparia, Salicornia bigelovii, Suaeda fruticosa

Khan and

Qaiser, 2006

Haloxylon stocksii Weber et al.,

2001

19

Salicornia bigelovii Glenn et al.,

1991

Products of

economic and

common use

(biofuels and

oil)

Aristida adscenscoinis, Calotropis procera, Haloxylo

spp., Imperata cylindrica, Juncus maritimus, Kochia

scoparia, Phragmites australis, Salicornia spp.,

Salsola spp., Suaeda spp.

Khan and

Qaiser, 2006

Chemicals

Acacia farnesiana, Aegiceras corniculata, Avicennia

marina, Casuarina obesa, Ceriops tagal, Eucalyptus

calmadulensis, Parkinsonia aculeate, Pithecellobium

dulce, Prosopis juliflora, Rhizophora mucronata,

Tamarix articulate

Khan and

Qaiser, 2006

Turfgrasses demonstrate a substantial range of tolerance levels to salinity, from salt-sensitive

to extremely salt-tolerant (halophytic) (Duncan and Carrow, 1999). Table 2.3 presents the

classification of turfgrasses in regards to their salt tolerance. Salinity tolerance of halophytic

turfgrass species, as compared with tolerances of conventional, non-halophytic turfgrass

species, is listed in Table 2.3. The list shows that turfgrass species range from being very

sensitive to very tolerant, and although some non-halophytes (such as Cynodon spp.) possess

reasonable salt tolerance, the halophytic species are most salt-tolerant. Thus, selections of

turfgrass-type cultivars from within halophytic species has been successful in providing salt

tolerant turfgrass options.

Screening experiments were conducted to determine the salinity threshold of salt-tolerant

turfgrasses; however, it is important to recognize that cultivars within species also differ in

salt tolerance. For example, for Z. matrella the cultivar Diamond is the most tolerant;

Crowne, DeAnza, El Toro, Emerald, JaMur, Marquis, Palisades, Royal and Victoria have

intermediate salinity tolerance; and Belair, Cavalier, Meyer, Omni, Sunburst and Zeon are the

least tolerant to salinity (Marcum et al., 1998; Qian et al., 1998).

Several factors can, however, complicate assessments of salinity tolerance. One complication

is that the growth stage when plants are exposed to salinity can also influence the response; as

examples, seedlings of Sporobolus airoides and Puccinellia distans are not as tolerant as

when in their mature stage (Hughes et al., 1975). A further consideration when halophytic

turfgrasses are evaluated is that assessments at low to moderate salinity levels do not reflect

their performance and sustainability over long periods of time. Halophytic turfgrasses should

be evaluated at higher salinity levels and in salt-affected lands (e.g. equivalent to sea water

20

level, ECw 55 dS m−1

) (Duncan and Carrow, 1999) and evaluations should last relatively long

periods to cover all seasons.

Table 2.3. Salinity tolerances of turfgrasses. List of salt tolerant turfgrasses and their salinity

threshold (50% decline in growth) defined based on the increase in ECe (Carrow and Duncan,

1998; USDA database) classification. * represent halophytic species. Notes: S. virginicus data

were from (Marcum and Murdoch, 1992), but the type measured was not of turfgrass quality;

a turf quality accession has recently been selected (D. Loch, pers. comm.).

Salinity Tolerance ECe (dS m-1

) Turfgrass

Very Sensitive <1.5

Annual bluegrass (P. annua), Colonial bentgrass (A.

capillaris), Rough bluegrass (P. trivialis), Centipedegrass

(E. ophiuroides) and Carpetgrass common (A. fissifolius).

Moderately Sensitive 1.6 – 3.0 Kentucky bluegrass (P. pratensis), seeded Champion and

Diamond Zoysia materlla.

Moderately Tolerant 3.1 – 6.0

Fine-leaf fescues (F. rubra), Hard Fescue (F. trachyphylla),

Bahiagrass (P. notatum), Buffalograss (B. dactyloides),

Blue grama (B. gracilis), Annual ryegrass (L. multiflorum)

and Creeping bengrass (Agrostis stolonifera).

Tolerant 6.1 – 10.0

Seaside bentgrass (A. palustris), common bermudagrass (P.

pratensis), Tall fescue (F. arundinacea), Zoysia japonica

(some)*, Perennial ryegrass (A. millefolium), Kikuyu (P.

clandestinum), Western wheatgrass (E. smithii) and Crested

wheatgrass (A. cristatum).

Very Tolerant 10.1 – 20.0 St. Augustinegrass (S. secundatum), Manilagrass (Zoysia

matrella)* and Bermudagrass (Cynodon spp).

Superior Tolerance >20.0

Seashore paspalum (P. vaginatum) (some)*, Saltgrass (D.

spicata)*, Alkaligrass (some) (P. distans)* and S.

virginicus*.

21

Among halophytic species used as turfgrasses, P. vaginatum is one of the most salt-tolerant

species also with high turfgrass quality; P. vaginatum tolerates irrigation with water of ECW

6–22 dS m-1

and survives irrigation with 40 dS m-1

(Semple et al., 2003; Carrow et al., 2005;

Lee et al., 2005). However, D. spicata is also highly salt tolerant (only 50% growth reduction

at ECW of 35 dS m-1

), and can survive with an ECW as high as 40 dS m-1

, but grows best at 20

dS m-1

(Leake et al., 2002), and D. spicata is a potential high quality turfgrass based on its

ability to retain good colour (Leake et al., 2002). S. virginicus has its optimal growth at

salinity levels between ECW 6-10 dS m-1

(Bell and O’Leary, 2003), members of this species

have been reported to survive irrigation with water of EC 55 dS m-1

(Gallagher, 1979).

Growth of halophytic grasses tends to decline at high salinity levels (200-300 mM NaCl)

(Flowers and Colmer, 2008), but some show optimal growth at moderate salinities (30-60

mM NaCl) accompanied by an increase in succulence, such as in Imperata cylindrica

(Hameed et al., 2009). For seashore paspalum (P. vaginatum) turfgrass types, growth (shoot

dry mass) of seven out of nine accessions were stimulated by 1-10% with saline irrigation

(ECW 10 dS m-1

) compared to the control (ECW 0 dS m-1

) (Lee et al., 2005). The most salt

tolerant one had also root growth stimulation under moderate salinity stress, followed by a

decline in root growth at higher salinity levels. Also for S. virginicus, shoot growth increased

by up to 13% of the control (1 mM NaCl) at salinity level up to 150 mM NaCl and then

declined to 67% of the control as salinity was raised to 450 mM NaCl (Marcum and

Murdoch, 1992). Growth stimulation of halophytes is likely to be attributed to the osmotic

adjustment increasing cell turgor. Growth reductions at high salinities are probably associated

with reduced turgor and the high energy cost of osmoregulation (Maathius et al., 1997), and

possible ion toxicity in cells and tissues.

Salinity stress reduces plant growth. This reduction is expressed, as a first response, by a

reduction in plant height or reductions in the number of leaves or shoots. As salinity stress

increases and plants suffer, morphological changes will appear (e.g. reduction in leaf area,

root length, and leaf wilting and tissue damage (called ‘firing’ for turfgrass leaves). There are

also other criteria used to assess plant growth, one is the relative growth rate, and its

components such as specific leaf area, unit leaf rate, leaf weight ratio, and leaf area ratio

(Subbarao and Johansen, 1994; Garnier, 1992). Salt tolerance in plants has usually been

22

expressed as the reduction in yield under certain salt concentration in the bulk soil compared

with yield reduction under non-saline conditions (Bernstein et al., 1974).

Shoots are generally more inhibited in growth by salinity than roots (Jamil et al., 2005). The

halophytic turfgrass Paspalum vaginatum demonstrated high tolerance to salinity levels of (~

24 dS m-1

) without shoot and root length being affected, but shoot and root fresh and dry

mass were severely affected (Pessarakli and Touchane, 2006). By contrast, substantial

declines occurred due to salinity in Cynodon dactylon where the shoot and root mass (more

than 6-fold decline in shoot DM and 4-fold in root), shoot and root length (3-fold decline in

shoot length and more than 1 for root) while no significant difference was found in

succulence (Pessarakli and Touchane, 2006).

Assessments of salinity tolerance in halophytic turfgrasses should be based on growth

responses under low, intermediate and high salinity levels of soil and/or irrigation water, as

compared with non-saline conditions. Assessment should not only be based on the

aboveground growth (i.e. leaves and stolons) but also should extend to belowground (i.e.

roots and rhizomes) assessments (Duncan and Carrow, 1999; Lee et al., 2004). Parameters

used to assess growth and performance of turfgrasses, differ to those used for grain crops.

Several parameters have been used to assess growth of turfgrasses such as: clipping mass,

time required to cover the soil surface, height and mass of the thatch layer, growth recovery

after stress, and root mass and distribution (Marcum, 1999; Carrow, 2004). Turfgrass quality

parameters (e.g. colour) should also be measured.

Root growth is reported to be stimulated up to 2-fold when salinity was increased from 15 to

35 dS m-1

in the halophytic turfgrasses Sporobolus virginicus and Distichlis spicata (Marcum

and Murdoch, 1992; Marcum et al., 2007), so that for Sporobolus virginicus root:shoot ratio

doubled at 60% seawater when compared with a non-saline control (Marcum and Murdoch,

1992). Such increases in root:shoot ratio are advantageous to plants growing under saline

environments as it increases water absorption by the root system which could be an adaptive

mechanism to tolerate salinity as it might improve exclusion of ions by roots as water enters

and thus decrease the uptake of potentially toxic ions (Yeo, 1981).

23

In summary, halophytic turfgrass species can tolerate saline irrigation water up to seawater

level (55 dS m-1

) and so have potential to grow in salt-affected lands. Marked differences in

salt tolerance exist among genera, species and varieties of various turfgrasses, so selecting

turfgrass species and accessions with high salinity threshold helps in establishing good

quality turfgrass areas (Carrow and Duncan, 1998). Halophytic turfgrasses could be used in

salt-affected areas where fresh water resources are limited. Shoot and root dry mass are likely

to decrease with increasing salinity, although the ratio of root:shoot can increase, and it will

also be important to assess colour responses to salinity as an additional criteria to growth.

2.3.1 Colour, a selection criteria of salt tolerance in turfgrasses

Colour is a key component of the aesthetic quality of turfgrass and is therefore an important

trait to evaluate. Turfgrass colour is an indicator of healthy development and higher rate of

photosynthetic activity (Martinello, 2005). Several reliable means to quantify turfgrass colour

are described in the literature, measuring the hue angle using chromometer (Barton et al.,

2009a,b), using digital image analysis (Richardson et al., 2001) and leaf chlorophyll content

as it significantly correlates with the turfgrass colour as shown in a study of 12 turfgrass

species (Rahman, 1983). Visual rating scales of turfgrass colour are subjective since these are

based on human judgment, yet a trained observer can be very accurate with visual ratings

provided he/she does not know the identity of treatments (Bell et al., 2009). A single

observer, or the same group of observers, should evaluate a trial until completion and keep a

photographic record of rating differences (Horst et al., 1984).

Reflectance is used to measure chlorophyll. The effects of different stresses on turfgrasses

can be assessed based on the sensitivity of changes in spectral reflectance of leaves in the

ranges 535 – 640 nm and 685 – 700 nm wavelengths, due to changes in chlorophyll

reflectance; absorbance in stressed leaves is lower than in non-stressed ones (Hendry et al.,

1987). As one example, reflectance at visible wavelength increased (i.e. absorbance

decreased) in wear-stressed leaves of Cynodon dactylon and Paspalum vaginatum (Trenholm

et al., 1999).

A comparison of the visible and infrared wavelengths showed that the visible wavelength is

good indicator of salinity stress which often increases the green spectrum (491-575 nm) near

24

550 nm and the red spectrum (647-760 nm) near 710 nm, with the effect on the red spectrum

being greater than that on the green (Carter, 1993). Reflectance from 550 nm and 700 nm

shows maximum sensitivity to a wide range of chlorophyll contents. In addition, the effects

of dehydration on terrestrial plants occur most strongly in the yellow region of the spectrum

(Carter, 1993). The key absorption signature occurs in the photosynthetically active region

(PAR) of the visible spectrum (400–700 nm), through the action of photosynthetic pigments,

but there are also further absorptions by water at longer wavelengths. The infrared

wavelengths (700-1200 nm) can be used to assess biomass of turfgrasses, and longer (1500 –

1800 and 2100 - 2300 nm) wavelengths are reported to assess leaf turgor pressure (Jensen et

al., 2000).

Leaf photochemical efficiency was estimated by measuring chlorophyll fluorescence (a ratio

of variable to maximum chlorophyll fluorescence), measuring the fluorescence of water

stressed plants is correlated with the passive remote sensing of reflectance changes at 531 nm

called as Physiological Reflectance Index (PRI) on both leaf and whole plant basis (Gamon et

al., 1990; Evain et al., 2004). To conclude, reflectance indicates the changes in plant

physiology due to environmental stress but these responses are not stress-specific, so that

there is no indication of which particular stress (i.e. salinity, high temperature, low

temperature, drought, nutrient deficiency or pests and light intensity) is the underlying cause

since all the response to these stresses are expressed in a same manner (Chapin, 1991;

Belkhodja et al., 1994). Nevertheless, such technologies provide an alternative for turfgrass

managers concerning the quick evaluation of turfgrasses (Trenholm et al., 1999; Bell et al.,

2000).

In studies of turfgrass responses to drought stress, a strong correlation was found between

canopy reflectance versus turfgrass quality and leaf firing varied with turfgrass species and

cultivars (Jiang and Carrow, 2005). Reflectance response to leaf dehydration in the near

infrared region was found to be consistent only when severe leaf dehydration happened

which alters cell structure. The wavelength at which turfgrass canopy reflectance is most

sensitive to water stress and whether variability in wavelength sensitivity exists between

species requires further investigation.

The length of exposure to saline irrigation is important for effects on turfgrass quality. A

study of ‘SeaDwarf’ P. vaginatum showed that whereby colour quality improved with time (8

25

weeks) under saline irrigation treatments by (35%, 19%, 30%, 36% and 27%) for irrigation

treatments of 9, 11, 14, 19 and 27 dS m-1

, but not for the 55 dS m-1

(sea water) when quality

declined by 60% (Berndt, 2007). The salinity problem may arise as a consequence of salt

accumulation reaching a level higher than the threshold tolerated by a turfgrass species, then

causing unavoidable stress to turfgrasses.

A study of P. vaginatum and D. spicata (halophytes) and C. dactylon, (non-halophyte)

showed that after increasing salinity of the irrigation water from 0 to 11 dS m-1

NaCl, colour

rating of the three species remained unaffected, however at 22 dS m-1

, colour of C. dactylon

and P. vaginatum dropped by 13% and 10%, respectively while for D. spicata remained

unchanged. At 33 dS m-1

, colour rating dropped by 25% and 22% in C. dactylon and P.

vaginatum, respectively and remained unchanged for D. spicata (Pessarakli et al., 2009). A

short-term study (6 days) of the halophytic turfgrass P. vaginatum, and the non-halophytes C.

dactylon, S. secundatum and E. ophiuroides irrigated with (19, 37 and 54 dS m-1

) showed that

P. vaginatum had a maximum reduction in colour quality of 18% at 54 dS m-1

after 6 days

whilst for C. dactylon, S. secundatum and E. ophiuroides were 30%, 60% and 100%,

respectively (Wiecko, 2003). In a different study, no visual deterioration of turfgrass colour

quality was noticed at 16 dS m–1

for 6 cultivars of P. pratensis when compared to the non-

saline control plots. At higher levels of salt (18 and 22 dS m–1

) colour rating declined (Poss et

al., 2010). These examples highlight that colour can be maintained at relatively high salinity

levels in some salt tolerant turfgrass species, but then colour declines as salinity stress

exceeds tolerable levels.

In addition to colour, general turfgrass quality traits (density, texture) are also important, and

these can be impacted on by environmental conditions (such as salinity) and also by

management practices (such as mowing). Examples are given briefly here for the species that

are the focus of the experimental work in this thesis. The halophytic turfgrass D. spicata

showed decreased turfgrass quality and shoot dry mass at 5 cm mowing height when

compared to mowing at 2.5 cm (Pessarakli and Kopec, 2010; Pessarakli, 2011). For

Paspalum vaginatum mown at < 2.5 cm, a finer textured dense turfgrass was produced and

tiller production also increased as mowing height was decreased (Fry and Huang, 2004);

however, at mowing height > 5 cm Paspalum vaginatum became spindly and showed

increased thatch production (Trenholm et al., 2003). No data are reported in the literature on

mowing height trials for S. virginicus.

26

In conclusion, turfgrass colour is a major component of turfgrass quality. Traditional methods

of determining turfgrass colour (i.e. visual scoring) are quick, yet not accurate to indicate the

turfgrass quality ratings, the variations amongst evaluators are sometimes higher than those

amongst different turfgrass cultivars. Recent technological advances in assessments of

turfgrass colour and surface stress responses will likely replace visual scoring as these

modern tools are implemented to measure quantify in salinity stress scientific research.

2.4 Interactions between salinity and environmental factors

Most environmental variables are expected to interact with salinity. An important example is

that air temperature and soil salinity values are highly inter-related; during winter

temperatures with low evapotranspiration and leaching events, salinity in the soil drops,

whereas during summer soil salinity increases and air temperatures are also highest at this

time (Ayars et al., 1998). It is essential, therefore, to explore interrelationships between

various environmental factors and salinity.

Environmental factors play a major role in determining how plants respond to salinity;

especially, air temperature, light and relative humidity (Maas, 1986). Under moderate

temperatures and humid environmental conditions, tolerance of turfgrass to salinity stress is

higher than compared with the under hot and dry conditions; conditions with increased ET

result in accumulation of the salts in the soil (Hoffman and Rawlins, 1971) and in leaves

(Glenn and Brown, 1998).

Turfgrass evapotranspiration (ET) is influenced by air temperature, wind, atmospheric water

vapour pressure, soil moisture tension, light intensity and duration (Huang and Fry, 1999).

The higher the ET, the lower soil water content will be and higher irrigation volumes are

required then to maintain ET. ET of turfgrass species differs widely due to turfgrass type (i.e.

C3 vs C4 grasses), a reduction of 25-30% water use in C4 turfgrasses was recorded when

compared with C3 grasses in the early summer season (Ford et al., 2006). Changes in soil

salinity will follow the change of soil moisture.

27

Salinity responses can be influenced by other environmental factors such as aeration,

temperature, and biotic stresses (disease). Turfgrasses vary not only in the rate at which they

tolerate salinity, but also in the surrounding environmental conditions that can influence the

measurable effects of salinity include waterlogging. Root zone salinity and waterlogging

greatly increase salt uptake by plants and reduces plant growth due to the ion toxicity if

compared with non-waterlogged plants (Barrett-Lennard, 2002).

Soil physical properties such as soil texture and bulk density can also affect the water use by

turfgrasses. A consequence of high bulk density in the subsoils (below 50 cm) is having

shallow root system (Jordan et al., 1993), the increase in bulk density increases the water

retention and decreases soil porosity, decrease ET (from 51.5 cm to 37.1 cm),

and restrict root

growth. Soil compaction in the top 3 cm increased the bulk density (from 1.27 g cm-3

to 1.34

g cm-3

) and moisture retention (O’Neil and Carrow, 1982; Sills and Carrow, 1983).

In summary, understanding the interaction between environmental conditions and salinity

tolerance of turfgrass species will assist in identifying species, cultivars and genotypes with

high quality along with their better adaptation to different saline environments.

2.4.1 Evaporative demand and turfgrass water use

This section discusses turfgrass water use and ways of measuring the water use. The use of a

crop coefficient in irrigation scheduling, and comparisons of water use between C4 and C3

turfgrasses, are given. Examples on variations between species and cultivars of turfgrasses in

terms of water use are discussed.

The definition of turfgrass water use represents the amount of water used for plant growth

plus the water lost through evaporation from the soil surface and transpiration from the plant

(Beard, 1973). ET and water use are considered to be comparable terms as there is no way of

separating between them under field conditions. Turfgrass water use and water use efficiency

are the way forward to achieve sustainable farming and landscaping by providing the plants

with their water requirements (Wareing and Phillips, 1981).

28

Several methods have been developed to estimate crop ET. Most methods use weather data to

provide an estimate of reference (or potential) evapotranspiration (ETo), often convert the

ETo to "actual" ET using a multiplicative factor known as a crop coefficient (Kc):

ET = Kc x ETo

Kc of the warm season turfgrass in Georgia, USA was 0.75 while for the cool season

turfgrass was 0.80 (Carrow, 1995), but another study by Aronson et al. (1987) noted a Kc of

1.0 would suffice all cool season grasses in Rhode Island. A study showed that Kc for warm

season grasses mowed at 1.6–2.5 cm rests in the range of 0.6–0.8 (Kneebone and Pepper,

1982). Areas of low recovery, low traffic and less fertilizer requirements are maintained at

the lower end of the range whilst the upper end of the Kc range would be appropriate for

areas of high quality turfgrass where fertilization regimes are high and rapid recovery is

required (Kneebone and Pepper, 1982). Kc is species and region specific, and they are useful

for conserving water on irrigated turfgrass surfaces through providing a convenient means of

determining turfgrass water use, and thereby, provide a method of calculating how much

water to apply with each irrigation event.

Warm season (i.e. C4) grasses are having ETo rates between 2 and 6 mm d-1

, while C3 grass

ET rates range from 3 to 8 mm d-1

at 60% ET (Beard, 1994), because the C4 grasses have

thicker cuticles which prevents the loss of water and concentrate CO2 in bundle sheath cells

and so can operate with lower stomatal conductance (Taiz and Zeiger, 1998). Carrow (1995)

found warm season bermudagrass, St. Augustine and zoysiagrass had ETa rates of 3.1, 3.3,

3.5 mm d-1

, respectively. Meyer and Gibeault (1987) noted minimum irrigation level of warm

season grasses from 40 to 63% ET. Importantly, turfgrass quality increased significantly as

irrigation increased (up to 55% ETo), but above these amounts of irrigation quality was not

further improved for bermudagrass or St. Augustinegrass, demonstrating that excess watering

is wasteful (Qian et al., 2000).

Evapotranspiration varies between species and locations. Warm season turf grasses, such as

zoysiagrass, bermudagrass, and buffalograss, have relatively low (< 6 mm d-1

)

evapotranspiration rates (Kim and Beard, 1988). A study of bermudagrass irrigated at 100,

80, and 60% of ET values estimated from modified Class A pan reported that at 60% ET,

colour quality was better than acceptable and not significantly different from bermudagrass

irrigated at 100% of ET (Meyer and Camenga, 1985). However, turfgrass water requirements

change frequently; influenced primarily by environmental conditions such as available soil

29

water, solar radiation, relative humidity, wind velocity, and temperature (Nishat et al., 2007).

ET increases with increased light levels, increased temperatures, lowered humidity, moderate

to high wind speeds, and long days (Carrow, 1995; Hunt et al., 1987), an increase of 2-7 m s-1

in wind speed increased ET by 20-50% over that in still-air (Chow, 1996).

The microclimate affects turfgrass water use, an increase of solar radiation, high air

temperatures and low relative humidity cause significant increase to turfgrass ET rate

(Feldhake et al., 1984; Kneebone and Pepper, 1982). Cultural practices including mowing

and fertilisation influence turfgrass water requirements, doubling the mowing height from 30

mm to 60 mm caused rapid increase in turfgrass water use within the first 6 weeks (Biran et

al., 1981; Kim and Beard, 1985; Parr et al., 1984). Plant and soil parameters are often

successfully used to modify the potential ETo, as defined by the Penman equation (Rosenberg

et al., 1983).

Weighing lysimeters are often used to measure plant water use. Several reports in the

literature studied a wide range of plant species and show the advantage of using weighing

lysimeters to study crop water requirements. Lysimeters are measured to detect changes in

the total weight after one day of irrigation (Short, 2002). Weighing lysimeters are useful to

measure ET of grasses and small size plants (Harsch et al., 2009). Another advantage of

weighing lysimeters is they enable measurements of leachate volumes as the plant is grown in

a container at the desired location and periodically weighed as well as having leachate

collection devices (Abdou and Flury, 2004). Designing lysimeters is crucial to account for the

micrometeorological variations between lysimeters and actual growing environment in the

sense of radiation influx and heat dissipation (Mukammal et al., 1971, Green et al., 1984).

Lysimeter measurements have established that turfgrass water use varies amongst different

genotypes (Kneebone and Pepper, 1982; Carrow, 1995). A long term study of water use by 7

turfgrasses indicated a range of water use within warm-season turfgrasses of 1.99 to 6.05 mm

day-1

while for the ET of cool-season turfgrasses rates varied from 1.40 to 6.22 mm day-1

(Carrow, 1995). In another study (Beard, 1985), Tifway Bermuda and Zoysia in Texas used

5.9 and 4.8-7.2 mm day-1

respectively, while common Bermuda in North Carolina used mm

day, and Paspalum, Buffalograss, Fescues, and Kentucky Bluegrass used 7, 5.3-7.3, 12.6, 7.2,

5 mm day-1

, respectively.

30

It is important to understand the seasonal water use over a period of repeated years rather

than relying only on short study periods. Seasonal water use differences can be attributed to

environmental conditions, plant species (C3 vs C4) and growth habit of turfgrasses.

Selecting Kc values for use in irrigation management should be the most suitable for both the

species and the location of interest. Water stress will affect turfgrass evapotranspiration rate,

growth rate, and visual quality.

In conclusion, water use of turfgrasses is influenced by environmental factors such as

temperature, wind, solar radiation, relative humidity, soil texture, and soil moisture. These

factors affect both plant transpiration and soil evaporation. Understanding both the

environmental factors influencing water use and the physiology of the target turfgrass species

is important for developing efficient irrigation strategies for turfgrass, especially when water

resources are restricted. Knowledge of how much water to apply by irrigation to replenish

water loss through turfgrass evapotranspiration is an important water management strategy.

2.5 Mechanisms of salt tolerance in plants

Plants employ several mechanisms to tolerate soil salinity and even maintain growth and

productivity. Salinity tolerance mechanisms vary between species (Gorham, 1995), ranging

from the ability to avoid accumulating of high ion concentrations in leaf cells (i.e.

‘exclusion’), to coping with high ion concentrations in tissues (Greenway and Munns, 1980).

Three major problems encountered by plants as a consequence to salinity are: (1) specific ion

toxicity (i.e. Na+, Cl

-), (2) maintaining a favorable cell turgor pressure, (3) acquiring the

essential nutrient ions (K+, NO3

-) in spite of the predominance of other chemically similar,

potentially toxic ions (Na+, Cl

-) in the root-zone.

Halophytic grass species such as (D. spicata, S. airoides and S. cryptandrus) are

distinguished in maintaining low toxic ion concentrations in their leaves. Halophytic grasses

control the rate of entry of Na+ into their leaves (Koyro and Stelzer, 1988; Marcum and

Murdoch, 1992; Wyn-Jones and Gorham, 2002) so that the leaves remain functional and

retain high quality colour (i.e. tissues are not damaged). Additionally, some halophytic grass

species (D. spicata and S. virginicus) secrete ions from leaves through salt glands (Marcum,

31

1999; Ramadan, 2001). Halophytes also synthesise organic solutes to adjust the internal

osmotic potential as the external one declines, so as to avoid water deficits (Ramadan, 2001).

These topics of ion regulation and organic solute accumulation, as related to osmotic

adjustment, are considered in the following sub-sections.

2.5.1 Na+ and Cl- exclusion at different levels in the plant body

Ion exclusion takes place at several stages along the ion transport pathway into and through

plants. At the root level, the soil solution flows into the hydrophilic walls of epidermal cells

and passes freely along the apoplast into the root cortex, exposing all the parenchyma cells to

soil solution so that a large membrane surface area is exposed for nutrient uptake (Kamula et

al., 1994). Examples of ion exclusion at root level of halophytes are shown in (Rozema et al.,

1981; Naidoo, 1994; Wu et al., 2005). All plants exclude Na+ at the root level but the

efficiency of exclusion varies amongst species and can also depend on the salinity level and

other factors (e.g. waterlogging) in the growing media. Concentrations of Na+ and Cl

- in the

xylem of plants are typically between 1-2% of the external concentration, while in halophytes

with salt glands these are up to 5% (Munns et al., 1983). Salt-tolerant non-halophytes restrict

Na+ uptake, but this can also cause problems in having sufficient osmotica to adjust their

osmotic pressure (Rozema et al., 1985).

Examples of ion exclusion for turfgrasses is the study by Marcum (2001) which showed that

Distichlis spicata and Cynodon dactylon are active excluders and maintained less than 15%

of the leaf sap Na+ when compared with Buchloë dactyloides. For Cl

-, exclusion differences

were also clearly evident with only 3% in the leaf sap of Distichlis spicata and 15% in

Cynodon dactylon to that in Buchloë dactyloides at a salinity level equivalent to seawater.

Plants control delivery of Na+ into the xylem. The apoplastic (i.e. the extracellular) pathway

consists of cell wall and extracellular spaces. Water and solutes can move from one location

to another within roots through the continuum of cell walls, at least until apoplastic barriers

such as the casparian strips are encountered (Harvey et al., 1981). Water and solutes must

then transverse a cell plasma membrane to enter a cell, after which solutes can move from

cell to cell via plasmodesmata. The exclusion mechanisms rely on the effective

32

impermeability of the cell membranes to ions, and selectivity of transporters against Na+. If

Na+ enters the protoplast, then it can be extruded across the plasmamembrane out from the

cytoplasm (e.g., cereals, Munns et al., 2006; halophytic shrub Atriplex nummularia, Munns

and Tester, 2008). Na+ exclusion is driven by the electrochemical H

+ gradient established by

the H+-ATPase, which in turn allows plasmamembrane Na

+/H

+ antiporters to couple the

passive movement of H+ inside the cells, along its electrochemical potential, to the active

extrusion of Na+ (Blumwald et al., 2000).

Several additional mechanisms maintain low Na+ concentrations in xylem, such as: uptake of

the ions into cells in the outer half of the root (Tester and Davenport, 2003; Davenport,

2007), efflux of the ions back to the soil solution (Munns and Tester, 2008), and resorption of

Na+ from the xylem transpiration stream and transport back into the root cells (Tester and

Davenport, 2003). Other exclusion mechanisms also take place and contribute to regulation

of leaf Na+ concentrations, such as: active transport out of the xylem through transfer cells;

this reduces the Na+ prior to its arrival into the leaves (Kramer, 1983; Flowers et al., 1986).

Transfer cells are located in the proximal part of the root was found to retain Na+, a

significant difference in Na+

concentration was found between these cells and the leaves of

beans (Kramer et al., 1977). Na+ and Cl

- translocation from the leaves through phloem is also

another proposed mechanism regulating leaf Na+ (Lessani and Marschner, 1978; Flowers,

1985), but details of its contribution in halophytes is lacking.

Exclusion of salts at the root uptake level is essential, as excess entry of toxic ions causes cell

injury and death (Munns, 2002). Plant species and varieties differ in regards to Na+ and Cl

-

uptake. The salt tolerant grasses are able to maintain low concentrations in the leaves through

regulating the ion uptake from the roots (Glenn et al., 1992; Flowers, 2004). Certain low-

affinity K+ channels showed a high selectivity for K

+ over Na

+, but other channels might let

Na+ enter. A study of barley roots revealed that there is high selectivity for K

+ over Na

+

(Wegner and Raschke, 1994) whereas low K+:Na

+ selectivity was measured in Arabidopsis

root cells. Depolarisation the plasma membrane in saline conditions might also alter ion

channel activities to mediate the efflux of K+ and the influx of Na

+ (Maathuis and Sanders,

1995; Munns and Tester, 2008).

33

Restriction of Na+ entry maintains high cytosolic K

+:Na

+ ratio (Glenn et al., 1999). The

K+:Na

+ ratio is a key requirement for plant growth in high salt (Glenn et al., 1999). K

+:Na

+

ratio in the grass halophytes is typically higher than that of glycophytes (Gorham et al., 1985;

Yeo, 1998). Nevertheless, even halophytes can suffer declines as demonstrated by a study of

Sporobolus virginicus with substantial decline in K+:Na

+ ratio within the shoots as a result of

increasing salt concentration from 0.1 dS m-1

to seawater (Marcum and Murdoch, 1992).

Halophytes are distinguished in regards to regulation of ion concentrations in leaf tissues,

although a wide range of Na+ concentrations occur in the leaves of halophytes. As examples,

1.4 mg Na+ g

-1 DM accumulated in the leaves of the grass Sporobolus virginicus irrigated

with saline water equivalent to seawater salinity (Bell and O’Leary, 2003), 12 mg Na+

g-1

DM

accumulated in leaf succulent species Atriplex amnicola, 16 mg Na+ g

-1 DM for Atriplex

hortensis and 54 mg Na+ g

-1 DM for Suaeda nudiflora at 9 dS m

-1 (Shekhawat et al., 2006),

and 175 mg Na+

g-1

DM in the stem-succulent Salicornia bigelovii at 12.8 dS m-1

(Pfister,

1999). Na+

concentration is relatively low in halophytic monocots with salt glands and is very

high in succulent dicots which are halophytes (Albert and Popp, 1977; Gorham et al., 1980).

The distinction of halophytes comes from their ability to utilise Na+ as an osmolyte to adjust

the internal osmotic potential. It is reported that substantial accumulation of Na+ and Cl

- up to

30 and 50% of the plant dry mass respectively occurs in some of the dicotyledonous species

(Gorham et al., 1980; Flowers et al., 1977). K+:Na

+ ratio was negatively correlated with

salinity tolerance among the 10 genotypes (Lazarus et al., 2011). In the monocotyledonous

halophytes, from Poaceae family in particular, these have higher K+:Na

+ ratio and relatively

low tissue water content as well (Flowers et al., 1977). The K+:Na

+ ratio was higher in leaves

if compared to roots of Sporobolus virginicus (Bell and O’Leary, 2003). K+ (inorganic) and

sugar (organic) are the major osmolytes within the monocotyledons (Jeschke, 1984).

