2002-ag-1672 (m. asif iqbal) ph.d. thesis...

Response of maize (Zea mays) to auxin producing plant growth promoting rhizobacteria under saline conditions

By

Muhammad Asif Iqbal

M.Sc. (Hons.) Agriculture 2002-ag-1672

A thesis submitted in partial fulfillment of the requirements for the

Degree of

DOCTOR OF PHILOSOPHY In

Soil Science

Institute of Soil & Environmental Sciences FACULTY OF AGRICULTURE

UNIVERSITY OF AGRICULTURE, FAISALABAD

2014

To,

The Controller of Examinations, University of Agriculture, Faisalabad.

We, Supervisory Committee certify that the contents and form of thesis

submitted by Muhammad Asif Iqbal, Regd. No. 2002-ag-1672 have been found

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

Examiner (s) for the award of degree.

SUPERVISORY COMMITTEE

CHAIRMAN (Dr. Muhammad Khalid)

MEMBER (Dr. Zahir Ahmad Zahir)

MEMBER (Dr. Rashid Ahmad)

DEDICATED

TO

My graceful and polite father

My most loving mother

My brothers and sister

Who live in my mind and soul

Whose love is more precious

Than pearls and diamonds

Who are those whom I say my own

Whose love will never change

Whose prayers will never die

&

Who are nearest, dearest and deepest

ACKNOWLEDGEMENTS

All praise to ALMIGHTY ALLAH alone, the most merciful and the most compassionate and his Holy Prophet ‘MUHAMMAD’ (Peace be upon him) the most perfect and exalted among and even of ever born on the surface of earth, who is, forever a torch of guidance and knowledge for the humanity as a whole.

The work presented in this manuscript was accomplished under the inspiring guidance, gorgeous assistance, constructive criticism and enlightened supervision of Dr. Muhammad Khalid, Professor, Institute of Soil & Environmental sciences, University of Agriculture, Faisalabad. His efforts toward the inculcation of the spirit of constant work and the maintenance of professional integrity besides other invaluable words of advice will always serve as beacon of light throughout the course of my life. I take this humblest opportunity to express my deepest sense of gratitude and thankfulness to him.

It is matter of great pleasure and honor for me to express my gratitude and appreciation to the members of my Supervisory Committee, Dr. Zahir Ahmad Zahir, Professor, Institute of Soil & Environmental sciences, University of Agriculture, Faisalabad and Dr. Rashid Ahmad, Department of Crop Physiology, University of Agriculture, Faisalabad, under whose kind and scholastic guidance, keen interest and constant encouragement, I completed my research. I am thankful to all my School teachers especially, Muhammad Mumtaz who helped and guided me during the entire course of my studies.

A deep sense of appreciation and special thanks to Dr. M. Usman, Dr. S.M. Shahzad, Dr. Naveed Iqbal and Dr. Shahid Hussain, without whom generous guidance, encouragement and immense help, I would have not been able to complete my work. Friends are the comrades of the battle, the battle to generate knowledge, sift myths and facts and to remove ambiguity. They were co–sharer of my struggle and my work. I express my thankful feelings for my friends, Dr. Ahmad Nawaz, Dr. Sana Ullah, Dr. Jawad ul Hassan, Habib Ali, Kashif Anjum, Muhammad Aon, M. Hamid Rasool, Mujahid Manzoor, Sami Ullah, Zeeshan Aslam and M. Kashif Malana.

In the end I want to convey my love and affection to the apples of my eye (Areeba, Hunzla, Shaheera, Minahil, Amna, Aisha, Uswa, Laiba, Meher, Affan, Mahad, Hassan, Abubakar, Ibrahim, Abdullah Talat, Usama and Ahmad Afroz). They were always been a source of inspiration for me. My mind always refreshed in their company and I was able to shed the burden of sorrows, distress and hopelessness. They were my cutest companions during this journey. I regard them as my deariest and precious findings. They will always remain in my heart and will regulate its beat when pressure of life will try to stop it and will serve as a source of light when shadows of time will try to blind my eyes. May Allah bless all these people with long, happy and peaceful lives (Ameen).

Muhammad Asif Iqbal

DECLARATION

I hereby declare that the contents of the thesis “Response of Maize (Zea mays) to Auxin

Producing Plant Growth Promoting Rhizobacteria under Saline Conditions” are product of

my own research and no part has been copied from any published source (except the

references, standard mathematical or genetic models/formulae/protocols, etc.). I further

declare that this work has not been submitted for award of any diploma/degree. The

university may take action if the information provided is found inaccurate at any stage. In

case of any default the scholar will be proceeded against as per HEC plagiarism policy.

Signature of the student: ___________________

Name: Muhammad Asif Iqbal

Regd. No. 2002-ag-1672

LIST OF CONTENTS

CH# TITLE Page#

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 5

3. MATERIALS AND METHODS 33

4. RESULTS 48

5. DISCUSSION 133

6. SUMMARY 147

LITERATURE CITED 150

LIST OF TABLES

No. TITLE Page1. Physico-chemical characteristics of soil used for pot trial 37 2. Physico-chemical characteristics of soil used for field trial 38 3 Growth of rhizobacterial strains in broth tubes after 3 days

under salt stress conditions 49

4 Growth of rhizobacterial strains in broth tubes after 3 days under salt stress conditions

50

5 Amount of IAA equivalents (µg mL-1) produced by the selected rhizobacterial strains

52

6 Effect of rhizobacterial inoculation on total plant dry weight of maize seedlings under salt stressed axenic conditions

60

7 Effect of rhizobacterial inoculation on shoot root ratio of maize seedlings under salt stressed axenic conditions

61

8 Effect of rhizobacterial inoculation (with and without L-TRP) on shoot root ratio of maize at different salinity levels

73

9 Characterization of selected rhizobacterial strains 132

LIST OF FIGURES

No. TITLE PAGE

1 Effect of rhizobacterial inoculation on shoot length of maize seedlings under salt

stressed axenic conditions. 54

2 Effect of rhizobacterial inoculation on root length of maize seedlings under salt

stressed axenic conditions 56

3 Effect of rhizobacterial inoculation on shoot dry weight of maize seedlings under salt


4 Effect of rhizobacterial inoculation on root dry weight of maize seedlings under salt


5 Effect of rhizobacterial inoculation on plant height of maize under salt stressed pot

conditions 64

6 Effect of rhizobacterial inoculation on fresh biomass of maize under salt stressed pot

conditions 66

7 Effect of rhizobacterial inoculation on root length of maize under salt stressed pot

conditions 68

8 Effect of rhizobacterial inoculation on root weight of maize under salt stressed pot

conditions 71

9 Effect of rhizobacterial inoculation on grains yield of maize under salt stressed pot

conditions 72

10 Effect of rhizobacterial inoculation on 1000-grain weight of maize under salt stressed

pot conditions 75

11 Effect of rhizobacterial inoculation on cob biomass of maize under salt stressed pot

conditions 76

12 Effect of rhizobacterial inoculation on chlorophyll content (SPAD values) of maize

under salt stressed pot conditions 78

13 Effect of rhizobacterial inoculation on electrolyte leakage of maize under salt

stressed pot conditions 80

14 Effect of rhizobacterial inoculation on photosynthetic rate in shoot of maize under

salt stressed pot conditions 82

15 Effect of rhizobacterial inoculation on transpiration rate of maize under salt stressed

pot conditions 83

16 Effect of rhizobacterial inoculation on stomatal conductance of maize under salt


17 Effect of rhizobacterial inoculation on substomatal CO2 concentation of maize under

salt stressed pot conditions 86

18 Effect of rhizobacterial inoculation on proline content of maize under salt stressed

pot conditions 88

19 Effect of rhizobacterial inoculation on total solube sugars of maize under salt


20 Effect of rhizobacterial inoculation on total phenolics of maize under salt stressed

pot conditions 91

21 Effect of rhizobacterial inoculation on ascorbate peroxidase vales of maize under salt


22 Effect of rhizobacterial inoculation on K+/Na+ of maize under salt stressed pot

conditions 95

23 Effect of rhizobacterial inoculation on nitrogen concentration in shoot of maize


24 Effect of rhizobacterial inoculation on phosphorous concentration in grains of maize


25 Effect of rhizobacterial inoculation on crude protein content in grains of maize


26 Effect of soil salinity on grain yield per plant of maize in pots 101

27 Effect of rhozobacterial inoculation on grain yield per plant of maize

in pots

101

28 Effect of soil salinity on total chlorophyll contents (SPAD values) of

maize in pots

102

29 Effect of rhozobacterial inoculation on total chlorophyll contents

(SPAD values) of maize in pots

102

30 Effect of soil salinity on proline contents of maize in pots 103

31 Effect of rhozobacterial inoculation on proline contents of maize in

pots

103

32 Effect of soil salinity on electrolyte leakage of maize in pots 104

33 Effect rhizobacterial inoculation on electrolyte leakage of maize in

pots

104

34 Effect of rhizobacterial inoculation on plant height of maize under salt stressed field

conditions 106

35 Effect of rhizobacterial inoculation on grain yield of maize under salt stressed field

conditions 107

36 Effect of rhizobacterial inoculation on fresh biomass of maize under salt stressed

field conditions 109

37 Effect of rhizobacterial inoculation 1000-grain weight of maize under salt stressed


38 Effect of rhizobacterial inoculation on straw yield of maize under salt stressed field

conditions 112

39 Effect of rhizobacterial inoculation on cob length of maize under salt stressed field

conditions 114

40 Effect of rhizobacterial inoculation on cob weight of maize under salt stressed field

conditions 115

41 Effect of rhizobacterial inoculation chlorophyll contnt (SPAD values) of maize under

salt stressed field conditions 117

42 Effect of rhizobacterial inoculation on proline content of maize under salt stressed


43 Effect of rhizobacterial inoculation on crude protein contents of maize under salt

stressed field conditions 120

44 Effect of rhizobacterial inoculation on phosphorous in grain of maize under salt


45 Effect of rhizobacterial inoculation on K+/Na+ of maize under salt stressed field

conditions 123

46 Effect of rhizobacterial inoculation on phenol content of maize under salt stressed


47 Effect of rhizobacterial inoculation on total soluble sugar of maize under salt


48 Effect of rhizobacterial inoculation on electrolyte leakage of maize under salt


49 Effect of rhizobacterial inoculation on SOD values of maize under salt stressed field

conditions 130

S1 Exprimental layout of field trials 39

S2 Pictorial representation of rhizobacterial inoculation at different salinity levels

under axenic conditions 62

S3 Pictorial representation of rhizobacterial inoculation on root growth at different

salinity levels in pot experiment. 69

ABSTRACT

Soil salinity is one of the most widespread agricultural problems which reduce the field and

crop productivity. Salinity disturbs the hormonal balance in plants which results in poor

growth. Use of plant growth promoting rhizobacteria (PGPR) is considered an economical

and environment-friendly approach to combat salinity stress. This study was carried out to

investigate the effect of PGPR on the growth, antioxidant status, physiological parameters

and mineral content of maize (Zea mays) in salt affected soils. Bacterial strains were isolated

from the rhizosphere of maize growing under salt affected soil conditions. These strains were

screened on the basis of auxin production and their ability to withstand salinity stress.

Nineteen rhizobacterial strains producing auxin were further screened for their growth

promoting activity under axenic conditions at 0, 4, 8, 12 and 15 dS m-1 salinity levels. Two

strains were selected after this study and their ability of growth promotion in the presence and

absence of L-TRP was tested alone and in combination under salt affected pot and field

conditions. Results showed that soil salinity reduced the plant growth, physiological

parameters, mineral nutrient uptake and yield of maize while antioxidant activity and proline

concentration was increased. Inoculation with PGPR strains under saline soils alleviated the

salinity effects on the antioxidant enzymes (APX and SOD), along with those on

photosynthesis, mineral content and growth in the absence and presence of L-TRP

application. Dual inoculation showed better results than sole inoculation. The results of the

present study highlight the significance of PGPR strains to alleviate the adverse effects of

salinity stress.

1

CHAPTER-1

INTRODUCTION

Maize (Zea mays L.) is a major food crop after wheat and rice in Pakistan. It is a

multiuse crop as it provides raw material to industry and feed to livestock and poultry. It

contributes more than 10% to agricultural production and 15% to agricultural employment in

the country (Khaliq et al., 2004). Maize is one of the most significant cereal crops both for

human food and animal feed and is grown under varying conditions throughout the world.

Being an important cereal crop, maize is grown on about one million hectares with a total

production of about 3.13 million tons and an average yield of 3.3 ton ha-1 (Anonymous,

2008). Due to its important role in food and feed of humans and animals it is vital to cause

improvements in agronomic characteristics to get good and high quality produce under salt

affected soil conditions.

Around the world, crop farming has to deal with varying biotic and climatic stresses

which limit the crop growth and productivity (Nemati et al., 2011). Among such hazards is

soil salinization that reduces the productivity of fertile lands (Lawlor, 2002). There is

progressive increase in barren lands due to accumulation of salts (Azooz, 2004) and more

than half of the cultivated land is expected to be affected by soil salinization till 2050

(Vinocur and Altman, 2005). Problems associated with soil salinity not only affect agriculture

but also disturb the biodiversity of the environment (Parida et al., 2003; Fernandez-Aunión et

al., 2010). Due to low rain fall and dry climatic conditions the problem of soil salinity is more

vigorous in arid and semi-arid environments. Presence of higher levels of sodium and

chloride cause negative effects on plants growth. Soil salinity causes two types of stresses

namely osmotic stress and ion toxicity in plant cells, thus upsetting the growth and

metabolism. Plants suffer various physiological changes due to soil salinity which include

changes in nutrient and water uptake (Munns, 2005; Parida and Das, 2005), reduction in

photosynthetic activity (Chavez et al., 2011), production of reactive oxygen species

(Dionisio-Sese and Tobita, 1998; Sgherri et al., 2007; Pérez-López et al., 2009, 2010) and

modification nitrogen uptake and metabolism (Munns, 2005). All these changes ultimately

affect the plant growth and performance (Gouia et al., 1994). The osmotic effects brought by

soil salinity can at first stage cause the disintegration of root membranes (Carvajal et al.,

2

1999) and lower the transpiration rate and photosynthetic activity (Parida and Das, 2005).

Along with the induction of the osmotic stress, ionic toxicity is caused by the salinity stress.

These toxic ions negatively affect the NO3- transporters, nitrogen reductase (NR) activity and

glutamine synthase (GS) activity (Flores et al., 2000).

To surmount this problem, numerous approaches of defense against salt stress have

been adopted. Some of these strategies include the development of salt tolerant plants through

conventional and genetic engineering tools, reclamation and amelioration of salt affected soils

(Vinocur and Altman, 2005; Valliyodan and Nguyen, 2006; Ashraf and Akram, 2009; Mittler

and Blumwald, 2010). During current years, a new biological approach has been developed to

shelter plants from salt stress in soil, is the use of plant growth-promoting rhizobacteria

(PGPR) (Yue, 2007). Such beneficial microorganisms have been renowned in the

maintenance of healthy root, facilitation of nutrient uptake and helping the plants to withstand

environmental stresses and increase plant growth (Reed and Glick, 2004). Positive impacts of

these PGPR include the production of phytohormones; auxin, cytokinins and gibberellins

(Garcia de Salamone et al., 2001), enhancing release of the nutrients (Nautiyal et al., 2000) as

well as preventing the deleterious effects of environmental stresses (Kloepper and Schroth,

1978). Against using higher doses of pesticides and fertilizers the use of PGPR is an

economic and environmental friendly approach that promises the improvement in crop

growth and productivity on sustained basis. Different inter cellular and intra cellular

microorganism colonize the plant roots (Gray and Smith, 2005). Beneficial bacteria and fungi

present in the rhizosphere have the potential to directly and indirectly enhance the plant

growth under stress conditions (Dimkpa et al., 2009). The direct mechanisms of plant growth

stimulation by these microorganisms include the production of phytohormones, biological

nitrogen fixation, production of siderophores and phosphate solubilization (Hayat et al., 2010;

Rodriguez and Fraga, 1999). By an indirect way, these bacteria protect the plants against

diseases through antibiotics production (Lugtenberg and Kamolova, 2009).

Plants commonly adapt to various types of biotic and abiotic stress by causing

changes in their root morphology (Potters et al., 2007). The plant hormones play a vital role

in altering the root morphology (Spaepen and Vanderleyden, 2011; Spaepen et al., 2007).

Hayat et al. (2010) reported that most of the plant growth-promoting bacteria possess the

ability to produce indole acetic acid (IAA). Different plants inoculated with such bacteria

3

showed an increase in root growth, lateral root formation, root hairs, which helped them to

withstand abiotic stress. Activity of ACC-deaminase enzyme that degrades the ACC, the

precursor of ethylene, is stimulated by the IAA produced by the bacteria (Glick, 2005). Under

salt affected soil conditions, ACC-deaminase activity of bacteria can be helping for better

growth and yield of crops. Positive effects of such phytohormone producing PGPR have been

documented in different crops such as wheat (Khalid et al., 2004; Egamberdieva, 2009) and

rice (Mirza et al., 2006).

Plant hormones play a vital role in regulating the protective response against different

abiotic and biotic stresses (Ansari et al., 2012). These phytohormones are involved in the

expression of various genes via synergistic and/or antagonistic actions (Reed and Glick,

2004). This phenomenon is known as signaling cross talk (Kraft et al., 2007). Plants can slot

in core developmental programs and swiftly adjust growth to mutable situations by

persuading hormone homeostasis (Wolters and Jurgens, 2009). Indole acetic acid is known to

play a vital role in growth and developmental aspects of plants such as development of

vascular tissues, lateral root formation, gravitropism, cell differentiation apical dominance as

well as directive of gene expression (Malamy and Ryan, 2001; Wang et al., 2001). Current

proofs point out that auxin also responds to salinity in crop plants and its signaling is down-

regulated under different stresses (Kazan and Manners, 2009). When plants faces various

environments stresses, the internal levels of phytohormones are decreased which are mostly

unsatisfactory for prime growth (De Salamone et al., 2005). The negative effect of salinity on

the germination of seed may be due to drop in the internal levels of phytohormones

(Zholkevich and Pustovoytova, 1993; Debez et al., 2001). Naqvi and Ansari (1974) reported

that transport of cytokinin from the root to shoot was decreased due to salinity stress.

Similarly, Itai et al. (1968) reported that from the maize coleoptiles tip the recovery of

diffusible auxins was decreased. Therefore, under soil salinity reduction in growth of plants

could be the result of changes in hormonal balance. The negative effects of salinity were

alleviated by the exogenous application of such plant growth regulators (Dhingra and

Varghese, 1985; Khan and Weber 1986; Prakash and Parthapasenan, 1990; Gul et al., 2000;

Saimbhi, 1993; Gul et al., 2000; Khan et al., 2004; Afzal et al., 2005). A significant

reduction in IAA contents was noted in rice plants due to soil salinity by Prakash and

Prathapasenan (1990). They reported application of GA3 partly mitigate the effect of salinity

4

on reducing IAA levels. This shows that salinity may influence hormone balances by

affecting plant growth and development. Nilsen and Orcutt (1996) also reported marked

reduction in IAA concentration of in five days old rice after salinity application. In tomato the

level of IAA was also decreased due to salinity (Dunlap and Binzel, 1996). Sakhabutdinova et

al. (2003) documented that soil salinization causes a progressive reduction in IAA contents of

plant roots. The growth inhibiting effect of salt stress have been found to be alleviated by the

priming of wheat seed with various plant growth regulators by other researchers (Sastry and

Shekhawa, 2001; Afzal et al., 2005). Increasing soil salinity caused a reduction in seed

germination while application of IAA and/or NAA proved helpful in lowering the adverse

effects of soil salinity on germination (Balki and Padole, 1982; Gulnaz et al., 1999).

Keeping in view the above mentioned discussion it is hypothesized that auxin (IAA)

producing PGPR may facilitate maize growth in saline conditions. This study was planned

with the following objectives:

o Isolation of PGPR from rhizosphere of maize growing under salt affected conditions

o Determination of in-vitro biosynthesis of auxin by these microbes

o Screening of isolates for their growth promoting ability for maize under salt affected

axenic conditions

o Evaluation of PGPR potential to promote growth and yield of maize in pot and field

under saline conditions

o Characterization and identification of the selected strains

5

CHAPTER-2

REVIEW OF LITERATURE

Soil salinity is worldwide agricultural problem. Most of the water present on earth

contains about 3% sodium chloride salt. Such high concentration of salts has resulted in vast

range of salt affected soils. Approximately 900 Mha of the land is affected by this menace.

Soil salinity is considered a major threat to agricultural productivity and food security

(Flowers and Yeo, 1995; Munns, 2002).

To get good quality and high produce of crop, people have been using different

selection and breeding techniques since ages. Different crops are selected with special

reference to environment and soil conditions. Shift in cultivation of crops has been noted in

the Tigris-Euphrates basin of ancient Mesopotamia (from wheat to barley, a relatively salt

tolerant crop) as those soils gradually turned into salt affected soils (Jacobsen and Adams,

1958). This problem will cause more serious threats to food security as the world population

increases especially in developing countries like Pakistan.

2.1 Problem of salinity in Pakistan and at world level

Naturally soil salinization is caused due to the upward movement of groundwater

which is then evaporated on the surface of soils leaving behind the salts which cause soil

salinity. Alongside natural causes of soil salinity, human activities also add to this problem.

