In The Name Of

Allah The Most Beneficent,

The Most Merciful

Physiological, biochemical and phytoremedial characterization

of acacia species for salt affected soils

By

Ghulam Abbas (2004-ag-1146)

M.Sc. (Hons.) Agriculture (Soil Science)

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

in

SOIL SCIENCE

INSTITUTE OF SOIL AND ENVIRONMENTAL SCIENCES FACULTY OF AGRICULTURE

UNIVERSITY OF AGRICULTURE FAISALABAD, PAKISTAN

2013

Declaration

I hereby declare that the contents of the thesis “Physiological, biochemical and

phytoremedial characterization of acacia species for salt affected soils” are

product of my own research and no part has been copied from any published source

(except the references, standard mathematical or biochemical models/ equations/

formulae/ protocols etc.). I further declare that this work has not been submitted for

award of any other diploma/degree. The university may take action if the information

provided is found inaccurate at any stage.

Ghulam Abbas

2004-ag-1146

To

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the supervisory committee, certify that the contents and form of the

thesis submitted by Ghulam Abbas Regd. No. 2004-ag-1146 have been found

satisfactory and recommend that it be processed for evaluation, by External

Examiner(s) for the award of the degree.

SUPERVISORY COMMITTEE:

Chairman : (Dr. Muhammad Saqib) Member : (Dr. Javaid Akhtar) Member :

(Dr. Shahzad M.A. Basra)

Dedicated

To

My Dear Parents

Who taught to me the best,

behaved with me the best &

wished for me the best

AACCKKNNOOWWLLEEDDGGEEMMEENNTTSS All the praises and thanks are for Almighty ALLAH, The Compassionate, The

Merciful, the source of knowledge and wisdom, Who has blessed me with good health.

After that, all praises are due to His Holy Prophet MUHAMMAD (Peace be upon him),

the most perfect and exalted among and of ever born on earth, who is for ever a torch of

guidance and knowledge for humanity as a whole.

I have the honor to express my deep sense of gratitude and indebtedness to my

honorable supervisor, Dr. Muhammad Saqib, Institute of Soil & Environmental

Sciences, University of Agriculture, Faisalabad under whose dynamic and inspiring

guidance as well as sympathetic attitude, I started my research work and was able to

prepare this manuscript.

I offer my sincere gratitude to Dr. Javaid Akhtar, Institute of Soil and

Environmental Sciences, Dr. Shahzad M.A. Basra, Department of Crop Physiology,

University of Agriculture, Faisalabad for their consistent and inspiring guidance and

kind cooperation for accomplishing the research work.

I feel heartiest gratitude to my dearest parents, sister, brothers and esteemed and

sincere friends Behzad, Sami, Saqib, Shakeel, G. Farid, Ijaz, Tasneem and Shahid who

supported me throughout my studies.

Ghulam Abbas

CONTENTS CHAPTER #

TITLE

PAGE #

1

INTRODUCTION

1

2

REVIEW OF LITERATURE

6

3

MATERIALS AND METHODS

26

4

RESULTS AND DISCUSSION

41

5

SUMMARY

111

LITERATURE CITED

114

LIST OF TABLES

S. No. Title Page

3.1 The characteristics of soils used for experiments 39

3.2 The characteristics of irrigation water used for experiments 40

4.3.1 Effect of acacia species on soil pHs 89

4.3.2 Effect of acacia species on soil electrical conductivity (ECe) 90

4.3.3 Effect of acacia species on soil sodium adsorption ratio (SAR)

90

LIST OF FIGURES

Figure No.

Title Page

No.

4.1.1 Effect of salinity on shoot fresh weight of acacia species

45

4.1.2 Effect of salinity on shoot dry weight of acacia species

45

4.1.3 Effect of salinity on root fresh weight of acacia species

46

4.1.4 Effect of salinity on root dry weight of acacia species 46

4.1.5 Effect of salinity on shoot length of acacia species

47

4.1.6 Effect of salinity on root length of acacia species

47

4.1.7 Effect of salinity on shoot Na+ concentration of acacia species

48

4.1.8 Effect of salinity on root Na+ concentration of acacia species

48

4.1.9 Effect of salinity on shoot K+ concentration of acacia species

49

4.1.10 Effect of salinity on root K+ concentration of acacia species

49

4.1.11 Effect of salinity on shoot Cl- concentration of acacia species

50

4.1.12 Effect of salinity on root Cl- concentration of acacia species

50

4.1.13 Effect of salinity on shoot K+: Na+ ratio of acacia species

51

4.1.14 Effect of salinity on root K+: Na+ ratio of acacia species

51

4.1.15 Correlation between shoot dry weight and shoot Na+ concentration 52

4.1.16 Correlation between shoot dry weight and shoot K+ concentration

52

4.1.17 Correlation between shoot dry weight and shoot Cl- concentration

52

4.2.1 Effect of salinity on shoot fresh weight of acacia species

60

4.2.2 Effect of salinity on shoot dry weight of acacia species

60

4.2.3 Effect of salinity on root fresh weight of acacia species

61

4.2.4 Effect of salinity on root dry weight of acacia species 61

4.2.5 Effect of salinity on shoot length of acacia species

62

4.2.6 Effect of salinity on root length of acacia species

62

4.2.7 Effect of salinity on SOD activity of acacia species

63

4.2.8 Effect of salinity on CAT activity of acacia species

63

4.2.9 Effect of salinity on POD activity of acacia species

64

4.2.10 Effect of salinity on membrane stability index activity of acacia species 64

4.2.11 Effect of salinity on tartaric acid release (after 14 days) of acacia

species 65

4.2.12 Effect of salinity on tartaric acid release (after 28 days) of acacia

species 65

4.2.13 Effect of salinity on malic acid release (after 14 days) of acacia

species 66

4.2.14 Effect of salinity on malic acid release (after 28 days) of acacia

species 66

4.2.15 Effect of salinity on citric acid release (after 14 days) of acacia

species 67

4.2.16 Effect of salinity on citric acid release (after 14 days) of acacia

species 67

4.2.17 Effect of salinity on shoot ash alkalinity of acacia species

68

4.2.18 Effect of salinity on shoot ash alkalinity of acacia species

68

4.3.1 Effect of salinity and water stress on plant height of acacia species

78

4.3.2 Effect of salinity and water stress on stem diameter of acacia species 78

4.3.3 Effect of salinity and water stress on number of leaves of acacia species 79

4.3.4 Effect of salinity and water stress on number of branches of acacia species 79

4.3.5 Effect of salinity and water stress on shoot fresh weight of acacia species 80

4.3.6 Effect of salinity and water stress on shoot dry weight of acacia

species 80

4.3.7 Effect of salinity and water stress on root fresh weight of acacia

species 81

4.3.8 Effect of salinity and water stress on root dry weight of acacia

species 81

4.3.9 Effect of salinity and water stress on root length of acacia species

82

4.3.10 Effect of salinity and water stress on number of nodules of acacia

species 82

4.3.11 Effect of salinity and water stress on nodule fresh weight of acacia

species 83

4.3.12 Effect of salinity and water stress on nodule dry weight of

acacia species 83

4.3.13 Effect of salinity and water stress on chlorophyll contents of acacia species 84

4.3.14 Effect of salinity and water stress on relative water contents of acacia species 84

4.3.15 Effect of salinity and water stress on leaf Na+ concentration of acacia species 85

4.3.16 Effect of salinity and water stress on stem Na+ concentration of acacia species 85

4.3.17 Effect of salinity and water stress on root Na+ concentration of acacia species 86

4.3.18 Effect of salinity and water stress on leaf K+ concentration of acacia

species 86

4.3.19 Effect of salinity and water stress on stem K+ concentration of

acacia species 87

4.3.20 Effect of salinity and water stress on root K+ concentration of acacia

species 87

4.3.21 Effect of salinity and water stress on leaf Cl- concentration of acacia

species 88

4.3.22 Effect of salinity and water stress on stem Cl- concentration of

acacia species 88

4.3.23 Effect of salinity and water stress on root Cl- concentration of acacia

species 89

4.4.1 Effect of salinity and sodicity on plant height of acacia species

98

4.4.2 Effect of salinity and sodicity on stem circumference of acacia

species 98

4.4.3 Effect of salinity and sodicity on number of leaves of acacia species 99

4.4.4 Effect of salinity and sodicity on number of branches of acacia species 99

4.4.5 Effect of salinity and sodicity on Na+ concentration of older leaves of acacia species 100

4.4.6 Effect of salinity and sodicity on Na+ concentration of younger leaves of acacia species 100

4.4.7 Effect of salinity and sodicity on K+ concentration of older leaves of acacia species 101

4.4.8 Effect of salinity and sodicity on K+ concentration of younger leaves

of acacia species 101

4.4.9 Effect of salinity and sodicity on Cl- concentration of older leaves of

acacia species 102

4.4.10 Effect of salinity and sodicity on Cl- concentration of younger leaves

of acacia species 102

4.4.11 Effect of acacia species on soil pH (0-30cm)

103

4.4.12 Effect of acacia species on soil pH (30-60cm)

103

4.4.13 Effect of acacia species on soil EC (0-30cm)

104

4.4.14 Effect of acacia species on soil EC (30-60cm)

104

4.4.15 Effect of acacia species on soil SAR (0-30 cm)

105

4.4.16 Effect of acacia species on soil SAR (30-60 cm)

105

4.4.17 Effect of acacia species on soil organic matter content (0-30 cm)

106

4.4.18 Effect of acacia species on soil organic matter content (30-60 cm)

106

4.4.19 Effect of acacia species on soil bulk density

107

4.4.20 Effect of acacia species on soil infiltration rate

107

ABSTRACT

Salinity is a major environmental stress which is reducing crop yields particularly in

arid to semi-arid zones. In Pakistan large cultivated land is affected by various degrees of soil

salinity and sodicity. There exists a great diversity among plant species for their salt

tolerance. Selection of plant species capable of growing on salt-affected soils using saline

waters is based on the capability of a crop to survive at higher levels of salinity and to

provide useful end product. Keeping these facts in view, four studies were planned to explore

the salinity tolerance and phytoremedial potential of two different acacia species viz Acacia

ampliceps and Acacia nilotica. In the first experiment, three week old seedlings of both

species were transplanted in half strength Hoagland nutrient solution having five treatments

(control, 100, 200, 300 and 400 mM NaCl). The data regarding growth and ionic composition

(Na+, K+ and Cl-) showed that A. ampliceps was more tolerant to salinity than A. nilotica. The

seedlings of A. nilotica could not survive at 400 mM NaCl due to ion toxicity. In another

solution culture experiment, the release of organic acids (citric acid, malic acid and tartaric

acid) and comparative oxidative stress tolerance of these two species was investigated. More

rhizosphere acidification and higher activities of antioxidants including, SOD, POD and CAT

enabled A. ampliceps to produce more shoot and root biomass. Both the species were further

studied under the combination of salinity and water stress in the pots where A. ampliceps

proved to be better tolerant to salinity whereas A. nilotica performed better under water

stress. In the final study these species were grown in the salt affected field and their growth

and ionic data were recorded after every six months for two years. The changes in the soil

chemical and physical properties were also determined at these intervals. The comparison of

both species indicated that A. ampliceps produced more biomass and caused more reduction

in the soil chemical properties like pHs, ECe and SAR as compared to A. nilotica, due to more

addition of organic matter and rhizosphere acidification .On the other hand the physical

properties like bulk density and infiltration rate were also improved more under A. ampliceps

than under A. nilotica. So it would prove to be a good source of wood and forge for livestock

at the same time rehabilitating barren lands.

1  

CHAPTER-1

INTRODUCTION

All plant species are encountered by various types of stresses during their life cycle.

Salinity is the main environmental stress at present which is reducing crop yields (Majeed et

al., 2010). About 7% of the global land area, 20% of the world’s cultivated land area and half

of the irrigated land is affected by salinity (Zhu, 2001). According to FAO (2003) salinity is

affecting about 2.1% of the world dry land agriculture. About 6.67 m ha area of Pakistan is

having the problem of soil salinization and approximately it is one third of the total cultivated

area of the country (Khan, 1998). Salinity is more observed in arid and semi-arid regions of

the world (Murtaza et al., 2011) due to low rainfall, high evapo-transpiration and elevated

temperature which are accompanied by poor water and soil management practices (Azevedo

Neto et al., 2006). About 75-80 % of the pumped ground water in Punjab province is unfit

for irrigation owing to high EC, SAR and RSC which is adversely affecting the yield of crops

(Ghafoor et al., 2001).

Salt-affected soils may be saline (excess soluble salts), sodic (excess exchangeable

sodium) or saline sodic in nature. More than half of the global salt affected area is comprised

of sodic and saline sodic soils (Beltran and Manzur, 2005; Tanji, 1990). Primarily the parent

material and secondarily the human activities are responsible for this salinity and sodicity

(Qadir et al., 2007). The sodicity causes slaking, swelling and dispersion of clay that may

cause surface crusting (Quirk, 2001). Therefore water permeability, aeration, water holding

capacity, penetration of roots, tillage and sowing operations in these soils are disturbed. On

the other hand, imbalanced availability of plant nutrients in both saline and sodic soils affects

plant growth (Naidu and Rengasamy, 1993; Qadir and Schubert, 2002). These types of

changes affect plant root, soil microbes and result in the reduction of the crop yield and

productivity.

Saline soils contain plenty of soluble salts which reduce plant growth mainly by

osmotic stress, ion toxicity and nutritional imbalances (Neumann, 1995). Due to salinity

stress, the levels of Na+ and Cl- are much exceeded than majority of the macronutrients, and

2  

for micronutrients the ratio is even more. Therefore, saline soils are expected to have low

activities of nutrient and excessive ratios of Na+/Ca2+, Na+/K+, Mg2+/Ca2+ and Cl-/NO3-

(Grattan and Grieve, 1999). Although Na+ is harmful for plants, K+ is one of the vital

elements needed by the plants in larger amount (Mahajan and Tuteja, 2005). It has important

role in protein synthesis, activation of enzymes, photosynthesis (Marschner, 1995) and

osmotic adustment (Mahajan and Tuteja, 2005). Therefore, under salinity stress a higher

K+/Na+ ratio is indispensable for regular performance of the plants and for maintaining their

resistance against salinity (Greenway and Munns, 1980). The plants with higher K+: Na+ ratio

are considered better tolerant to salinity stress (Saqib et al., 2005). If the concentration of the

salt is high, plant growth (Amirjani, 2010), photosynthesis, respiration (Marschner, 1995),

nodulation (Al-Shaharani and Shetta, 2011), chlorophyll contents (Lu et al., 2010) and

activities of varoius enzymes are badly affected (Sairam and Tyagi, 2004). The degree of

damage depends on harshness and duration of stress (Nawaz et al., 2010), the plant species

(Croser et al., 2001) and genotypes (Fung et al., 1998), type of salts (Lauchli and Grattan,

2007) and the growth stage of the plant under stress (Levitt, 1972).

On the other hand, salinity also results in the production of reactive oxygen species

(ROS) like hydrogen peroxide, superoxide, singlet oxygen and hydroxyl radicals. These

species are responsible for many lethal metabolic effects like lipid peroxidation, damage to

proteins and mutation to the structure of DNA molecule (Wang et al., 2003). Among various

ROS, hydrogen peroxide, superoxides, and hydroxyl radicals are mainly produced in the

cytosol, chloroplasts, mitochondria and the apoplastic spaces (Bowler and Fluhr, 2000;

Mittler, 2002). Membrane injury due to salinity is related to over production of these reactive

oxygen species (Shalata et al., 2001).

Nature has gifted plants with explicit mechanisms to detoxify these reactive oxygen

species. These comprise of the activation of the enzymes of antioxidative pathway like

superoxide dismutase, peroxidase, catalase and enzymes of the ascorbate- glutathione cycle

(Noctor and Foyer, 1998; Smirnoff, 2005). The concentration of non-enzyme antioxidants

including tocopherol, flavones, ascorbic acid and carotenoids is also increased (Johnson et

al., 2003). Superoxides are dismutased to H2O2 and molecular oxygen with the help of

superoxide dismutase (Giannopolitis and Ries, 1977). It is an important enzyme for

3  

mitigation of oxidative stress in plants. H2O2 and some organic hydroperoxides are broken to

water and oxygen with the help of catalase enzymes (Ali and Alqurainy, 2006). Plant species

vary greatly regarding increased activities of enzymatic and non-enzymatic antioxidants in

response to salinity (Zhang and Kirkham, 1995; Nayyar and Gupta, 2006). Similar

differences have also been found within cultivars of a same species (Bartoli et al., 1999). The

plants with higher antioxidant production capacity can better scavenge ROSs and are more

salt tolerant (Shalata and Tal, 1998; Garratt et al., 2002).

Water shortage is a serious abiotic stress that negatively affects the growth and

productivity of crops (Zlatev and Lidon, 2012). Availability of water is very crucial for

fulfilling the needs of present and future human beings and its scarcity is increasing day by

day due to climatic changes (Rosegrant and Cline, 2003). As a result of water deficit the

yield losses up to 50 % have been noticed for many crops (Bray et al., 2000; Wang et al.,

2003). Drought affects plants in many ways and at various levels of their growth and

development (Yordanov et al., 2000; Wentworth et al., 2006). The dehydration as a result of

water deficit is thought to be responsible for many changes in water relations, biochemical

and physiological processes and structure of membranes (Tuba et al., 1996; Sarafis, 1998;

Yordanov et al., 2003).

Plants have various strategies to cope with water deficit like escape, tolerance, and

avoidance of tissue and cell dehydration (Turner, 1986). Avoidance includes rapid

phenological development, increased stomata and cuticular resistance, changes in leaf area

and orientation and many other anatomical features (Jones and Corlett, 1992). Osmotic

adjustment is the main strategy for maintaining cell turgor under low water potential

conditions, which enables the plants to take up water and to maintain their metabolic activity

and growth (Martiınez et al., 2004). Drought tolerant plants try to maintain high relative

water content than sensitive ones by accumulating different osmoticums which lower their

water potential.

The release of H+ from plant roots lowers pH of rhizosphere (Marschner, 2003).

Leguminous plants have been shown to acidify their rhizosphere (Schubert et al., 1990a) by

releasing sufficient amount of H+ ions from their roots (Schubert et al., 1990b). This H+ ion

4  

extrusion is responsible for the increase in pH of the cytosol, which in turn hastens the

formation of organic anions. These organic anions are a gauge of how much hydrogen ions

are released in the rhizosphere that is called as ash alkalinity. This parameter has been

characteristically determined in many plants to evaluate their acidification capacity (Moody

and Aitken, 1997; Noble and Randall, 1999). The determination of the ash alkalinity in

sodicity adapted species might be helpful to screen out such plants which have the greater

capacity for dissolution of calcite by releasing more hydrogen ions in the root zone. It is

supposed that rhizosphere acidification may be helpful in reducing soil sodicity and will

positively correlate with salinity-sodicity tolerance of different plant species.

The use of salt-affected soils and waters is becoming a necessity because of

shrinking normal soil and water resources and increasing population. Since last hundred

years, many different techniques like chemical treatments, tillage operations, crop based

interventions, water-related methods, and electrical currents have been employed to reclaim

the salt affected soils. Among all these, chemical amendments were used most vastly (Oster

et al., 1999). The reclamation of the salt-affected soils through engineering or chemical

approach is very expensive. Many salt affected soils are not even treatable and this approach

is also not sustainable over long time (Qureshi and Barret- Lennard, 1998). Nevertheless, in

the present times, the most effective and low cost reclamation approach is the

phytoremediation, (Ghaly, 2002; Ilyas et al., 1993; Robbins, 1986a). Using this approach the

native source of calcium like calcite is made soluble with the root action and the calcium

released is used to replace sodium on the exchange sites. It is very cheap as compared to

chemical reclamation approach, which is out of the range of the poor farming community of

the developing countries (Qadir and Oster, 2004). The response of various plants for

phytoremediation of sodic and saline-sodic soils varies with species. The plant species which

produce more biomass and at the same time have higher salinity and sodicity tolerance

potential are considered better for reclamation purpose (Kaur et al., 2002; Qadir et al., 2002).

Acacia is an important salt tolerant plant genus which has great potential for

livestock and agroforestry (Mahmood et al., 2009). Acacia trees give many benefits like

fodder and shade for livestock, improvement of soil fertility through nitrogen fixation,

production of litter and stabilizing soils (Benninson and Paterson, 1993). Acacia is a pan

5  

tropical and subtropical genus found abundantly in Australia, Asia, Africa and America.

Acacia species have the ability to survive in a diverse range of environment. Most of the

species are well tailored to the semi-arid and savannah regions tolerating both high pH and

waterlogged conditions (Benninson and Paterson, 1993). Many acacia species can survive at

higher levels of salinity and sodicity so they may be helpful for the reclamation of the salt

affected soils (Thomson, 1986; Mahmood et al., 2010). This project has been conducted to

evaluate the comparative physiological, biochemical and phytoremedial characterization of

two acacia species of different origin for salt affected soils. The species used include Acacia

ampliceps and Acacia nilotica. The specific objectives of this project are as under:

1. Assessment of salinity tolerance potential of two different acacia species.

2. To study different physiological and biochemical mechanisms of salinity tolerance in

the selected acacia species.

3. To study the ameliorative effects of selected acacia species on the salt affected soils.

6  

CHAPTER-2

REVIEW OF LITERATURE

Salt affected soils have higher concentration of soluble salts or exchangeable sodium

affecting normal growth of most of the crops. Salt-affected soils include saline, sodic and

saline sodic soils. These types of soils are predominantly present in arid and semi-arid

regions of the globe. In these areas annual rainfall is less than evopotranspirational losses of

water. Under humid and sub humid conditions salt affected soils do occur but at a smaller

scale. Both soils and plants face different types of problems due to the presence of salts. This

chapter reviews the status of salt-affected soils and their potential utilization through

selection, characterization and introduction of salt tolerant plants. This chapter reviews the

status of salt-affected soils and their potential utilization through selection, characterization

and introduction of stress tolerant plants.