Salt exclusion is classified as a ‘salt avoidance mechanism’ (i.e. selective exclusion

mechanism). Salt resistance also includes internal salt tolerance mechanisms such as tissue

tolerance to specific toxic ions (e.g. Na+ and Cl

-) and tissue dehydration tolerance (Duncan

and Carrow, 1999). The cell plasmamembrane regulates the traffic of material into and out of

the protoplast, but within the protoplast a large central vacuole, an organelle that can occupy

as much as 90% of the protoplast’s volume, is also surrounded by a membrane (tonoplast)

and there again is regulation of ions between the cytosol and the contents of the vacuole

(Lalonde et al., 2004). Ions stored in the vacuole contribute to osmotic pressure in the cell

34

and also can no longer interfere with enzymes in the cytosol. This intracellular

compartmentation enables plants to accumulate high levels of the Na+ for osmotic adjustment

(Carden et al., 2003; Colmer et al., 2005).

2.5.2 Ion secretion via salt glands

Some halophytes have secretory structures, called salt glands, on their leaves. Salt glands

function to move ions from the symplast out to the leaf surface, after which the salts are

periodically washed off by rain, irrigation or in coastal marshes tides. These glands often

form a glistening crust of salt that covers the leaves of these plants. In effect, salt glands are

dump sites for excess salt absorbed in water from the soil and consequently help plants to

manage their internal salt loads, and thus adapt to life in saline environments.

Salt tolerance and salt gland activity in grasses is related to the actual density of salt glands

on the leaves, or the number of glands per unit leaf area. A positive correlation was found

between leaf salt gland density and salt tolerance (clipping yield at high salinity), also

between salt gland density and quality of zoysiagrass colour (Marcum et al., 2003); thus,

higher leaf salt gland density increases salt tolerance. Secretion rate correlates with external

salt concentration. Secretion of Na+ from leaves of S. virginicus doubled as a result of

increasing the NaCl concentration in the nutrient solution from 125 to 450 mM (Bell and

O’Leary, 2003).

In some species salt secretion can be stimulated by exogenous application of benzyladenine

(Waisel, 1972), and application of this chemical could therefore enhance performance on

saline sites. However, a study of Zoysia matrella found no significant effect of 6-

benzyladenine on enhancing secretion rate (Nakamae and Nakamura, 1982) and also did not

increase leaf chlorophyll during autumn after applications of 6-benzyladenine despite the

high density of salt glands in this species.

35

2.5.3 Osmotic adjustment

Osmotic stress is a result of increasing salinity outside the roots. The presence of various salts

in the irrigation water and/or soil lowers its osmotic potential. Salinity therefore causes a

“physiological drought” which happens when water uptake cannot meet evapotranspiration

(ET) demand. This can occur in saline soils even though moisture in the soil is adequate

(Ben-Gal et al., 2009). Physiological drought causes plant wilting (Gorham et al., 1985;

Munns and Termaat, 1986; Munns, 1993). Plant wilting is a sign of low quality turfgrass (Foy

et al., 1978).

Water movement occurs along gradients in water potential. Water moves “down gradient”

from areas of higher to lower water potential. Osmotic potential of distilled water is zero

MPa, and this potential becomes negative when the salt concentration increases so that, sea

water for example has a potential of approximately -2.5 MPa (Jefferies, 1981). When osmotic

potential is lower outside the cell than inside the cell, turgor loss and growth inhibition occur

(Flowers and Yeo, 1981). Some plants can prevent the decrease in cell turgor by osmotic

adjustment (OA), the accumulation of solutes within the cells to lower the internal osmotic

potential below that of the external medium. However, energy is required for this process and

the used energy will be at the expense of potential plant growth (Salisbury and Ross, 1992).

The sensitivity of some non-halophytes to salinity stems from their inability to adjustment

osmotically, which may result from either an insufficient uptake of salt ions or a lack of

synthesising organic solutes (Greenway and Munns, 1980). Osmotic adjustment allows the

plant to maintain turgor under low (i.e. very negative) soil water potentials by decreasing

cellular osmotic potential. Osmotic adjustment has been demonstrated in numerous grasses

(Dacosta and Huang, 2006; Qian and Fry, 1997) and usually involves the accumulation of

compatible solutes such as carbohydrates, amino acids, and glycinebetaine, as well as mineral

ions.

The movement of water in and out of plant cells (between the apoplast and symplast) is a

form of osmosis (Steudle and Peterson, 1998). However, plant cells are able to influence the

direction of water movement via changes in osmotic potential and/or cell wall elasticity

(Steudle et al., 1977). In addition, plant cells are able to control the rate of diffusion via the

36

production of water pores (Carpita et al., 1979; Verslues et al., 2006). However, under saline

and dry soils, the mentioned gradient will not be sufficient, so the solute potentials of plant

cells decreased for osmotic adjustment through accumulating various solutes within the cells

(Howell, 2001).

Turfgrass species show a diverse capacity for osmotic adjustment. As an example, a study of

four turfgrass species showed that the rank of grasses for magnitude of osmotic adjustment

was buffalograss (0.84 MPa), zoysiagrass (0.77 MPa), bermudagrass (0.60 MPa), tall fescue

(0.34 MPa) (Qian and Fry, 1997). This diversity in osmotic adjustment was consistent with

variation in growth and survival of these species as they differ in maintaining turgor pressure

and allowing the continuity of water absorption in soils with low moisture content. Osmotic

adjustment mechanisms are reported in halophytic turfgrasses: Distichlis spicata, Sporobolus

airoides (Marcum, 1999) and S. virginicus (Ramadan, 2001). However, a different study

showed that S. virginicus and D. spicata maintained less negative osmotic potential than salt

sensitive species (Marcum, 1999), and a similar low negative osmotic potential was also

reported for 8 D. spicata genotypes (Marcum et al., 2007). D. spicata successfully

established from rhizomes was able to tolerate a wide range of osmotic potentials to less than

-2.3 MPa, which is equivalent to 0.5 M NaCl (Pavlicek et al., 1977).

Plant water potential ( w) varies throughout the day; its lowest value was found to be at about

noon or early afternoon, then recovering to higher values in the evening. Daily rhythm in

plant water and osmotic potentials was reported by (Ustin et al., 1982) showing higher values

in the morning and evening and towards the midday across 3 halophytic plants (Spartina,

Scirpus and Salicornia). The cyclic w helps out in scheduling the irrigation systems to meet

plant water requirements. Irrigation using saline water should be commenced before both

shoots and roots of plants are suffering low w (Colthier and Green, 1994).

Cell elongation is the first major physiological function to be impaired by low tissue water

potential. Reducing tissue water potential ( ) of only 0.1 MPa can impact cell growth (Hsiao,

1973), most of the reduction happens during midday as a result of growth-limiting water

stress which will be expressed as plant wilt or stomatal closure, usually, the recovery to

predawn water potential takes place (Schulze, 1986; Witmer et al., 1998). Osmotic

adjustment helps to maintain the size and growth of plant cells during periods of low water

intake due to high salt concentrations. However, even if cell shrinks, osmotic adjustment still

37

benefit the cells as the accumulated solutes appear to protect the cellular enzymes against

reversible damage during cellular dehydration (Mullet and Whitsitt, 1996).

Synthesis of compatible solutes, low-molecular-mass compounds that are not cytotoxic, is an

important component of osmotic adjustment (Flowers and Yeo, 1988). The compatible

solutes do not interfere with biochemical reactions (Hasegawa et al., 2000) and these stabilise

the proteins and cellular structures, and decrease the osmotic potential of the cell (Yancey et

al., 1982) and consequently maintain turgor pressure and cell functions under low external

water potential. These organic solutes are: sugars, some amino acids (esp. proline),

glycinebetaine, and sugar alcohols (Naidu et al., 2000; Ghoulam et al., 2002). These organic

solutes, plus K+ in the cytosol, balance the osmotic potential of Na

+ and Cl

- stored in the

vacuole. Glycinebetaine and proline are the major contributors to osmotic adjustment of

turfgrasses (Marcum, 1999) and these solutes will be discussed in this review.

2.5.3.1 Glycinebetaine

Glycinebetaine (GB) is a quaternary ammonium compound and it occurs in many organisms

including plants and animals (Rhodes and Hanson, 1993). GB (a member of N-methyl

substituted amino acids) was found in different plant families (i.e. Chenopodia1ceae,

Chlorideae and Solanaceae) (McCue and Hanson, 1990; Gorham et al., 1985). Halophytic

plants contain high levels of GB (10-100 mol g-1

fresh mass) (Wyn-Jones, 1980). GB and

proline are located in the cytoplasm (Aspinall and Paleg, 1981; Wyn-Jones et al., 1984),

which occupies 10% of total cell volume (Dunn, 2007).

The accumulation of GB in salt tolerant plants and halophytes as a result of exposing plants

to high salt concentrations is well documented in the literature (Colmer et al., 1995; Rahman

et al., 2002; Ashraf and Foolad, 2007). Halophytic species obtained from salt marshes

contain high GB concentrations: Gnaphalium californicum, Cuscuta salina, Distichlis spicata

and Salicornia depressa have concentrations of 114, 107, 40 and 38 mol g-1

fresh mass

(respectively) whereas Triticum aestivum (salt-sensitive species) had only (3.4 mol g-1

fresh

mass) (Rhodes et al., 1987). GB concentrations in leaves have been correlated to the external

salinity among C4 turfgrasses such as: Bouteloua spp., Buchloë dactyloides, Cynodon spp.,

Distichlis spicata, Distichlis stricta, Eremochloa ophiuroides, Paspalum vaginatum,

38

Sporobolus virginicus, Stenotaphrum secondatum, and Zoysia spp. (Marcum and Murdoch,

1994; Marcum, 1999; Alshammary et al., 2004). In the above studies, GB increased as the

turfgrasses acclimated to the salinity stress whereas other solutes (e.g. proline) remained

unchanged in the first 3 weeks after salts were imposed.

The major role for GB is to adjust the cytoplasmic osmotic potential. Many other roles are

reported in the literature, for instance, GB protects cell membranes against heat

destabilisation (Jolivet et al., 1982), stabilises soluble proteins (Murata et al., 1992; Chen et

al., 2009), protects enzyme activity against salt inhibition (Pollard and Wyn-Jones, 1979;

Raza et al., 2006), and can stabilises the association of the extrinsic PSII complex proteins

which in turn protects the photosystem II in salt stressed plants (Murata et al., 1992; Horn et

al., 2006). In addition to a role in salt tolerance, GB is also involved in plant tolerance to

freezing stress (Einset et al., 2007).

2.5.3.2 Proline

Proline accumulates in both salt sensitive and salt-tolerant plants. Higher leaf and root proline

accumulation was found in salt-sensitive cultivars of cereals (up to 2-fold higher) than in salt-

tolerant cultivars (Chen et al., 2007; Colmer et al., 1995). In other species, however, proline

has been proposed to be of more importance than GB; proline accumulation in Aster

tripolium and barley increased in response to stresses while GB was stable (Goas et al., 1982;

Ahmad and Wyn-Jones, 1979). Proline concentration in the roots of salt-tolerant varieties of

barley was twice as high as that of salt-sensitive (Chen et al., 2007). The increase in proline

concentrations in tissues of the salt tolerant crop sugar beet was positively correlated with salt

tolerance (Ghoulam et al., 2002). Reported roles of proline include osmotic adjustment,

protection of enzymes and of proteins in cell membranes (Ashraf and Foolad, 2007).

A study of two halophytes, Suaeda maritima and Salicornia europaea showed an increase in

proline concentration with the increase in salinity reaching (25 µmol g-1

DM) more than 5-

fold increase in the concentration of proline in the leaves between the 0 and 510 mM NaCl in

the irrigation solution (Moghaieb et al., 2004). Similar increases in proline were also reported

earlier for Spartina patens (Ewing et al., 1995). Wide ranges of variation in proline

accumulation have been reported for turfgrass species. Marcum (1999) showed an increase in

39

proline concentration in D. spicata to 3 mM in leaves when under 300 mM NaCl irrigation

treatment. However, Cressa critica had 50% of proline concentration that accumulated in

Sporobolus helvolus (3.8 and 7.9 µg g-1

fresh weight) during summer season (Kasera and

Mohammed, 2010). Though leaf proline concentrations increased under salinity in most, but

not all species, with a maximum levels reached only 11 mM (S. virginicus) at 450 mM NaCl

which contributed to 8% of the osmotic adjustment where as glycinebetaine was 126 mM

which contributed to 93% of the external salinity (Marcum, 2001).

The increases in tissue proline concentrations should enhance cell functioning during salinity

stress. For Paspalum vaginatum turfgrasses in controlled environment, a high concentration

of tissue proline was correlated with re-growth rate after the recovery from salinity (20 dS m-

1). Proline has also been applied exogenously to improve the tolerance of commercial plants

such as tomato to salinity. Exogenous application maintained higher K+ concentration in the

leaves (Heuer, 2003) also decreased K+

efflux from barley roots (Cuin and Shabala, 2005).

In summary, proline contributes to the osmotic adjustment in many salt-tolerant species.

Proline concentration increases as salinity increases (Qian et al., 2000; Chen and Li, 2002).

Proline appears to be associated with salinity tolerance, yet proline acts less efficiently as a

compatible solute in Chloridoid grasses when compared with GB.

2.5.3.3 Sugars

Sugars (glucose, fructose, sucrose, fructans) function in osmotic adjustment (Parida et al.,

2002; Kerepesi and Galiba, 2000). Other functions of sugars are reported also such as carbon

storage, and free-radicle scavenging. However, a range of sugars and sugar alcohols

accumulate as a result of reduction in photosynthesis, so that a specific role in acclimation

can sometime be difficult to demonstrate (Munns et al., 1982).

Glucose concentration in the leaves of 9 different genotypes of P. vaginatum was lower than

fructose concentration with a range of 1.44–2.77 mg g−1

DM across six levels of salt in the

irrigation water (ECw 0, 10, 20, 30, 40 and 50 dS m-1

). The highest glucose content was found

at ECw 20 dS m-1

, with decreased glucose content at ECw 30 dS m-1

, followed by an increase

40

at >ECw 30. Sucrose exhibiting greater contribution to Ψs adjustment than glucose at ECw 50

dS m-1

.

Sucrose is reported to be the most abundant sugar for several halophytic grasses (Hester et

al., 2001). However, leaf sucrose content did not relate to salt tolerance between genotypes of

P. vaginatum; sucrose content in leaf tissues was the highest with a maximum accumulation

of 10.08–13.02 mg g−1

DM for the 9 genotypes of P. vaginatum (Lee et al., 2008). Similarly,

fructose and glucose also only showed minor differences between tolerant and intolerant

genotypes of P. vaginatum. Contributions of sugars to osmotic adjustment in 9 genotypes of

P. vaginatum under saline irrigation (ECw 50 dS m-1

) did not differ across the genotypes (Lee

et al., 2008) whereas made significant contribution to ospmotic adjustment in S. virginicus

(Marcum and Murdoch, 1992).

2.6 Conclusions and hypotheses for thesis

Several mechanisms are employed in turfgrasses to tolerate high levels of soluble salts in the

irrigation water and soil. Different mechanisms explain the variation in salinity tolerance

between species/cultivars, but the most important mechanism is regulation of leaf ion

concentrations, predominately by ion exclusion (but also with a contribution of excretion in

some halophytic species with salt glands). Salinity tolerance was also enhanced by increased

K+ concentration. High leaf K

+ increase salinity tolerance due to increased turgidity and also

maintenance of a favourable K+:Na

+ ratio for enzyme functioning. An overview of factors

associated with salinity tolerance within halophytic turfgrass species would suggest that key

mechanisms are: (i) less toxic ion concentrations in the leaf tissues, (ii) maintenance of high

tissue K+, as it positively correlates with leaf water content and presumably turgor pressure,

and (iii) synthesis of organic solutes.

Soil salinity can have a strong influence on several agronomic aspects related to turfgrass

quality and productivity. Several management practises have to be modified including

irrigation scheduling to help minimising the salt accumulation in the root zone and

consequently accumulating the potential toxic ions in the leaf tissues of turfgrasses. Although

non-halophytes attempt to exclude Na+ and Cl

-, even at moderate soil salinity (e.g. above 4

41

dS m-1

) these elements can reach concentrations in shoots that exceed tolerable levels for

glycophytes which reduces turfgrass quality and productivity. By contrast, the halophytic

species are employing mechanisms to exclude (or excrete) Na+ and Cl

-, as well as possibly to

tolerate these salts within their tissues (e.g. vacuolar compartmentation). Turfgrass quality

(represented by colour) is a major concern in turfgrass management, so it is necessary to

consider aspects related to long-term field studies addressing the quality of turfgrass in saline

environments.

The purpose of this thesis was to investigate the comparative physiology for salt tolerance in

three halophytic species (Distichlis spicata, Sporobolus virginicus and Paspalum vaginatum)

and a non-halophytic species (Pennisetum clandestinum) when maintained as turfgrasses. The

three halophytes provide options in addition to traditionally-used non-halophytes such as

Pennisetum clandestinum a species widely-used as turfgrass in Mediterranean climates. All

four species are C4 grasses. This thesis reports the performance of the four species under

saline and non-saline irrigation (2-year field experiment) in Chapter 3. The dose responses of

the four species to hyper saline irrigation (0, 125, 250, 500 and 750 mM NaCl added to

nutrient solution under glasshouse conditions) is described in Chapter 4. Chapter 5 includes

the effect of different levels of irrigation volumes on the growth and performance of

Paspalum vaginatum (2-year field experiment). Chapter 6 describes the response of Distichlis

spicata to hyper saline irrigation (0, 230, 450 and 680 mM NaCl added to nutrient solution

under glasshouse conditions) and also the ion distributions in different parts of the plant

(leaves, roots, apical rhizome, intermediate rhizome and basal rhizome) as well as sugar

accumulation in these parts. Chapter 7 provides a concluding discussion, including

implications for halophytic turfgrasses in salt-affected lands and priorities for future research.

The following hypotheses were tested:

In Chapter 3: Hypothesis 1 - the halophytic turfgrass species would grow better and retain

colour better than the non-halophyte under saline conditions. Hypothesis 2 - the major traits

contributing to salinity tolerance in the halophytic grasses would be an ability to maintain a

high ratio of K+:Na

+ in the leaf tissues (Zhu et al., 1998; Leigh and Wyn-Jones, 1984; Chow

et al., 1990) and maintain tolerable concentrations of the toxic ions (Na+ and Cl

-).

In Chapter 4: Hypothesis 3 - the three halophytic grasses will suffer less growth declines in

response to increasing salinity than the non-halophyte. Hypothesis 4 – the three halophytic

grasses will prevent large increases in Na+ and Cl

- concentrations in leaf tissues, as compared

42

to the non-halophyte, when external soil salinity increases. Hypothesis 5 - shoot growth

reductions under salinity will be correlated with increases in leaf tissue Na+ and/or Cl

-

concentrations, both in the halophytes and the non-halophyte. Hypothesis 6 – water use by

the halophytes will be maintained under saline irrigation, whereas water use by the non-

halophyte will decline markedly.

In Chapter 5: Hypothesis 7 - as the volume of irrigation water is increased, the salinity of the

soil solution in the root zone would be maintained at a level closer to that of the applied

water, owing to the greater leaching of salts into deeper soil layers. Hypothesis 8 - in

situations where salt accumulates in the root zone owing to insufficient leaching, P.

vaginatum would have decreased growth and colour quality in response to the salinity of soil

solution. Hypothesis 9 - decreases in the growth and quality of P. vaginatum with different

volumes of saline irrigation would be mediated by ion concentrations in the tissues (viz. high

concentrations of Na+ or Cl

- and/or low concentrations of K

+).

In Chapter 6: Hypothesis 10 - sugars are expected to accumulate in the rhizomes as salinity is

increased; although younger segments of the rhizomes (apical segment) could differ to the

basal segment. Hypothesis 11 - D. spicata restricts the concentrations of the potentially toxic

ions (Na+ and Cl

-) in the apical segment of the rhizomes (where growth occurs), whereas

these ions accumulate in the older portions of rhizomes. Hypothesis 12 - Distichlis spicata

protects its leaves under high salt concentrations by maintaining low concentrations of the

potentially toxic ions (Na+ and Cl

-) in the leaves.

43

Chapter 3

Irrigation of three halophytic turfgrasses with saline water: growth, turf

quality and physiology of salt-tolerance in field plots

3.1 Abstract

Alternate water resources, such as brackish/saline water, are needed to irrigate turfgrass, as

potable water resources are becoming increasingly limited and more expensive. This chapter

focuses on the responses of three halophytic turfgrass species (Paspalum vaginatum,

Sporobolus virginicus and Distichlis spicata) and one non-halophyte (Pennisetum

clandestinum) to irrigation with saline groundwater (ECw 13.5 dS m-1

) at a field site in south-

western Australia over two years, focusing mostly on the data from the second year. Plots

with saline irrigation were compared with control plots irrigated with non-saline water, both

at approximately 60% replacement of net evaporation, during the dry months. After

commencing saline irrigation, soil salinity (EC1:5) increased from 4 to 14 dS m-1

in the

topsoil, but declined back to the initial level when irrigation ceased during the rainy months.

Salt tolerance was assessed by measuring the dry mass of clippings and from turfgrass colour;

the responses of leaf ion concentrations and osmotic potential were also assessed. Compared

with non-saline irrigation water, with the non-halophyte P. clandestinum, saline irrigation

reduced the dry mass of clippings by 31%, whereas with the three halophytes reductions were

more modest (10% for P. vaginatum, 6% for S. virginicus) or clippings increased slightly (by

4% for D. spicata). Leaves of the three halophytes contained ~50% lower Na+ and Cl

–

concentrations than P. clandestinum. K+:Na

+ in the most salt-tolerant species in this study (D.

spicata) was 3-fold more than the least salt-tolerant species (P. clandestinum). Colour

(greenness) of the non-halophyte declined after commencing saline irrigation, whereas the

greenness was not impacted, or even improved, for the three halophytic turfgrasses. The

44

present study has demonstrated the feasibility of using saline irrigation water for the growth

of halophytic turfgrasses in south-western Australia.

3.2 Introduction

Turfgrass is an essential component of urban environments, providing areas for recreation

and sport, as well as having environmental benefits such as cooling and dust mitigation

(Beard and Green, 1994). However, water shortages are occurring in many urbanised areas

due to the overuse of resources and rapid population growth (Kjelgren et al., 2000), so the

use of fresh water for landscape irrigation is being reduced or, in some cases, curtailed.

Secondary saline water sources, such as various effluents and brackish waters, are

increasingly being used for turfgrass irrigation (Brede, 2000). In Australia, rising saline

groundwater under many rural towns (Ferdowsian et al., 1996) might provide an opportunity

for the increased use of saline water as irrigation for turfgrass. Moreover, as control of the

saline watertables in many towns in the wheatbelt of Western Australia includes pumping and

disposal to drainage lines and salt lakes (Salama et al., 1994), finding alternative uses for this

water would also reduce the problem of disposal.

Irrigation with saline water can result in salt accumulation in the soil (Schaan et al., 2003).

Salt concentrations in the soil solution can be expected to be higher in arid and semi-arid

regions than in temperate and cool-humid regions as evaporation is higher in the former than

the latter environments (Dudal and Purnell, 1986). The potentially toxic ions (i.e. Na+ and Cl

-

) can then enter plants and accumulate in tissues and cells, resulting in symptoms of tissue

damage, such as colour decline, necrosis, leaf tip burn (White and Broadly, 2001) and

decreased leaf area (Munns, 2002).

Sodicity is another problem that can be associated with salinity where clay is a major

component of the soil. Sodicity refers to the exchange by sodium by other cations, such as

calcium, magnesium and potassium, on the exchange sites of clay particles in the soil. This

leads to dispersion of the particles, the clogging of soil pores and a blocking of the movement

of water and gases, which causes additional adverse effects on plant roots (Grattan and Oster,

2003).

45

The use of saline water sources in urban landscapes requires turfgrass cultivars that are salt

tolerant. Substantial work has been done in the USA to screen current turfgrass species for

salinity tolerance (Carrow, 2006; Marcum, 2008). Levels of salinity associated with a 50%

decline in growth of turfgrasses range from ECe values < 1.5 to > 20 dS m-1

(Huck et al.,

2000; Semple et al., 2003), with the most salt tolerant species being halophytes (“salt-loving

plants”). Although types with higher salt tolerance are still needed, some halophytic species

have now been developed for use as turfgrasses (Marcum, 2008). Paspalum vaginatum

(seashore paspalum or saltwater couch) is the most intensively developed halophytic grass,

with several cultivars released (Duncan and Carrow, 2002). However, P. vaginatum is a

species that lacks salt glands, and its salt tolerance is not as great as some other halophytic

grasses (Marcum and Murdoch, 1994).

Other halophytic grasses that have potential as turfgrasses are Distichlis spicata (saltgrass)

and Sporobolus virginicus (marine couch); the former species is reputed to have higher salt

tolerance than P. vaginatum (Kopec and Marcum, 2001), while with the latter, some ecotypes

are able to survive in twice-strength sea water (Blits and Gallagher, 1991), Salt tolerant

turfgrass types have been selected from both these species (Loch and Lees, 2001; Marcum et

al., 2005). In addition to growth, turfgrass colour is a good indicator of turfgrass quality,

responding to environmental stresses and management practices (Dean et al., 1996), and this

character could also be important in the assessment of turfgrass salt tolerance.

As with all monocotyledons, salinity tolerance in turfgrasses has been associated with the

regulation of Na+

and Cl- in the leaves; that is, differences in ion ‘exclusion’ from the shoots

have been related to plant growth in comparative studies at the levels of genus (Marcum,

1999), species (Marcum et al., 1998), and cultivar/ecotype (Qian et al., 2001; Marcum and

Pessarakli, 2006). Typically, monocotyledonous halophytes, from the family Poaceae have a

high K+:Na

+ ratio (Flowers et al., 1977; Bell and O’Leary, 2003). In addition, some highly

salt-tolerant halophytic grasses have salt glands in the leaves that secrete excess Na+ and Cl

-

from the shoots (Amarasinghe and Watson, 1989); salt glands occur in grasses from the

genus Distichlis and Sporobolus (Amarasinghe and Watson, 1989; Marcum, 1999).

The objective of the present work was to study responses to irrigation with saline water of

three halophytic turfgrass species (Distichlis spicata, Sporobolus virginicus and Paspalum

vaginatum) and of the non-halophyte P. clandestinum. This comparative study of these three

46

halophytes and P. clandestinum (the most common turfgrass used in the public open spaces

of south-west of Australia) is of importance as P. clandestinum is considered to be only

moderately tolerant of salinity (Semple et al., 2003) and it shows salt-damage at key turfgrass

assets in the target region of the study. Another objective of the study was to compare the

physiological responses of the three halophytic grasses to salinity with the salt tolerant non-

halophyte P. clandestinum (Joo et al., 2008); all four plants are C4 species.

The following hypotheses were tested: 1) The three halophytic turfgrass species would

maintain better shoot growth (clippings) and retain colour better than the non-halophyte

under saline conditions. 2) The major traits contributing to salinity tolerance in the halophytic

grasses would be an ability to maintain a high ratio of K+:Na

+ in the leaf tissues (Zhu et al.,

1998; Chow et al., 1990) and maintain tolerable concentrations of the toxic ions Na+ and Cl

-.

3.3 Materials and methods

Site preparation, establishment and irrigation schedule

Plots were established at Wagin, Western Australia, GPS location 33o 18' 38.54" S and 117

o

20' 52.99". A drainage system was installed 1 m below the soil surface and around the blocks

to ensure that there was no lateral movement of saline water; this consisted of buried 50-mm

slotted pipe surrounded by a 10 cm layer of 12 mm gravel, topped by second 10 cm layer of 7

mm gravel. The effluent collected by this system was diverted to a nearby sump and disposed

in a salt lake a few kilometers away.

The grasses were established in the field plots as follows. Organic fertilizer (chicken manure)

was applied at a rate of 2 tonnes ha-1

, and the plots were then cultivated to a depth of 10 cm

using a rotary hoe and levelled. Rooted plantlets (which had been established from rhizomes

in trays of vermiculite for 2 months in a glasshouse) were then planted into the field plots on

16 October 2006. These plantlets were established using irrigation with scheme water;

sprinklers (MP2000 Rotator, supplied by Total Eden, Australia) were stationed at 3 m

intervals and connected to an irrigation controller (Irritrol, RD-900, supplied by Total Eden,

Australia). Fertilizer (Turf Special, a soluble NPK fertilizer with micronutrients, CSBP

Limited) was applied every 4 weeks during establishment at a rate equivalent to 30 kg ha-1

N,

decreasing to 15 kg ha-1

N after 13 February 2007. Mowing to a height of 30 mm commenced

47

on 7 December 2006; mowing occurred every second week during the irrigation season and

at monthly intervals outside this period.

A weather station (Mark 4, Environdata, Australia) was installed on the site. Parameters

measured at 15 minute intervals included: daily maximum and minimum air temperatures,

total precipitation (Figure 3.1), relative humidity, total solar radiation, average wind speed

and average wind direction. Evapotranspiration (ET) was calculated for the 24-hour period

between 9 am on successive days using the Penman-Monteith equation (Allen et al., 1998).

The rate of irrigation was adjusted manually every two weeks so that irrigation was applied at

approximately 60% of the estimated ET of the preceding 2-week period.

Soil physical properties

Soil texture

Soil texture (percent sand, silt and clay) was determined by suspending the soil in a

measuring cylinder of water and allowing the soil particles of different size to settle out of

solution at different times, with clay particles take the longest (Hunt and Gilkes, 1992). The

fractions were subsequently dried and weighed (see Bowman and Hutka, 2002). Based on

these measurements, the topsoil was classified using a soil texture triangle (Hunt and Gilkes,

1992) as silty clay loam whereas the subsoil was a clay loam (Table 3.1).

The soil bulk density was determined at depths to 50 cm using cores of 7 cm diameter and 25

cm height for the top-soil and further cores for the sub-soil; these were oven-dried at 105 oC,

weighed and the soil dry mass per unit volume was calculated. The bulk densities of the

topsoil and subsoil were ~1.6 and 1.9 g cm-3

, respectively (Table 3.1), measurements were

made before commencing irrigation and at the end of experiment.

Infiltration rate

Infiltration rates of water in the topsoil (0–25 cm) were measured in situ using a local-made

infiltrometer and also a double ring infiltrometer, according to the method of Bouwer (1986).

Five replicates were measured at the end of the experiment; yet field measurements were not

possible in the subsoil (25-50 cm) as clay in this layer caused sealing. In view of this, I

attempted to obtain measurements of hydraulic conductivity (Ks) using a laboratory method.

Intact cores (76 mm diameter, 76 mm height) were taken of both topsoil (0-25 cm) and

subsoil (25–50 cm) and transported to the laboratory. It was intended that the Ks of these

48

cores would be measured based on the volume of water that passed through each core at a

given pressure; however, the rates of infiltration were below the detection limits of the

method (Table 3.1). For comparison, George (1992) reported that soils in the Wagin district

are likely to have Ks values in the range 0.01 to 1.10 m day-1

.

Level of groundwater

Six piezometers were installed across the site to a depth of 2 m; the piezometers were sealed

into the subsoil with bentonite to ensure that no irrigation water accessed the piezometers

from the soil surface. Piezometers were capped to prevent evaporation and the groundwater

level was recorded at 2-4 week intervals using a plopper attached to a tape measure (Plop102,

Eviroequip, Perth, Australia).

Soil salinity

Soil salinity was measured by collecting soil samples from the topsoil (0–25 cm) and subsoil

(25–50 cm) using a 7 cm diameter soil auger at fortnightly intervals during the irrigation

seasons and at monthly intervals during periods without irrigation. All plots were measured

using the EM38 in the horizontal orientation and 5 plots of each treatment were selected for

physical sampling on the basis of EM38 readings (ECa values) that were representative of the

range of values across the site. Samples were placed into zip-lock bags, transported to the

laboratory, weighed, oven dried and weighed again to calculate the soil water content.

Subsamples of dry soil (20 g) were extracted with 100 mL of DI water, shaken mechanically

for 16 hours and then centrifuged at 3,000 rpm for 5 min. The electrical conductivity of the

samples (EC1:5) was measured using an EC meter (Cyberscan 100, Omega Scientific,

Australia) after the samples had been allowed to equilibrate to room temperature. The salinity

of the soil solution (ECsoil solution) was estimated from EC1:5 values using the equation

C1 x V1 = C2 x V2

where C1 is the EC1:5, V1 is the volume of the 1:5 extract (ie. 100 mL), C2 is the ECsoil solution

and V2 is the volume of soil water originally present in the 20 g of soil used to make the 1:5

extract. Conversion from EC1:5 to ECe is given in Appendix 3.2

49

Cation exchange capacity and exchangeable sodium percentage

The cation exchange capacity of the soil (CEC) in meq/100g was calculated as the sum of

exchangeable Ca2+

, Mg2+

, Na+, K

+ and Al

3+ from soil samples. These cations were extracted

by suspending 2 g soil in 20 mL of 0.1 M BaCl2 + 0.1 M NH4Cl for 2 hours, and then

filtering off and analysing the supernatant using a Spectrometer (ICP-AES, iCAP 6300,

Thermo Scientific, Waltham, MA, USA) following Gilman and Sumpter method in Rayment

and Higginson (1992). The exchangeable sodium percentage (ESP) was calculated as

follows:

ESP= Exchangeable {(Na) / (Ca+Mg+K+Na+Al)}(100).

Table 3.1. Soil physical properties measured in the topsoil (0–25 cm) and subsoil (25–50 cm)

prior to commencing the irrigation treatments. Values are means of 5 measurements ± SEM.*

Soil property Topsoil Subsoil

Bulk density* 1.6 ± 0.28 g cm-3

1.86 ± 0.03 g cm-3

Infiltration rate** 93 ± 46 mL min-1

Below limit of

detection

Soil texture Silty clay loam Clay loam

Clay content 18.0 ± 2.1% 29.0 ± 2.9%

Sand content 29.6 ± 1.9% 57.3 ± 3.3%

Silt content 42.4 ± 2.1% 12.2 ± 1.7%

* Data on bulk density reported for the final measurement as no significant difference

between these data and those before commencing irrigation.

