Characterization of Conocarpus Species for Salt Affected Soils

Home , Conocarpus erectus, Conocarpus lancifolius

Characterization of Conocarpus species for salt affected soils

AMJAD SAEED

1999-ag-1091

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

DOCTOR OF PHILOSOPHY

in FORESTRY

DEPARTMENT OF FORESTRY AND RANGE MANAGEMENT FACULTY OF AGRICULTURE UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2015

DECLARATION I hereby declare that the contents of the thesis, “Characterization of Conocarpus 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 and genetic models/equations/formulae/protocol 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.

Amjad Saeed Reg. No. 1999-ag-1091

To, The Controller of Examinations, University of Agriculture, Faisalabad. We, the supervisory committee, certify that the contents and form of thesis submitted by Mr. Amjad Saeed, Reg. No. 1999-ag-1091 have been found satisfactory and recommend that it be processed for evaluation by the external examiner(s) for the award of degree.

SUPERVISORY COMMITTEE:

1. Chairman: ------(Dr. Muhammad Tahir Siddiqui)

2. Member: ------(Dr. Irfan Ahmad)

3. Member: ------(Dr. Muhammad Saqib)

DEDICATIONS TO My Graceful and Polite FATHER (LATE), Who Was the First and Last For My Life PARENTS, BROTHER For Their Unlimited Love, Affection and Countless Prayers & to my friends and colleagues For Whom, Support and Patience during This Whole Course of Study

ACKNOWLEDGEMENT First of all I am highly grateful to Almighty “ALLAH” who blessed me health, thoughts and courage to complete this study. My special praises are for the HOLY PROPHET HAZRAT MUHAMMAD (SAWW), the greatest social reformer, who is forever a torch of guidance for the entire humanity. While wishing to acknowledge my well-wishers I am deeply indebted to Dr. Muhammad Tahir Siddiqui, Professor, Department of Forestry and Range Management, University of Agriculture, Faisalabad, for his excellent supervision and pains taking scrutiny of this study and writing of this manuscript. I offer my great sense of gratitude to Dr. Muhammad Irfan, Assistant Professor, Dr. Farukh Nawaz, Assistant Professor and Mr. Muhammad Asif Chohan, Lecturer, Department of Forestry and Range Management, University of Agriculture, Faisalabad, for providing valuable suggestions and boosting up my morale during the conduct of this study. I am highly indebted to Dr. Muhammad Saqib, Associate Professor, Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, for his constructive criticism and valuable suggestions to improve this manuscript and especially thanks for Rai Muhammad Rafique Senior Research Officer, PFRI Faisalabad, Ch. Riaz Virk Research Officer, PFRI Faisalabad and Mansoor Ali Research Officer, PFRI Faisalabad for their support and guidance on each step. No acknowledgement could ever adequately express my obligations to my affectionate and adoring parents and loving friends, especially thanks to, Mr. Nadeem Ahmad, Tajmal Husnain, Rizwan Asghar Cheema, Zulqarnain Khan, Dr. Gulfam Hassan, Waqas Chokus, Shahzad Kasuri, Ali Gujar, Waqar Ahmad, Hafeezur Rehman, Hamza Khalid, Shoib Shafqat, Imdad Hassan, M.Waqas Joyia, Haseeb Zafar and Shampy Baba who always raised their hands in prayers for me and without whom moral support, the present distinction would have merely been a dream. Finally, as is customary, the errors that remains, are mine alone.

CONTENTS

Chapter Title Page No.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 6

3 MATERIALS AND METHODS 24

4 RESULTS AND DISCUSSION 34

5 SUMMARY 85

LITERATURE CITED 87

LIST OF TABLES

Table No. Title PageNo. 3.1 Soil used for Experiment No. 2 33 3.2 Salt-affected sites used in Experiment No. 3 33 3.3 The characteristics of irrigation water used in the experiments 33

4.2.1 Effect of conocarpus species on soil pHs. FC: field capacity 63

Effect of conocarpus species on soil electrical conductivity (ECe). FC: 4.2.2 64 field capacity

Effect of conocarpus species on soil sodium adsorption ratio (SAR). 4.2.3 64 FC: field capacity

4.3.1 Effect of conocarpus species on soil organic matter percentage (% age) 78

4.3.2 Effect of conocarpus species on soil bulk density (g cm-3) 78

4.3.3 Effect of conocarpus species on soil infiltration rate (cm hr-1) 78

4.3.4 Effect of conocarpus species on soil pH 79

4.3.5 Effect of conocarpus species on soil ECe (dS m-1) 79

4.3.6 Effect of conocarpus species on soil SAR ((mmol L-1)1/2) 79

LIST OF FIGURES

Figure No. Title Page No. 4.1.1 Effect of salinity on shoot fresh weight of conocarpus species 36 4.1.2 Effect of salinity on shoot dry weight of conocarpus species 36 4.1.3 Effect of salinity on root fresh weight of conocarpus species 37 4.1.4 Effect of salinity on root dry weight of conocarpus species 37 4.1.5 Effect of salinity on shoot length of conocarpus species 38 4.1.6 Effect of salinity on root length of conocarpus species 38 4.1.7 Effect of salinity on number of branches of conocarpus species 39 4.1.8 Effect of salinity on necrosis % of conocarpus species 39 4.1.9 Effect of salinity on shoot Na+ concentration of conocarpus species 40 4.1.10 Effect of salinity on root Na+ concentration of conocarpus species 40 4.1.11 Effect of salinity on shoot K+ concentration of conocarpus species 41 4.1.12 Effect of salinity on root K+ concentration of conocarpus species 41 Effect of salinity on shoot K+ : Na+ concentration of conocarpus 4.1.13 42 species 4.1.14 Effect of salinity on root K+ : Na+ concentration of conocarpus species 42 Effect of salinity and water stress on shoot height of conocarpus 4.2.1 43 species. FC: field capacity Effect of salinity and water stress on root length of conocarpus species. 4.2.2 43 FC: field capacity Effect of salinity and water stress on stem diameter of conocarpus 4.2.3 49 species. FC: field capacity Effect of salinity and water stress on shoot fresh weight of conocarpus 4.2.4 49 species. FC: field capacity Effect of salinity and water stress on shoot dry weight of conocarpus 4.2.5 50 species. FC: field capacity Effect of salinity and water stress on root fresh weight of conocarpus 4.2.6 50 species. FC: field capacity Effect of salinity and water stress on root dry weight of conocarpus 4.2.7 51 species. FC: field capacity Effect of salinity and water stress on number of branches of conocarpus 4.2.8 51 species. FC: field capacity Effect of salinity and water stress on necrosis % of conocarpus species. 4.2.9 52 FC: field capacity Effect of salinity and water stress on stomatal conductance of 4.2.10 52 conocarpus species. FC: field capacity WS: water stress Effect of salinity and water stress on sub stomatal conductance of 4.2.11 53 conocarpus species. FC: field capacity Effect of salinity and water stress on photosynthetic rate of conocarpus 4.2.12 53 species. FC: field capacity Effect of salinity and water stress transpiration rate of conocarpus 4.2.13 54 species. FC: field capacity

Effect of salinity and water stress on water use efficiency of 4.2.14 54 conocarpus species. FC: field capacity Effect of salinity and water stress on memberane stability index of 4.2.15 55 conocarpusspecies.FC: field capacity Effect of salinity and water stress on chlorophyll contents of 4.2.16 55 conocarpus species. FC: field capacity WS: water stress Effect of salinity and water stress on water potential of conocarpus 4.2.17 56 species. FC: field capacity WS: water stress Effect of salinity and water stress on osmotic potential of conocarpus 4.2.18 56 species. FC: field capacity Effect of salinity and water stress turgur potential of conocarpus 4.2.19 57 species. FC: field capacity Effect of salinity and water stress on Na+ in shoot of conocarpus 4.2.20 57 species. FC: field capacity Effect of salinity and water stress on Na+ in root of conocarpus species. 4.2.21 58 FC: field capacity Effect of salinity and water stress Na+ concentration in leave of 4.2.22 58 conocarpus species. FC: field capacity Effect of salinity and water stress on K+ concentration in shoot of 4.2.23 59 conocarpus species. FC: field capacity Effect of salinity and water stress on root K+ concentration of 4.2.24 59 conocarpus species. FC: field capacity Effect of salinity and water stress on leave K+ concentration of 4.2.25 60 conocarpus species. FC: field capacity Effect of salinity and water stress on shoot K+ : Na+ of conocarpus 4.2.26 60 species. FC: field capacity Effect of salinity and water stress on root K+ : Na+ of conocarpus 4.2.27 61 species. FC: field capacity Effect of salinity and water stress on leaf K+ : Na+ of conocarpus 4.2.28 61 species. FC: field capacity Effect of salinity and water stress on SOD of conocarpus species. FC: 4.2.29 62 field capacity Effect of salinity and water stress on POD of conocarpus species. FC: 4.2.30 62 field capacity Effect of salinity and water stress on CAT of conocarpus species. FC: 4.2.31 63 field capacity 4.3.1 Effect of salinity and sodicity on plant height of conocarpus species 72 Effect of salinity and sodicity on stem circumference of conocarpus 4.3.2 72 species 4.3.3 Effect of salinity and sodicity on biomass of conocarpus species 73 Effect of salinity and sodicity on Na+ in shoot of conocarpus species. 4.3.4 73 FC: field capacity Effect of salinity and sodicity stress on Na+ in leaf of conocarpus 4.3.5 74 species. FC: field capacity 4.3.6 Effect of salinity and sodicity on K+ concentration in shoot of 74

conocarpus species. FC: field capacity Effect of salinity and sodicity K+ concentration in on leaf of 4.3.7 75 conocarpus species. FC: field capacity Effect of salinity and sodicity on shoot K+ : Na+ of conocarpus species. 4.3.8 75 FC: field capacity Effect of salinity and sodicity on leaf K+ : Na+ of conocarpus species. 4.3.9 76 FC: field capacity Effect of salinity and sodicity on SOD of conocarpus species. FC: field 4.3.10 76 capacity Effect of salinity and sodicity on POD of conocarpus species. FC: field 4.3.11 77 capacity Effect of salinity and sodicity on CAT of conocarpus species. FC: field 4.3.12 77 capacity

ABSTRACT Salinity is a major environmental stress which is reducing crop yields as well as tree growth particularly in arid and 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. Screening of plant species for salt-affected soils is based on their survival and relative growth on higher salinity levels. Keeping these facts in view, three studies were planned to explore the salinity tolerance and response on water stress of two different conocarpus species viz Conocarpus lancifolius and Conocarpus erectus. In the first experiment, four month old seedlings of both species were transplanted in half strength Hoagland nutrient solution having ,five treatments (control, 100, 200, 300 and 400 mMNaCl). The data regarding growth and ionic composition (Na+ and K+) showed that Conocarpus lancifolius was more tolerant to salinity than Conocarpus erectus. The seedlings of Conocarpus erectus and Conocarpus lancifolius survived at 400 mMNaCl and decreased in plant height and fresh weight due to higher salt concentration were 62%, 67% and 68%, 73%, respectively. Both the species were further studied under the combination of salinity and water stress in the pots at half and double field capacity, where Conocarpus lancifolius proved to be better tolerant to salinity and water stress. In the final study these species were grown in the salt affected field and their annual growth and ionic data were recorded 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 Conocarpus lancifolius produced more biomass and caused marked reduction in the soil chemical properties like pHs, ECe and SAR as compared to Conocarpus erectus, 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 Conocarpus lancifolius than under Conocarpus erectus. Results revealed that conocarpus species will prove to be a good source of timber, fuel wood and forage for livestock as well as rehabilitating barren lands.