For example, irrigation practices in arid and semi-arid regions of the world are major source

of soil salinization (Supper, 2003). These practices elevate the water level and ultimately the

evaporation. All over the world, approximately more than 800 million hectares of soil is

affected by this problem (FAO, 2005). This accounts for almost 6% of the total land area.

Major source of this soil salinity and/or sodication is natural, but a reasonable part of land is

salt affected due to irrigation practices and clearing of land.

Out of 1500 million ha land that is cultivated under dry land agriculture, 2% (32

million ha) is affected by soil salinity to different levels. Forty-five million ha land of

irrigated agriculture are also subjected to this menace (FAO, 2005). Soil salinity either caused

naturally or by human activities reduces the 1/3rd yield of world’s arable lands. High amounts

6

of salts in soils, taking into account both human made and naturally occurring salinization,

are responsible for yield reduction on one third of the global arable land.

Approximately 6.30 Mha of Pakistani land are under soil salinity problem (Alam et

al., 2000). Irrigation in Pakistan is mainly done through a vast network of canals which

extends up to over 62,400 km. Although 15% of the cultivated land of Pakistan receives

irrigation, yet the production from this area is more than twice of that of rain fed land. These

lands contribute to the world’s food production. These lands face a serious issue of soil

salinity which reduces their production capacity (Rahman, 1998). It is estimated that the

population of the world is going to increase 1.5 billion people in next two decades. With

increase in population and urbanization, the issue of food productivity and maintenance

becomes a serious threat to agriculture (Owen, 2001). There is need to find ways to ensure

food security on sustained basis but not at the cost of new land area which would ultimately

cause deforestation and changes in biodiversity. It is estimated that developed and developing

countries will need to boost their production up to 20 and 60%, respectively (Gruhn et al.,

2000; Cakmak, 2002). Under these situations, the problem of soil salinity becomes more

important.

2.2 Impact of salinity stress on plant growth and development

Plants face four types of stresses due to soil salinity which adversely affects the plant

growth and development (Greenway and Munns, 1980) i.e.

1. Osmotic stress (limited water supply)

2. Ionic stress Ion toxicity (Ion toxicity)

3. Impairment of nutritional balance (caused by elevated concentration of toxic ions such as

Na+and Cl-)

4. Damage caused by reactive oxygen species (ROS)

7

2.2.1 Osmotic stress (Limited water supply)

At first instance, uptake of water is reduced due to soil salinity. This situation

ultimately leads to reduced growth. This effect of salinity stress is referred to as osmotic

stress. The effects of drought and soil salinity are similar regarding osmotic stress. The

production of new plant leaves primarily depends upon the soil water potential. In the

growing parts of plants, salts are not accumulated to such a high concentration that can inhibit

the growth, but they are continuously translocated into the expending vacuoles. So the growth

of newly formed leaves is not directly affected by the accumulation of salts (Munns, 2005).

Decrease in root/shoot growth is mainly caused by the water shortage caused by salinity

stress (Munns, 2002). This hypothesis is supported by the fact that in the growing cells, the

Na+ and Cl- concentrations are below toxic levels. Hu and co-workers (Hu et al., 2005)

reported that Na+ concentration in growing cells (of wheat sown in 120 mM sodium chloride)

was 20 mM at maximum and 10 mM in fastly expanding tissues. The concentration of Cl-

was recorded 50 mM only. Similar findings regarding concentration of toxic ions have been

documented by other researchers in maize (Neves-Piestun and Bernstein, 2005) and barley

(Fricke, 2004). It was also reported (Munns et al., 2000) that osmotic stress caused by the soil

salinity outside the roots causes change in root/shoot water relations which induce hormonal

signals that control the elongation of cells. Severity of salinity mainly directs the growth

inhibition rate. At low levels of osmotic stress, growth of leaves and stem is inhibited while

roots keep on growing and elongating (Hsiao and Xu, 2000). Extent of growth retardation

caused by osmotic stress depends upon the particular plant part, response time and plant

species under consideration as well as nature of salt stress treatment (Ashraf, 1994).

2.2.2 Ionic Stress

When certain ions are taken up from the soil or water and accumulated in the plants

itself, they cause toxicity. This ion toxicity may be caused even if the severity of salinity is

less. Na+, Cl- and SO42- are mainly considered as the toxic ions from salinity point of view

which are responsible for the low crop yield. Crops vary in their sensitivity to soil salinity e.g.

most of the woody perennial plants and crops are sensitive (Abrol et al., 1988). Taken up salts

are accumulated in older leaves and they are continuously transported to the transpiring

leaves where the concentration of Na+ and Cl- rises to a toxic level and ultimately cause the

8

death of leaves. This injury is mainly caused because these ions are accumulated to such a

high level that the cells are unable to translocate them into vacuoles. This buildup of salts in

the cytoplasm hinders the activity of enzymes. On the other hand, these ions can also be

deposited in the cell walls and deprives them of water (Munns, 2005) but no such effect was

observed by Muhling and Lauchli (2002) in two maize cultivars having different salt

tolerance abilities. Tester and Davenport (2003) reported that concentration of Na+ in root

cytosole was 10-30 m M. Cytosolic concentration of leaves are not clear but are thought to be

less than 100 mM (Wyn Jones and Gorham, 2002). The toxic levels of Cl- are not well

understood. Salts must be excluded from the roots into the soil solution otherwise they will

accumulate to toxic levels. Amount of water that is transpired is about 50 times higher than

that retained in leaves of plants (Munns, 2005). Another study (Hussain et al., 2003)

compared the two wheat verities to exclude Na+ and prevent from leaf chlorosis and

increasing their yield. They reported on the basis of their results that in high sodium-variety,

older leaves showed injury symptoms and died earlier than low-sodium genotypes. This

genotype also showed an increase in yield of 20% at moderate salinity levels. They concluded

that Na+ exclusion is a beneficial trait of genotypes to withstand salinity stress. Accumulation

of Na+ ions negatively affects the seed germination. This is mainly due to that it disturbs the

water content of plants. It may also displace Ca2+ and take its place in vital cell wall binding

sites, which ultimately affect the cell wall formation and growth of plants (Xue et al., 2004).

Similarly Cl- concentration higher than 80 mM also cause negative effect on morphology of

plants, function of stomata is disturbed and leaves become thinner (Loreto and Bongi, 1987).

Cl- is highly mobile in water and taken up by the plants and ultimately transported to leaves

when Cl- is built up to a toxic level in leaves, it causes burning and drying of leaves. This

situation may lead to loss of early leaves and plants become defoliated as whole Marschner

(1995).

2.2.3. Impairment of nutritional balance

Buildup of high amount of salts in the vicinity of roots results in osmotic stress. This

condition causes alteration in water relation of plants, impairment of essential nutrients

uptake and buildup of toxic ions in plants. Owing to this situation, the metabolism of plants

and activity of various enzymes is badly disturbed (Lacerda et al., 2003). Negative effect of

9

toxic ions on the uptake of essential nutrients by plants causes serious imbalance of nutrients

within plants (McCue and Hanson, 1990; Karimi et al., 2005). The activity of various

enzymes is altered due to high Na+: K+ caused by the accumulation of high levels of Na+ than

K+ (Booth and Beardall, 1991). The least uptake of K+ due to high Na+ uptake leads to

potassium deficiency symptoms in plants which are at first noted as chlorosis and after time

as necrosis of plant parts (Gopal and Dube, 2003). Potassium plays a vital role in protein

synthesis, osmoregulation, enhancing photosynthesis and maintenance of turgor pressure

(Ashraf, 2004). Calcium and K+ are essential for proper functioning and integrity of cell

membranes (Wenxue et al., 2003). Selective uptake of K+ and its compartmentalization in

cells and transportation in roots plays a main role in the maintenance of appropriate levels of

K+ in plants under salt stress conditions (Carden et al., 2003). Soil salinity tolerance of plants

is judged by their ability to take up and maintain adequate amount of Ca2+ under salinity

stress (Unno et al., 2002). Kinraide (1998) reported that under salinity stress, Ca2+/Na+ ratio

decreased which lead to membrane disintegration and selectivity. Hasegawa et al. (2000)

reported that exogenic application of Ca2+ resulted better growth of plants under salinity

stress. This might be due to improved Ca2+/Na+ ratio. Calcium is also important as it helps in

osmotic adjustment by enhancing the accumulation of compatible solutes (Girija et al., 2002).

Calcium also protects the plants from salinity stress by maintaining the integrity of

membranes and protecting the plants against oxidative damage (Larkindale and Knight,

2002). Khadri et al. (2007) found an increase in K+/Na+ ratio due to the application of ABA.

2.2.4. Reactive oxygen species

When plants are exposed to certain biotic and abiotic stresses, the activity of reactive

oxygen species (ROS) is increased. These species cause peroxidation of lipids, mutation of

DNA and degradation of proteins (Pitzschke and Hirt, 2006). These species are produced

mainly in the mitochondria and chloroplast of the plant cells (Mittler, 2002). Although these

ROS cause oxidative damage to plant cells, they also cause signal transduction during stresses

and in response to pathogenic attacks (Torres and Dangle, 2005).

Increased production of these species during salinity stress causes injury to the

membranes (Shalala et al., 2001). Decrease in CO2 uptake due to the closure of stomata

results in the production of ROS in higher amounts. This increased production of ROS causes

10

a decrease in the concentration of NADP+ (Foyer and Noctor, 2003). These ROS degrade

photosystem II and D1 proteins which ultimately result in inhibition of photosynthetic

activity. Hydrogen peroxide is also accumulated due to NADPH activity and photorespiration

caused by stress and may decrease the activity of enzymes. H2O2 in the presence of metal

ions is changed into highly active (.OH) which has the ability to degrade the biomolecules

(Halliwell and Gutteridge, 1989).

2.3 Measures to enhance salt tolerance

Crop’s salt tolerance can be enhanced by causing variations in present crops. Notable

enhancements in salinity tolerance have been achieved through conventional selection and

breeding approaches (Ashraf, 2002), as well as through agronomic practices. Such agronomic

strategies include the use of exogenic use of phytohormones and inoculation of plant seeds

with plant growth promoting rhizobacterial.

2.3.1 Conventional crop breeding

Breeding of crops by using conventional breeding techniques provides an economic

and efficient way of inducing stress tolerance in crops than soil amelioration methods.

Response of plants to salt stress is influenced by the interaction of various environmental

factors and salt stress. That’s why it is of great importance to develop such genotypes that

have the ability to cope with adverse conditions of soil and climate (Ashraf and McNeilly,

2004; Sairam and Tyagi, 2004). Such crop genotypes have been established and are

commercially available for cultivation. Such varieties of wheat and rice have been developed

which are tolerant to salinity stress (Ashraf and McNeilly, 1990). A limited success is

achieved in developing such salt tolerant crops. This is due to the fact that these cultivars

have only been tested under ideal conditions by the breeders, and if these genotypes are not

competitive then there is no benefit in developing such tolerant crops (Yamaguchi and

Blumwald, 2005). Although many high yielding cultivars have been developed by using this

approach but only a few cultivars are known that are tolerant to salinity (Ashraf, 1994). The

main drawback of this approach is that it requires a long period of time and undesirable genes

are also transferred alongside the desired one. The transfer of desired alleles from

11

interspecific and inter generic sources is limited due to reproductive barriers (Apse and

Blumwald, 2000).

2.3.2 Genetic Engineering

Methods of conventional breeding are useful for inducing stress tolerance in different

crops but due to complex trait and environmental impacts, this technique is not very much

successful and effective. As compared with conventional breeding techniques, genetic

engineering is faster and attractive tool for imposing salinity stress tolerance. The induced

genes are directly or indirectly protected against the stresses (Bohnert and Sheveleva, 1998).

In transgenic plants, a few genes have been tried to test stress tolerance (Winicov,

1998). Different genes that encode antioxidant enzymes have been cloned and found in

transgenic plants (Allen, 1995). Torsethaugen et al. (1997) also reported enhanced oxidative

stress tolerance in plants of tobacco that overexpressed cytosolic ascorbate peroxidase. Zhang

et al. (2001) incorporated AtNHXl gene that encodes for Na+/H+ antiport in canola plants and

it showed enhanced growth compared with wild type plants subjected to salinity stress. The

betA gene from E-coli was used to transform a maize line. This gene encodes an important

enzyme i.e. choline dehydrogenase in the biosynthesis of glycine betaine (Quan et al., 2004).

The disadvantage of this technique may be that the character improvement is done at cell

which may not appear in whole plant (Ashraf, 1994).

2.3.3 Exogenous application of phytohormones

When plants are subjected to salinity stress, their indigenous level of hormones is

disturbed. Younis et al. (2003) reported that salinity stress resulted in the decreased level of

IAA and increase in ABA in maize shoots. Similarly, Fricke et al. (2004) found that salinity

stress caused the accumulation of ABA in barley leaves. Akhiyarova et al. (2005) also found

increased contents of ABA in all plant parts due to salinity stress. Boucaud and Unger (1976)

reported decreased in gibberellic acid and cytokinin contents and increased ABA contents

owing to salt stress. Similar changes in level of IAA in maize have also been reported (Ribaut

and Pilet, 1994). It can be deduced from all these results that retardation in growth of plants

due to salinity stress is caused by changes in internal hormonal balance of plants. The

balancing of hormonal levels in plants under salinity stress can be achieved by the exogenic

12

application of phytohormones. Parasher and Varma (1988) reported positive effect of GA3

application in wheat under salinity stress. Afroz et al. (2005) reported that GA3 application

enhanced the photosynthetic efficiency and nitrogen uptake of mustard plants and improved

the growth. Improvement in seed germination and plant seedling by the application of GA3

and kinetin has been reported by Satvir et al. (1998). IAA application enhanced the uptake of

mineral nutrients and lowered the uptake of Na+ which ultimately resulted in better growth of

maize plants (Darra and Saxena, 1973). Datta et al. (1997) also found seed treatment with

kinetin and IAA effective in mitigating the salinity stress in plants.

2.3.4 Use of plant growth-promoting rhizobacteria (PGPR)

It is well established plant hormones play a vital role in controlling plant growth and

development. Many workers have reported that inoculation with auxin producing plant

growth promoting rhizobacterial can improve the growth of crop plants under salt affected

soil conditions. Zahir et al. (2010) conducted a pot trial to investigate the effect of Rhizobium

phaseoli to improve the growth of mung bean under salt affected conditions. They reported

that salinity badly reduced the growth of mung bean while application of rhizobium and L-

TRP were found very effective in mitigating the negative effects of salinity. Their combined

application was more effective than their sole application. They concluded from their results

that this improvement in growth might be due to auxin production ability of these microbes.

They suggested that inoculation with rhizobium producing auxins may be a useful tool to

induce the salt tolerance in mung bean.

Nia et al. (2012) conducted a greenhouse experiment to test the effectiveness of

inoculation with Azospirillum strains isolated from saline or non-saline soil in alleviating the

salinity stress in wheat plants grown with irrigation water with different electrical

conductivities (ECw) of 0.7, 4, 8 and 12 dS m-1. Inoculation with the two isolates increased

salinity tolerance of wheat plants; the saline-adapted isolate significantly increased shoot dry

weight and grain yield under severe water salinity. The component of grain yield most

affected by inoculation was grains per plant. Plants inoculated with saline-adapted

Azospirillum strains had higher N concentrations at all water salinity levels.

13

Bacteria producing 1-aminocyclopropane-carboxylate (ACC) deaminase can be

helpful in mitigating salinity stress in plants (Glick et al., 1997). Ethylene, a plant hormone,

has been found to inhibit the elongation of plant roots (Abeles et al., 1992). ACC-deaminase

produced by PGPR can breakdown the ACC, the precursor of ethylene and in this way, lower

the ethylene concentration in plants under stress situation (Glick, 1995; Glick et al., 1998). By

lowering ACC levels in plants, these bacteria help those plants in ameliorating the stress and

to improve their growth (Shah et al., 1997; Glick et al., 2007).

The symbiotic relationship between rhizobia and legumes is disturbed due to salinity.

It is documented in many studies that nodulation and N2 fixation was significantly decreased

due to salinity stress (Rabie et al., 2005). Other authors reported a decrease of rhizobial

colonization and shrinkage of root hairs of soybean, common bean, and chickpea in salt stress

(Singleton and Bohlool, 1984; Zahran and Sprent, 1986; Elsheikh and Wood, 1990).

The study of Marcar et al. (1991) indicated that symbiotic properties were more

sensitive to salinity than plant growth. Thus, the development of salt-tolerant symbioses is an

absolute necessity to enable cultivation of leguminous crops in salt-affected soils (Lauter et

al., 1981; Yousef and Sprent, 1988; Velagaleti and Marsh, 1989).

In the rhizosphere, the synergism between various bacterial genera, such as between

Bacillus, Pseudomonas, and Rhizobium has been demonstrated to promote plant growth and

development (Bolton et al., 1990; Halverson et al., 1993). The coinoculation with

Pseudomonas spp. and Rhizobium spp. had enhanced nodulation and nitrogen fixation, plant

biomass, and grain yield in various leguminous species (Bolton et al., 1990; Dashti et al.,

1998; Goel et al., 2002; Tilak et al., 2006). We have also observed that root-colonizing

Pseudomonas strains improve rhizobia-legume interactions. Combined inoculations could be

an option to improve plant growth and increase nodule numbers and N content of Galega

species (personal communications). Plant growth-promoting bacteria can prevent the

deleterious effects of stressors from the environment (Lugtenberg et al., 2001; Egamberdieva,

2009). Hasnain and Sabri (1996) reported that inoculation of plants with Pseudomonas sp.

stimulated plant growth by reduction of toxic ion uptake and increases in auxin contents.

Another explanation for enhancement of nodule formation by the rhizobia in legumes might

be the production of hydrolytic enzymes such as cellulases by root colonizing Pseudomonas

14

strains, which could make penetration of rhizobia into root hairs or intercellular spaces of root

cells easier, leading to increased numbers of nodules (Sindhu and Dadarwal, 2001).

2.4 Auxins

Auxins are a very important group of plant hormones which regulate different aspect

of plant growth and development. There are different types of auxins present in plants among

them indole-3-acetic acid (IAA), indole-3-propanic acid (IPA) and indole-3-butyric acid

(IBA) are the common examples of naturally occurring auxins. In tissues of plants simple

auxins can bind with other organic molecules to form conjugates which can be stored in

plants (Frankenberger and Arshad, 1995). Tryptophan is an essential amino acid which can

play the role of precursor of auxins (IAA). Through different pathways tryptophan undergoes

into various biochemical changes which result in wide variety of intermediates indole-3-

acetamide (IAM), indole-3-acetonitrile (IAN), tryptamine (TAM), tryptophol (TOL) and

indole-3-ethanol. Among different types of auxins in indole-3-acetic acid (IAA) is the

functional unit and most active form of auxins (Davies, 1995; Frankenberger and Arshad,

1995; Yasmin et al., 2007; Ali et al., 2008; Sridevi and Mallaiah, 2008).

2.4.1 Sources of auxins

Like higher plants, different soil microbes also possess the ability to biosynthesize the

auxins. Owing to presence of various substrates as root exudates microbes present in the

rhizosphere are more active and efficient in producing auxin than those present in the bulk

(Asghar et al., 2004; Khalid et al., 2004; Zahir et al., 2005). In the rhizosphere, some of the

auxins are released by the plants in the form of root exudates, where rest of the auxins might

have been synthesized by the active rhizospheric microbes. Rhizobia are endogenous

microbes and due to close contact with the tissues of plants are very active in producing

auxins. Etesami et al. (2009) isolated 100 rhizobial isolates belonging to genera Rhizobium

leguminosarum var. phaseoli and Rhizobium leguminosarum var. viciae and selected the five

rhizobial isolates superior in IAA production in vitro. These isolates were used as inoculants

alone and in combination with L-TRP to study their effect on growth parameters of wheat.

Ghosh et al. (2008) reported the presence of higher amounts of indole-3-acetic acid (IAA) in

root nodules of Phaseolus mungo. Rhizobia biosynthesized large quantities of auxin (142 μg

mL-1) upon the addition of L-tryptophan to the inoculation medium. An increase in auxin

15

production was observed when mannitol (1%), L-asparagine (0.3 %) and thiamine

hydrochloride (1 μg mL-1) were added in the inoculation medium in addition to L-tryptophan

(2 mg mL-1). Erum and Bano (2008) isolated rhizobial strains from high altitudes of northern

Pakistan and studied their phytohormone production capabilities (auxins and gibberillins).

They reported that rhizobia isolated from five different locations of higher altitudes of

Pakistan were capable of producing IAA and GA. They also studied the beneficial effects of

these isolates upon inoculation on soybean crop. Sridevi and Mallaiah (2007) isolated twenty

six rhizobium strains from root nodules of Sesbania sesban collected from different regions

of Andhra Pradesh. All the rhizobium strains produced auxins but the maximum auxin

production was observed by only five strains in yeast extract mannitol (YEM) medium upon

the addition of 2.5 mg mL-1 L-tryptophan.