** In situ measurements of infiltration in the topsoil were made using a local-made

infiltrometer, whereas measurements of subsoil infiltration were attempted using extracted

intact soil cores taken to a laboratory; in the latter case infiltration was not detectable over 72

hours.

50

Turfgrass colour

A chromameter (Minolta, CR-310, Osaka, Japan) was used to measure the colour of the

turfgrass. The chromameter applies a pulse of light and measures reflected light in the red,

green, and blue spectral ranges, with the result expressed as the hue angle (for further details

see Barton et al., 2009). The chromometer measures three parameters (L*, a*, b*). L* refers

to the lightness of the reading, with values ranging from black = 0 to white = 100. A* refers

to the green/redness of the turfgrass colour with values ranging from -60 indicating green to

+60 indicating red-purple, and b* indicates the yellow/blueness of the turfgrass colour with

values ranging from 90 indicating yellow to 180 indicating blue (McGuire, 1992). The hue

angle [h˚ = tan-1

(b*/a*)] was calculated from a* and b* values (Lancaster et al., 1997), hue

angle has no unit but it is an indication of greenness. The device was calibrated using a

calibration plate (CR-A44 Minolta, Osaka, Japan) every 18 measurements following the

manufacturer’s instructions. Colour changes were quantified by recording three positions in

each plot, to obtain a plot mean.

Growth and tissue water content

The turfgrass clippings from the fortnightly and monthly cuttings described above were

weighed and a subsample was taken, weighed and oven-dried at 65 °C. The dried subsamples

were weighed, and the water content (mL g-1

DM) was calculated.

The accumulation of thatch (i.e. height of thatch plus shoots immediately after mowing) and

the thatch dry mass were measured on 6 May 2008; at this time the plants had experienced 45

days without irrigation following the second summer of irrigation. Three cores per plot were

taken using a stainless steel core sampler (72 mm diameter). These samples were also

weighed and oven dried.

Concentrations of Na+, K

+ and Cl

- in leaf tissues

Leaf samples were collected every 2 weeks during the irrigation period and every 4 weeks

during the rainy season. A small patch of turfgrass (30 cm2) was randomly chosen within

each plot, rinsed with DI water using a hand-held sprayer to wash off any surface ions, and

then allowed to dry for about 30 min. Leaf tissue samples were collected using scissors and

the fresh mass was recorded; samples were then wrapped in aluminium foil, frozen in dry ice,

transported back to the laboratory, stored at -80 oC and then freeze-dried. Dry mass was

recorded and samples were then ground in a ball and mill.

51

Samples of ground tissue (~ 0.1 g) were extracted in 10 mL of 0.5 M HNO3, and Na+ and K

+

were measured using a flame photometer (Jenway PFP7 Flame Photometer; Essex, UK) after

appropriate dilution of the extracts. For Cl- determinations, 3 mL of extract was diluted with

0.1 N HNO3 in 10% (v:v) acetic acid, and Cl-

was determined by the silver ion titration

method using a chloridometer (Buchler–Cotlove chloridometer 662201; Fort-Lee, NJ, USA).

A reference tissue with known ion concentrations was taken through the same procedures to

check reliability; recovery of Na+, K

+ and Cl

- were 91%, 93% and 97%, respectively. No

adjustments were therefore made to the data.

Sap osmotic potential

Leaf tissues were collected in air-tight cryovials and frozen in dry ice. Back in the laboratory

the samples were thawed while still in the sealed vials, squeezed in a press, and the osmotic

potential of the expressed sap was measured using an osmometer (F110, Fiske Associates,

Needham Heights, MA, USA).

Statistical analyses

Statistical analyses were conducted using Genstat for Windows 10th Edition (Genstat

software, VSN International, Hemel Hempsted, UK). Analysis of variance (ANOVA) was

used to identify significant differences between treatments and between species using a split-

plot design with irrigation treatment as the main treatment and species as the subplot. When

significant differences were found, Duncan’s multiple range test was used to calculate the

mean separations. The significance level was P < 0.05, unless stated otherwise.

3.4 Results

Sequence of irrigation in the experiment

The different turfgrasses varied in the time taken to establish. Eight weeks after planting,

plots of the fast growing species P. clandestinum and P. vaginatum had achieved complete

cover, whereas plots of S. virginicus and D. spicata had only achieved 80-85% cover. For this

reason, commencement of the first saline irrigation was delayed to 15 January 2007 and this

treatment only lasted 12 weeks in the first summer. In the second summer, saline irrigation

commenced on 27 September 2007 and extended to 7 April 2008, so the bulk of the data

reported here will be for the second irrigation season. Some indicative data (weather, soil

52

conditions, plant DM, leaf ions, osmotic potential and colour) from the peak of the first saline

irrigation period (26 February 2007) are given in Appendix 3.1. Table 3.2 shows these

parameters averaged over the second irrigation season. These data will be referred to in the

relevant sections below.

Table 3.2. Average data (weather, soil conditions, plant DM, leaf ions, osmotic potential and

colour) over the second irrigation season (27 September 2007 to 7 August 2008)*.

P. clandestinum P. vaginatum S. virginicus D. spicata

Minimum

Maximum

Non-saline

Saline

Non-saline

Saline

Non-saline

Saline

Non-saline

Saline

Non-saline

Saline

Non-saline

Saline

Non-saline

Saline

Non-saline 32.44 ± 3.60 52.76 ± 8.80 17.10 ± 3.18 23.56 ± 2.13

Saline 24.49 ± 1.53 46.47 ± 4.31 15.56 ± 2.13 23.88 ± 1.78

Non-saline 1.78 ± 0.10 1.93 ± 0.11 1.03 ± 0.06 0.95 ± 0.03

Saline 1.29 ± 0.10 1.63 ± 0.06 0.83 ± 0.04 0.73 ± 0.02

Non-saline 0.13 ± 0.03 0.15 ± 0.01 0.10 ± 0.02 0.08 ± 0.01

Saline 0.66 ± 0.03 0.64 ± 0.01 0.31 ± 0.01 0.34 ± 0.01

Non-saline 0.29 ± 0.04 0.29 ± 0.04 0.27 ± 0.05 0.64 ± 0.08

Saline 0.16 ± 0.02 0.33 ± 0.02 0.37 ± 0.04 0.49 ± 0.03

Non-saline 0.21 ± 0.05 0.21 ± 0.02 0.11 ± 0.03 0.11 ± 0.02

Saline 0.85 ± 0.03 0.62 ± 0.01 0.39 ± 0.04 0.41 ± 0.03

Non-saline 2.40 ± 0.29 2.47 ± 0.43 2.93 ± 0.28 8.88 ± 0.89

Saline 0.47 ± 0.22 0.59 ± 0.05 1.40 ± 0.25 1.50 ± 0.20

Non-saline -1.21 ± 0.14 -1.29 ± 0.11 -1.89 ± 0.30 -1.32 ± 0.21

Saline -1.87 ± 0.21 -1.71 ± 0.11 -1.94 ± 0.18 -2.07 ± 0.25

Non-saline 115.09 ± 0.43 117.07 ± 1.06 113.61 ± 0.68 119.37 ± 0.58

Saline 100.46 ± 2.19 114.41 ± 1.21 114.15 ± 0.45 118.16 ± 0.74

Leaf K:Na ratio

Leaf Sap Osmotic Potential (MPa)

Colour (Hue angle)

Clippings DM (gm-2

)

Leaf Na+

(mmol g-1

DM)

Leaf K+

(mmol g-1

DM)

Leaf Cl- (mmol g

-1 DM)

Leaf water content (mL g-1

DM)

Soil ECsoil solution (dS m-1

) (0 - 25 cm)6.42 ± 1.46

22.42 ± 5.16

Soil ECsoil solution (dS m-1

) (25 - 50 cm)6.60 ± 1.30

25.88 ± 5.68

Soil Water Content (0 - 25 cm)0.13 ± 0.01

0.12 ± 0.01

Soil Water Content (25 - 50 cm)0.10 ± 0.01

0.10 ± 0.03

Soil EC1-5 (dS m-1

) (0 - 25 cm)2.07 ± 0.30

6.96 ± 0.73

Soil EC1-5 (dS m-1

) (25 - 50 cm)1.53 ± 0.18

7.10 ± 1.07

EM38h (dS m-1

)7.80 ± 0.12

12.80 ± 0.24

Parameter TreatmentSpecies

Air temperature oC

6.6

43.7

*Temperature data are the mean ± SEM of 188 daily records. Soil salinity (EC1:5) and soil

water content data are the mean ± SEM of 5 measurements. Ion data, leaf sap osmotic

potential and colour data are the mean of 3 measurements ± SEM on 14 harvest days at ~ 2

week intervals across the irrigation season.

53

Weather conditions

Figure 3.1 shows the average weather conditions at the site over the 17-month period from

December 2006 to April 2008. The average maximum air temperature during February (the

hottest month) was 31oC in 2007 and 2008, and the average minimum temperature was ~16

oC. The average relative humidity (RH) during the summer (December – February) was 61%,

which increased to 82% in August, and then fell to 50% in spring (October). The rainfall was

353 mm in 2007, of which 40% fell in July and August. There was a positive correlation

between average air temperature and RH (r2 = 0.88; P = 0.03). The total evaporation (ETo)

was 1290 mm in the second irrigation period (27 September 2007 – 7 April 2008), while the

estimated applied water was 774 mm for the same period (data not presented).

2007 2008

Air

Tem

pera

ture

(oC

)

Rain

fall

(mm

)0

25

50

75

100

Dec Mar Jun Sep Dec Mar Jun

0

25

50

75

100

Figure 3.1. Weather trends at the field site in Wagin between December 2006 and May 2008.

A. Average monthly air temperature ( ) average monthly relative humidity (RH) (

) and total monthly precipitation (bars).

Soil salinity, soil moisture, sodicity and the possibility of waterlogging

Figure 3.2 compares the sequence of change in the average EC1:5 with the bulk conductivity

of the soil as measured with the EM38 (ECah – Figure 3.2A) and average gravimetric water

content of the soil (Figure 3.2B and C, respectively). After commencing the saline irrigation,

the EM38 (ECah) values increased within a few weeks, reaching a maximum of 13 dS m-1

in

the first summer (short irrigation season), declined during the rainy season to 8 dS m-1

,

increased to 16 dS m-1

in the second summer (long irrigation season) and then declined to 12

dS m-1

by the time the monitoring ceased (Figure 3.2A). In contrast, with plots irrigated with

non-saline water, ECah values remained in the range of 7-9 dS m-1

, a range of values similar

to that of plots irrigated with saline water after leaching during the rainy season.

54

The EM38 is a machine that measures the bulk conductivity of the soil at two depths: in its

vertical orientation about half of the reading comes from depths less than 80 cm, and in its

horizontal orientation about half of the reading comes from depths less than 40 cm (Bennett

et al., 2009). The machine was therefore used in its horizontal orientation to track the

variation in the bulk conductivity of the soil where turfgrass roots exist.

The summary of data from the first irrigation season shows that the EC1:5 in the soil was

affected by both irrigation salinity and depth in the soil profile, (Appendix 3.1) showed that

soil in the topsoil reached to the same salinity level as that in the irrigation water, whilst in

the subsoil was only 54%. K+:Na

+ ratio in the leaf tissues was as high as 2.5 times more in the

halophytic D. spicata to that in the non-halophytic P. clandestinum. Colour rating in the first

irrigation season was declined by 5% for P. clandestinum, whilst increased by 3%, for S.

virginicus did not change for D. spicata and P. vaginatum, under saline irrigation.

Soil salinity (EC1:5) values for the second irrigation season provide a sense of the dynamics of

these changes (Figure 3.2B). For the non-saline irrigation treatment, the EC1:5 of the topsoil

and subsoil was relatively low for all plots (~2-3 dS m-1

) and these low values persisted from

the start of the irrigation season (27 September 2007) until the date of final measurement (7

April 2008), assuring that there was no lateral movement of the salts from non-saline to saline

irrigated plots (plots were within non-saline blocks or in saline blocks, separated by a vertical

gravel-filled interceptor with drainage to a sump to minimise any risk of lateral water

movement between irrigation blocks- see Materials and Methods). With the saline irrigation

treatment, the EC1:5 value began to diverge from the controls by 19 November 2007, and the

difference in EC1:5 measurements between treatments reached a maximum of about 10 dS m-1

after ~6 months from the commencement of the saline irrigation treatment (Figure 3.2B). At

this time the EC1:5 of the topsoil in the salinised plots had increased 3-fold from the time of

the commencement of irrigation. Rainfall at the site occurred in February and April (Figure

3.1); consequently the EC1:5 values in the topsoil and subsoil began to fall again after

February, and values in the saline water treated plots had declined to 6 dS m-1

by the time the

saline irrigation had ceased (Figure 3.2B).

During the hottest period with highest ET, the salinity of the topsoil was lower than the

subsoil for the saline irrigation (P = 0.01); by contrast with the non-saline irrigation, the EC1:5

of the subsoil declined from 2.2 to 0.33 dS m-1

after commencing the non-saline irrigation,

55

reaching a value of 32% of the salinity of the topsoil after ~3 months of irrigation (Figure

3.2A).

EC

1:5

(dS

m-1)

0

4

8

12

16

Sep Dec Mar Jun

A

EC

ah(d

S m

-1)

2007 2008

Gra

vim

etr

ic s

oil

wate

r (%

DM

)

0

4

8

12

16

Dec Mar Jun Sep Dec Mar Jun

Non-saline Saline

0%

10%

20%

Sep Dec Mar Jun

B

C

Figure 3.2. Average EC1:5 (A), apparent electrical conductivity (B), and soil water content

(C) in turfgrass plots irrigated with non-saline water (open symbols) or saline groundwater

(ECw 13.5 dS m-1

) (closed symbols). The apparent electrical conductivity was measured using

an EM38 in the horizontal position (ECah). EC1:5 measurements were taken of the topsoil (0–

25 cm) (circles) and subsoil (25–50 cm) (squares) in 9 m2 field plots at Wagin, Western

Australia. Each ECah point is the mean of 28-31 values; EC1:5 and soil moisture values are the

mean of 5 replicates with each replicate being from an individual plot. The error bars denote

the SEM. The continuous line under the horizontal axes indicates the irrigation period; the

dotted line indicates the subsequent period without irrigation after the rainy season

56

commenced. The average LSD values (at 5%) for predicted means of EC1:5, ECah and soil

moisture are 1.39, 4.32, 0.01, respectively.

Encouragingly, EC1:5 data were related to the EM38 data (cf. Figure 3.2A and B). A

composite dataset of all EC1:5 and ECah measurements across all plots and times of

measurement over the 17 month period showed a positive linear relationship between the two

parameters with r2 = 0.80 (data not shown). Considering data at single measurement times,

the r2 values for this correlation reached a maximum of 0.96 during the irrigation period and

minimum of 0.64 during the rainy season when soil salinity was lower.

Soil water content varied across the irrigation period (Figure 3.2C). Values of soil water

content ranged from 8 to 17% of soil DM. There were significant differences between

sampling dates (P<0.001) but no significant effect of salinity of irrigation water on stored soil

moisture (P = 0.28). However, with non-saline irrigation there was 16% more soil water in

the topsoil (0–25 cm) compared with the subsoil (25–50 cm) (P<0.001) (Figure 3.2C). No

significant effects in soil water content were noted in the saline irrigated plots.

Data on the changes in soil sodicity over the course of the experiment are presented in Figure

3.3. Prior to the commencement of irrigation, the exchangeable sodium percentage (ESP) of

the topsoil (0-25 cm) was ~5% (indicating that this soil was not sodic), while the ESP of the

subsoil (25-50 cm) was ~6% (indicating that this soil could be categorised as being sodic).

With non-saline irrigation, the ESP of the topsoil increased slightly to ~6, but ESP values in

the topsoil irrigated with saline water decreased to ~4 by the end of the second irrigation

season. In the subsoil, the ESP decreased with time irrespective of the irrigation salinity,

reaching values of 3.0–3.5 by the end of the second irrigation season (Figure 3.3). Overall,

Ca2+

accounted for the largest proportion (43–45%) of exchangeable cations, while Na+

accounted for 13–14% of the total CEC. The concentration of exchangeable Al3+

ranged from

0.1– 1.0% of the total CEC (data not presented).

57

0

4

Initial S1 S2E

xch

angeable

So

diu

m P

erc

enta

ge (E

SP)

0

4

Initial S1 S2Irrigation

Season 1Irrigation

Season 2Initial

A

B

Figure 3.3. Exchangeable sodium percentage of topsoil (A), and subsoil (B), prior to

commencing the irrigation treatments (Initial; 23 Jan 2007) and during the first and second

irrigation seasons (17 Dec 2007 and 6 Apr 2008, respectively). Open bars represent the non-

saline irrigation and closed bars represent the saline irrigation. Values are the mean of 5

replicates with each replicate being an individual plot. Error bars denote the SEM. The dotted

line at ESP = 6 represents the threshold level for categorising a soil as being sodic in

Australia (Northcote and Skene, 1972). The LSD (P = 0.05) of the means is 0.81.

Previous studies have shown that waterlogging can interact with salinity, impacting on plant

ion relations and growth in saline environments (Barrett-Lennard, 2003). However,

measurements of shallow bores in the present work showed that the depth to water table did

not change significantly over the eight months of monitoring (P= 0.064); the average depth to

the water-table was 0.97 m from the soil surface at the end of the rainy season and 1.06 m at

the end of the second irrigation season. About 85% of the turfgrass root systems are typically

located in the upper 0.1 m of the soil profile (Short, 2002). It therefore seems unlikely that

waterlogging would have been a constraint affecting the ion relations or growth of the

turfgrasses during the experiments.

Growth

The dry mass (DM) of clippings produced by the four turfgrass species varied seasonally

(Figure 3.4). The relative performance of the species was therefore assessed on the basis of

the total DM of clippings across the whole irrigation season (Table 3.2). The most vigorous

species was the halophyte P. vaginatum, which produced an average of 46.5 and 52.8 g m-2

per harvest of dry clippings for the saline and non-saline irrigation treatments, respectively

58

(Table 3.2); these differences equated to a total of 960 and 1389 g m-2

, respectively over the

entire irrigation season. Relative to P. vaginatum, the vigour (DM production with non-saline

irrigation) of the other three species declined in the order: P. clandestinum > D. spicata > S.

virginicus, with total clipping DM production as percent of P. vaginatum of 61%, 45% and

32%, respectively (calculated from Table 3.2).

The pattern of growth of the species varied with time. Under non-saline irrigation the non-

halophyte P. clandestinum had its maximum growth on 11 February 2008, when the average

air temperature was 41.7 oC. By contrast, whilst under saline irrigation, the maximum growth

of this species occurred six weeks later, when the average air temperature had decreased to

~20 oC and the EC1:5 was ~3 dS m

-1 in the topsoil and subsoil (Figure 3.4A, Figure 3.2). For

P. vaginatum under non-saline irrigation, the highest growth occurred on 31 December 2007,

whilst under saline irrigation, the highest growth took place six weeks later, when the average

air temperature was 23 oC and the EC1:5 were 3 and 4 dS m

-1 in the topsoil and subsoil,

respectively. S. virginicus also achieved its highest growth under non-saline irrigation on 28

January 2008 whereas under saline irrigation highest growth was 8 weeks later when average

temperature declined by 8 oC to be ~20

oC even though the EC1:5 were 3 and 4 dS m

-1 in the

top and subsoil, respectively. D. spicata achieved its highest growth under both saline and

non-saline irrigation on 28 January 2008 when average temperature was 28 oC and the EC1:5

was ~ 3 dS m-1

in the topsoil and subsoil.

59

2007 2008 2007 2008

Clip

pin

gs D

M (g

m-2)

0

40

80

Sep Dec Mar Jun

A

0

40

80

Sep Dec Mar Jun

B

0

40

80

Sep Dec Mar Jun

C

0

40

80

Sep Dec Mar Jun

D

Figure 3.4. Dry mass (DM) (g m-2

) of clippings of four turfgrass species irrigated with non-

saline water ( ) or saline groundwater (ECw 13.5 dS m-1

) ( ). (A) P. clandestinum,

(B) P. vaginatum, (C) S. virginicus, and (D) D. spicata. Plants were grown in 9 m2 field plots

at Wagin, Western Australia. Plots were mown every 2 weeks. Values are mean of 3

replicates. Error bars denote the SEM. The continuous line under the horizontal axes

indicates the irrigation period; the dotted line indicates the subsequent period without

irrigation after the rainy season commenced. The average LSD (P =0.05) for the means is

2.02.

Analysis of variance showed that the amount of thatch produced varied between the species

(P = 0.008; Table 3.3). P. clandestinum produced the highest thatch (7.6 and 4.8 kg m-2

under

non-saline and saline irrigated conditions, respectively), and the amount of thatch production

declined in the order P. clandestinum > S. virginicus > P. vaginatum > D. spicata (Table

3.3). Although the saline irrigation decreased the production of thatch in P. clandestinum by

36%, it had no effect on the other three species (P = 0.35).

60

Table 3.3. Comparison of DM of the thatch layer of the four turfgrass species at the

conclusion of the trial. Three cores (70 mm in diameter) were taken from each of the 9 m2

plots. Cores were washed free of soil, roots were removed, and the thatch was dried at 65oC

and weighed.

1.89LSD (P < 0.05) species

2.60 ± 0.272.74 ± 0.34Distichlis spicata

3.80 ± 0.483.72 ± 0.87Sporobolus virginicus

3.70 ± 0.853.70 ± 0.57Paspalum vaginatum

4.83 ± 0.727.60 ± 1.91Pennisetum clandestinum

SalineNon-saline

Thatch dry mass (kg m-2)Species

Leaf ion concentrations

In general, the three halophytic turfgrasses were more effective than the non-halophytic

species in regulating Na+ concentrations in the leaves (Figure 3.5; Table 3.2). When irrigated

with non-saline water, the average concentration of Na+ in the clippings were 0.13, 0.15, 0.10

and 0.08 mmol g-1

DM for P. clandestinum, P. vaginatum, S. virginicus and D. spicata,

respectively (Table 3.2). Saline irrigation increased the average concentrations of Na+ in the

clippings of P. clandestinum and P. vaginatum to 0.66 and 0.64 mmol g-1

DM, respectively

whilst this treatment only increased Na+ concentrations to half this level in the clippings of

other two species S. virginicus and D. spicata (0.31 and 0.34, respectively - Table 3.2). The

average ratio of Na+ concentrations for saline plots to non-saline plots during the irrigation

period was 5.1, 4.4, 4.2 and 3.2 for P. clandestinum, P. vaginatum, D. spicata and S.

virginicus, respectively (calculated from Table 3.2). A significant difference was found

throughout the second season between the four species (P < 0.001), between treatments (P <

0.001) and for the interaction between species and treatment (P < 0.001).

61

Leaf

Na

+(m

molg

-1 D

M)

2007 2008

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

B

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

C

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

D

2007 2008

A

Figure 3.5. Concentrations of Na+

in the leaves of four turfgrass species irrigated with non-

saline water ( ) or saline groundwater (ECw 13.5 dS m-1

) ( ). (A) P. clandestinum,

(B) P. vaginatum, (C) S. virginicus, and (D) D. spicata. Plants were grown in 9 m2 field plots

in Wagin, Western Australia and were sampled every 2 weeks. Values are the mean of 3

replicates. Error bars denote the SEM. The continuous line under the horizontal axes

indicates the irrigation period; the dotted line indicates the subsequent period without

irrigation after the rainy season commenced. The average LSD (at 5.0%) for predicted means

is 0.073.

The seasonal variation in concentrations of K+ in the clippings are reported in Figure 3.6 and

average values for the second irrigation season are given in Table 3.2. Significant differences

were found in K+ concentration in the clippings among the four species (P < 0.001) and

between the two irrigation treatments (P < 0.001), but there was no significant interaction

between species and treatments (P < 0.175). When irrigated with non-saline water, the

average concentrations of K+ in the clippings were 0.27 to 0.29 mmol g

-1 DM with P.

clandestinum, P. vaginatum and S. virginicus, but more than double this concentration with

D. spicata (0.64 mmol g-1

DM) (Table 3.2). Saline irrigation decreased the average

concentrations of K+ in the clippings of P. clandestinum and D. spicata to 0.16 and 0.49

mmol g-1

DM, but increased average concentrations of K+ in P. vaginatum and S. virginicus

to 0.33 and 0.37 mmol g-1

DM (Table 3.4). Overall in the second irrigation period, the saline

62

treatment leaf increased K+ concentrations by 39% and 13% in S. virginicus and P.

vaginatum, respectively, whereas it decreased K+ concentrations by 24% and 45% in D.

spicata and P. clandestinum, respectively. In contrast to Na+, there was little clear seasonal

effect on K+ concentrations in the clippings (Figure 3.6).

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

Leaf

K+

(mm

olg

-1 D

M)

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

A B

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

C D

2007 20082007 2008

Figure 3.6. Concentrations of K+

in the leaves of turfgrass species irrigated with non-saline

water ( ) or saline groundwater (ECw 13.5 dS m-1

) ( ). Measurements were made

of (A) P. clandestinum, (B) P. vaginatum, (C) S. virginicus, and (D) D. spicata. Plants were

grown under field conditions in 9 m2 plots in Wagin, Western Australia and were sampled

every 2 weeks. Values are the mean of 3 replicates. Error bars denote the SEM. The

continuous line under the horizontal axes indicates the irrigation period; the dotted line

indicates the subsequent period without irrigation after the rainy season commenced. The

average LSD (at 5%) for predicted means is 0.079.

The general patterns in concentrations of Cl- in the clippings (Figure 3.7; Table 3.2) were

similar to those previously described for Na+, although Cl

- concentrations were either similar

to or slightly higher than Na+ concentrations (Table 4.2). Chloride concentrations in the leaf

tissues varied significantly between the four species (P < 0.001) and between the two

irrigation treatments (P < 0.001), and there was a significant interaction between treatment

and species (P < 0.001). When irrigated with non-saline water, the average concentrations of

Cl- in the clippings were 0.21 mmol g

-1 DM for P. clandestinum and P. vaginatum, and 0.11

63

mmol g-1

DM for S. virginicus and D. spicata (Table 3.2). Saline irrigation increased the

average concentrations of Cl- in the clippings to 0.85, 0.62, 0.39 and 0.41 for P.

clandestinum, P. vaginatum, S. virginicus and D. spicata, respectively (Table 3.4).

Leaf

Cl-

(mm

olg

-1 D

M)

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

A

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

B

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

C

0.0

0.4

0.8

1.2

Sep Dec Mar Jun

D

2007 2008 2007 2008

Figure 3.7. Concentrations of Cl- in the leaves of turfgrass species irrigated with non-saline

water ( ) or saline groundwater (ECw 13.5 dS m-1

) ( ). Measurements were made

of (A) P. clandestinum, (B) P. vaginatum, (C) S. virginicus, and (D) D. spicata. Plants were

grown under field conditions in 9 m2 plots in Wagin, Western Australia and were sampled

every 2 weeks. Values are the mean of 3 replicates. Error bars denote the SEM. The

continuous line under the horizontal axes indicates the irrigation period; the dotted line

indicates the subsequent period without irrigation after the rainy season commenced. The

average LSD (at 5%) for predicted means is 0.062.

The average leaf K+:Na

+ ratio (Table 3.2) under non-saline irrigation was 2.4 to 2.9 for P.

clandestinum, P. vaginatum and S. virginicus, and 8.9 for D. spicata. With saline irrigation

this ratio decreased to 0.47, 0.59, 1.40 and 1.50, respectively. D. spicata and S. virginicus

maintained an adequate K+:Na

+ ratio towards the end of the saline irrigation season.

64

Leaf water content and osmotic potential

Tissue water was highest in the leaves of P. vaginatum, and P clandestinum under non-saline

conditions with average values of 1.93 and 1.78 mL g-1

DM; in contrast, S. virginicus and D.

spicata had average tissue water contents of 1.03 and 0.95 mL g-1

DM. (Table 3.2).Tissue

water content showed a significant difference (P=0.0002) between species and also between

treatments (P=0.009) (Figure 3.8). In general, the percentage decrease in leaf water content

with saline irrigation were in the order P clandestinum > D. spicata > S. virginicus > P.

vaginatum.

The osmotic potentials of the extracted leaf sap have been graphed in Figure 3.9, with the

seasonal average in Table 3.2. There was no significant effect of species on the osmotic

potential of expressed leaf sap (P=0.25), but saline irrigation treatment caused leaf osmotic

potential to become significantly more negative than with the non-saline irrigation treatment

(P < 0.004). The average leaf osmotic potentials over the second summer with non-saline

irrigation were -1.21, -1.29, -1.89 and -1.32 for P. clandestinum, P. vaginatum, S. virginicus

and D. spicata, respectively. With irrigation under saline conditions, these declined to -1.87,

-1.71, -1.94 and -2.07, respectively (Table 3.2). The difference in average leaf osmotic

potential between non-saline and saline irrigation appeared to be affected by species; the

greatest difference was for D. spicata (0.75 Mpa), and this difference decreased in the order

P. clandestinum (0.66 MPa) > P. vaginatum (0.42 MPa) > S. virginicus (0.05 MPa). Six

weeks after concluding the irrigation treatments, the sap osmotic potential of the saline

irrigated plants had returned to relatively similar levels to the non-saline irrigated plants.

65

2007 2008 2007 2008

Le

af w

ate

r co

nte

nt (

mL

g-1

DM

)

0.0

1.0

2.0

3.0

Sep Dec Mar Jun

0.0

1.0

2.0

3.0

Sep Dec Mar Jun

0.0

1.0

2.0

3.0

Sep Dec Mar Jun

A B

D

0.0

1.0

2.0

3.0

Sep Dec Mar Jun

C

Figure 3.8. Leaf water content (mL g-1

DM) of leaves of turfgrass species irrigated with non-

saline water ( ) or saline groundwater (ECw 13.5 dS m-1

) ( ). (A) P. clandestinum,

(B) P. vaginatum, (C) S. virginicus, and (D) D. spicata. Plants were grown under field

conditions in 9 m2 plots at Wagin, Western Australia. Plots were mown every 2 weeks.

Values are the mean of 3 replicates. Error bars denote the SEM. The continuous line under

the horizontal axes indicates the irrigation period; the dotted line indicates the subsequent

period without irrigation after the rainy season commenced. The average LSD (at 5%) for

predicted means is 0.21.

66

-4

-3

-2

-1

0

Sep Dec Mar Jun

-4

-3

-2

-1

0

Sep Dec Mar Jun

C

Leaf

sap o

sm

otic p

ote

ntial (M

Pa)

-4

-3

-2

-1

0

Sep Dec Mar Jun

-4

-3

-2

-1

0

Sep Dec Mar Jun

D

A B

2007 2008 2007 2008

Figure 3.9. Leaf osmotic potential (MPa) of turfgrass species irrigated with non-saline water

( ) or saline groundwater (ECw 13.5 dS m-1

) ( ). (A) P. clandestinum, (B) P.

vaginatum, (C) S. virginicus, and (D) D. spicata. Plants were grown in 9 m2 field plots in

Wagin, Western Australia. Values are the mean of 3 replicates. Error bars denote the SEM.

The continuous line above the horizontal axes indicates the irrigation period; the dotted line

indicates the subsequent period without irrigation after the rainy season commenced. The

average LSD (at 5%) for predicted means is 0.49.

Turfgrass colour

Colour of the turf was assessed on the basis of hue angle; values less than 90 represent a

brown colour, and good colour was indicated by the extent of hue angle readings greater than

this. The colour of the turfgrass has been graphed in Figure 3.10, with the seasonal average in

Table 3.2. There was a significant interaction between species and irrigation treatment (P <

0.001). Under non-saline conditions all species were green with average hue angles between

115 and 119 (Table 3.2). With saline irrigation, the non-halophyte (P. clandestinum) turned

yellow with brown leaf tips within 4 weeks, and the average hue angle decreased from 115 to

100. In contrast, with the three halophytic species greenness was maintained under saline

irrigation with changes in hue angle of +0.5 to -2.7. Impressively, the greenness of D. spicata

67

under saline irrigation was higher than the greenness of the other 3 species even when

irrigated with non-saline water (Table 3.2).

The seasonal data (Figure 3.10) showed another interesting feature of the three halophytic

species. During autumn when average temperatures had decreased to 15 oC in the day and 5.4

oC at night, the halophytic species which had been exposed to prior irrigation with saline

water were substantially greener those plants that had been irrigated with non-saline water.

The best example was D. spicata in which the hue angle was 22 higher during the first two

months in autumn (following saline irrigation) than in plants previously exposed to non-

saline irrigation water (Figure 3.10).

Hue a

ngle

(gre

enness)

2007 2008

80

100

120

Sep Dec Mar Jun

80

100

120

Sep Dec Mar Jun

80

100

120

Sep Dec Mar Jun

80

100

120

Sep Dec Mar Jun

DC

BA

2007 2008

Figure 3.10. Hue angle (greenness) of four turfgrass species irrigated with non-saline water

( ) or saline groundwater (ECw 13.5 dS m-1

) ( ). (A) P. clandestinum, (B) P.

vaginatum, (C) S. virginicus, and (D) D. spicata. Plants were grown in 9 m2 field plots in

Wagin, Western Australia and were sampled every 2 weeks. Values are the mean of 3

replicates, with 3 measurements per plot mean. Error bars denote the SEM. The continuous

line under the horizontal axes indicates the irrigation period; the dotted line indicates the

subsequent period without irrigation after the rainy season commenced. The average LSD (at

5%) for predicted means is 10.62.