CHAPTER-1 INTRODUCTION

Salinization, climate alteration, diminishing agriculture land (due to industrialization, urbanization, and desertification) and doubling of world population in the next 50 years and reducing resource availability are serious threats for food security (Roy and Chakraborty, 2014; Iqbal et al., 2014; Malik, 2012; Reguera, et al., 2012). Salt stress (afflict negative impacts on plant growth and development) cause profit reduction due to morpho- physiological, anatomical and biochemical modifications in crops and trees (Díaz et al., 2010; Levitt, 1972) along-with hindrance in expressing their full genetic potential (Vahdati and Lotfi, 2013). ). Salinity is the excess of soluble salt in soil and inland water resources that cause reduction in plant’s growth and development. The common salts lead to salinity + 2+ 2+ - 2- 3- + - include Na , Ca and Mg (Cations) and Cl , SO4 and HCO (anions). Na and Cl have the most lethal effects on plant development and growth (Yadav et al., 2011; Leske and Buckley 2003). 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). Firstly the parent material and secondly the human activities are responsible for salinity and sodicity (Qadir et al., 2007). Salinity is one of the main environmental stresses at present which, hamper crop yields (Majeed et al., 2010). Salinity is more conspicuous in arid and semi-arid regions of the world (Murtaza et al., 2011) due to low rainfall, high evapo-transpiration and temperature, usually accompanied by poor water and soil management practices (Azevedo Neto et al., 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 (Zhu, 2001). According to FAO (2003) salinity is affecting about 2.1% of the world dry land agriculture. Pakistan is primarily an arid and semiarid country and out of total 23 million ha cultivated lands, 6.8 million ha are affected by soil and water salinity ranging from slightly saline sodic (0.2 m ha), moderately saline (2.3 m ha), highly saline (1.5 m ha) and very high saline (2 m ha). About 75-80 % of the pumped ground water in Punjab province of Pakistan is unfit for irrigation owing to high EC, SAR and RSC which adversely affect the crop production (Ghafoor et al., 2001). Furthermore, approximately 14% of the

1 entire irrigated areas are affected by salinity. Likewise, it is increasing @ 40,000 hectares, annually (Khan, 2007; Batool et al., 2014; Iqbal et al., 2009). Salt stress is an alarming threat for arable land and fresh water reservoirs (wetlands, lakes, streams and rivers) worldwide, principally in arid and semi-arid zones (Payen et al. 2014). Salt stress leads to osmotic stress (water deficit), ionic imbalance, specific ionic toxicity, deterioration of soil structure, weakening of plant defense potential and enhancing susceptibility to biotic stresses, alteration in hormonal status and modification of membrane’s structure and composition. Plants under saline environment may grow slowly or die that depends on salt sensitive plant species, stress intensity and time (days, months) depending on specific salt level. (Gao et al., 2014; Jbir-Koubaa et al., 2014; Ödemişa and Çalişkan, 2014; Suzuki et al., 2014; Batool et al., 2014; Wang et al., 2013; Yadav et al., 2011). The sodicity may cause surface crusting, due to which water permeability, aeration, water holding capacity, penetration of roots, tillage and sowing operations in such soils are disturbed, and affect normal plant growth (Naidu and Rengasamy, 1993; Quirk, 2001; Qadir and Schubert, 2002). 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). 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 various enzymes are badly affected (Sairam and Tyagi, 2004). Although Na+ is harmful for plants, but K+ is one of the vital elements needed by the plants in large amount (Mahajan and Tuteja, 2005). Potassium plays important role in protein synthesis, activation of enzymes, photosynthesis and osmotic adjustment (Mahajan and Tuteja, 2005). Therefore, under salinity stress a higher K+/Na+ ratio is indispensable for normal performance of the plants and maintaining adequate 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). 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

2 structure of DNA molecule (Wang et al., 2003). Nature has gifted plants with explicit mechanisms to detoxify these reactive oxygen species. These comprise of the activation o f the enzymes of antioxidative pathway like superoxide dismutase, peroxidase, catalase and enzymes of the ascorbate- glutathione cycle (Noctor and Foyer, 1998; Smirnoff, 2005).

These are important enzymes 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). 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). The plants with higher antioxidant production capacity can better scavenge ROSs and are more salt tolerant (Shalata and Tal, 1998; Garratt et al., 2002). Plants have various strategies to cope with salt and water stress like escape, tolerance, and avoidance of tissue and cell dehydration (Turner, 1986). Furthermore, plants are categorized into two types on the basis of adaptation to salinity: halophytes and glycophytes. Halophytes are the salt-resistant/tolerant plants have strong salt tolerance genetic base and complete their life cycles in excessively high soluble salt (125 to 5,000 parts per million) containing soils or waters. Pakistan has 410 halophyte species out of 2500 species. However, glycophyte group includes salt sensitive/susceptible plants lacking salt tolerance genetic basis and they can only bloom in areas having abundant fresh water along-with soils containing soluble salts less than 125 ppm. Water is very essential 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).Water shortage is a serious abiotic stress that negatively affects the growth and productivity of crops (Zlatev and Lidon, 2012). As a result of water deficit the yield losses have been surged up to 50 % for many crops and trees (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). Excessive water also affect the soil and crop environment, through the depletion of oxygen, leading to reduced root respiration and other imperative plant processes, as well as

3 the production and accumulation of phytotoxic compounds, such as ethylene, in plant roots and soil. Saturated soil conditions alter the soil’s redox potential, favoring loss of nitrogen and production of ions that are toxic under certain soil conditions. These factors combine to obstruct plant growth and cause significant yield losses. There are several factors that influence the magnitude and impact of excess water stress on growing crops and tree health including: soil type, plant species, plant growth stage, temperature, day length and duration of the stress. Under conditions of excess water, lack of oxygen (O2) changes the soil and crop environment. Oxygen diffuses in water 10000 times more slowly than in air, resulting in changes in nutrient availability and microbial activity, reduced plant respiration and energy production and the accumulation of compounds in roots and soil that may become toxic to plants. In waterlogged soil, diffusion of gases through soil pores is so strongly inhibited by their water content that it fails to match the needs of growing roots. Slow oxygen influx is the principal cause of injury to roots, and the shoots they support. 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 these, chemical amendments were often used on saline soils (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, (Ilyas et al., 1993; Robbins, 1986a). 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. It is 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).

Conocarpus has two species of family Combretaceae as Conocarpus erectus and Conocarpus lancifolius which are shrub and tree, respectively.Conocarpus erectus is an evergreen shrub native to Florida's mangrove forest ecosystem in North America. It is found on the edges of salt flats, rock lands of the Florida Keys, edges of hammocks, borders of fresh and brackish marshes in South Florida. Conocarpus erectus can tolerate severe desert heat where summer temperatures may reach up to 47°C and can also grow in soils of very low fertility. Conocarpus erectus are source of food for wildlife and also protect the soil during storm and help to fix dunes (Popp et al., 1989). Conocarpus erectus is also planted as ornamental plant in yards, streets, parking lots and parks (Gilman and Watson, 1993). Conocarpus erectus is a folk remedy for catarrh, anemia, diarrhea, conjunctivitis, diabetes, fever, headache, gonorrhea, hemorrhage, orchids, swellings, prickly heat, and syphilis (Morton, 1981). Conocarpus lancifolius is native to coastal and riverine areas of Somalia, Djibouti, and Yemen. It is found throughout the Horn of Africa, the Arabian Peninsula, and South Asia. The wood of Conocarpus lancifolius is durable and used to make posts for turnery, railroad ties, fuel, buildings, boats, and charcoal. Its bark and leaves are also used in tannery. Conocarpusis reported to be a soft, non-toxic and attractive plant to feed animals because its green residues, branches and shoots are used as fodder (Suleiman et al., 2005.The present project was enacted to assess the injurious effects of salt and water stress on Conocarpus species. No detailed work has been done in Pakistan on easily propagated Conocarpus species especially on its growth response against salinity and water stress. Proposed project would help scientists as well as foresters in encountering salt and water stress. It will also be helpful in reclamation of salt affected soils and increase the tree cover in Pakistan. Following are the objectives of the present study, Objectives:  Assessment of salinity tolerance potential of Conocarpus species.  Investigation and clarification of morpho-physiological, biochemical and ionic attributes for salt and water stress in Conocarpus species.  To determine the ameliorative as well as comparative effects of Conocarpus species on the physicochemical properties of salt affected soils.

CHAPTER-2 REVIEW OF LITERATURE

Soils suffering from elevated quantities of soluble salts especially that of exchangeable sodium destroy the natural growth of a number of crops and trees. Saline, sodic and saline sodic soils come under the umbrella of salt-affected soils. Climate, specifically arid and semi-arid, has a far- reaching effect on the formulation of salt-affected soils across the globe. Such kind of climate offers less annual rainfall which, in turn, increases evapo-transpirational losses leading to increased salt concentration in soil. Salts affect equally to both plants and soils resulting in declined annual production of crops and trees. This chapter elaborates the potential use of salt-affected soils for the proper selection, characterization and apposite introduction of salt tolerant plants. 2.1 Salinity standing and its general prevalence Salinity exists as a potential threat to the optimal growth of plants worldwide. It is a source of continuous peril for over 100 countries (Massoud, 1981). More than 4,000,000 square kilometer area of the world is more or less affected by salinity (FAO, 2006). Nearly, 23% soils across the world are saline ones and 37% are sodic ones. According to Beltran and Manzur, (2005) about 830 million hectares of land is under salinity. Nearly, 6% of all the land in the Asia-Pacific region is under the stress of salinity. Three countries including Pakistan, Australia, and Thailand occupy 6.8% of the total world area of which 10% is salt- affected. According to FAO, (2006) ranging Australia is on third position with 254,000 square kilometers, Pakistan eighth and Thailand on forty-fifth position in salt effected countries. Nearly, 7% of the world’s area is salt-affected; 20% cultivated area of the world and half of the irrigated land is affected by salinity (Zhu, 2001). According to Saboora et al. (2006) salinity is on rise at a rate of 10% per year across the world. In countries like Pakistan, salinity and sodicity are the two gravest issues having dire consequences for agriculture. Total cultivated area of Pakistan is about 22.94 mha; 18.78 mha is irrigated; 6.67 mha is salt- affected (Khan, 1998). Nearly, 75-80% underground water used for irrigation in Pakistan is brackish viz having high EC, SAR and RSC (Ghafoor et al., 2001).

2.2 Potential hazards of Salts on Plants According to Greenway and Munns (1980), salinity induces four stresses in plants: (a) osmotic stress leading to water shortage (b) specific ion toxicity due to excess of Na+ and - + -3 -3 Cl (c) nutritional disruption resulting in deficiency of K , NO and PO4 (d) antagonistic effect of macromolecules owing to higher levels of reactive oxygen species. 2.2.1 Osmotic regulations in plants There occurs decrease in water retention capacity of plants when faced with salinity which induces a sharp decline in the growth of plants. It is known as osmotic stress. Water deficit marked by salinity is quite similar to that of caused by drought (Nawaz et al., 2010). The formation of new leaves is dependent on the water potential of the soil solution. Cells with fast growth have the ability to store large amounts of salts in their expanding vacuoles; salts are rarely stored in the cytoplasm, which cause a reduction in the growth of new leaves (Munns, 2005). During the early growth stages of trees, water deficit causes more damage to roots and shoots compared to salt specific stresses (Munns, 2002). It is evident from the fact that concentrations of Na+ and Cl- remains below the poisonous levels at early stages. It was revealed through a trial conducted on wheat that expanding leaf tissues absorbed only 20 and 50 mM of the sodium and chloride, respectively from a solution with a total concentration of 120 mM NaCl (Hu et al., 2005). Fricke et al. (2004) made a similar observation by noting that 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. Such an elevated concentration of sodium, instead of damaging plant cells, was indeed beneficial in adjusting osmotic potential of the cells faced with salinity. Given the above observations, it may be safely assumed that the fast growth of the cells might be a potential factor in keeping lower concentrations of the salts within the cells. The growth of shoot cells is found to be regulated through hormonal signals caused by the osmotic effect of the salt exterior to roots (Munns et al., 2000). However, decline in plant growth faced with salinity might be attributed to the degree of stress, because osmotic stress is not extreme, root growth continues while, shoot growth hinders (Hsiao and Xu, 2000). Damage owing to osmotic stresses in contingent upon 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 Salts like sodium, chloride and sulphate when absorbed in greater amounts are the major source of specify ion toxicity. This phenomenon can either result in partial loss of crop growth or a complete loss of an entire crop. Different types of plant species respond in unique manners to specific ion toxicity as most of the crops and woody plants having sensitivity for this toxicity (Abrol et al., 1988). The elevated concentration of Na+ against other cations was the potent reason of decline in the growth of Acacia ampliceps (Mahmood et al., 2010). Higher concentration of salts is mainly found in older leaves. If salt accumulation does not stop for a long time, it lead to necrosis of leaves. It only results when the salt concentration is too high to be stored in the vacuole of the cell. Under such conditions, higher concentration of salts starts building up in cytoplasm where they disrupt the functioning of enzymes and other proteins. Parallel to this phenomenon, high buildup of salts may cause cell dehydration due to their accumulation in the cell walls (Munns, 2005). Plants cope with these salts either through restricting their entry at all or ejecting salts from cytoplasm. Concentration of Na+ in the cytoplasm of the roots ranges from 10-30 mM (Tester and Davenport, 2003). Its concentration in the cytoplasm of leaf is not precisely known but is assumed to be much lower than 100 mM (Wyn Jones and Gorham, 2002). Plants exercise various mechanisms to exclude the excessive salts through the roots failing to which leads to gradual accumulation of salts in the cells. Husain et al. (2003) conducted a trial over the different genotypes of wheat crop to assess their tolerance to Na+. Leaf injury was found more in the wheat genotype having elevated concentration of sodium in the older leaves and reduced the yield. Loreto and Bongi (1987) revealed that chloride concentration over 80mM in total tissue water induces gravechanges in the plant morphology, e.g. stomata loses their grip necessary to face the environmental changes and leaves fail to maintain proper thickness. Chloride moves at a fast rate in soil as well as in plant. Excess of chloride results in burning of the leaves, necrosis of leaf tissues initially starts from the tips and then spreads to the whole leave as the intensity of the stress increases. Marschner (1995), found that greater levels of chloride induce burning of the leaves and leads to gradual defoliation of the whole plant.