2.1 Extent of salinity problem

Salinity is a worldwide problem. Total land surface of the world is about 13.2×109

ha, out of which 7×109 ha are arable and 1.5×109 ha are cultivated (Massoud,1981). More

than 100 countries of the world are facing the problem of soil salinity. More than 4,000,000

square kilometer area of the world is affected to some extent by salinity (FAO, 2006). About

23% of the world cultivated land is saline and 37% is sodic in nature. About 830 million

hectares are having salinity menace globally (Beltran and Manzur, 2005). On an average

almost 6% of all land in the Asia-Pacific region is affected by salinity. Pakistan, Australia,

and Thailand together have 6.8% of the world’s land area but are having more than 10% of

the world’s land affected by salinization. With respect to absolute size affected, Australia is

on third position with 254,000 square kilometers, Pakistan eighth and Thailand on forty-fifth

(FAO, 2006).

About 7% of the global land area, 20% of the world’s cultivated land area and half of

the irrigated land is affected by salinity (Szabolcs, 1994; Zhu, 2001). According to Saboora

et al. (2006) salinity is increasing at a rate of 10% per year throughout the world. Salinity and

sodicity are most serious soil issues in Pakistan. Total cultivated area of Pakistan is about

7  

22.94 mha and irrigated agriculture is practiced on about 18.78 mha. Salt affected area of

Pakistan is about 6.67 mha (Khan, 1998). About 75-80% of the pumped ground water in

Punjab province is unfit for irrigation owing to high EC, SAR and RSC which is adversely

affecting the yield of crops (Ghafoor et al., 2001).

2.2 Effects of salts on plants

According to (Greenway and Munns, 1980) salinity causes following four types of

stresses for plants (a) osmotic stress resulting in water deficit (b) specific ion toxicity caused

by higher levels of Na+ and Cl- (c) nutritional imbalance resulting in the deficiency of major

nutrients like K+, NO3- and PO43- (d) harm to macromolecules due to over production of

reactive oxygen species.

2.2.1 Osmotic effect

Under salinity stress the ability of the plants to take up water is reduced which results

in the reduction of growth of the plant. This type of water stress is called osmotic stress.

Salinity induced water deficit is very similar to that of drought (Nawaz et al., 2010). The

water potential of the soil solution regulates the formation of new leaves and this is very

similar for drought stressed plants. As fastly growing cells can store higher levels of salts in

their expanding vacuoles, so salts are seldom accumulated in the cytoplasm to reduce the

growth of the new leaves (Munns, 2005). Shoot and root growth is more affected due to

water stress as compared to salt specific effect in early days of stress (Munns, 2002). This

fact is evident from the concentrations of the Na+ and Cl- below the poisonous levels. For

instance, in an experiment wheat was grown in 120 mM NaCl, and the concentrations of the

sodium and chloride in the expanding leaf tissues were only 20 and 50 mM respectively (Hu

et al., 2005). Likewise, Neves-Piestun and Bernstein (2005) reported that sodium and

chloride were only 40 mM in the most swiftly increasing tissues of maize growing in 80 mM

NaCl, and these concentrations of sodium and chlorides do not seem to have strong

association with the growth reduction under that salinity level. Fricke et al. (2004) noticed

that the concentration of sodium in the mesophyll and epidermal cells of the barley was just

38 and 49 mM respectively, after 24 h exposure to 100 mM NaCl. This much concentration

of sodium instead of damage was somewhat useful in osmotic adjustment of the cells to

8  

salinity. The fast growth of the cells might be helpful in maintaining the lower concentrations

of the salts within the cells. Water relations of the shoot showed that hormonal signals caused

by the osmotic effect of the salt exterior to roots are regulating the growth of the cells

(Munns et al., 2000). Growth reduction due to salinity mainly is dependent on the magnitude

of the stress. At moderate osmotic stress, root growth is not much affected whereas the

growth of the shoot is reduced (Hsiao and Xu, 2000). Osmotic damage depends on many

factors like duration of the stress, plant species, type of cell and tissues and the way in which

the stress is applied (Ashraf, 1994; Munns et al., 2000).

2.2.2 Specific ion toxicity

This type of toxicity is mainly due to sodium, chloride and sulphate ions taken up in

larger amounts than usual. Reduction in crop growth and ultimately the complete loss of the

crop may occur due to this toxicity effect. There is a great genetic variability among plant

species for this type of stress with most of the crops and woody plants having sensitivity for

this problem (Abrol et al., 1988). Mahmood et al. (2010) concluded that the main reason of

growth reduction in case of Acacia ampliceps was the higher accumulation of Na+ as against

of other cations. Mostly the salt are accumulated in the older leaves of plants. If this salt

build up is continued for a considerable period of time it may result in the death of the leaves.

This happens when the salt concentration is too high to be stored in the vacuole of the cell. In

such a case the excessive salts go to cytoplasm where they interfere with the normal

functioning of the enzymes. On the other hand, they cause cell dehydration by being

accumulated in the cell walls (Munns, 2005). In order to avoid this effect plant either tries to

restrict the salt entry in the body or reduces the amount of the salts in their cytoplasm.

Sodium concentration in the cytoplasm of the roots ranged from 10-30 mM (Tester and

Davenport, 2003). The concentration of the sodium in the cytoplasm of the leaf is not exactly

known but is thought to be much lower than 100 mM (Wyn Jones and Gorham, 2002). The

toxic concentration of the chloride is not clearly understood. Exclusion of the salt is made

possible from the roots and if this is not done the salts will gather in the cell with the passage

of time. Husain et al. (2003) performed an experiment to study the effect of sodium on leaf

injury and concluded that the wheat genotype with more sodium in the older leaves suffered

from chlorosis more and produced low yield as compared to the genotype with less sodium

9  

accumulation at low salinity level but at higher levels of salinization there was not much

difference in the yield of the both genotypes.

Loreto and Bongi (1987) found that if the chloride concentration exceeds 80mM in

total tissue water, it will cause changes in the plant morphology, response of the stomata to

the environmental changes is reduced and thickness of the leaves is also reduced. The

movement of the chloride in the soil and plant is quite rapid. The higher concentration of the

chloride cause burning of the leaves, leaf tissues are dried initially from the tips and spread to

the whole leave as the intensity of the stress increases. According to Marschner (1995) higher

concentration of chloride cause burning of the leaves and with the passage of time the entire

plant becomes defoliated.

2.2.3 Nutritional imbalance

Unnecessary buildup of the salts cause disturbance in the water relations of the plants

which result in the limited uptake and utilization of the important nutrients. As a result

metabolic activities of the cell and functioning of the enzymes is disturbed (Munns, 2002;

Lacerda et al., 2003). Nutrients and salt interaction causes deficiencies and imbalances of the

major nutrients (McCue and Hanson, 1990). Imbalance of the ions occurs due to higher

accumulation of sodium and chloride and as a result limited uptake of the other mineral

nutrients like potassium, calcium and manganese (Karimi et al., 2005). Elevated Na+: K+

ratio causes enzyme inactivation and affects normal metabolism of the plant (Booth and

Beardall, 1991). More uptakes of sodium and chloride causes reduction in the uptake of

potassium and symptoms like in case of potassium deficiency do occur (Gopa and Dube,

2003). Hyper accumulation of Na+ suppressed the uptake of K+, Ca2+ and Mg2+ in Acacia

ampliceps (Mahmood et al., 2010). Potassium is vital for protein formation, osmoregulation,

cell turgor maintenance and photosynthesis (Freitas et al., 2001; Ashraf, 2004). K+ along

with Ca2+ are necessary for maintaining the integrity and proper working of the cell

membranes (Wenxue et al., 2003). Sufficient amount of K+ in plant cell under salinity

depends on the selective uptake of the K+ and discriminatory compartmentation of K+ and

Na+ in the shoots (Munns et al., 2000; Carden et al., 2003). The regulation of the calcium

within the plant under salt stress is a crucial parameter of the plant salinity tolerance (Soussi

10  

et al., 2001). Salinity reduces the Ca2+/Na+ ratio in soil solution, which has harmful effects on

membrane properties, causing displacement of membrane bound Ca2+ and ultimately the

integrity and selectivity of the membranes is lost (Kinraide, 1998). Exogenous application of

Ca2+ is found to reduce the damages of salinity to plants, most probably by keeping better

K+/Na+ selectivity (Hasegawa et al., 2000). Decreased K+ uptake in response to salinity was

also observed earlier (Khalil et al., 2012; Marcar et al., 1991). The plants with higher K+:

Na+ ratio are considered better tolerant to salinity stress (Saqib et al., 2005).

2.2.4 Oxidative stress

Salinity stress causes the hyper production of reactive oxygen species (ROS) such as

hydrogen peroxide, superoxide, singlet oxygen and hydroxyl radicals. Phytotoxic reactions

like lipid peroxidation, damage to proteins and mutation to DNA are caused by these ROS

(Wang et al., 2003; Pitzschke et al., 2006). The main sites of the production of these ROS

within the cell are cytosol, chloroplasts, mitochondria, and the apoplastic space (Bowler and

Fluhr, 2000; Mittler, 2002). On the other hand these ROS also have some positive roles in the

cell like signal transduction (Torres and Dangl, 2005). Injury to membranes due to salinity is

also connected to the over production of the ROS (Shalata et al., 2001). The higher levels of

ROS damage the D1 protein of PS II and cause photoinhibition. Increased Photorespiration

under stress and NADPH activity also contributes to increase in hydrogen peroxide

accumulation, which reduces the activity of the enzymes by oxidizing their thiol groups.

2.3 Salt tolerance of plants

The capability of the plants to grow and complete their life cycle under highly saline

environment is called salt tolerance. There is a great genetic diversity among plant species

and genera regarding salinity tolerance (Flowers et al., 2007; Greenway and Munns, 1980).

Most of the modern day plants are salt sensitive or glycophytes. On the other hand some

plants are inhabitant of saline conditions and are called salt tolerant or halophytes. Due to

some specific anatomical, morphological or avoidance mechanisms the halophytes can

tolerate higher levels of salinity (Flowers et al., 2007). There is a variety of physiological

and biochemical mechanisms in plants which enable them to grow and survive in saline

11  

environment. Different biochemical mechanisms are additive or synergistic in nature

(Iyengar and Reddy, 1996).

2.3.1 Ion regulation and compartmentalization

For proper growth and reproduction under both non saline and saline situation uptake

of ions and their compartmentalization is very vital (Adams et al., 1992b). Ionic homeostasis

is interfered due to salt stress. Higher cytoplasm concentration of salts is not tolerable by

both glycophytes and halophytes. So in order to carry on normal metabolism the salts are

either compelled to vacuoles or they are compartmentalized in various tissues and organs

(Zhu, 2003). Marcar et al. (1991) ranked three acacia species for salt tolerance in the order:

A. ampliceps > A. auriculiformis > A. mangium. The tolerance of A. ampliceps was related to

its comparatively low shoot Na+ and Cl- concentration. Na+/H+ antiporter which is a salt

induced enzyme is responsible for Na+ removal out of cytoplasm and its storage in the

vacuole (Apse et al., 1999). Under salinity stress, plants try to keep higher potassium level in

the cytoplasm as compared to sodium. This is made possible by regulating the activity of Na+

and K+ transporters and of H+ pumps that generate the driving force for transport (Zhu et al.,

1993). It is supposed that AtNHX1 may play a role in pH regulation and K+ homeostasis in

the specialized cells.  Exogenous application of Ca2+ reduces the toxicity of salinity by

keeping higher K+/Na+ selectivity (Liu and Zhu, 1997). Salt secretion and exclusion are two

other mechanisms of tolerance. Secretion of salts is made possible by specialized structures

known as salt glands. These glands secrete salts (particularly NaCl) from their leaves and

help in maintaining its low concentration in the cell (Hogarth, 1999). Exclusion of salt occurs

at root level to normalize the salt concentration of their leaves (Levitt, 1980). 

2.3.2 Biosynthesis of compatible solutes 

Compatible solutes are low-molecular weight compounds synthesized in the

cytoplasm to balance the higher ionic concentrations in the vacuoles of the cell. They are

called compatible because they do not interfere with normal metabolic processes (Hasegawa

et al., 2000; Zhifang and Loescher, 2003). Their main functions in the cell are protection of

important cellular structures, osmotic balance which make the influx of water possible and

scavenging of ROS (Hasegawa et al., 2000). These osmolytes are produced under stress

12  

conditions due to metabolic diversion into unique biochemical reactions. These compatible

solutes include mainly proline (Singh et al., 2000), glycine betaine (Wang and Nil, 2000),

sugars (Kerepesi and Galiba, 2000), and polyols (Bohnert et al., 1995).

A significant amount of assimilated CO2 is converted into polyols which have many

important functional roles like compatible solutes, molecular chaperones, and scavenging of

reactive oxygen species (Bohnert et al., 1995). Different types of sugars like glucose,

fructose, sucrose and starch are accumulated in response to any type of stress (Parida et al.,

2002). The main functions which they perform are osmoprotection, osmotic adjustment,

storage of carbon and detoxification of ROS. Salinity induced hyper production of these

sugars is observed in many plants (Khatkar and Kuhad, 2000; Singh et al., 2000). Various

nitrogen containing compounds are accumulated in plants in response to salt stress and are

involved in stress tolerance of the plants (Mansour, 2000). For example the level of glycine

betaine was found to be increased in many plants under salinity stress (Khan et al., 1998;

Wang and Nil, 2000). Likewise the accumulation of proline was also observed as a nontoxic

and protective osmolyte in response to salinity (Jain et al., 2001).

2.3.3 Production of antioxidants

Antioxidants are those compounds which detoxify the free radicals produced under

any type of stress. Their activities are increased many folds under stress conditions. There is

a strong relation between the levels of these compounds and salt tolerance of plants (Mittova

et al., 2003). These may be enzymes or non-enzymes. Enzymes include superoxide

dismutase (SOD), catalase (CAT) and various peroxidases (POD). Non enzymes are

tocopherol (vitamin E) carotene (vitaminA) and ascorbic acid (vitamin C). Superoxide

dismutase (SOD) converts superoxide radical to H2O2 whereas catalase and a variety of

peroxidase catalyze the breakdown of H2O2 (Chang et al., 1984). Because of higher oxygen

levels during photosynthesis (Steiger et al., 1977) these reactive species are mostly produced

within the chloroplasts (Asada and Takahashi, 1987). Increased activities of SOD under salt

stress conditions was previously observed indicating its role in salinity tolerance of various

plant species (Khan and Panda, 2008; Mittal et al., 2012; Hu et al., 2011). Chen et al. (2011)

observed that salt-tolerant wheat cultivar exhibited more SOD activity as compared to

13  

sensitive cultivar. Morais et al. (2012) observed that A. longifolia was better adapted to saline

conditions than Ulex europaeus, owing to its better anti-oxidants activities particularly of

catalase (CAT). Other workers also found increased acitivties of this enzyme in response to

salinity like Patel and Saraf (2013), Sekmen et al. (2012) and Mittal et al. (2012) in J.

curcas L., G. oblanceolata and B. juncea, respectively. Zhang et al. (2013) observed that

the activities of POD in the stems of B. papyrifera were significantly increased under salinity

stress whereas in case of leaves and roots it was decreased.

2.3.4 Induction of plant hormones

Salinity stress also results in increased contents of plant hormones like abscisic acid,

cytokinins (Thomas et al., 1992; Aldesuquy, 1998) and jasmonates. Salinity-induced genes

are changed due to abscisic acid. Abscisic acid (ABA) induced genes were found to be

involve in salinity tolerance of rice (Gupta et al., 1998). Positive effects of ABA on

photosynthesis, growth and translocation of assimilates are well documented under salinity

stress (Popova et al., 1995). Higher level of ABA under salt stress results in more Ca2+

uptake and in this way increase the integrity of membranes, enabling the plants to regulate

ion fluxes (Chen et al., 2001). According to Gomez Cadenas et al. (2002) ABA reduces

ethylene production and shedding of leaves under salinity in citrus by reducing the uptake of

Cl- in the leaves. Jasmonates are also considered important in salinity tolerance of plants. It is

observed that salt-tolerant varieties of tomato have more jasmonate level than salt-sensitive

ones (Hilda et al., 2003).

2.4 Water stress (water deficit) and salinity

Water shortage is a serious abiotic stress that negatively affects the growth and

productivity of crops (Zlatev and Lidon, 2012). During plant growth, drought may be long

lasting in those climatic areas where water availability is low and erratic as a result of

changes in weather situation (Harb et al., 2010). Availability of water is very crucial for

fulfilling the needs of present and future human beings and its scarcity are increasing day by

day due to climatic changes (Rosegrant and Cline, 2003). As a result of water deficit the

yield losses up to 50 % have been noticed for many crops (Bray et al., 2000; Wang et al.,

2003). The impact of any environmental stress on the plants is estimated by the duration and

14  

strength of that particular stress factor and the genetic ability of the target plant to cope that

stress (Zlatev and Lidon, 2012). Drought affects plants in many ways and at various levels of

their growth and development (Yordanov et al., 2000; Wentworth et al., 2006).

Both salinity and water stress exert similar effects on plants (Munns, 2002), but it is

unclear that what is the response of the plants when both of these stresses are acting together

(Meiri 1984; Homaee et al., 2002). In the field we cannot escape the water shortage because

the water content of the soil changes from place to place and with different times of the

seasons. So plant roots are suffering from some degree of both these stresses at different

places in different seasons (Homaee et al., 2002). For instance, plant faces water shortage in

the upper part of the root zone whereas salinity stress may be operating in the lower part of

the root zone.

It is obvious that the combined effect of both of these is more alarming than the either

alone but it is difficult to say that which one of them is reducing the growth more as

compared to the other due to environmental variability. According to Meiri (1984) matric

potential (due to water stress) resulted in more reduction in the growth of shoots of bean than

did the osmotic effect (due to salt stress). Shalhevet and Hsiao (1986) also observed more

effect of water stress than salinity in case of pepper and cotton at equal decline in water

potential of soil. From a thermodynamic point of view, even though matric and osmotic

components act in an additive manner, the kinetic factors like water uptake and transpiration

should also be considered (Maas and Grattan, 1999).

Plant reaction to these stresses is different for high and low evaporative demand

because movement of water to plant roots from nearby soil is under the control of matric

forces and not the osmotic forces. Drying of the soil with a corresponding reduction in matric

potential reduces the water flow to roots in a non-linear manner (Homaee et al., 2002).

Instead when soil salinization is increased, at given water content, water potential is reduced

but the rate of water movement towards roots is not affected. Furthermore under salinity

stress, the cortical cells of roots have some capability to adjust the osmotically that would

allow the water to rush into the root cells. Similar outcomes were found by Shalhevet and

Hsiao (1986) who noticed that water-stress resulted in more reduction in leaf water potential

15  

of cotton and pepper as compared to salinity at the same level of soil-water potentials.

Moreover it was found that there was less osmotic adjustment in leaves in case of water

shortage than salinity. In case of water stress turgor pressure of leaves is reduced which

results in less transpiration, less assimilation of CO2 and as a result less growth of the plants.

The difference between matric and osmotic effects varies with plant type, root-length density

and evaporative demand of the atmosphere.

Plants have various strategies to cope with water deficit like escape, tolerance, and

avoidance of tissue and cell dehydration (Turner, 1986). Avoidance includes rapid

phenological development, increased stomata and cuticle resistance, changes in leaf area,

orientation and many other anatomical features (Morgan, 1984; Jones and Corlett, 1992).

Osmotic adjustment is the main strategy for maintaining cell turgor under low water potential

conditions, which enables the plants to take up water and to maintain their metabolic activity

and growth (Gunasekera and Berkowitz, 1992; Martiınez et al., 2004). Drought tolerant plant

try to maintain high relative water content than sensitive ones by accumulating different

osmotica which lower their water potential.

2.5 Reclamation of salt affected soils

The reclamation of sodic and saline sodic soils is done by providing a  readily

accessible source of calcium for the replacement of excessive sodium from the exchange

sites. Excessive supply of good quality water is then applied to leach down this replaced

sodium out of the root zone. For this process to be accomplished successfully, we need to

have ample quantity of water, its unrestricted flow thorough the soil profile and a good

natural or synthetic drainage system (Gupta and Abrol, 1990; Oster et al., 1999). The

majority of the sodic and saline-sodic soils are having a source of calcium in the form of

calcite (CaCO3), which is found at varying depths in the soil profile. This calcite might be an

ingredient of the parent material or it may be formed in soil by the process of precipitation

and may act as a cementing agent for the soil particles. Owing to its very low solubility the

natural dissolution of calcite is not sufficient to provide ample quantities of calcium for an

effective reclamation to occur. Dolomite is another calcium mineral found in the sodic and

saline sodic soils but its solubility is even less than that of calcite. The minerals of calcium

16  

with more solubility are not frequently present in the soils (Suarez and Rhoades, 1982). As a

result, for the successful reclamation of sodic and saline sodic soils we have to apply the

chemical amendments having calcium source (Gupta and Abrol, 1990; Oster et al., 1999).

One such amendment is gypsum which provides calcium on its dissolution in the soil

solution and the other is sulfuric acid which enhances the solubility of calcite to release

sufficient quantity of calcium in soils. Chemical approach of soil reclamation is having some

constrains like (a) inferior quality of amendments having lot of impurities; (b) their limited

supply when required by the farmers and (c) their high cost which is out of reach of the poor

farming community (Qadir et al., 2007).