68

3.5 Discussion

Salinity of the soil solution

The saline irrigation treatment applied in this experiment affected plant growth because of its

impact on the salinity of the soil solution. This discussion therefore commences with a

description of the changes that occurred in the salinity of the soil solution; the calculation is

made assuming that all the salt contributing to the EC1:5 (Figure 3.2B) is dissolved in the soil

moisture (Figure 3.2C). Seasonal trends in the estimated salinity of the soil solution have

been graphed in Figure 3.11 and the treatment averages are reported in Table 3.2.

Under non-saline conditions, the average ECsoil solution was ~6.5 dS m-1

in both the topsoil and

subsoil over the second irrigation season (Table 3.2). However, with saline irrigation, the

average ECsoil solution in the topsoil and subsoil increased gradually reaching 43 and 38 dS m-1

,

respectively. In the first 8 weeks after commencing saline irrigation the ECsoil solution doubled,

and after 20 weeks it was 4-times its initial value. It is particularly noteworthy that because of

evaporation of the applied water, with the saline treatment, the salinity of the soil solution

reached concentrations in the root zone that were more than 3 times the ECW (Figure 3.11).

This effect was not unique to the saline treatment; the non-saline treatment had an ECw of

0.31 dS m-1

and the ECsoil solution was 7.6 dS m-1

at the beginning of the irrigation season, and

towards the end of the experiment, the ECsoil solution had increased to 8.4 dS m-1

.

2007 2008

EC

So

il s

olu

tio

n(d

S m

-1)

0

25

50

75

Sep Dec Mar Jun

A

Figure 3.11. Estimated salinity of the soil solution in turfgrass plots irrigated with non-saline

water (open symbols) or saline groundwater (ECw 13.5 dS m-1

) (closed symbols). These

values have been calculated assuming that the salts making up the EC1:5 (Figure 3.2B)

contributed to the salinity of the soil solution by being dissolved in the soil water (Figure

3.2C). Measurements were taken of the topsoil (0–25 cm) (circles) and subsoil (25–50 cm)

(squares) in 9 m2 field plots at Wagin, Western Australia. Values are the mean of 5 replicates

with each replicate being an individual plot. Error bars denote the SEM. The continuous line

under the horizontal axes indicates the irrigation period; the dotted line indicates the

69

subsequent period without irrigation after the rainy season commenced. The average LSD (at

5%) for predicted means is 8.54.

Plant growth and colour (Hypothesis 1)

In this study, growth was assessed by measuring the DM of shoot clippings; this seemed

justified for two reasons: (a) root growth could not be measured easily in a non-destructive

way during the course of the experiment, and (b) shoot growth is more sensitive to salinity

than root growth (Shalhevet et al., 1995; Bernstein et al., 2001).

It was hypothesised that the three halophytic turfgrass species would maintain better shoot

growth and retain colour better than the non-halophyte (P. clandestinum) under saline

conditions. Overall the results of the present study did not support hypothesis 1 in regards to

maintaining growth under saline irrigation; in the event, the halophyte P. vaginatum and the

non-halophyte P. clandestinum had higher DM of clippings under saline conditions than the

other two halophytes (D. spicata and S. virginicus). However, the results did support the

retention of green colour in halophytic species; the average colour remained unchanged for

the three halophytic species but the hue angle declined by 13% in P. clandestinum. Based on

greenness, species grown under saline irrigation decreased in the order: D. spicata > S.

virginicus > P. vaginatum > P. clandestinum (Table 3.2).

This section of the discussion will discuss two strategies to achieve high growth under

conditions of salinity, increasing plant vigour and increasing salinity tolerance, and also

elaborate on the quality of turfgrass species under salinity. Ecophysiologists widely report the

effects of abiotic stress on growth, in terms of impacts on vigour (growth under non-stressed

conditions) and tolerance (the ratio of growth under saline conditions to growth under non-

stressed conditions). In theory, a plant could grow strongly under saline conditions because it

had high vigour, high tolerance to salinity or a combination of both traits. In the present

work, it had been expected that the most rapidly growing species under non-saline conditions

would be the non-halophyte P. clandestinum, however the halophytic species P. vaginatum

had 63% more DM than P. clandestinum and far higher growth than the other two species

showing that P. vaginatum was the most vigorous of the studied species.

As expected, the four species differed substantially in salt tolerance. The halophytic species

D. spicata and S. virginicus were the most salt tolerant species with average ratios of DM

70

under saline irrigation to DM under non-saline irrigation of 0.92 in D. spicata and 0.83 in S.

virginicus. In contrast, the more vigorous P. vaginatum and P. clandestinum had average

ratios of DM under saline irrigation to DM under non-saline irrigation of 0.77 and 0.60 and

were ranked third and fourth, respectively, in salinity tolerance (calculated from data in Table

3.2). Overall, in the present work species salt tolerance (ratio of clipping DM under saline

conditions to clipping DM under non-saline conditions) decreased in the order D. spicata

(1.01) > S. virginicus (0.91) > P. vaginatum (0.88) > P. clandestinum (0.74) for the studied

species (calculated from data in Table 3.2).

The different degrees of growth reduction in response to salinity can help in classifying these

species for use as turfgrass surfaces under saline irrigation. In high traffic areas, I would

recommend the fast growing species P. vaginatum, whereas for road sides and parks, D.

spicata and S. virginicus would seem suitable, since the colour of the three species did not

decline with saline irrigation.

Ion relations (Hypothesis 2)

The second hypothesis was that the two major traits contributing to salinity tolerance in the

halophytic grasses would be an enhanced ratio of K+:Na

+ in the leaf tissues and their ability

to exclude the toxic ions (Na+ and Cl

-). All grasses are essentially excluders as they have

mechanisms to exclude ions at the root level; yet I discuss here the exclusion at the leaf tissue

level. It will be argued here, that the hypothesis is supported; both the K+:Na

+ ratio and (Na

+,

Cl-) exclusion were linked to salinity tolerance. D. spicata and S. virginicus have shoot

exclusion mechanisms and presumably regulate ion entry via roots (Marcum, 1999), whereas

P. vaginatum have well-controlled root exclusion mechanisms (Lee et al., 2007).

In the present work, I have argued that salt tolerance can be assessed as the ratio of clipping

DM under saline to clipping DM under non-saline conditions, and that the 4 species therefore

decreased in salt tolerance as follows: D. spicata > S. virginicus > P. vaginatum > P.

clandestinum. The link between this assessment of tolerance and K+:Na

+ ratio is clear; under

saline irrigation, the average K+:Na

+ in the clippings were 1.5, 1.4, 0.59 and 0.47 for D.

spicata , S. virginicus, P. vaginatum and P. clandestinum, respectively (Table 3.2).

Different turfgrass species employ different mechanisms to tolerate salinity. The maintenance

of a high ratio of K+:Na

+ seems to be an important physiological mechanism for D. spicata

71

and P. vaginatum to tolerate the high salt concentrations. High ratios of K+:Na

+ have been

reported to keep cells functioning, enabling protein synthesis and the regulation of stomatal

opening and closure (Zhu et al., 1998; Chow et al., 1990).

Both toxic ions (Na+ and Cl

-) increased with saline irrigation, a range of 3-fold more in D.

spicata and S. virginicus, 4-fold more in P. vaginatum whereas for the non-halophyte, the

increase was 5-fold. However, the concentration of these toxic ions in S. virginicus and D.

spicata was 50% of that in the non-halophyte species, and for P. vaginatum, it was 73%

(Table 3.2).

The case for stating that salt tolerance was related to the exclusion of Na+ and Cl

- from the

tissues is strong (but not as strong) as for the role of K+:Na

+. With saline irrigation, the

respective average Na+ and Cl

- concentrations (mmol g

-1 DM) increased in the order S.

virgincus (0.31 and 0.39) < D. spicata (0.34 and 0.41) < P. vaginatum (0.64 and 0.62) < P.

clandestinum (0.66 and 0.85), an order that was not exactly identical to the order of

increasing salt tolerance (Table 3.2).

Results of the present study regarding ion concentrations in leaf tissues (Na+ and Cl

-) of 3

halophytic turfgrass species vs non-halophytic are in agreement with those reported by

(Marcum and Murdoch; 1992; Naidoo and Naidoo, 1998; Bell and O’Leary, 2003; Marcum

et al., 2007; Lazarus et al., 2011) whereby salt-tolerant grasses controlled the accumulation

of Na+ and Cl

- in the leaf tissues under saline irrigation, yet the accumulation did not exceed

levels required for osmotic adjustment.

The K+:Na

+ ratio is a key requirement for plant growth in high salt (Glenn et al., 1999).

K+:Na

+ ratio in the grass halophytes is typically higher than that of glycophytes (Gorham et

al., 1985; Yeo, 1998), however significant variations have been reported on the negative

correlation between salinity tolerance and K:Na ratio among the 10 genotypes of D. spicata

(Lazarus et al., 2011). Nevertheless, even halophytes can suffer declines as demonstrated by

a study of Sporobolus virginicus with substantial decline in K+:Na

+ ratio within the shoots as

a result of increasing salt concentration from 0.1 dS m-1

to seawater (Marcum and Murdoch,

1992).

72

The distinction of some halophytes comes from their ability to utilise Na+ as an osmolyte to

adjust the internal osmotic potential. It is reported that substantial accumulation of Na+ and

Cl- up to 30 and 50% of the plant dry mass respectively occurs in some of the dicotyledonous

species (Gorham et al., 1980; Flowers et al., 1977). In the monocotyledonous halophytes,

from Poaceae family in particular, these have higher K:Na ratio and relatively low tissue

water content as well (Flowers et al., 1977). The K+:Na

+ ratio was higher in leaves if

compared to roots of Sporobolus virginicus (Bell and O’Leary, 2003). K+ (inorganic) and

sugar (organic) are the major osmolytes within the monocotyledons (Jeschke, 1984).

Growth after saline irrigation was ceased upon rainfall

In the present study, 2 weeks after ceasing both irrigation treatments (saline and non-saline),

the plots received 38 mm of rain and the minimum and maximum air temperatures declined

by 3 and 4 oC, respectively. Over this time, the ECsoil solution decreased by 37% and 15% in the

topsoil and subsoil; these conditions led to a doubling in the growth (clipping DM) of the

non-halophyte P. clandestinum compared with its growth during the final two weeks of saline

irrigation. In contrast, the halophytes (P. vaginatum and S. virginicus) had 82% of clipping

DM and 91% for D. spicata compared with growth during the final two weeks of saline

irrigation. With P. clandestinum plots previously irrigated with saline water had 5% more

growth than those irrigated with non-saline water during the recovery period of 2 weeks.

Results in this regards indicate that the recovery appeared to be more pronounced in the non-

halophyte P. clandestinum as it can recover by doubling its growth as fast as in 2 weeks after

salinity stress, the fast recovery indicates some acclimation/adaptation to salinity conditions,

whereas the halophytes continued their previous good performance.

3.6 Conclusion

There is potential to save fresh water resources through allocating the saline water resources

to irrigate halophytic turfgrass species like: D. spicata, S. virginicus and P. vaginatum, these

species grew with less thatch accumulation and high colour retention with salinity levels up

to one fourth seawater (salinity of seawater is 55 dS m-1

) without causing degradation in soil

physical and chemical properties, or a rising water table, provided that subsurface drainage

network was installed. Stimulation of turfgrass growth under saline conditions did not occur

in the halophytic species and some of them (D. spicata and S. virginicus) were clearly slow

growing species. Use of slow-growing species should not be a concern as many of the

73

turfgrass amenities are used in public places and low traffic areas thus fast growth and

recovery is not needed; instead it is recommended to have slow growing species to minimise

the turfgrass maintenance requirements (i.e. salt-tolerant rather than vigorous species).

Potentially toxic ions (Na+ and Cl

−) can be absorbed by plant roots and translocated to leaves,

where they accumulate in sensitive plants. This accumulation leads to leaf discolouration and

reduction of colour quality, however with high salt-tolerant species, a better mechanism of

ion exclusion was employed to maintain the low Na+ and Cl

−, and retain high K

+:Na

+ ratio.

74

3.7 Appendices

Appendix 3.1. Average data (weather, soil and plant DM, leaf ions, osmotic potential and

colour) over the first irrigation season (15 December 2006 to 5 August 2007)*.

P. clandestinum P. vaginatum S. virginicus D. spicata

Minimum

Maximum

Non-saline

Saline

Non-saline

Saline

Non-saline

Saline

Non-saline 0.13 ± 0.02 0.22 ± 0.04 0.10 ± 0.02 0.07 ± 0.01

Saline 0.97 ± 0.1 0.66 ± 0.01 0.51 ± 0.03 0.41 ± 0.05

Non-saline 0.49 ± 0.03 0.57 ± 0.12 0.55 ± 0.06 0.42 ± 0.05

Saline 0.54 ± 0.04 0.52 ± 0.02 0.47 ± 0.06 0.56 ± 0.12

Non-saline 0.22 ± 0.02 0.13 ± 0.05 0.13 ± 0.02 0.15 ± 0.01

Saline 1.07 ± 0.12 0.50 ± 0.13 0.37 ± 0.02 0.40 ± 0.14

Non-saline 3.76 ± 0.1 2.59 5.5 ± 0.1 0.60

Saline 0.56 0.79 0.92 1.37

Non-saline -1.1 ± 0.03 -1.4 ± 0.05 -1.8 ± 0.02 -1.5 ± 0.05

Saline -2.6 ± 0.33 -2.2 ± 0.17 -2.6 ± 0.12 -2.8 ± 0.13

Non-saline 119.7 ± 2.6 122.0 ± 1.9 113.0 ± 2.8 123.4 ± 3.5

Saline 115.3 ± 1.3 119.7 ± 2.7 119.0 ± 1.6 127.8 ± 0.7

Leaf Na+

(mmol g-1

DM)

EM38h (dS m-1

)

Soil EC1-5 (dS m-1

) (25 - 50 cm)

Soil EC1-5 (dS m-1

) (0 - 25 cm)

Colour (Hue angle)

Leaf K:Na ratio

Leaf Cl- (mmol g

-1 DM)

Leaf K+

(mmol g-1

DM)

Leaf Sap Osmotic Potential (MPa)

ParameterSpecies

41.2

7.9 ± 0.1

Treatment

1.0Air temperature

oC

12.5 ± 0.3

3.8 ± 0.1

13.8 ± 1.1

2.8 ± 0.1

4.6 ± 0.4

*Temperature data are the mean ± SEM of 87 daily records. Soil salinity and soil water

content data are the mean ± SEM of 5 measurements. Ion data, leaf sap osmotic potential and

colour data are the mean of 3 measurements ± SEM on harvest days at ~ 2 week intervals

across the irrigation season.

Appendix 3.2. Approximate relationship between ECe and EC1:5 for different soil textures

(modified from Duncan and Carrow, 1998; Anderson et al., 2007)*.

Soil Texture ECe : EC1:5

Sand, loamy sand ECe = 15 X EC1:5

Sandy loam ECe = 12 X EC1:5

Clay loam ECe = 9 X EC1:5

Clay ECe = 6 X EC1:5

Soil containing gypsum will have different conversion factors.

75

Appendix 3.3. Chemical analysis of the saline irrigation water. Measurements were taken

every month and the presented data are the averages of 3 data sets measured during the

second irrigation season.

Test Unit Value Test Unit Value

EC dS/m 15.38 Mg mg/L 180

pH 6.5 Mn mg/L 0.28

Water

temperature oC 20.2 NO2 mg/L <0.02

Ag mg/L <0.005 NO3 mg/L 0.01

Al mg/L <0.005 Na mg/L 2570

Alkalinity mg/L 315 Ni mg/L <0.01

As mg/L <0.001 P_SR mg/L <0.01

Ba mg/L 0.068 P_total mg/L <0.01

Be mg/L <0.001 Pb mg/L <0.0005

CO3 mg/L <1 S mg/L 130

Ca mg/L 344 SO4 mg/L 390

Cd mg/L <0.002 Sb mg/L <0.05

Cl mg/L 4540 Se mg/L <0.05

Cr mg/L <0.001 SiO2 mg/L 65

Cu mg/L <0.005 Solid_su mg/L 6

F mg/L 1.2 Sr mg/L 2.2

Fe mg/L 0.064 TDS sum mg/L 8200

HCO3 mg/L 385 TDS_180C mg/L 8000

Hg mg/L <0.0001 TOC mg/L 1

K mg/L 13.6 Zn mg/L 0.01

76

77

Chapter 4

Responses of tissue ions and growth of four turfgrass species to

increasingly saline conditions

4.1 Abstract

A glasshouse experiment was conducted to evaluate responses of tissue ion concentrations

and growth of four turfgrass species to increasing NaCl salinity in sand culture irrigated with

a nutrient solution. The species evaluated were a non-halophyte Pennisetum clandestinum,

and three halophytes Paspalum vaginatum, Sporobolus virginicus and Distichlis spicata.

Saline irrigation (0, 125, 230, 500 and 750 mM NaCl added to the nutrient solution)

commenced after full coverage of the pots, for 60 days. Growth of the non-halophyte P.

clandestinum was vigourous in control conditions, but was progressively retarded by

increasingly saline conditions. The halophyte P. vaginatum was intermediate in vigour and

also suffered growth reductions under salinity, whereas the two other halophytes were of low

vigour but maintained low growth rates in saline conditions. Growth stimulation by salinity

was only noted in the belowground growth (roots and rhizomes) for S. virginicus. Leaf Na+

and Cl- concentrations under saline conditions (e.g. 230 mM NaCl) were highest in P.

clandestinum, whereas the three halophytes maintained lower concentrations of these ions in

leaves: S. virginicus (32% of the Na+ in P. clandestinum), D. spicata (53%) and P. vaginatum

(67%). Declines in leaf K+:Na

+ ratio as salinity increased were correlated with aboveground

growth in the four species (R2=0.97). An outdoors weighing lysimeter experiment showed

water use by the four species ranged from 51-63 % of Epan, and it declined by 22% at ECw

78

13.5 dS m-1

in P. clandestinum, whereas declines were much less in S. virginicus (9%), P.

vaginatum (5%), and D. spicata was not affected. In summary, salinity tolerance of

halophytic turfgrass species was demonstrated in controlled conditions, with tolerance

apparently associated with ability to regulate leaf ion concentrations.

4.2 Introduction

Use of halophytes as turfgrasses in saline areas requires an understanding of the responses of

the various available genotypes to increasing levels of salinity. Assessments of salt tolerance

of non-halophytic turfgrasses are available in the literature (e.g. Marcum, 2008), but few

studies have addressed salinity tolerance of halophytic turfgrasses, with the notable exception

of P. vaginatum and D. spicata (Marcum, 1999; Carrow et al., 2005; Marcum et al., 2005;

Marcum et al., 2007).

Assessments of growth and quality of turfgrasses targeted for saline areas should include

non-saline, low, intermediate, and high salinity levels (Duncan and Carrow, 1999). In

particular, information is needed on the responses of halophytic turfgrasses to hypersaline

conditions. Screening for salinity tolerance under controlled environmental conditions is

more efficient than under field conditions (Shannon and Noble, 1990), as salinity is spatially

uneven in fields and salinity also changes according to the soil water content (Rengasamy,

2002). Ultimately, however, genotypes need to be tested for performance in field conditions

(Royo and Aragues, 1999).

High concentrations of ions in the root zone reduces soil water potential, which consequently

reduces the water availability to plants (Rengasamy, 2002). When excess ions enter the

shoots, growth reductions can also result from ion toxicity in plant tissues (Munns and Tester,

2008). Halophytes, in contrast to non-halophytes, can tolerate saline conditions owing to a

suit of physiological traits, amongst which regulation of internal ion composition, ion

compartmentation, and osmotic adjustment appears to be features of major importance

(Flowers and Colmer, 2008).

Salt tolerance in non-halophytic turfgrasses is limited, such as for P. clandestinum

(Harivandi, 1984; Rogers et al., 2005), for Zoysia pacifica (Marcum, 2008), so that use of

halophytic species has been proposed for sites with saline soils/saline irrigation water

79

(Carrow et al., 2005). The present work focuses on three halophytic turfgrasses. P. vaginatum

tolerates saline irrigation with ECw of 6–22 dS m-1

and survives irrigation with 40 dS m-1

(Semple et al., 2003), with others also reporting tolerance of irrigation with ECw 41 dS m-1

(Duncan and Carrow, 1999). D. spicata is highly salt tolerant (only 50% growth reduction at

ECw of 35 dS m-1

), and can survive irrigation with ECw as high as 40 dS m-1

, but grows best

when irrigated with ECw of ~20 dS m-1

(Leake et al., 2002). Salt tolerance in D. spicata has

been attributed partly to the presence of salt-excreting glands on its leaves (Alshammary et

al., 2004; Marcum et al., 2007). S. virginicus is a third halophytic turfgrass option now

available, as a turf type was selected in Australia (D. Loch, pers. comm.). S. virginicus was

reported to tolerate ECw of 55 dS m-1

(Gallagher, 1979), but the accession tested was not a

turf type. Salt tolerance of S. virginicus is attributed to tissue ion regulation, partly due to the

presence of salt-excreting glands on its leaves and discrimination between Na+ and K

+ during

transport to shoots (Bell and O’Leary, 2003). Despite this available background knowledge,

direct comparisons of the tolerances of these three halophytic turfgrasses were lacking; so,

this was the objective of the experiments described here.

Two experiments were conducted. The main experiment evaluated NaCl dose-responses of

four species Distichlis spicata, Sporobolus virginicus, Paspalum vaginatum (three

halophytes) and Pennisetum clandestinum (non-halophyte). Above-ground and below-ground

dry mass, tissue ions, tissue water content and osmotic potential were evaluated over a wide

range of NaCl concentrations in sand culture. A second experiment used outdoor weighing

lysimeters to measure turfgrass water use by the four species. The lysimeters contained a

relevant field soil and were irrigated with either scheme water or saline groundwater (same

soil and water as used in Chapter 3). Turfgrass water use varies amongst different species and

genotypes (Kneebone and Pepper, 1982; Carrow, 1995), and can change as influenced by

environmental stress (Nishat et al., 2007), including salinity, so that the water use data

provide additional insights into functioning of the four species under semi-field conditions.

The following hypotheses were tested: 1) The three halophytic grasses will suffer less growth

declines in response to increasing salinity than the non-halophyte. 2) The three halophytic

grasses will prevent large increases in Na+ and Cl

- concentrations in leaf tissues, as compared

to the non-halophyte, when external soil salinity increases. 3) Shoot growth reductions under

salinity will be correlated with increases in leaf tissue Na+ and/or Cl

- concentrations, both in

80

the halophytes and the non-halophyte. 4) Water use by the halophytes will be maintained

under saline irrigation, whereas water use by the non-halophyte will decline markedly.

4.3 Materials and methods

Experiment 1

The grasses were propagated from rhizomes in a coarse white sand and irrigated with a

nutrient solution containing (mM): 4.7 K2SO4, 9.3 CaCl2, 5.0 Na2SO4, 1.0 MgSO4, 0.05 Fe

EDDHA (‘Sequestrene 138’), 0.7 Ca(NO3)2, 0.3 K2HPO4, 0.2 NH4H2PO4; and (µM): 25

H3BO3, 2 MnSO4, 2 ZnSO4, 0.5 CuSO4, 0.5 Na2MoO4. The nutrient solution was adjusted to

pH 6.6, using KOH. The nutrient solution contained Ca2+

(10 mM) at a concentration

equivalent to that in seawater, such that growth responses in the higher-salinity treatments

would be determined by direct Na+ and Cl

- effects, rather than Na-induced Ca

2+ deficiency

(Greenway and Munns, 1980; Kopittke and Menzies, 2005). No symptoms of Ca2+

deficiency

were observed during the experiment.

Plants were established in 10 L pots for 3 months inside a glasshouse (July, August and

September 2008; Perth, Western Australia). Air temperatures in the glasshouse were,

respectively, 0, 31.2 and 20.8 oC for the minimum, maximum and average temperature. Pots

contained a coarse gravel layer of 4 cm height topped with 3 cm height of fine gravel and 10

cm height of coarse white sand; the gravel layers enabled daily flushing of the pots without

sand erosion. Irrigation volume of 350 mL of nutrient solution was applied daily thought the

entire experiment. Shoots were clipped to a uniform size (30 mm height) prior to

commencing the NaCl treatments, and initial samples were taken on 25th

September, 2008.

Five treatments were imposed: control (no NaCl in addition to that in the basal solution), 125,

230, 500 and 750 mM NaCl), with four replicates of each treatment x species combination in

a randomised complete block (RCB) design.

Treatments were imposed by 50 mM NaCl increments in the complete nutrient solution every

day, so that the highest NaCl level was reached on 5th

October 2008, and these levels were

maintained for 60 days by daily irrigations with the appropriate solution, and the final harvest

was taken on 4th

December 2008. Treatments continued for 60 days after the highest salt

concentration (750 mM NaCl) had been reached. Plants were not clipped during the treatment

period.

81

Quality of turfgrass was assessed by visual scores on a scale from 1 to 7, with 7 representing

dark green turfgrass and 1 representing brown colour (1 = dead, 3 = marginal, 5 = fully

acceptable, 7 = best possible). At harvests, leaf tissues were rinsed with de-ionised water and

left to dry for ~15 minutes; this procedure washed off any secreted salts or salts accumulated

on leaf surfaces from irrigation. Sub-samples of leaf tissues were excised and put into1.5 ml

air-tight cryo-vials, frozen in dry ice, and then stored at -80 oC prior to measurements of leaf

sap osmotic potential. Samples were thawed while still in the sealed vials, squeezed in a

press, and the osmotic potential of the expressed sap was measured using an osmometer

(F110, Fiske Associates, Needham Heights, MA, USA). After the final harvest of plants in

this experiment, plants were separated into aboveground tissues (leaves and shoots) and

belowground tissues (roots and rhizomes), both tissues were measured. The four species are

rhizomatous species while P. vaginatum, P. clandestinum and S. virginicus also form stolons.

Leaf tissue samples were collected using scissors and the fresh mass was recorded; samples

were then wrapped in aluminium foil, frozen in dry ice, transported to the laboratory, stored

at -80 oC and then freeze-dried. Dry mass was recorded and samples were then ground in a

ball and mill. Belowground organs were washed free of sand by three washes in de-ionised

water containing mannitol at a concentration of 0.0, 41.9, 77.09, 167.6 or 251.39 g L-1

so that

the osmotic concentration of mannitol equalled the osmotic concentration of the respective

irrigation treatments with 0, 125, 230, 500 and 750 mM NaCl; 5 mM CaSO4 was also added

to the washing solution.

Relative growth rate (RGR) was calculated as: RGR = (lnW2 - lnW1) / (t2 - t1) where W1 is

the dry mass at the initial harvest and W2 is the dry mass (DM) at the final harvest, t2 is the

date of the final sampling and t1 is the date of the initial sampling. Fresh masses of plant

material were measured, frozen in liquid N2 prior to being freeze-dried, and then dry masses

were recorded. Tissue water content was calculated from the fresh and dry mass data. Dried

samples were then ground to a fine powder in a ball and mill, and subsamples used for

measuring ions (described below).

Ions (Na+, K

+ and Cl

–) were extracted from ~0.1 g of ground tissue in 10 mL of 0.5 M HNO3

in plastic vials. The samples were shaken for 48 h in the dark at 30oC. Diluted extracts were

analysed for Na+ and K

+ using a flame photometer (Jenway PFP7 Flame Photometer; Essex,

UK), and Cl– was measured using a chloridometer (Buchler–Cotlove chloridometer 662201;

82

Fort-Lee, NJ, USA). The reliability of the ion measurements was confirmed using a reference

plant tissue sample with known concentrations of the three ions. The recovery of the ions

from the reference material was 91% - 96%; no adjustments were made to the data presented.

Experiment 2

This experiment focused on the water use of the four grasses in weighing lysimeters (91 cm

high x 15 cm diameter, PVC tubes with a bottom fitted containing a central drainage point).

Lysimeters contained soil from the field site described in Chapter 3, packed (top 80 cm of

soil) to the same bulk density as at the site (1.6 g cm-). The lysimeters were located in an

outdoor area on the UWA campus, Crawley, Western Australia, and irrigated daily at 100%

replacement of net evaporation measured on-site with a Class-A Evaporation Pan. Monthly

average maximum, minimum and mean air temperatures are given for Perth City (Appendix

4.2).

Two irrigation treatments were imposed, saline (ECw 13.5 dS m-1

) groundwater obtained

from the field site at Wagin, Western Australia, and described in Chapter 3, and a non-saline

treatment of domestic tap water. The experiment had three replicates of each species by

irrigation combination, being a total of 24 lysimeters. Water use was measured on five

occasions over three months, dates were: 10 January, 28 February, 1 March, 13 March and 19

March 2008. Evapotranspiration (ET) values on those dates were 8.36, 7.43, 8.53, 5.84 and

8.0 mm, respectively.

Water use (WU) from the lysimeters was calculated as:

WU= (M1-M2) - D

where:

M1 is the mass of the lysimeter immediately after irrigation

M2 is the mass of lysimeter 24-h after M1, and

D is the mass of the drainage water.

These data on volume of water used were then converted to a surface area basis, so that ET

(mm) of turfgrass could be compared with class-A pan evaporation during the same 24 h

period.

83

A chromameter (Minolta, CR-310, Osaka, Japan) was used to measure the colour of the

turfgrass in the weighing lysimeters. The chromameter applies a pulse of light and measures

reflected light in the red, green, and blue spectral ranges, with the result expressed as the hue

angle (for further details see Barton et al., 2009). The chromometer measures three

parameters (L*, a*, b*). L* refers to the lightness of the reading, with values ranging from

black = 0 to white = 100. A* refers to the green/redness of the turfgrass colour with values

ranging from -60 indicating green to +60 indicating red-purple, and b* indicates the

yellow/blueness of the turfgrass colour with values ranging from 90 indicating yellow to 180

indicating blue (McGuire, 1992). The hue angle [h˚ = tan-1

(b*/a*)] was calculated from a*

and b* values (Lancaster et al., 1997). The device was calibrated using a calibration plate

(CR-A44 Minolta, Osaka, Japan) every 18 measurements following the manufacturer’s

instructions. Colour changes were quantified by recording three positions in each lysimeter to

obtain a mean value then used to represent each experimental unit.

.

Each lysimeter was clipped weekly during the study (January, February and March 2008).

The turfgrass clippings were weighed and a subsample was taken, weighed and the oven-

dried at 65 °C and the DM was recorded.

Statistical analyses of data

Analysis of variance was performed using Genstat 10th

Edition (VSN International Ltd).

Treatment effects, species differences, and species by treatment interactions were judged to

be significantly different at P < 0.05.

4.4 Results

Growth

The non-halophyte P. clandestinum and the two halophytes (P. vaginatum and D. spicata)

had almost similar (~ 0.04 g g-1

d-1

) relative growth rates (RGR) in the non-saline controls,

whereas S. virginicus had half the RGR of the other two halophytes. Irrigation with 125 mM

NaCl in the nutrient solution caused mortality of the leaves of P. clandestinum by the end of

the experiment, under 750 mM NaCl irrigation treatment the mortality occurred earlier (in the

second week) and at 500 mM NaCl by the third week (Appendix 4.1). The absolute growth in

the aboveground tissues of P. clandestinum was the highest (66 g DM at 0 mM NaCl) and

84

had declined by 80% at 125 mM NaCl treatments (Figure 4.1A), and for the belowground

tissues it was 42 g DM and this declined to just 23% at 750 mm NaCl (Figure 4.1B).

The absolute growth of P. vaginatum in the aboveground tissues was the second highest at 33

g DM for the 0 mM NaCl treatment and 18 g DM for 125 mM NaCl, and its growth under

750 mM NaCl was half of the no-salt treatment. For the belowground tissues, growth of P.

vaginatum was the second highest after P. clandestinum under 0 mM NaCl and double its

value at the other 4 treatments. S. virginicus had aboveground DM values ranging from 10 g

for the no-salt to ~ 6 g for the maximum salt treatment (750 mM NaCl), the average

belowground DM to the aboveground DM was 4-fold more across all treatments (Appendix

4.3). D. spicata had the lowest aboveground tissues of 7.5 g DM at no-salt treatment and it

declined to 50% at the maximum salinity level. The average belowground DM to the

aboveground DM was 4-fold more across all treatments (Appendix 4.3).

Growth as percentage of control showed that there were no growth stimulations at any

salinity level in the aboveground tissues, however the belowground DM doubled in the

halophyte S. virginicus at 125 mM NaCl whilst decrease the belowground DM at higher

concentration. The two-way ANOVA test for the belowground DM showed differences

amongst species (P =0.007), treatments (P =0.008) and also for the interaction between

species and treatments (P =0.003). All levels of salts reduced aboveground growth (P

<0.001) in P. vaginatum and P. clandestinum and were not affected in S. virginicus and D.

spicata.

The general trend of the relative growth rate data (Figure 4.1E,F) showed that the order of

aboveground growth was P. vaginatum > D. spicata > S. virginicus > P. clandestinum. For

the belowground growth the order was D. spicata > P. vaginatum > S. virginicus > P.

clandestinum with exception of S. virginicus being the second highest at higher salt

concentrations (250 and 500 mM NaCl). The aboveground RGR was declining as salinity

increased in the irrigation solution, however the belowground RGR declined much less and

even showed the above discussed stimulation effect on S. virginicus.

85

0

20

40

60

80

0 200 400 600 800

0

20

40

60

80

0 200 400 600 800

Tis

sue D

M (

g)

NaCl added to the irrigation solution (mM)

DM

% o

f contr

ol

Rela

tive G

row

th R

ate

(g.g

-1d

-1)

0%

50%

100%

150%

0 200 400 600 800

0%

50%

100%

150%

0 200 400 600 800

0%

2%

4%

0 200 400 600 800

B

C D

F

A

0.00

0.02

0.04

0.06

0 200 400 600 800

E

Figure 4.1. Effect of NaCl added to nutrient solution on the dry mass (DM) (A,B), DM as %

of control (C,D) and relative growth rate (E,F) in the aboveground (left-side graphs) and

belowground (right-side graphs) of perennial grasses grown in irrigated sand culture under

glasshouse conditions for 60 days (Experiment 1). The grasses were: P. clandestinum (

), P. vaginatum ( ), S. virginicus ( ), and D. spicata ( ). Values are the

average of 4 replicates. Error bars denote the SEM. P values for the aboveground DM were

0.04 (species), 0.007 (treatment) and 0.18 (species x treatment); LSDs for DM were 12.2

(species) and 13.6 (treatment). P values for the belowground DM were 0.007 (species), 0.003

(treatment) and 0.008 (species x treatment); the LSDs were 5.7 (species), 6.4 (treatment) and

12.7 (species x treatment).