2.2.3 Nutritional Disruptions Interface of nutrients and salts results in deficiencies and imbalances of the major nutrients (McCue and Hanson, 1990). Unevenness in the uptake of nutrients emerges because of higher accumulation of sodium and chloride and in return, it limits the uptake of potassium, calcium and manganese (Karimi et al., 2005). Super fulvous accumulation of the salts proves to be detrimental for the water relations of the plants which not only limits nutrient uptake but also their utilization. It affects metabolism of cells and working of the enzymes (Munns, 2002; Lacerda et al., 2003). Higher Na+: K+ ratio yields enzyme inactivation and disruptsusual metabolism of the plant (Booth and Beardall, 1991). Excessive uptake of Na+ and Cl- reduces the uptake of K+, hence, causing its deficiency (Gopa and Dube, 2003). Hyperaccumulation of Na+ suppresses the absorption of K+, Ca2+ and Mg2+ in Acacia ampliceps (Mahmood et al., 2010). Potassium facilitates protein formation as it deals with osmoregulation, cell turgor maintenance and photosynthesis (Freitas et al., 2001; Ashraf, 2004). Potassium and calcium are of prime importance for maintaining the composition and normal functioning of the cell membranes (Wenxue et al., 2003). Ample concentration of K+ in plant cells exposed to saline conditions is contingent upon the selective uptake of the K+ and discriminatory compartmentalization of K+ and Na+ in the shoots (Munns et al., 2000; Carden et al., 2003). The regulation of calcium within the plant under salt stress is a crucial parameter of the plants tolerance to salinity (Soussi et al., 2001). Reduction in the Ca2+/Na+ ratio due to salinity has damaging effects on membrane tissues resulting in to the displacement of membrane bound Ca2+ and resultantly the structure and selectivity of the membranes is lost (Kinraide, 1998). Exogenous application of Ca2+ may cause a reduction in the damages caused by salinity in plants, most probably by keeping better K+/Na+ selectivity (Hasegawa et al., 2000). Decreased K+ uptake in response to salinity was also observed earlier by Marcar et al., (1991) and Khalil et al., (2012). The plants with higher K+: Na+ ratio are considered better tolerant to salinity stress (Saqib et al., 2005). 2.2.4 Oxidative upheavals One of the demerits of salinity is that it leads to the hyper production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, single oxygen and hydroxyl radicals. Production of these ROS occurs mainly within the cytosol, chloroplasts,

9 mitochondria and the apoplastic space (Bowler and Fluhr, 2000; Mittler, 2002). Phototoxicreactions like lipid per oxidation, denaturationof proteins and abrupt changes in DNA results due to the over production of ROS (Wang et al., 2003; Pitzschke et al., 2006). Deformation of membranes due to salinity is also linked to the over production of the ROS (Shalata et al., 2001). Although, ROS are detrimental to plants, yet these also have some positive impacts in the cell like signal transduction (Torres and Dangl, 2005). Elevated concentrations of ROS harm the D1 protein of PS II and become a source of photo inhibition. Increased Photorespiration under stress and NADPH activity also contributes to raise hydrogen peroxide accumulation, which reduces the activity of the enzymes by oxidizing their thiol groups. 2.3.1 Drought Stress Water availability 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). 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). 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 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). Both salinity and water stress exert similar effects on plants (Munns, 2002), separately under combined affects, both the affects are not clear (Meiri 1984; Homaee et al., 2002). It is imperative to explore this avenue as the plant may experience water shortage in the upper zone, while salinity may be operating in the lower part of the root zone (Homaee et al., 2002). Meiri (1984) noted higher affect of matric potential (due to water stress) on reduction of growth of shoots of bean than the osmotic effect (due to salt stress). Similarly, Shalhevet and Hsiao (1986) also observed more effect of water stress than salinity in case of pepper and cotton, associated with equal decline. From thermodynamic point of view, even though

10 metric and osmotic components act in an additive manner, the kinetic factors like water uptake and transpiration should also be considered. 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, thecortical cells of roots have some capability to adjust 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 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. Drought tolerant plants try to maintain high relative water content than sensitive ones by accumulating different osmotica which lower their water potential. 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 stomatal and cuticle 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). 2.3.2 Waterlogged Soils and Plants The soil and crop environment by causing depletion of oxygen, leading to reduced root respiration and other vital plant processes, as well as the production and accumulation of phytotoxic compounds, such as ethylene, in plant roots and soil. Saturated soil conditions change the soil’s redox potential, favoring loss of nitrogen and production of ions that are toxic under certain soil conditions. These factors when combined hamper plant growth and

11 cause significant yield losses. It is self-evident that waterlogging of the soil or deeper submergence occur when water enters soil faster than it can drain away under gravity. There is mounting evidence that, in several parts of the World, inputs of water are growing. One cause may be climate change. (Prudhomme and Svensson., 2002).Greater fluxes of river water resulting from mountain deforestation are also being observed in many parts of the World, with loss of mountain forest and wetlands playing a major part in heightening peaks of river outflow. An overall outcome would be increase in frequency of flooding of lowland regions such as the lower reaches of the River Rhine in Europe and the Euphrates delta of Bangladesh and West Bengal. These are highly populated areas but also contain much productive farmland where satellite imaging has recorded many major flooding events. While it is damage to human life and property that attracts most media coverage, flash flooding can devastate vegetation of poorly adapted species especially farm crops. In developing countries especially, threatens the well-being of many people who depend on locally produced food. Intensive and large-scale irrigation of farmland can also increase the incidence of waterlogging of the soil. Water tables can rise as a result. This is especially in heavily irrigated dry regions, such as Sindh province in the Indus valley of Pakistan. Here, 50 to 60 years ago, the water table was 4 m below ground. By 1984, it was less than 1.6 m over most of the irrigated regions, the rising water table was laden heavily with salts. The resultant environmental catastrophe has led to launch a multi-million dollar drainage project (the Left Bank Outfall Drainage Project) of immense scale but bringing in controversy and environmental concern. The problem is exacerbated by the flatness of the topography that inevitably slows the rate of lateral drainage. Change in land use has also resulted in various environmental catastrophic in the world, as conversion of meadow land to arable farming andexpanding urbanization of the landscape also creates large expanses of non-absorbing hard surface that accumlates rainwater to its periphery via surface run-off or underground drainage systems(Van Der Ploeg et al., 2002).. There are several factors that influence the magnitude and impact of excess water stress on growing crops, including: soil type, plant species, plant growth stage, temperature, day length and duration of the stress. Under excess water conditions, it is the lack of oxygen

(O2) that changes the soil and crop environment. Oxygen diffuses in water 10000 times more slowly than in air, resulting in changes in nutrient availability and microbial activity, reduced

12 plant respiration and energy production and the accumulation of compounds in roots and soil that may become toxic to plants. In waterlogged soil, diffusion of gases through soil pores is so strongly inhibited that it fails to match the needs of growing roots. A slow oxygen influx is the principal cause of injury to roots, and the shoots they support. Thus, restricted release of ATP so vital to support life processes may be at least as important a reason for death of flooded root tips as slow ATP production (Greenway and Gibbs, 2003) conclude that early cell death can be avoided, if small amounts of available energy are successfully re-diverted to permit synthesis of certain critical ‘anaerobic’ proteins (e.g., alcoholic fermentation enzymes), that support glycolysis and fermentation and help to prevent excessive acidification of the cytoplasm and vacuole and maintain membrane integrity.

Like oxygen, carbon dioxide (CO2) and ethylene (C2H4) gases diffuse more slowly through water than through air, and accumulate around plant roots as a result. Ethylene is a root growth inhibitor with varying effects on different crops. Barley, which is highly sensitive to ethylene toxicity, experiences root death at relatively low concentrations. Several other crops will respond to a buildup of ethylene by initiating survival mechanisms, such as production of secondary roots and upward growth of roots in search of oxygen. Potatoes respond to ethylene by increasing the size of their tuber pores (lenticels) for increased air exchange. Legumes are indirectly affected by ethylene through its inhibition of N-fixing rhizobium formation and function. Although an absence of oxygen is usually fatal to growing root tips, surprisingly small amounts of external oxygen (e.g. 0.006 – 0.01 mol m-3 in solution) are able keep them alive (note: water in equilibrium in air contains approximately 0.25 mol m-3 of oxygen at 25 ºC). Growth arrest and death arise principally because, demand for ATP exceeds the supply (b) self-poisoning by products of anaerobic metabolism. These and related aspects are examined below. Anaerobic roots generate ATP mainly by glycolysis. This pathway also feeds pyruvic acid into ethanolic fermentation (and also into lactic acid fermentation, especially in the first hours of anoxia before the cytosol acidifies. ATP generation capability of glycolysis/fermentation depends on a ready supply of glucose and its precursors. Sugar shortage caused by anaerobic arrest of starch breakdown and sugar unloading in roots can thus shorten the duration of survival. Such ability is due, in

13 part, to its possession of an anaerobically inducible gene coding for α-amylase, the enzyme principally responsible for degrading starch to a range of sugars. Anaerobic roots may also die from self-poisoning by products of anaerobic metabolism; the most notable toxin being excess protons that acidify the cytoplasm and vacuole (Gerendás and Ratcliffe, 2002). In support of this notion, roots of pea, black eyed peas and navy beans, which collapse particularly quickly when anoxic, acidify their cytoplasm more rapidly than do longer-lived anoxic maize, soybean or pumpkin root tips. The sources of the extra protons within the cell have proved difficult to identify (Gerendás and Ratcliffe, 2002). Another possible toxin is acetaldehyde. In alcoholic fermentation, activity of the enzyme that converts acetaldehyde to ethanol (alcohol dehydrogenase - ADH) usually exceeds that of the enzyme that promotes acetaldehyde production from pyruvic acid (pyruvate decarboxylase - PDC). Normally, this state of affairs ensures low sub-toxic concentrations of acetaldehyde in anoxic cells. However, after such tissue is returned to air, this control is sometimes lost and plant tissue typically generates a burst of acetaldehyde that could be damaging (Boamfa et al., 2003). Another potential toxin is nitric oxide (Dordas et al. 2003). 2.4 Reclamation of salt affected soils The reclamation of sodic and saline sodic soils is done by providing 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. 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

14 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). 2.5 Phytoremediation of salt affected soils 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). 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 (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 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. Secondly, 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 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. 2.6 Mechanisms behind phytoremediation Phytoremediation of calcareous sodic and saline-sodic soils helps in increasing the solubility rate of native calcite via various processes in the rhizospher which result in increasing levels of calcium in the soil solution. Following processes are responsible for the phytoremediation of the salt-affected soils. 2.6.1 Physical impacts of roots Plant roots are crucial for soil structure maintenance and the formation of macro pores 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 Newton, 2003). Moreover roots cause different changes by removing entrapped air from larger pores and generate alternate wetting and drying cycles. In the rhizospher 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 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 increased 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

16 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 density in a five year period. It was supposed that these changes might be due to the wide spread and deep root system of the grown grass (Malik et al., 1986). Biological drilling by deep roots is thought to be a substitute to deep tillage for the reclamation of dense subsoils (Elkins et al., 1977). Macrospores 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. 2.6.2 Uptake of salts by shoots Hyper accumulators like halophytes have the ability to gather higher amounts of salts and Na+ in their different parts including shoots. The salts taken up by plants during their growth on salt affected soils are removed with the harvesting of the plant species. 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 considerably high quantity of salt but it is not enough to play a major role in the phytoremediation 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

17 leaching of salts and Na+ out of the rhizosphere instead of removal by harvesting the above ground plant materials. 2.6.3 Partial pressure of Carbon dioxide in the root zone

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. The dissolution of calcite is thought to be a function of

CO2 in the rhizospher. First of all CO2 is dissolved in water and it is converted into H2CO3. 2+ This acid reacts with CaCO3 and Ca ions are released or the other possibility is that H2CO3 + - + 2+ is dissociated in to H and HCO3 and the reaction of H with CaCO3 releases Ca ions (Nelson and Oades, 1998). Under flooded soil conditions this partial pressure is even more increased (Ponnam peruma, 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 rhizospher 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 result 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 pressure of CO2 in the rhizospher. 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 sogham 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

18 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. 2.6.4. Release of protons from plant roots Proton (H+) release is thought to be a process responsible for rhizospher 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 rhizospher is increased (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 + rhizospher of different N2-fixing plant species (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 rhizospher 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 rhizospher and are called as ash alkalinity. This parameter has been characteristically determined in many crops and trees to evaluate their acidification capacity (Noble and Randall, 1999; Noble et al., 1996). 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 rhizospher. 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

19 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. 2.7 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 environment. Different biochemical mechanisms are additive or synergistic in nature (Iyengar and Reddy, 1996). 2.7.1 Ion Regulation and Compartmentalization For proper growth and reproduction under both non saline and saline situations 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

20 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 At NHX1 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. Scretion of salts is made possible by specialized structures known as salt glands. These glands screte salts (particularly NaCl) from their leaves and help in maintaining its low concentration in the cell. Furthermore, exclusion of salt occurs at root level to normalize the salt concentration of their leaves (Levitt, 1980). 2.7.2 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 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 osmo- protection, 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). 2.7.3 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, 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 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 activities 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. 2.7.4 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

22 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). 2.8 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), 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.), 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.

