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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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