86

Tissue ion relations

The overall trend in (Figure 4.2A,B) showed that D. spicata and S. virginicus had

consistently lower concentrations of the toxic ions (Na+ and Cl

-) than P. vaginatum, this

suggests that either the belowground organs of the two species differ in their ability to

exclude Na+ and/or that these two species excrete ions via their glands.

A comparison of the Na+ in the aboveground biomass shows that the non-halophyte P.

clandestinum showed sharp increases in aboveground Na+ (8-fold more at 125 mM NaCl

treatment and 16-fold more at 250 mM NaCl) which might have caused leaf firing and leaf

senescence (Figure 4.2 and Appendix. 4.1); ion concentration was measured for dead leaves

of P. clandestinum at higher salt concentrations (500 and 750 mM NaCl). By contrast, the

three halophytes: P. vaginatum, S. virginicus and D. spicata maintained low tissue Na+ at

lower salinity levels (up to 230 mM NaCl), but as salinity increased substantial increases in

the aboveground Na+ occurred for P. vaginatum whilst the S. virginicus and D. spicata

remained unchanged. It is therefore considered that the higher salinity tolerance observed for

S. virginicus and D. spicata is due largely to its ability to exclude and/or secrete Na+ from its

leaves (Marcum, 2006).

A two-way ANOVA of the ion concentrations revealed that salinity significantly

progressively increased in both Na+

and Cl- concentrations in the above and belowground

tissues, but for P. vagintum Na+ and Cl

- had greater increase in shoots compared to roots

(Figure 4.2 A,B, E, F).

87

NaCl added to the irrigation solution (mM)

Tis

sue

io

n c

on

ce

ntr

ation

s (

mm

olg

-1D

M)

Na

+K

+

B

D0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 800

A

0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 800

C

B

D

E F

Cl-

Figure 4.2. Effects of concentration of NaCl added to the nutrient solution on the

concentrations of Na+ (A,B), K

+ (C,D) and Cl

- (E,F) in the aboveground tissues (left-side

graphs) and belowground tissues (right-side graphs) of P. clandestinum ( ), P.

vaginatum ( ), S. virginicus ( ), and D. spicata ( ). Plants were grown in

irrigated sand culture under glasshouse conditions for 60 days. Values are the average of 4

replicates. Error bars denote the SEM. P values for Na+, K

+ and Cl

- in the aboveground was <

0.001; the LSDs are 0.04, 0.04 and 0.09 respectively. P values for Na+, K

+ and Cl

- in the

belowground was < 0.001; the LSDs are 0.03, 0.03 and 0.05, respectively.

In addition to ion toxicity, ion imbalances can also influence plant growth and tolerance of

salinity. In this study, K+:Na

+ ratio declined in the aboveground biomass, the ratio ranges

between 10 for the non-halophyte (P. clandestinum), for the halophytes, 15 for P. vaginatum,

30 for S. virginicus and 3 for D. spicata at 0-added NaCl while it was 3 for S. virginicus and

1 for the other 3 species at 125 mM NaCl. The external K+:Na

+ ratio in the irrigation solution

was 1, 0.07, 0.04, 0.02 and 0.01 for 0, 125, 230, 500 and 750 mM NaCl concentration.

88

Table 4.1. Effects of concentrations of NaCl added to the nutrient solution on the K+:Na

+

ratio in the aboveground mass and belowground mass of the four turfgrass species. Plants

were grown in irrigated sand culture under glasshouse conditions for 60 days. Values are the

average of 4 replicates.

Portion

Concentration

(mM NaCl) P. clandestinum P. vaginatum S. virginicus D. spicata

Belowground

tissues

0 9.7 15.4 29.8 2.7

125 0.9 0.8 3.1 1.0

230 0.5 0.4 2.2 0.5

500 0.3 0.2 0.8 0.4

750 0.2 0.1 0.7 0.4

Aboveground

tissues

0 3.1 15.7 4.8 13.4

125 0.6 1.6 1.2 1.3

230 1.1 0.9 0.8 0.7

500 0.4 0.6 0.6 0.7

750 2.0 0.5 0.5 0.4

Correlations were established between the growth as % of control and the tissue ion

concentrations (Na+, K

+, Cl

- and K

+:Na

+) as they were significant (Figure 4.3 and Appendix

4.5), other correlations (absolute DM to ion concentrations were not significant and therefore

not shown. Correlations showed that increasing Na+ and Cl

- concentration in the aboveground

tissues corresponds to the reduction in growth as a % of non saline control, also the reduction

in K+

concentration in the aboveground correlates with the reduction in growth (Figure 4.3),

equations and R2 are given in (Appendix. 4.5). Detailed introduction of the correlations is

given in the discussion section of this chapter.

89

Gro

wth

% c

ontr

ol

,,,,,

.

0%

25%

50%

75%

100%

0 0.25 0.5 0.75 1

0%

25%

50%

75%

100%

0 0.25 0.5 0.75 1

0%

25%

50%

75%

100%

0 0.25 0.5 0.75 1

K+ mmol g -1 DM

Cl- mmol g -1 DM

B

D

F

Na+ mmol g -1 DM

0%

25%

50%

75%

100%

0 0.5 1 1.5 2

0%

25%

50%

75%

100%

0 5 10 15 20

0%

25%

50%

75%

100%

0 5 10 15 20

0%

25%

50%

75%

100%

0 5 10 15 20

Cl- mmol mL-1 H2O

K+ mmol mL-1 H2O

Na+ mmol mL-1 H2O

A

C

E

G

K+:Na+ ratio

Figure 4.3. Correlations between of the internal ion concentrations and aboveground tissues

% of control for: P. clandestinum ( ), P. vaginatum ( ), S. virginicus ( ), and D. spicata

( ). Graphs on left side (A,C,E,G) are showing the ion concentrations given in mmol mL-1

whilst graphs (B,D,F,H) are given in mmol g-1

DM. Equations and R2 values are given in

Appendix 4.3.

90

Tissue water content

The tissue water content of the non-halophyte P. clandestinum was 1.83 mL g-1

DM for the

non-saline control then dropped to 1.56 mL g-1

DM at 125 mM NaCl, at 250 mM NaCl was

1.41 mL g-1

DM, beyond this concentration P. clandestinum leaves went brown. Tissue water

was the highest in the aboveground tissues of P. vaginatum but still ranged from 2.16 mL g-1

DM for the lowest salt concentration to 0.50 mL g-1

DM in the highest concentration,

followed by S. virginicus with a range of 1.91 - 0.28 mL g-1

DM, then D. spicata with a range

of 0.72 – 0.13 mL g-1

DM. For the belowground tissues at the lowest salinity level P.

clandestinum had the highest tissue water content 3.54 mL g-1

DM then dropped by 26% at

125 mM NaCl, at the later concentration the four species had similar water content in (2.56

mL g-1

DM), beyond this concentration, the belowground tissue water content increased by

(30%, 75% and 85%) in D. spicata, P. clandestinum and P. vaginatum, respectively whilst

declined by (42%). in S. virginicus.

91

Tis

sue W

ate

r C

onte

nt

(mL

g-1

DM

)

NaCl added to the irrigation solution (mM)

0

1

2

3

0 200 400 600 800

A

0

2

4

6

0 200 400 600 800

B

Figure 4.4. Effect of concentration of NaCl added to the nutrient solution on the tissue water

content of: (A) aboveground biomass and (B) belowground DM of P. clandestinum ( ),

P. vaginatum ( ), S. virginicus ( ), and D. spicata ( ). Plants were grown

in irrigated sand culture under glasshouse conditions for 60 days. Values are the average of 4

replicates. Error bars denote the SEM. P values for the aboveground tissue water content

were <0.001 for both species and treatments while for the interaction 0.398; LSDs values

were 0.33, 0.36 and 0.72 for species, treatment and the interaction, respectively. P values for

the belowground tissue water content were 0.19, 0.002 and 0.49 for species, treatments and

the interactions, respectively; LSDs values were 1.26, 1.41 and 2.82 for species, treatment

and the interaction, respectively.

Leaf sap osmotic potential

The increase in salt levels reduced the leaf sap osmotic potential (Figure 4.5) of the non-

halophytic P. clandestinum (from -1.0 to -2.40 MPa). By contrast, leaf osmotic potentials in

the three halophytes did not decline to such negative values; at 500 mM NaCl, the leaf sap

osmotic was (-0.8 to -1.5 MPa).

92

NaCl added to the irrigation solution (mM)

Leaf s

ap

osm

otic

pote

ntial (

MP

a)

-4

-3

-2

-1

0

0 200 400 600 800

Figure 4.5. Effect of concentration of NaCl added to the nutrient solution on the sap osmotic

potential of leaves of four perennial grasses grown in irrigated sand culture in a glasshouse

for 60 days (Experiment 1). The grasses were: P. clandestinum ( ), P. vaginatum

( ), S. virginicus ( ), and D. spicata ( ). Values are the average of 4

replicates. Error bars denote the SEM. P value is < 0.001 for both species and treatments,

LSD values are 1.951 and 2.147, respectively.

Colour quality

Visual scoring of the non-halophyte (P. clandestinum) had a collapse in quality with saline

irrigation from 5 to 2, the grass turned brown beyond 250 mM NaCl. By contrast, the

halophytic species S. virginicus and D. spicata maintained high scores (5-6) during the saline

irrigation period for the various concentrations applied, whereas P. vaginatum had a score of

4.

93

NaCl added to the irrigation solution (mM)

Tu

rf q

ua

lity (

vis

ual sco

rin

g)

0

2

4

6

8

0 200 400 600 800

Figure 4.6. Turfgrass quality rated visually in response to five salinity treatments to: P.

clandestinum ( ), P. vaginatum ( ), S. virginicus ( ), and D. spicata

( ). Turfgrass quality scores based on a scale from 1 to 7, where 7 equals best score

and 1 representing brown colour.Values are the average of 4 replicates.

Water use (experiment 2)

Water use declined following the saline irrigation. Reduction in water use was significantly

different between species whereby the non-halophyte had the highest reduction when

irrigated with saline water compared to non-saline (31% reduction), followed by P.

vaginatum (21%), then D. spicata (13%) and finally the least reduction in water use was for

S. virginicus (7%).

Comparison of the growth under saline versus non-saline conditions showed that saline

irrigation decreased total clipping DM by 33%, 10%, 23% for P. clandestinum, P. vaginatum

and D. spicata, respectively whereas growth of S. virginicus remained unchanged (P value

was <0.001 for species, P=0.003 for irrigation treatments and P<0.001 for the interaction

between species and irrigation treatment).

94

Table 4.2. Effect of saline irrigation water (ECw of 13.5 dS m-1

) on the average leaf weekly

clippings DM per lysimeter and water use of four turfgrass species grown in 16 L lysimeters.

Lysimeters were outdoors and water use was measured on selected days over 3 months.

Values are the average of 3 replicates. Error bars denote the SEM. Water use measurements

were taken 3 days after clipping of the grasses. Epan values were 8.4, 7.4, 8.5, 5.8 and 8.0

mm day-1

.

Species Treatment Growth

(g DM week-1

)

Water use

(% Epan)

P. clandestinum Non-saline 1.48 ± 0.14 62% ± 1%

Saline 0.99 ± 0.05 51% ± 2%

P. vaginatum Non-saline 2.16 ± 0.22 61% ± 2%

Saline 1.93 ± 0.29 56% ± 1%

S. virginicus Non-saline 2.08 ± 0.07 63% ± 2%

Saline 2.12 ± 0.17 60% ± 1%

D. spicata Non-saline 1.34 ± 0.23 53% ± 5%

Saline 1.03 ± 0.13 52% ± 3%

Colour quality (experiment 2)

Greenness of the non-halophyte (P. clandestinum), measured as hue angle, had decreased on

average by 12% with saline irrigation compared to non-saline (Figure 4.7). By contrast, the

three halophytic species all maintained high hue angle under saline irrigation when compared

to non-saline; no significant difference was also found between saline or non-saline

irrigation. Moreover, no significant difference was found between colour of the three

halophytic species.

95

2008

Hue a

ngle

(G

reenness)

200720082007

80

100

120

Dec Jan Feb

80

100

120

Dec Jan Feb

80

100

120

Dec Jan Feb

80

100

120

Dec Jan Feb

C

A B

D

Figure 4.7. Hue angle (greenness) of (A) P. clandestinum, (B) P. vaginatum, (C) S.

virginicus and (D) D. spicata irrigated with: non-saline water ( ) and saline groundwater

(ECw 13.5 dS m-1

) ( ). Plants were grown in 16 L lysimeters with readings of shoot

colour being taken weekly. Values are the mean of 3 replicates, were each replicate consisted

of measurements taken at three random positions in each lysimeter. Error bars denote the

SEM. P values for the hue angle were 0.005, 0.02 and <0.001 for species, treatments and the

interaction; LSDs values were 4.99, 2.61 and 6.11 for species, treatment and the interaction,

respectively.

4.5 Discussion

Growth

Salinity decreased the dry mass (DM) of aboveground tissues of the non-halophyte compared

to the halophytic species (Figure 4.1A,C,E) as hypothesised in this study; hypothesis 1. The

declines in growth may be due to the Na+ and Cl

- concentrations in the aboveground tissues

reaching inhibitory levels. However, for the belowground tissues, D. spicata increased in DM

by 14% at the treatment of 125 mM NaCl and belowground DM of S. virginicus increased by

60% at 230 mM NaCl the (Figure 4.1B,D,F). It has been also noticed by (Marcum, 2006) that

the root growth as percentage of control of halophytes such as S. virginicus and D. spicata

increased by 13% and 8% with 125 mM NaCl treatment. The stimulatory effect on growth of

96

halophytes irrigated with saline water was also reported in (Alshammary et al., 2004; Flowers

and Colmer, 2008; Munns and Tester, 2008; Redondo et al., 2008). The stimulation might be

related to the preferential allocation of dry matter to roots (Kage et al., 2004) and likely it

facilitates the adaptation to salinity to counter low external water potential by increasing plant

absorptive area and maintaining water balance (Alshammary et al., 2004). Stimulation of root

growth under moderate salinity is much more common in salt-tolerant grasses and halophytes

and could reach 2-10 times more than that of glycophytes (Poljakoff-Mayber, 1988), resulting

in increased root/shoot ratios, and therefore increased water absorption/transpiration area.

The aboveground tissues of the non-halophyte species was reduced by salinity. Factors such

as the accumulation of the excessive salts through the transpiration stream likely caused the

growth reduction in the vigorous species P. clandestinum and probably also for P. vaginatum;

their RGR was the highest under no-salt treatment. For the species with lower vigour (i.e. D.

spicata and S. virginicus), these showed high salt tolerance as the aboveground DM was

unchanged when salinity was increased. Data from the total growth (aboveground and

belowground DM) showed the same trend too with minor change in DM of the halophytic

species (Appendix 4.4). Tissue ion concentrations are discussed in the next section, but

importantly this study found that under high salt levels, D. spicata and S. virginicus

controlled the Na+ and Cl

- accumulation in leaves better than the higher Na

+ and Cl

- in the

third halophytic species P. vaginatum. The high toxic ion concentration in P. vaginatum

occurred despite a higher tissue water content (Lee et al., 2005).

Ion concentrations (Na+, K

+ and Cl

-)

Correlations were established in this study between growth as % of control and the ion

concentrations in aboveground tissues on both tissue water basis (Figure 4.3A,C,E,G) and dry

mass basis (Figure 4.3B,D,F,H). Growth reductions were correlated to K+ concentration in

the aboveground tissues for the halophytes and non-halophyte; the non-halophytic species (P.

clandestinum) accumulated higher K+ under non-saline conditions than the halophytes;

however, halophytes maintained adequate K+ (~0.4 mmol g

-1 DM) in saline treatments. The

presented correlations revealed that Na+ caused drastic growth reduction in P. clandestinum

and P. vaginatum where Cl- accumulation caused a gradual growth reduction in these two

species with increasing the external salt concentrations, with exception of P. vaginatum at

low-moderate salinity levels (<500 mM NaCl) in which the tissue water content would have

diluted the ions and lead to growth continuity. Thus, overall hypothesis 3 that shoot growth

97

reductions under salinity will be correlated with increases in leaf tissue Na+ and/or Cl

-

concentrations, both in the halophytes and the non-halophyte, was supported by the present

study.

D. spicata and S. virginicus have demonstrated varying abilities to exclude Na+ and Cl

- from

the leaf tissues, this supports the second hypothesis. Cell membranes appear to be selective

against Cl- (Maas, 1993) besides the exclusion of Na

+ from the xylem and subsequent

sequestering in old tissues of either the root or the stem (Maas, 1993). When ion

concentrations were expressed on a dry mass basis, however, there was no clear distinction

between halophytes and non-halophyte species in regards to growth reduction. When

expressed in terms of water content, however, the differences between halophytes and the

non-halophyte became clear. The more distinctive criteria of salinity tolerance appears to be

in Na+, Cl

- concentrations and K

+:Na

+ ratio in leaf tissues; however, these important criteria

as yet are not known to have a uniform manner in which they influence salinity tolerance

(Lee et al., 2007). K+:Na

+ ratio correlated positively with visual scores of the four species

(Appendix 4.6D); species with high K+:Na

+ ratio had the better visual appearance (Appendix

4.6D).

K+ is essential for plant growth and development and also have a role in osmotic adjustment

to maintain low water potential of plant tissues (Wang et al., 2004; Marcum et al., 2007). K+

concentration in leaves and K+:Na

+ ratio in both above and belowground tissues decreased

with increases in soil salinity. K+:Na

+ ratio was higher in the belowground tissues than in

aboveground tissues which indicates that roots and rhizomes have the ability to select K+ to

keep the nutrient balance. The inorganic ions play the most important role in leaf osmosis

under salt stress (Niu et al., 1995). With the increase in NaCl concentration, the concentration

of Na+ and Cl

- increases gradually but the concentration of K

+ declines (Luttge and Smith,

1984).

The higher accumulation of Na+ and Cl

- under high salinity levels (250 mM NaCl and above)

may further decrease the osmotic potential to absorb more water from soils, which was

favourable to the plant growth at salinity up to 250 mM NaCl. However, high salinity

reduced plant growth and accumulation of Na+ and Cl

- in P. clandestinum and P. vaginatum

had further increased, which might then be toxic to plant tissues if ion concentrations

exceeded tolerable levels.

98

Data of Na+ and Cl

- indicate that D. spicata and S. virginicus have better ion exclusion

(and/or exclusion) mechanisms (Marcum et al., 2007), while P. vaginatum must have better

tissue tolerance than P. clandestinum as the later turned to brown (Figure 4.6) due to ion

toxicity (Figure 4.2 and also reduction of turfgrass quality (Appendix 4.3A,C). Despite that P.

vaginatum accumulated high concentrations of Na+ and Cl

- compared to other halophytic

species (D. spicata and S. virginicus), yet it retained high colour rating which shows a

distinction in tissue tolerance to this toxic ion.

In summary, the glasshouse experiment showed that NaCl treatments decreased the

concentration of K+ and increased Na

+ in the above-ground tissues of the non-halophyte. The

three halophytic species did not show similar increase in Na+ concentration in their tissues,

subsequently avoid plant mortality and decline in growth and quality. Other mechanisms also

were employed such as ion compartmentalisation and salt extrusion, in particular with S.

virginicus and D. spicata as these two species have salt glands (Flowers et al., 1977; Flowers

and Yeo, 1986; Marcum and Murdoch, 1992; Bell and O’Leary, 2003; Marcum et al., 2007).

Water use - Experiment 2

Irrigation with saline water (13.5 dS m-1

) in this experiment reduced the water use of the non-

halophyte species greatly (31%), whilst for halophytes the reductions were more modest (7 –

21% of those in the non-saline irrigation); this supports the 4th

hypothesis. Differences

between water use of turfgrass species are noted, where the non-halophyte uses more water

under non-saline water whilst the halophytes water use is not changed by salinity up to 13.5

dS m-1

. Variations in water use of turfgrass species is noted for C4 grasses when irrigated

with 50-60% ET as recommended to maintain growth and colour (Short and Colmer, 1999).

Other responses to salinity

The non-halophyte growth responses to increasing salinity in P. clandestinum were direct

upon imposing the saline irrigation (125 mM NaCl) to the plant. These responses include

increasing leaf osmotic potential (more negative) than the halophytic species followed by

decline in colour quality, specific ion toxicity and consequently leaf firing with increasing

salinity then decreasing water uptake and tissue water content over time (Hanson et al.,

1979). At higher salinity treatments growth ceased (Figure 4.1A,B, Figure 4.5).

99

Na+ can be stored in older tissues of roots, rhizomes and stems, thereby contributing to lower

ion concentrations in the leaf tissues of S. virginicus (Marcum, 1999; Bell and O’Leary,

2003; Ramadan and Flowers, 2004) and in D. spicata (Hansen et al., 1976; Marcum, 1999;

Alshammary et al., 2004; Yensen and Biel, 2006), so that these halophytic species reduce the

toxic ion concentration in the leaf tissues. Both D. spicata and S. virginicus have salt glands

which is an efficient mechanism to lower the internal ion concentrations (Marcum 1999;

Ramadan, 2001; Marcum, 2008 ). The higher the saline irrigation solution, the more efficient

secretion rate (Torello and Rice, 1986; Marcum and Murdoch, 1994; Qian et al., 2001), in S.

virginicus it ranges from (166.5 µmol g dry mass–1

d–1

when the external salinity was 125 mM

NaCl to 336.7 µmol g–1

dry mass d–1

when the external salinity was 450 mM NaCl (Bell and

O’Leary, 2003), the low leaf Na+ and Cl

- concentrations in these halophytes may therefore be

a consequence of the availability of salt glands.Studies of turfgrass species showed that shoot

ion exclusion correlated with secretion efficiency from salt glands in bermudagrass leaves

(Marcum and Pessarakli, 2006) and zoysiagrass (Marcum et al., 1998) and Chloridoid grasses

(Marcum, 1999), but salt gland secretion rates were beyond the scope of the present study.

In the current study, due to the long growth periods used (60 days of growth after imposing

the treatments) without frequent clipping to the leaf tissues, it is likely that the growth of

turfgrasses represented by (relative growth rate, Figure 4.1E,F) was reduced over the course

of the experiment as a result of both osmotic effects and salt-accumulation effects.

4.6 Conclusions

Tissue Na+, K

+ and Cl

- concentrations and K

+:Na

+ ratios showed differences between salinity

treatments, and species. The studied cultivars of D. spicata and S. virginicus had consistently

lower Na+ and Cl

- concentrations and higher K

+:Na

+ ratio in leaf tissues compared with the

other two species, and particularly at lower NaCl in the irrigation solutions. Low K+:Na

+ ratio

is a good indicator to salinity stress in both the aboveground biomass and belowground, so

long as the concentration of NaCl in the irrigation solution did not exceed 230 mM NaCl., at

higher salinity treatments the concentration of K+ reduced due to ion selectivity and the high

Na+ concentration strongly inhibited K

+ uptake and accumulation and presumably has

subsequent affects on the integrity and functionality of the cell membranes. Lower K+:Na

+

ratio as salinity increased indicated that very large imbalances were occurring (Gorham et al.,

1990) at which may affect cell metabolism and thus restrict growth; there seems to be a

100

critical level of K+:Na

+ ratio favours the salt tolerance of plants as reported for barley 0.6 and

wheat 0.34 (Chauhan et al., 1980). Long term exposure to high saline levels and continual

accumulation of Na+ and Cl

- may result in eventual mortality for species without leaf ion

secretion or the capacity for efficient ion compartmentalization into vacuoles. The most salt-

tolerant species were in order of D. spicata, S. virginicus, P. vaginatum and finally P.

clandestinum.

4.7 Appendices

750 500 250 125 0

NaCl in the irrigation solution (mM)

Appendix 4.1. Visual effect of the addition of NaCl to the nutrient solution on the

appearance of P. clandestinum (top rowm of pots), P. vaginatum (row of pots second from

top), S. virginicus (row of pots third from top), and D. spicata (row of pots at bottom).

Treatments had been imposed for 59 days in sand culture pots in a glasshouse. Photo shows

the four turfgrass species and the five NaCl treatments.

101

Appendix 4.2. Average monthly maximum, minimum and mean air temperature for Perth

city (Bureau of Meteorology)

Month Average Maximum

oC

Average Minimum

oC

Average Mean

oC

November 2007 29 21 14

December 2007 28 21 15

January 2008 34 26 19

February 2008 33 26 20

March 2008 30 23 17

Appendix 4.3. Proportion of root DM to shoot DM for P. clandestinum, P. vaginatum, S.

virginicus, and D. spicata when irrigated with different concentrations of saline water.

Root:Shoot Ratio

Irrigation

Treatment

(mM NaCl)

P. clandestinum P. vaginatum S. virginicus D. spicata

0 0.63 1.03 2.86 3.76

125 1.56 2.06 3.04 4.82

250 1.85 2.35 6.28 3.27

500 2.63 1.97 4.61 5.06

750 2.07 3.10 4.74 6.35

102

To

tal g

row

th D

M (

g)

NaCl added to the irrigation solution (mM)

0

25

50

75

100

125

0 200 400 600 800

Appendix 4.4. Effect of NaCl added to nutrient solution on the total dry mass (DM) for the

above and belowground growth of perennial grasses grown in irrigated sand culture under

glasshouse conditions for 60 days (Experiment 1). The grasses were: P. clandestinum (

), P. vaginatum ( ), S. virginicus ( ), and D. spicata ( ). Values are the

average of 4 replicates.

103

Appendix 4.5. Equations and R2 values for the correlations between of the internal ion concentrations (Na

+, K

+ and Cl

-) on DM basis and the

aboveground tissue DM % of control for: P. clandestinum, P. vaginatum, S. virginicus, and D. spicata. Correlations between the internal ion

concentrations and the absolute DM were insignificant and not reported here. Graphs are given in Figure 4.3.

Species Na+ K

+ Cl

- K

+:Na

+ ratio

P. clandestinum y = 2.2664x

2 - 3.1391x + 1.3312.

R² = 1

y = -0.0612x + 0.9173

R² = 0.6539

y = -0.766x + 1.0242

R² = 0.6305

y = 0.2214x2 - 0.5753x + 0.6552

R² = 1

P. vaginatum y = 0.7611x

2 - 1.7761x + 1.0043.

R² = 0.8627

y = 0.0502x2 - 0.7192x + 2.687

R² = 0.8901

y = 0.8115x2 - 1.7318x + 0.9519

R² = 0.799

y = -0.0139x2 + 0.2734x + 0.1405

R² = 0.9954

S. virginicus y = 0.4494x

2 - 0.9251x + 1.0149.

R² = 0.8948

y = 0.0893x2 - 0.4491x + 1.1082

R² = 0.9222

y = -0.0047x2 + 0.0761x + 0.558

R² = 0.6096

y = -0.0981x2 + 0.6253x + 0.2588

R² = 0.9822

D. spicata y = 0.0312x

2 - 0.2753x + 1.03.

R² = 0.9502

y = 0.0179x2 - 0.5035x + 4.0419

R² = 0.9198

y = 0.0131x2 - 0.1821x + 1.0542

R² = 0.9629

y = -0.0342x2 + 0.5111x + 0.2869

R² = 0.7983

104

Vis

ual S

co

res

y = -4.3714x2 + 7.1507x + 3.4583R² = 0.1336

y = 70.059x2 - 61.29x + 15.086R² = 0.8236

y = -63.035x2 + 49.698x - 3.6364R² = 0.986

y = -53.277x2 + 60.208x - 10.049R² = 0.9254

0

2

4

6

8

0 0.5 1 1.5 2

y = -23.257x2 + 10.238x + 5.6354R² = 0.853

y = 0.9983x2 - 3.9514x + 5.6609R² = 0.8409

y = -1.1021x2 - 2.5598x + 6.4278R² = 0.918

y = -17.663x2 + 1.015x + 6.9346R² = 0.9122

0

2

4

6

8

0 0.5 1 1.5 2

P. clandestinum

P. vaginatum

S. virginicus

D. spicata

y = -0.0008x2 + 0.1295x + 4.3588R² = 0.9483

y = -0.01x2 + 0.3799x + 1.4184R² = 1

y = -0.0035x2 + 0.2779x + 1.8957R² = 0.9062

y = -0.0002x2 + 0.0634x + 4.3613R² = 0.601

0

2

4

6

0 5 10 15 20

y = -13.333x2 + 11.766x + 4.0379R² = 0.9813

y = -1.114x2 - 0.2342x + 5.1297R² = 0.9893

y = -0.8487x2 - 3.2743x + 6.5149R² = 0.9723

y = -6.945x2 + 5.034x + 6.0222R² = 0.7912

0

2

4

6

8

0 0.5 1 1.5 2

Na+ mmol g -1 DM K+ mmol g -1 DM

C

A B

D

Cl- mmol g -1 DM K+:Na+ ratio

Appendix 4.6. Correlations between ion concentrations (A) Na+ (B) K

+ (C) Cl

- given in

mmol g-1

DM and (D) K+:Na

+ ratio on the visual scoring of: P. clandestinum ( ), P.

vaginatum ( ), S. virginicus ( ), and D. spicata ( ).

105

Chapter 5

Volume of saline irrigation controls salinity of the soil solution, growth

and quality of the halophytic turfgrass Paspalum vaginatum

5.1 Abstract

This chapter evaluated the effects of the volume of saline irrigation on salt accumulation

in the root zone and the growth of the halophytic turfgrass Paspalum vaginatum in the

field. The salinity of the irrigation water was 13.5 dS m-1

and four levels of irrigation

were applied (40, 60, 80, and 100% of replacement to net evaporation). At the highest

and lowest rates of irrigation, the estimated salinities of the soil solution (ECsoil solution)

were ~2.6-fold and ~5-fold higher, respectively, than the EC of the applied water (ECw).

Growth was greatest at the highest irrigation level and declined as irrigation volume was

reduced. The average leaf Na+ and Cl

- concentrations increased, whereas the average leaf

K+ decreased by 50%, as irrigation volume was reduced. Leaf sap osmotic potential was

more negative, as low as -2.4 MPa. at the lower irrigation level. Turfgrass colour

increased with increasing irrigation volume, with exception of the 40% replacement

treatment. Irrigating P. vaginatum with saline water (ECw 13.5 dS m-1

) at 80%

replacement of net evaporation maintained the growth of high quality turfgrass by

maintaining the salinity of the soil solution within the range tolerated by P. vaginatum.

The present findings will help in developing effective management to improve the use of

alternative irrigation water resources while maintaining good quality turfgrass.

106

5.2 Introduction

About one-third of the domestic water that is currently used worldwide is applied to

gardens and turfgrasses (Smith, 1998). As competition for fresh water resources

increases, the use of salt-tolerant turfgrass species and cultivars irrigated with brackish or

saline water may become an important strategy for reducing the pressure on fresh water

resources (Huck et al., 2000; Carrow and Duncan, 2000; Barnett et al., 2005).

Salt concentrations in the soil solution depend both upon the amount of salt in the soil as

well as the soil water content; as soil water content decreases, ions become more

concentrated (Suarez, 2001). Given this, the salinity of the soil solution can increase

when the soil dries out between irrigations or rainfall events, and it can vary greatly

between the upper and lower layers of the root zone (Sheng and Xiuling,, 1997). Thus,

monitoring the salinity of the soil solution is crucial for the sustainable use of saline

water in irrigation, although salt and water dynamics in soils are hard to monitor, difficult

to predict (Dinar et al., 1991) and both can vary temporally and spatially (horizontally

and vertically) (Rhoades et al., 1989). Irrigation water quality and volumes applied are

the most important drivers of the salinity of the soil solution, because evapotranspiration

can cause the drying of the soil and salt accumulation in the upper soil layers where

turfgrass roots grow.

When using saline water sources, frequent and adequate irrigation to prevent the buildup

of salts in the soil can play an important role in controlling salts in the root zone (De

Villiers et al., 1997), but the development of drainage systems, and the disposal of this

drainage water can have adverse off-site impacts. As an example, a 3-year monitoring

study of Distichlis palmeri irrigated with saline drainage water (ECw = 15 dS m-1

)

showed that daily irrigation was required to maintain growth (Yensen, 2002), turfgrasses

are irrigated daily during the growing season, therefore it is expected not to accumulate

salts.

107

Irrigation scheduling is part of turfgrass best management practice: it encourages the

downward flux of soil water to control root zone salinity, and at sufficiently high rates

could decrease salt concentrations in the soil solution to levels equivalent to the ECw

(Carrow and Duncan, 1998; Sevostianova et al., 2011; Duncan et al., 2000). Turfgrasses

have an advantage over other crops in the use of saline irrigation water (Murphy and

Murphy, 2006) as turfgrass swards create a better environment for rainfall penetration

and their complete canopy closure minimizes evaporation from the soil surface (Maas et

al., 1982); both factors would reduce salt accumulation in the uppermost soil layers.

Paspalum vaginatum is a salt tolerant turfgrass species (Carrow, 1995) that can be

irrigated with brackish and saline water with ECw values up to 40 dS m-1

(Semple et al.,

2003), provided salts are periodically leached from the root zone to levels of salt

accumulation beyond the tolerance threshold of ECw 54 dS m-1

(Duncan et al., 2000). P.

vaginatum employs mechanisms to tolerate salinity such as: selective ion uptake by the

roots and accumulation of ions in vacuoles within plant cells for osmotic adjustment

(Leonard, 1983).