Bhowmick and Basu (1987) reported that Rhizobium sp. isolated from legume host

produced auxin but a significant difference in auxin biosynthesis by the strains was observed

depending upon the culture conditions. Bhowmick and Basu (1986) reported that large

amounts of auxin were produced by the Rhizobium sp. from the root nodules of Sesbania

grandiflora upon the addition of L-tryptophan in the culture medium. Auxin production could

be promoted more than 76% by supplementing the medium with mannitol, sodium molybdate

and Triton X-100. Basu and Ghosh (1998) found that root nodules of a monocotyledonous

tree, Roystonea regia, produced high quantities (16.9 μg g-1 fresh mass) of auxin. Root

nodules contained higher amounts of tryptophan (1555.1 μg g-1 fresh mass) which might have

been microbially converted into auxin. The Rhizobium sp. isolated from the root nodules of R.

regia produced high amount of auxin in tryptophan supplemented medium. Dullart (1970)

quantified the auxin present in root nodules and roots of Alnus glutinosa (L.)

spectroflurometrically. More auxins were present in nodules than in roots. Kaneshiro et al.

(1990) conducted experiments on two different strains of Bradyrhizobium japonicum (USDA

110 and 26) which are different in biological nitrogen fixation and auxin-biosynthesis in

soybean. These strains produced effective nodules and symbiont 26 contained 0.3–1.1 μg

IAA g-1 nodular mass. Infection of nodules by tryptophan catabolic variants 4b and 20d,

isolated from B. japonicum strain also extracted two to four folds more auxin (2–3.9 μg g–1)

while nodules infected with B. japonicum strains USDA 110 and 123 produced significantly

less auxin. Yasmin et al. (2007) screened fifteen rhizobacterial isolates from sweet potato

16

rhizosphere for in vitro auxin biosynthesis with and without L-TRP, solubilization of

phosphates and N-fixing ability and reported that all the isolates efficiently produced auxin in

L-TRP supplemented medium. Most of the isolates were effective in biological nitrogen

fixation and phosphate solubilization while differed in plant growth promoting activities.

Asghar et al. (2000) tested 100 rhizobacterial isolates from rapeseed for in vitro auxin

biosynthesis which ranged between 0.13 to 14.4 μg IAA mL-1 of the culture medium. Auxin

production increased many folds in supplementation with filter sterilized L-tryptophan.

Sarwar and Kremmer (1995a) tested the in vitro auxin-producing capabilities of 16 cultures

belonging to Xanthomonas, Pseudomonas, Agrobacterium spp. and Alcaligens and four

Pseudomonas spp. They observed more auxin biosynthesis in rhizosphere isolates than non-

rhizosphere isolates. Sarwar and Kremmer (1995) also concluded from another study that 16

out of 70 isolates were capable of metabolizing L-TRP into auxin in the range of 4.0-86.1 μg

IAA mL-1 of the liquid medium. Spaepen et al. (2009) reported that interaction of rhizobia

with rhizosphere results in the production of plant hormones like auxins in addition to many

other compounds. Although most of the rhizobia have the ability to produce auxins (IAA), yet

the role of auxin in the legume rhizobium symbiosis is not clear. To test the effect of auxin on

rhizobial gene expression, a transposon (mTn5gusA-oriV) mutant library of Rhizobium etli,

enriched for mutants that show differential gene expression under microaerobiosis and/or

addition of nodule extracts as compared with control conditions, was screened for altered

gene expression upon addition of auxin. Four genes were regulated by auxin which appeared

to be involved in plant signal processing, motility or attachment to plant roots, clearly

demonstrating a distinct role for auxin in legume-Rhizobium symbiosis.

2.4.2 Substrate dependent biosynthesis of auxins by other soil micro biota

Other than rhizobia some other microbes such as bacteria, fungi and actinomycetes

when provided with suitable precursor also possess the ability to produce the auxin. Certain

rhizobacterial strains isolated from wild plants have been reported to produce auxins and

these strains have also potential to enhance the growth of wheat (Ali et al., 2009). These

strains when supplemented with precursor L-TRP produced auxin in the range of 0.6-8.22 μg

mL-1 IAA. These researchers proposed that growth enhancement of wheat by these strains

were due to their ability to produce auxin. Gas chromatography and mass spectrometric (GC-

17

MS) analysis revealed that auxin was produced by bacterial strains in the range of IAA in

supplementation with L-Trp. Auxin produced as a result of plant microbe interactions

significantly increased growth of T. aestivum. Nassar et al. (2005) isolated auxin producing

yeast (Williopsis saturnus) from the rhizosphere of maize. These yeasts were used to

investigate their ability to produce auxin in vitro and their potential to enhance the growth of

maize in axenic and green house conditions. These strains produced many fold higher level of

auxins when supplemented with L-TRP as compared to controls where no augmentation with

L-TRP was done. Growth of maize seedlings when inoculated with these strains was also

significantly improved in growth room and green house experiments. Significant increases in

shoot and root dry weight and lengths were observed when maize seedlings were inoculated

with W. saturnus in the presence of L-TRP. These strains did not produce detectable amounts

of gibberellins or cytokinins. In another experiment of the same study another strain of

endophytic fungi R. glutinis which had similar root colonization ability but did not possess

the ability of auxin production was unable to promote growth of maize seedlings. These

findings highlight the significance of substrate dependent auxin biosynthesis and its role in

plant growth promotion.

Tami and Yaacov (1992) documented that higher concentrations of L-TRP in the

culture of Azospirillum brasilense Sp7 produced toxic effects which were revealed in the

form of decrease in growth, changes in colour of colonies, changes in DNA and alterations in

path of protein synthesis. IAA biosynthesis is affected by the age of culture and concentration

of added L-TRP. It was found that IAA production was linear vs. time after the addition of

tryptophan by pulse injection and younger cultures produced higher amounts of IAA as

compared to older cultures. Thus IAA production from tryptophan and its eventual release in

growth medium of A.brasilense Sp7 might be a way out through which this bacterial strain

overcomes the toxic concentrations of tryptophan. Tao et al. (2008) demonstrated the

enhanced production of indole-3-pyruvic acid from an, amino transferase, Trp transaminase

(TAA1) from L-TRP.

Although auxin production by soil microbes positively influence the growth and

development of plants in most cases, but there are some pathogenic species of microbes

which have auxin production abilities but they cause highly negative effect on plant growth

18

and health. Vandeputte et al. (2005) reported that an auxin producing gram positive

pathogenic bacterial strain Rhodococcus fascians, produced leaf galls in plants possibly due

to auxin production. Regulation of auxin production is controlled by plasmid encoded genes.

Intermediates of indole -3-acetic acid present in cells of Rhodococcus fascians indicate that

this strain used IPA pathway as their biosynthesis while possibly limited the indole-3-ethanol

pathway. In this study, auxin production is directly correlated with leaf galls by these

pathogenic bacteria.

Ali et al. (2008) isolated 16 bacterial strains from different plant species.

Identification of these strains was carried out by using 16S rRNA gene sequencing. After this

these strains were tested for their abilities to produce auxin and for their potential to enhance

the growth of Vigna radiata (L.). Auxin production by these strain ranged from 0.60 to 3.0 μg

IAA mL-1 in the presence of L-TRP. Auxin production was confirmed by the gas

chromatography and mass spectrometric GC-MS analysis. Inoculation with B. megaterium

MiR-4 (61%) and B. pumilus NpR-1 strains resulted in significant improvements in all tested

growth parameters. Positive correlation of auxin production and various parameters of growth

was drawn such as shoot length (r = 0.495*, P = 0.05), number of pods (r = 0.498*, P = 0.05)

and grain weight (r = 0.537*, P = 0.05) at full maturity under greenhouse conditions. Results

of this study suggested that auxin production by these strains was such a trait that can be

exploited for increased production of Vigna radiata (L.).

2.4.3 Biochemistry of microbial production of auxins

On the basis of their intermediates i.e. indole-3- acetamide (IAM), indole-3-pyruvate

(IPyA), tryptamine, and indole-3-acetonitrile (Patten and Glick, 1996), plants and

microorganisms produce auxin by using different pathways that may be tryptophan dependent

or independent (Bartel, 1997). Disease causing bacteria cause hyperplasia by auxin

production which alters the appearance and development of plants. Morris (1995) reported

that Agrobacterium tumefaciens, Agrobacterium rhizogenes, Pseudomonas savastanoi and

Erwinia herbicola carry out biosynthesis of auxin through the IAM pathway this was

confirmed by the analysis of iaaM gene from DNA sequencing.

19

Disease induction can be controlled by reducing the supply of tryptophan and

inhibiting the activity of iaaM as IAA production requires iaaM gene. E. herbicola pv.

gypsophilae uses IAM and IPyA pathways for the biosynthesis of auxin. IAM pathway

controls the size of gall while IPyA pathway regulates the epiphytic fitness (i.e. plant growth

monitoring) (Hutcheson and Kosuge, 1985). It is like a universal truth those bacteria that

cause diseases in plants and those that help the plants in better growth (i.e. PGPR) both

biosynthesize the auxin. Auxin production by the plant growth promoting rhizobacteria

improves the nutrient uptake of the inoculated plants which helps in better growth of these

plants. Beneficial bacteria mostly synthesize the auxins by IPyA pathway (Costacurta and

Vanderleyden, 1995). Azospirillum brasilense a plant growth promoting rhizobacterial strain

can produce auxin by one tryptophan-independent pathway and two tryptophan-dependent

pathways (IAM and IPyA) (Prinsen et al., 1993).

Badenoch-Jones et al. (1983) reported that most species of Rhizobium can produce

auxin. It is documented in many studies that alterations in levels of phytohormones such as

auxin and cytokinins are the basic requirements for nodulation (Mathesius et al., 1998). On

the roots of alfalfa pseudo nodules are produced due to inhibition of auxin polar transport

(Hirsch and Fang, 1994). In rhizobia nod gene is expressed by plant produced flavonoids

which produce Nod factors (lipo-chitin oligosaccharides or LCOs) and results in inhibition of

auxin transport in legumes (Mathesius et al., 1998). In a transgenic white clover plant,

expression of the auxin-responsive reporter constructs GH3: gusA was rapidly and transiently

down regulated after inoculation by Rhizobium leguminosarum bv. trifolii which was

subsequently up regulated at the site of nodule initiation (Mathesius et al., 1998). These

studies predicted that local lowering of the auxin balance is a prerequisite for the initiation of

nodule primordia. Prinsen et al. (1991) reported that auxins production by Rhizobium is

stimulated by flavonoids to make Nod factors (lipochitino-oligosaccahride compounds). Co-

inoculation of Rhizobium and Azospirillum can increase nodulation (Dullaart, 1970; Volpin et

al., 1996). Inoculation with A. brasilense resulted in significant increase in activity of nod-

inducer of crude alfalfa root exudates because these exudates possessed flavonoid compounds

compared with those of un-inoculated plants (Volpin et al., 1996). Root nodules possess more

auxin compared with un-inoculated roots and active auxin biosynthesis takes place in

functional nodules (Badenoch-Jones et al., 1983). It is concluded that increased auxin levels

20

in nodules are resulted from rhizobia because a mutant of Bradyrhizobium japonicum that

produces 30-fold more auxin than the wild-type strain exhibited higher nodulation efficiency

(Kaneshiro and Kwolek 1985). Inoculation with mutant B.japonicum strains overproducing

IAA produced higher quantities of auxin in comparison with wild-type bacteroids (Hunter,

1989) predicted that increased auxin biosynthesis in nodules is of bacterial origin.

Badenoch-Jones et al. (1983) detected the radioactivity in all tissues after hours of

high specific activity [(3)H]IAA application to the apical bud of intact pea (Pisum sativum L.

cv Greenfeast) plants. More radio activity was detected in the nodules than root and more in

functional nodules than in non-functional nodules. Thin layer chromatography (TLC) analysis

confirmed the presence of IAA and indole-3-acetylaspartic acid (IAAsp) in effective nodules

and also the involvement of auxin metabolism in functional root nodules. Williams and

Signer (1990) demonstrated the biosynthesis of auxin by Rhizobium meliloti Rm1021 in

alfalfa when supplemented with tryptophan. Mutants which did not utilize tryptophan analogs

still produced indole-3-acetic acid but were Fix (-) as these bacteria did not fully differentiate

into the nitrogen fixing bacteriods. These mutants possessed essential genes which were

tightly linked to a dominant streptomycin resistance locus. Kepinski (2008) concluded from

experiments that auxin, in spite of being simple molecule, plays a crucial role in plant growth

and development. Auxin regulated gene expression by promoting the ubiquitin-dependent

proteolysis of the Aux/IAA family of transcriptional repressor proteins. Mode of action of

auxin is binding the TIR1/AFB auxin receptor F-box proteins thereby enhancing their

recruitment of Aux/IAA repressors for SCF-mediated ubiquitination. TIR1 is an integral

constituent of an SCF-type E3 ubiquitin ligase and is thus the prototype of an entirely

different receptor in which the generic mechanism for ubiquitination is regulated by the

binding of a small molecule. The auxin-mediated destabilization of Aux/IAAs genes mediates

the expression of Auxin Response Factors (ARF) family of DNA-binding transcription

factors.

2.5 Indole acetic acid (IAA) producing rhizobacteria

Many important plant-microbial interactions center on the production of auxins, IAA

being the main plant auxin. The IAA is responsible for the division, expansion and

differentiation of plant cells and tissues and stimulates root elongation. The ability to

21

synthesize IAA has been detected in many rhizobacteria as well as in pathogenic, symbiotic

and free living bacterial species (Costacurta et al., 1995; Tsavkelova et al., 2006).

At present, auxin synthesizing rhizobacteria are the most well-studied phytohormone

producers (Tsavkelova et al., 2006; Spaepen et al., 2007). These rhizobacteria synthesize IAA

from tryptophan by different pathways, although it can also be synthesized via tryptophan-

independent pathways, though in lower quantities (Spaepen et al., 2007). Phytopathogenic

bacteria mainly use the indole acetamide pathway to synthesize IAA, which has been

implicated in tumor induction in plants. It is not clear whether it is used by beneficial

bacteria. In contrast, the indole pyruvic acid pathway appears to be the main pathway present

in plant growth promoting beneficial bacteria (Patten and Glick, 2002).

Azospirillum is the most studied indole acetic acid producer among all plant growth

promoting rhizobacteria (Dobbelaere et al., 1999). Other bacteria belonging to different

genera i.e. Aeromonas (Halda-Alija, 2003), Azotobacter (Ahmad et al., 2008), Bacillus

(Swain et al., 2007), Burkholderia (Halda-Alija, 2003), Enterobacter (Shoebitz et al., 2009),

Pseudomonas (Hariprasad and Niranjana, 2009) and Rhizobium (Ghosh et al., 2008) have

also been found to possess IAA production ability. Inlole acetic acid producing bacteria on

inoculation increase the germination of seed, stimulate growth of root, alter the root system

architecture and improve the biomass of root. Tsavkelova et al. (2007) have reported

improvement in seed germination of orchid seed (Dendrobium moschatum) by inoculating

with an auxin producing Mycobacterium sp. Along with stimulation of root growth, these

bacterial species also enhance the growth of tubers in tuber crops. Swain et al. (2007) applied

Bacillus subtilis as a suspension on the surface of Dioscorea rotundata L. and found

improvements in growth parameters as compared with control plants.

2.5.1 Impact of microbially derived auxins on plant growth and development under

normal soil conditions

Auxins produced by the microbes augment the suboptimal levels of auxins in plants

and help in regulating normal functioning of plants. Ali et al. (2009) investigated the effect of

different auxin producing PGPR on the growth and development of wheat crop (Inqalab-91)

auxin production by these strains was confirmed by GC-MS analysis. It was found that auxin

22

production these strains ranged 0.6-8.22 μg IAA mL-1 in L-TRP supplemented media. A

significant positive correlation was observed between auxin production by bacterial strains

and endogenous IAA content of T. aestivum for GC-MS (r = 0.62; P = 0.05) and colorimetric

analysis (r = 0.69; P = 0.01). Likewise, highly significant positive correlation for shoot length

(r = 0.637; P = 0.01) and shoot fresh weight (r = 0.63; P = 0.01) was observed with auxin

production under gnotobiotic conditions. Inoculation with these bacterial isolates increased

shoot length (up to 29.16%), number of tillers (97.35%), spike length (25.20%) and seed

weight (13.70%) at harvesting.

IAA producing rhizobacterial strains had the potential to increase the endogenous

levels of auxin in wheat and its growth. Bacterial strains isolated from the rhizosphere of wild

plants can be used for enhancing the growth and production of agronomic crops. Patten and

Glick (2002) reported that beneficial auxin producing PGPR had the potential to improve

plant growth while IAA producing pathogenic bacteria caused tumors in plants by using the

indole-3-acetamide pathway. In order to investigate the role of IAA produced by bacteria on

enhancing the host plant root growth, an IAA deficient mutant Pseudomonas putida GR12-2

was developed. For this purpose ipde gene was isolated. Ipde gene is involved in encoding an

important enzyme in indole-3-pyruvic pathway via indole pyruvate decarboxylase was

developed by using bacterial cultures were potentially capable of increasing endogenous IAA

content and growth of T. aestivum var. Inqalab-91. Microbial strains associated with wild

herbaceous flora can be effectively used for improving growth and yield of agronomically

important crops. Pattens and Glick (2002) reported that many plant-associated bacteria

synthesize IAA while tumors (out growths which are formed due to auxin overproduction)

are induced in plants as a result of IAA production by phytopathogenic bacteria, mainly via

the indole-3-acetamide pathway. Upon inoculation with wild type Pseudomonas putida

GR12-2 canola seedlings roots were increased 30-50% as compared to those seedlings that

were inoculated with IAA deficient bacteria.

It is highlighted from these studies that IAA produced by bacterial strains affects the

growth and development of roots of host plants. In hydroponic study, Gravel et al. (2007)

used different bacterial and fungal strains to investigate their effect on the growth of tomato

plants. Pseudomonas putida and T. atroviride improved fruit yields in rock wool and in

23

organic medium as a result of microbial biosynthesis of auxins in supplementation of L-Trp,

tryptamine and tryptophol @ 200 mg mL-1 in the culture medium. With increasing

concentration of L-TRP the root/shoot fresh weights of tomato plants were increased by

inoculation with both of these strains. It was also found that IAA (0-10 mg mL-1) application

along with bacterial inoculation significantly improve the root growth. The detrimental effect

of auxin (IAA) on root elongation might had been reduced due to reduced ethylene

biosynthesis from its precursor ACC by microbial transformation of IAA in the rhizosphere

and/or by ACC deaminase activity contained in both microorganisms.

Perrine et al. (2004) tested the IAA biosynthesis by using an isotope dilution GCMS

assay of IAA and L-Trp in the culture supernatant of Rhizobium leguminosarum bv. trifolii

strains for increasing or inhibiting rice plant growth. Results revealed higher quantities of

auxin by rhizobial strains supplemented with tryptophan in BIII minimal medium compared

to BIII media alone. Rhizobial strains synthesized very low quantities of auxin required for

inhibiting rice growth thus proving that auxin produced by rhizobial strains had no role in

limiting rice growth and development. Pedraza et al. (2006) used nine strains of nitrogen

fixing bacteria efficient in IAA biosynthesis in tryptophan supplemented media. Auxin

production was the highest (16.5-38 µg IAA mg-1 protein) by Azospirillum strains while

Glucon acetobacter and P. stutzeri biosynthesized lower quantities of auxins (1 - 2.9 µg mg-1

protein) in supplementation with tryptophan. Inoculation with B. megaterium MiR-4, B.

pumilus NpR-1 and B. subtilis TpP-1, under axenic conditions increased shoot length by 56,

47 and 46% over un-inoculated control. Bacillus megaterium MiR-4 and B. pumilus NpR-1

inoculation increased shoot fresh weight by 61 and 38%, respectively, over control. There

was highly significant positive correlation between auxin production and growth parameters

of Vigna radiata (L.) under axenic and greenhouse conditions. This implies that auxin-

producing Bacillus spp. can be effectively utilized for enhancing the yield and of Vigna

radiate (L.).

Bellamine et al. (1998) demonstrated the effect of exogenous application of auxin on

poplar shoots, raised in vitro, induced to root by incubation on an auxin (NAA) containing

medium for 7h. Shoots were kept in an auxin-free medium for 13 days post treatment.

Ninteen-seven percent (97%) of the treated shoots produced roots. Application of auxin

24

inhibitors (POAA-phenoxyacetic acid; PBA-2-phenoxy-2-methylpropionic acid; PCIB-2-

(pchlorophenoxy)-2-methylpropionic acid) into auxin-free medium after the 7h induction by

NAA (α-naphthaleneacetic acid), completely or severely inhibited rooting. (PCIB, PBA and

POB are anti-auxin compounds). The exclusion of calcium from the auxin-free medium

reduced the percentage of rooting by 42%. The inhibition was still higher in the presence of a

calcium chelator EGTA (ethyleneglycol-O, Ό-bis (2-aminoethyl ether)-N, N, Ń, Ń, tetraacetic

acid). Application of lanthanum chloride (calcium channel blocker) to the auxin-free medium

completely inhibited rooting. These results confirmed the involvement of endogenous auxin

and calcium application in the later stages of the adventitious root formation.