CHAPTER-3 MATERIALS AND METHODS The research work has been conducted in the wire house of the Institute of Soil and Environmental Sciences and at Shorkot Irrigated Plantation, Punjab. This research has been conducted to evaluate the comparative salt tolerance and phytoremedial potential of two conocarpus species. Three different experiments were conducted to achieve the specific objectives of this research project: 3.1 Experimental techniques 3.1.1 Study- 1: Effect of different salinity levels on growth and ionic composition of Conocarpus erectus and Conocarpus lancifolius Plant source The plants raised in polythene bags of conocarpus species of almost same age and size were collected from nursery of Punjab Forestry Research Institute, Faisalabad. Growth conditions: This experiment was conducted in wire house of the Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad. The healthy seedlings of four month old plants of two conocapus species were transferred by removing ball of earth of polythene bags in thermo pore 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 after every week. The design of experiment was CRD factorial with four replicates. One week after 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 duration by the addition of NaOH or HCl. Measurements: After ten weeks of salinity development, plants were harvested. Data about shoot fresh weight, root fresh weight, and shoot and root lengths were noted. The roots and shoots were separately oven dried at 75○C for 48 hours. The dry root and shoot weights were also 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

24 water and used for ionic analysis. Na+ and K+ were determined using the flame photometer. The details of plant analysis are given in section 3.4. 3.1.3 Study-2: Response of Conocarpus erectus and Conocarpus lancifolius to salinity in combination with water stress Growth conditions: A pot culture study was conducted to determine the effect of salinity and water stress on the two conocarpus 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 nearby 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, 30 and 40 dS m-1) were developed in the respective treatment of pots by adding measured amount of NaCl before filling the soil in the pots. In control no salt was added. Six month old nursery plants in polythene bags of both the species were transplanted in these pots. After the establishment of the plants, water stress (half of field capacity and double of field capacity) was applied till end of the experiment. The water characteristics were given in Table 3.3 and soil characteristics were mentioned in Table 3.1. Measurements: Soil samples were taken from each pot and analyzed for different properties (pHs, ECs and SARs) before planting the plants and after harvesting the trees. After one year of salinity development, plants were harvested. Data of shoot freshweights, root fresh weights, shoot and root lengths, collar diameter, necrosis% and No. of branches was noted. The roots and shoots were separately oven dried at 75○C for 48 hours. The dry root and shoot weights were also recorded. Before harvesting, parameters regarding gas exchange i.e. photosynthetic rate (A), transpiration rate (E), intrinsic CO2 concentration (Ci), stomatal conductance (gs) and photosynthetic water use efficiency (WUE) were measured by using CIRAS-3 (PP System, Amesbury, MA, USA) with PLC3 universal leaf cuvette, measuring both sides of the flag leaves. Cuvette was provided light via light emitting diodes (LED) and with a photon flux of -2 -1 -1 1000 µmol m s , ambient leaf temperature and 390 µmol mol CO2. Chlorophyll contents with SPAD value and activities of antioxidants enzymes like superoxide dismutase (SOD), peroxidases (POD) and catlase (CAT) were determined. Activity of SOD was determined on the basis of its ability to inhibit the photoreduction of nitrobluetetrazolium (NBT) as

25 described by Giannopolitis and Ries (1977). To determine activities of catalase (CAT) and peroxidase (POD) the method of Chance and Maehly (1955) was used. The leaf water potential (ѱw) was determined by using Scholander type pressure chamber apparatus and osmotic potential (ѱs) by using vapor pressure Osmometer. The turgor potential (ѱp) was measured by the determination of difference between water potential (ѱw) and osmotic potential (ѱs) values. The dried and grounded 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 of Na+ and K+ were determined by using the flame photometer.The data was statistically analyzed and interpreted. 3.1.4 Study-3: Comparative role of Conocarpus erectus and Conocarpus lancifolius in the phytoremediation of the salt affected soil Growth conditions: A field experiment was conducted to determine the role of Conocarpus erectus and Conocarpus lancifolius in the reclamation of salt affected soil at Shorkot Irrigated Plantation, Punjab. Six month old healthy plants from nursery of both the plant species were transplanted in the pits already made in the field for this purpose at spacing of 10 x 6 on three different sites of low (site-III), medium (site-I) and high (Site-II) salinity levels as illustrated in Table.3.2. Plants were irrigated with canal water as characterized in Table 3.3. Measurements: Plant growth parameters like plant height, stem diameter and number of branches were taken after every six months for two years. For ionic composition of leaves 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 was also determined. Standard procedures were applied to analyze and interpret the data using appropriate experimental design following (Steel et al. 1997). The details of soil, water and plant analysis are given in sections 3.2, 3.3 and 3.4, respectively.

3.2 Soil analysis 3.2.1 Particle-size analysis Hydrometer method (Bouyoucos, 1962) was followed for particle size analysis. Forty grams of air dried 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 after and contents of cylinder were shaken manually by means of a metal plunger. When uniform suspension was obtained, plunger was taken out and after 4 minutes hydrometer reading (HR1) was recorded. Shaking procedure was repeated by removing hydrometer ensuring minimum disturbance and second hydrometer reading (HR2) was recorded after 2 hours. Since hydrometer is calibrated at temperature of 68 oF (20 oC), the o HR1 and HR2 were corrected for temperature variation (for each degree above 20 C, 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 correct 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. Sampler was carefully removed so as to preserve its contained soil with natural structure and packing with maximum possible care. The soil extending each end of the sampler was trimmed with a strong edge knife and was transferred to weighing pot. It was dried in an oven at 105 oC until constant weight was achieved. 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)]

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 (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. 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 settle for 30 minutes. Solution was 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 Na Fusing 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 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 saturated paste extracts (ECe) Suction pump was used to obtain extract from the saturated soil paste. Electrical conductivity of the saturated 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)] 2- - 3.2.6. Carbonate and bicarbonate (CO3 + HCO3 )

Saturated extract was titrated against 0.01 N H2SO4 using phenolphthalein indicator 2- to colorless end point for CO3 . 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 2- -1 CO3 (mmolc L ) = × 1000 mL of sample taken

2- Where a is mL of H2SO4 used during titration for CO3

(b – 2a) × Normality of H2SO4 - -1 HCO3 (mmolc L ) = × 1000 Volume of sample taken (mL) - Where b = mL of H2SO4 used for HCO3 determination. 3.2.7. Calcium + Magnesium (Ca2+ + Mg2+) Saturated 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 2+ 2+ -1 Ca + Mg (mmolc L ) = × 1000 mL of sample taken 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 + -1 concentration of soluble Na in mmolc L was calculated by using the following formula: ppm concentration × dilution factor + -1 Na (mmolc L ) = 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 + -1 Na in mmolc L was calculated by the help of formula given: ppm concentration × dilution factor + -1 K (mmolc L ) = 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

-1 Where the concentration of soluble cations was in mmolc L 3.3 Water analysis 2- Samples of irrigation water were collected in clean plastic bottles. The EC, CO3 , - - 2- 2+ 2+ + HCO3 , Cl , SO4 , Ca + Mg , Na and SAR of water were determined in the same way as used for soil saturation extract. 3.4 Plant Analysis 3.4.1 Sodium and potassium 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 H2O2as gently added. The tubes were placed again into the digestion block until fumes were produced for 20 minutes. Again digestion tubes were removed from digestion. Above step was repeated until the cool material became colorless. After digestion

30 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-prepared standard solutions using reagent grade salt of NaCl and KCl, respectively. 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 Sairamet 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 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 nitrobluetetrazolium (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 nitrobluetetrazolium (NBT) reduction. Activity of

31 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 the 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) 20mMguaiacol, 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 were determined by the method of Bradford (1976) using bovine serum albumin as standard. 3.5 Water related Analysis 3.5.1. Leaf Water Potential ѱw(-MPa) The third leaf from the top (fully expanded youngest leaf of conocarpus was excised between 6:30 am to 8:30 am for the determination of leaf water potential (ѱw) by using Scholander type pressure chamber. The measurement of this parameter was carried out before harvesting the trees. 3.5.2. Osmotic Potential ѱs(-MPa) The leaf which was used for the determination of leaf water potential (ѱw) was frozen in a freezer below -20oC for more than 7 days. Afterwards, the frozen leaf material was thawed and sap from the material was extracted by pressing with the help of glass rod. This sap was directly used for the determination of osmotic potential (ѱs) by using vapor pressure Osmometer. 3.5.3. Turgor Potential ѱp(MPa) The turgor potential (ѱp) was measured by the determination of difference between water potential (ѱw) and osmotic potential (ѱs)values. Ѱp= ѱw-ѱs

3.6. Statistical Analysis Standard procedures were applied to analyze the data using appropriate experimental design (Steel et al. 1997) and the means were compared using least significant difference (LSD) test. Detail of statistical analysis is given in Chapter four under individual experiments. Table 3.1 Soil used for Experiment No. 2 Soil Characteristics Values Units Texture Sandy loam Saturation 29 % age pHs 7.62 -1 ECe 3.30 dS m SAR 12.43 (mmol L-1)1/2 Table 3.2 Salt-affected sites used in Experiment No. 3 Soil Site-I Site-II Site-III Units Characteristics Compartment-50 Compartment-39 Compartment-32 Texture Sandy clay loam Sandy clay loam Sandy clay loam Saturation 27 32 27 % age pHs 7.73 7.78 8.07 -1 ECe 23.60 31.65 17.19 dS m TSAR 69.42 73.83 62.13 (mmol L-1)1/2 Organic matter 0.81 0.68 .91 % Bulk density 1.62 1.68 1.42 g cm-3 Infiltration rate 0.38 0.33 .44 cm hr-1

Table 3.3 The characteristics of irrigation water used in the experiments Characteristics Used in pots (Exp. No.2) Used on salt-affected field (Exp. No.3) EC (dS m-1) 1.34 1.28 TSS (mmol L-1) 13.4 12.20 SAR (mmol L-1)1/2 7.43 8.55 RSC (mmol L-1) Nil Nil

CHAPTER-4 RESULTS AND DISCUSSION

4.1 Study- 1: Effect of different levels of salinity on growth and ionic composition of conocarpus species 4.1.1 RESULTS 4.1.1.1 Morphological Parameters The difference between species, treatments and their interaction was significant for both these parameters. The increasing levels of salinity resulted in the corresponding decline in shoot fresh and dry weights of both the conocarpus species (Fig. 4.1.1 & 4.1.2). C. lancifolius and C. erectus survived even at 400 mM NaCl, whereas C. lancifolius performed better. C. lancifolius and C. erectus showed 68% and 73% decrease in shoot fresh weight, and decreasing trend was observed in case of shoot dry weight. 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 incresing salt concentration in the growing medium. Like in shoot growth salinity resulted in the corresponding decline in the root growth at 400 mM NaCl (Fig. 4.1.3 & 4.1.4). The affect of increase in salinity levels resulted in decline in both root fresh and dry weights. Reduction in fresh and dry weights of root was 69% and 71% in C. erectus and 63% and 66% decrease was recorded in C. lancifolius. The length of both shoot and root also decreased due to salinity stress (Fig. 4.1.5 & 4.1.6). Maximum decrease in shoot height was 67% at salinity level of 400 mM NaCl in C. erectus as compared to control, whereas root length increased up to 200 mM NaCl and then decrease was noted in 300 mM NaCl and 400 mM NaCl, respectively. This indicated that with increase in salinity level there was more reduction in shoot and root length of C. erectus than C. lancifolius. Moreover results showed similar decreasing trend in case of number of branches, i.e 88% decrease in C. lancifolius and 89% decline in C. erectus were recorded at 400 mM NaCl salt concentration (Fig. 4.1.7). However, the species differed significantly from each other. C. erectus produced more number of necrotic leaves with increase in salinity as compared to C. lancifolius. Maximum necrotic leaves 80% in C. lancifolius and 84% in C. erectus were recorded at 400 mM NaCl (Fig. 4.1.8). This indicated negative effect of increase in salinity level and there was more reduction in number obranches in C. erectus than that of C. lancifolius whereas, necrosis percentage was contrary to number of branches in response to increase in salt concentration.

4.1.2 Ionic Parameters Salinity significantly increased the shoot and root Na+ concentration in both the species (Fig. 4.1.9 & 4.1.10). The comparison of both the species in various treatments showed that in control treatment, both the species exhibited almost equal concentration of Na+. However, in all the other treatments, C. lancifolius accumulated significantly lower Na+ as compared to C. erectus in both shoot and root. The difference between both the species was the maximum at 400 mM NaCl. In this treatment, the maximum Na+ concentration (5.6 mmol g-1dwt) was found in the shoot of C. erectus. At the same salinity level, shoot Na+ concentration was 5.37mmol g-1dwt in C. lancifolius. The shoot Na+ concentration in this treatment was 5.51 and 4.82 mmol g-1dwt for C. erectus and C. lancifolius, respectively (Fig. 4.1.11 & 4.1.12). The concentration of K+ as against of Na+ decreased significantly at each increasing level of salinization. In control treatment K+ concentration was the maximum, whereas at 400 mM NaCl, it was the minimum. The comparison of species at each salinity level showed that C. lancifolius accumulated more K+ in both organs as compared to C. erectus. At 400 mM NaCl, shoot K+ concentration was 2.20 mmol g-1dwt in C. lancifolius, where as in case of C. erectus, it was 1.96 mmol g-1dwt (Fig. 4.1.13 & 4.1.14). The K+: Na+ ratio was the highest in control treatment and decreased with increasing salinity levels. This ratio was minimum in 400 mM NaCl treatment. Conocarpus lancifolius has higher K+: Na+ ratio than C. erectus for both root and shoot under saline conditions (Fig. 4.1.13 & 4.1.14).