The present work evaluated the effect of four levels of irrigation with saline water (ECw

13.5 dS m-1

) on the growth and quality of P. vaginatum in field plots during the irrigation

period in a Mediterranean-type environment. Salt accumulation in the soil as a

consequence of irrigating with different volumes of saline water was examined, and the

physiological responses of P. vaginatum, such as tissue ion concentrations, tissue water

content, and turfgrass quality were studied. It was hypothesised that:

1. Increasing the volume of irrigation water will maintain salinity of the soil solution in

the root zone at a level closer to that of the applied water, owing to the greater leaching of

salts into deeper soil layers.

2. In situations where salinity accumulates in the root zone owing to insufficient leaching,

P. vaginatum would have adversely decreased growth and colour quality in response to

the salinity of soil solution.

108

3. Decreases in the growth and quality of P. vaginatum with different volumes of saline

irrigation would be mediated by ion concentrations in the tissues (viz. high

concentrations of Na+ or Cl

- and/or low concentrations of K

+).

5.3 Materials and Methods

Site establishment

Plots were established as previously described (see Chapter 3) on 16 October 2006 at

Wagin, Western Australia, GPS location 33o 18' 38.54" S and 117

o 20' 52.99" E. As

previously described, the plots were planted with rooted plugs and irrigated with non-

saline water until the plots were fully covered (15 December 2006). Mowing commenced

4 weeks after planting at a height of 30 mm every fortnight during the irrigation season

and every month during the period with rainfall (i.e. no irrigation), as the temperature

was also cooler and growth declined. Fertilizer (Turf Special, a soluble NPK fertilizer

with micronutrients, CSBP Limited; for chemical analysis see Appendix 5.1) was applied

every 4 weeks at a rate equivalent to 30 kg N ha-1

. The soil type was a clay loam and has

been described in the Materials and Methods of Chapter 3.

Meteorological data were measured using a weather station (Mark 4, Environdata,

Australia) located on-site. Parameters measured hourly were air temperature, relative

humidity, wind speed, wind direction, solar radiation and rainfall. Values of net

evaporation were calculated using the Penman-Monteith equation (Allen et al., 1998).

Maximum and minimum daily air temperature, relative humidity, rainfall and

evapotranspiration data are presented in Figure. 5.1A.

Four irrigation treatments (100%, 80%, 60% and 40% replacement of net evaporation)

were imposed by watering daily during the irrigation seasons: December 2006 to April

2007 and September 2007 to April 2008. There were 3 replicates of each treatment in a

completely randomized design (12 plots in total) each with an area of 16 m2. The total

calculated ET0 over the second irrigation season (reported in this chapter) was 1290 mm,

109

and the applied irrigation water amounts were 516, 774, 1032 and 1290 mm for the 40%,

60%, 80% and 100% treatments, respectively.

Irrigation system

The plots were irrigated using a pop-up sprinkler system (MP2000 Rotator, supplied by

Total Eden, Australia), controlled by irrigation controller (Irritrol, RD-900, supplied by

Total Eden, Australia). The source of saline irrigation water was the local groundwater.

Irrigation took place before sunrise to minimize the absorption of ions from the water by

the turfgrass leaves (Haynes and Goh, 1977; Mass et al., 1982). Irrigation volumes were

adjusted every 2 weeks, based on the average daily net evaporation of the previous week.

This experiment was conducted adjacent to the experiment discussed in chapter 3

whereby P. vaginatum was evaluated under the same saline water and also non-saline

water irrigated with 60% replacement of net evaporation. Comparisons are often made in

this chapter in regards to growth and ion concentrations with reference to the non-saline

treatment from Chapter 3.

Level of watertable

Six piezometers (2 m long each) were installed on-site to monitor the watertable; these

were sealed with bentonite clay to ensure there was no leakage from the surface water. A

plopper on a tape measure (Plop102, Eviroequip, Perth, Australia) was used every

fortnight to measure the height of the watertable from the soil surface.

Soil salinity measurements

Soil salinity was measured at fortnightly intervals during irrigation and monthly during

times with no irrigation, by collecting soil samples at two depths, 0–25 cm (topsoil) and

25–50 cm (subsoil) from each plot, using a 7 cm diameter soil auger. Samples were wet

weighed, oven dried at 105oC and weighed again to calculate soil water content.

Measurements of soil EC1:5 were conducted for samples (20 g) of dry soil incubated with

100 ml of de-ionized water (DI), shaken for 16 hours, and then centrifuged at 3000 rpm

for 5 min. Values of EC1:5 were measured using (Cyberscan 100, Omega Scientific,

110

Australia) and then converted to ECsoil solution by multiplying the EC1:5 value (C1) by the

volume of water in the 1:5 dilution (V1) and dividing by the volume of water in the 20 g

soil sample as measured (V2); that is, solving for C2 using this equation: C1 x V1 = C2 x

V2.

Soil salinity was also assessed every fortnight using the EM38 (Geonics, Mississauga,

ON, Canada) by measuring the same 3 locations in each plot with the instrument in a

horizontal position (EM38ah).

Plant growth

Turfgrass clippings were collected from each plot individually (mowing fortnightly),

weighed and a subsamples were taken, weighed and oven dried at 65 °C. The dried

subsamples were weighed. The ratio of subsample dry mass/ subsample fresh mass, and

the total fresh mass, were used to estimate the total plot clippings dry mass.

Concentration of ions

Leaf tissues for analyses of ions and sap osmotic potential were sampled from random

locations in each plot, in the morning immediately prior to mowing. These small areas

were rinsed with DI water to wash any salts from the surface of leaves, left to dry for

approx. 30 mins, then leaf samples were excised using scissors. The leaf samples were

placed in Al-foil envelopes, weighed and frozen in dry-ice for transport to the laboratory,

stored at -80 oC, freeze-dried, then weighed again. Leaf water content was calculated as:

water content (mL g-1

dry mass) = (fresh mass – dry mass) / (dry mass). Samples were

then ground using a ball mill.

Ground tissues were extracted in 10 mL of 0.5 M HNO3, and appropriate dilutions were

measured for Na+ and K

+ by flame photometry (Jenway PFP7 Flame Photometer; Essex,

UK). For Cl- determination, the extract was diluted with 0.1 N HNO3 in 10% (v:v) acetic

acid, and Cl- was determined by the silver ion titration method with an automatic

chloridometer (Buchler–Cotlove Chloridometer). A reference tissue with known ion

concentrations was taken through the same procedures to check the reliability of analyses

111

of every batch of samples; the recoveries of Na+, K

+ and Cl

- were 91%, 93% and 97%,

respectively. No adjustments were made to the data presented.

For measurements of leaf sap osmotic potential, tissues were collected in air-tight vials

and frozen in dry ice for transport to the laboratory. For analyses, samples were thawed,

squeezed in a simple press and the expressed sap was measured using an osmometer

(F110, Fiske Associates, Needham Heights, MA, USA). Sap osmotic potential was

converted from mOsmol to MPa.

Turfgrass colour

A chromameter (Minolta, CR-310, Osaka, Japan) was used to quantify the colour of the

turfgrass in situ at 3 random locations in each plot by measuring the light reflected by the

turfgrass surface at various wavelengths; the relative changes in colour were quantified

by recording the hue angle (cf. Barton et al., 2009). The device was calibrated once every

18 measurements. Values of the hue angle less than 100 are considered to represent an

unacceptable colour for turfgrasses based on visual observations (Barton et al., 2009).

Statistical analysis

Statistical analysis of the data was performed using Genstat 10th

Edition. All the acquired

data were represented by an average of the three replicate measurements and standard

errors (SE). In cases where more than one measurement was taken per plot, the plot mean

was calculated and this value was used as one replicate. An ANOVA test for repeated

measurements was used to test the significance of treatments at the 5% level. Duncan’s

multiple test was used to test the significance of the interaction between treatments.

5.4 Results

Data from the first year (short irrigation season) are summarised in Appendix 5.2 to

illustrate the effect of different levels of irrigation on the measured parameters during the

hottest period (26 February 2007) about six weeks after commencing the irrigation

treatments. The main results presented in this chapter are from the second irrigation

112

season (September 2007 to April 2008), as this was a longer treatment period and the

turfgrass plots had all fully established. Data averaged over the second irrigation season

have been summarised in Table 5.1.

Table 5.1. Average data (weather, soil conditions, plant DM, leaf ions, osmotic potential

and colour) over the second irrigation season (27 September 2007 to 7 April 2008).

Saline water (ECw 13.5 dS m-1

) was applied to plots of P. vaginatum at 40%, 60%, 80%

and 100% replacement of ETo. The average daily maximum and minimum temperatures

over this period were 31 and 16 oC, respectively.

40% 60% 80% 100%

Air temperature oC

EM38h (dS m-1

)

Soil EC1-5 (dS m-1

) (0 - 25 cm) 9.2 ± 1.2* 9.11 ± 1.0* 7.49 ± 0.9* 7.39 ± 1.1*

Soil EC1-5 (dS m-1

) (25 - 50 cm) 10.1 ± 1.9* 9.3 ± 1.2* 8.8 ± 1.2* 7.2 ± 1.0*

Gravimetric Soil Water Content (%

dry soil), (0 - 25 cm)0.12 ± 0.01* 0.13 ± 0.01* 0.13 ± 0.01 0.14 ± 0.01*

Gravimetric Soil Water Content (%

dry soil), (25 - 50 cm)0.10 ± 0.01 0.11 ± 0.01 0.12 ± 0.02 0.12 ± 0.01

Soil ECsoil solution (dS m-1

) (0 - 25 cm) 49.3 ± 1.3 39.9 ± 3.3 38.6 ± 8.8 32.9 ± 5.5

Soil ECsoil solution (dS m-1

) (25 - 50 cm) 64.2 ± 12.3 52.3 ± 10.6 40.6 ± 6.3 37.6 ± 6

Clippings DM (gm-2

) 2 weeks 76.2 ± 10.4* 64.6 ± 5.4* 69.2 ± 10.5* 84.5 ± 8.9*

Leaf Na+

(mmol g-1

DM) 0.60 ± 0.01 0.60 ± 0.03 0.55 ± 0.01 0.54 ± 0.05*

Leaf K+

(mmol g-1

DM) 0.34 ± 0.01 0.31 ± 0.03 0.26 ± 0.01 0.26 ± 0.01

Leaf Cl- (mmol g

-1 DM) 0.56 ± 0.01* 0.51 ± 0.01* 0.44 ± 0.01* 0.45 ± 0.02

Leaf K+:Na

+ ratio 0.57 ± 0.1* 0.51 ± 1* 0.47 ± 0.6 0.48 ± 0.02

Leaf Sap Osmotic Potential (MPa) -2.4 ± 0.1* -2.0 ± 0.1* -2.1 ± 0.2 -2.0 ± 0.08

Colour (Hue angle) 105.6 ± 2.1 104.5 ± 1.3* 107.9 ± 0.7* 109.2 ± 2.6*

ParameterTreatment

23.4

15 ± 0.3

Temperature data are the mean ± SEM of 188 daily records. Soil salinity (EC1:5) and soil

water content data are the mean ± SEM of 3 measurements. Ion data, leaf sap osmotic

potential and colour data are means of 3 measurements ± SEM on 14 harvest days at ~ 2

week intervals across the irrigation season. * denotes significance at P<0.05.

113

0

25

50

75

100

0

10

20

30

Dec Mar Jun Sep Dec Mar

2007 2008

Air

Tem

p. (

oC

) E

vap

(m

m)

EC

ah (d

S m

-1)

A

5

10

15

20

Dec Mar Jun Sep Dec Mar Jun

B

Rain

fall

(mm

)

Figure 5.1. Relationship between weather conditions and the apparent electrical

conductivity of the plots between December 2006 and April 2008. (A). Weather data;

maximum air temperature ( ), minimum air temperature ( ), average daily

evapotranspiration ( ) and rainfall (bars). (B). Average apparent electrical

conductivity (ECah) readings in turfgrass plots irrigated with saline groundwater (ECw

13.5 dS m-1

) at four different levels of irrigation. Values are the mean of 3 replicates

where a replicate is a plot mean. Error bars denote ± SEM. ECah measurements were

taken every 2 weeks over the experimental period using the EM38 in the horizontal

position. Three positions were recorded in each 16 m2 plot; values are the average of the

four irrigation treatments. The line beneath the x-axis indicates the irrigation season

(continuous line) or rainy season without irrigation (dotted line).

Soil salinity, soil moisture and the estimated salinity of the soil solution

Soil salinity responded to the irrigation treatments. Data on salinity of the soil measured

by the EM38 in the horizontal position revealed a significant difference between

treatments (P = 0.03; data not shown), data on the seasonal changes in ECah are given in

(Figure 5.1B). The rank of average ECah values across treatments over the second

114

irrigation season was: 80% > 60% > 100% > 40% replacement of net evaporation. Soil

water content also differed between irrigation treatments, with water content being lowest

in the 40% treatment and highest in the 100% treatment.

Measurements of salinity in extracted soil cores (EC1:5 measurements) are reported in

Table 5.1 and Figure 5.2A (topsoil) and Figure 5.2B (subsoil). With saline irrigation, the

EC1:5 values of the top soil increased reaching 9.12, 9.11, 7.94 and 7.39 dS m-1

for

treatments of 40%, 60%, 80% and 100% replacement of net evaporation, respectively,

whereas for subsoil the EC1:5 values were 10.1, 9.31, 8.81 and 7.21 dS m-1

. The

maximum salinity was noted with the lowest irrigation treatment (40%) in the subsoil

(EC1:5 17.9 dS m-1

) (Figure 5.2B). The results from Duncan’s multiple test showed

significant differences between the four treatments, as an example between the 40% and

60%, also between the 40% and 80% replacement of net evaporation.

The average soil moisture content (gravimetric) were 13 % for the topsoil and 11.3 % for

the subsoil (Table 5.1), but soil moisture content changes during the season. As expected,

the average soil moisture content was in the order of 100% > 80% > 60% > 40%

replacement of net evaporation.

The estimated salinity in the soil solution is reported in Table 5.1 and Figure 5.2E,F.

These data suggest that the salinity of the soil solution is inversely related to the irrigation

volume. In topsoil (0–25 cm), the average salinity of the soil solution (ECsoil water) was

3.2, 2.1, 2.0 and 2.3-fold higher than the salinity (ECw) of the irrigation water (13.5 dS m-

1 ) for 40%, 60%, 80% and 100% treatments, respectively. In the subsoil (25–50 cm)

these effects were even more extreme with low irrigation volumes, with 5.2, 4.0, 2.6 and

2.3-fold higher average ECsoil water values, respectively, than in the irrigation water (ECw)

(Table 5.1).

The seasonal variation in estimated salinity of the soil solution is given in Figure 5.2E

and F. These data show that soil salinity increased during the summer with saline

irrigation, but there was a subsequent decline during the rainy period when irrigation was

115

not applied. During the irrigation season, the soil solutions became more concentrated

than the irrigation water. Between 14 January and 8 April 2008, in the topsoil the ECsoil

solution was ~2.6 times the EC of the applied water (ECw) when the irrigation water was

supplied at 100% of net evaporation.

116

2007 2008

EC

1:5

(dS

m-1

)

0

5

10

15

20

Sep Dec Mar Jun

T1 T2 T3 T4

0

5

10

15

20

Sep Dec Mar Jun

5%

10%

15%

20%

Sep Dec Mar Jun

5%

10%

15%

20%

Sep Dec Mar Jun

0

30

60

90

Sep Dec Mar Jun

Estim

ate

d E

Cso

ilw

ate

r (d

S m

-1) A F

0

30

60

90

Sep Dec Mar Jun

Gra

vim

etr

ic s

oil

wate

r conte

nt (%

)

2007 2008

A B

C D

E

Figure 5.2. Average EC1:5 (A, B), soil water content (C, D), and estimated salinity of the

soil solution (E, F) in turfgrass plots irrigated with saline groundwater (ECw 13.5 dS m-1

)

at four irrigation levels: 40% ( ), 60% ( ), 80% ( ) and 100% ( )

replacement of daily net evaporation. Soil cores were collected from topsoil (0–25 cm;

left-side graphs) and subsoil (25–50 cm; right-side graphs). Values of ECsoil solution (E, F)

were estimated from EC1:5 measurements (A, B) and measurements of the soil water

content (C, D). Values are means of 3 replicates. Error bars indicate ± SEM. The line

beneath the x-axis indicates the irrigation season (continuous line) or rainy season

without irrigation (dotted line). Data are from the second irrigation season.

The maximum ECsoil solution occurred in the hottest period during February 2008, reaching

a value of 82 dS m-1

in the topsoil (0-25 cm) of the 40% replacement treatment (Figure

117

5.2F). Significant regressions were found between the 2-weeks average air temperature

and the ECsoil solution during the irrigation seasons. Correlations between ECsoil solution and

ET are presented in (Appendix 5.6).

y = 5.21x - 67.05

R2 = 0.87

y = 7.04x - 93.59

R2 = 0.95

0

25

50

75

100

0 10 20 30

Top soil Sub-soil

Air Temperature (oC)

Estim

ate

d E

Cso

ilw

ate

r (d

S m

-1)

Figure 5.3. Relationships between the average ECsoil solution of the four irrigation

treatments in the topsoil and subsoil to average air temperature over the prior 2 weeks.

Measurements were taken every fortnight during the second irrigation season.

Leaching of salts commenced after the second irrigation season ended and seasonal

rainfall began (Figure 5.2 A,B,E,F). Salinity in the subsoil began to decline about 4

weeks after irrigation ceased (Figure 5.2 F).

Growth (clippings)

Analysis of variance showed that there was a strong effect (P < 0.001) of the level of

irrigation on the production of clippings. The highest production was for the highest level

of irrigation and production decreased as the level of irrigation decreased. The difference

in production between irrigation levels was significant (P <0.001), also there is highly

significant difference in the clipping DM across months as P. vaginatum is a C4 and

requires warm weather (Turgeon, 1991). There was a 6-fold increase in clipping DM

under 60% treatment between 22 October and 11 February 2008 as the temperature range

increased from 5.4 – 19.1 oC to 14.9 – 38

oC. Furthermore, a 2-way ANOVA showed that

there was a difference in the interaction between treatment and season (P = 0.017); plots

118

receiving higher irrigation levels during the summer, produced more clippings than those

with no irrigation during the winter when clipping production was reduced.

The ANOVA test showed a significant difference in clipping DM between treatments,

however regression tests showed no significant correlation between ECsoil solution and the

clippings DM (g m-2

). The highest clipping DM for the second irrigation season was in

the order: 100% > 40% > 80% > 60% replacement of net evaporation (Figure 5.4).

Clip

pin

g D

M (

g m

-2)

0

100

200

Sep Dec Mar Jun

Cum

ula

tive c

lippin

g D

M (

kg m

-2)

0

4

8

12

Sep Dec Mar Jun2007 2008

A

B

Figure 5.4. Effect of irrigation volume on the above ground growth of Paspalum

vaginatum. A. Clipping dry mass at each mowing (DM; g m-2

) B. Cumulative clipping

dry mass (DM; kg m-2

) over the second irrigation season. P. vaginatum was irrigated

with: 40% ( ), 60% ( ), 80% ( ) and 100% ( ) replacement of

daily net evaporation. Plants were grown in 16 m2 plots in Wagin, Western Australia, and

irrigated with saline groundwater (ECw 13.5 dS m-1

). Values are the mean of 3 replicates.

Error bars denote ± SEM. Plots were mown every 2 weeks. The line beneath the x-axis

indicates the irrigation season (continuous line) or non-irrigated season (dotted line).

119

Tissue ion concentrations

The concentrations of Na+ and Cl

- in the leaf tissues increased after irrigation with saline

water commenced (c.f. pre-irrigation leaf ion concentrations in caption with data in

Figure 5.5). Compared with the initial values just prior to commencement of saline

irrigation, the maximum increases in tissue ions with saline water were 7-fold for Na+

and 13-fold for Cl- (Figure 5.5A,C). By contrast, tissue K

+ decreased by up to 90%

(Figure 5.5B). A comparison of K+ concentration in the leaf tissues of P. vaginatum

irrigated with saline water at 60% replacement of net evaporation with that in the

adjacent plots irrigated with non-saline water at the same volumes (Chapter 3) showed

that there was a 50% decrease in K+ concentration as a result of saline irrigation (0.6 –

0.3 mmol g-1

DM during the hottest period, 26th

February 2008) (cf. Figure 5.4 with

Figure 3.6B from Chapter 3). For Na+ and Cl

-, there was a significant increase in tissue

concentrations over time (P= 0.01) in the saline irrigated plots compared with those

irrigated with non-saline water (cf. Figure 5.4A, C with Figure 3.5B, Figure 3.7B from

Chapter 3).

The level of irrigation had no significant effect on leaf Na+ concentration (Table 5.1,

Figure 5.5A). Na+ concentration did not change at 40, 60 and 80% treatments and

declined by 30% at 100% treatment. However, the interaction between the sampling date

and the irrigation treatment was significant (P < 0.001) as the Na+ concentration in the

leaf tissues increased to 0.6 mmol g-1

DM at 40% replacement of net evaporation, during

the hottest period (26th

February 2008) where the net evaporation was at its maximum

value 8.72 mm d-1

.

The decline in K+ concentration was significant with irrigation treatment (P =0.02), with

sampling date (P < 0.001) and for the interaction between the sampling date and

irrigation treatment (P < 0.001) (Figure 5.5B). P. vaginatum was unable to maintain the

starting concentration of K+ in the leaf tissues during saline irrigation.

Irrigation treatments did not significantly affect the leaf Cl- concentration (P = 0.47). The

concentration of Cl- in the leaf tissues was highest at the first sampling after the

120

commencement of saline irrigation (0.7-0.8 mmol g-1

DM) and then remained below 0.56

mmol g-1

DM (Figure 5.5C). The sampling date affected the Cl- concentration (P < 0.001)

as increased upon commencing the irrigation treatments then decreased over time. The

average Cl-:Na

+ % in leaves was 94, 88, 90 and 100% for the 40, 60, 80 and 100%

replacement of net evaporation, respectively.

121

Ion c

oncentr

ations in leaf

tissues (

mm

olg

-1D

M)

2007 2008

0

0.5

1

Sep Dec Mar Jun

40% 60% 80% 100%

0

0.5

1

Sep Dec Mar Jun

0

0.5

1

Sep Dec Mar Jun

C

B

A

Na

+K

+C

l-

Figure 5.5. Concentrations of ions in the leaf tissues of P. vaginatum: (A) Na

+, (B) K

+

and (C) Cl-. Plants were irrigated with: 40% ( ), 60% ( ), 80% ( ) and

100% ( ) replacement of net evaporation, were grown in 16 m2 plots at Wagin,

Western Australia, and were irrigated with saline groundwater (ECw 13.5 dS m-1

). Values

are the mean of 3 replicates. Error bars denote ± SEM. Leaves were rinsed with DI water

prior to sampling. Data are from the second irrigation season. Ion concentrations in the

leaves before the start of the saline irrigation (mmol g-1

DM) were: 0.10 for Na+, 0.66 for

K+ and 0.18 for Cl

-. The line beneath the X-axis indicates the irrigation season

122

(continuous line) or non-irrigation season (dotted line). The average LSD values (at 5%)

for predicted means of Na+, K

+ and Cl

- are 0.04, 0.036 and 0.07, respectively.

Leaf sap osmotic potential

Analysis of variance showed that the leaf sap osmotic potential (Table 5.1, Figure 5.6)

was significantly different for leaf tissues from plots with different volumes of irrigation

(P = 0.006). Duncan’s multiple test showed that the lowest irrigation level (40%) had a

lower (more negative) osmotic potential than the higher irrigation treatments (60, 80 and

100% - Table 5.1), and the latter three higher treatments were not significantly different

from each other. The interaction between sampling date and irrigation treatment was also

significant (P < 0.001).

The leaf sap osmotic potential data were consistent with the osmotic potential of the

external solution (cf. Lang, 1967), given the ECsoil solution (Figure 5.2C), and comparing

these data sets with the calculated water potential and the osmotic coefficient (Appendix

5.5). It was found that the topsoil osmotic potential was -2.2 MPa with 40% treatment, -

1.76 MPa with 60% and 80% whereas -1.32 MPa with 100%. For subsoil the osmotic

potential was -2.64 MPa with 40%, -2.20 MPa for 60% and -1.76 MPa for 80 and 100%

treatments.

The average leaf water content was at its lowest value under 40% replacement of net

evaporation (1.98 mL g-1

DM). The more the irrigation volume, the higher the tissue

water content; the increase in tissue water content was 3%, 18% and 27%, for irrigation

volumes of 60%, 80% and 100% replacement of net evaporation, respectively (Figure

5.7).

123

Leaf

sap o

sm

otic p

ote

ntial (M

Pa)

2007 2008

-3

-2

-1

0

Sep Dec Mar Jun

Figure 5.6. Leaf sap osmotic potential (MPa) of P. vaginatum irrigated with: 40%

( ), 60% ( ), 80% ( ) and 100% ( ) replacement of net

evaporation. Plants were grown in 16 m2 plots in Wagin, Western Australia, and irrigated

with saline groundwater (ECw 13.5 dS m-1

). Values are the mean of 3 replicates. Error

bars denote ± SEM. Leaf tissues were collected in air-tight vials and frozen in dry ice.

Data are from the second irrigation season. Line above the graph indicates the irrigation

season (continuous line) or non-irrigation season (dotted line). The average LSD value (at

5%) for predicted mean of leaf sap osmotic potential is 0.08.

Tis

sue w

ate

r conte

nt

(mL

g -1

DM

)

Replacement of net evaporation (%)

y = 0.5134x + 1.7559R² = 0.9666

1.5

2

2.5

40% 60% 80% 100%

Figure 5.7. Correlation between irrigation volumes % replacement of daily net

evaporation and the average tissue water content over the second irrigation season. Plants

were grown in 16 m2 plots in Wagin, Western Australia, and irrigated with saline

groundwater (ECw 13.5 dS m-1

). Values are the mean of 3 replicates over 14 sampling

occasions. Plots were mown every 2 weeks.

124

Leaf t

issue w

ate

r co

nte

nt (

mL

g-1

DM

)

2007 2008

0

1

2

3

4

Sep Nov Jan Feb Apr Jun

Figure 5.8. Leaf tissue water content (mL g

-1 DM) of P. vaginatum irrigated with: 40%

( ), 60% ( ), 80% ( ) and 100% ( ) replacement of net

evaporation. Plants were grown in 16 m2 plots in Wagin, Western Australia, and irrigated

with saline groundwater (ECw 13.5 dS m-1

). Values are the mean of 3 replicates. Error

bars denote ± SEM. Plots were mown every 2 weeks. Data are from the second irrigation

season. Line beneath the X-axis indicates the irrigation season (continuous line) or non-

irrigation season (dotted line).

Turf colour

The higher the hue angle means the greener is the grass. Watering volume had no

significant effect (P = 0.09) on turfgrass hue angle (Table 5.1, Figure 5.9). The average

hue angle over the second irrigation season showed with increasing the replacement to

net evaporation, hue angle increased, with exception of 40% treatment. The average hue

angle was decreased by 1% at 60% when compared to 40% treatment, whereas increased

by 2 and 3% with 80 and 100% treatments, respectively.

P. vaginatum has been reported as a fast recovering species after saline irrigation; it

recovered its high quality colour after saline irrigation ceased and there was no

significant difference between the level of irrigation and the final recovery in the colour.

However, in the first phase of recovery in the rainy season (April and May 2008), the

plots that had previously received the lowest irrigation level (40%) retained highest

colour, followed by 60%, 80% and finally 100%, and values at these times were

125

significantly different with P < 0.03 (Figure 5.9). The interaction between treatments and

time of the experiment showed a significant difference (P =0.04).

As previously noted, the different levels of irrigation caused variations in the salinity of

the soil solution. Analysis of correlations showed that there were significant relationships

between the ECsoil solution and the colour at 40%, 60%, 80% and 100% replacement of net

evaporation with R2 of 86%, 99%, 88% and 74%, respectively over the second irrigation

season (Appendix 5.4).

90

110

130

Sep Dec Mar Jun

Hue a

ngle

(gre

enness)

2007 2008

Figure 5.9. Hue angle (greenness) of P. vaginatum irrigated with: 40% ( ), 60%

( ), 80% ( ) and 100% ( ) replacement of net evaporation. Field plots

(16 m2) at Wagin in Western Australia, were irrigated with saline groundwater (ECw 13.5

dS m-1

). Values are the mean of 3 replicates (each replicate being a plot mean). Error bars

denote ± SEM. Each replicate consisted of measurements taken at three random positions

in each plot during the second irrigation season (long season).

126

5.5 Discussion

The three hypotheses proposed in the Introduction are related in terms of impacts on soil

and plants as illustrated schematically in Figure 5.10.

Low volume of

saline irrigation

Increased salinity of

soil solution

Hypothesis 1

Cause

ConsequencesIncreased Na+ and

Cl-; decreased

K+:Na+

Hypothesis 3

Decreased growth

and colour

Hypothesis 2

Figure 5.10. Causes and consequences of saline irrigation to highlight the 3 hypotheses

in this study.

Soil salinity (Hypothesis 1)

It was hypothesized “Increasing the volume of irrigation water will maintain salinity of

the soil solution in the root zone at a level closer to that of the applied water, owing to the

greater leaching of salts into deeper soil layers”. It was found that applying high volumes

of saline irrigation (100% replacement of net evaporation) did indeed decrease the

average salinity of the soil solution (ECsoil solution) (Figure 5.11; Table 5.1) in the root-zone

if compared to low volume (40%); the decreases were by 30% and 60% in the topsoil and

subsoil, respectively. The rank of the treatments based on the average highest salinity in

the soil solution over the second irrigation season was: 40% > 60% > 80% > 100%

replacement of net evaporation for both the top and subsoil; therefore the hypothesis was

supported.

127

As noted above, irrigation amounts of 40% and 60% replacement of net evaporation lead

to salt accumulation; this degraded the colour of P. vaginatum (Figure 5.9, Table 5.1).

Although there was salt accumulation in the soil during the dry season at low irrigation

volumes (40% and 60% replacement of net evaporation), the rainy season was sufficient

to leach the salts from the top 50 cm and turfgrass colour recovered within 2 weeks of

terminating the irrigation (Figure 5.9). Other workers have also found similar effects of

seasonal rainfall. In a Mediterranean-type climate, high rainfall (618 mm) in winter was

sufficient to desalinise the soil profile in the top (0 -1.2 m) by reducing the ECe from 4.27

to 2.32 dS m-1

(Ben-Hur et al., 2001). In arid and semi-arid region, a 7-year research

study using saline water for irrigation (ECw ranging from 6 to 18.8 dS m-1

) proved that

saline drainage water with ECw up to 12.2 dS m-1

could be used for irrigating field crops

without yield reduction (pearl millet and sorghum) or without degrading the soil texture

(increasing sodicity and/or reducing water holding capacity) provided leaching is taken

into account; in this study leaching took place during the rainy season, the average

rainfall over the period of the study was 650 mm and the average annual pan evaporation

was 2500 mm (Sharma and Rao, 1998).

Increasing the irrigation volume from 40% to 60% replacement of net evaporation

reduced ECsoil solution by 18% (topsoil) and 14% (subsoil), and at 80% replacement of net

evaporation the reduction in ECsoil solution was 23 and 37%, and at 100% replacement of

net evaporation reductions were 34 and 42% in the top and subsoils, respectively. Given

the short-term monitoring (4 weeks) after terminating the irrigation treatments and

knowing that plots received 28.5 mm rain during this period, the reduction in ECsoil solution

was correlated with previous irrigation treatment whereby plots irrigated with 100%

replacement had the highest reduction in ECsoil solution and plots irrigated with 40%

replacement had the lowest reduction in ECsoil solution (Appendix 5.3). To prevent salt

accumulation due to irrigating with saline water alone in long term, adequate irrigation

volume should be applied to enhance leaching of the excess salts (Rhoades et al., 1973).

The applied irrigation water in excess of turfgrass water requirement can provide

adequate leaching and improved the downward movement of salts from the root zone

128

(Bakkar et al., 2010). Soil moisture during the irrigation season was between 11.3–13

gravimetric soil water content (%), which corresponds to 60–80% of the field capacity.

This level of moisture is likely to be adequate to the growth of turfgrasses, as there are

previous reports of the maintenance of turf quality under intensive management

conditions with irrigation at 80% replacement of net evaporation (Carrow, 1995).

Growth and colour quality (Hypothesis 2) and ion relations (Hypothesis 3)

The second and third hypotheses were that in situations where salinity accumulates in the

root zone owing to insufficient leaching, P. vaginatum would have adversely decreased

growth and colour quality in response to the salinity of soil solution (Hypothesis 2),

decreases in the growth and quality of P. vaginatum with different volumes of saline

irrigation would be mediated by ion concentrations in the tissues (viz. high

concentrations of Na+ or Cl

- and/or low concentrations of K

+ (Hypothesis 3).

The average effects of irrigation treatment on ECsoil solution were variable which made it

difficult to track the effects of irrigation treatment through ion relations and growth;

however these effects were much clearer when the values (ECsoil solution, Na+ and Cl

- in the

clippings, and clipping DM) were calculated for each individual plot, and these factors

were correlated with each other (Figure 5.11). When data at the individual plot level

were correlated:

The average ECsoil solution was negatively correlated with the replacement of net

evaporation (Figure 5.11; P = 0.013.).

The average concentrations of both Cl- and Na

+ in the clippings were positively

correlated with ECsoil solution (Figure 5.11 B and C; P = 0.012 and 0.039

respectively), and

The concentration of Cl- in clippings was negatively correlated with clipping DM

(Figure 5.11D; P = 0.004).

These correlations clearly establish the attribution of growth effects (consequences),

through Cl- (and Na

+) concentrations in leaves, salt concentrations in the soil solution, to

the rates of replacement of net evaporation (the ultimate cause). Hypotheses 2 and 3 are

therefore supported.