Hussain et al. (1988) used 10 different concentrations of IAA sprays and investigated

their effect on the growth and nodulation of Leucaena leucocephala. They used 10 sprays per

week. After each spray they observed the girth and height of plants. After eight and half

months plants were harvested and weights of roots, nodules and numbers were recorded. The

plant species were also analyzed for their nutrient concentrations. At 50 ppm IAA the

parameters were significantly enhanced. Higher doses of IAA caused negative effects on the

growth and their values were found even lower as compared with controlled plants. Rafael et

al. (1989) used root stocks of dwarf apple verities and studied their root response and levels

of indole-3-acetic acid. They used Linsmaier-Skoog medium which was supplemented with

benzyladenine (BA), indole-3-butyric acid (IBA), and 1, 3, 5-trihydroxybenzoic acid (PG) to

grow the apple root stock. Lepoivre medium was used to investigate the effect of IBA and PG

on root growth and development. M9 and M26 produced 80-100% root response respectively

by using IBA. Before root inducing treatment in shoots of both root stocks free and conjugate

auxins concentrations were evaluated by using HPLC. These results were reconfirmed by

GC-MS. It was revealed from the results that the lower part of M26 contained 2.8 times

higher contents of free auxins than similar tissues in M9. 298 and 263.7 ng per gram fresh

weight of free auxins was recorded in epical parts of M26 and M9. M9 contained

significantly higher amounts of conjugated auxins than M26, which means that most of the

auxins M9 were found in conjugated form. Due to different free auxins concentrations both

strains showed variable rooting response.

25

2.5.2 The role of bacterial phytohormones in plant growth promotion in saline soils

The mechanism through which plant growth promoting rhizobacteria improve the

growth and development of host plant include the mobilization of nutrients (Lugtenberg et al.,

2002; Lugtenberg and Kamilova, 2004) and production of phytohormones (Lifshitz et al.,

1987; Frankenberger and Arshad, 1995). The microbial synthesis of plant growth regulators is

an important factor in soil fertility. Gibberellin and indole acetic acid are secondary

metabolites, which are important biotechnological products, obtained commercially from

bacteria and fungi for the agricultural and horticultural industry (Patten and Glick, 2002).

Indole acetic acid (IAA) is the most common natural auxin found in plants. IAA is involved

in physiological processes such as cell elongation and tissue differentiation (Taiz and Zeiger,

1991; Frankenberger and Arshad, 1995) and has also been associated with the plant growth-

promoting effect of numerous rhizospheric microorganisms (Patten and Glick, 2002).

It is hypothesized that negative effect of salinity on seed germination may be due to

impairment of levels of plant growth hormones (Zholkevich and Pustovoytova, 1993;

Jackson, 1997; Debez et al., 2001). In some earlier studies, it is reported that movement of

cytokinins from root to shoot and from coleoptile tips of maize and the recovery of diffusible

auxins is limited due to salinity stress (Itai et al., 1968). Use of PGRs such as gibberellins,

auxins and cytokinins has been found very effective in removing the deleterious effects of

salinity stress (Gul et al., 2000; Khan et al., 2001, 2004; Afzal et al., 2005; Prakash and

Parthapasenan, 1990). Exogenous application of these PGRs caused improvements in growth

of plants under salinity stress (Saimbhi, 1993). Priming of wheat seed with these PGRs have

been found very effective and alleviating the growth retarding effects of salinity stress (Singh

and Dara, 1971; Datta et al., 1998; Sastry and Shekhawa, 2001; Afzal et al., 2005). In a study

conducted by Sakhabutdinova et al. (2003) showed that salinity stress caused a progressive

decline in the IAA contents in plant root system. Under such conditions exogenous

application of phytohormones supplies additional natural hormones that help the plants for

normal growth and development under salt affected situations (Kabar, 1987; Afzal et al.,

2005).

It is reported in many studies that plant growth promoting rhizobacteria (PGPR)

which can produce phytohormones on inoculation with plants can alleviate the dangerous

26

effects of salinity stress and thus improve the crop growth (Lindberg et al., 1985;

Frankenberger and Arshad, 1995). Egamberdieva (2009) reported that IAA producing strains

improved the germination percentage up to 79% and mitigated the negative effects of salinity

on wheat due to their ability to produce auxin. These strains had the ability to produce auxin

even in saline conditions. In another study Hasnain and Sabri (1996) found that Pseudomonas

sp. reduced the uptake of toxic ions such as Na+ and increased the auxin level of wheat plants.

Phytohormone production by other PGPR has also been demonstrated by other researchers

(Zimmer et al., 1995) and their on growth promotion has been reported (Frankenberger and

Arshad 1995). Plant growth promoting rhizobacteria help the plants by supplying the

additional phytohormes which can mitigate the harmful effects of salty (Egamberdieva,

2009). Keeping in view such results it can be concluded that such phytohormones producing

rhizobacteria can be used for better growing of crops in saline conditions without doing any

genetic changes in plants.

Along with acting as plant growth regulators and stimulating the growth of plant, IAA

also increases the activity of ACC synthase. ACC synthase produces more ACC, which is

transformed by the action of ACC oxidase into ethylene (Mayak et al., 1999). The ACC

produced at higher levels in plants may be exuded out due to concentration gradient and is

hydrolyzed by the ACC-deaminase produced by PGPR. It can concluded from this

mechanism the final effect of ethylene production and its effect on root growth primarily

depends upon balance ACC deaminase and IAA produced by bacterial strain. Plants show

different responses to IAA application. Its effects are determined by the initial root

elongation. The growth of plants which have slow root elongation is restricted by the

application of phytohormones at any concentration, while plants which have fast elongating

roots show increase in elongation by the use of plant growth regulators at low concentrations

(Pilet and Saugy, 1987).

It is conclude that different species of ACC-deaminase producing plant growth

promoting rhizobacteria produce variable amounts of IAA. Interaction of ethylene and IAA

was explained in a model proposed by Glick et al. (1998). Such bacteria not only lower the

concentration of ethylene and relieve the plant from stress but also minimize the synthesis of

auxin response factor (ARF). In this way the bacteria mitigate the effect of stresses and

27

improve the growth and development of plants. However as the synthesis of ARF is

enhanced, production of ACC is also increased which ultimately checks the synthesis of

ARF. Through this feedback mechanism, ethylene limits its production by its own.

Plants are immovable creation, but they can highly modify their physiology which

enables them to withstand different environmental situations. This is due to that, they have

highly active meristem and they can also produce different organs due to embryogenesis

(Wolter and Jurgens, 2009). Plants use different defensive mechanisms such as changes in

root morphology to cope stresses (Malamy, 2005). Hormonal homeostasis is involved in

morphogenesis, cell division and elongation. IAA is the physiologically most active auxin in

plants and its importance in plant growth and development is reflected by the fact that still no

fully auxin deficient mutant in plants is developed. Auxin is mainly produced in meristems of

aerial parts and roots (Teale et al., 2006). Minute changes in auxin concentration play a vital

role in plant growth and development. Especially under stress situations two phenotypic traits

such as formation of lateral roots and branching are altered by changes in auxin level (Potters

et al., 2007).

Higher number of indole acetic acid producing plant growth promoting rhizobacterial

strains was found in plant tissues as revealed by analysis for bacterial existence (Spaepen et

al., 2007). A significant increase in lateral root formation and root hair was recorded in

different species of plants owing to inoculation with such beneficial bacterial strains (Dimkpa

et al., 2009). The beneficial effects of Azospirillum on the growth and development of roots

are reported in many studies. Plants roots inoculated with Azospirillum show morphological

changes mainly due to the production of plant growth regulators such as auxins. Auxin is the

most important hormone among all the hormones (Spaepen et al., 2007). Evidences of

involvement of auxin in root proliferation are documented in many studies. El-Khawas and

Adachi (1999) conducted a hydroponic study to investigate the effect of A. brasiliense on the

root growth of rice plants. They reported on the basis of their results that root length, surface

area of roots, root dry matter, lateral roots and number of root hairs was increased compared

with control plants. IAA applied exogenously to plants also showed similar responses as of

inoculation with Azospirillum (Remans et al., 2008). Low phytohormone producing and

28

having nitrogen fixing ability mutant of A. brasiliense could not increase the root growth as

compared with control plants (Kundu et al., 1997).

Depending upon the relationship between indole acetic acid and ACC, the positive

effects of IAA on root growth can be either direct or indirect through the reduction of

ethylene levels (Lugtenberg and Kamilova, 2009). When plant faces any stress such as

salinity, ethylene regulates the plant growth and causes reduction in root shoot growth. It is

found that under stress conditions ethylene is produced in two phases in plants. At first phase

minute amount of ethylene stimulates the stress response genes in plants. After one to three

days of stress application higher amount of ethylene is produced which results in retardation

of growth caused negative effect on plants such as chlorosis, senescence and abscission

(Glick et al., 2007).

ACC-deaminase producing rhizobacteria lower the ethylene production in plants by

degrading its precursor ACC into 2-oxybutanate and ammonia. Thus in this way these

bacteria reduce the synthesis if auxin response factor (Glick et al., 2007; Kang et al., 2010).

Under stress conditions ACC may be released from the plants due to concentration gradient,

which is consumed by the ACC-deaminase producing rhizobacteria for their growth. ACC-

deaminase production by plant growth promoting rhizobacteria is a beneficial trait for the

plants and PGPR itself for the normal growth, as the concentration of ACC is lowered in

plants and PGPR can use ACC as a metabolite (Glick et al., 1998). Under various

environmental stresses these ACC-deaminase producing bacteria have been practically found

efficient in promoting the growth and development of plants. Mayak et al. (2004) found that

ACC-deaminase producing PGPR Achromobacter piechaudii significantly improved the root

and shoot fresh/dry matter of tomato plants. Saravanakumar and Samiyappen (2007)

compared the effect of ACC deaminase producing PGPR strain and those lacking ACC

deaminase activity. They found that ACC deaminase producing PGPR strains improve the

salinity resistance in growth of the plants as compared to the control plants inoculated with

ACC deaminase lacking strain. In another study, Siddikee et al. (2010) also reported that 14

halo tolerant bacterial strains caused amelioration of salinity stress in canola plant mainly due

to activity of ACC-deaminase. Maize plants inoculated with Pseudomonas fluorescens

showed increased root and shoot growth. Effect of drought stress on the ripening, growth and

29

yield of pea was also revealed due to the ACC deaminase production. Nadeem et al. (2010)

documented improved growth of wheat under salt stress due to inoculation with ACC

deaminase producing plant growth rhizobacteria. Bianco and Defez (2009) observed the

response of Medicago plants inoculation with Sinorhizobium meliloti strains under high salt

stress condition. They reported that IAA overproduction showed remodulation of

phytoharmones in different plant tissues. Higher levels of auxins were found in roots and

nodules of plants with reduction in IAA level in shoot was compared in relation to those

plants which were inoculated with wild type 1021 strain (Mt–1021). Ethylene resisting genes

were also observed through transcriptional analysis. The results showed that Mt – RD64 did

not show induction of this pathway under salt stress which is an indication that plants were

rescued from stress damages.

2.6 Other mechanisms of growth promotion by PGPR

2.6.1 Accumulation of protective compounds

Accumulation of nitrogen containing compounds (NCC) such as amino acids,

proteins, quaternary ammonium compounds and polyamines have been correlated with

improved growth and yield under different stresses such as salinity and drought stress.

Szabados and Savoure (2009) reported a very high concentration of proline in cells under salt

and drought stress as compared to normal soil conditions. This is due to enhanced production

and less breakdown of such compounds under stressful conditions. In different studies in

which transgenic plants were used elaborate that metabolism of proline has a complex effect

on development of stress responses. Proline acts as a compatible solute in plants and thereby

reserves the carbon and nitrogen. In some other studies it is also reported that proline acts has

an anti-oxidative activity and serves as a scavenger of ROS. Proline also stabilizes the

architecture of proteins and increases the activity of various enzymes by acting as molecular

chaperones. When inoculated in the plants it controls the cystolic pH and controls the redox

potential of cells. Increased biosynthesis and accumulation of proline due to the inoculation

with various rhizobacterial strains was observed in different plants. The production and

accumulation of such solutes is carried out as expanse of energy (41 moles of ATP) and

reduction in growth. These alterations help the plant to survive and recover under different

stress conditions such as salinity stresses. Bianco and Defez (2009) reported 2 fold increase in

30

proline contents of shoots of Medicago plant as a result of inoculation with Mt-64 strain.

These increased levels of proline were positively correlated with reduced symptoms of

senescence as necrosis, chlorosis and drying.

2.6.2 Biosynthesis of antioxidative enzymes

Plants produce different reactive oxygen species such as super oxide (O2-), hydrogen

peroxide (H2O2), hydroxyl radical (OH) and singlet oxygen (102) in different metabolic

activities as byproducts in various cellular compartments (Apel and Hirt 2004). These

reactive oxygen species are harmful and cause oxidative damage to the DNA, proteins and

lipids. As during photosynthesis in the chloroplasts internal concentrations of oxygen are

very high so these portions are highly prone to oxidative damage by reactive oxygen species.

Under normal conditions these oxygen species are continuously removed by antioxidant

component and plant faces no damage. But under stressful environment the production of

these reactive species is elevated and they are accumulated in the different parts due to

imbalance between their productions and scavenge. Among different ROS scavenges

mechanism, SOD, APX and CAT are the major mechanisms. Different antioxidants such as

ascorbic acid and glutathione are present at high concentration in chloroplasts and other

compartments of cells. These play a vital role in defending the plant from oxidative damage

caused by ROS (Miller et al., 2010). Balance between antioxidants is necessary for the

detoxification of different reactive oxygen species (Mittler, 2002). Kohler et al. (2010)

reported that salinity stress was mitigated due to the induction of antioxidant enzymes in

plants inoculated with PGPR strains. Under medium and high salt stress plants inoculated

with strain showed increased biomass than the control plants. Bianco and Defez (2009) also

reported that plants inoculated with Mt-RD64 strain showed less signs of oxidative damage.

This improvement was mainly due to the enhanced activity of antioxidant enzymes.

Sandhya et al. (2010) conducted a study to investigate the effect of five drought

tolerant PGPR strains on the growth and antioxidative activity of maize. The results of the

study revealed that the activity of antioxidant enzymes was lower in inoculated plants as

compared with the uninoculated plants. In barley plants the antioxidative activity was also

reduced. A study conducted by Omer et al. (2009) revealed that under salinity stress activity

31

of antioxidant enzymes such as catalase and peroxidase increase significantly in two

cultivars of barley. On the other hand when these plants were inoculated with Azospirillum

baselines, they showed lowering in the activity of such enzymes, which removed the

deleterious effects of salinity and enhanced the growth and yield of barley plants under salt

effected conditions.

2.6.3 Enhancement of nutrients uptake

Different adaptive mechanisms that plants adopt to avoid or tolerate different stresses

define the survival and productivity of these plants under stresses. Many research studies

report that mineral status plays a crucial role in adapting the harsh environmental stress

conditions. Abiotic stresses disturb the mineral uptake and then accumulation in plants and

external application of these nutrients can alleviate the stress symptoms on plants.

Phosphorus after the nitrogen is the 2nd most important mineral nutrient for growth of plants.

It is the part of various biochemical compounds such as nucleotides, nucleic acid,

phosphoproteins and phospholipids. Under various conditions of salinity stress uptake and

accumulation of phosphorus is reduced and plants develop deficiency symptoms (Martinez

and Lauchlie 1994). Under salt affected soil conditions lowering the availability of P may be

due to the ionic strength that results in the reduced activity of phosphate. Reduced solubility

of Ca-P minerals adsorption of P may be other processes that lower the P availability under

salt effected soil conditions. In soil amount of soluble P is very minimum (less than 1ppm)

(Hinsinger, 2001). Cells can uptake different forms of P but HPO4-2 or HPO4

-1 are the main

forms of P that are adsorbed. In soil P is in the organic and inorganic forms. Like other

mineral nutrients such as K+1, Fe+2, Zn+2 and Cu+2, its mobility in soil is very low (Hayat et

al., 2010). Plant growth promoting rhizobacteria convert the insoluble form of P, both present

in organic and inorganic forms into soluble forms that are available to the plants. Different

plant growth promoting rhizobacterial strains that belong to different genera can solubilize

insoluble compounds of phosphorous. Generally these strains produce some organic acids

that can solubilize the mineral phosphates (Rodriguez and Fraga, 1999). These organic acids

cause acidification in the cells of microbes and thus surrounding environment. In this way P

is released by proton substitution for Ca+2. Many plant growth promoting genera such as

32

Pseudomonas, Erwinia, Rhizobium and Bacillus have been found to produce such organic

acids (Rogriguez and Fraga, 1999).

Soil salinity poses serious threat to crop farming throughout the world. A number of

physical, chemical and biological approaches have been employed to combat this menace.

Soil inhabiting bacteria are also recently being used for this purpose. These bacteria use

different mechanisms to help the plants to withstand salinity stress. Among such bacteria are

those which are capable of producing auxin. These bacteria have been tested on different

crops under normal soil conditions. Very scarce information is available that reports the

efficacy of these bacteria under salt affected soil conditions. Keeping in view the findings of

previous research a comprehensive study was planned to evaluate the effectiveness of auxin

producing rhizobacteria for enhancing the growth, physiology and yield of maize in salt


33

CHAPTER-3

MATERIALS AND METHODS

The research work comprised of series of experiments. These were conducted in the

laboratory, growth room, wire house and farmer fields. Large numbers of rhizobacterial

strains were isolated from the rhizosphere of maize plants growing under salt affected soil

conditions. Axenic trials were carried out to screen the strains of rhizobacteria on account of

growth improving potential with maize as test crops. The performances of the screened

isolates were further evaluated under salt affected soil conditions in pots and fields. The

procedures adopted during the whole experimentation are discussed in detail as under:

3.1 Isolation of PGPR

Plant growth promoting rhizobacteria were isolated from the rhizosphere of maize (cv.

Neelam) growing in the salt affected soil (Sandy clay loam, ECe; 7.2 d Sm-1) from the semi-

arid region of Faisalabad, Punjab, Pakistan by dilution plate technique using general purpose

media (GPM). Fast growing colonies were selected and further purified. Isolated

rhizobacteria were stored in refrigerator at 4 0C for further experimentation.

3.2 Preparation of inoculum

Sterilized general purpose media was inoculated with each rhizobacterial culture and

incubated at 28 + 1 0C for 3 days in shaking incubator at 100 rpm. Fresh inoculum were

prepared for each experiment and diluted to get uniform population (108-109 CFU mL-1) by

measuring optical density (OD 0.5) using spectrophotometer at wavelength 535 nm.

3.3 Seed inoculation

The inoculum for PGPR strain was prepared by culturing the strain on 250 mL of liquid

medium in a 500-mL Erlenmeyer flask incubated for 72 h at 29 oC and 100 rpm. Maize seeds

(Monsanto-919) were surface sterilized with 70% ethanol for 2 min, treated with 2% sodium

hypochlorite for 5 min, and followed by three washings with sterilized distilled water. For

inoculation, PGPR inoculum of selected strains, having cell suspension of 107-108 CFU mL-1,

were injected into sterilized peat (150 mL kg-1 of seed, seed to peat ratio 4:1 w/w). Maize

seed dressing was done with inoculated peat mixed with 10% sterilized sugar solution. In

34

case of un-inoculated control, the seeds were coated with the sterilized peat treated with

sterilized broth and 10% sterilized sugar solution but without bacterial inocula. While for co-

inoculation (1:1 ratio) equal volume of both strains was used.

3.4 Osmotic adjustment test

The isolated strains were tested according to the method as described by Zahir et al.

(2010) for their ability to withstand salinity stress. For this purpose, strains were grown in test

tubes on different salinity levels (original, 10, 20, 30 dS m-1). Fifteen milliliters of broth was

taken in test tubes and autoclaved. The tubes were inoculated with the isolated strains and

incubated at 28 + 1 0C and the absorbance was measured at (λ) 540 nm by using

spectrophotometer.

3.5 Measurement of Auxin Production

Auxin production measurement was carried out according to the method described by

the Sarwar et al. (1992). Twenty milliliters of sterilized General Purpose media (GPM) was

taken in Erlenmeyer flasks of 100 mL. Five milliliters solution of 5% L-TRP after filter

sterilization (0.2 μm membrane filter, Whatman No.2) was applied to the medium to prepare

a media with final concentration of 1.0 g L-1

. Inoculation of the flask contents was done with

0.1 mL of 3-day old broth culture (OD 0.5 (108

-109

CFU mL-1

). After proper plugging, the

flasks were incubated for 48 hours using shaking incubator adjusted at 28 + 2 °C and 100

rpm. A flask containing all the materials except inocula was kept as control for comparison.

The experiment was carried out with three replications. After incubation, the contents in the

flask were filtered using Whatman No.2 filter paper. For measuring IAA equivalents, 3 mL of

filtrate was taken in test tubes and 2 mL of Salkowski reagent (2 mL 0.5 M FeCl2 + 98 mL

35% HClO4) was added to it. The mixture in the tubes was allowed to stand for 30 minutes

for colour development. Standard curve was used for the comparison to calculate auxin

production by the selected isolates. Colour was also developed in the standard solutions of

IAA. The intensity of the colour was measured at (λ) 535 nm by using a spectrophotometer.

Rhizobacterial cultures giving higher auxin production were selected for jar experiments.

35

3.6 Screening of rhizobacterial strains for growth promoting potential

A jar experiment was conducted in the growth room under controlled conditions to

screen rhizobacterial strains for their root/ shoot growth promoting ability. For these

experiments 500g sieved sand was filled in the jars. Jars were wrapped with paper and

autoclaved twice. Different salinity levels (EC 0, 4, 8, 12 and 15 dS m-1) were developed in

the jars by using sodium chloride (NaCl) salt. Surface disinfection of maize seeds was done

by rinsing and stirring maize seeds for 3 minutes in a sterile flask with 5% sodium

hypochlorite. After three minutes the seeds were removed from the sodium hypochlorite

solution and washed with sterile water. Surface disinfected seeds were dipped for 10 minutes

in the broth of respective rhizobacterial strains for inoculation. Three inoculated seeds of each

strain were sown in each jar. To meet the water and nutritional requirements of the seedlings,

half-strength Hogland solution (Hogland and Arnon, 1950) was applied to jars. The jars were

placed randomly following completely randomized design in growth room with three repeats.