2.6 Phytoremediation of salt affected soils

In comparison to the chemical approach there is another way for reclamation of sodic

and saline sodic soils which is called as phytoremediation because of involvement of plants

in this technique (Kumar and Abrol, 1984; Mishra et al., 2002; Qadir et al., 2002; Robbins,

1986a). Via this approach the native source of calcium like calcite is made soluble with the

root action and the calcium released is used to replace sodium on the exchange sites. The

level of salinization in the soil solution during phytoremediation helps to keep the soil in a

proper structure and aggregation which assists the movement of water through the soil profile

and increases the process of reclamation (Oster et al., 1999).

The response of various plants for phytoremediation of sodic and saline-sodic soils is

different for different species. The plant species which produce more biomass and at the

same time have the potential to tolerate higher levels of salinity and sodicity are considered

better for reclamation purpose (Kaur et al., 2002; Qadir et al., 2002). In phytoremediation the

farmer has not to spend huge amount of rupees to buy chemical amendments instead he is

expected to get benefit from the farm level products of the grown trees and grasses. For the

soil of moderate levels of salinity and sodicity and if the irrigation water is available in

abundant quantity, the effectiveness of phytoremediation is almost as much as might be

expected with the application of gypsum. The effectiveness of chemical amendments is

restricted to only the zone of their application (Ilyas et al., 1993; Qadir et al., 1996a;

Robbins, 1986b). When the reclamation was completed in that zone thereafter it went to

17  

some more depth within the soil depending on the degree of calcium saturation of the

exchange sites in comparison to that of sodium (Oster and Frenkel, 1980; Suarez, 2001). On

the other hand phytoremediation was found to be effective throughout the whole root zone

depending on the plant type, root structure and rooting depth (Akhter et al., 2003; Ilyas et al.,

1993). Deep-rooted crops having tap root system like alfalfa have shown more promising

results as compared to other plants (Ilyas et al., 1993).

Nutritional troubles are very common in sodic soils, like deficiency of many nutrients

and the higher concentrations of sodium and chloride (Naidu and Rengasamy, 1993).

Phytoremediation plays an important role in the provision of many nutrients for the

succeeding crops. Qadir et al. (1997) found that the levels of phosphorus, zinc, and copper

were increased with phytoremediation which might be due to the exudation of roots and

dissolution of calcite having coating of some nutrients. The levels of these nutrients were

decreased in those plots which were having no crop and were treated with gypsum.

2.7 Mechanisms involved in phytoremediation

Phytoremediation of calcareous sodic and saline-sodic soils helps in increasing the

solubility rate of native calcite via various processes in the rhizosphere which result in

increasing levels of calcium in the soil solution. Following processes are responsible for the

phytoremediation of the salt-affectd soils.

2.7.1 Physical impacts of roots

Plant roots are crucial for soil structure maintenance and the formation of macropores

is also carried on by plant roots within the soil. In this way they increase the porosity of soil

by making structural cracks or biopores (Czarnes et al., 2000; Oades, 1993; Pillai and

McGarry, 1999; Yunusa and Newton, 2003). Moreover roots cause different changes by

removing entrapped air from larger pores and generate alternate wetting and drying cycles. In

the rhizosphere due to the production of hyphae of fungus and polysaccharides soil

aggregation is enhanced (Boyle et al., 1989; Tisdall, 1991). Some roots behave like tillage

tool by growing through compact soil layers and make soil very conducive for cropping

(Elkins et al., 1977). Leaching of the desorbed Na+ from the exchange site out of the root

18  

zone is also facilitated by root action. In this regard deep rooted crops are more important

because they can survive under higher levels of salinity and sodicity during

phytoremediation. This fact was verified by Tisdall (1991) who found that deep-rooted

perennial grasses and legumes did improve the soil structure and hydraulic properties of

sodic soils were also improved (Akhter et al., 2004; Ilyas et al., 1993). Ilyas et al. (1993)

found that Alfalfa grown on a saline sodic soil increase the saturated hydraulic conductivity

two times in one year growth. Cresswell and Kirkegaard (1995) concluded that deep-rooted

crops like alfalfa in a mixed cropping systems as a prospective biological drilling approach to

increase permeability of the subsoil.

Akhter et al. (2004) found that Kallar grass grown on saline sodic soil resulted in

considerable increase in soil porosity, water holding capacity and reduction in bulk desity in

a five year period. It was supposed that these changes might be due to the widespread and

deep (Malik et al., 1986) root system of the grown grass. Biological drilling by deep roots is

thought to be a substitute to deep tillage for the reclamation of dense subsoils (Elkins et al.,

1977). Macropores are formed by deep root growth and their subsequent decomposition

increases the diffusion of different gases and water movement within the soil. These benefits

are harvested by the succeeding crops (Cresswell and Kirkegaard, 1995; Elkins, 1985).

Mishra and Sharma (2003) monitored the performance of two leguminous tree species i.e.

Prosopis juliflora and Dalbergia sissoo and they found a reduction in the bulk density and an

incresase in the porosity of the soil at the end of the experimental period. The positive

changes in the soil were considered due to the addition of organic matter which enhanced soil

aggregation and developed an appropriate soil structure.

2.7.2 Uptake of salts by shoots

The salts taken up by plants during their growth on salt affected soils are removed

with the harvesting of the plant species. Hyper accumulators like halophytes have the ability

to gather higher amounts of salts and Na+ in their different parts including shoots. Hyder

(1981) found that atriplex species grown in rangelands accumulated the salts ranging from

130 to 270 g Kg-1 of their weight. When this species was grown on a salt affected soil the

salt concentration was found up to 390 g Kg-1 (Malcolm et al., 1988). Although this is a

19  

considerably high quantity of salt but it is not enough to play a major role in the

phytoemediation of highly salt affected soils alone. However, when leaves are shed from the

plant species most of the salt taken up by plant is deposited again in the soil. This type of salt

removal is not very significant when the irrigation water is also salty in nature.

Gritsenko and Gritsenko (1999) found that Na+ which was taken up in the above

ground portions of many plant species was 2-20% of the total amount of the salt taken up by

the plants. Qadir et al. (2003b) observed that the amount of the Na+ removed by the

harvesting of the shoots of the alfalfa crop was only 1-2% of the total Na+ removed.

Consequently, the major contributor for decreasing sodicity with phytoremediation is the

leaching of salts and Na+ out of the rhizosphere instead of removal by harvesting the

aboveground plant materials.

2.7.3 Carbon dioxide Partial pressure in the root zone

The dissolution of calcite is thought to be a function of CO2 in the rhizophere. First of

all CO2 is dissolved in water and it is converted into H2CO3. This acid reacts with CaCO3 and 

Ca2+ ions are released or the other possibility is that H2CO3 is dissociated in to H+ and HCO3-

and the reaction of H+ with CaCO3 releases Ca2+ ions. In aerobic soils, the partial pressure of

CO2 is enhanced up to 1 kPa, which is equal to about 1% of the soil air by volume (Nelson

and Oades, 1998). Under flooded soil conditions this partial pressure is even more increased

(Ponnamperuma, 1972) because under such conditions the movement of the CO2 to the

atmosphere is restricted. So the preservation of CO2 in the soil enhances the partial pressure

of this gas in the soil.

Likewise, the partial pressure of CO2 in the rhizosphere is  increased due to root

respiration when the soil is under any crop (Robbins, 1986a). In non-calcareous soils, an

increase in CO2 level may results in the production of H+ ions with resultantly a reduction in

soil pH. On the other hand, such reduction in pH is not found in case of calcareous soils

(Nelson and Oades, 1998), because of the solubility of calcite which increases the pH (Van

den Berg and Loch, 2000). So, the solubility of  calcite in calcareous salt affected soils is

governed by higher partial pressure of CO2 which provides ample quantity of calcium for the

reclamation process. Respiration of roots is not the only means which affects the partial

20  

pressure of CO2 in the rhizosphere. It is also affected by production of CO2 from oxidation of

plant root exudates and organic matter decomposition and production of organic acids by soil

organisms, which are helpful in dissolution of calcite. All these processes are helpful in the

release of Ca2+ which can replace Na+ at a relatively much rapid rate than that would have

occurred at normal CO2 partial pressure in the atmosphere. So the leaching of Na+ from the

soil profile during phytoremediation should be done when plants are growing at faster rate so

that the full benefit of the calcite dissolution can be taken due to enhanced CO2 partial

pressure. Bauder and Brock (1992) evaluated alfalfa, barley, and sordan alone and in blend

with surface-applied chemical agents in order to alleviate the effects of saline sodic water.

They came to the conclusion that the plants which are having more CO2 production capacity

result in more acidification of soil and resultantly more removal of Na+ out of the profile.

Thus, such crops can reduce the requirement of water for leaching and drainage volume.

2.7.4. Release of protons from plant roots

Proton  (H+) release is thought to be a process responsible for rhizosphere

acidification. It is well documented that when plants are ammonium fed, they acidify their

root zones but in case of nitrate nutrition pH of the rhizosphere is increased (Marschner and

Romheld, 1983; Schubert and Yan, 1997). Moreover the leguminous crops which carry on

nitrogen fixation also decrease the pH of their  rhizospheres (Schubert et al., 1990b).

Substantial H+ release has been noticed in the rhizosphere of different N2-fixing plant species

(Marschner and Romheld, 1983; Schubert et al., 1990a). The discharge of H+ ions in the

rhizosphere of leguminous plants is responsible for the dissolution of calcite with a resultant

release of Ca2+. The liberation of H+ ions in the rhizosphere causes an electrochemical

gradient. When plant uptake cations, the extrusion of H+ ions is enhanced via fractional

depolarization of the membrane potential, which further helps in active H+ pumping

(Schubert and Yan, 1997). This H+ ion extrusion is responsible for the increase in pH of the

cytosol, which in turn hastens the formation of organic anions. These organic anions are a

measure of net H+ ion release in the rhizosphere and are called as ash alkalinity (Jungk,

1968). This parameter has been characteristically determined in many crops and trees to

evaluate their acidification capacity (Noble and Randall, 1999; Noble et al., 1996).

21  

Noble and Nelson (2000) in an inclusive determination of ash alkalinity of 106 plant

species in the semiarid tropics of northern Australia, found a range of values from 25 to 347

cmolc kg-1 for Themeda triandra and Brunoniella acaulis, respectively. This ash alkalinity

varies to a great deal among species, whereas there is only a small variability within a

species. It is supposed that there is a relation between adjustment to a specific agro ecotype

and the net amount of ash alkalinity which is released by a plant species. For instance,

Calliandra calothyrsus, wet tropics species had very minute ash alkalinity (44 cmolc kg-1) as

against of Stylosanthes seabrana, a semiarid tropics species, which had a very high value of

ash alkalinity (125 cmolc kg-1). It is supposed that plant species of high base saturation might

have more ash alkalinity and so the more release of H+ ions in their rhizosphere.

Consequently, the measurement of ash alkalinity in species that are adapted to sodic soil

conditions could be used to select the most appropriate species to enhance the rate of calcite

dissolution through H+ release in the root zone. Noble et al. (1997) found that the removal of

the all above ground portion of the legume /grass base system resulted in more net acid

addition rate as compared to that pasture system where the removal of above ground part was

not done. This evidence clearly demonstrated the association of greater rate of acid addition

with highly exploitive production systems. In a lysimeter study on a calcareous sodic soil,

Qadir et al. (2003a) found that the alfalfa without additional N supply only relying on

nitrogen fixation removed more Na+ from the exchange sites as compared to that which was

supplied with ammonium nitrate. It suggests that the amelioration rate of calcareous sodic

soils could be increased by means of crop management conducive for the release of greater

amount of CO2 and H+ in the root zone. In addition, using appropriate N2-fixing crops as a

phytoremediation tool has the advantage of enhanced availability of N in the soil for the post

amelioration crops.

2.8 Plants capable of phytoremediation

Many types of plants including crops, shrubs, trees, and grasses have been used in

phytoremediation of salt affected soils. Some triumphant examples include kallar grass

(Malik et al., 1986), sesbania (Qadir et al., 2002), alfalfa (Ilyas et al., 1993), bermuda grass

(Oster et al., 1999) and sordan grass (Robbins, 1986a). Some shrub species from the genera

Atriplex and Maireana (Barrett-Lennard, 2002), Kochia scoparia L. (Garduno, 1993),

22  

Portulaca oleracea L. (Grieve and Suarez, 1997) and Glycyrrhiza glabra L. (Kushiev et al.,

2005). A number of tree plantations have been grown on sodic and saline-sodic soils. These

include: T. arjuna. (Jain and Singh, 1998), P. juliflora. (Bhojvaid and Timmer, 1998), D.

sissoo, A. nilotica (L.) (Kaur et al., 2002), A. ampliceps (Mehmmod et al., 2009, 2010),

Parkinsonia aculeate L. and P. cineraria (L.), Druce (Qureshi and Barrett-Lennard, 1998),

Sesbania sesban (L.) (Singh, 1989), and Leucaena leucocephala (Qureshi et al., 1993).

Farrington and Salama (1996) suggested tree plantation as the best and long term option for

controlling dryland salinity.

2.9 Salt tolerance and phytoremedial potential of acacia species

Singh and Gill (1990) conducted an experiment using different tree species like P.

juliflora, Acacia nilotica (L.), Eucalyptus tereticornis, Albizia lebbeck (L.) and Terminalia

arjuna. They found a significant decrease in pH and increase in OM, P and K of surface 0.15

m soil. Marcar et al. (1991) in a sand culture experiment evaluated the salinity tolerance

potential of three acacia species i.e A. ampliceps, A. auriculiformis and A. mangium. On the

basis of relative reduction in plant dry weight and nodulation species were ranked in the

order A. ampliceps > A. auriculiformis > A. mangium. The better adaptation of A. ampliceps

was linked with its less shoot Na+ and Cl- concentrations. A. ampliceps and A. auriculiformis

survived at 400 mM NaCl whereas A. mangium died at this salinity level.

Garg (1998) determined phytoremedial capacity of four different tree species: Acacia

nilotica, Dalbergia sissoo, Prosopis juliflora and Terminalia arjuna. It was found that

Dalbergia sissoo and Prosopis juliflora produced more biomass and reduce sodicity of the

soil more as compared to other species. Moreover there was an increase in organic C of the

soil as well. Dagar et al. (2001) observed changes in soil properties on which three tree

species were grown for seven years. Tamarix articulata ameliorated the soil by reducing the

values of ESP and pH followed by Prosopis juliflora and Acacia nilotica. Increase in organic

carbon was also observed. These changes were more prominent in upper soil layer than the

lower one. The role of silvopastoral systems for improving soil organic matter content and

microbial activity for effective management of sodic soils was analysed by Kumar et al.

(2002). The silvopastoral system comprised of tree species of Acacia nilotica, Dalbergia

sissoo and Prosopis julifloraalong with grass species of Desmostachya bipinnata and

23  

Sporobolus marginatus. Nitrogen mineralization rate was more in silvopastoral systems

compared to ‘grassonly’system. Soil organic matter was linearly related to microbial biomass

carbon, soil N and nitrogen mineralization rates. On the basis of improvement in soil OM,

and enhanced availability of soil N in the tree + grass systems, agroforestry could be

implemented for improving the fertility status of highly sodic soils.

Ramoliya and Pandey (2002) studied the effect of soil salinity on emergence, growth

and physiology of Acacia nilotica. The salinity levels were 4.1, 6.2, 8.1, 9.9, 12.2 and 14.3

dS m-1. An inverse association was found between germination of seeds and salinity levels.

No germination was found when soil salinization surpassed 12.2 dS m-1. Both shoot and root

growth was reduced in response to increasing salinity levels. Yokota (2003) compared five

acacia species, A. ampliceps, A. salicina, A. ligulata, A. holosericea, and A. mangium in

solution culture for their salt tolerance and found that A. ampliceps was more tolerant and

survived even at 428 mM NaCl where all the other species died. Madsen and Mulligan

(2006) conducted a trial to observe the emergence and growth responses of Eucalyptus

citriodora, Eucalyptus camaldulensis, Eucalyptus populnea provenances and Acacia salicina

to various EC levels. Acacia salicina was the only species to have seedling emergence and

survival at 20 dS m-1 and the dry weight reduction was less obvious for this species than for

any of the eucalyptus. Acacia salicina performed significantly better at these salinity levels

than the eucalyptus species.

Shirazi et al. (2006) conducted a field study to observe the growth and nutrients

contents of some salt tolerant multipurpose tree species (A. ampliceps, A. stenophylla, A.

nilotica, Eucalyptus camaldulensis, and Conocarpus lancifolius) under saline conditions. A.

ampliceps had the maximum survival percentage followed by Conocarpus lancifolius, A.

nilotica, A. stenophylla and Eucalyptus camaldulensis. The plant height was the maximum in

A. nilotica followed by Eucalyptus camaldulensis and A. ampliceps. The analysis of leaves

showed that A. nilotica had the maximum contents of macro and micro nutrients so it can

play an important part in enhancing the fertility of the salt affected soils and can also give

good economic returns from the marginal lands. Tewari et al. (2006) compared the growth of

Dalbergia sissoo and Acacia nilotica seedlings on various levels of sodicity and salinity. The

increasing levels of salinity and sodicity reduced the growth and dry weight of both species.

24  

However, this reduction was relatively greater in D. sissoo than in A. nilotica. Root: shoot

ratio and root weight ratio in both the species increased with increasing sodicity and salinity

levels. On the other hand relative growth rate and net assimilation rate was decreased. Mehari

et al. (2006) conducted a glasshouse experiment to evaluate the salinity tolerance potential of

two acacia species i.e A. tortilis and A. nilotica. They found both species highly tolerant and

even survived at 300 mM NaCl but A. nilotica was more tolerant than the other species.

Ashraf et al. (2008) studied the growth performance of five species of Acacia, i.e.,

Acacia ampliceps, A. stenophylla, A. machonochieana, A.sclerosperma, and A.nilotica in the

salt effected field having salinity in the range of 4-25 dS m-1. They found that A. nilotica and

A. ampliceps were more promising as compared to other species. A. ampliceps grew well in

medium to high salinity levels whereas Acacia sclerosperma and A. machanochieana were

found alive in low salinity patches. The maximum plant height and stem diameter was found

in case of Acacia nilotica. There was an increase in the contents of soil organic matter, N, P,

and K at the end of the experiment. The SAR and EC showed a decreasing trend particularly

in case of Acacia nilotica.

Hardikar and Pandey (2008) studied the effects of salinity on, growth, water status

and mineral uptake of Acacia senegal (L.). Salinity reduced the water potential of tissues,

which caused an internal water deficit to plants. The growth of the seedlings was

significantly decreased with increasing salinity. There were no active mechanisms to control

net uptake of Na+ and its transport to shoot tissues. The contents of N, P, K and Ca were also

considerably reduced. Mahmood et al. (2009) applied combined stress salinity and sodicity to

Acacia ampliceps. Plant growth parameters were reduced considerably with increasing levels

of salinity and sodicity. Moreover the plants improved the soil health through decreasing ECe

and SAR of the soil.

Mahmood et al. (2010) assessed the sodicity tolerance potential of Acacia ampliceps

to various levels of sodicity ranging from 10-70 (mmol L-1)1/2. Plants could not survive in

SAR of 60 and 70 (mmol L-1)1/2. The obtained results showed that with increasing the levels

of sodicity sodium concentration was increased in the leaves whereas those of potassium,

calcium and magnesium decreased with increased levels of sodicity. Compartmentation and a

25  

wider K+: Na+ ratio were considered as the main mechanism for plant survival. Abari et al.

(2011) studied the germination response of two acacia species under various concentrations

NaCl and KCl and found the germination of A. oerfota at 350 mM NaCl. On the other hand

A. tortilis did not germinate at 300 mM NaCl. Basavaraja et al. (2011) grew Acacia nilotica

on a sodic soil for ten year period and found that there was a remarkable decline in the

sodicity level of the soil. Na+ was leached down from the exchange sites and was

accumulated in the lowr soil depths. The concentrations of the extractable Ca, Mg and K

were increased throughout the profile depth. Organic carbon, cation exchange capacity and

the availability of the major nutrients was also increased.

Morais et al. (2012) evaluated the salt tolerance of two legumes species; Ulex

europaeus, and Acacia longifolia. Salt stress significantly reduced the plant height and the

dry weight in A. longifolia whereas in U. europaeus the effect was not significant. The root:

shoot ratio and root mass ratio increased as a result of increasing salinity in A. longifolia but

not in case of U. europaeus. The invasive species (A. longifolia) respond better to salinity as

a result of more activities of CAT and GR and a higher K+/Na+ ratio. Kumar et al. (2012)

grew different trees species to analyze its effects on the survival, growth and biomass

production. The results demonstrated that clonal Eucalyptus, Terminalia arjuna, Tamarix

articulata, Dalbergia sissoo and Acacia ampliceps were tolerant to both salinity and

waterlogging. Soil salinity and sodicity were reduced particularly in 0-30 cm soil depths.

It can be concluded from the literature that the salinity has many detrimental effects

on plant growth. The nature has gifted plants with various strategies to cope with higher

concentrations of salts. The salt affected soils can be reclaimed with different approaches but

the phytoremediation is the most suitable approach currently available. The literature leads to

think that the acacia species may have tremendous genetic diversity regarding their salt

tolerance and phytoremedial potential. The present studies are planned to assess the salinity

tolerance potential of Acacia ampliceps and Acacia nilotica by studying different

physiological and biochemical mechanisms and to determine their comparative

phytoremediation potential for the salt affected soils.

26  

CHAPTER-3

MATERIALS AND METHODS

The research work reported in this thesis has been conducted in the wire house of the

Institute of Soil and Environmental Sciences and at Proka farm, University of Agriculture

Faisalabad, Pakistan. This research has been conducted to evaluate the comparative salt

tolerance and phytoremedial potential of two acacia species. Four different experiments as

given below were conducted to achieve the specific objectives of this research project:

3.1 Experimental techniques

3.1.1 Study- 1: Effect of different levels of salinity on growth and ionic composition of

Acacia ampliceps and Acacia nilotica

Seed source

The seeds of Acacia species were collected from Forest Research Institute, Peshawar

and Nuclear Institute of Agriculture and Biology, Faisalabad.