C. lancifolius C. erectus 160

140 a

120 b

100 c cd d 80 e 60 e ef fg Shootfresh weight (g plant‐1) 40 g 20

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig.4.1.1 Effect of salinity on shoot fresh weight of conocarpus species

C. lancifolius C. erectus 40 a 35

b )

1 30 -

c 25 cd cd d 20

Shoot plant dry (g weight Shoot 10 e e e 5 e

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig.4.1. 2 Effect of salinity on shoot dry weight of conocarpus species

C. lancifolius C. erectus 60

a a a

) 1 - ab a 40 ab ab

30 bc

20 c

Root plant (g weight freshRoot c 10

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig. 4.1.3 Effect of salinity on root fresh weight of conocarpus species

C. lancifolius C. erectus 14 abcd a

12 a 1) - 10 ab abc bcde 8

6 bcde cde de

4 Root Root dryweight (g plant e

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig. 4.1.4 Effect of salinity on root dry weight of conocarpus species

C. lancifolius C. erectus 120 a

100 ab

bc c c 80

d de 60 ef f 40 Shoot length(cm)Shoot g

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig.4.1. 5 Effect of salinity on shoot length of conocarpus species

C. lancifolius C. erectus 60

c ab 50 d a c de 40 f bc ef 30 g

Rootlength(cm) 20

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig.4.1. 6 Effect of salinity on root length of conocarpus species

C. lancifolius C. erectus 40

35 a a a 30 ab 25 b bc 20

15 No. branches No. of

cd 10 d

5 d d

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig.4.1. 7 Effect of salinity on number of branches of conocarpus species

C. lancifolius C. erectus 100 a 90 b 80 70 c 60 d 50 40

Leaves necrosis (%) Leavesnecrosis 30 e e 20 f 10 f f f 0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl Axis Title

Fig.4.1. 8 Effect of salinity on necrosis % of conocarpus species

C. lancifolius C. erectus 6 a

5 b b c

dw) 4 cd 1 1 - de ef fg 3

g in Shoot (mgg in Shoot

+ 2 Na h 1

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl Axis Title

Fig.4.1. 9 Effect of salinity on shoots Na+ concentration of conocarpus species

C. lancifolius C. erectus 7

a 6 a abc ab

5 bc

dw) cd

1 1 - 4 e de e

in R00ts in (mg f + +

Na 2

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig.4.1. 10 Effect of salinity on roots Na+ concentration of conocarpus species

C. lancifolius C. erectus 8 a

7 b

6 bc

dw) 1 - 5 de ef ef 4 fg fg

3 g

in Shoot (mgg in Shoot

+ + K 2

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig. 4.1.11 Effect of salinity on shoot K+ concentration of conocarpus species

C. lancifolius C. erectus 8 a

7 b 6

dw) bc 1 1 - 5 cd de 4 ef ef f 3

f in Roots in (mgg

+ f K 2

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig.4.1. 12 Effect of salinity on root K+ concentration of conocarpus species

C. lancifolius C. erectus 4.5 a 4

3.5 b

3 +

Na 2.5 bc + + :

cd Shoot K Shoot 1.5 de de 1 de e e e 0.5

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig.4.1. 13 Effect of salinity on shoot K+: Na+ concentration of conocarpus species

C. lancifolius C. erectus 4 a 3.5

+ 2.5

Na b + + : 2 bc

Roots K Roots 1.5 cd

1 de de e e e 0.5 e

0 Control 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl

Fig.4.1. 14 Effect of salinity on root K+: Na+ concentration of conocarpus species

4.1.4 Discussion This study indicated salinity tolerance potential of the two conocarpus species.However there was a distinct difference between the tested species used regarding the salinity tolerance. Shoot fresh and dry weights of both the conocarpus species declined with the increasing levels of salinization and the maximum reduction was noticed at 400 mM NaCl concentration. Salinity induced reduction in shoot growth in trees 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 principel 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). The ionic composition revealed that shoot and root Na+ of both the species increased significantly in response to increasing salinity levels with more accumulation in C. erectus than that of C. lancifolius. This higher accumulation of Na+ in the tissues corresponds to the lower growth and dry weights of C. erectus. 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+ 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 in conformity to present study. Inverse correlation between the shoot growth and Na+ concentration was recorded. 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 in case of present experiment, where C. lancifolius owing to higher K+: Na+ ratio showed better salt tolerance than C. erectus. The higher Na+ and lower K+ and K+: Na+ resulted in more reduction of root and shoot growth of C. erectus more as compared to C. lancifolius.

4.1.5 Conclusion Although salinity had a detrimental effect on all the growth parameters of both the conocarpus species, moreover there was a distinct genetic difference between the tested species. On the basis of better ionic homeostasis C. lancifolius had more growth and survival at higher salinity levels than that of C. erectus. 4.2 Study- 2: Response of Conocarpus erectus and Conocarpus lancifolius to salinity in combination with water stress 4.2.1 RESULTS 4.2.1.1 Morphological attributes

Salinity caused significant reduction in plant height and number of branches of both the species however when salinity was combined with water stress (1/2x field capacity), considerable reduction was observed as compared to control as well as water stress conditions (2x field capacity), as illustrated in Fig.4.2.1 & 4.2.3. Number of branches were recorded 38 (maximum) at control condition in C. lancifolius and 7 (minimum) when salinity and drought applied at 40 dSm-1 and in case of C. erectus 43 (maximum) at control condition and 13 (minimum) when salinity and drought applied at 40 dSm-1(Fig.4.2.8). Plants obtained 38 cm (minimum) height when both salinity and water stress (1/2 field capacity) were applied on C. erectus. Shoot fresh and dry weight was reduced when salinity was combined with water stress (1/2xfield capacity), the reduction was more as compared to salinity and water stress (2xfield capacity) as illustrated in Fig.4.2.4 & 4.3.5. Collar diameter decreased, with increase in salinity and water stress, collar diameter was decreased. Maximum decrease in collar diameter in C. lancifolius and C. erectusis 38% and 47% and 51% and 60% was recorded when salinity is applied with (2xfield capacity) and (1/2xfield capacity), respectively. The combined effect of both treatments and the interaction of species with water stress was significant for shoot fresh and dry weights. Shoot fresh weight declined 67%, and 64% in C. erectus and C. lancifolius respectively, when salinity (40 dSm-1) was applied with drought. The comparison of both the species indicated that C. lancifolius produced more shoot fresh and dry weight under salinity and water stress. Root growth also reduced significantly due to salinity and excessive water stress. Marked species effect was non-significant, however, the effect of both treatments and the

44 interaction of species with water stress were 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 observed for root length. Root length was 32% decreased in C. lancifolius at water stress (2xfield capacity) was combined with salinity (40 dSm-1) and there was 2% increase at water stress (1/2xfield capacity), when combined with salinity (40 dSm-1). The comparison of both the species indicated that C. lancifolius produced more root fresh and dry weight under salinity stress in drought conditions. However, when water stress (1/2xfield capacity) was combined with salinity, C. lancifolius produced more fresh and dry weight than C. erectus. When water stress (2xfield capacity) was combined with salinity both the species showed trend of retarded growth of root. Because of salt stress, number of branches decreased in C. lancifolius up to 67% in salinity and excessive water and 81% in salinity and drought. C. erectus showed 49% decline response in salinity and excessive water and 69% decrease in drought and salinity (40 dSm- 1). C. lancifolius performed better in salinity and excessive water stress as compared to salinity and drought conditions, however when compared with control conditions growth and development of both the species declined. C. erectus has 9% increase in necrosis percentage as compared to control and similarly, C. lancifolius got 4% boost in necrosis percentage as compared to control when salinity applied with drought (Fig. 4.2.9). 2.2.2 Physiological attributes Chlorophyll decreased in response to both stresses (Fig. 4.2.16), however the interactive effect was more detrimental than either of the individual stress. The effect of species, water stress, salinity and the interaction of species with water stress was found significant for chlorophyll contents. The comparison of both the species indicated that C. lancifolius produced more chlorophyll contents under salinity and water stress. When excessive water stress was combined with 10 dS m-1 salinity, C. erectus showed 45.07 spad value and C. lancifolius 54.37 spad value. When excessive water stress was combined with 20, 30 and 40 dS m-1 salinity, both the species showed attributes as 49.37, 45.37 and 42.47 spad values in C. lancifolius and 42.9, 40.07 and 38.67 spad values in C. erectus. Analysis of Chlorophyll contents revealed values i.e 47.03, 44.37, 42.03 and 36.5 spad values in C. lancifolius and 41.63, 38.9, 37.63 and 33.70 spad values in C. erectus at 10, 20, 30 and 40 dS m-1 salinity when combined with drought. Salinity and drought reduced the leaf water

45 potential with highest negative value -2.26 MPa was observed in C. erectus (Fig. 4.2.17). Osmotic potential reduced 58% at 40 dSm-1, when water stress applied at ½ field capacity as compared to control ( (Fig. 4.2.18). Salinity and water stress have negative effect on photosynthetic rate (Fig.4.2.12). Photosynthetic rate of C. erectus and C. lancifolius was decreased by enhanced in salinity and water stress. At control level, mean photosynthetic rate was maximum i.e 6.31 µmol m- 2sec-1 and 8.69 µmol m-2sec-1 respectively. The maximum decrease in photosynthetic rate was 70% and 71% in C. lancifolius and C. erectus respectively was observed at drought and 40 dS/m salinity as compared to control level. Water use efficiency and vapor pressure density in C. lancifolius and C. erectus was decreased by increased in salinity and water stress (Fig.4.2.14). Water use efficiency and vapor pressure density decreased 49%, 32% and 50%, 35% at salinity level of 40 dS/m water stress (1/2xfield capacity) respectively, as compared to control. Transpiration rate of C. lancifolius and C. erectus was decreased by increased in salinity and water stress were applied (Fig.4.2.13). The maximum decrease was observed in transpiration rate 50% and 67% at salinity level of 40 dS/m water stress (1/2xfield capacity) as compared to control in C. lancifolius and C. erectus respectively. Stomatal conductance of C. lancifolius and C. erectus was decreased in response to increased salinity and water stress (Fig.4.2.10). At control level, stomatal conductance was maximum with mean value of 226 mmol m-2sec-1 and 180 mmol m-2sec-1 of C. lancifolius and C. erectus. The maximum decrease in stomatal conductance 26% and 47% was observed at salinity level of 40 dS/m water stress (1/2xfield capacity) as compared to control, where as maximum increase in sub stomatal conductance 18% and 24% respectively, was observed at salinity level of 40 dS/m water stress (1/2xfield capacity) as compared to control 4.2.2.3 Ionic composition Salinization of the growing medium significantly increased the Na+ concentration in the leaf, stem and root of both the species. Water stress in combination with salinity, further increased Na+ concentration in both the species (Fig. 4.2.20, 21 & 22). The effect of species, water stress and salinity was found significant in all the three plant parts (leaf, stem and roots). The comparison of both species in various treatments showed that C. lancifolius accumulated significantly lower Na+ as compared to C. erectus in leaf, shoot and root.

The concentration of K+ decreased significantly at each increasing level of salinization. Water stress in combination with salinity further decreased K+ in both the species. However, water stress alone had no effect on K+ concentrations (Fig.4.2.23, 24&25). The comparison of both species in various treatments showed that C. lancifolius accumulated significantly higher K+ as compared to C. erectus in leaf, stem and root in the treatments with significant difference between the species. In present experiment the maximum Na+ contents in leaf were 5.24 mmol/g dw at salinity and water stress level of 40 dS/m. 4.2.2.4 Biochemical attributes The membrane stability index decreased in response to salinity which indicated the oxidative damage and toxic effect of salt (Fig.4.2.15). C. lancifolius showed more index value which differed significantly from C. erectus in both stress treatments. To mitigate the oxidative damage, the concentration of antioxidant enzymes increased with increasing salt stress. The activity of superoxide dismutase (SOD) increased in both species in response to salinity and this increase was 39% and 20%, in C. lancifolius and C. erectus, respectively (Fig.4.2.29). 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.4.2.31). Like other enzymes the activity of peroxidase (POD) also increased in response to salinity in both species (Fig.4.2.30). At 40 dSm-1 salinity level and water stress (1/2xfield capacity) POD activity was significantly more in case of C. lancifolius. 4.2.2.5 Soil properties For determination of various soil properties normal soil was taken and the required salinity levels were developed in the respective treatment pots by adding calculated amount of NaCl. Prior to 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 4.2.1-3). It was observed that there was slight reduction in these soil parameters which indicated the phytoremedial potential of both the species.