Jars were placed in a growth chamber at 25 ± 1 8C set at a 16 h light and 8 h dark period, and

the light intensity was set to 350 mmol m–2 s–1.After twenty days of germination, the

seedlings were measured for root/shoot length, fresh and dry weight.

3.7 Pot experiment

On the basis of results from the ‘‘Jar trial,’’ two strains (M4 and M7) giving high mean

values for the growth parameters of seedlings were selected and further evaluated for

improving the growth and yield of maize (Hybrid Monsantto-919) plants with and without L-

TRP by conducting a pot trial in a wire house. Soil used to pot the seeds was analyzed for

physicochemical characteristics (Table 1). Surface-disinfected seeds of maize were inoculated

(as discussed above) with 4 days old cultures of selected rhizobacterial strains. L-tryptophan

solution was applied in the inoculation media. For the uninoculated control, seeds were

coated with the sterilized (autoclaved) peat treated with sterilized broth. Salinity levels of 4,

8, 12 and 15 dS m–1 were maintained (using NaCl salt) in addition to the non-saline control

(original EC, 1.63 dS m–1). For this, the calculated amount of solid salt (NaCl) was mixed in

the soil with a mechanical mixture before filling the pots. Three inoculated/uninoculated,

non-germinated maize seeds were sown in each pot containing 12 kg of soil per pot at all the

5 EC levels which was thinned to one plant, 15 days after germination. There were 3

36

replications of each treatment. Pots were arranged in the wire house at ambient light and

temperature according to a completely randomized design. Recommended doses of N, P, and

K fertilizers (175:100:50 kg ha–1) were applied in each pot. Phosphorus and potassium were

applied as a basal dose, while nitrogen was applied in splits. Tube well water was applied to

the pots for irrigation whenever needed. Data regarding growth and yield parameters were

collected using standard procedures.

3.8 Field experiments

Field experiments were conducted to further confirm the efficacy of selected rhizobacterial

strains for improving growth and yield of maize at farmer fields, 72/GB, Faisalabad-Pakistan

(73-74 E, 30-31.5 N,) for two consecutive years. Soil samples were collected, air dried, mixed

thoroughly, sieved (2.0 mm), and analyzed for physico-chemical characteristics (Table 2).

The soil was analyzed for saturation percentage, pH, electrical conductivity (ECe), cation

exchange capacity (CEC), sodium adsorption ratio (SAR), organic matter, total N, available

P, and extractable K (Table 2) using standard procedures. Inoculated and non-inoculated

(non-germinated) (as described above) seeds of maize (Hybrid Monsantto-919) were sown in

the well-prepared field at 120 kg ha-1 by maintaining a row to row distance of 75 cm with a

plot size of 20 m2. L-tryptophan solution was applied in the inoculation media. The

composition of microbial community of maize was not tested at any time during the

experiment. Phosphorus and potassium were applied as a basal dose, while nitrogen was

applied in splits. The experiments were laid out in randomized complete block design with 3

replications. Plants were irrigated with good quality canal water (electrical conductivity, 0.08

dS m–1; sodium adsorption ratio, 0.2 (mmol L–1)1/2, and residual sodium carbonates, 0.04

mequiv. L–1) meeting the irrigation quality criteria for crops in the area. Growth and yield

contributing parameters were recorded after maturity. Grain and straw samples were analyzed

for nitrogen, phosphorus, and potassium content.

3.9 Soil analyses

Various physical and chemical characteristic of the soil samples were determined

using the procedures illustrated by US Salinity Laboratory Staff (1954) if not mentioned

afterward.

37

Table 1: Physico-chemical characteristics of soil used for pot trial

Characteristics Units Value

Sand % 51.5

Silt % 23.2

Clay % 25.3

Textural Class Sandy Clay Loam

Saturation percentage % 33.1

pH --- 7.8

ECe dS m-1 1.63

CEC Cmolc kg-1 5.12

Organic matter % 0.64

Total nitrogen % 0.07

Available phosphorus mg kg-1 8.33

Extractable potassium mg kg-1 138

38

Table 2: Physico-chemical characteristics of soil used for field trial

Year I Year II Textural Class Sandy clay loam Sandy clay loam

pH 7.6 7.8

ECe (dS m-1

) 6.7 6.8

Organic matter (%) 0.63 0.65

Total nitrogen (%) 0.05 0.04

Available phosphorus (mg kg-1

) 3.93 3.82

Extractable potassium (mg kg-1

) 84.2 78.8

39

Figure S1: Exprimental layout of field trials

40

3.9.1 Particle size analysis

Forty gram of air-dried soil passed through a 2 mm sieve was taken in a 600-mL

plastic beaker. Sodium hexa meta phosphate (2%) was added at 40 mL as dispersing agent

and the soil was left overnight. The suspension of soil was stirred with mechanical stirrer,

poured in a 1-L calibrated cylinder and brought to volume with water. Readings for sand and

silt plus clay were taken by means of Bouyoucos hydrometer following Moodie et al. (1959).

By using International Textural Triangle, soil textural class was dogged.

3.9.2 Saturation percentage (SP)

Saturated paste of soil was made by taking 250 g in a beaker and putting distilled

water whilst stirring the sample using spatula. A portion of the prepared saturated paste of

soil was taken in a tarred china dish. Sample was weighed, dried to constant weight at 1050C

by using an oven and then again weighed. Saturation percentage of the soil sample was

computed using following formula (Method 27a).

Saturation percentage (SP) = (Loss in weight on drying) X 100/ (Weight of the oven dry soil)

3.9.3 Electrical conductivity (ECe)

Saturated paste of soil was transferred into a filter funnel. Vacuum was applied using

a vacuum pump. The saturated extract was collected in a bottle (Method 3a). Using digital

Conductivity Meter, electrical conductivity of the extract was dogged by (Method 3a and 4b).

3.9.4 pH of saturated soil paste (pHs)

After preparation, the saturated soil paste was allowed to stand for 1 h. pH Meter

(Kent EIL Model 7015) was employed to find out the pH of the paste after carefully

standardizing it with standards of 4.0 and 9.2 pH (Method 21a).

3.9.5 Organic matter

One gram of air dried soil sample was weighed in an Erlenmeyer flask of 500-mL

capacity. Solution of 1N potassium dichromate at 10 mL per sample was added by means of a

pipette. Twenty milliliter of sulfuric acid (concentrated) was added to it. The sample was

mixed by shaking and left for 30 minutes. Distilled water at150 mL and 0.5 N ferrous sulfate

solution at 25 mL was added into the sample and the excess was titrated using 0.1N solution

41

of potassium permanganate to pink end point. The same procedure was followed for running

a blank (Moodie et al., 1959).

3.9.6 Total nitrogen

For the determination of total nitrogen, sulfuric acid digestion method of Gunning and

Hibbard’s was used for soil sample digestion and for the distillation of ammonia into 4%

boric acid, macro Kjeldhal’s apparatus was used (Jackson, 1962).

3.9.7 Available phosphorus

Five grams of soil were taken in 250 mL Erlenmeyer flask. Solution of 0.5 M Sodium

bicarbonate was added at 100 mL and the suspension was given 30 minutes shaking. The

extract was taken by filtering the solution and clear extract was taken in volumetric flask at 5

mL Color developing reagent was also added to the flask at 5 mL and completed the volume

using distilled water. By using spectrophotometer, the readings of the soil samples and

standards were recorded at 880 nm wavelengths. Actual P concentration was detected using

standard curve prepared for standards following Watanabe and Olsen (1965).

3.9.8 Extractable potassium

Five grams of soil were taken in centrifuge tube (50 mL capacity) accompanied by 33

mL of ammonium acetate (1N of pH 7.0) solution. After centrifugation, the supernatant were

filtered and taken in 100 mL volumetric flask. The same procedure was repeated two more

times. Jenway PFP-7 Flame photometer was used to determine the potassium (Method 1la).

3.10 Plant analyses

After 60 days fresh leaf samples were collected from the pot and field studies and

analyzed for proline, sodium, potassium and relative water content. At maturity, plants were

harvested and data about growth and yield parameters were recorded. Grain samples were

analyzed for nitrogen and phosphorous.

3.10.1 Measurement of physiological parameters

The physiological parameters i.e. photosynthetic rate (A), stomatal conductance of

water (gs) and transpiration rate (E) was measured by portable infra-red gas analyzer (IRGA).

These parameters were taken in morning (09.00-12.00) at photosynthetic photon flux density

42

of 1200-1400 µ mol m-2 s-1 (Ben-Asher et al., 2006) two fully expanded leaves from one plant

in each experiment unit were selected for the measurement of data regarding above

parameters.

3.10.2 Chlorophyll content (SPAD value)

Chlorophyll content (SPAD value) in leaf of maize plant were measured using

chlorophyll meter SPAD-502 (Minolta, Minolta Co, Ltd, Uk) as described by Hussain et al.

(2000).

3.10.3 Relative water content

Relative water content (RWC) of leaves was determined using the following formula

as described by Mayak et al. (2004).

Relative water content (RWC) = Fresh weight- Dry weight/Fully turgid weight-Dry weight

The fully turgid weight of leaf was taken after putting it in 100 % humidity in the dark

at 4 oC for 48 h.

3.10.4 Electrolyte leakage

Electrolyte leakage (EL) was measured following the protocol described by

Jambunathan (2010). For this purpose, leaf discs were transferred in 5 mL de-ionized water

and electrical conductivity (EC) (R1) was recorded with EC meter (Jenway Conductivity

Meter Model 2052) after 4 h incubation at 28 ± 1 0C and 100 rpm in orbital shaking

incubator. The same samples were autoclaved at 121 0C for 20 min to determine EC (R2).

%EL = (EC before autoclaving R1/ EC after autoclaving R2) X 100

3.11 Analysis of stress-related metabolites 3.11.1 Total soluble sugars

Total soluble sugars were measured by anthrone reagent (0.2 %) following Sadasivam

and Manickam (1992). A reaction mixture (10 mL) consisting of 200 µL leaf extract (1 g leaf

homogenized in de-ionized water), 1,800 µL DI water and 8 mL anthrone reagent was heated

43

for 10 min in boiling water and cooled in ice bath to stop the reaction. Absorbance was

measured at 630 nm and total soluble sugar concentration (µg mL-1) was calculated using

glucose standard curve.

3.11.2 Proline content

Proline content was measured following Bates et al. (1973). A reaction mixture

consisting of leaf extract (1 g leaf homogenized with 3 % sulphosalicylic acid), ninhydrin

acid and glacial acetic acid were heated at 100 0C for 1 h in water bath. The reaction was

stopped by cooling in ice bath and absorbance was recorded at 520 nm after mixture

extraction with toluene. Proline content (µmol g-1) was calculated following standard curve of

L-proline.

3.12Enzymatic/non enzymatic antioxidant activity assays

Ascorbate peroxidase (APX) activity was determined by tracking the ascorbate

reduction through H2O2 with decrease in spectrophotometer absorbance at 290 nm (Nakano

and Asada, 1981). Two milliliters reaction mixture consisting of 20 µL crude leaf extract, 660

µL ascorbic acid solution, 660 µL potassium phosphate buffer (pH 7.0, 50 mM) and 660 µL

H2O2 was used to measure APX activity. Decrease in absorbance was monitored for 3 min

just after the addition of H2O2. Enzyme activity was calculated in the form of µmol ascorbate

min-1 g-1 leaf fresh weight. (Elavarthi and Martin, 2010)

3.13 NP analyses

3.13.1 Digestion

The method of Wolf (1982) was followed for digestion of the plant samples. Plant

sample (dried and ground) was taken in digestion tubes at 0.1 g. Two milliliters of

concentrated sulfuric acid were poured into the tubes. The sample was placed in laboratory at

room temperature and left over night. Then 1 mL H2O

2 (A.R. grade 35%) was added into

digestion tube by the side and was swiveled. The tube was heated up to 350 0C after

mounting it in a digestion block for 20 minutes. After cooling, 1 mL of H2O

2 was added and

again heated the tube for 20 minutes. Same procedure was continued awaiting colorlessness

44

of the material. Clear extract was made 50 mL by means of distilled water. The acquired

extract was stored and used for subsequent estimation of N, P and K as under:

3.13.3 Nitrogen determination

Kjeldhal’s method was used to determine the total nitrogen from the plant samples.

Five milliliter of digested sample was taken in Kjeldhal flask. Forty percent solution of

sodium hydroxide was added at 10 mL and the flask was connected to the Kjeldhal

distillation unit immediately and started distillation. Distillate was collected in 100 ml conical

flask already having 5 mL solution of 2% boric acid accompanied by some mixed indicator’s

drops. Distillation was stopped when the distillate was about 40-50 mL in the collecting flask

which was then titrated with standardized 0.01 N sulfuric acid. The titration volume was

recorded.

3.13.4 Phosphorus determination

Volumetric flask (50 mL capacity) was taken and 5 mL of the digested material was

added to it along with Barton Reagent at 10 mL. After making 50 mL total volume, the

sample was allowed to stay for half an hour. Readings of the blank, standards and samples

were recorded by spectrophotometer at 410-nm wavelength. Calibration curve was prepared

for standards and the phosphorus contents of the samples were determined from this

calibration curve.

Barton Reagent

The reagent was acquired by combining A and B solutions and making volume up to

1 L by following the procedure of Ashraf et al. (1992).

a) Solution A

Ammonium molybdate at 25 gram was dissolved in 400 mL of distilled water.

b) Solution B

Three hundred milliliter of boiling water was used to dissolve 1.25 grams of

ammonium metavenadate. After cooling the solution, HNO3

(concentrated) was added at 250

mL and again cooled at room temperature.

45

3.13.5 Potassium determination

Flame photometer (Jenway PFP-7) was used to determine potash from the extracted

samples. First of all, KCl standards of 2, 4, 6, 8 and ppm were prepared and then by plotting

the flame photometer reading against the respective K concentration there developed a

standard curve. Flame photometer readings of the samples were compared with calibration

curve and the actual K concentration in the samples were computed.

3.14 Characterization of the selected isolates

3.14.1 Root colonization assay

Surface sterilized seeds of maize and wheat inoculated with 3-day-old rhizobacterial

broth culture of the selected strains were sown in sterilized sand filled glass jars system

modified from Simon et al. (1996). The jars were sited under controlled conditions for 10

days in growth room at 25±1 0C. The sand was soaked with autoclaved half-strength

Hoagland solution. All the treatments were replicated four times. After 10 days the seedlings

were uprooted and roots were detached. To estimate root colonization by the selected strains,

one centimeter of root tip from each jar was cut off and placed in sterilized conical flask

having sterilized water. The flasks containing root suspension were shaken vigorously for 30

minutes in shaking incubator at 100 rpm. One milliliter material of each treatment was taken

in sterilized Petri plate and autoclaved Yeast Manitol Agar (YMA) media was spread onto it.

The Petri plates were incubated for 4 days at 28±1 0C. Bacterial colony forming units were

counted in each Petri plate by using colony counter.

3.14.2 Phosphate solubilization assay

The method of Mehta and Nautiyal (2001) was used to assess the potential of the

selected rhizobial strains to solubilize inorganic phosphate qualitatively. Phosphate growth

media i.e. NBRI-PBB (National Botanical Research Institute Phosphate Bromophenol Blue)

was used. A loop full of each culture (OD 0.5 (108

– 109

CFU mL-1

) was placed at three

places on petri plates and incubated for 7 days at 28 ± 1 °C. After seven days the colonies

showing clearing zone around them were characterized as positive for phosphate

solublization. Each treatment was repeated three times in the assay.

46

3.14.3 Exopolysaccharide (EPS) production assay

Broth culture (3-day-old) of the strains (OD 0.5 108

– 109

CFU mL-1

) was applied on

the Petri plates having RCV-glucose media (Ashraf et al., 2004) at five places. The plates

were incubated for four days at 28°C ± 1 after which the Petri plates were examined for

mucoid growth of the inoculated strains. Mucoid colonies were considered as positive in EPS

production.

3.14.4 Chitinase activity assay

The strains were characterized for chitinase activity following Chernin et al. (1998).

Yeast mannitol agar media containing 0.2% (w/v) chitin was prepared, autoclaved and poured

into Petri plates. Loop full of the broth culture of the selected strains (OD 0.5 108

– 109

CFU

mL-1

) was applied on Petri plates at five places. Petri plates were incubated for 5 days at 28 ±

1 °C. Halos formation around rhizobacterial colonies was judged and the strains showing

these halos were characterized as positive for chitinase activity.

3.15 Identification of selected strains

The selected strains were identified by using the BIOLOG® identification system

(Microlog System release 4.2 Biolog Inc., USA). The BIOLOG® identification system has

been found equally as reliable for identificstion as 16s RNA (Flores-Vargus and O, Hara,

2006). For identification on Biolog system the isolates were grown on Biolog universal

growth medium. Cells from the agar plates were separated with a sterile swab. The swab was

twirled and pressed against the inside surface of the tube on the dry glass above the fluid line

to break up clumps and release cells. The swab was moved up and down the wall of the tube

until the organisms mixed with the fluid and became a homogenous, clump free suspension.

The turbidity was adjusted to appropriate value for each isolate. This suspension was poured

into the multi-channel pipette reservoir. Eight sterile tips were fastened securely onto the 8-

channel repeating pipette. Tips were primed and 150 µL inoculum was poured into each well.

Micro plate was covered with its lid and incubated at 30 0C for 6 hours. After incubation,

micro plate was loaded to micro plate reader (Biolog) and identified putatively by microlog

software.

47

3.16 Statistical analysis

Treatment data were compared with ANOVA ( analysis of variance) according to completely

randomized design in pot trial and randomized complete block design for field trial using the

software SPSS version 18.0, ( Steel et al., 1996) and treatment means were compared using

Duncan’s Multiple Range Tests (p≤0.05) (Duncan, 1955). Selection of efficient rhizobacterial

strains was done by using the principal component scoring method using MIITAB software.

48

CHAPTER-4

RESULTS

When plants are subjected to abiotic stress such as soil salinity, their endogenous level of

hormones is disturbed which results in poor growth of plant. In the present study, we isolated

number of rhizobacterial strains from the rhizosphere of maize plants growing under salt-

affected soil conditions. These strains were tested for their ability to tolerate salinity stress by

using osmoadaptation assay. Out of these strains, most tolerant strains were selected and their

auxin production ability in the presence and absence of L-TRP was measured. Strains which

tolerated salinity and produced sufficient amounts of auxin were selected to test their ability

of growth promotion of maize seedlings under salt stressed axenic conditions. Two most

efficient strains (M4 and M7) were selected for further evaluation in pot and field trials to

improve the growth and yield of maize under salt stressed conditions.

4.1 Osmoadaptation assay

The ability of isolated rhizobacterial strains to produce auxin was tested by carrying

out the osmoadaptation assay. The selected strains were subjected to grow at four salinity

levels i.e. original, 10, 20 and 30 dS m-1 developed by using sodium chloride. The growth of

rhizobacterial strains was assessed by measuring the optical density (OD at 540 nm) after

three days of incubation. It was found from the results that all the strains have variable ability

to withstand salinity stress. Most of the strains were tolerant of salinity stress and did not

showed significant decrease in growth with increasing salinity. Maximum optical density at

highest salinity level was observed by the strain MA10 and followed by MA12 and MA9.