Growth conditions:

This experiment was conducted in wire house of the Institute of Soil and

Environmental Sciences, University of Agriculture Faisalabad. The healthy nursery of three

week old plants of two acacia species was transferred in thermo pole sheets with holes in

them floating on 25 L capacity plastic tubs. These tubs were filled with ½ strength

Hoagland’s solution (Hoagland and Arnon, 1950). Proper aeration was provided by bubbling

air through the nutrient solution eight hours a day. The solution was changed weekly. The

design of experiment was CRD factorial with four replicates. After one week of

transplantation, different salinity levels (control, 100, 200, 300 and 400 mM) were developed

step wise with NaCl. The pH of the experiment was monitored daily and maintained at 6.0 +

0.5 throughout the experiment by the addition of NaOH or HCl.

27  

Measurements:

After eight weeks of salinity development plants were harvested. At harvest data of

shoot fresh weight, shoot dry weight, root fresh and dry weights, and shoot and root lengths

was noted. The roots and shoots were separately oven dried at 75○C for 48 hours. The dry

root and shoot weights were recorded.The dried and ground shoot and root samples were

digested with H2SO4 and H2O2 following the method of Wolf (1982). After digestion volume

was made 50 ml with distilled water and used for ionic analysis. Na+ and K+ were determined

using the flame photometer whereas Cl- was determined with chloride analyser. The details

of plant analysis are given in section 3.4.

3.1.2 Study- 2: Organic acid release and oxidative stress tolerance of Acacia ampliceps

and Acacia nilotica in response to salinity

Growth conditions:

This experiment was conducted at the Institute of Soil and Environmental Sciences,

University of Agriculture Faisalabad. The healthy nursery of three week old plants of Acacia

ampliceps and Acacia nilotica was transferred in thermo pole sheets with holes in them

floating on 25 L capacity plastic tubs. These tubs were filled with ½ strength Hoagland’s

nutrient solution (Hoagland and Arnon, 1950). Proper aeration was provided by bubbling air

through the nutrient solution eight hours a day. The solution was changed weekly. The design

of experiment was CRD factorial with four replicates. After one week of transplantation,

different salinity levels (control, 100 and 200 mM) were developed step wise with NaCl. The

pH of the experiment was monitored daily and maintained at 6.0 + 0.5 throughout the

experiment by the addition of NaOH or HCl.

Measurements:

After four weeks of salinity development plants were harvested and data of shoot

fresh and dry weights, root fresh and dry weights, shoot and root lengths were noted.

Membrane stability index (MSI) and activities of antioxidants enzymes including superoxide

dismutase (SOD), peroxidases (POD) and catlase (CAT) were determined. Activity of SOD

was determined by its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) as

28  

described by Giannopolitis and Ries (1977). For activities of catalase (CAT) and peroxidase

(POD) the method of Chance and Maehly (1955) was used. Organic acids including citric

acid, malic acid and tartaric acid were analyzed using the methods detailed ahead in section

3.4.5.3. Na+ and K+ were determined using the flame photometer whereas Cl- was

determined with chloride analyser. The details of plant analysis are given in section 3.4.

3.1.3 Study-3: Effect of water stress on the response of Acacia ampliceps and Acacia

nilotica to salinity.

Growth conditions:

A pot culture study was conducted to investigate the effect of salinity and water stress

on the two acacia species. Experiment was conducted in the wire house of the Institute of

Soil and Environmental Sciences, University of Agriculture Faisalabad. Normal soil was

collected from an agricultural field and analyzed for physicochemical properties. Soil was

passed through 2 mm sieve and filled in pots @ of 12 kg per pot. The required salinity levels

(control, 10, 20 and 30 dS m-1) were developed in the respective treatment pots by adding

calculated amount of NaCl before filling the soil in the pots. In control no salt was added.

Three month old nursery plants of both the species were transplanted in these pots. After the

establishment of the plants water stress was applied during the period of the experiment in

pots under water stress treatment. The water characteristics are given in Table 3.2.

Measurements:

Plant growth data like plant height, stem diameter, number of branches and number of

leaves were recorded after four months. Chlorophyll contents were determined before

harvesting. After harvesting, relative water contents (RWC) were determined following

method detailed ahead in section 3.4.2. Nodule number and their fresh and dry weights were

determined. The plants were separated into root, stem and leaves after harvesting. Ionic

concentrations of Na+, K+ and Cl- were determined in these parts. Soil samples were taken

from each pot and analyzed for different properties including pHs, ECe and SAR. The details

of soil, water and plant analysis are given in section 3.2 and 3.3 and 3.4.

29  

3.1.4 Study-4: Comparative role of Acacia ampliceps and Acacia nilotica in the

phytoremediation of the salt affected soil

Growth conditions:

A field experiment was conducted to investigate the role of Acacia ampliceps and

Acacia nilotica in the reclamation of salt affected soil at Proka farm, University of

Agriculture Faisalabad. Three month old healthy nursery of both the plant species was

transferred in the pits already made in the field for this purpose. Plants were irrigated with

saline water as characterized in Table 3.2.

Measurements:

The plants of both acacia species were grouped into two categories i. e. good

and poor, depending on their health at the age of six months. Plant growth data like plant

height, stem circumference, number of branches and number of leaves were taken after every

six months for two years. For ionic composition two types of leaves (older and younger)

were taken and used for ionic analysis. Soil samples taken from different depths in the root

zone of the selected plants were analyzed for various properties like pHs, ECe and SAR. The

soil organic matter content and the change in the physical properties of soil like bulk density

and infiltration rate were also determined. The details of soil, water and plant analysis are

given in section 3.2 and 3.3 and 3.4.

3.2 Soil analysis

3.2.1 Particle-size analysis

Hydrometer method (Bouyoucos, 1962) was followed for particle size analysis. Forty

grams of air dry soil was taken in a 400 mL beaker, 40 mL of 2% sodium hexametaphosphate

[(NaPO3)6] solution was added, mixture was transferred to dispersion cup and stirred for 10

minutes. The contents of dispersion cup were washed into 1000 mL graduated cylinder

having 1L capacity within 36+ 2 cm height. The volume was made up to 1000 mL with

distilled water and hydrometer was placed in cylinder. Hydrometer was removed and

contents of cylinder were shaken manually by means of a metal plunger. When uniform

30  

suspension was obtained, plunger was taken out and after 4 minutes hydrometer reading

(HR1) was recorded. Shaking procedure was repeated by removing hydrometer with

minimum of disturbance and second hydrometer reading (HR2) was recorded after 2 hours.

Since hydrometer is calibrated at temperature of 68 oF (20 oC), the HR1 and HR2 were

corrected for temperature variation (for each degree above 20oC, added a factor of 0.3 to the

reading and for each degree less than 20oC, subtracted a factor of 0.3 from the reading to get

corrected hydrometer reading) and designed as CHR1 and CHR2, respectively. Calculations

involved are:

Silt + clay (%) = [(CHR1)100] / (weight of soil)

Clay (%) = [(CHR2)100] / (weight of soil)

Silt (%) = % (silt + clay) - (%) clay

Sand (%) = 100- [% silt + %clay]

Soil textural class was determined using USDA textural triangle.

3.2.2 Bulk density (ρb)

Bulk density was determined by core method (Klute, 1986). A core sampler with

double ring set of known volume was allowed to penetrate into soil, enough to fill the

sampler, but not to compress the soil in the confined space of the core sampler. Then sampler

was carefully removed so as to preserve its contained soil with natural structure and packing

as nearly as possible. The soil extending each end of the sampler was trimmed with a strong

edge knife and was transferred to weighing pot. It was dried it in an oven at 105 oC until

constant weight and weighed again. The bulk density (ρb) was calculated with the formula as

under:

ρb (Mg m-3) = [Oven dry mass of soil (g)]/[Bulk volume of the soil sample (cm3)]

31  

3.2.3. Water infiltration rate

The water infiltration rate was measured with the help of double-ring infiltrometer

(Klute, 1986). Two metallic rings were driven 5 cm into the soil using a level metallic plank

and a hammer. The diameter of inner ring was 30 cm and that of outer ring was 60 cm. The

height of inner and outer rings was 30 cm. A graduated scale, reading in cm and mm, was

fixed on inner side of the inner ring. A polythene sheet was used to line the bottom of the

inner ring to avoid soil disturbance while pouring water. The inner and outer rings were then

filled up to the same depth with tube well water, which was and/or to be used for irrigation.

The polythene sheet was then quickly and gently removed and the initial reading of the water

level in the inner ring was recorded. Then after 5, 10, 20, 30 minutes readings were taken and

finally at a regular intervals of 20 minutes till the intake rates became steady.

3.2.4 Organic matter

Soil organic matter (OM) was determined following the method described by Walkly-

Black (Jackson, 1962). For this purpose, one gram of soil was swirled in 10 mL of 1.0 N

K2Cr2O7 solution, and 20 mL of concentrated H2SO4, was added. It was mixed and allowed

to stand for 30 minutes. Then diluted to about 200 mL with distilled water and titrated

against FeSO4.7H2O to dull green end point in the presence of 0.5 g NaF and 30 drops of

diphenylamine as indicator.

Organic matter was calculated by the following formula:

OM (%) = [(Vblank - Vsample) x M x 0.69] / [Wt. of soil (g)], where

Vblank = Volume (mL) of FeSO4.7H2O used in blank

Vsample = Volume (mL) of FeSO4.7H2O used to titrate the sample

M = Molarity of FeSO4.7H2O solution

0.69 = 0.003 x 100 x (100/74) x (100/58), where

0.003 = me wt. of carbon

32  

100 = to convert OM in %

100/58 = Factor to convert carbon to OM

100/72 = Recovery factor for carbon

3.2.4. pH of saturated soil paste (pHs)

For pHs determination, 300 g soil was saturated with distilled water, wet soil mass

was allowed to stand overnight and pHs was recorded by Jenco Model 671P pH meter.

3.2.5. Electrical conductivity of soil saturaed paste extracts (ECe)

Suction pump was used to obtain extract from the saturated soil paste. Electrical

conductivity of the saturation extract was determined with the help of TOA, CM-14P

conductivity meter. Sodium hexametaphosphate (0.1% solution) was added @ 1 drop per 25

mL of the extract to prevent any precipitation of salts during storage of extract. The EC meter

was calibrated with 0.01 N KCl solution. Cell constant (k) was calculated by the formula:

K = [1.4118 dS m-1] / [EC of 0.01 N KCl solution (dS m-1)]

3.2.6. Carbonate and bicarbonate (CO32- + HCO3

-)

Saturation extract was titrated against 0.01 N H2SO4 using phenolphthalein indicator

to colorless end point for CO32-. To the same sample, methyl orange indicator was added and

titrated against 0.01 N H2SO4 to pinkish yellow end point for HCO3-.

2a × normality of H2SO4

CO32- (mmolc L-1) = × 1000

mL of sample taken

Where a is mL of H2SO4 used during titration for CO32-

33  

(b – 2a) × Normality of H2SO4

HCO3- (mmolc L-1) = × 1000

Volume of sample taken (mL)

Where b = mL of H2SO4 used for HCO3- determination.

3.2.7. Chloride (Cl-)

Saturation extract was titrated against 0.005 N AgNO3 solution using K2CrO4

indicator to a brick red end point and chloride was calculated as under:

mL of AgNO3 × normality of AgNO3

Cl- (mmolc L-1) = × 1000

mL of sample taken

3.2.8. Sulphate (SO42-)

Sulphate was calculated by difference:

SO42- (mmolc L-1) = TSS – (CO3

2- + HCO3- + Cl-), all expressed as mmolc L-1.

3.2.9. Calcium + Magnesium (Ca2+ + Mg2+)

Saturation extract was titrated against 0.01 N EDTA (Versinate solution) in the

presence of NH4Cl + NH4OH buffer solution using eriochrome black T indicator to a bluish

green end point.

mL of EDTA used × Normality of EDTA

Ca2+ + Mg2+ (mmolc L-1) = × 1000

mL of sample taken

34  

3.2.10. Sodium (Na +)

A series of NaCl standard solutions (2, 4, 6, 8, 10, 12, 14 and 16 ppm Na+) was used

to standardize the Sherwood- 410 Flame Photometer. Sample readings were recorded and

concentrations (ppm) were calculated from regression equation obtained by plotting

concentration of standards against their readings from flame photometer. The actual

concentration of soluble Na+ in mmolc L-1 was calculated from the formula below:

ppm concentration × dilution factor

Na+ (mmolc L-1) =

eq. wt. of Na+

3.2.11. Potassium (K+)

A series of KCl solutions (2, 4, 6, 8, 10, 12 and 14 ppm K+) were used to standardize

the Sherwood- 410 Flame Photometer. Sample readings were recorded and concentrations

(ppm) were calculated from regression equation obtained by plotting concentration of

standards against their readings from flame photometer. The actual concentration of soluble

Na+ in mmolc L-1 was calculated from the formula below:

ppm concentration × dilution factor

K+ (mmolc L-1) =

eq. wt. of K+

3.2.12. Sodium adsorption ratio (SAR)

Sodium adsorption ratio was calculated by the formula:

Na+

SAR =

[(Ca2+ + Mg2+) / 2]1/2

35  

Where the concentration of soluble cations was in mmolc L-1

3.3 Water analysis

Samples of irrigation water were collected in clean plastic bottles. The EC, CO32-, HCO3

-, Cl-

, SO42-, Ca2+ + Mg2+, Na+ and SAR of water were determined in the same way as for soil

saturation extract.

3.3.1 Residual sodium carbonate (RSC)

It was calculated by the equation:

RSC (mmolc L-1) = (CO32- + HCO3

-) – (Ca2+ + Mg2+)

Where all ions are expressed in mmolc L-1

3.4 Plant Analysis

3.4.1 Sodium potassium and chloride

The roots and shoots were separately oven dried at 75○C for 48 hours. The dry root

and shoot weights were recorded. The dried and ground shoot and root samples were

digested with H2SO4 and H2O2 following the method of Wolf (1982). For this purpose the

dried ground material was placed in digestion tubes, 10 mL of conc. H2SO4 was added and

incubated over night at room temperature. Then 2 mL of H2O2 (35% A. R. grade extra pure)

was poured down through the sides of the digestion tubes and was rotated. Tubes were placed

in a digestion block and heated up to 350 oC until fumes were produced and continued to heat

for another 30 minutes. Digestion tubes were removed from the block and allowed to cool.

Then 2 mL of H2O2 were gently added. The tubes were placed again into the digestion block

until fumes were produced for 20 minutes. Again digestion tubes were removed. Above step

was repeated until the cool material became colorless. After digestion volume was made 50

ml with distilled water and used for ionic analysis. The ionic concentration for Na+ and K+ in

plant samples was determined by Sherwood- 410 Flame Photometer with the help of self-

36  

prepared standard solutions using reagent grade salt of NaCl and KCl respectively. Chloride

was determined with Sherwood- 926 chloride analyzer.

3.4.2 Measurment of relative water contents (RWC)

For relative water contents, young leaf samples were weighed (0.5 g) as fresh weight

and immediately hydrated to full turgidity (Sairam et al., 2002). After 4 hours, samples were

taken out of distilled water and turgid leaves were quickly dried with filter paper to remove

surface water and immediately weighed to obtain fully turgid weight (TW). These samples

were oven dried at 65°C for 48 hours to determine dry weight (DW). Relative water contents

were determined by the following formula:

RWC= [(FW-DW)/ (TW-DW)]

3.4.3 Measurement of membrane stability index (MSI)

The membrane stability index was determined by estimating the ions leaching from leaf

tissue into distilled water by the method described by Sairam et al. (2002). Fresh leaf sample

(0.2 g) was taken in test tubes containing double distilled water in two sets. One set of test

tubes was kept in water bath at 40°C temperature for 30 minutes and its electrical

conductivity was recorded (C1) using an electrical conductivity meter. Second set of test

tubes was kept at 100°C in boiling water for 15 minutes and its electrical conductivity (C2)

was also recorded. MSI was calculated using the following formula:

MSI = (1-C1/C2) ×100

3.4.4 Activities of antioxidant enzymes

Superoxide dismutase (SOD)

Catalase (CAT)

Peroxide (POD)

For extracting antioxidant enzymes, 0.5 g fresh leaf samples were ground using a tissue

grinder in 5 mL of 50 mM cold phosphate buffer (pH 7.8) placed in an ice bath. The

37  

homogenate was centrifuged at 15000 x g for 20 min at 4 °C. The supernatant was used for

determination of antioxidant enzymes.

3.4.4.1 Superoxide dismutase (SOD)

SOD activity was determined by measuring its ability to inhibit the photoreduction

of nitroblue tetrazolium (NBT) using the method as described by Giannopolitis and Ries

(1977). The reaction mixture (3 ml) contained 50 µM NBT, 13 mM methionine, 1.3 µM

riboflavin, 50 mM phosphate buffer (pH 7.8), 75 nM EDTA and 20 to 50 µl of enzyme

extract. The test tubes containing the reaction solution were irradiated under light (15

fluorescent lamps) at 78 µmol m-2 s-1 for 15 min. The absorbance of the irradiated solution

was recorded on UV-VIS-spectrophotometer at 560 nm. One unit of SOD activity was

defined as the amount of enzyme required for 50% inhibition of nitroblue tetrazolium (NBT)

reduction. Activity of each enzyme was expressed on protein basis and protein contents of

the extract were determined by the method of Bradford (1976) using bovine serum albumin

as standard.

3.4.4.2 Catalase (CAT) and Peroxidase (POD)

For the determination of activities of peroxidase (POD) and catalase (CAT), method

of Chance and Maehly (1955) was used with some modifications. CAT reaction mixture (3

ml) contained 5.9 mM H2O2, 50 mM phosphate buffer (pH 7.0) and 0.1 ml enzyme extract.

The reaction was initiated by addition of the enzyme extract. The changes in absorbance of the

reaction mixture were recorded after every 20 seconds at 240 nm. One unit enzyme activity

was defined as change in absorbance of 0.01 units per minute. The POD reaction mixture

contained (3 ml) 20 mM guaiacol, 50 mM phosphate buffer (pH 5.0), 40 mM H2O2, and 0.1

ml enzyme extract. The changes in absorbance of the reaction mixture were recorded every

20 s at 470 nm. One unit of POD activity was defined as an absorbance change of 0.01 units

per min. Activity of each enzyme was expressed on protein basis and protein contents of the

extract was determined by the method of Bradford (1976) using bovine serum albumin as

standard.

38  

3.4.5 Determination of ash alkalinity and organic acids

3.4.5.1 Ash alkalinity

Ash alkalinity was measured following the method of Jungk (1968) with minor

modifications. Ground samples (0.05g) were taken in porcelain crucible. After adding 2.5ml

of 0.1M NaOH, crucible was heated on sand bath till dryness and dry ashed at 520 oC in a

muffle furnace overnight. After cooling 10 ml of 0.1M HCl was added to this ash in two

splits and boiled for 1 minute on hot plate and the material was transferred in 100 ml

volumetric flask. The flask was again heated till boiling and volume was made after cooling

the contents. Excess acid in 50 ml of this solution was back titrated with 0.1 M NaOH and

reading was used to calculate ash alkalinity.

Ash alkalinity (mmol NaOH/100g dwt) = Volume of 0.1M HCl×2×100

3.4.5.2 Root exudate collection

Collection of root exudates and their analysis for organic acids was done following

the method of Fang et al. (2008) with some changes in the chromatographic conditions. The

root exudates were collected on 14th and 28th days after the NaCl treatment. After 2 hours of

exposure to light in the morning, the plants were taken from the tubs and washed three times

with distilled water. The plant roots were placed in a conical flask with 100 mL de-ionized

water. After 2 hours, the plants were returned to the solution in tubs. The exudate solution

was then filtered and concentrated with the help of rotary evaporator. The exudates were re-

dissolved in 10 mL de-ionized water and transferred to test tubes for high pressure liquid

chromatography (HPLC) analysis.

3.4.5.3 Analysis of organic acids in root exudates

Prior to the sample measurement, the root exudate solution was filtered with a 0.45

mm filter film. The high pressure liquid chromatography (HPLC-10A, SHIMADZU) was

used to isolate organic acids in root exudates and determine their contents. The

chromatographic conditions were: Column: Shim-Pack CLC-ODS (C-18), (25 cm×4.6mm, 5

µm); Mobile phase: Isocratic: H3PO4 (6×10-3 mol/L), (pH 2.1). Injection volume: 20 mL;

39  

Flow rate: 0.7 mL/min; Column temperature: RT; Detector: SPD 10-A, UV- Visible at 210

nm. Determination of organic acids was done through the standard reagent method and the

contents of these organic acids were determined by the peak area method.

3.5. Statistical Analysis

Standard procedures were applied to analyze the data using appropriate experimental

design following (Steel et al. 1997) and the means were compared using least significant

difference (LSD) test. Any specific detail of statistical analysis is given in chapter four under

individual experiments.