200.00 C. lancifolius C. erectus 180.00 a de 160.00 b f bc 140.00 cd 120.00 f e 100.00 f b e d e 80.00 g f 60.00 g Shoot (cm) height h 40.00 20.00 0.00

Fig. 4.2.1 Effect of salinity and water stress on shoot length of conocarpus species. FC: field capacity 90 C. lancifolius C. erectus a a 80 ab a bc 70 ab 60 bc d d cd de de 50 d de 40 e ef g 30 fg

20 Rootlenght (cm) 10 0

Fig. 4.2.2 Effect of salinity and water stress on root length of conocarpus species. FC: field capacity

1.20 a C. lancifolius C. erectus b 1.00 c de d b b 0.80 f ef c g 0.60 g d de e h

0.40 f g Diameter Diameter (cm)

0.20

0.00

Fig. 4.2.3 Effect of salinity and water stress on collar diameter of conocarpus species. FC: field capacity 180 a C. lancifolius C. erectus 160

140 b 120 c c b 100 cd b de 80 ef c fg cd gh cde 60 de Shoot freshweiight (g) h ef 40 f

Fig. 4.2.4 Effect of salinity and water stress on shoot fresh weight of conocarpus species. FC: field capacity

35 C. lancifolius C. erectus a 30

25 b c 20 c b cd b de 15 ef c fg cd gh cde de 10 h ef

Shootweight dry (g) f 5

Fig. 4.2.5 Effect of salinity and water stress on shoot dry weight of conocarpus species. FC: field capacity 50 C. lancifolius C. erectus a 45 a ab 40 bc bc 35 a 30 cd a ab de 25 de bc bc 20 cd cd cde 15 de e 10 Rootfreshweight (g) 5 0

Fig. 4.2.6 Effect of salinity and water stress on root fresh weight of conocarpus species. FC: field capacity

9 C. lancifolius C. erectus a 8 a ab 7 bc bc 6 a cd 5 a ab de de 4 bc bc cd cd 3 cde de e

Rootdry weight (g) 2

Fig. 4.2.7 Effect of salinity and water stress on root dry weight of conocarpus species. FC: field capacity

50 a C. lancifolius C. erectus 45 ab abc 40 bcd cd 35 de ef de 30 c d 25 f de e 20 f f

No. ofNo. branches g 15 g g 10 5 0

Fig. 4.2.8 Effect of salinity and water stress on number of branches of conocarpus species. FC: field capacity

60 C. lancifolius C. erectus a 50

40 a b

30 c Neccrosis %

20 b d e d c f d c g 10 f ef ef e e

Fig. 4.2.9 Effect of salinity and water stress on necrosis % of conocarpus species. FC: field capacity 250 a C. lancifolius C. erectus

) a

1 1 b -

S b

c 2 2

- 200 c c d d d e f e 150 g f g h i 100

50 STOMATAL CONDUCTANCE (MOL M (MOL STOMATAL CONDUCTANCE 0

4.2.10 Effect of salinity and water stress on stomatal conductance of conocarpus species. FC: field capacity

C. lancifolius C. erectus

) 1 1

- 350 S

2 a

- a ab bcd a bc M abc ab d cd 2 300 cde bcd e ef def ef gh f

250

(ΜMOLCO 2 200

150 STOMATAL STOMATAL CO

- 100 SUB 50

Fig.4.2.11 Effect of salinity and water stress on sub stomatal conductance of conocarpus species. FC: field capacity

10 C. lancifolius C. erectus

9 a

1 1 )

- S

2 8 - b b 7 c

6 cd d c 5 e c ef f 4 d g de 3 ef h f f

2 PHOTOSYNTHETIC RATE RATE M (ΜMOLPHOTOSYNTHETIC 1

4.2.12 Effect of salinity and water stress on photosynthetic rate of conocarpus species. FC: field capacity

C. lancifolius C. erectus 6

)

1 -

S 5

2 -

b b 4 bc cd de cd de 3 def ef efg fg fg fg gh g 2 h h

1 TRANSPIRATION RATE M (MMOLTRANSPIRATION

Fig. 4.2.13 Effect of salinity and water stress transpiration rate of conocarpus species. FC: field capacity

1.6 C. lancifolius C. erectus a 1.4 ab bc c d 1.2 bc c de ef 1 d de fg g ef 0.8 fg g g 0.6 g

0.4 WATER USE EFFICIENCY (PN/E) EFFICIENCY USE WATER 0.2

Fig. 4.2.14 Effect of salinity and water stress on water use efficiency of conocarpus species. FC: field capacity

90 a C. lancifolius C. erectus 80 ab ab bcd 70 ab ab abc abc bcd 60 de ab

50 cde cde 40

30 de e e e

20 e MEMBRANE STABILITY INDEX STABILITY (%) MEMBRANE 10

Fig. 4.2.15 Effect of salinity and water stress on membrane stability index of conocarpus species. FC: field capacity

70 C. lancifolius C. erectus

60 a a b bc 50 bc cd cd bcd de de cde bcde ef ef f def ef 40 f

20 CHOLOROPYHL CONTENT (SPAD) CONTENT CHOLOROPYHL 10

Fig. 4.2.16 Effect of salinity and water stress on chlorophyll contents of conocarpus species. FC: field capacity

2.5 fg C. lancifolius C. erectus

2 de d

h c c a

1.5 b a ab MPA)

- g f

ef e 1 cd cd b d

0.5 WATER POTENTIAL ( POTENTIAL WATER

Fig. 4.2.17 Effect of salinity and water stress on water potential of conocarpus species. FC: field capacity

3.5 C. lancifolius C. erectus g 3 i h e e 2.5 f

d g d MPA)

- b b 2 d f a a c e 1.5 c

OSMOTIC POTENTIAL ( POTENTIAL OSMOTIC 0.5

Fig. 4.2.18 Effect of salinity and water stress on osmotic potential of conocarpus species. FC: field capacity

1.2 C. lancifolius C. erectus

1 a abc ab abc ab bcd cd 0.8 abc bc abc abc abc cd bc 0.6 d cd c d 0.4

0.2 TUGURPOTENTIAL (MPA)

Fig. 4.2.19 Effect of salinity and water stress turgor potential of conocarpus species. FC: field capacity

C. lancifolius C. erectus 8 a 7 b 6 a b ab c b b

dw) 5 1 1 - bc cd c 4 b d de d 3 e

e in Shoot in Shoot (mgg + 2 Na f 1

Fig. 4.2.20 Effect of salinity and water stress on Na+ in shoot of conocarpus species. FC: field capacity

6 a C. lancifolius C. erectus a ab

5 b ab b

dw)

1 1 - bc bc 4 bcd bc cde cd de cd 3 b

in in Leaves(mgg e + + e

Na 2

1 f

Fig. 4.2.21 Effect of salinity and water stress on Na+ in root of conocarpus species. FC: field capacity 9 C. lancifolius C. erectus a 8 ab bc 7 c cd 6 ab a de abc abc e

dw) 5 bcd 1 1 - cd f de 4 e e 3 f

in in Roots(mgg + +

Na 1

Fig. 4.2.22 Effect of salinity and water stress Na+ concentration in leave of conocarpus species. FC: field capacity

9.00 C. lancifolius C. erectus 8.00 a 7.00

ab abc bc

dw) 1

- 6.00 bcd cde bcd 5.00 cde def 4.00 def def def ef ef ef ef

in in (mgg Shoot 3.00 + + f f K 2.00 1.00 0.00

Fig. 4.2.23 Effect of salinity and water stress on K+ concentration in shoot of conocarpus species. FC: field capacity 10 a C. lancifolius C. erectus 9

8 abc ab bcd b 7

dw) bc cde cd

1 1 6 - 5 cde def ef ef de 4 f f de e 3 e

2 in Leaves Leaves (mgg in

+ 1 K 0

Fig. 4.2.24 Effect of salinity and water stress on root K+ concentration of conocarpus species. FC: field capacity

8 a C. lancifolius C. erectus 7

6 b bc

dw) bc

1 1 5

- bcd cde cd cde 4 def ef ef 3 ef ef f def ef ef f

in in Roots (mgg 2

+ K 1

Fig. 4.2.25 Effect of salinity and water stress on leave K+ concentration of conocarpus species. FC: field capacity 9 a C. lancifolius C. erectus 8

5 +

Na 4 + : + b bc 3 dc 2 bcd Shoot K cd cd d cd d d d d 1 d d d d d 0

Fig. 4.2.26 Effect of salinity and water stress on shoot K+: Na+ of conocarpus species. FC: field capacity

5 C. lancifolius C. erectus 4.5 b 4 bc bc 3.5 b + 3

Na 2.5 b + : + b bc K 2 b 1.5 b b b b bc bc 1 bd c Leaves b c 0.5 0

Fig. 4.2.27 Effect of salinity and water stress on root K+ : Na+ of conocarpus species. FC: field capacity

4.5 a C. lancifolius C. erectus 4 3.5 3

2.5 +

Na 2 b d + : + 1.5 bc c cd 1 cd cd cd d cd d

RootsK d d 0.5 d cde d d 0

Fig. 4.2.28 Effect of salinity and water stress on leaf K+ : Na+ of conocarpus species. FC: field capacity

30 a C. lancifolius C. erectus 25 b a c b b 20 d e c f d g 15 e e h h f 10 i

SOD (U/mg SOD (U/mg protien) 0

Fig. 4.2.29 Effect of salinity and water stress on SOD of conocarpus species. FC: field capacity

25 C. lancifolius C. erectus a

ba 15 a c b b d 10 e c c d f f 5 e de POD (U/mg (U/mg POD protien) f h g 0

Fig. 4.2.30 Effect of salinity and water stress on POD of conocarpus species. FC: field capacity

80 C. lancifolius C. erectus a a 70 a b b b bc 60 c c d d 50 e e e 40 f g 30 h CAT (U/mg protien) (U/mg CAT 20 i

Fig. 4.2.31 Effect of salinity and water stress on CAT of conocarpus species. FC: field capacity

Table 4.2.1 Effect of conocarpus species on soil pHs FC: field capacity Treatments Initial values C. lancifolius C. erectus (After 1yr) (After 1yr) Control 7.62±0.06 7.60±0.06 7.61±1.89

10 dS/ m+2XFC 7.80±0.06 7.75±1.60 7.73±0.08

20 dS/ m+2XFC 7.73±1.01 7.75±1.45 7.68±0.03

30 dS/ m+2XFC 8.55±1.03 8.51±1.82 8.49±2.89

40 dS/ m+2XFC 8.44±1.02 8.34±1.72 8.31±1.32

10 dS/ m+1/2XFC 8.33±0.05 8.23±0.03 8.28±0.09

20 dS/ m+1/2XFC 8.34±0.05 8.25±1.73 8.31.85

30 dS/ m+1/2XFC 8.45±0.08 8.33±2.30 8.52±1.90

40 dS/ m+1/2XFC 7.44±0.06 7.42±2.45 7.4±1.64

Table 4.2.2 Effect of conocarpus species on soil electrical conductivity (ECe). FC: field capacity Treatments Initial values C. lancifolius C. erectus (After 1yrs) (After 1yrs) Control 4.30±1.42 4.04±0.68 4.13±0.52 10 dS/ m+2XFC 10±1.38 7.24±2.73 8.28±2.13 20 dS/ m+2XFC 20±3.68 14.45±5.88 16.61±3.75 30 dS/ m+2XFC 30±6.39 23.32±8.60 25.2±6.28 40 dS/ m+2XFC 40±4.30 31.15±1.83 33.20±1,19 10 dS/ m+1/2XFC 10±1.14 8.14±0.66 9.02±0.49 20 dS/ m+1/2XFC 20±1.43 16.59±2.50 17.73±2.06

30 dS/ m+1/2XFC 30±5.14 24.40±6.16 26.78±4.77

40 dS/ m+1/2XFC 40±7.53 33.02±6.71 34.33±0.78

Table 4.2.3 Effect of conocarpus species on soil sodium adsorption ratio (SAR). FC: field capacity Treatments Initial values C. lancifolius C. erectus (After 1yr) (After 1yr) Control 6.43±1.41 5.33±1.59 5.47±1.17

10 dS/ m+2XFC 26.7±3.8 21.64±4.34 23.36±3.26

20 dS/ m+2XFC 34.72±3.64 31.21±0.46 33.1±2.10

30 dS/ m+2XFC 43±4.41 35.71±3.5 37.1±9.59

40 dS/ m+2XFC 58±3.38 48.2±0.46 5131±2.10

10 dS/ m+1/2XFC 29.52±3.20 23.56±1.58 26.37±1.15

20 dS/ m+1/2XFC 37.5±3.71 33.8±3.19 35.4±2.10

30 dS/ m+1/2XFC 45±5.32 36.1±1.57 39.9±4.22

40 dS/ m+1/2XFC 61.4±4.34 50.9±5.5 53.8±3.89

4.2.4 Discussion Increasing levels of salt in the growing medium caused significant reduction in plant height, collar diameter and number of branches. However, additive effect of water stress, caused more reduction as compared to individual stress on growth parameters. The comparison of both the species depicted that the effect of salinity on these parameters was more in the case of C. erectus. Similar observations have also been reported by El Atta et al. (2012) and EI-Juhany and Aref (2005). Shoot and root fresh and dry weights were found to follow similar reduction trend alone aswell as combined with water stress. The reduction was more in C. erectus as compared to C. lancifolius. The comparison of both the species also indicated that C. lancifolius produced more shoot and root fresh and dry weights under salinity and water stress environment. Results are in conformity with finding of 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. Marked reduction in shoot weight was due to spontaneous reduction in stem height, diameter 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). C. erectus produced more root length than that of C. lancifolius. This trend indicates that conocarpus 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 retaeded 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 succeeding levels and have impact on all features of plant metabolism. Chlorophyll contents also followed decreaing trend in response to both applied stresses, however, the interactive effect was more detrimental than the either of the individual stress. 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 mainly attributed to damage to chloroplasts caused by reactive oxygen species (Smirnoff, 1995).