49

Table 3: Growth of rhizobacterial strains in broth culture after 3 days under salt stress

conditions

Strain Original 10 dS m-1 20 dS m-1 30 dS m-1

MA-1 1.21 ± 0.10* 0.89 ± 0.04 0.97 ± 0.02 0.97 ± 0.23

MA-2 1.26 ± 0.20 1.05 ± 0.06 1.00 ±0.12 0.75 ± 0.12

MA-3 1.08 ± .01 1.11 ± 0.01 0.98 ± 0.12 1.00 ± 0.08

MA-4 1.00 ± 0.08 1.11 ± 0.36 1.08 ± 0.02 0.78 ± 0.04

MA-5 0.70 ± 0.03 1.40 ± 0.12 0.70 ± 0.05 0.87 ± 0.03

MA-6 0.81 ±0.03 0.94 ± 0.05 0.89 ± 0.04 0.83 ± 0.07

MA-7 0.97 ±0.02 0.85 ± 0.02 0.84 ± 0.17 0.77 ± 0.26

MA-8 0.63 ±0.01 1.14 ± 0.06 0.93 ± 0.11 0.78 ± 0.17

MA-9 0.52 ± 0.01 0.64 ± 0.12 0.40 ± 0.15 0.63 ± 0.21

MA-10 0.87 ± 0.07 0.51 ± 0.03 0.50 ± 0.03 0.68 ± 0.02

MA-11 1.05 ± 0.04 0.62 ± 0.21 0.69 ± 0.02 0.79 ± 0.15

MA-12 0.95 ± 0.03 0.60 ± 0.20 0.64 ± 0.03 0.75 ± 0.01

MA-13 1.48 ± 0.04 1.38 ±0.06 1.04 ± 0.04 0.98 ± 0.23

MA-14 1.43 ± 0.06 0.77 ± 0.08 1.50 ± 0.23 0.65 ± 0.08

MA-15 1.04 ± 0.09 0.50 ± 0.03 1.02 ± 0.14 1.25 ± 0.01

Values are means of three replicates ± Standard error (SE)

50

Table 4: Growth of rhizobacterial strains in broth culture after 3 days under salt stress

conditions

Strain Original 10 dS m-1 20 dS m-1 30 dS m-1

MA-16 0.99 ± 0.07* 0.20 ± 0.01 1.07 ± 0.09 0.83 ± 0.11

MA-17 0.04 ± 0.04 0.07 ± 0.05 0.09 ± 0.14 0.08 ± 0.14

MA-18 1.02 ± 0.01 0.78 ±0.10 0.94 ± 0.05 1.15 ± 0.07

MA-19 0.68 ± 0.01 0.84 ±0.03 0.80 ± 0.20 0.91 ± 0.05

MA-20 1.30 ± 0.01 1.22 ± 0.08 1.06 ± 0.06 1.10 ± 0.12

MA-21 1.08 ± 0.03 1.26 ± 0.17 1.09 ± 0.16 1.03 ± 0.01

MA-22 0.80 ± 0.01 0.78 ± 0.11 0.67 ± 0.08 0.59 ± 0.06

MA-23 0.86 ± 0.01 0.78 ± 0.02 0.82 ± 0.02 0.81 ± 0.08

MA-24 0.92 ± 0.02 0.87 ± 0.06 0.77 ± 0.01 0.85 ± 0.23

MA-25 1.05 ± 0.02 1.01 ± 0.07 1.21 ± 0.12 0.96 ±0.11

MA-26 0.63 ± 0.02 0.66 ± 0.04 0.62 ± 0.11 0.67 ± 0.06

MA-27 0.81 ± 0.01 0.80 ± 0.15 0.77 ± 0.03 0.78 ± 0.05

MA-28 0.58 ± 0.01 0.54 ± 0.13 0.58 ± 0.05 0.57 ±0.12

MA-29 0.75 ±0.02 0.82 ± 0.02 0.79 ± 0.07 0.63 ± 0.16

Values are means of three replicates ± Standard error (SE)

51

4.2 Auxin production assay

The data in Table 5 shows auxin production ability of the rhizobacterial strains. It is

evident from the data that all rhizobacterial strains were able to produce auxin (IAA

equivalents) to varying degrees, both in the presence and absence of L-TRP. However, the

auxin production was enhanced by applying the precursor L-TRP.

Rhizobacterial strain MA-11 produced maximum IAA equivalents when

supplemented with L-TRP. The minimum auxin production was recorded by strain MA-21.

The data also suggested that these strains were not over auxin producing, but produced

moderate amounts of IAA equivalents.

After conducting auxin production assay, eleven strains were selected for further screening in

growth room (axenic) experiment. The selected strains were renamed as follows.

MA1 → M1

MA2 → M2

MA3 → M3

MA4 → M4

MA5 → M5

MA7 → M6

MA11 → M7

MA13 → M8

MA14 → M9

MA19 → M10

MA20 → M11

52

Table 5: Amount of IAA equivalents (µg mL-1) produced by the selected rhizobacterial

strains

Strains With

L-TRP

Without

L-TRP Strains

With

L-TRP

Without

L-TRP

MA-1 9.23 d* 6.11 gh MA-14 5.56 a 3.09 op

MA-2 7.25 b 5.61 lm MA-15 4.61 c 0.53 q

MA-3 10.19 g 7.07 jk MA-18 2.25 j 2.02 k

MA-4 13.26 g 7.19 g MA-19 5.12 b 2.08 jk

MA-5 6.76 e 5.68 i MA-20 4.61 c 1.63 l

MA-6 2.53 i 2.48 i MA-21 1.18 no 0.91 p

MA-7 8.13 d 4.53 f MA-23 2.95 h 1.40 mn

MA-8 3.07 gh 0.98 op MA-24 2.06 jk 1.11 op

MA-11 19.19 g 12.08 jk MA-25 3.25 g 1.45 lm

MA-13 6.12 b 3.52 q

Means sharing similar letter(s) do not differ significantly

53

4.3 Jar trial (Growth room study)

The selected rhizobacterial strains which were found having the ability of auxin

production were further screened to access their ability to promote growth of maize seedlings

under salt affected axenic conditions. The results of the experiment showed that salinity had a

repressive effect on the growth of maize but inoculation with auxin producing PGPR strains

improved the growth of maize with varying degree.

4.3.1 Shoot length

The Figure 1 demonstrates the effect of salinity and inoculation with rhizobacterial

strains on the shoot length of maize. It was found that shoot length significantly decreased

with increasing salinity levels. Maximum decrease in shoot length was recorded at EC 15 dS

m-1. At this level maximum increase in shoot length was observed by the strain M4 followed

by strain M7 both in the absence and presence of L-TRP as compared to their respective

uninoculated control. At EC 4, 8, and 12 dS m-1 strain M4 was the best performer regarding

improvement in shoot length, where it caused 88.12, 57.67 and 45.43% increase in the

absence and 49.89, 56.71 and 51.73% increase in the presence of L-TRP as compare to their

uninoculated controls. Other strains were also effective in improving the shoot length but

most of them showed non-significant increases in shoot length as compared to their control.

Under normal conditions all the strains improved the shoot length both in the absence

and presence of L-TRP. However along with L-TRP application the performance of strains

was enhanced. Under these conditions strain M6 was among the front runners in improving

the shoot length in the absence and presence of L-TRP. Strains M2, M3, M5, M8, M9, M10

and M11 in the absence of L-TRP showed non-significant results as compared to

uninoculated control at normal conditions.

54

5

15

25

35

45

55

65

Without L-TRP With L-TRP Without L-TRP With L-TRP Without L-TRP With L-TRP Without L-TRP With L-TRP Without L-TRP With L-TRP

EC (Original) dS m-1 EC 4 dS m-1 EC 8 dS m-1 EC 12 dS m-1 EC 15 dS m-1

Shoo

t le

ngth

(cm

)

Control M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11

Figure 1: Effect of rhizobacterial inoculation on shoot length of maize seedlings under salt stressed axenic conditions.

55

4.3.2 Root length

Figure 2 demonstrate the results regarding root length of maize seedlings. A marked

reduction in root length of maize seedlings subjected to salinity stress was recorded. A 22, 32,

35 and 55% decrease in root length was observed at EC 4, 8, 12 and 15 dS m-1. However,

rhizobacterial inoculation was found effective in mitigating the deleterious effects of salinity

and caused significant improvements in root length at all salinity levels.

At 15 dS m-1, strain M4 performed better than the rest of the strains and caused 57 and

69% increase in root length in the absence and presence of L-TRP, respectively, over

uninoculated control. Most of the other strains showed non-significant increases in the root

length. At EC 4, 8 and 12 dS m-1, strains M4, M7 and M1 were the leaders in enhancing the

root length both in the presence and absence of L-TRP. Under normal conditions, strains M1

and M3 performed better than the rest of the strains. In the absence of L-TRP, M2, M6 and

M10 showed non-significant increases in root length as compared to uninoculated control.

4.3.3 Shoot dry weight

The Figure 3 shows that salinity significantly reduced the shoot dry weight at all

salinity levels. At EC 15 dS m-1 maximum reduction in shoot dry weight was recorded.

Rhizobacterial inoculation was found effective in improving the shoot dry weight to varying

degrees, but most of the strains showed non-significant increases in shoot dry weight.

Maximum increase in shoot dry weight was recorded by strain M4 both in the absence and

presence of L-TRP application, respectively, followed by strain M7. In the presence of L-

TRP strain M4 also performed better than the rest of the strains at other salinity levels and

caused 25, 36 and 49% increase in shoot dry weight of maize seedlings at 4, 8 and 12 dS m-1,

respectively, as compared to respective uninoculated control.

Under normal conditions all the strains significantly improved the shoot dry weight

both in the absence and presence of L-TRP. Strains M1, M4 and M6 were among the leaders

in improving the shoot dry weight of maize under normal conditions. Maximum increase in

this case was recorded by strain M6 which caused 45 and 52% increase in shoot dry weight of

maize seedlings over its respective uninoculated control.

56

0

10

20

30

40

50



Roo

t le

ngt

h p

er p

lan

t (c

m)


Figure 2: Effect of rhizobacterial inoculation on root length of maize seedlings under salt stressed axenic conditions.

57

0.05

0.15

0.25

0.35

0.45

0.55

0.65



Shoo

t dr

y pe

r pl

ant

(g)


Figure 3: Effect of rhizobacterial inoculation on shoot dry weight of maize seedlings under salt stressed axenic conditions.

58

4.3.4 Root dry weight

It is evident from the Figure 4 that salinity stress had a repressive effect on root dry

weight and it declined with increasing salinity levels. Inoculation with different rhizobacterial

strains improved the root dry weight to varying levels at all tested salinity levels. Under

normal conditions maximum increase in root dry weight was recorded with strain M6 both in

the absence and presence of L-TRP. This strain was followed by strain M1 and M4. Strain

M9 was found least effective among all the strains in improving the root dry weight of maize

seedlings.

Maximum reduction in root dry weight was observed at the highest salinity level i.e.

15 dS m-1. At this level, strain M7 was found most effective in enhancing the root dry weight

of maize seedlings which caused 45 and 57% increase in the absence and presence of L-TRP,

respectively, as compared to its uninoculated control. Strain M7 was followed by strain M4,

M1 and M6 regarding improvement in root dry weight. Rest of the strains showed non-

significant increase in root dry weight as compared to uninoculated control.

4.3.5 Total plant dry weight

Total plant dry weight significantly decreased with increasing salinity levels (Table

6). At 15 dS m-1, maximum increase in total plant dry weight was observed by the strains M4

and M7. At EC 4, 8, and 12 dS m-1, strain M4 performed better than rest of the strains

regarding improvement in total plant dry weight. Other strains were also effective in

improving the total plant dry weight but most of them showed non-significant increases in

total plant dry weight as compared to their control.

Under normal conditions all the strains improved the total plant dry weight both in the

absence and presence of L-TRP. However along with L-TRP application the performance of

strains was enhanced. Under these conditions strain M6 was among the front runners to

improve the total plant dry weight in the absence as well as in the presence of L-TRP.

59

0.10

0.20

0.30

0.40

0.50

0.60



Roo

t dr

y pe

r pl

ant

(g)


Figure 4: Effect of rhizobacterial inoculation on root dry weight of maize seedlings under salt stressed axenic conditions.

60

Bacterial strains

Total plant dry weight (g)

Normal (1.6 dS m-1) 4 dS m-1 8 dS m-1 12 dS m-1 15 dS m-1

L-TRP (-) L-TRP (+) L-TRP (-) L-TRP (+) L-TRP (-) L-TRP (+) L-TRP (-) L-TRP (+) L-TRP (-) L-TRP (+)

Control 0.61±0.013 0.66±0.010 0.47±0.017 0.51±0.012 0.32±0.009 0.37±0.019 0.26±0.032 0.30±0.008 0.20±0.063 0.23±0.059

M1 0.92±0.018 1.03±0.015 0.74±0.063 0.86±0.059 0.53±0.060 0.63±0.005 0.43±0.003 0.48±0.015 0.36±0.032 0.41±0.010

M2 0.79±0.007 0.88±0.026 0.61±0.004 0.71±0.003 0.46±0.008 0.54±0.026 0.40±0.017 0.44±0.009 0.25±0.001 0.28±0.019

M3 0.84±0.032 0.94±0.009 0.57±0.015 0.66±0.032 0.43±0.029 0.52±0.017 0.34±0.004 0.38±0.059 0.24±0.029 0.27±0.003

M4 0.99±0.011 1.11±0.060 0.84±0.017 0.96±0.053 0.61±0.063 0.72±0.012 0.49±0.008 0.56±0.001 0.41±0.032 0.46±0.015

M5 0.90±0.011 1.01±0.017 0.71±0.019 0.81±0.004 0.53±0.017 0.63±0.009 0.33±0.009 0.36±0.019 0.26±0.060 0.29±0.004

M6 1.05±0.027 1.17±0.059 0.76±0.003 0.88±0.017 0.52±0.059 0.61±0.008 0.42±0.004 0.48±0.017 0.35±0.009 0.39±0.017

M7 0.91±0.063 1.01±0.060 0.78±0.017 0.90±0.026 0.55±0.017 0.66±0.032 0.48±0.029 0.54±0.012 0.42±0.001 0.47±0.012

M8 0.84±0.004 0.94±0.032 0.66±0.005 0.76±0.010 0.50±0.001 0.59±0.053 0.39±0.060 0.43±0.059 0.27±0.010 0.31±0.059

M9 0.73±0.059 0.81±0.001 0.61±0.017 0.70±0.019 0.43±0.008 0.51±0.012 0.34±0.019 0.39±0.005 0.27±0.009 0.30±0.005

M10 0.76±0.015 0.85±0.004 0.55±0.008 0.63±0.029 0.42±0.017 0.50±0.060 0.36±0.008 0.40±0.063 0.28±0.026 0.31±0.029

M11 0.79±0.010 0.88±0.026 0.63±0.003 0.72±0.001 0.46±0.015 0.54±0.026 0.35±0.003 0.40±0.017 0.27±0.009 0.30±0.012

Table 6: Effect of rhizobacterial inoculation on total plant dry weight of maize seedlings under salt stressed axenic conditions.

61

Bacterial strains

Shoot root ratio

Normal (1.6 dS m-1) 4 dS m-1 8 dS m-1 12 dS m-1 15 dS m-1

L-TRP (-) L-TRP (+) L-TRP (-) L-TRP (+) L-TRP (-) L-TRP (+) L-TRP (-) L-TRP (+) L-TRP (-) L-TRP (+)

Control 1.90 2.01 2.23 2.34 2.07 1.66 1.92 1.60 1.87 1.77

M1 1.41 1.47 1.61 1.61 1.46 1.29 1.39 1.33 1.57 1.60

M2 1.89 1.96 2.06 2.06 1.72 1.45 2.88 2.77 2.66 2.71

M3 1.30 1.35 2.08 2.08 2.36 2.05 2.59 2.43 2.99 3.05

M4 1.47 1.53 1.55 1.55 1.55 1.35 1.49 1.43 1.57 1.59

M5 1.47 1.53 1.64 1.64 1.67 1.52 1.49 1.42 2.82 2.88

M6 1.46 1.52 1.64 1.64 1.55 1.35 1.37 1.31 1.65 1.68

M7 1.46 1.52 1.53 1.53 1.64 1.44 1.41 1.35 1.58 1.61

M8 1.88 1.96 2.57 2.58 1.64 1.38 1.90 1.82 1.25 1.28

M9 1.37 1.42 1.58 1.58 1.47 1.32 1.10 1.06 1.69 1.73

M10 1.97 2.05 1.52 1.52 1.53 1.32 1.82 1.74 1.67 1.70

M11 1.68 1.75 1.82 1.82 2.69 2.38 2.70 2.56 2.54 2.60

Table 7: Effect of rhizobacterial inoculation on shoot root ratio of maize seedlings under salt stressed axenic conditions.

62

Figure S2: Pictorial representation of rhizobacterial inoculation at different salinity levels under axenic conditions

63

4.4 Pot trial

4.4.1 Plant height

The results of the pot trial revealed that salinity had a repressive effect on plant height

(Figure 5), regardless of inoculation treatment. It was evident from the results that with

increasing salinity levels there was progressive decline in plant height. However,

rhizobacterial inoculation with M4, M7 or both seemed promising in alleviating the salinity

stress which was reflected in case of improvement in plant height. Rhizobacterial inoculation

in single and dual combinations improved plant height at all salinity levels. The Effect of

rhizobacterial inoculation was pronounced when strains were tested along with L-TRP

application.

Salinity stress significantly reduced the plant height at all levels. Maximum reduction

in plant height (71%) was noted at 15 dS m-1. In case of single strain inoculation strain M4

performed better than strain M7; M4 and M7 increased plant height up to 51 and 27%,

respectively, over their respective uninoculated controls. When both these strains were tested

in the presence of L-TRP, further increase in plant height was recorded i.e. M4 and M7

increased up to 54 and 29%, respectively, over control. However, combined application of

strain M4 and M7 gave the best results regarding the improvement in plant height at all

salinity levels both in the presence and absence of L-TRP application. At 15 dS m-1, an

increase up to 74% by M4 and M7 was recorded, in the absence and presence of L-TRP,

respectively, over uninoculated control regardless of L-TRP.

64

ik

m

r

s

ghj

m

r

s

d-g d-gi

m

q

d degh

n

q

f-h gh

k

n

r

d-f e-g

j

o

r

c

a

c

l

p

ba

c

m

p

0

20

40

60

80

100

120

140

160

180

200

Original 4 8 12 15 Original 4 8 12 15

Without L-TRP With L-TRP

Pla

nt H

eigh

t (c

m)

Control M4 M7 M4XM7

Figure: 5 Effect of rhizobacterial inoculation (with and without L-TRP) on plant height of maize at different salinity levels (original = 1.63,

4, 8, 12 and 15 d Sm-1). Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average of

three replicates. Control (Uninoculated), M4 (Pseudomonas fluorescens), M7 (Serratia proteamaculans).

Salinity levels (dS m-1)

65

4.4.2 Fresh biomass

The data (Figure 6) about the Effect of rhizobacterial inoculation on fresh biomass of

maize grown under salt affected soil conditions revealed that salinity had adverse effect on

the fresh biomass and it decreased with increasing salinity. However, improvement in fresh

biomass was observed due to inoculation with rhizobacterial strains.

Sole inoculation was effective under normal and at all salinity levels. Maximum

reduction (45%) in fresh biomass was recorded at highest salinity level. Maximum increase in

fresh biomass at salinity 15 dS m-1 was observed by strain M4 in the presence and absence of

L-TRP and that increase was 39 and 31%, respectively, over its respective uninoculated

control. Inoculation with stain M7 gave non-significant results with control.

Co-inoculation of M4xM7 performed even better than sole inoculation of strain M4

and M7 at all salinity levels. At the highest salinity level i.e.15 dS m-1 an increase of 35 and

33% in fresh biomass as compared to respective uninoculated control was recorded by

inoculation with M4xM7 in the absence and presence of L-TRP application, respectively. The

results of co-inoculation were statistically non-significant as compared to single inoculation

treatments. Co-inoculation was also effective at lower salinity levels and under normal soil

conditions. At 12 dS m-1, increase in fresh biomass due to co-inoculation with M4xM7 was

46 and 43%, over respective uninoculated control when it was tested with and without L-TRP

application, respectively. At normal soil conditions, combination M4xM7 resulted 22.17%

increase in the absence of L-TRP and 34% increase in fresh biomass over respective

uninoculated control along with L-TRP application.

66

e-h f-i

k-ml-n

o

d-g d-g

kll-n

o

d-f d-ff-i

h-j

l-n

d-f d-fe-h

g-j

kl

d-f d-ff-j

jk

no

d-f d-ff-i

ij

m-o

c

a

def-i

l-n

b

ccd

e-h

l-n

0

50

100

150

200

250

300

350



Fre

sh B

iom

ass

(g)

Control M4 M7 M4XM7

Figure: 6 Effect of rhizobacterial inoculation (with and without L-TRP) on fresh biomass of maize at different salinity levels (original =

1.63, 4, 8, 12 and 15 d Sm-1). Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is

average of three replicates. Control (Uninoculated), M4 (Pseudomonas fluorescens), M7 (Serratia proteamaculans).


67

4.4.3 Root length

The results regarding root length (Figure 7) demonstrate that soil salinity

progressively decreased the root length with increasing soil salinity level, however, the

rhizobacterial inoculation with and without L-TRP application was found effective in

reducing the adverse effects of salinity on root length. At EC 15 dS m-1, 75% reduction in root

length was recorded. Sole inoculation with strain M4 without L-TRP caused 81% increase in

root length over respective uninoculated control at EC 15 dS m-1.

Inoculation with strain M4 Along with L-TRP application caused 6, 30, 35 and 39%

increase in root length at 4, 8, 12 and 15 dS m-1, respectively. Sole inoculation with strain M7

also significantly improved root length at all salinity levels. Combined application of M4 and

M7 (M4xM7) performed better than their sole inoculation at both L-TRP applications. An

increase of 33 and 35% in root length at 15 dS m-1 was recorded by the inoculation of

M4xM7 in presence and absence of L-TRP application, respectively.

68

lmn

op

tu

v

klm

no

tu

v

e-gg-i

jk

q

tu

b-dd-f

ij

p

tu

ghij

k

qr

u

c-fg-i

k

q

tu

b bc

fg

mn

st

a a

b-e

n

rs

0

10

20

30

40

50

60



Roo

t Len

gth

(cm

)

Control M4 M7 M4XM7

Figure: 7 Effect of rhizobacterial inoculation (with and without L-TRP) on root length of maize at different salinity levels (original = 1.63,



.


69

Figure S3: Pictorial representation of rhizobacterial inoculation on root growth at different salinity levels in pot experiment.

70

4.4.4 Root weight

Data regarding the root weight (Figure 8) reveal that soil salinity progressively

decreased the root weight with increasing soil salinity level. However, the rhizobacterial

inoculation effectively improved the root weight under saline conditions. Inoculation

performed even better when it was tested along with L-TRP application. At EC 15 dS m-1,

up to 69% reduction in root weight was recorded. Sole inoculation with strain M4, without

L-TRP application caused 44% increase in root length over respective uninoculated

control at EC 15 dS m-1. L-TRP application alone also improved the root weight.