129

y = -28.011x + 61.453

R² = 0.475

0

20

40

60

80

40% 60% 80% 100%

ECsoil solution (dS m-1)

Replacement of net Evaporation

Cause

Consequences

Clippin

gs D

M (

g m

-2)

Leaf

Cl-

(mm

olg

-1D

M)

y = 0.0042x + 0.3388

R² = 0.3618

0

0.2

0.4

0.6

0.8

0 20 40 60 80

y = 0.0037x + 0.309

R² = 0.4822

0

0.2

0.4

0.6

0.8

0 20 40 60 80

y = -1.4999x + 1.405

R² = 0.4518

0

0.25

0.5

0.75

1

0 0.2 0.4 0.6 0.8

Leaf

Na

+(m

molg

-1D

M)

ECsoil solution (dS m-1)

EC

so

ils

olu

tion

(dS

m-1

)

Leaf Cl- (mmol g -1 DM)

A

B C

D

Figure 5.11. Correlations for cause and consequences of amount of saline irrigation

between: (A) Replacement of net evaporation and ECsoil solution, (B) ECsoil solution and leaf

Na+, (C) ECsoil solution and K

+, (D) leaf Cl

- and clipping dry mass for the 3 replicates of

each treatment; 40% (▲), 60% (●), 80% (■) and 100% (□).

Why were these effects not clearer from inspecting treatment averages? The causes of the

variation in plots around the treatment averages are not known. Variation in the site in

other studies has been attributed to variation in soil fertility, for example if less fertilizer

is leached away in areas with less irrigation (Cahn et al., 1994; Yao et al., 2009).

130

With regards to surface greenness, it was not possible to establish clear links between the

net replacement of evapotranspiration, average ECsoil solution, and average Cl- and Na

+

concentrations in the clippings (cf. Figure 5.11). On a single plot basis, greenness was not

correlated with ECsoil solution, Na+ and Cl

- concentrations in the clippings, or clippings dry

mass (data not presented). The reasons for this are not known.

One possible reason for this lack of correlation with other parameters is that colour was

strongly affected by seasonal effects, and that these masked other effects. The effect of

time on the turfgrass colour was significant (P < 0.001), and likely caused by changes in

seasonal conditions more so than the irrigation treatments. During the winter (non-

irrigated) season, low air temperatures were associated with a decline in the colour of P.

vaginatum, which is a C4 species. Such “winter declines” are well-documented in the

literature for C4 turfgrasses, and these decreases occur because of several factors

including reduction in carbohydrate reserves (Dunn and Nelson, 1974).

The literature shows that P. vaginatum can adapt quickly to salinity stress during the

irrigation season by increasing the tissue water content to maintain turgor pressure, also

by accumulating inorganic and organic solutes during the stress period of high salt

concentrations in the soil solution (Lee et al., 2007, Lee et al., 2008). It is therefore likely

that organic solutes were effective during the irrigation season as the salinity in the soil

solution increased whilst P. vaginatum maintained the growth and quality.

Surprisingly, after terminating the irrigation treatments, the clipping DM from plots that

received the lowest irrigation treatment (40%) then produced the highest DM, followed

by those previously at 60%, 80% and 100% replacement of net evaporation. This effect

could be a consequence of the higher concentration of the organic solutes (mainly

glycinebetaine and proline) that had accumulated in plants with lowest irrigation level

which made them the fastest to recover (Lee et al., 2008). Another possibility might be

that under the higher irrigation treatments (60%, 80% and 100% replacement of net

evaporation) there may have been more leaching of the nutrients from the soil, and

131

growth was therefore lower than in the plots with 40% replacement of net evaporation

(Allen et al., 1998).

P. vaginatum takes up ions to increase the osmotic level in their tissues, which in return

permits water to move from soil into tissues to avoid the physiological osmotic stress

(Flowers et al., 1977). Toxic concentration of Na+ in plant tissues ranges from 0.5 – 1.25

mmol g-1

DM (Munns et al., 1983). However, the maximum Na+ concentration in P.

vaginatum was 0.75 mmol g-1

DM. Moreover, increasing tissue water content as salinity

increases, diluted the toxic effect of Na+ and Cl

-; a significant relationship between tissue

water content and irrigation volume is reported in Figure 5.7. Lee et al. (2005) reported

that the increase in tissue water content in P. vaginatum would have maintained turgor

pressure and this was the major mechanism to sustain growth of P. vaginatum under high

levels of salts (ECw 20 dS m-1

).

5.6 Conclusions

This study helps define the required irrigation levels when using saline groundwater (13.5

dS m-1

) in a field situation; information essential to achieve high quality turfgrass.

The higher the irrigation amount applied, the lower the salinity in the soil solution

of the topsoil.

Salinity of the subsoil was significantly higher than the topsoil when irrigated

with saline water due to leaching effect, however at higher irrigation level (100%

replacement of net evaporation), there was no significant difference between the

top and subsoil.

Growth represented by clipping dry mass (DM) was correlated with irrigation

level, the higher the irrigation the more the clipping DM. (The exception was at

40% replacement of net evaporation, possibly due to site specific variation in

fertility).

Leaf water content was correlated with the level of irrigation, the higher the

irrigation level the higher the leaf water content, which revealed that P. vaginatum

132

was restricted in water uptake at salinity in the soil solution which is equivalent to

the salinity in sea water (~55 dS m-1

).

Significant correlation between the potentially toxic ions (Na+, Cl

-) concentration

in the leaf tissues and salinity of the soil solution, consequently there was

significant correlation between Cl- concentration and the clipping DM.

Higher levels of irrigation resulted in less negative osmotic potential since salinity

in the soil solution was lower, this lead to less lowering the requirements for

osmotic adjustment.

P. vaginatum was able to retain high colour quality under saline irrigation (ECw

13.5 dS m-1

), even with substantial concentrating of salt in the soil at low

irrigation volumes with little leaching. There was no significant difference

between treatments in respect of greenness, demonstrating the suitability of P.

vaginatum for use with this saline water source.

133

5.7 Appendices

Appendix 5.1. Chemical analysis of the fertiliser as provided by the manufacturing

company. Units are w/w (%).

Fertiliser N P K S Ca Cu Zn Mn Fe Mg B

Turf Special

12.3 1.8 6.2 16.0 4.0 0.10 0.10 0.4 0.50 trace trace

Appendix 5.2. Summary of average results from the first season of irrigation (15

December 2006 to 5 August 2007).

0.10117.1 ± 1.1117.8 ± 0.4116.8 ± 0.7114.3 ± 1.3Colour (Hue angle)

0.25-2.0 ± 0.04-1.9 ± 0.09-2.1 ± 0.06-2.1 ± 0.06Leaf Sap Osmotic Potential (MPa)

0.360.8 ± 0.050.9 ± 0.080.8 ± 0.030.9 ± 0.1Leaf K:Na ratio

0.450.2 ± 0.10.3 ± 0.10.4 ± 0.020.2 ± 0.1Leaf Cl- (mmol g-1 DM)

0.260.5 ± 0.050.5 ± 0.020.4 ± 0.010.5 ± 0.06Leaf K+ (mmol g-1 DM)

0.130.6 ± 0.030.6 ± 0.040.5 ± 0.020.6 ± 0.03Leaf Na+ (mmol g-1 DM)

0.3084.4 ± 14.380.1 ± 5.977.4 ± 2.162.7 ± 2.6Clippings DM (gm-2) 2 weeks

0.0314.3 ± 0.213.7 ± 0.313.3 ± 0.712.0 ± 0.3EM38ah (dS m-1)

0.526.0 ± 0.47.2 ± 0.37.6 ± 1.16.5 ± 0.7Soil EC1-5 (dS m-1) (25 - 50 cm)

0.118.8 ± 0.49.1 ± 0.311.8 ± 0.711.7 ± 1.7Soil EC1-5 (dS m-1) (0 - 25 cm)

100%80%60%40% P value

TreatmentParameter

0.10117.1 ± 1.1117.8 ± 0.4116.8 ± 0.7114.3 ± 1.3Colour (Hue angle)

0.25-2.0 ± 0.04-1.9 ± 0.09-2.1 ± 0.06-2.1 ± 0.06Leaf Sap Osmotic Potential (MPa)

0.360.8 ± 0.050.9 ± 0.080.8 ± 0.030.9 ± 0.1Leaf K:Na ratio

0.450.2 ± 0.10.3 ± 0.10.4 ± 0.020.2 ± 0.1Leaf Cl- (mmol g-1 DM)

0.260.5 ± 0.050.5 ± 0.020.4 ± 0.010.5 ± 0.06Leaf K+ (mmol g-1 DM)

0.130.6 ± 0.030.6 ± 0.040.5 ± 0.020.6 ± 0.03Leaf Na+ (mmol g-1 DM)

0.3084.4 ± 14.380.1 ± 5.977.4 ± 2.162.7 ± 2.6Clippings DM (gm-2) 2 weeks

0.0314.3 ± 0.213.7 ± 0.313.3 ± 0.712.0 ± 0.3EM38ah (dS m-1)

0.526.0 ± 0.47.2 ± 0.37.6 ± 1.16.5 ± 0.7Soil EC1-5 (dS m-1) (25 - 50 cm)

0.118.8 ± 0.49.1 ± 0.311.8 ± 0.711.7 ± 1.7Soil EC1-5 (dS m-1) (0 - 25 cm)

100%80%60%40% P value

TreatmentParameter

134

Replacement of net evaporation

Red

uction in E

Cs

oil

so

lutio

n

y = 0.1286x - 0.0562

R² = 0.9486

0%

10%

20%

30%

40%

50%

40% 60% 80% 100%

Appendix 5.3. Changes in the average ECsoil solution between irrigation on and irrigation

off. Samples were taken 4 weeks before and 4 weeks after terminating the saline

irrigation. Plots received 28.5 mm rain during this period after termination of the saline

irrigation.

Hue a

ng

le (g

reenness)

ECsoil water (dS m-1)

y = 0.0034x2 - 0.4146x + 121.81R² = 0.8592

y = 0.0101x2 - 1.0376x + 133.09R² = 0.9919

y = -0.0045x2 - 0.0431x + 118.49R² = 0.8797

y = 0.0058x2 - 0.3739x + 117.83R² = 0.7442

90

100

110

120

130

0 25 50 75 100

80%

100%

40%

60%*

*

*

Appendix 5.4. Correlations between greenness and ECsoil solution (dS m

-1) under four

irrigation levels 40% ( ), 60% ( ), 80% ( ) and 100% ( ). * on R2

values denote significance level at P<0.05.

135

Appendix 5.5. Summary of water potentials in MPa for NaCl molalities and temperatures

used in calculating the soil osmotic potential (after Lang 1967).

ET (mm)

Estim

ate

d E

Cs

oil

solu

tion

(dS

m-1)

0

25

50

75

0 2 4 6 8 10

Appendix 5.6. Scatter plots between evapotranspiration (mm) and ECsoil solution (dS m

-1)

under four irrigation levels 40% ( ), 60% ( ), 80% ( ) and 100%

( ). None of the R2 values were significant at P<0.05.

0°C 7.5°C 15°C 25°C 35°C

0.00 0.00 0.00 0.00 0.00 0.00

0.20 -0.84 -0.86 -0.88 -0.92 -0.95

0.50 -2.07 -2.14 -2.20 -2.28 -2.36

0.70 -2.90 -3.00 -3.09 -3.21 -3.33

1.00 -4.17 -4.32 -4.46 -4.64 -4.82

1.50 -6.36 -6.61 -6.84 -7.13 -7.41

1.70 -7.26 -7.55 -7.82 -8.17 -8.49

1.80 -7.73 -8.04 -8.33 -8.70 -9.04

1.90 -8.19 -8.53 -8.84 -9.24 -9.60

2.00 -8.67 -9.03 -9.36 -9.78 -10.16

Water potential (MPa)

TemperatureNaCl

Morarity

136

137

Chapter 6

Salinity tolerance of Distichlis spicata: ion concentrations within segments

of rhizomes, and in roots and leaves

6.1 Abstract

Distichlis spicata is a halophytic species with potential use as a high quality turfgrass in

salt-affected lands. D. spicata employs several physiological mechanisms to tolerate saline

irrigation; ion exclusion and the potential role of tissue sugars as osmotica were studied for

different tissue types (leaves, roots, rhizomes). Growth of D. spicata declined in response

to high salinity levels. D. spicata maintained relatively low Na+ and Cl

- concentrations in

rhizomes (0.5 mmol g-1

DM) under 680 mM NaCl. Variations in ions and total sugars

concentrations were evident in different portions of the rhizomes; the apical portions

contained 50% less sugar than in basal segments. For leaves, retention of high

concentrations of K+

(0.5 mmol g-1

DM) was evident even at the highest salt treatment. It is

concluded from this study that D. spicata employs different concentrations of ions and

sugars in different tissues when exposed to increasing levels of external salinity.

138

6.2 Introduction

Salinity has adverse effects on plants due to osmotic effects and ion toxicity effects (Munns

and Tester, 2008). Salts in the rhizosphere cause osmotic stress to the plants at the initial

stages of their growth (Munns, 2008) followed by toxicity stress as Na+ and Cl

- accumulate

to high concentrations in leaf tissues that exceed tolerance threshold levels (Ungar, 1977).

Tissue tolerance threshold levels can differ amongst species (Munns and Tester, 2008), and

would be a component of the overall salinity tolerance threshold level of salts in irrigation

water or soils at which yield first declines (Maas and Hoffman, 1977). Salinity can also

reduce K+ uptake in plants (Naidoo, 1987; Niu et al., 1995), being a problem since K

+ is

essential in the cellular fluids for metabolic processes in plants (Marschner, 1995). In

addition to poor growth, high salinity can also cause leaf necrosis, which in the case of

turfgrasses has been termed “leaf firing” and this tissue damage impacts on colour and thus

decreases the quality of turfgrasses (Duncan and Carrow, 2005).

Halophytes possess combinations of several mechanisms to tolerate high concentrations of

Na+ and Cl

- in the soil solution, depending on the species these mechanisms include: 1) salt

‘exclusion’ from roots and leaves (i.e. regulation of the rate of entry of ions), 2) salt

excretion through salt glands or salt bladders, 3) leaf or stem succulence, 4) ion

compartmentalisation into vacuoles (contributing also to osmotic adjustment), 5) synthesis

of compatible organic solutes for cytoplasmic osmotic adjustment, and 6) maintenance of

K+ uptake. Accumulation of soluble sugars has also been proposed to contribute to osmotic

potential and thus salinity tolerance in some species (Munns et al., 1983; Kerepesi and

Galiba, 2000). Sugars can play significant roles in structural stability of macromolecules

(Peterbauer and Richter, 2001). The concentrations of sugars, however, varied dramatically

amongst species in response to salinity, increasing in non-halophyte species but decreasing

in halophytes (Wyn-Jones and Gorham, 1983; Pedersen et al., 2006; Colmer et al., 2008).

Various halophytes have been used as forages species on saline areas (Aronson, 1985;

Khan and Qaiser, 2006) and some halophytes have potential use as landscaping plants for

salt-affected areas (DePew and Tillman, 2006). Distichlis spicata is a rhizomatous

139

halophytic C4 grass with potential use as a turfgrass. Rhizomes are underground vegetative

stems and each has multiple nodes that will initiate growth, so that the growth pattern

typically consists of main rhizomes with several lateral (or branching) ones. The

availability of rhizomes is a characteristic of interest to turfgrass breeders (Porter, 1958),

establishing turf sites using shredded rhizomes is an efficient method (Cockerham, 2008;

Barton et al., 2006; Reummele et al., 1993). Distichlis spicata has low ability to produce

seeds (Pavlicek et al., 1977; U.S. Forest Service, 2004), and germinability can be as low as

3% for the inland subspecies (Qian et al., 2006), so that propagation normally uses

rhizomes (Pessarakli and Kopec, 2008; Pessarakli et al., 2009). The only published

research on D. spicata rhizomes concluded that rhizome fragments were able to tolerate a

wide range of osmotic stresses (to less than -2.8 MPa) as well as cold storage prior to

planting, were capable of sprouting in any season, and had optimum growth when soil

temperatures were 25 oC (Pavlicek et al., 1977). Previous studies of halophytic grasses

have focused on shoot or root growth; few data are available on responses of rhizomes to

salinity. Moreover, rhizomes are often bulked with roots to evaluate below-ground to the

above-ground portions (e.g. experiments on Sporobolus virginicus, Bell and O’Leary,

2003). Studies of the responses of rhizomes to salinity should aid understanding of D.

spicata under saline conditions.

Previous studies have investigated the responses of D. spicata to increasing salinity by

studying the above growth versus the below growth, but have not evaluated the role of

rhizomes as energy reservoir (Marcum, 1999; Alshammary et al., 2004). These earlier

studies showed that shoot growth of D. spicata was not reduced as salinity increased from

control to 23 dS m-1

and root growth was stimulated at salinity levels ranging from 5 to 23

dS m-1

. The experiments presented in this chapter evaluated how D. spicata responds to

salinity in terms of growth rates, ion accumulation in the roots, shoots and rhizomes, and

tissue sugar levels. The hypotheses tested were: 1) Sugars are expected to accumulate in the

rhizomes as salinity is increased; although younger segments of the rhizomes (apical

segment) could to the basal segment. 2) D. spicata restricts the concentrations of the

potentially toxic ions (Na+ and Cl

-) in the apical segment of the rhizomes (where growth

occurs), whereas these ions accumulate in the older portions of rhizomes. 3) Distichlis

140

spicata protects its leaves under high salt concentrations by maintaining low concentrations

of the potentially toxic ions (Na+ and Cl

-) in the leaves.

6.3 Materials and Methods

The study was conducted in a glasshouse at The University of Western Australia, Perth,

Australia. Sand culture pot experiments were irrigated with a complete nutrient solution

(described below). The experiment was planted from rhizomes in 3 L pots filled with

coarse white sand over a 3 cm layer of gravel in the bottom of each pot. Each pot was

irrigated (250 mL per day) with a nutrient solution containing (mM): 4.7 K2SO4, 9.3 CaCl2,

5.0 Na2SO4, 1.0 MgSO4, 0.05 Fe EDDHA (‘Sequestrene 138’), 0.7 Ca(NO3)2, 0.3 K2HPO4,

0.2 NH4H2PO4; and (µM): 25 H3BO3, 2 MnSO4, 2 ZnSO4, 0.5 CuSO4, 0.5 Na2MoO4. The

nutrient solution was adjusted to pH 6.6, using KOH. The nutrient solution contained 10

mM Ca2+

(listed above) a concentration approximately equivalent to that in seawater, such

that growth responses in the higher-salinity treatments would be determined by direct Na+

and Cl- effects, rather than Na

+-induced Ca

2+ deficiency (Greenway and Munns, 1980;

Kopittke and Menzies, 2005). No symptoms of Ca2+

deficiency were observed during the

experiment.

Plants were established for 3 months inside a glasshouse (October, November and

December 2007; Perth, Western Australia). Air temperatures in the glasshouse were,

respectively, 31.3 and 15.3 oC for the average maximum and minimum temperatures. The

experiment then commenced in December (see below). Data on air temperature, sand

temperature and relative humidity during the experiment are given in Figure 6.1A.

Shoots were clipped to a uniform size (30 mm height) prior to commencing the NaCl

treatments, and initial samples were taken on 14th

December 2007. Four treatments were

imposed: control (0 mM NaCl in addition to that in the basal solution), 230, 450 and 680

mM NaCl). The controls had 10 mM Na+ and 18.6 Cl

- in the basal nutrient solution as

141

supply of some Na+ and Cl

- is essential for many halophytes (Flowers et al., 1977). There

were four replicates of each treatment in a randomised complete block (RCB) design.

Treatments were imposed by 50 mM NaCl increments, so as to avoid sudden stress (Koch

et al., 2007). Salt was added in the complete nutrient solution every second day except for

weekends, so that the highest NaCl level was reached on 18th

January 2008. Treatments

continued for 90 days after the highest salt concentration (680 mM NaCl) had been

reached. The final harvest was taken on 18 April 2008.

Air

Tem

p. o

CS

oil te

mp

era

ture

oC

0%

20%

40%

60%

80%

100%

0

10

20

30

40

50

Jan Feb Mar Apr

0

10

20

30

40

50

Jan Feb Mar Apr

B

A

2008

RH

%

Figure 6.1. Summary of (A) average daily air temperature ( ), average daily relative

humidity RH ( ), and (B) soil temperature, in the glasshouse during the experiment.

After reaching the final concentrations, all pots were watered daily by applying enough

solution to leach each pot. Water-holding capacity of the sand when fully drained was 12%

(gravimetric soil water content).

142

Leaf water content and osmotic potential

Leaves were washed with DI water to remove the secreted salts from the salt glands and

left to dry for 30 min prior to excision. Leaf water content was measured as the difference

between fresh mass determined for a sub-sample of fresh leaves and dry mass determined

after the sample had been wrapped in Al-foil, placed on dry ice, stored at -80 oC, and then

freeze-dried. Sap osmotic potential was measured on sub-samples sealed into air-tight

cryovials and frozen in dry ice, stored at – 80 oC, and then sap was expressed from

freeze/thawed tissues using a simple press, and analysed with a freezing-point depression

osmometer (F110, Fiske Associates, Massachusetts).

Roots and the below-ground rhizomes were rinsed in 10 mM CaSO4 in DI water with

mannitol to provide the same osmotic potential as in the four treatments (leachate was

collected and measured) and to maintain the same Ca2+

concentration in the nutrient

solution. Rhizomes were split into three segments: apical the first 6 cm of the rhizome,

basal the last 6 cm, and in between (remaining middle segments). Tissue samples were

wrapped in Al-foil, placed on dry ice, stored at -80 oC, and then freeze-dried. The

remaining tissue samples were dried in an oven at 65oC. Measurements were taken of

shoot, rhizome and root dry mass (DM).

Relative growth rate (RGR) was calculated as: RGR = (lnW2 - lnW1) / (t2 - t1) where W1

is the dry mass at the initial harvest and W2 is the dry mass (DM) at the final harvest, t2 is

the date of the final sampling and t1 is the date of the initial sampling.

Ion concentrations

Ion concentrations (Na+, K

+ and Cl

–) were measured in freeze-dried samples ground to a

fine powder. To extract the ions, 10 ml of 0.5 M HNO3 was added to 0.1 g of ground tissue

(exact amounts of acid and ground tissue were recorded) in vials and the samples were

shaken for 48 h in darkness at 30oC. Diluted extracts were analysed for Na

+ and K

+

(Jenway PFP7 Flame Photometer), and for Cl– (Buchler-Cotlove Chloridometer). Values

were validated using a reference plant tissue sample with known concentrations of Na+, K

+

143

and Cl– (Broccoli 85, ASPAC, 2000). The recovery of the ions from the reference material

was 91% - 95%; no adjustments were made to the data presented.

Sugar concentrations

Sugars in the various freeze-dried tissues were extracted in 80% (v/v) ethanol, refluxed

with boiling for 20 minutes, twice, and determined as hexose units using anthrone (Yemm

and Willis, 1954) and a spectrophotometer (Shimadzu, UV-240, Tokyo, Japan). The

recovery of glucose spiked into some samples before the extraction was used to check the

reliability of the procedure, with recovery of 93% (no adjustments were made to the data

presented).

Soil salinity

The leachate for all pots was collected every fortnight (Figure 6.2). EC of the leachate was

recorded using an EC electrode and meter (Jenway, 4020, Jen-way, England). Sand core

samples from all pots at the end of the experiment were collected using a 3 cm diameter

and 7 cm long PVC tube. Samples were weighed for wet mass, then dried at 105 oC for 48

hours then reweighed to measure the dry mass, so that water content in the sand could be

calculated. Subsamples of 4 g dry mass were taken to measure EC1:5 in the sand by adding

20 ml DI water, shaking mechanically for 16 h, centrifuged at 2000 rpm for 5 min, and left

to equilibrate with room temperature, and then EC was measured. Data are given in

Appendix 6.4A,B).

144

EC

of le

ach

ate

(dS

m-1

)

Sampling date (in 2008)

0

20

40

60

80

100

120

14-Jan 11-Feb 10-Mar 7-Apr

Figure 6.2 Changes in EC of the leachate (dS m-1

) during the course of the experiment,

values are means of 4 replicates. EC of the irrigation treatments were 3.5 dS m-1

( ),

23 dS m-1

( ), 45 dS m-1

( ) and 68 dS m-1

( ).

Statistical analysis of data

Two-way ANOVA was performed using Genstat 10th

Edition (VSN International Ltd).

Treatment effects, tissue type, and rhizome segments by treatment interactions were judged

to be significantly different at P < 0.05.

6.4 Results

Growth

In non-saline conditions, the rhizomes and roots were both of larger dry mass than the

leaves, and RGR of roots exceeded that of rhizomes and of leaves (Figure 6.3). The

response to salinity varies in the different plant segments, growth as % of control in roots

and apical rhizomes was stimulated with 230 mM NaCl irrigation treatment. However no

growth stimulation was noted in the DM and RGR. Plants survived, albeit with slow

growth and considerable leaf damage (see below), even at the highest salinity of 680 mM

NaCl (Figure 6.3).

145

Growth (i.e. DM at final sampling) declined by 50% at 450 mM NaCl, compared to

control, for the roots, apical rhizomes and leaves, whereas growth remained unchanged for

the middle and basal segment of the rhizomes; mature rhizomes grew by producing more

branches. The ANOVA for the dry mass as % of control revealed a significant effect of

salinity levels on growth as a whole (P < 0.001) and also significant differences between

the different segments of the plants (P < 0.001), the interaction between salinity level and

plant segment was significant too (P < 0.001).

The ANOVA of the DM data showed a significant difference between salinity levels (P <

0.001) and between the different segments of the plants (P < 0.001), the interaction

between salinity level and plant segment was significant too (P < 0.001). The ANOVA of

the relative growth rate showed a significant difference between salinity levels (P < 0.001)

and between the different segments of the plants (P < 0.001), but the interaction between

salinity level and plant segment was insignificant (P < 0.40).

DM

% t

o c

on

tro

l tr

ea

tme

nt

0%

50%

100%

150%

200%

0 200 400 600 800

B

0

20

40

0 200 400 600 800

Tis

su

e D

M (

g)

A

Re

lative

Gro

wth

Ra

te (

g.g

-1d

-1)

0.00

0.02

0.04

0 200 400 600 800

C

0%

50%

100%

150%

200%

0 200 400 600 800

D

DM

% t

o c

on

tro

l tr

ea

tme

nt

(rh

izo

me

se

gm

en

ts)

NaCl (mM)

0%

50%

100%

150%

200%

0 200 400 600 800

Figure 6.3. Effect of concentration of NaCl added to the nutrient solution on the leaves

( ), rhizomes ( ) and roots ( ) of D. spicata. Effects are measured as (A) dry

mass (DM) (B) growth (DM) as % of control (C) relative growth rate (D) growth (DM) as

% of control in the apical rhizomes ( ), middle rhizome ( ) and basal rhizome

146

( ). Plants were grown under 0–680 mM NaCl in irrigated sand culture under

glasshouse conditions for 120 days. Values are the mean of 4 replicates. Error bars denote

the SEM. The LSD (at 5%) of DM means is 1.87.

D. spicata produced more rhizomes and roots mass than leaves mass under non-saline

conditions (Figure 6.3). The ratio of rhizome:leaf and root:leaf initially increased as salinity

was increased reaching ~2-fold more at 230 mM NaCl, then root:leaf ratio declined

whereas rhizome:leaf ratio increased (Figure 6.4). At 450 mM NaCl and above, rhizome

growth declined and the ratio of rhizome:leaf did not differ from that under 0 mM NaCl. At

higher levels of salinity (450 and 680 mM NaCl) the growth of Distichlis spicata declined

significantly (Figure 6.3).

NaCl (mM)

0

1

2

0 200 400 600 800

Rhizome/Leaf Root/Leaf

Ra

tio

in D

M

Figure 6.4. Ratios of rhizome to leaf dry mass ( ) and root to leaf dry mass ( )

of Distichlis spicata irrigated with nutrient solutions containing 0–680 mM NaCl. Values

are the mean of 4 replicates and are for final harvest, taken 90 days after salinities reached

their final concentration. Error bars denote the SEM.

The diameter of the main rhizome ~2 cm and the length after growth in sand culture for 90

days irrigation with 680 mM NaCl , although in pots of diameter 15 cm, was 71 cm (data

not shown), as the rhizome had grown in circles against the wall of the pot.

Tissue water content in D. spicata varied significantly for different segments, being lower

in leaves than in rhizomes. Water contents in rhizome and in leaves declined with

increasing salinity, but in the case of rhizomes at or above 450 mM NaCl the water content

147

either remain unchanged (apical rhizome) or even increased (middle and basal rhizomes)

(Figure 6.5).

0

1

2

3

0 200 400 600 800

Tis

sue w

ate

r conte

nt (m

Lg

-1D

M)

NaCl (mM) Figure 6.5. Effect of different salinity levels: (0, 280, 450 and 680 mM NaCl) on tissue

water content of leaves ( ), apical rhizome ( ); the first 6 cm, middle rhizome

( ) and basal rhizomes ( ) the last 6 cm, of Distichlis spicata. Values are means

of 4 replicates and are for final harvest (± standard error). The initial water content was

2.59 and 4.04 mL g-1

DM for leaves and bulk rhizomes. The LSD (at 5%) for comparisons

of means is 0.014.

Ion relations

The increase in salt concentration of the irrigation solution resulted in significant increase

of Na+ concentrations in the tissues of Distichlis spicata (P<0.001). Increases in tissue Na

+

were almost linear increases as external NaCl was raised, with R2 0.99 for both the leaves

and rhizomes and 0.92 for the roots. At 450 mM NaCl the Na+ concentration in the leaves

was >6-fold more than the non-saline control (Figure 6.6A). Significant difference was

found between the different tissues (P<0.001) and also the interaction between plant tissue

type and salt concentration (P<0.001). The highest Na+ concentration was found in the

roots followed by leaves then rhizomes up until 450 mM NaCl, at higher salt level (680

mM NaCl) Na+ concentration in the leaves exceeded that in the roots.

The concentration of Cl- in tissues followed the same trend as Na

+, but since the non-saline

controls already contained higher Cl- than Na

+, the magnitude of the increases in tissue Cl

-

148

concentrations were less; for example, at 450 mM NaCl it was 2.5-fold more than in the

controls (Figure 6.6C). Significant differences were found between tissue types within

salinity treatments, and the interaction between salt treatment and the different tissue

segments was also significant (P<0.001). Concentrations of Na+ and Cl

- were in order of

leaves > roots > rhizomes (Appendix 6.1A,C). Within rhizomes, the order for the tissue ion

concentrations was apical segment > middle > basal (Appendix 6.2A,C).

K+ concentration was high in leaves and rhizomes of controls (low NaCl in nutrient

solution) and then declined at higher levels of NaCl. The K+ concentration in rhizomes and

leaves of plants at 450 mM NaCl was 70% of that in plants under non-saline conditions

(Figure 6.6B). The decline was significant amongst different plant segments, different

salinity treatments and also the interaction between both (P<0.001). In the case of roots, K+

was ~50% of its concentration in the rhizomes and leaves in the non-saline controls and K+

did not decline in roots under the three salt treatments (Appendix 6.2B). This maintenance

of K+ in roots was in contrast to K

+ in the three rhizome segments all of which declined

(Appendix 6.3A,B,C). The insensitivity of root K+ to salinity also contrast with the changes

in Na+ and Cl

- in roots: increases of 6-fold for Na

+ and 2-fold for Cl

- were noted at 450 mM

NaCl when compared to the non-saline control.

The investigation of ion concentrations in different segments of rhizome showed that

average Na+ concentration in the apical segments of the rhizome was 60% more of that in

the basal and middle segments across all the NaCl treatments. The average Cl- was 25%

more in the apical rhizomes than in the basal and middle segments across NaCl treatments

(Figure 6.7).

149

0

1

2

0 200 400 600 800

0

1

2

0 200 400 600 800

0

1

2

0 200 400 600 800

NaCl in the irrigation solution (mM)

Ion

co

nce

ntr

atio

n (m

molg

-1D

M)

Na

+K

+C

l-

A

B

C

Figure 6.6. Concentrations of Na+, K

+ and Cl

- in tissues of Distichlis spicata: leaves

( ), mean of the three rhizome segments ( ) and roots ( ). Plants were

irrigated (250 mL per day) with nutrient solutions containing 0–680 mM NaCl. Values are

the mean of 4 replicates and are for final harvest. Error bars denote the SEM. The average

LSD (at 5%) for means of Na+ is 0.07, K

+ is 0.02 and Cl

- is 0.1.

150

Ion

co

nce

ntr

atio

n (m

molg

-1D

M)

Na

+K

+C

l-

0

1

2

0 200 400 600 800

0

1

2

0 200 400 600 800

0

1

2

0 200 400 600 800

A

C

B

NaCl (mM) Figure 6.7. Concentrations of Na

+, K

+ and Cl

- in rhizome tissues of Distichlis spicata:

Apical rhizome ( ), middle rhizomes ( ) and basal rhizome ( ). Plants were

irrigated (250 mL per day) with nutrient solutions containing 0–680 mM NaCl. Values are

the mean of 4 replicates and are for final harvest, taken 90 days after salinities reached their

final concentration. Error bars denote the SEM. The average LSD (at 5%) for means of Na+

is 0.08, K+ is 0.02 and Cl

- is 0.11.

151

Sugars

The concentration of sugars declined in roots, and also in basal and middle segments of

rhizomes when growth was stimulated at low salt concentrations (stimulation means more

rhizomes became mature over time), the concentration of sugars in the apical segment of

rhizome and in leaves did not change. The maximum sugar concentration was noted in the

basal rhizomes and roots under 680 mM NaCl, in these segments the growth was at its

lowest rate (Figure 6.8; Figure 6.3A,C), so that sugar was accumulating. No significant

correlation was noted between the increase in sugar and the decline in growth with

exception of growth in the apical segments of rhizome (Appendix 6.1).

The lowest sugar concentrations were in the apical rhizomes followed by leaves under the

lowest and highest NaCl treatments; sugars might have increased at intermediate salinity as

growth was presumably inhibited more than photosynthesis, whereas at the highest salinity

the declines could result from greater inhibition of photosynthesis restricting supply of

sugars. However, this is still debatable as other reports showed that sugar concentration

increased under salinity stress (Muscolo et al., 2003).