Present results indicated that C. erectus is more sensitive to water stress (drought) as compared to excessive water stress. Salinity significantly increased the Na+ concentration in the leaf, stem and root of both species. Water stress in combination with salinity, further increased Na+ concentration in both the species, in drought condition as compared to salinity and excessive water. C. lancifolius accumulated significantly lower Na+ as compared to C. erectus in leaf, stem and root. Similar results regarding increased ionic concentration in response to salinity were 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 salt tolerant behavior known as ion exclusion, which, 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 K+ concentration in both the species. C. lancifolius accumulated significantly higher K+ as compared to C. erectus in leaf, stem and root. Decreased K+ uptake in response to salinity and water stress have been reported by Khalil et al., 2012; Marcar et al., 1991. The K+ has a key role in salt tolerance and uptake of K+ is decreased in the presence of Na+ (Fox and Guerinot, 1998). The reduction of K+ concentration was found in all the three components which indicated that Na+ suppressed K+ uptake. The higher concentration of Na+ in case of C. erectus indicated that ion exclusion was poorly operative in conocarpus species so it had low K+ concentration as compared to C. lancifolius. 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. C. lancifolius showed higher index value than C. erectus in both stress treatments which showed less membrane damage in this species. To mitigate the oxidative damage, the concentration of antioxidant enzymes increased 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

66 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 C. lancifolius which is relatively more tolerant than C. erectus. It may be concluded that SOD have crucial role in modulating the salt tolerance of the two conocarpus 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 increased in both the species but higher, in case of C. lancifolius than C. erectus under salt and water stress conditions. The increased activity of catalase seems to be responsible for the detoxification of H2O2 more in case of C. lancifolius than C. erectus. 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. Some other researchers Patel and Saraf (2013), Sekmen et al. (2012) and Mittal et al. (2012) in J. curcas L., G. oblanceolata and B. juncea respectively also found increased acitivties of this enzyme in response to salinity.

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. This showed that at higher salinity level C. lancifolius was more involved in the detoxification of H2O2 than C. erectus.Increased activity of POD was also found by Wang et al. (2009). Zhang et al. (2013) observed that the activities of POD in the B. papyrifera stem were significantly increased under salinity stress whereas in case of leaves and roots it was decreased. Morais et al. (2012) reported 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 C. lancifolius is positively correlated with its alt tolerance. 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 C. lancifolius caused more amelioration as compared to C. erectus.

4.2.5 Conclusion This study explored the genetic potential of both conocarpus species for two very important abiotic stresses i.e. salinity and water shortage. Researcher observed that C. lancifolius has more tolerance for salinity stress than C. erectus. On the other hand the former species was relatively sensitive to water stress than the later one. Based on the results the results it can be recommended that if there is facing dual problem of excessive water and water shortage then C. lancifolius can be grown for the rehabilitation of marginal lands. 4.3 Study-3: Comparative role of Conocarpus lancifoliusand Conocarpus erectus in the phytoremediation of the salt affected soil 4.3.1 Results 4.3.1.1 Morphological attributes The plant growth parameters including plant height, stem diameter, above ground biomass were determined. The plants of both conocarpus species were raised on three different sites having high (site-II), medium (site-I) and low (site-III) saline conditions differed significantly regarding growth parameters. The plant height, stem circumference and aboveground biomass was found maximum on site-III with low salinity after 24 months of transplantation. After two years, data regarding different growth parameters showed that C. lancifolius performed better as compared to C. erectus in saline environment. The plant height of conocarpus species indicated that it was decreased significantly under salt stress (Fig. 4.3.1, 2, & 3). The highest reduction in plant height as 78.67 cm in C. erectus and 398 cm in C. lancifolius was recorded on site-II having highest value of Ece, followed by 189 cm, 261 cm and 476 cm, 561cm in C. erectus and C. lancifolius on site-I and site-III, respectively. Both the species showed reduction in plant height which aggraveted with increase in salt stress. Furthermore, the interaction between salinity and species was significant (p≤0.05). On all three sites, reduction in diameter of C. lancifolius was recorded as 4.45 cm, 3.59 cm and 3.29cm on site-III, site-I and site-II, respectively, while the highest decrease was noted in C. erectus on site-I, site-II and site-III as 0.5933 cm, 0.5333 cm and 0.7333cm, respectively (Fig. 4.3.1, 2, & 3). In the same way salt stress level on site-II (highest level of salinity) showed 36% reduction in aboveground biomass as compared to site-III (low level of salinity) in C. erectus and 26% reduction in aboveground biomass as compared to site-III (low level of salinity) in C. lancifolius.Furthermore, 25% reduction in C. erectus and 17%

68 decrease in C. lancifolius was observed in site-I (medium level of salinity), as compared to site-III (low level of salinity) illustrated in Fig. 4.3.1, 2, & 3. 4.3.1.2 Ionic composition Plants exposed to various salinity levels exhibited a significant enhancement in their leaf sodium (Na+) contents (Fig. 4.3.5). Results showed that leaves of C. lancifolius accumulated less Na+ contents as compared to C. erectus. Under site-III conditions, minimum Na+ activity was recorded in C. lancifolius (2.97 mg/g D.Wt.) while maximum was observed in C. erectus (3.77 mg/g D.Wt.). Comparison of species regarding Na+ contents in shoots indicated that C. erectus had 53% increase on site-II as compared to site-III, while 67% increase was observed in C. lancifolius on site-II as compared to site-III. Moreover, data presented in (Fig. 4.3.5) revealed that 33% increase in C. erectus on site-I was recorded as compared to site-III. Salt stress had less percentage increase in leaf Na+ contents as compared to shoot. A comparison of ionic composition showed a significantly maximum K+ concentration in shoot as 5.71 mmol g-1 dwt in C. lancifolius on site-III was noted, whereas minimum K+ concentration was recorded in C. erectuson on site-II (Fig. 4.3.6&7). In C. lancifolius K+ concentration in leaf was 4.52 mmol g-1 dwt on site-III and 2.42 mmol g-1dwt K+ concentration in C. erectus on site-II (Fig. 4.3.6&7 ). Maximum K+: Na+ was recorded in C. lancifolius as 2.32 on site-III and minimum (0.62) in shoot of C. erectuson in site-II (Fig. 4.3.8). Ionic composition in field condition of both conocarpus species showed that C. lancifolius is more tolerant to salinity as compared to C. erectus. 4.3.1.3 Biochemical attributes The activity of superoxide dismutase (SOD) increased in both species in response to salinity, which was maximum (22.94U/mg Proteins) at site-II and minimum (13.09 U/mg Proteins) at site-III (Fig.4.3.10). Plants response to different salinity levels showed a significant increase of SOD in the leaves (Figure 4.3.10). Results showed C. lancifolius increased with increase in salinity levels in CAT activity as 67.94, 60.48 and 54.34 U/mg Proteins on site-II, site-I and site-III, respectively. Data revealed that C. erectus response on site-III, 31% more as compared to site-III and 12% boost on site-II, as compared to site-III (Figure 4.3.12). C. erectus had minimum (5.39 U/mg Proteins) activity on site-III and maximum (9.41U/mg Proteins) value on site-II of POD, while C. lancifolius performed on site-I, site-II and site-III as 14.81, 18.76 and 9.77, respectively (Figure 4.3.11).

4.3.1.4 Chemical properties of the soil Soil analysis of the field showed that it was a saline sodic field as it had values of pHs, ECe and SAR. Within each plot these parameters were having different values, 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. Trees consume various salts and consequently ameliorate these soil parameters. With the passage of time the pHs, ECe and SAR of soil slowly decreased and this reduction was more in case where C. lancifolius plants were raised as compared to C. erectus (Table. 4.3.4-6). Comparison of species showed that pHs of the soil fluctuates little bit with decreasing trend as increase in - salinity levels. The highest reduction was observed in Ece of soil where C. erectus (8.4 dSm 1) was raised and decreased where C. lancifolius (7.067 dSm-1) was planted on site-III. Data recorded after two years on site-II, where C. erectus was raised, showed 12% decrease in Ece as compared to initial value, whereas C. lancifolius showed decline of 20%, as compared to the initial value. Both the species showed reduction in soil Ece after planting the conocarpus species. Furthermore, the interaction between salinity and species was significant (p≤0.05). Under different sites, reduction in soil SAR, after planting the conocarpus species, C. lancifolius depicted results as 20.99 mmolL-1 cm, 25.99 mmolL-1 and 33.52 mmolL-1 on site- III, site-I and site-II respectively, while less decrease was noted in C. erectus on site-I, site-II and site-III as 28.64 mmolL-1 cm, 39.20 mmolL-1 and 22.23 mmolL-1 (Table 4.3.6). Similarly salt stress level on site-II (highest level of salinity) showed 15% reduction in soil SAR after two years of planting of C. erectus whereas, C. lancifolius showed 21% decrease in soil SAR after two years (Table 4.3.6). The maximum reduction in pHs, ECe and SAR was observed in of C. lancifolius on site-III. This reduction was minimum in case of C. erectus on site-II having high salinity. The comparison of C. erectus and C. lancifolius indicated that latter have more ameliorative role as compared to former. 4.3.1.5 Soil organic matter content The leaf litter addition and its decomposition over the time added significant amount of organic matter (OM) in the soil. Marked addition was noted on site-III. The increase in OM content was more in the case of C. lancifolius and it was least in the case of C. erectus (Table. 4.3.1). Data regarding organic matter indicated that the highest increase in OM, was recorded in C. lancifolius under site-III was 0.95 %, followed by 0.92 % in C. erectus, while

70 least increase was determined on site-II in C. lancifolius (0.73%) and in in C. erectus (0.71%). 4.3.1.6 Bulk density and infiltration rate There was marked importance in the physical properties of the soil i.e. bulk density and infiltration rate 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 C. lancifolius on site-III after 24 months of tree plantation. This reduction was minimum in case of C. erectus on site-II (Table 4.3.2). On the other hand, C. lancifolius plants showed maximum increase in infiltration rate after two years, compared to C. erectus plants (Table.2-3). Bulk density of soil where conocarpus species were planted indicated significant decrease, under higher levels of salt stress (Fig. 4.3.1, 2, & 3). The highest reduction in bulk density (29%) of soil was obseved on site II, where C. erectus was raised and 23% decrease in bulk density of soil, where C. lancifolius was planted, on site II. Both the species showed reduction in bulk density with increase in salinity levels as 24% and 16% in C. erectus on site I and site-III, respectively. Results revealed that 22% and 14% decrease in C. lancifolius on site I and site-III respectively (Fig. 4.3.1, 2, & 3). Mutual interaction between salinity and species was also significant (p≤0.05). On three sites, infiltration rate increased i.e 0.58 cmh-1 on site-III, which is 31% more as compared to the initial value two year before planting of C. lancifolius and minimum increase (9%) was noted on site-II, where C. erectus was raised (Fig. 4.3.1, 2, & 3).

C. lancifolius C. erectus a 600

b 500 c

400

300

200 b

b b plant height (cm) 100

0 Site I Site II Site III

Fig. 4.3.1 Effect of salinity and sodicity on plant height of conocarpus species

C. lancifolius C. erectus 5 a 4.5

4 b 3.5 c

2.5

1.5 diameters diameters (cm)

1 de d d 0.5

0 Site I Site II Site III

Fig. 4.3.2 Effect of salinity and sodicity on stem circumference of conocarpus species

C. lancifolius C. erectus 6000 a

5000

b 4000 b

3000

c 2000 c

c Above ground biomass Above groundbiomass (gms)

1000

0 Site I Site II Site III

Fig. 4.3.3 Effect of salinity and sodicity on above ground biomass of conocarpus species

6 C. lancifolius C. erectus

a 5 ab bc

dw) cd 1 1

- de

e 3

in Shoot in (mgg 2

+ Na

0 Site I Site II Site III

Fig.4.3.4 Effect of salinity and sodicity on Na+ in shoot of conocarpus species. FC: field capacity

C. lancifolius C. erectus 6 ab a c 5 cd de

4 dw)

1 1 e -

in in (mgg leaves

+ + Na 1

0 Site I Site II Site III

Fig. 4.3.5 Effect of salinity and sodicity stress on Na+ in leaf of conocarpus species. FC: field capacity

7 C. lancifolius C. erectus a 6 cd

5 bc ab cb

dw) 4

1 b -

in Shoot in (mgg + + K 1

0 Site I Site II Site III

Fig. 4.3.6 Effect of salinity and sodicity on K+ concentration in shoot of conocarpus species. FC: field capacity

C. lancifolius C. erectus 9

8 a

7 ab

dw) 6

1 1 bc -

5 c c c 4

in leaves (mgg 3

+ K 2

0 Site I Site II Site III

Fig. 4.3.7 Effect of salinity and sodicity K+ concentration in leaf of conocarpus species. FC: field capacity

C. lancifolius C. erectus 3

a 2.5

2 b

+ Na

+ + : bc 1.5 cd

Shoot K Shoot cd 1 d

0.5

0 Site I Site II Site III

Fig. 4.3.8 Effect of salinity and sodicity on shoot K+: Na+ of conocarpus species. FC: field capacity