In the presence of L-TRP application, inoculation with stain M4 showed up to

32% increase in root weight at EC 15 dS m-1. Similar improvement in root weight was

also observed at other salinity levels. Sole inoculation with strain M7 also significantly

improved root weight at all salinity levels both with and without L-TRP application. As

compared with single strain inoculation, combined application of M4 and M7 (M4xM7)

performed better at both L-TRP applications. An increase up to 58 and 78% in root weight

at 15 dS m-1 was recorded by M4xM7 in presence and absence of L-TRP application,

respectively, over their respective uninoculated controls.

4.4.5 Grain yield

Figure 9 demonstrates, that the rhizobacterial inoculation with and without L-TRP

application was found effective in improving the grain yield of maize under salt stress. It

is evident from the data that salinity adversely reduced the grain yield. At EC 15 dS m-1 no

grain formation was observed. At 12 dS m-1, 83% reduction in grain yield was recorded.

Sole inoculation with strain M4 without L-TRP caused 20.6, 31.1 and 41% increase in

grain yield over respective uninoculated control at EC 4, 8 and 12 dS m-1, respectively.

Along with L-TRP application, up to 33% increase in grain yield was recorded by

inoculation with strain M4 at 12 dS m-1. In case of single strain inoculation strain M4

performed better than M7. At both L-TRP applications, combined application of M4 and

M7 (M4xM7) performed better than their sole inoculation. An 89 and 77% increase in

grain yield at 12 dS m-1 was recorded by M4xM7 in presence and absence of L-TRP

application respectively, over their respective uninoculated controls.

71

ijk

mn

st

u

gh

i-k

lm

rstu

cdcd

h-j

n-p

st

bccd

g-i

m-o

q-s

deef

jk

o-q

st

cdde

i-k

n-p

st

ab a

fg

lm

p-r

a a

de

l

o-q

0

5

10

15

20

25

30

35

40

45

50



Roo

t wei

ght

(g)

Control M4 M7 M4XM7

Figure: 8 Effect of rhizobacterial inoculation (with and without L-TRP) on root weight of maize at different salinity levels (original = 1.63,




72

ghi

j

o

fghi

j

o

c-e c-efg

m

cd cdde

l

de egh

n

c-e c-egh

mn

aba

c

l

ab ab

k

0

10

20

30

40

50

60

70

80

90

100



Gra

in Y

ield

(g)

Control M4 M7 M4XM7

Figure 9: Effect of rhizobacterial inoculation (with and without L-TRP) on grain yield of maize at different salinity levels (original = 1.63, 4,

8, 12 and 15 d Sm-1). Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average of



73

L-TRP Strains

Salinity Levels (dS m-1)

1.63 4 8 12 15

Wit

hou

t Control 4.17 5.10 5.64 11.11 15.09

M4 3.42 3.49 4.83 4.30 8.87

M7 3.00 4.08 1.46 4.72 5.84

M4 X M7 2.34 0.55 1.41 1.70 4.00

Wit

h

Control 4.10 4.87 5.46 10.84 13.28

M4 3.33 3.34 4.74 4.01 8.53

M7 2.92 3.96 1.49 4.00 5.22

M4 X M7 1.99 0.52 1.37 1.80 3.81

Table 8: Effect of rhizobacterial inoculation (with and without L-TRP) on shoot root ratio of maize at different salinity levels (original =



74

4.4.6 1000-grain weight

The results showing the effect of salinity and rhizobacterial inoculation on 1000-grain

weight of maize are presented in the Figure 10. The data demonstrates that soil salinity

progressively decreased the 1000-grain weight with increasing soil salinity level, however the

rhizobacterial inoculation with and without L-TRP application improved the 1000-grain

weight. At EC 12 dS m-1, 51% reduction in 1000-grain weight was recorded. In the absence

of L-TRP, sole inoculation with strain M4 caused up to 8, 22 and 37% increase in 1000-grain

weight over respective uninoculated control at EC 4, 8 and 12 dS m-1, respectively.

The results of inoculation improved when it was carried out along with L-TRP

application. In the presence of L-TRP strain M4 caused 10, 21 and 34% increase in 1000-

grain weight at 4, 8 and 12 dS m-1, respectively. Both in the presence and absence of L-TRP,

sole inoculation with M7 also positively improved 1000-grain at all salinity levels. Combined

application of M4 and M7 (M4xM7) performed better than their sole inoculation at both L-

TRP applications. Up to 59 and 57% increase in 1000-grain at 12 dS m-1 was recorded by

M4xM7 in presence and absence of L-TRP application, respectively.

4.4.7 Cob biomass

Figure 11 shows the effects of salinity and rhizobacterial inoculation on cob biomass

of maize. The data demonstrates that soil salinity caused a significant reduction in the cob

biomass however, the rhizobacterial inoculation significantly increased the cob biomass.

Combined application of both strains showed better results than their sole inoculation. The

effect of inoculation was enhanced when it was used along with L-TRP application. Only L-

TRP application also positively improved the cob biomass. At EC 12 dS m-1, 8% reduction in

cob biomass was recorded. Sole inoculation with strain M4 without L-TRP caused up to 6, 10

and 38% increase in cob biomass over respective uninoculated control at EC 4, 8 and 12 dS

m-1, respectively.

At 4, 8 and 12 dS m-1, M4 strain along with L-TRP application caused up to 7, 10 and

39% increases in cob biomass, respectively. Sole inoculation with M7 also positively

improved cob biomass at all salinity levels both with and without L-TRP application. M4 and

75

h-k j-l

pq

r

h-k i-l

p

r

eff-h

mn

pq

c-efg

m

pq

efg-j

m-o

q

de

g-i

m-o

q

abcd

l

no

a a

kl

o

0

50

100

150

200

250



1000

Gra

in W

eigh

t (g

)

Control M4 M7 M4XM7

Figure 10: Effect of rhizobacterial inoculation (with and without L-TRP) on 1000-grain weight of maize at different salinity levels

(original = 1.63, 4, 8, 12 and 15 d Sm-1). Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range

test. Data is average of three replicates. Control (Uninoculated), M4 (Pseudomonas fluorescens), M7 (Serratia proteamaculans).


76

fgh-j

k

m

cd deg-i

k

de efh-j

k

cdef

ij

l

defg

j

l

a ac

f-h

ap

cd

g-j

c cdg-i

k

0

20

40

60

80

100

120

140

160

180



Cob

bio

mas

s (g

)

Control M4 M7 M4XM7

Figure: 11 Effect of rhizobacterial inoculation (with and without L-TRP) on cob biomass of maize at different salinity levels (original =




77

M7 applied in combination (M4xM7) showed better results than their sole inoculation at

both L-TRP applications. An increase up to 55 and 57% in cob biomass at 12 dS m-1 was

recorded by M4xM7 in presence and absence of L-TRP application, respectively.

4.4.8 Chlorophyll contents (SPAD Values)

The results regarding chlorophyll contents (Figure 12) indicate that the salinity

significantly reduced the chlorophyll contents at all levels. However inoculation/co-inoculation

reduced the effects of salinity and improved the chlorophyll contents both in the presence and

absence of L-TRP application. At 4, 8, 12 and 15 dS m-1, a significant decrease of 8, 23, 24 and

26%, respectively, in chlorophyll contents was recorded as compared with uninoculated controls.

Single inoculation improved the chlorophyll contents at all tested salinity levels both in

the presence and absence of L-TRP. In the absence of L-TRP, stain M4 performed better than

M7 and increased the chlorophyll contents up to 17, 35, 11 and 14% at 4, 8, 12 and15 dS m-1

respectively, over its respective uninoculated control. Maximum increase in chlorophyll contents

in the absence of L-TRP was recorded by coinoculation with M4XM7 which gave up to 34.86,

64.74, 30.84 and 10.58% increase at 4, 8, 12, and 15 dS m-1 respectively, over its respective

uninoculated control. Separate application of L-TRP also caused positive improvement in

chlorophyll contents but these results were non-significant as compared to uninoculated control.

Along with L-TRP application co-inoculation performed better than sole inoculation with

M4 and M7. Maximum increase in chlorophyll contents was recorded up to 24, 55, 28 and 13%

at 4, 8, 12 and 15 dS m-1, respectively, by M4xM7 over uninoculated control. Single inoculation

with M4 and M7 also significantly improved the chlorophyll contents at all salinity levels as

compared with uninoculated control. The results of M4 and M7 inoculation along with L-TRP

were statistically non-significant as compared to one another at all tested salinity levels except at

EC 15 dS m-1.

78

f-hij

m-o

q

r

e-hhi

l-n

q

r

cd c-ed-g

k-n

p

cc-e c-f

jk

no

c-e c-ee-h

l-n

p

bcc-e c-e

lm

p

a a a

g-i

k-m

aab ab

e-h

kl

0

10

20

30

40

50

60



SPA

D c

hlor

ophy

ll v

alue

s Control M4 M7 M4XM7

Figure: 12 Effect of rhizobacterial inoculation (with and without L-TRP) on SPAD chlorphyll values of maize at different salinity levels

(original = 1.63, 4, 8, 12 and 15 d Sm-1). Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range



79


The effect of inoculation/combined inoculation on the electrolyte leakage of maize

grown under salt affected soil conditions is shown in Figure 13. The data demonstrate that

electrolyte leakage increased due to exposure to salinity. At EC 15 dS m-1, up to 120%

increase in electrolyte leakage was noted, as compared with uninoculated control.

Electrolyte leakage decreased due to single inoculation/coinoculation with

rhizobacterial strains both in the absence and presence of L-TRP. However, inoculation/co-

inoculation gave better results when it was carried out along with L-TRP application.

Maximum reduction in electrolyte leakage was recorded by coinoculation with M4xM7 that

was up to 39, 37, 38 and 82% at 4, 8, 12 and 15 dS m-1, respectively over respective un-

inoculated control. Along with L-TRP application sole inoculation also significantly reduced

the electrolyte leakage at all salinity levels. As compared with strain M7 and M4 showed

greater reduction in electrolyte leakage. The results of single inoculation were statistically

non significant with each other.

Inoculation/coinoculation in the absence of L-TRP application significantly reduced

the electrolyte leakage. In this case M4xM7 combination gave maximum reduction in

electrolyte leakage that was up to 40, 35, 40 and 43% at 4, 8, 12 and 15 dS m-1, respectively

as compared with uninoculated control. L-TRP application as alone treatment was also found

effective in reducing the electrolyte leakage at all salinity levels.

80

k-m

hi

ef

b

a

o-q

jk

ji

c

a

rs

n-pk-m

hi

e

t

p-rm-o

i-j

ef

qr

k-n k-m

hi

c

t

p-rl-o

f-h

d

trs

n-p

jk

gh

u

tpr

k-n

hi

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2



Ele

ctro

lyte

leak

age

(dS/

m)

Control M4 M7 M4XM7

Figure 13: Effect of rhizobacterial inoculation (with and without L-TRP) on electrolyte leakage of maize at different salinity levels (original

= 1.63, 4, 8, 12 and 15 d Sm-1). Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is



81

4.4.10 Photosynthetic rate

Photosynthetic rate was adversely affected by the soil salinization (Figure 14).

However inoculation/coinoculation was found promising in improving the photosynthetic rate

of maize plants. Application of L-TRP further enhanced the efficiency of inoculation. Up to

73% decrease in photosynthetic rate was noted at EC 15 dS m-1. Similar decrease in

photosynthetic rate was also observed at other salinity levels.

In the presence of L-TRP, M4xM7 caused maximum increase up to 143% in

photosynthetic rate at EC 15dS m-1. Single inoculation with M7 in the presence of L-TRP

caused up to 57, 92, 86 and 98% increase at 4, 8, 12 and 15dS m-1 respectively, as compared

to respective uninoculated control. Single inoculation/coinoculation in the absence of L-TRP

also significantly improved the photosynthetic rate but combined inoculation gave better

results than sole inoculation. M4xM7 combination enhanced photosynthetic rate up to 90% at

15 dS m-1 in the absence of L-TRP, respectively over respective un-inoculated control.

Separate application of L-TRP also caused significant improvement in photosynthetic rate as

compared to control.

4.4.11 Transpiration rate

Figure 15 reveals that soil salinization had a negative effect on transpiration rate.

However, inoculation/coinoculation mitigated the adverse effects of salinity and improved the

transpiration rate at all salinity levels. A 9, 21, 51 and 69% decrease in transpiration rate was

observed at EC 4, 8, 12 and 15 dS m-1, respectively. Separate application of L-TRP also

caused significant improvement in transpiration rate compared to control.

Maximum increase up to 90, 96, 92 and 125% increase in transpiration rate at 4, 8, 12

and 15 dS m-1, respectively, was recorded by M4xM7 in the presence of L-TRP. Single

inoculation with M7 in the presence of L-TRP caused 73, 75, 95 and 100% increase at 4, 8,

12 and 15 dS m-1, respectively, as compared to respective un-inoculated control. Inoculation

in the absence of L-TRP also significantly improved the transpiration rate. M4xM7

combination in the absence of L-TRP enhanced transpiration rate up to 90% at 15 dS m-1,

respectively over respective un-inoculated control. Separate application of L-TRP also caused

significant improvement in transpiration rate compared to control.

82

kl

m-o

p-r

tu u

i-k

l-n

o-r

s-u s-u

c-e

h-j

kl

m-q

r-t

b-d

f-h

jk

m-o

o-r

e-g

g-j

kl

qr rs

d-f

g-i

i-k

n-r o-r

ab

b-e

g-j

lm

n-r

aa-c

e-g

jk

m-p

0

5

10

15

20

25

30



Pho

tosy

nthe

tic

rate

Control M4 M7 M4XM7

Figure 14: Effect of rhizobacterial inoculation (with and without L-TRP) on photosynthetic rate (µmol m-2 S-1) of maize at different salinity levels

(original = 1.63, 4, 8, 12 and 15 d Sm-1). Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test.

Data is average of three replicates. Control (Uninoculated), M4 (Pseudomonas fluorescens), M7 (Serratia proteamaculans).


83

n-qp-r

r-t

uv

v

m-p n-qp-r

s-u

v

g-ji-k

l-n

r-t

v

d-he-i

i-k

n-q

tu

c-gf-i

j-l

p-s

uv

a-db-e

e-i

l-o

r-t

a-c

c-g

h-k

o-r

tu

aab

b-f

k-m

q-s

0

1

2

3

4

5

6

7



Tra

nspi

rati

on r

ate

Control M4 M7 M4XM7

Figure 15: Effect of rhizobacterial inoculation (with and without L-TRP) on transpiration rate (mmol m-2 S-1) of maize at different salinity

levels (original = 1.63, 4, 8, 12 and 15 d Sm-1). Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range



84

4.4.12 Stomatal conductance

Severe reduction in the stomatal conductance of maize was recorded due to soil

salinity (Figure 16). On the other hand, inoculation was found promising in improving the

stomatal conductance of maize plants. Combined application of strains performed better than

their separate inoculation. L-TRP application further enhanced the performance of

inoculation. A decrease up to 9, 21, 51 and 69% in stomatal conductance was recorded at EC

4, 8, 12 and 15 dS m-1, respectively.

In the presence of L-TRP maximum increase up to 125% in stomatal conductance

was observed by M4xM74 at 15 dS m-1. In the absence of L-TRP, inoculation/combined

inoculation also significantly improved the stomatal conductance. M4xM7 combination

enhanced stomatal conductance up to 90% at 15 dS m-1, over respective un-inoculated

control. Similarly at EC 8 and 12 dS m-1, an increase up to 81 and 87% was recorded by

inoculation with M4xM7.

4.4.13 Substomatal CO2 concentration

Soil salinization caused a negative effect on substomatal CO2 concentration (Figure

17). However inoculation/combined-inoculation in the absence and presence of L-TRP was

found promising in improving the transpiration rate of maize plants. At EC 4, 8, 12 and 15 dS

m-1, up to 5, 18, 32 and 40% decrease in transpiration rate as compared with control was

recorded, respectively.

M4xM7 in the presence of L-TRP caused maximum increase up to 9, 3, 9, and 8%

increase in substomatal CO2 concentration at 4, 8, 12 and 15 dS m-1. Single inoculation with

strain M4 in the presence of L-TRP caused up to 6, 3, 7, 6 and 7% increase at 4, 8, 12 and

15dS m-1 respectively, as compared with respective un-inoculated control.

Inoculation/coinoculation in the absence of L-TRP also significantly improved the

transpiration rate.

85

e-ih-k

k-p

j-o

p-r

b-e

b-f

e-i

j-n

qr

a-d

d-g h-m

l-p

o-r

ab

b-gf-l

k-r

qr

e-k

d-i

h-o

l-r

o-r

ab

b-g

f-l

k-r

qr

a-c

b-g

f-l

j-r

m-r

a

b-f

d-j

j-r

n-r

0

0.05

0.1

0.15

0.2

0.25



Stom

atal

con

duct

ance

Control M4 M7 M4XM7

Figure 16: Effect of rhizobacterial inoculation (with and without L-TRP) on stomatal conductance (mmol m-2 S-1) of maize at different salinity




86

d-hf-i

k-m

p-r

t

c-f

e-g

h-j

o-qr-t

c-f

e-g j-l

n-p

st

c-f

d-ff-h

o-q

h-k

c-e

e-h

h-j

m-or-t

ab

c-e

i-k

o-q

i-k

a-c

d-f

h-j

m-o

r-t

a

b-d

g-j

l-n

g-j

0

0.05

0.1

0.15

0.2

0.25



Subs

tom

atal

CO

2co

ncen

trat

ion

(vpm

)Control M4 M7 M4XM7

Figure 17: Effect of rhizobacterial inoculation (with and without L-TRP) on substomatalCO2 concentration of maize at different salinity levels




87

4.4.14 Proline contents

Proline acts as an osmolyte, so increased concentration of proline is produced in

plants under stress condition. Figure 18 represents that proline concentration increased in

plants subjected to salt stress. The maximum increase (96%) in proline contents as compared

with control was recorded in plants subjected to salinity at 15 dS m-1. Inoculation with

rhizobacterial strains caused a significant reduction in proline contents over uninoculated

controls both in the presence and absence of L-TRP. Along with L-TRP sole inoculation with

strain M4 caused maximum reduction (15%) in proline contents at EC 15 dS m-1 over

respective uninoculated control. At 4, 8 and 12 dS m-1, strain M4 caused 24, 21 and 17%

decrease in proline contents, respectively, over respective uninoculated control. M4xM7

along with L-TRP caused 33, 45, 23 and 22% decrease in proline contents at 4, 8, 12 and 15

dS m-1, respectively, over respective uninoculated control. Without L-TRP application

maximum decrease in proline contents at EC 15 dS m-1 was recorded by M4xM7 which was

24% less as compared to respective uninoculated control. Sole inoculation with M4 and M7

caused positive reduction in proline contents, both in the presence and absence of L-TRP.

4.4.15 Total soluble sugars

Figure 19 shows the effect of inoculation/combined-inoculation on the total soluble

sugars of maize grown under salt affected soil conditions. The data demonstrate that

concentration of soluble sugars was increased due to exposure to salinity. Upton 5, 7, 13 and

24% increase in soluble sugars was noted at 4, 8, 12 and 15 dS m-1, respectively, as compared

to uninoculated control.

Soluble sugars contents were decreased due to inoculation/co-inoculation with

rhizobacterial strains both in the absence and presence of L-TRP. However inoculation/co-

inoculation gave better results when it was carried out along with L-TRP application.

Maximum reduction in total soluble contents was recorded by co-inoculation with M4xM7

that was up to 30, 30, 34 and 32% at 4, 8, 12 and 15 dS m-1, respectively over respective un-

inoculated control. Along with L-TRP application sole inoculation also significantly reduced

the total soluble sugars at all salinity levels. As compared with strain M7, M4 showed greater

reduction in soluble sugar contents.

88

r

e-gcd

b

a

rs

f-hde

bca

tu

mni-l

h-j

d-f

tu

o-ql-n

h-j

d-f

u

k-mh-k gh

cd

tu

m-oi-l

gh

de

u

qn-p

l-n

gh

v

rpq

lmh-j

0

0.5

1

1.5

2

2.5

3



Prl

ine

Con

tent

s (µ

mol

g-1

FW


Figure 18: Effect of rhizobacterial inoculation (with and without L-TRP) on proline contents of maize at different salinity levels (original =




89

c-fb-d bc

b

a

j-m j-mh-k

g-jd-g

h-mg-k g-j

e-h

c-e

op l-pm-o m-o

i-mg-j f-i f-h

d-g

b-d

m-o m-ok-o k-o

h-l

m-o l-o l-oj-m

h-m

q q pq pqo-q

0

0.2

0.4

0.6

0.8

1

1.2

1.4



Tot

al so

lubl

e su

gars

(µg

g-1F

W)

Control M4 M7 M4XM7

Figure 19: Effect of rhizobacterial inoculation (with and without L-TRP) on total soluble sugars of maize at different salinity levels (original =




90

Inoculation/co-inoculation also significantly reduced the soluble sugar contents in the absence

of L-TRP application. In this case combination M4xM7 gave maximum reduction in soluble

sugars that was up to 29, 31, 31 and 30% at 4, 8, 12 and 15 dS m-1, respectively as compared

to uninoculated control. L-TRP application as alone treatment was also found effective in

reducing the total soluble sugar concentration at all salinity levels as compared to control.