Table 3.1 The characteristics of soils used for the experiments

A. Normal soil used for Exp. No. 3

Soil Characteristics Values Units

Texture Sandy loam

Saturation %age 27 %

pHs 7.44 8.68

ECe 2.31 dS m-1

SAR 6.43 (mmol L-1)1/2

40  

B. Salt-affected soil used for Exp. No. 4

Soil Characteristics Values Units

Texture Sandy clay loam

Saturation %age 32 %

pHs 8.86a,8.62b

ECe 13.9a, 11.72b dS m-1

SAR 58.71a, 52.5b (mmol L-1)1/2

organic matter 0.47a, 0.34b %

Bulk density 1.62 g cm-3

Infiltration rate 0.32 cm hr-1

a: 0-30 cm soil depth b: 30-60 cm soil depth

Table 3.2 The characteristics of irrigation water used for the experiments

Characteristics Used in pots

(Exp. No.3)

Used on salt-affected soil

(Exp. No.4)

EC (dS m-1) 1.34 2.55

TSS (mmol L-1) 13.4 25.5

SAR (mmol L-1)1/2 7.43 11.2

RSC (mmol L-1) Nil Nil

41  

CHAPTER-4

RESULTS AND DISCUSSION

4.1 Study- 1: Effect of different levels of salinity on growth and ionic composition of

Acacia ampliceps and Acacia nilotica

4.1.1 Introduction

Soil salinity is a major environmental issue that is responsible for the reduction of

yield of many crops (Majeed et al., 2010). It is of particular importance for arid and semi-

arid areas of the world (Murtaza et al., 2011). About, 7% of the global land area and

approximately 20% global cultivated land is affected by salinity (Zhu, 2001). About 6.67 m

ha area of Pakistan is having the problem of soil salinization and approximately it is one third

of the total cultivated area of the country (Khan, 1998).

The growth of most of the plants is badly affected due to salinity (Taiz and Zeiger,

2006) and if the level of salinity is too high the growing plants may be eliminated totally

(Garg and Gupta, 1997). The provision of all the nutrients to plant is made through roots so

the reaction of roots to salinity is very vital for understanding the whole plant response to

salinity (Khalil et al., 2012). It is quite clear that plant cells, tissues and organs display varied

response to harsh environmental conditions at different levels of their growth (Munns, 1993).

High cytoplasmic solute concentrations cannot be tolerated by most of the plants, so in order

to carry on routine metabolic functions, plants have to dump higher concentrations of salts in

their vacuoles or they have to restrict them in older tisssues (Munns and Tester, 2008).

Potassium ion is very vital for many cell functions like cell enlargement, osmoregulation and

homeostasis (Schachtman et al., 1997). The plants with higher K+: Na+ ratio are considered

better tolerant to salinity stress (Saqib et al., 2005). Inspite of decline in growth due to

salinity, there is marked genotypic difference within plant species regarding their sensitivity

to higher concentrations of salts (Saqib et al., 2006).

According to Saboora et al. (2006) it is expected that salinity will increase at a rate of

about 10% per year around the globe. Grassland cover and ultimately the feed production for

animals is reduced due to salinization in the areas of arid and semiarid climate (El-kharbotly

42  

et al., 2003). It is a great demand of the time that we should look for such plant species

which are naturally gifted with high salinity tolerance potential and at the same time can give

reasonable economic yield from such baren lands.

Acacia has substantial production capacity in a variety of livestock and agro forestry

systems. It is a good source of fuel wood, commercial gum and its leaves and pods can be fed

to animals. The other benefits include fodder and shade for animals, construction and

fencing, enhancement of soil fertility by fixing atmospheric N2 and stabilization of soil

structure through litter production (Benninson and Paterson, 1993). According to Aslam

(2006) the most simple and economical way of controlling soil salinity is the plantation of

trees. The objective of this study was to evaluate the salinity tolerance potential of two acacia

species for their growth and ionic relationship, so that the most promising specie can be

recommended for salt affected lands.

4.1.2 Methodology

This experiment was conducted in the green-house of the Institute of Soil and

Environmental Sciences, University of Agriculture Faisalabad, Pakistan. Seeds of two acacia

species (A. nilotica and A. ampliceps) were collected from Forest Research Institute

Peshawar, Pakistan and Nuclear Institute of Agriculture and Biology, Faisalabad, Pakistan.

The seeds were sown in iron trays having two inch layer of sand washed with distilled water.

These sown seeds were kept moistened with water and with nutrient solution after seedling

emergence. After three weeks, the seedlings were transplanted in foam plugged holes in

polystyrene sheets floating over nutrient solution in 25L plastic tubs.

After one week of transplantation, different levels of salinity i.e. 100, 200, 300 and

400 mM NaCl were developed by the addition of calculated amount of NaCl in two

increments (one per day) in the salinity treatment tubs, whereas no salt was added in control.

The pH of the solution was maintained at 6.0±0.5 with dilute NaOH or HCl and the solution

was changed weekly during the period of study. After eight weeks, the plants were harvested

and the data regarding shoot and root fresh weights and the respective lengths were recorded.

The roots and shoots were separately oven dried at 75○C for 48 hours. The dry root and shoot

weights were recorded. The dried shoot and root samples were ground in grinding machine

43  

and digested with H2SO4 and H2O2 following the method of Wolf (1982). After digestion

volume was made up to 50 ml with distilled water and used for ionic analysis.

The ionic concentration of Na+ and K+ in plant samples was determined by Sherwood- 410

Flame Photometer with the help of self-prepared standard solutions using reagent grade salt

of NaCl and KCl respectively. Chloride was determined with Sherwood- 926 chloride

analyzer. Data of the experiment were subjected to analysis of variance in completely

randomized design with factorial arrangements (Steel et al. 1997). The significance of

differences among treatments and genotypes was determined using LSD test.

4.1.3 RESULTS

4.1.3.1 Shoot and root growth

The increasing levels of salinity resulted in the corresponding decline in shoot fresh

and dry weights of both the acacia species (Fig. 4.1.1 & 4.1.2). The difference among

species, treatments and their interaction was significant for both these parameters. A.

ampliceps survived even at 400 mM NaCl whereas A.nilotica could not survive at this

salinity level (data not shown). In control treatment, both species did not differ significantly

however in the rest of the treatments they differed significantly from each other and this

difference widened with more salt concentration in the growing medium. Like shoot growth

salinity resulted in the corresponding decline in the root growth as well (Fig. 4.1.3 & 4.1.4).

The main effects as well as their interaction were significant for both root fresh and dry

weights. Reduction in fresh and dry weights of root was more in the case of A. nilotica than

A. ampliceps. The length of both shoot and root was also decreased due to salinity stress (Fig.

4.1.5 & 4.1.6). Although treatments have significant effect, however the species did not

differ significantly from each other. A. nilotica produced more root and shoot length up to

200 mM NaCl level. However in 300 mM NaCl treatment, A. ampliceps showed more length

of both root and shoot. This indicated that with increase in salinity level there was more

reduction in shoot and root length of A. nilotica than A. ampliceps.

4.1.3.2 Shoot and root ionic composition

Salinity significantly increased the shoot and root Na+ concentration in both the

species (Fig. 4.1.7 & 4.1.8). The individual as well as interactive effects of treatment and

44  

species were found significant. The comparison of both the species in various treatments

showed that in control treatment, both the species have similar concentration of Na+. In all

the other treatments, A. ampliceps accumulated significantly lower Na+ as compared to A.

nilotica in both shoot and root. The difference between both the species was the maximum at

300 mM NaCl. In this treatment, the maximum Na+ concentration (2.3 mmol g-1dwt) was

found in the shoot of A. nilotica. At the same salinity level shoot Na+ concentration was 1.72

mmol g-1dwt in A. ampliceps. The root Na+ concentration in this traeatment was 2.86 and 2.1

mmol g-1dwt for A. nilotica and A. ampliceps respectively. Salinity significantly increased

the shoot and root Cl- concentration in both the species (Fig. 4.1.11 & 4.1.12). The individual

as well as interactive effects of treatment and species were found significant. In all the

salinized treatments, A. ampliceps accumulated significantly lower Cl- as compared to A.

nilotica in both shoot and root. At 300 mM NaCl the maximum Cl- concentration in the shoot

of A. nilotica was 2.62 mmol g-1dwt. At the same salinity level shoot Cl- concentration was

2.1 mmol g-1dwt in A. ampliceps. At this salinity level the root Cl- concentration was 3.21

and 2.47 mmol g-1dwt for A. nilotica and A. ampliceps respectively. The concentration of K+

as against of Na+ decreased significantly at each increasing level of salinization (Fig. 4.1.9 &

4.1.10). The individual effects of treatments and species as well as their interaction were

found significant. In control treatment K+ concentration was the maximum where as in 300

mM NaCl, it was the minimum. The comparison of species at each salinity level showed that

A. ampliceps accumulated more K+ in both organs as compared to A. nilotica. At 300 mM

NaCl, shoot K+ concentration was 0.55 mmol g-1dwt in A. ampliceps where as in case of A.

nilotica it was 0.29 mmol g-1dwt. At this salinity level the root K+ concentration was 0.35

and 0.19 mmol g-1dwt for A. ampliceps and A. nilotica, respectively.

The K+: Na+ ratio was the maximum in control treatment and decreased with

increasing salinity levels. This ratio was the minimum in 300 mM NaCl treatment. Acacia

ampliceps has higher K+: Na+ ratio than A. nilotica for both root and shoot under saline

conditions (Fig. 4.1.13 & 4.1.14). There was a strong negative correlation of Na+ and Cl-

concentration with shoot dry weight of both the acacia species (Fig. 4.1.15 & 4.1.17). On the

other hand a strong positive correlation was found in the case of shoot K+ concentration and

shoot dry weight of both the species (Fig. 4.1.16).

45  

         

 

              

            Fig.4.1.1 Effect of salinity on shoot fresh weight of acacia species                                

 

              

              Fig.4.1. 2 Effect of salinity on shoot dry weight of acacia species                              

a

b

d

f

a

c

e

g

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. niloticaShoot fresh weight (g plant‐1) 

a

b

d

f

a

c

e

g

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Shoot dry weight (g plant‐1) 

46  

          

         Fig. 4.1.3 Effect of salinity on root fresh weight of acacia species                              

 

      

     Fig. 4.1.4 Effect of salinity on root dry weight of acacia species                              

a

b

d

f

a

c

e

g

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. niloticaRootfreshweight (g plant‐1) 

a

b

d

f

a

c

e

g

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Root dry

weight (g plant‐1) 

47  

                     

Fig.4.1. 5 Effect of salinity on shoot length of acacia species                              

 

       

     Fig.4.1. 6 Effect of salinity on root length of acacia species                              

b

c

d

e

a

bc

d

f

0

5

10

15

20

25

30

35

40

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Shootlength(cm) 

b

c

d

e

a

bc

d

f

0

5

10

15

20

25

30

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Rootlength(cm) 

48  

  

Fig.4.1. 7 Effect of salinity on shoot Na+ concentration of acacia species                              

 

 

Fig.4.1. 8 Effect of salinity on root Na+ concentration of acacia species                             

f

e

d

b

f

d

c

a

0

0.5

1

1.5

2

2.5

3

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Shoot Na+

concentration (mmol g‐1dwt)

g

f

d

b

g

e

c

a

0

0.5

1

1.5

2

2.5

3

3.5

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Root Na+

concentration (mmol g‐1dwt)

49  

          

          Fig. 4.1.9 Effect of salinity on shoot K+ concentration of acacia species                              

             

            Fig.4.1. 10 Effect of salinity on root K+ concentration of acacia species                              

a

b

d

e

a

c

e

f

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Shoot K+concentration (mmol g‐1dwt)

a

b

d

e

a

c

e

f

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Root K+concentration (mmol g‐1dwt)

50  

          

         Fig.4.1. 11 Effect of salinity on shoot Cl‐ concentration of acacia species                              

         

        Fig.4.1. 12 Effect of salinity on root Cl‐ concentration of acacia species                              

g

f

d

b

g

e

c

a

0

0.5

1

1.5

2

2.5

3

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. niloticaShoot Cl‐concentration (mmol g‐1dwt)

g

f

d

b

g

e

c

a

0

0.5

1

1.5

2

2.5

3

3.5

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Root Cl‐concentration (mmol g‐1dwt)

51  

                    

                    Fig.4.1.13 Effect of salinity on shoot K+: Na+ ratio of acacia species                              

                   

                   Fig.4.1.14 Effect of salinity on root K+: Na+ ratio of acacia species                              

a

b

def

a

c

ef

0

1

2

3

4

5

6

7

8

9

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Shoot K+:N

a+

a

b

de

a

c

ee

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Control 100 mM NaCl 200 mM NaCl 300 mM NaCl

A. ampliceps A. nilotica

Root K+: N

a+

52  

                   

Fig.4.1.15 Correlation between shoot dry weight and shoot Na+ concentration

                

Fig.4.1.16 Correlation between shoot dry weight and shoot K+ concentration

                

Fig.4.1.17 Correlation between shoot dry weight and shoot Cl- concentration

 

y = ‐0.2938x + 0.7638**R² = 0.9931

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.5 1 1.5 2 2.5

Shoot Na+ (mmol g‐1dwt)

Shoot dry weight (g plant‐1)

y = 0.647x ‐ 0.0264**R² = 0.9247

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 0.2 0.4 0.6 0.8 1 1.2 1.4

Shoot dry weight (g plant‐1)

Shoot K+ (mmol g‐1dwt)

y = ‐0.2527x + 0.7577**R² = 0.9883

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.5 1 1.5 2 2.5 3

Shoot dry weight (g plant‐1)

Shoot Cl‐ ( mmol g‐1 dwt)

53  

4.1.4 Discussion

This study indicated good salinity tolerance potential of the two acacia species

however there was a distinct genetic difference between the species used regarding the

salinity tolerance. Shoot fresh and dry weights of both the acacia species declined with the

increasing levels of salinization and the maximum reduction was noticed at 300 mM NaCl

concentration. At 400 mM NaCl salinity level, A. nilotica could not survive however the

seedlings of A. ampliceps were found alive. On relative basis, shoot dry weight was

decreased up to 68 and 85% as compared to control in A. ampliceps and A. nilotica

respectively. Salinity induced reduction in shoot growth in acacia was also found by Farooq

et al. (2010) and Yokota (2003). Reduction in the growth of plants due to salinity is mainly

attributed to three principle factors i.e osmotic effects, ion toxicity and deficiency of

necessary nutrients (Munns and Tester 2008). These factors are operative at both cellular and

whole plant level and affect all the metabolic activities of plants (Garg and Gupta, 1997).

Like shoot growth, the growth of root was also decreased to a great extent due to

salinized treatments as compared to control. On relative basis, reduction in root dry weight as

compared to control was 80 and 61% in A. nilotica and A. ampliceps, respectively. Reduction

in root growth due to salinity was also found by and Hardikar and Pandey (2008) and

Mahmood et al. (2010). At higher levels of salinity both root formation (Kramer, 1983) and

their elongation (Garg and Gupta, 1997) is reduced to a greater extent. The physical growth

parameters like fresh and dry weights of both root and shoot are usually related to salinity

tolerance potential of all plants at their early stages of growth so they are the tools of

selection criteria for salinity tolerance (Larcher, 1995). One thing is noteworthy that the

length of both root and shoot was more in A. nilotica than A. ampliceps in control as well as

in 100 and 200 mM NaCl concentrations. However in these treatments the weight of both

root and shoot was more in A. ampliceps than A. nilotica. The reason is that the leaves of A.

ampliceps are much fleshy and heavier than those of A. nilotica and the roots of A. ampliceps

have more lateral growth as compared to A. nilotica. In 300 mM NaCl level there was sever

reduction in length of both root and shoot but A. ampliceps produced more shoot and root

length than A. nilotica. Genetic variability in acacia was also observed by Marcar et al.

(1991) who found in a sand culture experiment that A. ampliceps and A. auriculiformis

survived at 400 mM NaCl whereas A. mangium died at this salinity.

54  

The ionic composition revealed that shoot and root Na+ and Cl- concentration of both

the species increased significantly in response to increasing salinity levels with more

accumulation in A. nilotica than A.ampliceps. This higher accumulation of Na+ in the tissues

corresponds to the lower growth and dry weights of A. nilotica. These results are also

supported by the findings of Marcar et al. (1991). The buildup of poisonous ions in plant

tissues is thought to be the major factor of decline in growth under salinity stress (Muscolo et

al., 2003). There was a strong negative correlation of Na+ and Cl- concentration with shoot

dry weight. On the other hand a strong positive correlation was found in the case of shoot K+

concentration and shoot dry weight. The findings of Khalil et al. (2012) are very much

similar to ours as he also found an inverse correlation between the shoot growth and Na+

concentration. This larger concentration of Na+ in tissues causes nutrient imbalance, osmotic

effects and specific ion toxicity (Arzani, 2008).

Moreover K+ has a key role in salt tolerance where uptake of K+ is decreased by Na+

(Fox and Guerinot, 1998). In our experiment, the reduction of K+ concentration was found in

both root and shoot which indicated that Na+ repressed the uptake of K+. Decreased K+

uptake in response to salinity was also observed by (Khalil et al., 2012; Marcar et al., 1991).

The plants with higher K+: Na+ ratio are considered better tolerant to salinity stress (Saqib et

al., 2005) and same was the case in our experiment where A. ampliceps owing to higher K+:

Na+ ratio showed better salt tolerance than A. nilotica. The higher Na+, Cl- and lower K+ and

K+: Na+ resulted in the reduction of root and shoot growth of A. nilotica more as compared to

A. ampliceps.

4.1.5 Conclusion

Although salinity had a detrimental effect on all the growth parameters of both the

acacia species, there was a distinct genetic difference between the tested species. On the basis

of better ionic homeostasis A. ampliceps had more growth and survival at higher salinity

levels than A. nilotica.

 

 

 

 

 

55  

4.2 Study- 2: Organic acid release and oxidative stress tolerance of Acacia ampliceps

and Acacia nilotica in response to salinity  

4.2.1 Introduction

Salinity causes many morphological, physiological, biochemical and molecular

disorders in plants. If the concentration of the salt is too high, plant growth (Amirjani, 2010),

photosynthesis, respiration (Marschner, 1995), nodulation (Al-Shaharani and Shetta, 2011)

and activities of varoius enzymes are badly affected (Sairam and Tyagi, 2004). The degree of

damage depends on harshness and duration of stress (Nawaz et al., 2010), the plant species

(Croser et al., 2001) the genotypes (Fung et al., 1998), type of salts (Lauchli and Grattan,

2007) and the growth stage of the plant under stress (Levitt, 1972).

Salinity results in the production of reactive oxygen species like hydrogen peroxide,

superoxide, singlet oxygen and hydroxyl radicals. These species cause very harmful effects

in the cell like lipid peroxidation, damage to proteins and changes in the DNA molecule

(McCord, 2000, Wang et al., 2003). Among various ROS, hydrogen peroxide, superoxides,

and hydroxyl radicals are mainly produced in the cytosol, chloroplasts, mitochondria, and the

apoplastic spaces (Bowler and Fluhr, 2000; Mittler, 2002). Membrane injury due to salinity is

related to over production of these reactive oxygen species (Shalata et al., 2001). Due to

osmotic stress stomata are closed leading to reduced supply of CO2 for photosynthesis and

over production of superoxide in chloroplast which leads to photoinhibition and

photooxidation and resultantly cell damage (Ashraf, 2009).

Nature has gifted plants with explicit mechanisms to detoxify these reactive oxygen

species. These comprise of the activation of the enzymes of antioxidative pathway like

superoxide dismutase, peroxidase, catalase and enzymes of the ascorbate- glutathione cycle

(Noctor and Foyer, 1998; Smirnoff, 2005). The concentration of non-enzyme antioxidants

including tocopherols, flavones, ascorbic acid and carotenoids is also increased (Johnson et

al., 2003). Superoxides are dismutased to H2O2 and molecular oxygen with the help of

superoxide dismutase (Giannopolitis and Ries, 1977). It is an important enzyme for

mitigation of oxidative stress in plants. H2O2 and some organic hydroperoxides are broken to

water and oxygen with the help of catalase enzymes (Ali and Alqurainy, 2006). Peroxidases

are also involved in the conversion of detoxification of hydrogen peroxide.

56  

Plant species vary greatly regarding increased activities of enzymatic and non-

enzymatic antioxidants in response to salinity (Zhang and Kirkham, 1995; Nayyar and Gupta,

2006). Similar differences have also been found within cultivars of a same species (Bartoli et

al., 1999). The detoxification potential of an antioxidant is affected by the type of species,

stress intensity and the plant growth stage at the time of stress. Salinity stress in plants

usually results in the over production of antioxidants in plants and the plants with higher

antioxidant production capacity can better scavenge ROSs and are more salt tolerant (Shalata

and Tal, 1998; Garratt et al., 2002).

The release of H+ from plant root is considered to lower pH of rhizosphere

(Marschner, 2003). Leguminous plants have been shown to acidify their rhizosphere

(Schubert et al., 1990a) by releasing sufficient amount of H+ ions from their roots (Schubert

et al., 1990b). This H+ ion extrusion is responsible for the increase in pH of the cytosol,

which in turn hastens the formation of organic anions. These organic anions are a gauge of

how much hydrogen ions are released in the rhizosphere and are called as ash alkalinity. This

parameter has been characteristically determined in many plants to evaluate their

acidification capacity (Moody and Aitken, 1997; Noble and Randall, 1999). The

determination of the ash alkalinity in sodicity adapted species might be helpful to screen out

such plants which have the greater capacity for dissolution of calcite by releasing more

hydrogen ions in the root zone. It is hypothesized that oxidative stress tolerance and

rhizosphere acidification will positively correlate with salt tolerance of different plant

species. Acacia is an important genus with good salt tolerance potential and ability to

rehabilitate salt-affected lands.The main objective of this study is to investigate the

comparative oxidative stress tolerance and rhizosphere acidification potential of the two

acacia species under salinity stress.

4.2.2 Methodology

This experiment was conducted at the Institute of Soil and Environmental Sciences,

University of Agriculture Faisalabad. The healthy nursery of three week old plants of Acacia

ampliceps and Acacia nilotica was transferred in thermo pole sheets with holes in them

floating on 25 L capacity plastic tubs. After one week of transplantation, different salinity

levels (control, 100 and 200 mM) were developed step wise with NaCl. The pH of the

57  

experiment was monitored daily and maintained at 6.0 + 0.5 throughout the experiment by

the addition of NaOH or HCl. After four weeks of salinity development plants were

harvested and data of shoot fresh and dry weights, root fresh and dry weights, shoot and root

lengths were noted. Membrane stability index (MSI) and activities of antioxidant enzymes

like superoxide dismutase (SOD), peroxidases (POD) and catlase (CAT) were determined.