The 2-way ANOVA test showed highly significant difference between salinity treatments

in respect of sugar concentration (P<0.002), and also (P<0.001) between different plant

segments, however, the difference in sugars for the interaction of salinity and plant

segments was not significant (P<0.08).

152

NaCl (mM)

Sug

ar co

nce

ntration

(mg

g-1

DM

)

0

200

400

0 200 400 600 800

Figure 6.8. Concentrations of total sugars in tissues of Distichlis spicata: leaves ( ),

apical rhizome ( ), middle rhizomes ( ), basal rhizome ( ) and roots (

). Plants were irrigated (250 mL per day) with nutrient solutions containing 0–680 mM

NaCl. Values are the mean of 4 replicates and are for final harvest, taken 90 days after

salinities reached their final concentration. Error bars denote the SEM. The average LSD

(at 5%) for predicted means is 69.4.

6.5 Discussion

Growth and tissue sugar concentrations (Hypothesis 1)

Distichlis spicata in this study was highly tolerant to hypersaline conditions; at 680 mM

NaCl, shoots, rhizomes and roots grew over a 90-day period of continuous irrigation with

saline water. Growth reduction is observed in plants irrigated with the high salt levels.

Distichlis spicata grows at high external salinity levels by accumulating solutes to adjust

the internal osmotic potential of halophytes (Flowers et al., 1977) and secreting toxic ions

(Marcum et al., 2007).

This study showed that D. spicata had the highest growth as a percentage of control in the

apical rhizomes (50% increase) at 230 mM NaCl to the controls (Figure 6.3D), whereas the

average growth for bulk rhizomes was (9% increase) and root growth was (19% increase).

At higher salt level (450 mM NaCl), only growth of apical rhizomes were stimulated by

28% as compared with the non-saline controls. Previous reports showed variability

153

amongst different genotypes of D. spicata (Marcum et al., 2005; Marcum et al., 2007);

however, a tolerant genotype can be expected to have more than one adaptation mechanism

(Singh et al., 2009).

Results showed that sugar and growth (as a percentage of control) in the apical rhizomes

have a significant negative correlation with R2 of 0.99 (Figure 6.3B,D; Figure 6.8;

Appendix 6.1). The highest growth (RGR) and least sugar concentration in the apical

rhizomes compared to other plant segments, indicate that the justify the apical rhizomes

must use solutes other than sugars to adjust osmotically under high levels of salinity,

therefore hypothesis 1 (the apical segment of rhizome could have less sugar accumulation

to the basal segment) is accepted.

D. spicata is a slow growing species under hypersaline conditions (680 mM NaCl) (Figure

6.3C), the relative growth rate decreased significantly. The general decrease in growth rate

of Distichlis spicata is a response to the high salt concentration and could be due to the

combination of factors such as decreased leaf water content, Na+

and Cl-toxicity, and

decreased K+ concentration.

Variations in temperature, light, humidity, or nutrients affected the salinity tolerance of D.

spicata (Kemp and Cunningham, 1981). However, salinity threshold of D. spicata is

reported to be 9.8 dS m-1

(Aschenbach, 2006), 25 dS m-1

(El-Haddad and Noaman, 2001)

and 32 dS m-1

(Gulzar and Khan, 2002). The wide range in tolerance to salinity is attributed

to the prevail environmental conditions in these studies as some were conducted in

glasshouses and others in open field; increase the irrigation frequency and/or high relative

humidity increase the tolerance of D. spicata . Also, inland collections of D. spicata are

less tolerant than the costal/salt-marsh collections. No significant difference in root DM

was found at higher salinity up to 450 mM NaCl (Sagi et al., 1997). The overall growth of

D. spicata demonstrated a typical monocotyledon halophyte were growth declined as

salinity increased.

154

Ion concentrations (Hypothesis 2 – Rhizome portions)

This section will discuss ion concentrations in light of the tissue water content within

rhizome segments. The water content of the plant tissues was the highest in order of apical

rhizome > middle rhizome > basal rhizome. This trend indicates less dilution of ions

occurred in the basal rhizome compared to other meristematic tissues (apical rhizomes),

subsequently helped the apical rhizomes to continue growing and to avoid ion toxicity.

This is the first time that rhizomes are studied in segments and monitored for ions

concentrations, sugar and water content. It is therefore concluded that the reduced water

content in the basal and middle segments of rhizomes is supporting mechanism to tolerate

salinity (Glenn and O'Leary 1984; Glenn 1987).

Based on the reduced tissue water content in the basal and middle rhizome and the high

water content in apical rhizomes, In addition to the lower ion concentrations (DM basis) in

the apical rhizomes, these apical tissues also had a higher water content than the middle

and basal rhizomes, so that consequently ions would also be somewhat diluted in

concentration on a water basis in the apical tissues (Na+

and Cl-), I accept hypothesis 2 (D.

spicata controls the concentration of the potentially toxic ions (Na+ and Cl

-) in the apical

segment of the rhizomes (where growth occurs), whereas these ions accumulate in the older

portions of rhizomes).

Ion concentrations (Hypothesis 3 – Leaves)

It was hypothesised that D. spicata protects its leaves under low-high salt concentrations by

maintaining low concentrations of the potential toxic ions in the leaves. Results of the

present study illustrated the ability of D. spicata to control Na+ and Cl

- in the leaf tissues

lower than their concentrations in root tissues (Figure 6.6A,C). This is consistent with leaf

tissues being more sensitive to these toxic ions than roots (Munns and Termaat, 1986).

These low leaf tissue ion concentrations could result from restricted ions having been

transported to the leaf tissues, and also excretion via salt glands. Na+

concentration is

relatively low in leaves of halophytic monocots with salt glands compared to those in

halophytic dicots (Albert and Popp, 1977; Gorham et al., 1980). Only at high salt

concentrations (680 mM NaCl), was the concentration of the toxic ions in the leaf tissues

155

more than those in roots (Figure 6.6A,C; Appendix 6.2). Previous studies also showed that

the halophytic grass Sporobolus virginicus when irrigated with saline water equivalent to

seawater salinity only 1.4 mg Na+ g

-1 DM accumulated in the leaves (Bell and O’Leary,

2003). Previous reports found that the efficiency of salt secretion by salt glands increases as

the external salt concentration increases (Rozema et al., 1981; Ramadan, 1998; Marcum

and Pessarakli, 2006).

Additionally, maintaining high concentration of K+ takes place at different levels within the

D. spicata. Leaves and rhizomes have ability to maintain 70% of the K+ whereas roots lost

50% of K+ at the highest salinity level when compared to the control treatment. This may

cause metabolic disorders and shows how important is the maintenance of K+ when salts

imposed. Many researchers have observed that salinity reduces the K+ accumulation in salt

stressed plants, and this is usually attributed to the competition of K+ and Na

+ during

uptake (reducing K+ influx) and increasing K

+ efflux (Flowers and Colmer, 2008).

Based on the ability of D. spicata to protect its leaves under high external salt

concentrations (< 680 mM NaCl) by maintaining low concentrations of the potentially

toxic ions (Na+ and Cl

-). I found that the reduced tissue water content in the leaves along

with compartmentation of the toxic ion to rhizomes and roots maintained the growth (DM)

of leaves when compared to roots and rhizomes, therefore hypothesis 3 is supported.

The increase in cytoplasmic osmotic potential due to the accumulation of compatible

solutes is accompanied by water influx into the cell, thus providing the turgor necessary for

cell expansion (Wyn-Jones and Storey, 1981; Yoshiba et al., 1997). Most cellular osmotic

adjustment occurs in the vacuole using primarily inorganic ions (Na+ and Cl

-), but the

cytoplasmic osmotic potential must also be adjusted and this is achieved due to the

accumulation of compatible solutes (Wyn-Jones and Storey, 1981). The lowering of

osmotic potential (i.e. more negative) is accompanied by water influx into the cell, thus

providing the turgor necessary for cell expansion (Yoshiba et al., 1997).

156

The sugar concentration increased following the decline in growth at moderate-to-high

salinity levels (Bell and O’Leary, 2003). Sugar accumulation in the middle and basal

segments of rhizomes and in leaves of salinised plants in the present study was likely due

to the reductions in their growth exceeding reductions in net photosynthesis. Comparative

studies of salt sensitive and tolerant plants demonstrated that salt sensitive species with

greater growth inhibition accumulate more sugar than the tolerant ones (Dubey and Singh,

1999). The present study indicates that D. spicata is highly tolerant of long-term exposure

to gradual increase of salinity. However, D. spicata is known to accumulate glycinebetaine

(Marcum, 1999; Murphy et al., 2003), so the sugar measurements here are only part of the

overall composition of organic solutes.

6.6 Conclusions

Ion exclusion is predominant salt avoidance mechanism in glycophytes (Greenway and

Munns, 1980), but some authors have also suggested that reduced leaf water content in

expanded cells/tissues with elasticity can decrease the requirement for ion uptake for

osmotic adjustment (Glenn, 1987), so that tissues can further restrict the uptake of toxic

ions. By contrast, others consider that increased tissue water content can ‘dilute’ toxic ions

(e.g. succulent halophytes, Flowers et al. 1977) and a similar effect might have occurred in

the apical rhizomes of plants in the present study in which increased water content in their

tissues could have diluted somewhat the concentrations of Na+ and Cl-.

D. spicata protects its leaves within medium-high salt levels by sequestering the toxic ions

to the roots and rhizomes even at 680 mM NaCl by storing these toxic ions in roots and

rhizomes. An important discovery from this research is that rhizomes maintain their growth

when the salts increase in the root zone. The exploration of different segments of the

rhizome could result in the identification of the optimal propagation under saline

environment by using the apical rhizomes than the currently used shredded rhizomes based

on the distinction of apical rhizome segments to relocate ions and also to maintain high

tissue water content. The existence in plants of quantitative variations in the physiological

trait of sugar accumulation in response to salt stresses has suggested it is a possible

157

consideration as a selection criterion for breeding programs. For future research, I

recommend to quantify the different types of sugars in the segments of rhizomes and also

to study the sugar/ion concentrations under sudden massive changes in salinity of the

irrigation water. Studying other organic solutes (glycinebetaine and potentially proline) will

add detailed understanding on the contribution of different organic solutes to the osmotic

adjustment. Such research will broaden the use of D. spicata in coastal areas as these areas

are subjected to sudden changes in salinity.

D. spicata is recommended for hypersaline environments (Marcum, 2008). The slow

growth rate of D. spicata as shown in this study supports the fact that D. spicata is a

potential high salt-tolerant turfgrass with low-maintenance requirements. The studied

turfgrass species is widely accepted as high quality turfgrass even though its growth rate is

low (2%) at equivalent to seawater level, but this species it is worthy of future turfgrass

research for use on low-traffic saline sites.

158

6.7 Appendices

Basal r

hiz

om

es

Clipping DM (g)

y = -19.84x + 97.717R² = 0.9998

0

25

50

75

100

0 1 2 3

0

100

200

300

0 10 20 30

0

50

100

150

0 1 2 3

A

B

C

Sug

ar (

g g

-1D

M)

Mid

dle

rh

izom

es

Ap

ical r

hiz

om

es

Appendix 6.1. Correlation between the average sugar concentration and average growth

(DM) of (A) apical rhizome, (B) middle rhizome and (C) basal rhizome. Data points are for

the 3 salt treatments. Values are the mean of 4 replicates at the final harvest.

159

NaCl (mM)

Ion

co

nce

ntr

atio

n (m

molg

-1D

M)

Leaves

Rh

izo

mes

Ro

ots

y = 0.0022x + 0.0266R² = 0.9858

y = -0.0005x + 0.5982R² = 0.9959

y = 0.0018x + 0.1809R² = 0.9358

0

0.5

1

1.5

2

0 200 400 600 800

Na

K

Cl

y = 0.0011x + 0.0472R² = 0.9854

y = -0.0004x + 0.436R² = 0.9432

y = 0.0009x + 0.2045R² = 0.9716

0

0.5

1

1.5

2

0 200 400 600 800

Na

K

Cl

y = 0.0017x + 0.1171R² = 0.9203

y = -5E-05x + 0.206R² = 0.1222

y = 0.0015x + 0.2351R² = 0.9686

0

0.5

1

1.5

2

0 200 400 600 800

Na

K

Cl

C

B

A

Appendix 6.2. Correlation between the average Na+ ( ), K

+ ( ) and Cl

- ( )

on the average growth (DM) of (A) leaves, (B) rhizome and (C) roots, data are for the four

treatments. Values are the mean of 4 replicates at the final harvest.

160

NaCl (mM)

Ion

co

nce

ntr

atio

n (m

molg

-1D

M)

Ap

ical r

hiz

om

eM

idd

le rh

izom

eB

asal r

hiz

om

e

y = 0.0014x + 0.0735R² = 0.9993

y = -0.0005x + 0.5549R² = 0.9688

y = 0.001x + 0.259R² = 0.9829

0

0.5

1

1.5

2

0 200 400 600 800

Na

K

Cl

y = 0.0009x + 0.0667R² = 0.9331

y = -0.0004x + 0.3878R² = 0.952

y = 0.0007x + 0.1641R² = 0.9161

0

0.5

1

1.5

2

0 200 400 600 800

Na

K

Cl

y = 0.001x + 0.0015R² = 0.9672

y = -0.0004x + 0.3645R² = 0.8726

y = 0.001x + 0.1905R² = 0.9244

0

0.5

1

1.5

2

0 200 400 600 800

Na

K

Cl

C

B

A

Appendix 6.3. Correlation between the average Na+ ( ), K

+ ( ) and Cl

- ( )

on the average growth (DM) of (A) apical rhizome, (B) middle rhizome and (C) basal

rhizome, data are for the four treatments. Values are the mean of 4 replicates at the final

harvest.

161

y = -0.0244x2 + 3.2373x - 2.3963

R² = 0.9981

0

20

40

60

80

100

120

0 20 40 60 80

y = -19.223x2 + 97.206x - 27.657

R² = 0.7246

0

20

40

60

80

100

120

0 0.5 1 1.5 2 2.5 3

A

B

ECw (dS m-1)

EC1:5 (dS m-1)

EC

leach

ate

(dS

m-1)

Appendix 6.4. (A) Correlation between salinity of irrigation water (ECw) and salinity of

the leachate (also an ECw) and (B) correlation between soil salinity EC1:5 and salinity of the

leachate. Values are means of 4 replicates, leachate was taken 2 days before the final

harvest. Error bars denote the SEM.

162

163

Chapter 7

Concluding Comments

7.1. Overview

The experimental chapters (Chapters 3-6) in this thesis have investigated various aspects

of the growth, quality and physiology of salt tolerance in four species used as turfgrass

(Pennisetum clandestinum, Paspalum vaginatum, Sporobolus virginicus and Distichlis

spicata) and the implications of irrigating these with saline water (ECw 13.5 dS m-1

under

field conditions and higher salinity levels in glasshouse experiments) on soil salinity and

turfgrass performance. This thesis has contributed knowledge on the salinity tolerance

levels and mechanisms for tolerance in the three halophytic species (P. vaginatum, S.

virginicus and D. spicata) compared with the non-halophyte (P. clandestinum).

7.2. Tested hypotheses

Twelve hypotheses have been tested in four experimental chapters; Table 7.1 lists these

hypotheses and the conclusion on each hypothesis (√ for hypothesis supported and × for

hypothesis rejected).

164

Table 7.1 Summary of hypotheses tested and main findings as related to each hypothesis.

Chapter Hypotheses Conclusion

3

1. The halophytic turfgrass species would grow and

retain colour better than the non-halophyte under saline

conditions. Found: The halophyte P. vaginatum and the non-

halophyte P. clandestinum were more vigourous and so had

higher DM of clippings under saline field conditions than D.

spicata and S. virginicus; colour was unchanged for the three

halophytic species but declined in P. clandestinum under

saline irrigation.

× Growth

√ Colour

2. The major criteria for salinity tolerance in the

halophytic grasses would be to maintain a high ratio of

K+:Na

+ in the leaf tissues and maintain tolerable

concentrations of toxic Na+ and Cl

-. Found: High K

+:Na

+

ratio and low Na+ and Cl

- were related to the salinity

tolerance (growth as % of control) of the 4 species.

√

4

3. The halophytic grasses would suffer less growth

declines in response to increasing salinity than the non-

halophyte. Found: Shoot mass was reduced more than

belowground mass with increasing salinity of the external

solution; reductions in growth were less pronounced in the

halophytes than the non-halophyte.

√

4. The halophytic grasses would have smaller increases

in Na+ and Cl

- concentrations in leaf tissues, compared

with the non-halophyte, when external soil salinity

increases. Found: D. spicata and S. virginicus had better

abilities to maintain lower Na+ and Cl

- in the leaf tissue under

a range of external salinity levels, presumably salt exclusion

played a major role but salt excretion via glands would also

have contributed to the low leaf Na+ and Cl

- concentrations in

these two halophytes (excretion was not studied).

√

165

5. Shoot growth reductions under salinity would be

correlated with increases in leaf tissue Na+ and/or Cl

-

concentrations, both in the halophytes and the non-

halophyte. Found: Shoot growth reductions under salinity

were positively correlated with increases in leaf tissue Na+

and Cl- concentrations, both in the halophytes and the non-

halophyte. Growth reductions were also correlated with

decreased leaf K+:Na

+ ratio.

√

6. Water use by the halophytes would be maintained

under saline irrigation, but would decline markedly in the

non-halophyte. Found: Irrigation with saline water reduced

the water use of the non-halophyte substantially, whereas for

the three halophytes the reductions were modest.

√

5

7. As the volume of irrigation water is increased, the

salinity of the soil solution would be maintained at a level

closer to that of the applied water, owing to the greater

leaching of salts into deeper soil layers. Found: Salinity of

the soil solution decreased in the order of 40% > 60% > 80%

> 100% replacement irrigation of net evaporation.

√

8. In situations where salinity accumulates in the root

zone owing to insufficient leaching, P. vaginatum would

have decreased growth and colour quality in response to

the salinity of soil solution. Found: correlational analysis

showed that clipping dry mass declined as irrigation was

reduced, but colour was not systematically affected by

irrigation treatment.

√

9. Decreases in the growth and quality of P. vaginatum

with different volumes of saline irrigation would be

mediated by ion concentrations in the tissues, i.e. high

concentrations of Na+ or Cl

- and/or low concentrations of

K+. Found: The decreases in growth with decreased irrigation

√

166

(Hypothesis 8) were associated with increase in ECsoil solution

and Cl- concentration in the clippings. Na

+ concentration was

not significantly correlated with growth reduction.

6

10. In D. spicata, sugars are expected to accumulate in

the rhizomes as salinity is increased; although younger

segments of the rhizomes (apical segment) could differ to

the basal segment. Found: Total sugars in the apical

rhizomes declined, whereas they increased in the non-

growing older basal rhizome segments.

√

11. D. spicata restricts the concentrations of the

potentially toxic ions (Na+ and Cl

-) in the apical segment

of the rhizomes (where growth occurs), whereas these

ions accumulate in the older portions of rhizomes. Found:

Concentrations of Na+

and Cl- were lower in the apical

rhizome, whereas these ions accumulated in the older basal

portions of rhizomes.

√

12. D. spicata protects its leaves under high salt

concentrations by maintaining low concentrations of the

potentially toxic ions (Na+ and Cl

-) in the leaves. Found:

Concentrations of Na+ and Cl

- in the leaf tissues were lower

than in root tissues, also the leaves maintain higher K+

concentrations.

√

7.3. Reflections on experimental outcomes

Soil salinity

The present thesis has adopted the position that it is the salinity of the soil solution that

drives growth and quality of the studied species. The relationship between ECe, soil

moisture and the salinity of the soil solution has been developed into an approximate rule

167

of thumb by researchers from the USA, where the salt concentration of the soil solution is

~twice the ECe at field capacity and about 4 times the ECe at permanent wilting point

(Maas and Hoffman, 1977). In the present work, I have directly estimated the salinity of

the soil solution from EC1:5 readings and soil water content measurements.

Changes in soil salinity were evaluated in field plots in a Mediterranean-type climate;

ECsoil solution was ~6.5 dS m-1

prior to saline irrigation and increased gradually to ~ 40 dS

m-1

by mid-summer (Figure 3.11, Chapter 3), and growth of the non-halophyte (P.

clandestinum) was reduced. Growth of one of the halophytes (P. vaginatum) was also

reduced. By contrast, growth of S. virginicus and D. spicata were not impeded. Colour

remained unchanged by saline irrigation for the three halophytic species, but it declined

in P. clandestinum (Figure 3.10, Chapter 3). In addition, the four species differed in

vigour; P. vaginatum was the most vigorous of the studied species. When irrigation with

saline water ceased during autumn, the non-halophyte showed recovery whereas the

halophytes continued their previous good performance. The thesis also investigated the

quality of turfgrass species under range of salinities at levels up to 750 mM NaCl (Figure

4.6, Chapter 4). Understanding the ability of species to withstand extreme salinities helps

turfgrass managers to introduce species into turfgrass amenities given that soil salinity is

dynamic and plants respond to the salinity of the soil solution.

An illustration of the effect of salinity on plant vigour and salinity tolerance, with two

main causes for decreased growth and colour quality in saline conditions, is shown in

Figures 7.1 and 7.2. Salinity of the soil solution causes increases in toxic ions in plant

tissues which consequently reduce growth and quality in the non-halophytic species.

Figure 7.1 shows schematically that high ECsoil solution can be expected to have two kinds

of consequences for plant growth (cf. Greenway and Munns, 1980). Firstly, as the soil

dries, the salt concentration increases resulting in decreasing osmotic potential, which in

addition to the matric potential further decreases the total soil water potential (Turner,

1974). These two factors cause a “physiological drought”. Secondly, the high Na+ and Cl

-

168

can also cause ionic toxicity (excessive Na+ and Cl

- in tissues) and an imbalance between

K+ and high Na

+ in the cells (Munns and Tester, 2008).

A) Adverse water relations

B) Ion toxicity

Figure 7.1 Conceptual

model presenting (A)

responses of plants to

the adverse water

relations, and (B)

responses to ion toxicity

(Na+ and Cl

-) when

irrigated with saline

water. Model is

developed after

Greenway and Munns

(1980).

169

Figure 7.2 Conceptual model

presenting responses of some

halophytes and non-halophyte

turfgrasses when irrigated with saline

water. Model includes responses

discussed in experimental chapters and

literature review of this thesis.

170

Growth and colour of turfgrass

Reporting the responses of plants to salinity, in terms of the impact on vigour (growth

under non-saline conditions) and tolerance (the ratio of growth under saline conditions to

growth under non-saline conditions) adds to best selection criteria of wide range of plant

species for various uses (turfgrasses, forages and field crops) in saline situations. In

theory, a plant could grow strongly under saline conditions because it had high vigour,

high tolerance to salinity, or a combination of both traits (Richards, 2008). The

halophytic turfgrass species D. spicata, S. virginicus and P. vaginatum retained high

quality colour after commencing the saline irrigation whereas the non-halophyte (P.

clandestinum) suffered leaf firing and after time turned brown (Chapters 3 and 4). The

tolerance of the four turfgrass species to hypersaline environments (Chapter 4)

demonstrated the potential to use these halophytic species with alternative irrigation

water resources while maintaining good quality turfgrass, confirming earlier work on P.

vaginatum (Carrow et al., 2005) but adding new knowledge for S. virginicus and D.

spicata.

Irrigation volumes

Soil salinity increased as irrigation volume was decreased. The field evaluation of P.

vaginatum when irrigated with saline water at four volumes (40, 60, 80 and 100%

replacement of net evaporation) showed at 100% replacement of net evaporation a

reduction of 30-60% in soil salinity compared to that under the lowest irrigation volume

(40% replacement). Leaching of the soluble salts from the root zone through applying

high irrigation volumes (80 and 100% replacement of net evaporation) was effective in

removing excess salts from the upper zone of the soil. In addition, it was also clear in

Chapters 3 and 5 that soil salinity increased during the irrigation season then after

terminating the irrigation upon autumn rainfall, soil salinity decreased. Salt leaching

requires, in addition to high irrigation volumes or seasonal rainfall, that a suitable

drainage system is installed, and especially when the soil texture is clay (Carrow et al.,

2005).

171

Water use

The outdoors lysimeter experiment studied turfgrass water use. Irrigation using saline

water (13.5 dS m-1

) reduced the water use of the non-halophyte by 31%, being more

inhibitory than for the three halophytes which showed more modest reductions (7 – 21%

of those in the non-saline irrigation) (Table 4.2 of Chapter 4). Variations in water use

under saline conditions are attributed to the ability of the plants to osmotically adjust, and

maintain the growth of leaves and roots (Beard, 1985; Kneebone et al., 1992). In the

cases of S. virginicus and D. spicata growth rates were low. This lower growth

presumably enabled these species to maintain low ET rates. The lower ET rates are a trait

useful for water conservation (Barnett et al., 2005). In addition, the slower growth in

these two species means they will likely be best suited to low traffic areas where wear

and also maintenance requirements are less than in high traffic areas.

Stimulation by salinity of root growth over shoot growth was found in Chapter 4; this is

considered to be a mechanism employed to tolerate salinity (Kage et al., 2004). Increased

roots relative to shoot size might help counter low external water potential, by increasing

plant absorptive area and maintaining water balance (Donovan and Gallagher, 1985;

Alshammary et al., 2004). The study of D. spicata in the glasshouse also showed

increased root:shoot ratio in saline conditions (Appendix 4.3 of Chapter 4). Of the studied

species, it seems that P. vaginatum, S. virginicus and D. spicata with greater salinity

tolerance stimulate the growth of roots and rhizomes relative to shoots when the soil

water becomes saline. In contrast, the non-halophyte species P. clandestinum suffered

reductions in growth in both the belowground and aboveground tissues.

Dose response to salinity

The controlled environment (glasshouse) provided an opportunity to study the turfgrass

responses to a wide range of salt concentrations. D. spicata and S. virginicus tolerated

salinities up to 750 mM NaCl. Growth was studied for both the aboveground tissues

(leaves and stolons) and the belowground tissues (roots and rhizomes). The declines in

growth with saline irrigation were associated with high Na+ and Cl

- concentrations in the

aboveground tissues reaching inhibitory levels (Pessarakli et al., 2006). However, for the

172

belowground tissues, D. spicata increased by 14% at the treatment of 125 mM NaCl and

S. virginicus increased by 60% at 230 mM NaCl. D. spicata and S. virginicus controlled

the Na+ and Cl

- accumulation in leaves better than the higher Na

+ and Cl

- in the third

halophytic species P. vaginatum. The high (presumably more toxic) ion concentrations in

P. vaginatum occurred despite a higher tissue water content. Halophytes maintained

adequate K+ (~0.4 mmol g

-1 DM) in saline treatments, whereas K

+ declined in the non-

halophyte (Figures 4.2 of Chapter 4).

Use of saline water (at seawater levels or even above) to grow turfgrasses was supported

by the findings of Chapter 5 where the halophytic S. virginicus and D. spicata retained

high colour quality even at 750 mM NaCl. Even though the growth declined, using these

species might provide an advantage to some turfgrass amenities and road side verges, as

these require low maintenance. Successful seawater irrigation depends on the use of

halophytes able to survive and retain good colour at high level of salts in the soil (Mudie,

1974; Glenn et al., 1991).

Ion relations

There was considerable similarity in the effects of external salinity on ion concentrations

in the leaves between the different experimental chapters (Figure 7.3). Salt tolerance

amongst the four studied turfgrasses could be attributed to the exclusion (and possible

excretion) of potentially toxic ions from the leaf tissues, so that leaves contained lower

Na+ and Cl

-, and higher K

+ concentration. Toxic ions (Na

+ and Cl

-) increased in

concentration in the leaf tissues of the four species when irrigated with saline water

(Figure 7.3), however, mechanisms of ion exclusion (or excretion) enabled the halophytes

to maintain lower Na+ and Cl

−, and retain higher K

+:Na

+ ratio than the non-halophyte (P.

clandestinum). On average, S. virginicus and D. spicata (the two halophytic species that

possess salt glands) contained ~50% less Na+ and Cl

- in their leaves than P.

clandestinum.

A comparison of the Na+ in the aboveground biomass (Chapter 4) shows that the non-

halophyte P. clandestinum had increases in aboveground Na+ (8-fold more at 125 mM

173

NaCl and 16-fold more at 250 mM NaCl) which might have caused leaf firing and leaf

senescence. By contrast, the three halophytes, P. vaginatum, S. virginicus and D. spicata,

maintained low tissue Na+ at lower salinity levels (up to 230 mM NaCl), but as salinity

increased substantial increases in the aboveground Na+ occurred for P. vaginatum whilst

levels in S. virginicus and D. spicata remained almost unchanged. It is therefore

considered that the high salinity tolerances observed for S. virginicus and D. spicata were

due largely to their abilities to exclude and/or secrete toxic ions (Na+ and Cl

-) from the

leaves (Marcum, 2006). The low concentration of Na+ was evident despite the slower

growth of D. spicata and S. virginicus, whereas the faster-growing halophyte (P.

vaginatum) had higher Na+ and Cl

- concentrations. These higher concentrations in P.

vaginatum would have resulted from higher uptake and/or from the lack of salt glands on

this species and therefore no ion excretion.

Leaf water content is an adaptive mechanism to tolerate salinity in some halophytic

turfgrasses (Lee et al., 2008). In this study, only P. vaginatum had high leaf water content

when compared to D. spicata and S. virginicus which presumably contributed to the

ability of P. vaginatum to tolerate the higher concentrations (dry mass basis) of toxic ions

compared to their concentration in the other two halophytic species. The higher tissue

water content in P. vaginatum would have diluted the ion concentration in leaves (cf.

Grieve and Fujiyama, 1987).

Another trait contributing to salt tolerance, at least in D. spicata (Chapter 6), might be

localisation of Na+ and Cl

- in old rhizomes to maintain tolerable levels in the younger

rhizomes, and thus maintain growth under high salinity levels. The young (apical

rhizome) had the lowest concentration of Na+ and Cl

-.

Potassium (K+) is an essential macronutrient for plants, and it has a role as an osmoticum

to maintain the low water potential of plant tissues (Wang et al., 2004). This study shows

that K+ concentration in leaves and shoots was higher than in roots and rhizomes. The

reduction in K+ concentration in the non-halophyte correlates with the increase in soil

salinity. High K+ concentration contributes to plant tissue K

+:Na ratio, of importance for

174

avoiding toxic ion effects. K+:Na

+ ratio in leaves was further decreased with the increase

of soil salinity but in roots and rhizomes was more stable except for the extremely high

saline conditions. Many salt tolerant plants exhibit greater K+:Na

+ selectivity than salt-

sensitive plants. In this thesis, the higher K+:Na

+ ratios were for the most salt tolerant

species (D. spicata and S. virginicus) and is thought to be a significant salt tolerance

adaption (calculated from Fig. 7.3).

Osmotic potential

Osmotic adjustment has been identified as a salinity tolerance mechanism in halophytes

(Flowers et al., 1977). Osmotic adjustment enables the maintenance of cell turgor, salt-

tolerant grasses adjust osmotically at minimal rate if compared to salt sensitive grasses at

same salinity level (Marcum, 1999). In the present study (Chapters 3 and 4), two species

(S. virginicus and D. spicata) maintained low tissue Na+ and Cl

- concentrations and high

turfgrass quality under salinity stress, but had low leaf sap osmotic potential (more

negative). In contrast, P. clandestinum that did not perform well under salinity stress, had

high tissue ions and therefore high apparent levels of osmotic adjustment. In addition to

the ion concentration, the organic solutes contribute to the osmotic adjustment in grasses;

as one example, glycinebetaine increased in Sporobolus virginicus at 450 mM NaCl

external salinity (Marcum, 2001). The overall implication is that a particular species may

employ an important attribute, such as osmotic adjustment, but that one mechanism,

alone, will not necessarily predict comparative performance between species.

Furthermore, also within the same species, the same mechanism (osmotic adjustment)

might change as per the level of salinity stress (low vs high external salinity).

175

0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 8000

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 8000

0.5

1

1.5

2

0 200 400 600 8000

0.5

1

1.5

2

0 200 400 600 800

Ion

co

nce

ntr

atio

ns

in le

af t

issues

(m

molg

-1D

M)

Na+

K+

Cl-

NaCl (mM)

P. clandestinum P. vaginatum S. virginicus D. spicata

0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 800

0

0.5

1

1.5

2

0 200 400 600 800

Figure 7.3 Comparison of Na+, K

+ and Cl

- concentrations in leaf tissues of Pennisetum clandestinum (first column), Paspalum vaginatum (second

column), Sporobolus virginicus (third column) and Distichlis spicata (fourth column) across Chapters. Leaf tissue ion concentrations are graphed

against the estimated salinity of the soil solution in the field study (Chapter 3) and salinity of the nutrient solution used in the glasshouse

experiment (Chapter 4). Continuous lines represent the glasshouse study (Chapter 4) and the dotted lines represent the field experiment (Chapter

3). Values of ECsoil solution (dS m-1

) were converted to NaCl (mM) assuming that 1 dS m-1

~ 10 mM NaCl.

176

7.4. Conclusions

Salt tolerances of the four species studied were in the order: D. spicata, S. virginicus, P.

vaginatum and then P. clandestinum. The three halophytes performed well when irrigated

with saline groundwater (13.5 dS m-1

) at a field site with a hot, dry summer environment.

Salt tolerance was also demonstrated under controlled conditions at higher levels of

salinity. The three species differed in vigour. Irrigation volume dose-response trials in the

field established that P. vaginatum can maintain high colour quality even though salt

accumulation in the soil can be considerable, but growth had declined. D. spicata and S.

virginicus are also expected to maintain high growth and colour quality under high

irrigation volumes (80 and 100% replacement of net evaporation) based on the results

from the field experiment when irrigated at 60% replacement and also their superior

tolerance to salinity in the controlled environment experiment.

177

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