C. lancifolius C. erectus 1.8 a 1.6

1.4 b

1.2 c

+ 1

Na cd + : + 0.8 e de

0.6 Leaf K

0.4

0.2

0 Site I Site II Site III

Fig. 4.3.9 Effect of salinity and sodicity on leaf K+ : Na+ of conocarpus species. FC: field capacity

C. lancifolius C. erectus 30

a 25 b c 20 b b 15 e

SOD (U/mg SOD (U/mg protien) 5

0 Site I Site II Site III

Fig. 4.3.10 Effect of salinity and sodicity on SOD of conocarpus species. FC: field capacity

C. lancifolius C. erectus 25

b 15 c d 10 e

5 POD (U/mg POD (U/mg protien)

0 Site I Site II Site III

Fig. 4.3.11 Effect of salinity and sodicity on POD of conocarpus species. FC: field capacity

C. lancifolius C. erectus 80 a b 70 c d

e 60 f

20 CAT (U/mg CAT (U/mg protien) 10

0 Site I Site II Site III

Fig. 4.3.12 Effect of salinity and sodicity on CAT of conocarpus species. FC: field capacity

Table 4.3.1 Effect of conocarpus species on soil organic matter percentage (% age) Treatments Initial values C. lancifolius C. erectus (After 2yrs) (After 2yrs) Site – I 0.81±1.09 0.87±0.71 0.83±1.61

Site – II 0.68±2.44 0.73±2.95 0.71±1.65

Site – III 0.91±1.84 0..95±1.04 0..92±3.24

Table 4.3.2 Effect of conocarpus species on soil bulk density (g cm-3) Treatments Initial values C. lancifolius C. erectus (After 2yrs) (After 2yrs) Site – I 1.62±1.46 1.51±0.46 1.58±2.10

Site – II 1.68±1.39 1.63±3.5 1.67±9.59

Site – III 1.42±0.41 1.34±0.46 1.35±2.10

Table 4.3.3 Effect of conocarpus species on soil infiltration rate (cm hr-1) Treatments Initial values C. lancifolius C. erectus (After 2yrs) (After 2yrs) Site – I 0.38 0.47±0.41 0.43±0.46

Site – II 0.33 0.39±2.25 0.40±3.5

Site – III 0.44 0.58±1.14 0.52±0.46

Table 4.3.4 Effect of conocarpus species on soil pH Treatments Initial values C. lancifolius C. erectus (After 2yrs) (After 2yrs) Site – I 7.73±1.85 7.65±1.73 8.71.85

Site – II 7.78±1.90 8.73±2.30 8.72±1.90

Site – III 8.07±1.06 7.92±2.45 7.94±1.64

Table 4.3.5 Effect of conocarpus species on soil ECe (dS m-1) Treatments Initial values C. lancifolius C. erectus (After 2yrs) (After 2yrs) Site – I 23.60±0.76 17.14±0.66 19.02±0.49

Site – II 31.65±1.66 24.59±2.50 26.73±2.06

Site – III 17.19±1.77 14.40±6.16 15.78±4.77

Table 4.3.6 Effect of conocarpus species on soil SAR ((mmol L-1)1/2) Treatments Initial values C. lancifolius C. erectus (After 2yrs) (After 2yrs) Site – I 49.42±2.5 43.8±3.19 45.4±2.10

Site – II 53.83±1.3 46.1±1.5 49.9±2.22

Site – III 42.13±0.58 32.9±3.5 34.8±3.89

4.3.4 Discussion

The nursery plants were transplanted to the field at the age of 6 months on three different sites with different salinity levels in different compartments of Shorkot Irrigated Plantation. 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 C. lancifolius (90%) than C. erectus (82%). The plants were irrigated with available canal water and as grew taller, they become vigorous at spacing of 10x6 feet. Based on the visual health, three plants were selected for data collection, planted at spacing of. There was recording of more height and collar diameter, in C. lancifolius than of C. erectus after 24 months of transplantation. Overall comparison of both species indicated that C. lancifolius produced significantly more height, diameter and above ground biomass than C. erectus. Similar type of growth and survival pattern was also observed by Ashraf et al. (2008) while studying the growth performance of tree species in the salt affected field having salinity in the range of 4-25 dS m-1. Number of branches was more in case of C. erectus due to its bushy nature as compared to C. lancifolius. However the number of leaves was consistently more in C. erectus than C. lancifolius. Leaf ionic composition showed a significantly higher Na+ concentration in the shoot and leaves of C. erectus. On the other hand, these leaves had the minimum concentration of K+. This type of ionic composition corresponds to more accumulation of Na+ in shoot and leaves of C. erectus and less K+ as compared to C. lancifolius leaves and shoots. Maintaining higher K+ or lower Na+ in C. lancifolius 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). Moreover, under salinity stress plants try to restrict accumulation of toxic ions like Na+ and Cl- in their leaves so that leaf fall can alleviate these harmful ions. This is another biological 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). To mitigate the oxidative damage, the concentration of antioxidant enzymes increases with increasing salt stress. In the present field experiment SOD activity increased under saline environment. Increased activities of SOD under salt stress conditions were previously

80 observed by many researchers like Khan and Panda, 2008; Mittal et al., 2012 and Hu et al.,

2011. Catalase (CAT) is an important enzyme which converts H2O2 to H2O and O2 (Chen et al., 2011). The activity of catalase increased in both the species with more increase in case of C. lancifolius than C. erectus with increase in salinity. The increased activity of catalase seems to be responsible for the detoxification of H2O2 more in case of C. lancifolius than C. erectus. 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. 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. This shows that at higher salinity levelC. lancifolius was more involved in the detoxification of H2O2 than C. erectus. Increased activity of POD was also found by Wang et al. (2009).

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 on site II having highest level of salinity. Similar results were found by Basavaraja et al. (2010). A gradual decrease in pHs, Ec and SAR was observed in both species with increase in their growth. However, reduction was more in case of C. lancifolius as compared to C. erectus. Phytoremediation requires some 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 2+ water and converted into H2CO3. This acid reacts with CaCO3 and Ca ions are released + - + 2+ H2CO3 is dissociated in to H and HCO3 and the reaction of H with CaCO3 releases Ca 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 may collectively help in dissolution of calcite. All these processes are help 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) elucidated that plants having more CO2 production capacity result in more acidification of soil and resultantly more removal of Na+ out of the

81 profile. Release of Protons (H+) and organic acids from plant roots is thought to be 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 rhizosphere of C. lancifolius as compared to C. erectus might be responsible for more dissolution of calcite with a resultant release of Ca2+ and ultimately more reduction in SAR. The reduction in pH value 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 of C. lancifolius and C. erectus 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 which was more evident for C. lancifolus than C. erectus. 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) 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 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 plants of C. lancifolius due to more litter 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 Conocarpus plantation. 4.3.5 Conclusions As a result of increasing population pressure and shrinkage of normal soil and water resources, utilization of salt-affected soils and waters for the agriculture productivity. This study showed that the management and utilization of saline waters on salt-affected lands can be done successfully grow salt tolerant Conocarpus species. The comparison of both species indicated that C. lancifolius produced more biomass and provided amelioration in the soil properties than C. erectus.

CHAPTER-5 SUMMARY

Salt-affected soils may be categorised as saline, sodic or saline-sodic in nature. Higher concentration of soluble salts or exchangeable sodium in salt affected soils affecting normal growth of most of the crops and trees. 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. Nevertheless, 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.

Drought spell are determintal for sustained plant growth and vigour. Scarcity of water is increasing day by day due to climatic changes, which negatively affects the growth and productivity of crops and trees. Excessive water also affect the soil and crop environment, through the depletion of oxygen, leading to reduced root respiration and other imperative plant life processes, as well as the production and accumulation of phytotoxic compounds, such as ethylene, in plant roots and soil. As a result of salt and water stress huge yield losses in crops and retarded growth of trees have been noticed.

Reclamation of the salt affected soils have been in vouge using different techniques like, chemical amendments, tillage operations, crop based interventions, water effiency related methods, and electrical currents. However, in recent times, the most effective and low cost reclamation approach is the phytoremediation. Through 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. Many tree species are salt tolerant and have great potential for timber, livestock and agro forestry. Conocarpus species can survive

83 at higher levels of salinity and sodicity, and considered helpful for the reclamation of the salt affected soils.

Present project has been conducted for determining physiological, biochemical and phytoremedial characterization of two conocarpus species of different origin for salt affected soils. The species used included Conocarpus erectus and Conocarpus lancifolius.

The specific objectives of the project are the followings:

 Assessment of salinity tolerance potential of conocarpus species.

 Investigation and clarification of morpho-physiological, biochemical and ionic attributes for salt and water stress in conocarpus species.

 Study of the ameliorative and comparative effects of conocarpus species on the physicochemical properties of salt affected soils.

To articulate the above objectives in mind, three studies were carried out. In the first experiment, four month old seedlings of both species were transplanted in half strength Hoagland nutrient solution having four treatments (control, 100, 200, 300 and 400 mM NaCl). After 10 weeks, the plants were harvested and the data regarding shoot and root fresh weights and the respective plant 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 ionic composition (Na+ and K+) showed that C. lancifolius was more tolerant to salinity than C. erectus. The seedlings of C. erectus could not survive at 400 mM NaCl due to ion toxicity.

Both the conocarpus species were further studied under the combination of salinity and water stress in the pots. The salinity levels (control, 10, 20, 30 and 40 dS m-1) were developed in the respective treatment pots by adding measured amount of NaCl. After the establishment of the plants water stress (half of field capacity and double of field capacity) was applied during entire duration of the experiment. Soil samples were taken from each pot and analyzed for different properties (pHs, ECs and SARs) before transplanting and after harvesting the trees. Chlorophyll content were determined before harvesting the plants at end of the year. Data of shoot fresh and dry weights, root fresh and dry weights, shoot and root lengths, stem diameter and No. of branches were noted.The results demonstrated that C.

84 lancifolius produced more shoot and root biomass and better responded to salinity and water stress with double of field capacity. Postharvest soil analysis showed negligible changes in the soil properties.

Before harvesting, parameters regarding gas exchange i.e. photosynthetic rate (A), transpiration rate (E), intrinsic CO2 concentration (Ci), stomatal conductance (gs) and photosynthetic water use efficiency (WUE) were measured by using CIRAS-3 (PP System, Amesbury, MA, USA).The maximum decrease in photosynthetic rate was 70% and 71% in C. lancifolius and C. erectus, respectively at drought and 40 dS/m salinity as compared to control level.The maximum decrease was observed in Water use efficiency and vapor pressure density 49%, 32% and 50%, 35% at salinity level of 40 dS/m+ water stress (1/2xfield capacity) as compared to control. Marked decrease was observed in transpiration rate 50% and 67% at salinity level of 40 dS/m+ water stress (1/2xfield capacity) as compared to control.The maximum decrease in stomatal conductance 26% and 47% was observed at salinity level of 40 dS/m and water stress (1/2xfield capacity) as compared to control, where as maximum increase in sub stomatal conductance 18% and 24% was observed at salinity level of 40 dS/m+ water stress (1/2xfield capacity) as compared to control.

The leaf water potential (ѱw) was determined by using Scholander type pressure chamber apparatus and osmotic potential (ѱs) by using vapor pressure Osmometer. The turgor potential (ѱp) was measured by the determination of difference between water potential (ѱw) and osmotic potential (ѱs) values. The salinity and drought imposed has greatly reduced the leaf water potential and highest negative value -4.48 MPa was observed in C. erectus. Osmotic potential reduced at 40 dSm-1and water stress applied at half field capacity (58%) as compared to control.

Antioxidants enzymes like superoxide dismutase (SOD), peroxidases (POD) and catlase (CAT) were also determined. The activity of superoxide dismutase (SOD) increased in both species in response to salinity and this increase was 39% and 20% respectively, for C. lancifolius and C. erectus at 40 dSm-1 salinity level and water stress (1/2xfield capacity). POD activity was maximum in case of C. lancifolius as compared to C.erectus.

Ionic concentrations of Na+ and K+ were determined in leaves and shoot, root and leave parts. Water stress(1/2xfield capacity) in combination with salinity, Na+ concentration increased in both the species and more increase was noted in C. erectus. The comparison of both species in various treatments showed that C. lancifolius accumulated significantly lower Na+ as compared to C. erectus in leaf, shoot and root and in case of K+ showed that C. lancifolius accumulated significantly higher K+ as compared to C. erectus in all parts. Postharvest soil analysis showed non-significant changes in soil pH, whereas Ec and SAR showed significant response in soil properties after one year.

Third final study, these species were raised on three different sites having diverse levels of salinity in the salt affected field and their growth, ionic and biochemical properties were recorded after two years. The changes in the soil chemical and physical properties were also determined. The comparison of both species indicated that C. lancifolius produced more biomass and caused more reduction in the soil chemical properties like pHs, ECe and SAR as compared to C. erectus, 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 of C. lancifolius than C. erectus.

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 C. lancifolius showed more tolerance than C. erectus due to its better physiological and biochemical mechanisms of salt tolerance. On the other hand C. lancifolius provided more phytoremediation of the saline-sodic soil, which could be grown on such lands as a good source of timber, fuel wood and forage for livestock, simultaneously rehabilitating barren lands.

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