Root exudates were collected for the analysis of organic acids i.e citric acid, malic acid and

tartaric acid. Shoot and root ash alkalinity was also measured.

4.2.3 RESULTS

4.2.3.1 Shoot and root growth

Shoot fresh and dry weights were reduced in response to salinity in case of both the

species (Fig.4.2.1 & 4.2.2). The difference among species, treatments and their interaction

were significant for both these parameters. Under both salinity levels A. Ampliceps produced

more shoot fresh and dry weight than A. nilotica. The difference between both the species

was more at 200 mM than at 100 mM NaCl salinity. Root fresh and dry weights were also

decreased with increasing NaCl concentration in the growing medium (Fig.4.2.3 & 4.2.4).

The main effects as well as their interaction were significant for both root fresh and dry

weights. Decrease in root fresh and dry weight was more in the case of A. nilotica than A.

Ampliceps under both salinized treatments. The length of both shoot and root was also

decreased due to salinity stress (Fig.4.2.5 & 4.2.6). Although treatments and species have

significant effect, their interaction was found non significant for shoot and root lengths. A.

nilotica produced more root and shoot length than A. ampliceps under control and 100 mM

NaCl level. However in 200 mM NaCl treatment, both the species were statistically similar.

This showed that relative decrease in length of both root and shoot was more in the case of A.

nilotica than A. ampliceps.

4.2.3.2 Antioxidants and membrane stability index

The increasing salt concentration in the growing medium caused oxidative damage to

the cell membranes. The membrane stability index decreased in response to salinity which

indicated the oxidative damage and toxic effect of salt (Fig.4.2.10). The effect of salinity,

species and their interaction was significant. A. Ampliceps showed more index value and it

differed significantly from A. nilotica in both stress treatments. To mitigate the oxidative

58  

damage, the concentration of antioxidant enzymes was increased with increasing salt stress.

The activity of superoxide dismutase (SOD) increased in both species in response to salinity

and this increase was more for A. ampliceps than A. nilotica (Fig.4.2.7). Regarding this

enzyme the individual as well as interactive effects were found significant. Both the species

differed significantly in both salinity treatments regarding the activity of SOD. The activity

of catalase (CAT) also increased with salinity in both the species however the interaction of

species with treatment was found non-significant (Fig.4.2.8). The difference between both

the species was significant only at 200 mM NaCl concentration. Like other enzymes the

activity of peroxidase (POD) was also increased in response to salinity in both species

(Fig.4.2.9). At lower salinity level (100 mM NaCl) POD activity was significantly more in

case of A. nilotica. On the other hand at higher level of salinization (200 mM NaCl), A.

ampliceps showed more POD activity than A. nilotica.

4.2.3.3 Ash alkalinity and organic acids

Significantly increased quantity of ash alkalinity was found in response to salinity in

roots and shoots of both the species (Fig.4.2.17 & 4.2.18). The relative increase in ash

alkalinity was more in case of root than shoot. The interaction of species with treatments was

found non-significant in case of root ash alkalinity. Both the species differed significantly in

stress treatments for both shoot and root ash alkalinity values. Regarding organic acids, it

was observed that with increase in salinity the release of these acids was also enhanced. In

case of tartaric acid, the release was more in case of A. ampliceps, wether the samples were

taken after 14 or 28 days. (Fig.4.2.11 & 4.2.12). The individual as well as interactive effects

were found significant. Both the species differed significantly under both salinity levels. The

malic acid release was more in case A. nilotica after 14 days however both the species

differed only at 200 mM NaCl concentration (Fig.4.2.13). The analysis of exudates after 28

days showed that the release of malic acid was more in the case of A. ampliceps than A.

nilotica (Fig.4.2.14) and both the species differed significantly in both salinized treatments.

Regarding citric acid it was noticed that its release was more in the case of A. ampliceps in

case of both samplings (Fig.4.2.15 & 4.2.16). The individual as well as interactive effects

were found significant. The difference between both the species was non significant at 100

mM NaCl, however their difference was significant at 200 mM NaCl concentration for both

sampling times. The overall comparison of both acacia species showed that A. ampliceps

59  

released more organic acids as compared to A. nilotica. The release of tartaric acid was more

in both species in 2nd exudation. The malic acid was more released in 2nd exudation in case of

A. ampliceps whereas in case of A. nilotica it was less in 2nd exudation. The citric acid release

was decreased in 2nd exudation in case of both species.

 

60  

                            

                         Fig. 4.2.1 Effect of salinity on shoot fresh weight of acacia species                              

                          

                           Fig. 4.2.2 Effect of salinity on shoot dry weight of acacia species                              

a

b

d

a

c

e

0

0.5

1

1.5

2

2.5

3

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

Shoot freshweight (g plant‐1) 

a

b

c

a

c

d

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

Shoot dry weight (g plant‐1) 

61  

              

              Fig. 4.2.3 Effect of salinity on root fresh weight of acacia species                             

              Fig. 4.2.4 Effect of salinity on root dry weight of acacia species                              

a

a

c

a

b

d

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

Root fresh weight (g plant‐1) 

a

b

c

a

c

d

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

Root dry weight (g plant‐1) 

62  

                          

                           Fig. 4.2.5 Effect of salinity on shoot length of acacia species                              

  

                           Fig. 4.2.6 Effect of salinity on root length of acacia species                              

           

b

c

d

a

bc

d

0

5

10

15

20

25

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

Shootlength(cm) 

b

c

d

a

bc

d

0

2

4

6

8

10

12

14

16

18

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

Rootlength(cm) 

63  

 

              

Fig. 4.2.7 Effect of salinity on SOD activity of acacia species                              

              Fig. 4.2.8 Effect of salinity on CAT activity of acacia species                              

 

 

e

c

a

e

d

b

0

1

2

3

4

5

6

7

8

9

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

SOD activity (U m

g‐1protein)

d

c

a

d

c

b

0

10

20

30

40

50

60

70

80

90

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

CATactivity (U m

g‐1protein)

64  

 

                         

                        Fig. 4.2.9 Effect of salinity on POD activity of acacia species                              

   

                      Fig. 4.2.10 Effect of salinity on membrane stability index of acacia species                              

e

d

a

e

c

b

0

1

2

3

4

5

6

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

POD activity (U m

g‐1protein)

a

b

c

a

c

d

0

10

20

30

40

50

60

70

80

90

100

Control 100 mM NaCl 200 mM NaCl

A. ampliceps A. nilotica

Mem

brane stability index (%)

65  

Fig. 4.2.11 Effect of salinity on tartaric acid release (after 14 days) of acacia species                              

               Fig. 4.2.12 Effect of salinity on tartaric acid release (after 28 days) of acacia species                              

d

b

a

d

c

b

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Control 100 mM NaCl 200 mM NaCl

A.ampliceps A.nilotica

Tartaric acid (µmol h

 ‐1  g ‐1

  root fw

t)

e

c

a

e

d

b

0

0.01

0.02

0.03

0.04

0.05

0.06

Control 100 mM NaCl 200 mM NaCl

A.ampliceps A.nilotica

Tartaric acid (µmol h

 ‐1  g ‐1

  root fw

t)

66  

Fig. 4.2.13 Effect of salinity on malic acid release (after 14 days) of acacia species                              

Fig. 4.2.14 Effect of salinity on malic acid release (after 28 days) of acacia species                             

d

c

b

d

c

a

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Control 100 mM NaCl 200 mM NaCl

A.ampliceps A.nilotica

Malic acid (µmol h

 ‐1  g ‐1

  root fw

t)

d

b

a

d

c

b

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Control 100 mM NaCl 200 mM NaCl

A.ampliceps A.nilotica

Malic acid (µmol h

 ‐1  g ‐1

  root fw

t)

67  

      

Fig. 4.2.15 Effect of salinity on citric acid release (after 14 days) of acacia species                              

          

       Fig. 4.2.16 Effect of salinity on citric acid release (after 28 days) of acacia species                              

d

c

a

d

c

b

0

0.1

0.2

0.3

0.4

0.5

0.6

Control 100 mM NaCl 200 mM NaCl

A.ampliceps A.nilotica

Citric acid (µmol h

 ‐1  g ‐1

  root fw

t)

d

c

a

d

c

b

0

0.1

0.2

0.3

0.4

0.5

0.6

Control 100 mM NaCl 200 mM NaCl

A.ampliceps A.nilotica

Citric acid (µmol h

 ‐1  g ‐1

  root fw

t)

68  

Fig. 4.2.17 Effect of salinity on shoot ash alkalinity of acacia species  

                            

Fig. 4.2.18 Effect of salinity on root ash alkalinity of acacia species                              

d

b

a

d

c

b

0

50

100

150

200

250

300

350

Control 100 mM NaCl 200 mM NaCl

A.ampliceps A.nilotica

Shoot ash alkalinity(m

mol N

aOH/100gdwt)

d

c

a

dd

b

0

50

100

150

200

250

300

Control 100 mM NaCl 200 mM NaCl

A.ampliceps A.nilotica

Root ash alkalinity(m

mol N

aOH/100gdwt)

69  

4.2.4 Discussion  

The results of all the studied parameters indicated that there was a distinct difference

between both the species regarding their response to salinity. Fresh and dry weight of both

root and shoot were decreased in both species, although A.nilotica showed more decline in

these parameters than A. ampliceps. Similarly decrease in shoot and root growth have

previously been reported in acacia species including Acacia holosericea (Yokota, 2003),

Acacia senegal (Hardikar and Pandey, 2008) and Acacia ampliceps (Marcar and Crawford,

2011). The length of both shoot and root also decreased due to salinity stress. A. nilotica

produced more root and shoot length than A. ampliceps under control and 100 mM NaCl

level. However in 200 mM NaCl treatment, both the species did not differ significantly from

each other. This shows that relative decrease in length of both root and shoot was more in

case of A. nilotica than A. ampliceps. Decreased shoot length match the findings of Al-

Shaharani and Shetta (2011) for two acacia species, and Amirjani (2010) for Glycine max.

Reduction in plant growth under saline conditions is due to osmotic and ionic effects

(Greenway and Munns, 1980; Nawaz et al., 2010).

Membrane injury due to salinity is related to over production of the reactive oxygen

species (Shalata et al., 2001). The membrane stability index decreased in response to salinity

which indicated the oxidative damage and toxic effect of salt on the structure of the

membranes. A. ampliceps showed significantly more index value than A. nilotica in both

stress treatments which shows less membrane damage in this species. To mitigate the

oxidative damage, the concentration of antioxidant enzymes increases with increasing salt

stress. In the present study SOD activity has been increased under saline environment.

Increased activities of SOD under salt stress conditions was previously observed indicating

its role in salinity tolerance of various plant species (Khan and Panda, 2008; Mittal et al.,

2012; Hu et al., 2011). Chen et al. (2011) observed that salt-tolerant wheat cultivar exhibited

more SOD activity as compared to sensitive cultivar. These results match our findings as we

also found more SOD activity in A. ampliceps which is relatively more tolerant than A.

nilotica. The difference between both the species was less at 100 mM NaCl concentration but

at higher salinity level (200 mM NaCl) A. ampliceps showed significanly higher SOD

activity than A. nilotica. So it can be concluded that SOD have crucial role in modulating the

70  

salt tolerance of the two acacia species. SOD dismutates superoxide radicals to H2O2 and O2

in the cytosol, mitochondria and chloroplast.

Catalase (CAT) is an important enzyme which converts H2O2 to H2O and O2 (Chen et

al., 2011). The activity of catalase was increased in both the species with more increase in

case of A. ampliceps than A. nilotica under stress conditions. The difference between both the

species was non-significant at 100 mM NaCl, however they differed significantly at 200 mM

NaCl concentration. The increased activity of catalase seems to be responsible for the

detoxification of H2O2 more in case of A. ampliceps than A. nilotica. Morais et al. (2012)

conducted a three months experiment under controlled conditions with four concentrations of

NaCl (0, 50, 100 and 200 mM) and found that A. longifolia was better adapted to saline

conditions than Ulex europaeus, owing to its better anti-oxidants activities particularly of

catalase. Other workers also found increased acitivties of this enzyme in response to salinity

like Patel and Saraf (2013), Sekmen et al. (2012) and Mittal et al. (2012) in J. curcas L., G.

oblanceolata and B. juncea respectively.

Peroxidise (POD) is also involved in the detoxification of H2O2. Both the species

showed enhancement in the activity of this antioxidant in response to salinity. At 100 mM

NaCl concentration, the activity of POD was significantly more in the case of A. nilotica but

at 200 mM NaCl concentration, A. ampliceps showed more POD activity than A. nilotica.

This shows that at higher salinity level A. ampliceps was more involved in the detoxification

of H2O2 than A. nilotica. Increased activity of POD was also found by Wang et al. (2009).

Zhang et al. (2013) observed that the activities of POD in the stems of B. papyrifera were

significantly increased under salinity stress whereas in case of leaves and roots it was

decreased. Morais et al. (2012) observed no change in the POD activity in A. longifolia and

Ulex europaeus in response to salinity. These three enzymes including SOD, CAT and POD

are thought to be the main components of plants oxidatitve stress tolerance. The salt induced

hyper production of these three enzymes in A. ampliceps is positively correlated with its alt

tolerance.

The ash alkalinity increased in both shoot and root in both the species however the

relative increase in ash alkalinity was more in the case of root than shoot. The comparison of

both species indicated that ash alkalinity was more in the case of A. ampliceps than A.

71  

nilotica. Ash alkalinity is gauge of how many hydrogen ions are released from the plants

(Jungk, 1968). More ash alkalinity in case of A. ampliceps indicated the more acidification

potential of this species as compared to other. The extrusion of H+ ions from roots of various

plants has been found previously under various stresses. For example Tang et al. (2001)

observed that roots of Medicago truncatula L. released more protons under P deficient

conditions. Legume plants dependent on nitrogen fixation uptake more cations than anions

and resultantly release more H+ ions in their rhizosphere (Raven et al., 1990; Tang and

Rengel, 2001). However there is a great difference within species regarding proton release.

With increasing the levels of salinity the release of organic anions i.e citric acid, malic acid

and tartaric acid was also increased. The release of tartaric acid and citric acid was more in

the case of A. ampliceps for both the sampling times. The malic acid release was more in the

case A. nilotica after 14 days however after 28 days the release was more in the case of A.

ampliceps than A. nilotica. The findigs of Chen and Lin, (2010) are in accordance with our

results. They found enhanced concentration of various organic acids in tomato roots in

response to NaCl stress and relate them with salt tolerance of tomato. Organic acids release is

considered as one of the crop stress tolerance mechanism (Larsen, 1998). Some special types

of proteins are involved in organic acid release under stress conditions (Ryan et al., 1995).

According to Qadir et al. (2005) rhizosphere acidification may be helpful in reducing soil

sodicity by dissolving native lime and hence positively correlate with salinity- tolerance of

different plant species. We also observed that A. ampliceps had more exudation of three

studied acids and had more ash alkalinity; hence it is better tolerant to salinity as compared to

A. nilotica.

4.2.5 Conclusion

It can be inferred from this study that salinity caused oxidative damage in both

species and A. ampliceps responded in a better way to this damage due to more production of

antioxidant enzymes. On the other hand in response to increasing salinity levels both

leguminous species caused rhizoshere acidification A. ampliceps had more exudation of

organic acids and ash alkalinity; hence it is better tolerant to salinity and have more potential

for reclamation of salt affected soils as compared to A. nilotica.

72  

4.3 Study-3: Effect of water stress on the response of Acacia ampliceps and Acacia

nilotica to salinity.

4.3.1 Introduction

Drought or water deficit is a serious abiotic stress that negatively affects the growth

and productivity of crops (Zlatev and Lidon, 2012). During plant growth, drought may be

long lasting in those climatic areas where water availability is low and erratic as a result of

changes in weather situation (Harb et al., 2010). Availability of water is very crucial for

fulfilling the needs of present and future human beings and its scarcity are increasing day by

day due to climatic changes (Rosegrant and Cline, 2003). As a result of water deficit the

yield losses up to 50 % have been noticed for many crops (Bray et al., 2000; Wang et al.,

2003). The impact of any environmental stress on the plants is estimated by the duration and

strength of that particular stress factor and the genetic ability of the target plant to cope that

stress (Zlatev and Lidon, 2012). Drought affects plants in many ways and at various levels of

their growth and development (Yordanov et al., 2000; Wentworth et al., 2006).

The dehydration as a result of water deficit is thought to be responsible for many

changes in water relations, biochemical and physiological processes and structure of

membranes (Tuba et al., 1996; Sarafis, 1998; Yordanov et al., 2003). The availability of

water determines the allocation pattern of photosynthates to different organs. The relative

allocation of photosynthates to roots is increased under mild water shortage conditions (Gorai

et al., 2010). In this way the water uptake capacity of the plants is enhanced. Ramos et al.

(1999) concluded that fresh plant mass is more reduced than dry biomass under water deficit

conditions which is due to disturbed water relations.

Plants have various strategies to cope with water deficit like escape, tolerance, and

avoidance of tissue and cell dehydration (Turner, 1986). Avoidance includes rapid

phenological development, increased stomata and cuticular resistance, changes in leaf area,

orientation and many other anatomical features (Jones and Corlett, 1992). Osmotic

adjustment is the main strategy for maintaining cell turgor under low water potential

conditions, which enables the plants to take up water and to maintain their metabolic activity

and growth (Martiınez et al., 2004). Drought tolerant plants try to maintain high relative

73  

water content than sensitive ones by accumulating different osmotica which lower their water

potential.

The capacity to maintain high relative water content (RWC) values under drought

was observed in drought tolerant bean cultivars (Zlatev, 2005) and in Astragalus

gombiformis Pom. and Medicago sativa L. (Gorai et al., 2010). Water deficit caused a

reduction in chlorophyll content in chickpea cultivars (Mafakheri et al., 2010) and in

sunflower varieties (Manivannan et al., 2007). This reduction is mainly attributed to damage

to chloroplasts caused by active oxygen species (Smirnoff, 1995). The leguminous plants

have been observed to show low nodulation and nitrogen fixation capacity under drought

stress (Pimratch et al., 2008). In nodules water shortage increases soluble sugars and

decreases the solute potential in cells maintaining the turgor at a low water potential (Furlan

et al., 2012). Acacia tree, like other xerophytic plants has the capability to defy drought and

survive under arid environments by conserving water. This can be done either by reducing

water loss or by escalating water absorption by various morphological and physiological

adaptations (Aref and El-Juhany, 1999). Minimizing the exposed leaf surface area leads to

limited water loss via transpiration from the plant. Reduction in leaf area is achieved by

inhibiting leaf initiation (Ibrahim, 1995) or reducing leaf size (Ibrahim et al., 1998) or

enhancing leaf senescence and consequently leaf shedding (Begg, 1980). Roots show a high

degree of morphological plasticity that enables them to cope with shortage of water

(Kummerow, 1980). Production of thin and deep penetrating roots by the water-stressed

plants is considered an important mechanism for more water uptake (Aref and El-Juhany,

1999).

To cope with water scarcities and to fulfill the increasing water demand for crop

husbandry, the use of low quality water will become inevitable around the globe. A lot of

studies have been done with either water stress or salt stress separately, probably for the sake

of convenience. However, the most realistic approach would be the use of salinity and water

stress together, as this would simulate conditions that are very much similar to the field under

arid conditions (Ramoliya and Pandey, 2002). The effect of higher salt concentrations in soils

on the plant growth is basically through soil solution. Hence, it is supposed that soils having

low moisture content may affect plant growth more than wet soils. Also, occurrence of

frequent droughts is almost a regular phenomenon in arid saline regions. Ultimately, effect of

74  

salts on the plant growth should be studied under wet and dry soil conditions. The

information regarding plant growth and development under such natural environments could

be used as a mean for identification of plant species for the afforestation of arid and saline

ecosystems.

4.3.2 Methodology

A pot culture study was conducted in the wire house of the Institute of Soil and

Environmental Sciences, University of Agriculture Faisalabad, to investigate the effect of

salinity and water stress on the two acacia species. Normal soil was collected from an

agricultural field and analyzed for physicochemical properties. Soil was passed through 2

mm sieve and filled in pots @ of 12 kg per pot. The required salinity levels (control, 10, 20

and 30 dS m-1) were developed in the respective treatment pots by adding calculated amount

of NaCl before filling the soil in the pots. Healthy three month old nursery plants were

transplanted in these pots, one plant in each pot. After the establishment of the plants in the

initial two weeks, water stress was applied during the period of the experiment. The plants

under water stress were not irrigated for fifteen days at an alternate interval of fifteen days

(i.e. fifteen days water stress and fifteen days normal irrigation). The plants under no water

stress treatment pots or during the days of no water stress in water stress treatment pots were

irrigated regularly so as to keep the soil moisture levlel to 70% of the soil water holding

capacity. Plant growth data like plant height, stem diameter, number of branches and number

of leaves were taken after four months. Chlorophyll contents were determined before

harvesting. After harvesting, relative water contents (RWC) were determined. Nodule

number and their fresh and dry weights were also determined. The plants were separated into

root, stem and leaves after harvesting. Ionic concentrations of Na+, K+ and Cl- were

determined in these parts. Postharvest soil samples were taken from each pot and analyzed

for different properties including pHs, ECe and SAR.

4.3.3 RESULTS

4.3.3.1 Shoot and root growth

Salinity caused significant reduction in plant height and stem diameter of both the

species however when salinity was combined with water stress, more reduction was observed

75  

as compared to individual stress for both the growth parameters (Fig.4.3.1&4.3.2). The effect

of salinity, water level and species was significant for both these parameters. The interaction

of species with both treatments was also found significant. A. nilotica produced significantly

more plant height in all the treatments except that of 30 dS m-1. Number of leaves and

branches were also more in the case of A. nilotica than A. ampliceps in all the treatments

except for branches at 30 dS m-1 (Fig.4.3.3 & 4.3.4). Regarding both these parameters the

main effects were significant and interaction of species with salinity was also significant for

number of leaves. Shoot fresh and dry weigh was reduced due to salinity and when salinity

was combined with water stress, the reduction was more as compared to salinity alone

(Fig.4.3.5 & 4.3.6). The effect of both treatments and the interaction of species with water

stress was significant for shoot fresh weight however the species effect was non-significant.

In case of shoot dry weight again the species effect was non-significant however the

treatment effect and the interaction of both treatments with species was significant. The

comparison of both the species indicated that A. ampliceps produced more shoot fresh and

dry weight under salinity stress. However under water stress and when water stress was

combined with 10 dS m-1 salinity, A. nilotica produced more fresh and dry weight than A.

ampliceps. When water stress was combined with 20 and 30 dS m-1 salinity both the species

did not differ significantly from each other.

Root growth was also reduced significantly due to salinity and water stress (Fig.4.3.7,

8 & 9). The species effect was non-significant however the effect of both treatments and the

interaction of species with water stress was significant for root fresh and dry weights. The

effect of both treatments and species as well as the interaction of both treatments with species

was significant for root length. The comparison of both the species indicated that A.

ampliceps produced more root fresh and dry weight under salinity stress. However under

water stress and when water stress was combined with 10 dS m-1 salinity, A. nilotica

produced more fresh and dry weight than A. ampliceps. When water stress was combined

with 20 and 30 dS m-1 salinity both the species did not differ significantly from each other. A.

nilotica produced significantly more root length in all the treatments except that of 30 dS m-1.

In this treatment both the species did not differ significantly from each other.

76  

4.3.3.2 Nodule formation

Number of nodules and their fresh and dry weights were decreased as a result of

salinity and water stress (Fig. 4.3.10, 11 &12). The effect of both treatments and the

interaction of species with both treatments was significant for nodule numbers. In case of

nodule fresh and dry weights, the species effect was non-significant however the treatments

effect and the interaction of species with water stress was significant. The comparison of

both the species indicates that A. ampliceps produed more number and weight of nodules

under salinity stress alone. However under water stress and when water stress was combined

with 10 dS m-1 salinity, A. nilotica showed more nodulation as compared to A. ampliceps.

When water stress was combined with 20 and 30 dS m-1 salinity both the species had similar

nodulation behavior.

4.3.3.3 Physiological attributes

Chlorophyll and relative water contents were decreased in response to both stresses

(Fig. 4.3.13&4.3.14), however the interactive effect was more detrimental than the either

individual stress. The effect of species, water stress, salinity and the interaction of species

with water stress was found significant for chlorophyll contents. In case of relative water

content, the main effects and the interaction of species with both treatments was significant.

The comparison of both the species indicates that A. ampliceps produced more chlorophyll

and relative water contents under salinity stress alone. However under water stress and when

water stress was combined with 10 dS m-1 salinity, A. nilotica showed more values of both

these parameters. When water stress was combined with 20 and 30 dS m-1 salinity both the

species did not differ significantly from each other regarding both these attributes.

4.3.3.4 Ionic composition

Salinization of the growing medium significantly increased the Na+ concentration in

the leaf, stem and root of both species. Water stress in combination with salinity, further

increases this concentration in both the species. However water stress alone had no effect on

Na+ concentrations (Fig. 4.3.15, 16 & 17). The effect of species, water stress and salinity was

found significant for all the three parts however the interaction of salinity and water stress

was significant for stem and root only. The comparison of both species in various treatments

77  

showed that A. ampliceps accumulated significantly lower Na+ as compared to A. nilotica in

leaf, stem and root.

The concentration of K+ decreased significantly at each increasing level of

salinization Water stress in combination with salinity further decreased this concentration in

both the species. However water stress alone had no effect on K+ concentrations (Fig.4.3.18,

19 &20).The effect of species, water stress and salinity was found significant for all the three

parts however the interaction of salinity and water stress was significant for root only. The

comparison of both species in various treatments showed that A. ampliceps accumulated

significantly higher K+ as compared to A. nilotica in leaf, stem and root in the treatments

with significant difference between the species.

Salinity significantly increased the Cl- concentration in the leaf, stem and root of both

the species. Water stress in combination with salinity, further increases this concentration in

both the species. However water stress alone had no effect on Cl- concentrations (Fig. 4.3.21,

22 & 23). The main effects as well as the interaction of salinity and water stress were found

significant for all the three parts. The comparison of both species in various treatments

showed A. nilotica accumulated significantly higher Cl- as compared to A. ampliceps in leaf,

stem and root.

4.3.3.5 Soil properties

Normal soil was taken and the required salinity levels were developed in the

respective treatment pots by adding calculated amount of NaCl. Before the transplantation of

the plants in the pots soil samples were taken and analyzed for pHs, ECe and SAR. After the

harvesting of the plants again soil was analyzed for the above mentioned properties (Table

4.3.1-3). It was observed that there was slight reduction in these soil parameters which

indicated the phytoremedial potential of both the species.

 

78  

 

Fig. 4.3.1 Effect of salinity and water stress on plant height of acacia species. WS: water stress                            

 

Fig. 4.3.2 Effect of salinity and water stress on stem diameter of acacia species. WS: water stress                                      

 

 

 

cd

d‐f

ghhi

fgf‐h

ii

j

a

bc

e‐g

i

ab

c‐e

fg

i

0

20

40

60

80

100

120

140

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.niloticaPlant height (cm)

b

cdde

gh

effg

gh

i

a

b

d

h

bc

d

ef

hi

0

0.2

0.4

0.6

0.8

1

1.2

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Stem

 diameter (cm

)

79  

 

Fig. 4.3.3 Effect of salinity and water stress on number of leaves of acacia species. WS: water stress                             

 

 

 

Fig. 4.3.4 Effect of salinity and water stress on number of branches of acacia species. WS: water 

stress                            

eef e‐g

fg

ef ef

fgg

a

b

c

cd

ab

b

cd

d

0

50

100

150

200

250

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.niloticaNumber of leaves plant‐1

bc

cdd

e

cd

de

e

f

a

ab

c

e

ab

bc

cd

e

0

2

4

6

8

10

12

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Number of branches plant‐1

80  

 

Fig. 4.3.5 Effect of salinity and water stress on shoot fresh weight of acacia species. WS: water stress                            

 

Fig. 4.3.6 Effect of salinity and water stress on shoot dry weight of acacia species. WS: water stress                            

a

b

d

f

de

f

h

a

c

e

g

bcd

f

h

0

10

20

30

40

50

60

70

80

90

100

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Shoot freshweight (g plant‐1) 

a

b

de

gh

deef

h

i

a

cd

fg

i

bc

de

gh

i

0

2

4

6

8

10

12

14

16

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Shoot dry

weight (g plant‐1) 

81  

 

Fig. 4.3.7 Effect of salinity and water stress on root fresh weight of acacia species. WS: water stress                            

 

 

Fig. 4.3.8 Effect of salinity and water stress on root dry weight of acacia species. WS: water stress                            

abc

c‐e

h

e

g

h

i

ab

de

fg

i

cd

ef

gh

i

0

5

10

15

20

25

30

35

40

45

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Root freshweight (g plant‐1) 

a

bccd

hi

e‐ggh

i

j

ab

c‐e

fg

j

cd

d‐f

hi

j

0

1

2

3

4

5

6

7

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Rootdry weight (g plant‐1) 

82  

 

Fig. 4.3.9 Effect of salinity and water stress on root length of acacia species. WS: water stress  

                          

 

Fig. 4.3.10 Effect of salinity and water stress on number of nodules of acacia species. WS: water 

stress                             

 

cd

d‐f

fg

gh

efe‐g

h

i

a

bc

de

h

ab

c

ef

h

0

10

20

30

40

50

60

70

80

90

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Root length (cm

)

a

b

de

f‐h

cd

ef

hi

k

a

bc

fg

ij

b

cd

gh

jk

0

5

10

15

20

25

30

35

40

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Number of nodulesplant‐1

83  

 

 

Fig. 4.3.11 Effect of salinity and water stress on nodule fresh weight of acacia species. WS: water 

stress                             

 

Fig. 4.3.12 Effect of salinity and water stress on nodule dry weight of acacia species. WS: water 

stress                            

a

bc

ef

gh

cd

f

h

j

a

d

g

i

b

de

h

ij

0

50

100

150

200

250

300

350

400

450

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Nodule fresh weight ( mg plant‐1)

a

bc

de

g

cd

ef

g

h

a

d

f

h

b

de

g

h

0

10

20

30

40

50

60

70

80

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Nodule dry weight (m

g plant‐1)

84  

 

 Fig. 4.3.13 Effect of salinity and water stress on chlorophyll contents of acacia species. WS: water 

stress                            

 

Fig. 4.3.14 Effect of salinity and water stress on relative water contents of acacia species. WS: water 

stress                             

a

c

d

g

d

ef

g

h

b

d

fg

h

c

de

fg

h

0

10

20

30

40

50

60

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.niloticaChlorophyllcontents (SPAD)

aab

ef

gh

de

fg

ij

abcd

h

ij

bc

de

i

j

0

10

20

30

40

50

60

70

80

90

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Relative water content (%

)

85  

 

Fig. 4.3.15 Effect of salinity and water stress on leaf Na+ concentration of acacia species. WS: water 

stress                             

 

Fig. 4.3.16 Effect of salinity and water stress on stem Na+ concentration of acacia species . WS: water 

stress                           

h

fg

d

c

h

ef

d

b

h

ef

c

a

gh

e

c

a

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.niloticaLeaf Na 

+concentration(m

mol  g‐1dwt)

h

g

e

c

h

fg

d

b

h

f

d

ab

h

ef

cd

a

0

0.5

1

1.5

2

2.5

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Stem

 Na+

  concentration (mmol  g‐1dwt)

86  

 

 Fig. 4.3.17 Effect of salinity and water stress on root Na+ concentration of acacia species. WS: water 

stress                         

Fig. 4.3.18 Effect of salinity and water stress on leaf K+ concentration of acacia species. WS: water 

stress                            

i

h

f

c

i

g

d

b

i

h

e

ab

i

f

cd

a

0

0.5

1

1.5

2

2.5

3

3.5

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Root Na+

concentration (mmol  g‐1dwt)

a

b

ef

i

a

cd

fg

j

a

c

gh

jk

a

de

h

k

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Leaf K

+ concentration (mmol  g‐1dwt)

87  

 

 

Fig. 4.3.19 Effect of salinity and water stress on stem K+ concentration of acacia species. WS: water 

stress                             

 

Fig. 4.3.20 Effect of salinity and water stress on root K+ concentration of acacia species. WS: water 

stress                           

a

b‐d

e

fg

b

de

fg

ij

ab

c‐e

f

hi

bc

e

gh

j

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Stem

K+concentration (mmol  g‐1dwt)

a

bc

d

e

ab

d

ef

gh

ab

cd

e

fg

ab

d

f

h

0

0.1

0.2

0.3

0.4

0.5

0.6

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

RootK+concentration (mmol  g‐1dwt)

88  

 

 

Fig. 4.3.21 Effect of salinity and water stress on leaf Cl‐ concentration of acacia species. WS: water 

stress                        

 

Fig. 4.3.22 Effect of salinity and water stress on stem Cl‐ concentration of acacia species. WS: water 

stress                            

i

h

f

e

i

gh

d

b

i

gh

e

cd

i

fg

c

a

0

0.5

1

1.5

2

2.5

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Leaf Cl‐concentration (mmol  g‐1dwt)

h

g

e

d

h

ef

d

bc

h

fg

d

b

h

ef

cd

a

0

0.5

1

1.5

2

2.5

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Stem

Cl‐concentration (mmol  g‐1dwt)

89  

 

 

 

Fig. 4.3.23 Effect of salinity and water stress on root Cl‐ concentration of acacia species. WS: water 

stress                             

Table 4.3.1 Effect of acacia species on soil pHs. WS: water stress

Treatments  Initial values  A. ampliceps  A. nilotica 

Control 7.44±0.04  7.4±0.06  7.42±1.65 

10 dS/ m 7.85±0.05  7.8±2.60  7.83±0.09 

20 dS/ m 8.34±1.03  8.25±1.73  8.31±1.85 

30 dS/ m 8.55±1.02  8.45±2.80  8.52±1.90 

WS 7.44±0.06  7.42±2.45  7.4±1.64 

10 dS/ m+WS 7.86±0.02  7.83±1.59  7.85±0.08 

20 dS/ m+WS 8.33±1.01  8.29±2.75  8.31±1.86 

30 dS/ m+WS 8.55±1.03  8.51±1.82  8.53±2.89 

g

f

e

c

g

e

d

b

g

f

d

b

g

e

c

a

0

0.5

1

1.5

2

2.5

3

3.5

4

Control 10 dS/ m 20 dS/ m 30 dS/ m WS 10 dS/ m+WS 20 dS/ m+WS 30 dS/ m+WS

A.ampliceps A.nilotica

Root Cl‐concentration (mmol  g‐1dwt)

90  

Table 4.3.2 Effect of acacia species on soil electrical conductivity (ECe) 

Treatments  Initial values  A.ampliceps  A nilotica 

Control  2.31±0.12  2.24±0.68  2.28±0.52 

10 dS/ m  10±1.29  8.92±2.73  9.22±2.13 

20 dS/ m  20±2.58  18.0±5.88  18.7±3.75 

30 dS/ m  30±5.15  28.81±8.60  29.2±6.28 

WS  2.31±0.18  2.27±0.66  2.25±0.49 

10 dS/ m+WS  10±1.16  9.45±2.50  9.48±2.06 

20 dS/ m+WS  20±3.57  18.17±6.16  18.96±4.77 

30 dS/ m+WS  30±4.14  28.07±6.71  28.43±7.00 

Table 4.3.3 Effect of acacia species on soil sodium adsorption ratio (SAR) 

Treatments  Initial values  A ampliceps  A.nilotica 

Control 6.43±1.41  5.33±1.59  5.47±1.17 

10 dS/ m 19.5±3.82  18.2±5.64  18.6±4.26 

20 dS/ m 37.43±1.64  33.2±10.46  35.1±7.10 

30 dS/ m 50.1±4.41  47.7±15.5  48.1±9.59 

WS 6.43±1.40  5.56±1.58  5.37±1.15 

10 dS/ m+WS 19.5±2.41  18.1±6.19  18.4±4.10 

20 dS/ m+WS 37.43±5.81  35.1±11.57  35.9±7.22 

30 dS/ m+WS 50.1±7.84  46.9±15.5  47.8±9.89 

91  

4.3.4 Discussion

Increasing levels of salt in the growing medium caused significant reduction in plant

height, stem diameter, number of leaves and branches. However when salinity was combined

with water stress, more reduction was observed as compared to individual stress for these

growth parameters. The comparison of both the species demonstrated that the effect of

salinity on these parameters was more in the case of A. nilotica. On the other hand under

water stress alone and in combination with salinity levels, the reduction was more in the case

of A. ampliceps. In the past some other workers also had similar observations like El Atta et

al. (2012) and EI-Juhany and Aref (2005). Shoot and root fresh and dry weights were reduced

due to salinity and when salinity was combined with water stress, the reduction was more in

the later case. The comparison of both the species indicated that A. ampliceps produced more

shoot and root fresh and dry weights under salinity stress. However under water stress and

when water stress was combined with salinity, A. nilotica produced more weight of both

shoot and root than A. ampliceps. Such decline in shoot and root weight in response to

salinity and water stress was also observed by Ramoliya and Pandey (2002) EI-Juhany and

Aref (2005) and El Atta et al. (2012).

Many features of plant growth are vulnerable to water stress at cellular (Hsiao, 1973) as

well as whole-plant (Kramer and Kozlowski, 1979) levels. Large reduction in shoot weight was

due to simultaneous reduction in stem height, diameter, number of leaves and branches.

Kozlowski (1982) reported that water deficit reduces leaf area by inhibiting initiation of leaves

as well as their succeeding enlargement. The reduction in root weight was less as compared

to shoot weight which indicated the morphological flexibility of root systems that facilitate

them to survive under variable soil conditions (Kummerow, 1980). A. nilotica produced more

root length than A. ampliceps. These results indicate that this species has an inclination for

rapid root extension which confirms the survival of this species in severe dry habitats

(Etherington, 1987; Pandey and Thakarar, 1997). Shoot and root growth of both the species

was also reduced due to saline treatments which could be the result of osmotic effect

(causing water deficit), ion toxicity and nutrional imbalance (Garg and Gupta, 1997). These

harmful effects are operative on the cellular as well as on higher levels of organization and

have impact on all features of plant metabolism (Kramer, 1983).

92  

Nodulation was decreased in response to both salinity and water stress. The

comparison of both the species indicates that A. ampliceps produed more number and weight

of nodules under salinity stress alone. However under water stress and when water stress was

combined with lower level of salinity i.e. 10 dS m-1, A. nilotica showed more nodulation as

compared to A. ampliceps. When water stress was combined with higher salinity levels both

the species had similar nodulation behavior. The reduction in nodulation and nitrogen

fixation capacity of leguminous plants has been observed under drought stress (Pimratch et

al., 2008) as well as salinity (Al-shaharani and Shetta, 2011).

Chlorophyll and relative water contents were decreased in response to both stresses

however the interactive effect was more detrimental than the either individual stress. Salinity

alone was more detrimental for A. nilotica and water stress was more harmful for A.

ampliceps In the past water deficit caused a reduction in chlorophyll content in chickpea

(Mafakheri et al., 2010) and sunflower (Manivannan et al., 2007). This reduction is mainly

endorsed to damage to chloroplasts caused by reactive oxygen species (Smirnoff, 1995).

According to Ramoliya and Pandey (2002) high relative water content of leaves is thought to

be an adaptation to xeric habitat. The higher relative water content values in response to

drought stress were noticed in drought tolerant bean (Zlatev, 2005) and in Astragalus

gombiformis Pom. and Edicago sativa L. (Gorai et al., 2010). Our results are in confirmation

with these observations as we also found that A. nilotica which is relatively drought tolerant

showed higher relative water content than A. ampliceps which is more sensitive to water

stress.

Salinity significantly increased the Na+ and Cl- concentration in the leaf, stem and root

of both species. Water stress in combination with salinity, further increased this

concentration in both the species. A. ampliceps accumulated significantly lower Na+ and Cl-

as compared to A. nilotica in leaf, stem and root. Similar results regarding increased ionic

concentration in response to salinity was also observed by Marcar et al. (1991) and Khalil et

al. (2012). The buildup of poisonous ions in plant tissues is thought to be the major factor of

decline in growth under salinity stress (Muscolo et al., 2003). The comparison of three

components i.e. root, stem and leaf showed that the concentration of these ions was

maximum in root followed by stem and leaf. This type of ion accumulation is a salt tolerant

93  

behavior known as ion exclusion and it is positively related with salt tolerance of various

plant species.

The concentration of K+ decreased significantly at each increasing level of

salinization and water stress in combination with salinity further decreased this concentration

in both the species. A. ampliceps accumulated significantly higher K+ as compared to A.

nilotica in leaf, stem and root. Decreased K+ uptake in response to salinity (Khalil et al.,

2012; Marcar et al., 1991) and water stress was previously also observed. The K+ has a key

role in salt tolerance and uptake of K+ is decreased by Na+ (Fox and Guerinot, 1998). The

reduction of K+ concentration was found in all the three components which indicated that

Na+ repressed the uptake of K+. The higher concentration of Na+ in case of A. nilotica

indicated that ion exclusion was poorly operative in this species so it had low K+

concentration as compared to A. ampliceps.

Pre and post-harvest soil analysis indicated that there was some reduction in the soil

parameters i.e. pHs, ECe and SAR which indicated the phytoremedial potential of both the

species. However A. ampliceps caused more amelioration as compared to A. nilotica.

4.3.5 Conclusion

This study explored the genetic potential of both acacia species for two very

important a biotic stresses i.e. salinity and water shortage. We observed that A. ampliceps has

more tolerance for salinity stress than A. nilotica. On the other hand the former species was

relatively sensitive to water stress then the later one. So summarizing the results it can be

recommended that if we are facing the dual problem of salinity and water shortage than we

should grow A. nilotica for the rehabilitation of marginal lands.

94  

4.4 Study-4: Comparative role of Acacia ampliceps and Acacia nilotica in the

phytoremediation of the salt affected soil

4.4.1 Introduction

Degradation of soil due to salinity and sodicity is a key environmental issue which is

effecting agricultural production and sustainability, predominantly in arid and semiarid parts

of the world (Pitman and Lauchli, 2002; Qadir et al., 2006). The sodicity causes slaking,

swelling and dispersion of clay that may cause surface crusting (Quirk, 2001). Therefore

water permeability, aeration, water holding capacity, penetration of roots, tillage and sowing

operations in these soils are disturbed. On the other hand, imbalanced availability of plant

nutrients in both saline and sodic soils affects plant growth (Naidu and Rengasamy, 1993;

Qadir and Schubert, 2002). These types of changes affect plant root, soil microbes and result

in the reduction of the crop yield and productivity.

The use of salt-affected soils and waters is becoming a necessity because of shrinking

normal soil and water resources and increasing population. The reclamation of the salt-

affected soils through engineering or chemical approach is very expensive. Many salt

affected soils are not even treatable and this approach is also not sustainable over long time

(Qureshi and Barret- Lennard, 1998).

Since last hundred years, many different techniques like chemical treatments, tillage

operations, crop based interventions, water-related methods, and electrical currents have been

employed to reclaim the salt affected soils. Among all these, chemical amendments were

used most effectively (Oster et al., 1999). Nevertheless, in the present times, the most

effective and low cost reclamation approach is the phytoremediation (Ghaly, 2002; Ilyas et

al., 1993; Robbins, 1986a). Using this approach the native source of calcium like calcite is

made soluble with the root action and the calcium released is used to replace sodium on the

exchange sites. It is very cheap as compared to chemical reclamation approach, which is out

of the range of the poor farming community of the developing countries (Qadir and Oster,

2004).

The response of various plants for phytoremediation of sodic and saline-sodic soils

varies with species. The plant species which produce more biomass and at the same time

95  

have higher salinity and sodicity tolerance potential are considered better for reclamation

purpose (Kaur et al., 2002; Qadir et al., 2002).

The effectiveness of chemical amendments is restricted to only the zone of their

application (Ilyas et al., 1993; Qadir et al., 1996a). On the other hand phytoremediation was

found to be effective throughout the whole root zone depending on the plant type, root

structure and rooting depth (Akhter et al., 2003; Ilyas et al., 1993; Robbins, 1986b). Deep-

rooted crops having tap root system like alfalfa have shown more promising results as

compared to other plants (Ilyas et al., 1993). After biological reclamation the availability of

the nutrients is also increased. Singh and Gill (1990) observed that there was a significant

decrease in pH and increase in organic matter content, available P and K of surface 0.15 m

soil reclaimed with different trees like P. juliflora, Acacia nilotica, Eucalyptus, Albizia

lebbeck and Terminalia arjuna. It has been observed that raising salt tolerant woody plants

along with shrubs and grasses is not only cheaper but also profitable and long lasting practice

(Qureshi et al., 1993). Many tree and grass species have the ability not only to reclaim salt

affected soils but also have potential economic value (Aslam, 2003). This experiment was

conducted to evaluate the comparative growth performance of both acacia species and their

effect on the physicochemical properties of the saline sodic soil which was receiving salty

water.

4.4.2 Methodology

This field experiment was conducted to investigate the role of Acacia ampliceps and

Acacia nilotica for the reclamation of salt affected soil at Proka farm, University of

Agriculture Faisalabad. Three months old healthy nursery of the selected plant species was

transferred in the pits made in the field for this purpose. Plants were irrigated with saline

water. Plant growth data like plant height, stem circumference, number of branches and

number of leaves were taken after every six months for two years. For ionic composition two

types of leaves (older and younger) were taken and used for ionic analysis. Soil samples

taken from different depths were analyzed for various properties like pHs, ECe and SAR.

The soil organic matter content and the change in the physical properties of soil like bulk

density and infiltration rate were also determined.

96  

4.4.3 Results

4.4.3.1 Plant growth

The plant growth parameters including plant height, stem circumference, no. of leaves

and no. of branches were determined after every six months. The plants of both acacia

species were grouped in to two categories i. e. good and poor, depending on their health. The

plant types differed significantly regarding growth parameters (Fig. 4.4.1, 2, 3&4). The plant

height and stem circumference was found maximum in the good plants of A. nilotica after 24

months of transplantation. These two parameters were minimum in the poor plants of the

same species. Number of branches was initially more in case of good plants of A. nilotica but

after one year the more spreading nature of A. ampliceps resulted in the production of more

branches as compared to the A. nilotica. Number of leaves was more in case of A. nilotica as

compared to A. amplpiceps throughout the growth period for both good and poor plants.

4.4.3.2 Ionic composition

There were significant differences among plant type and leaf type regarding Na+, K+

and Cl- concentrations (Fig. 4.4.6-10). A comparison of the leaf ionic composition showed a

significantly higher Na+ and Cl- concentration in the older leaves of poor plants of A.

nilotica, whereas the young leaves of good plants of A. ampliceps had the minimum

concentration of these ions. On the other hand, the K+ concentration was maximum in the

young leaves of A. ampliceps good plants and it was minimum in the older leaves of A.

nilotica.

4.4.3.3 Chemical properties of the soil

Soil analysis of the field further showed that it was a saline sodic field owing to high

values of pHs, ECe and SAR. Within each plot there was difference regarding these

parameters which was indicated by the difference in the growth of the plants. All the plants

were of same size at the time of transplantation but later on some grow more fastly as

compared to others. The growing trees have an ameliorative effect on these soil parameters.

With time the pHs, ECe and SAR slowly decreased and this reduction was more in the case of

good plants as compared to poors (Fig. 4.4.11-16). The maximum reduction in pHs, ECe and

SAR was observed in case of good plants of A. ampliceps. This reduction was minmum in

97  

the case of poor plants of A. nilotica. The comparison of A. nilotica and A. ampliceps

indicated that A. ampliceps was having more ameliorative role as compared to A. nilotica.

The comparison of depths showed that the ameliorative effect was more pronounced in upper

soil depth as compared to lower one.

4.4.3.4 Soil organic matter content

The leaf litter addition and its decomposition over the time added significant amount

of organic matter (OM) in the soil. The large spreading of the good plants of A. ampliceps

caused more addition of OM and the shading of the soil might have resulted in its minimum

decomposition. The increase in OM content was more in the case of good plants of A.

ampliceps and it was least in the case of poor plants of A. nilotica. The upper soil layer has

more OM content as compared to lower one (Fig. 4.4.17&18).

4.4.3.5 Bulk density and infiltration rate

The physical properties of the soil i.e. bulk density and infiltration rate was also

improved with the passage of time due to ameliorative effect of plant roots. The bulk density

decreased and infiltration rate increased over time. The decrease in bulk density was the

maximum in the case of good plant of A. ampliceps after 24 months of tree plantation. This

reduction was least in case of poor plants of A. nilotica (Fig. 4.4.19). On the other hand the

good plants of A. ampliceps showed maximum increase in infiltration rate after two years

against the poor plants of A. nilotica (Fig. 4.4.20).

4.4.3.6 Water analysis

The available water used for raising the plant species at the experimental site was

tube well water. The analysis shows that it is saline sodic water (Table 2) as it was having

higher values of EC and SAR than the permissible limits.

 

 

 

 

98  

      

        Fig. 4.4.1 Effect of salinity and sodicity on plant height of acacia species  

                                

         Fig. 4.4.2 Effect of salinity and sodicity on stem circumference of acacia species                             

Where G: good plants P: poor plants

0

100

200

300

400

500

600

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)Plant height (cm)

0

5

10

15

20

25

30

35

40

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Stem

 circumference (cm

)

99  

Fig. 4.4.3 Effect of salinity and sodicity on number of leaves of acacia species                            

 

Fig. 4.4.4 Effect of salinity and sodicity on number of branches of acacia species                            

                                 Where G: good plants P: poor plants 

0

100

200

300

400

500

600

700

800

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)Number of leaves

0

50

100

150

200

250

300

350

400

450

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Number of branches plant‐1

100  

 

Fig. 4.4.5 Effect of salinity and sodicity on Na+ concentration of older leaves of acacia species                            

 

Fig. 4.4.6 Effect of salinity and sodicity on Na+ concentration of younger leaves of acacia species                            

Where G: good plants P: poor plants

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Na+

concentration (mmol  g‐1dwt)

0

0.5

1

1.5

2

2.5

3

3.5

4

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Na+

concentration (mmol  g‐1dwt)

101  

         

         Fig. 4.4.7 Effect of salinity and sodicity on K+ concentration of older leaves of acacia species                            

     

    Fig. 4.4.8 Effect of salinity and sodicity on K+ concentration of younger leaves of acacia species                             

Where G: good plants P: poor plants

0

0.5

1

1.5

2

2.5

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)K+concentration (mmol  g‐1dwt)

0

0.5

1

1.5

2

2.5

3

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

K+concentration (mmol  g‐1dwt)

102  

Fig. 4.4.9 Effect of salinity and sodicity on Cl‐ concentration of older leaves of acacia species                            

Fig. 4.4.10 Effect of salinity and sodicity on Cl‐ concentration of younger leaves of acacia species                            

Where G: good plants P: poor plants

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Cl‐concentration (mmol  g‐1dwt)

0

0.5

1

1.5

2

2.5

3

3.5

4

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Cl‐concentration (mmol  g‐1dwt)

103  

Fig. 4.4.11 Effect of acacia species on soil pH (0‐30cm)                          

Fig. 4.4.12 Effect of acacia species on soil pH (30‐60cm)                          

Where G: good plants P: poor plants

8

8.1

8.2

8.3

8.4

8.5

8.6

8.7

8.8

8.9

9

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

pHs(0‐30 cm)

8.25

8.3

8.35

8.4

8.45

8.5

8.55

8.6

8.65

8.7

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

pHs(30‐60 cm)

104  

Fig. 4.4.13 Effect of acacia species on soil EC (0‐30cm)                          

Fig. 4.4.14 Effect of acacia species on soil EC (30‐60 cm)                          

Where G: good plants P: poor plants

0

2

4

6

8

10

12

14

16

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

ECe(dS m

‐1) (0‐30 cm)

0

2

4

6

8

10

12

14

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

ECe(dS m

‐1) (30‐60 cm

105  

Fig. 4.4.15 Effect of acacia species on soil SAR (0‐30 cm)                          

Fig. 4.4.16 Effect of acacia species on soil SAR (30‐60 cm)                          

Where G: good plants P: poor plants

0

10

20

30

40

50

60

70

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

SAR (mmolL‐1)1/2(0‐30 cm)

0

10

20

30

40

50

60

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

SAR (mmolL‐1)1/2(30‐60 cm)

106  

Fig. 4.4.17 Effect of acacia species on soil organic matter content (0‐30 cm)                          

Fig. 4.4.18 Effect of acacia species on soil organic matter content (30‐60 cm)                          

Where G: good plants P: poor plants

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Organic m

atter (%

) (0‐30 cm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Organic m

atter (%

) (30‐60 cm)

107  

Fig. 4.4.19 Effect of acacia species on soil bulk density   

Fig. 4.4.20 Effect of acacia species on soil infiltration rate   

Where G: good plants P: poor plants

1.35

1.4

1.45

1.5

1.55

1.6

1.65

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Bulk den

sity (g cm

‐3)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

6  Months 12 Months 18 Months 24 Months

A. ampliceps (G) A. nilotica (G)

A. ampliceps (P) A. nilotica (P)

Infiltration rate(cm hr‐1)

108  

4.4.4 Discussion

The nursery plants were transplanted to the field at the age of 4 months. Most of the

plants of both species survived however few of them could not face the harsh environmental

conditions. The survival rate was more for A. ampliceps (90%) than A. nilotica (82%). The

plants were irrigated with available tube well water which was saline sodic in nature. With

the passage of time the plants get established in these hard conditions and performed well

which demonstrates their salt tolerance. Based on the visual health, the plants were grouped

into two classes i.e. good and poor. Among various growth parameters, plant height and stem

circumference were maximum in case of good plants of A. nilotica. At the same time the

values of these parameters were minimum in poor plants of A. nilotica after 24 months.

Overall comparisom of both species indicated that A. nilotica produced significantly more

height and circumference than A. ampliceps. This type of growth and survival pattern was

also found by Ashraf et al. (2008) while studying the growth performance of five acacia

specie in the salt effected field having salinity in the range of 4-25 dS m-1. Number of

branches was more in the case of A. nilotica during initial growth but later on the bushy

nature of A. ampliceps produced more branches than A. nilotca. However the number of

leaves was consistently more in A. nilotica than A. ampliceps. This was true for both good

and poor plants.

Leaf ionic composition showed a significantly higher Na+ and Cl- concentration in

the older leaves of poor plants of A. nilotica. On the other hand these leaves had the

minimum concentration of K+. This type of ionic composition corresponds to the general

appearance of the plant i.e. the poor plants accumulated more Na+ and Cl- and less K+ as

compared to those of good plants. Maintaining higher K+ or lower Na+ in A. ampliceps would

have helped it to grow better than its counterpart. The maintenance of lower Na+ and higher

K+ concentration within cell is considered an important determinant of salt tolerance

(Qureshi and Barret- Lennard, 1998, Saqib et al. 2005). More over under salinity stress plants

try to restrict toxic ions like Na+ and Cl- in their older leaves so that on shedding of these

leaves they can get rid of these harmful ions. This is another strategy which plants use to

survive under severe saline conditions. The disturbed ion and water homeostasis may cause

molecular damage, stunted growth and even death of the plants (Zhu, 2001).

109  

The use of poor quality water adversely affects the soil quality and plant growth

(Qadir and Schubert, 2002). As discussed earlier salinity and sodicity of soil and water

adversely affects plant growth and development. However, the plants differ in their salt

tolerance and the survival and growth of the selected plants under these water and soil

conditions demonstrates the salinity and sodicity tolerance of these plants. The occurrence of

poor and good plants and difference in their leaf ionic composition shows inter and intra

species difference for salinity and sodicity tolerance in the studied plants. Earlier Akhtar and

Hussain (2008) have also found differences for salt tolerance among the plant species.

The growing plants have remedial effect on soil chemical properties like pHs, ECe and

SAR. Among these parameters maximum reduction was found in case of SAR. Similar

results were found by Basavaraja et al. (2010). With the passage of time these parameters

slowly decreased and this reduction was more in case of good plants as compared to poors.

For phytoremediation to be carried out there must be a source of calcium in the soil.

The dissolution of calcite is thought to be a function of CO2 in the rhizophere: First of all

CO2 is dissolved in water and it converted into H2CO3. This acid reacts with CaCO3 and Ca2+

ions are released or the other possibility is that H2CO3 is dissociated in to H+ and HCO3- and

the reaction of H+ with CaCO3 releases Ca2+ ions (Qadir et al., 2007). This soluble Ca2+

replaces Na+ on exchange sites (Robbins, 1986b). The replaced Na+ is either leached down

with percolating water or is taken up by plant roots (Qadir et al., 1997). Respiration of roots,

production of CO2 from oxidation of plant root exudates and organic matter decomposition

and production of organic acids by soil organisms are collectively helpful in dissolution of

calcite. All these processes are helpful in the release of Ca2+ which can replace Na+ at a

relatively much rapid rate than that would have occurred at normal CO2 partial pressure in the

atmosphere (Qadir et al., 2007).

Bauder and Brock (1992) concluded that crops which are having more CO2

production capacity result in more acidification of soil and resultantly more removal of Na+

out of the profile. Release of Protons (H+) and organic acids from plant roots is thought to be

a process responsible for rhizosphere acidification. The leguminous crops which carry on

nitrogen fixation also decrease the pH of their rhizospheres (Schubert et al., 1990b). The

more release of H+ ions along with various organic acids (as seen in study 2) in the

110  

rhizosphere of A. ampliceps as compared to A. nilotica might be responsible for more

dissolution of calcite with a resultant release of Ca2+ and ultimately more reduction in SAR.

The reduction in pHs was the result of Na+-Ca2+ exchange and the subsequent leaching of

Na+ (Qadir et al. 1996). It was also observed that due to leaching of soluble salts there was a

reduction in ECe of the upper soil layer. The comparison A. ampliceps and Acacia nilotica

showed that the former tree species has caused more reduction in these soil characteristics as

compared to later one.

The physical properties of the soil also improved with time and again this

improvement was more for A. ampliceps than A. nilotica. These results are in line with

Akhter et al. (2004) who found that Kallar grass grown on saline sodic soil resulted in

considerable increase in soil porosity, water holding capacity and reduction in bulk density in

a five year period. Likewise Mishra and Sharma (2003) monitor the performance of two

leguminous tree species i.e. Prosopis juliflora and Dalbergia sissoo and they found a

reduction in the bulk density and an increase in the porosity of the soil at the end of the

experimental period. The positive changes in the soil were considered due to the addition of

organic matter which enhanced soil aggregation and developed an appropriate soil structure.

The organic matter content also increased with time and this increase was more in case of

good plants of A. ampliceps due to more liter production and shading of the soil. Ashraf et al.

(2008) also observed an increase in organic matter content of the saline sodic soil which was

under acacia plantation.

4.4.5 Conclusions

As a result of increasing population and shrinking normal soil and water resources,

we are compelled to utilize salt-affected soils and waters for the agriculture productivity.

This study shows that the management and utilization of saline waters on salt-affected lands

can be done successfully by growing salt tolerant acacia species. The comparison of both

species indicated that A. ampliceps produce more biomass and caused more amelioration in

the soil properties than A. nilotica, so it would prove to be a good source of fuel wood and

forge for livestock as well as a plant for phytoremediation of salt-affected soils.

111  

CHAPTER-5

SUMMARY

Salt affected soils have higher concentration of soluble salts or exchangeable sodium

affecting normal growth of most of the crops. Salt-affected soils may be saline, sodic or

saline- sodic in nature. These types of soils are predominantly present in arid and semi-arid

parts of the world. In these areas annual rainfall is less than evapotranspiration losses of

water. As a result net movement of water is upward which results in the deposition of various

types of salts in the upper soil layer. Plants face different types of problems due to the

presence of these salts. Salinity poses many challenges to plant growth and development

including high levels of soluble salts, deficiencies of micronutrient and reduced water uptake.

Disturbed ion homeostasis leads to molecular damage, stunted growth and even death of the

plants. On the other hand, the sodicity causes slaking, swelling and dispersion of clay that

may cause surface crusting. Therefore water permeability, aeration, water holding capacity,

penetration of roots, tillage and sowing operations are highly disturbed in these types of soils.

Availability of water is very crucial for fulfilling the needs of present and future generations

and its scarcity are increasing day by day due to climatic changes. As a result of water deficit

huge yield losses have been noticed in case of many crops.

Reclamation of the salt affected soils have been done using different techniques like

chemical treatments, tillage operations, crop based interventions, water-related methods, and

electrical currents. However, in recent times, the most effective and low cost reclamation

approach is the phytoremediation. In this approach the native source of calcium like calcite is

made soluble with the root action and the calcium released is used to replace sodium on the

exchange sites. The response of various plants for phytoremediation of sodic and saline-sodic

soils varies with species. Most of the acacia species are highly salt tolerant plant and have

great potential for livestock and agroforestry. Many acacia species can survive at higher

levels of salinity and sodicity, so they may be helpful for the reclamation of the salt affected

soils.

This project has been conducted for the physiological, biochemical and

phytoremedial characterization of two acacia species of different origin for salt affected soils.

112  

The species used include Acacia ampliceps and Acacia nilotica. The specific objectives of

the project are the followings:

1. Assessment of salinity tolerance potential of two different acacia species.

2. To study different physiological and biochemical mechanisms of salinity

tolerance in the selected acacia species.

3. To study the ameliorative effects of selected acacia species on the salt affected

soils.

Keeping the above objectives in mind, four studies were planned. In the first

experiment, three week old seedlings of both species were transplanted in half strength

Hoagland nutrient solution having five treatments (control, 100, 200, 300 and 400 mM

NaCl). After eight weeks, the plants were harvested and the data regarding shoot and root

fresh weights and the respective lengths were recorded. The roots and shoots were separately

oven dried and their dry weights were also recorded. The dried shoot and root samples were

digested and used for ionic analysis. The data regarding growth and ionic composition (Na+,

K+ and Cl-) showd that A. ampliceps was more tolerant to salinity than A. nilotica. The

seedlings of A. nilotica could not survive at 400 mM NaCl due to ion toxicity.

In another solution culture experiment, the plants of both species were exposed to 100

and 200 mM NaCl concentration. After four weeks of salinity development plants were

harvested and physical growth data were noted. Membrane stability index (MSI) and

activities of antioxidants enzymes like superoxide dismutase (SOD), peroxidases (POD) and

catlase (CAT) were determined using their standard methods. Root exudates were collected

for the analysis of organic acids i.e citric acid, malic acid and tartaric acid. Shoot and root ash

alkalinity was also measured. The results indicated that more rhizosphere acidification and

higher activities of antioxidants enabled A. ampliceps to produce more shoot and root

biomass and better respond to salinity. Both the species were further studied under the

combination of salinity and water stress in the pots. The salinity levels (control, 10, 20 and

30 dS m-1) were developed in the respective treatment pots by adding calculated amount of

NaCl. After the establishment of the plants water stress was applied during the period of the

experiment. Plant growth data like plant height, stem diameter, number of branches and

number of leaves were taken after four months. Chlorophyll and relative water contents

113  

(RWC) were determined following standard method. Nodule number and their fresh and dry

weights were determined. The plants were separated into root, stem and leaves after

harvesting. Ionic concentrations of Na+, K+ and Cl- were determined in these parts.

Postharvest soil samples were taken from each pot and analysed for different properties (pHs,

ECe and SAR). The results demonstrated that A. ampliceps proved to be better tolerant to

salinity whereas A. nilotica performed better when salinity was combined with water stress.

Postharvest soil analysis showed non-significant changes in the soil properties.

In the final study these species were grown in the salt affected field and their growth and

ionic data was recorded after specific time intervals for two years. The changes in the soil

chemical and physical properties were also determined at different intervals. The comparison

of both species indicated that A. ampliceps produced more biomass and caused more

reduction in the soil chemical properties like pHs, ECe and SAR as compared to A. nilotica,

due to more addition of organic matter and rhizospher acidification. On the other hand the

physical properties like bulk density and infiltration rate were also improved more in case A.

ampliceps than A. nilotica.

On the basis of results of the presented studies, it can be concluded that salinity

caused a significant reduction in growth of both the species however A. ampliceps showed

more tolerance than A. nilotica due to its better physiological and biochemical mechanisms

of salt tolerance. On the other hand, A. nilotica performed better when salinity was combined

with water stress due to its xerophyte nature. A. ampliceps caused more phytoremediation of

the saline sodic soil so it could be grown on such lands as a good source of fuel wood and

forge for livestock at the same time rehabilitating barren lands.

114  

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