4.4.16 Total phenol contents

Data regarding the total phenol contents is presented in Figure 20. The data

demonstrate that concentration of phenol contents was increased due to exposure to salinity.

However, inoculation/coinoculation lowered the phenol contents were decreased due to with

rhizobacterial strains. Inoculation gave better results when it was carried out along with L-

TRP application. Maximum reduction in phenol contents (42%) over respective un-inoculated

control were recorded by M4xM7 at 15 dS m-1. Along with L-TRP application sole

inoculation also significantly reduced the phenol contents at all salinity levels. Strain M4

showed greater reduction in phenol contents as compared with strain M7.

In the absence of L-TRP application, M4xM7 combination gave maximum reduction

in soluble sugars that was up to 45% at 15 dS m-1, as compared with respective uninoculated

control. L-TRP application as alone treatment was also found effective in reducing the phenol

contents at all salinity levels as compared to control.

91

b-d b-d bc

c-f

a

d-g c-f c-gb-e

b

f-k e-j e-id-e

b

j-o i-o i-oh-m

d-h

i-n h-m h-mb-e

b-e

n-p n-p m-o l-og-l

l-o k-o j-nb-d

b-g

q pq pq o-ql-o

0

0.5

1

1.5

2

2.5



Tot

al p

heno

lics

(µg

g-1F

W)

Control M4 M7 M4XM7

Figure 20: Effect of rhizobacterial inoculation (with and without L-TRP) on total phenolics of maize at different salinity levels (original =




92

4.4.17 Ascorbate peroxidase (cAPX)

Figure 21 shows the effect of inoculation/coinocualtion on the ascorbate peroxidase of

maize grown under salt affected soil conditions. The data demonstrate that activity of

ascorbate peroxidase increased under salinity stress. An increase of 5, 7, 13 and 24% in

ascorbate peroxidase was noted at 4, 8, 12 and 15 dS m-1, respectively as compared to

uninoculated control.

Ascorbate peroxidase activities were decreased due to inoculation/co-inoculation with

rhizobacterial strains both in the absence and presence of L-TRP. However

inoculation/coinoculation gave better results when it was carried out along with L-TRP

application. Maximum reduction in activity was recorded by co-inoculation with M4xM7 that

was 30, 30, 34 and 32% at 4, 8, 12 and 15 dS m-1, respectively over respective un-inoculated

control. Along with L-TRP application sole inoculation also significantly reduced the

ascorbate peroxidase at all salinity levels. Strain M4 showed greater reduction in ascorbate

peroxidase activity as compared with strain M7. Inoculation/combined-inoculation also

significantly reduced the ascorbate peroxidase activity in the absence of L-TRP application.

In this case M4xM7 combination gave maximum reduction that was up to 30, 31, 31 and 30%

at 4, 8, 12 and 15 dS m-1 respectively, as compared to uninoculated control. L-TRP

application as alone treatment was also found effective in reducing the ascorbate peroxidase

activity.

93

e-hd-g

c-ea-c

ab

f-h e-g

e-gd-f

d-f

g-ie-g

d-f

b-d

a-c

h-j h-j h-j h-j g-if-h f-h

d-f

a-c

a

0.610.55

0.65 0.67 0.69

0

0.2

0.4

0.6

0.8

1

1.2



cAP

X (m

M a

scor

bate

min

-1g-1


Figure 21: Effect of rhizobacterial inoculation (with and without L-TRP) on cAPX activity of maize at different salinity levels (original =




94

4.4.18 K:Na

The results of the inoculation on the K:Na are demonstrated in Figure 22. It is clear

from the data that salinity stress significantly decreased the K:Na in maize plants at all

salinity levels. Upto 9, 42, 57 and 61% decrease in K:Na was recorded at 4, 8, 12 and 15 dS

m-1, respectively, as compared with control. However, inoculation/coinoculation enhanced the

K:Na both in the absence and presence of L-TRP application. Maximum improvement in

K:Na was caused by co-inoculation with M4xM7 along with L-TRP application. This

increase in K:Na was up to 52, 69, 90 and 87% at 4, 8, 12 and 15 dS m-1 respectively, over

respective uninoculated control. Sole inoculation with M4 and M7 along with L-TRP also

performed better and maximum increase in this case was recorded by strain M4 which caused

up to 31, 46, 60 and 75% increase in K:Na at 4, 8, 12 and 15 dS m-1 respectively, over


Inoculation/coinoculation also improved the K:Na even in the absence of L-TRP

application. Co-inoculation with M4xM7 caused an increase up to 47, 75, 88 and 87% at 4, 8,

12 and 15 dS m-1, respectively as compared to respective uninoculated control. Separate

application of L-TRP also positively improved the K:Na but this improvement was

statistically non significant as compared to respective control.

4.4.19 Nitrogen in shoot (%)

The results regarding the Effect of rhizobacterial inoculation on the nitrogen content

in shoot of maize of maize under salt affected soil conditions revealed that salinity had a

repressive effect on nitrogen content (Figure 23). It was evident from the results that with

increasing salinity levels there was progressive decline in nitrogen content. However

rhizobacterial inoculation seemed promising in alleviating the salinity stress which was

reflected in case of improvement in nitrogen content. Rhizobacterial inoculation in single and

dual combinations improved nitrogen content at all salinity levels. The Effect of

rhizobacterial inoculation was pronounced when strains were tested along with L-TRP

application.

95

ikl

u

vv

hijk

tu

vv

d-f fg

l-np-r

s-u

c c-e

kl

n-qr-u

fggh

m-pq-t

u

cde-g

k-m

q-su

b b

ij

m-oq-s

a

b

hi

kl

o-q

0

0.5

1

1.5

2

2.5

3

3.5



K/N

aControl M4 M7 M4XM7

Figure 22: Effect of rhizobacterial inoculation (with and without L-TRP) on K+/Na+ of maize at different salinity levels (original = 1.63, 4, 8,

12 and 15 d Sm-1). Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average of three

replicates. Control (Uninoculated), M4 (Pseudomonas fluorescens), M7 (Serratia proteamaculans).


96

i

k

p

qr

f-i

j

n

pr

d-f a e-i

jk

op

c-e cd d-h

j

no

d-h d-h g-i

kl

p

c-e d-g d-g

k

p

b b bc

hi

a

b bc-e

lm

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6



Nit

roge

n in

sho

ot (

%)

Control M4 M7 M4XM7

Figure 23: Effect of rhizobacterial inoculation (with and without L-TRP) on nitrogen concentration in shoot of maize at different salinity levels




97

Salinity stress significantly reduced the nitrogen content at all tested salinity levels.

Maximum reduction in nitrogen content (71%) was noted at 15 dS m-1. In case of single strain

inoculation, M4 performed better than strain M7 and increased nitrogen content up to 51 and

27%, respectively. But when both these strains were tested in the presence of L-TRP further

increase in nitrogen content was noted i.e. M4 and M7 caused increase up to 53 and 29%,

respectively. However combined application of M4 and M7 gave the best results regarding

the improvement in plant height at all salinity levels both in the presence and absence of L-

TRP application. At 15dS m-1, an increase up to 74 and 73% was recorded in the absence and

presence of L-TRP respectively over respective uninoculated control by inoculation with

M4xM7.

4.4.20 Phosphorous concentration in grains (%)

As shown by the Figure 24 the phosphorous contents of maize plants were

significantly reduced by the soil salinity. Up to 17.54, 35.08 and 45.61% decrease in

phosphorous concentration was observed by salinity at 4, 8, and 12 dS m-1.

Sole and combined inoculation with M4 and M7 resulted significant increase in

phosphorous concentration under normal as well as salt affected soil conditions both in the

presence and absence of L-TRP application. Co-inoculation with M4xM7 caused maximum

increase in phosphorous concentration in grain when it was used in the absence and presence

of L-TRP application. In the presence of L-TRP 43, 57 and 53% increase and in the absence

of L-TRP a 42, 51 and 48% increase in phosphorous concentration was recorded at 4, 8 and

12 dS m-1, respectively, as compared with uninoculated control by this inoculation.

Sole inoculation with M4 and M7 also significantly enhanced the phosphorous contents.

Maximum increase in this case recorded by inoculation with M4 when it was tested along

with L-TRP, which resulted up to 25, 31 and 28% more phosphorous contents in maize grain

at 4, 8 and 12 dS m-1, respectively as compared with uninoculated control. Separate

application of L-TRP was also found effective in enhancing the P contents but this increase

was statistically non significant as compared to control at all tested salinity levels.

98

f

gh

i

j

d-f

gh

i

j

deef

gh

i

cdd-f

g

i

def

h

i

cdd-f

gh

i

a

bc

f

h

a

b

d-f

gh

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8



P in

gra

in (%


Figure 24: Effect of rhizobacterial inoculation (with and without L-TRP) on phosphorous concentration in grains of maize at different salinity




99

4.4.21 Crude protein contents

As shown by the Figure 25 the protein contents of maize grains were significantly

reduced by the soil salinity. Upto 7, 24 and 53% decrease in crude protein concentration as

compared with control was observed by salinity at 4, 8, and 12 dS m-1.

Sole and combined inoculation with M4 and M7 resulted a significant increase in

protein contents under normal as well as salt affected soil conditions both in the presence and

absence of L-TRP application. Co-inoculation with M4M7 caused maximum increase in

protein concentration in grain when it was used in the absence and presence of L-TRP

application. In the presence of L-TRP a 12, 11 and 60% increase and in the absence of L-

TRP, a 17, 28 and 56% increase in crude protein concentration was recorded at 4, 8 and 12 dS

m-1, respectively as compared with unioculated control by this inoculation.

4.5 Correlation analysis

The different coefficient of correlation between salinity, inoculation and different parameters

were correlated and significance of correlation was performed at p < 0.05 by using SPSS

software. A significant negative correlation was found between salinity and grain yield (Fig.

26) while a positive correlation was found between salinity and proline contents at different

salinity levels (Fig. 30). A positive correlation between inoculation and grain yield (Fig. 27)

was recorded while a negative correlation between inoculation and proline contents was

observed at different salinity levels (Fig. 31).

100

ghj

m

q

de

j

l

q

cd ef

j

o

bcde

h

n

ef ef

k

p

cdfg

ij

o

b b

hi

n

a bef

l

0

2

4

6

8

10

12

14



Cru

de p

rote

in c

onte

nts

(%)

Control M4 M7 M4XM7

Figure 25: Effect of rhizobacterial inoculation (with and without L-TRP) on crude protein contents in grains of maize at different salinity




101

Figure 26: Effect of soil salinity on grain yield per plant of maize in pots

Figure 27: Effect of rhozobacterial inoculation on grain yield per plant of

maize in pots

R²=0.3727

R²=0.286

R²=0.3095

R²=0.1683

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

0 2 4 6 8 10

1.63

4

8

12

15

R²=0.1933R²=0.1729

R²=0.1864

R²=0.1758

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

0 5 10 15

Control

M4

M7

M4xM7

102

Figure 28: Effect of soil salinity on total chlorophyll contents (SPAD

values) of maize in pots

Figure 29: Effect of rhizobacterial inoculation on total

chlorophyllcontents (SPAD values) of maize in pots

R²=0.3189

R²=0.2164

R²=0.2138

R²=0.2441

R²=0.2

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0 2 4 6 8 10

1.63

4

8

12

15

R²=0.206

R²=0.1582

R²=0.1808

R²=0.2116

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0 2 4 6 8 10 12

Control

M4

M7

M4xM7

103

Figure 30: Effect of soil salinity on proline contents of maize in pots

Figure 31: Effect of rhizobacterial inoculation on proline contents of

maize in pots

R²=0.3248

R²=0.3835

R²=0.3557

R²=0.2208

R²=0.2801

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 2 4 6 8 10

1.63

4

8

12

15

R²=0.1543

R²=0.1474

R²=0.147

R²=0.1588

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 2 4 6 8 10 12

Control

M4

M7

M4xM7

104

Figure 32: Effect of soil salinity on electrolyte leakage of maize in pots

Figure 33: Effect of rhizobacterial inoculation on electrolyte leakage of

maize in pots

R²=0.6303

R²=0.5117

R²=0.4527

R²=0.2925

R²=0.2303

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0 2 4 6 8 10

1.63

4

8

12

15

R²=0.1188

R²=0.1131

R²=0.1197

R²=0.0909

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0 2 4 6 8 10 12

Control

M4

M7

M4xM7

105

4.5 Field trial

In order to confirm the results of the pot trial under natural salt affected soil

conditions field trial were conducted for two consecutive years at a farmer field 72 GB,

Satiana Road, Faisalabad. The results of the field trial are described below.

4.5.1 Plant height

The results regarding the Effect of rhizobacterial inoculation on plant height of maize

are demonstrated in Figure 34. It was revealed from the data that plant height was improved

with bacterial inoculation in the absence and presence of L-TRP application. In the year I,

maximum increase in plant height (43%) was found in case of M4xM7 combination over un-

inoculated control. Single strain inoculation with M4 and M7 also significantly increased the

plant height up to 31 and 28%, respectively as compared as compared with uninoculated

control. Rhizobacterial inoculation along with L-TRP further enhanced the plant height. In

this case M4xM7 combination showed 37% increase over respective un-inoculated control.

Sole application of M4 and M7 along with L-TRP significantly improved plant height up to

26 and 25% respectively, over respective un-inoculated control. In 2nd year experiment,

maximum increase in plant height was recorded by M4xM7 combination both in the presence

and absence of L-TRP. Combined application of rhizobacterial strains improved plant height

up to 31 and 33% in the absence and presence of L-TRP respectively, as compared with


4.5.2 Grain yield

The results of Effect of rhizobacterial inoculation on the grain yield of maize grown

under salt affected soil condition are represented in Figure 35. In the first year experiment, a

maximum increase in grain yield was recorded up to 41 and 35% by M4xM7 when it was

tested with out and along with L-TRP application respectively, as compared with

uninoculated control. Single inoculation with M4 and M7 also significantly enhanced the

grain yield up to 33 and 30% respectively, over respective un-inoculated control. These

strains along with L-TRP application improved grain yield up to 23 and 20% respectively,

over respective un-inoculated control. Sole L-TRP application also significantly improved the

grain yield.

106

Figure 34: Effect of rhizobacterial inoculation (with and without L-TRP) on plant height of maize under salt affected field conditions. Bars showing similar

letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average of three replicates. Control (Uninoculated), M4 (Pseudomonas

fluorescens), M7 (Serratia proteamaculans)

107

Figure 35: Effect of rhizobacterial inoculation (with and without L-TRP) on grain yield of maize under salt affected field conditions. Bars

showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average of three replicates. Control

(Uninoculated), M4 (Pseudomonas fluorescens), M7 (Serratia proteamaculans)

108

In the second year trial co-inoculation with M4xM7 without L-TRP application

showed 49% improvement in grain yield over un-inoculated control while up to 50% increase

in grain yield over un-inoculated control was observed when M4xM7 combination was used

along with L-TRP. Single inoculation with M4 and M7 without L-TRP improved grain yield

up to 36 and 30% respectively, as compared with uninoculated control. Along with L-TRP

application sole inoculation with M4 and M7 showed increase up to 36 and 35% respectively,

over their respective uninoculated control.

4.5.3 Fresh biomass

Data (Figure 36) showed that single and dual inoculation significantly improved the

fresh biomass both in the presence and absence of L-TRP application.

In the year I, experiment, single inoculation with M4 and M7 in the absence of L-TRP

enhanced fresh biomass up to 44 and 43%, respectively, over un-inoculated control. While

M4xM7 showed a maximum increase (50%) when used in the absence of L-TRP. Along with

L-TRP application M4 and M7 improved fresh biomass of maize up to 37 and 25%

respectively, while in this case maximum increase (35%) was recorded by M4xM7, as

compared with uninoculated control.

In the second year trial, maximum increase in fresh biomass up to 51 and 36% was

recorded by the M4xM7 combination in the absence and presence of L-TRP respectively,

over their respective un-inoculated controls. Sole inoculation with M4 and M7 also

significantly improved the fresh biomass.

109

Figure 36: Effect of rhizobacterial inoculation (with and without L-TRP) on fresh biomass of maize under salt affected field conditions.

Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average of three replicates.

Control (Uninoculated), M4 (Pseudomonas fluorescens), M7 (Serratia proteamaculans)

110

4.5.4 1000 grain weight

The data regarding 1000 grain weight is presented in Figure 37. Maximum

improvement in 1000 grain weight up to 24 and 26% over their respective controls was seen

in case of M4xM7 in 1st and 2nd year respectively, when it was applied along with L-TRP.

Without L-TRP application M4xM7 showed an increase in 1000 grain weight up to 25% in

year I and up to 26% in year II. In year I, sole inoculation with M4 and M7 without L-TRP

application improved 1000 grain weight up to 19% and 25% respectively, over respective un-

inoculated control. L-TRP application further enhanced the inoculation and both strains (i.e.

M4 and M7) shoed up to 18 and 19% increase in 1000 grain weight, respectively, over their

respective controls.

In the second year experiment, sole inoculation with M4 and M7 in the absence of L-TRP

gave up to 20 and 21% significant increase in 1000 grain weight respectively, over their

respective un-inoculated control. While along with L-TRP application up to 18 and 19%

increase in 1000 grain weight was recorded by inoculation with M4 and M7, respectively,

over uninoculated control.

4.5.5 Straw yield

Results of straw yield (Figure 38) depicted that sole inoculation and dual application

of rhizobacterial strains significantly enhanced the straw yield both in the absence and

presence of L-TRP in both years. Sole inoculation with M4 and M7 in the absence of L-TRP

significantly enhanced the straw yield up to 52 and 53% respectively, in first year and up to

51 and 49% respectively, over their respective un-inoculated control in the second year

experiment. Along with L-TRP application, the maximum increase (29%) in straw yield was

observed by single inoculation with M4 over its respective control in year I. while in 2nd year

experiment maximum increase (21%) was also recorded by inoculation with M4.

In first year co-inoculation with M4xM7 in the absence and presence of L-TRP

application resulted in a significant increase up to 56 and 35%, respectively, over their

respective un-inoculated control. While in 2nd year, an increase up to 50 and27% was

observed by M4xM7 when it was tested in the presence and absence of L-TRP, respectively.

111

Figure 37: Effect of rhizobacterial inoculation (with and without L-TRP) on 1000-grain weight of maize under salt affected field conditions.



112

Figure 38: Effect of rhizobacterial inoculation (with and without L-TRP) on straw yield of maize under salt affected field conditions. Bars



113

4.5.6 Cob length

The results (Figure 39) of cob length showed that single and combined inoculation

with M4 and M7 significantly improved the cob length both in the presence and absence of L-

TRP application.

Without L-TRP application increase in cob length varied from 27.24 to 40.95% in

year I experiment and from 30 to 44% in the year II experiment. The maximum increase in

cob length without the application of L-TRP was recorded up to 40 and 44% by M4xM7 co-

inoculation over respective un-inoculated control in year I and year II, respectively.

Along with L-TRP application the improvement in cob length ranged from 14 to 29%

in year I and from 14 to 25% in year II. M4xM7 co-inoculation gave maximum improvement

in cob length over its respective control.

4.5.7 Weight per cob

Results regarding the Effect of rhizobacterial inoculation on cob weight are demonstrated in

the Figure 40.

In the year I trial, the maximum increase (53 and 46%) in cob weight over respective

un-inoculated control was recorded in case of M4xM7 co-inoculation in the absence and

presence of L-TRP, respectively. Sole inoculation with M4 and M7 in the absence of L-TRP

gave 42 and 41% increase in cob weight over their respective un-inoculated control. Along

with L-TRP application maximum increase (32%) was recorded by inoculation with strain

M4, as compared with uninoculated control.

In the 2nd year experiment, single inoculation with M4 and M7 in the absence of L-

TRP gave up to 40 and 36% increase in cob weight respectively, over respective un-

inoculated control. Along with L-TRP application M4 and M7 showed significant increase in

cob weight up to 33 and 31% respectively, over their un-inoculated control. The maximum

increase in cob weight up to 50 and 47% was recorded in case of M4xM7, when it was tested

without and along with L-TRP application, respectively.

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Figure 39: Effect of rhizobacterial inoculation (with and without L-TRP) on cob length of maize under salt affected field conditions. Bars



115

Figure 40: Effect of rhizobacterial inoculation (with and without L-TRP) on weight per cob of maize under salt affected field conditions.



116

4.5.8 Total chlorophyll contents (SPAD values)

The results regarding total chlorophyll contents of maize grown under salt affected

soil condition are presented in Figure 41. It is clear from the results that the inoculation/ co-

inoculation significantly improved the total chlorophyll contents of maize both in the absence

and presence of L-TRP application.

In 1st year experiment, co-inoculation with MxXM7 showed maximum improvement

in total chlorophyll content (23 and 25%) over its respective un-inoculated control in the

presence and absence of L-TRP application, respectively. Single strain inoculation with M4

and M7 also significantly improved the total chlorophyll contents with and without L-TRP

application. The mere application of L-TRP also improved chlorophyll contents but this

improvement was statistically non-significant as compared to control where no L-TRP was

applied.

In the 2nd year experiment, the improvement in chlorophyll ranged from 15 to 27%

over un-inoculated control in case when inoculation/co-inoculation was tested without L-TRP

application. On the other hand, inoculation/co-inoculation along with L-TRP application

improved chlorophyll from 15 to 25% over respective uninoculated control. Separate

application also non- significantly improved total chlorophyll contents of maize.

4.5.9 Proline contents

Under stress condition plants produce osmolytes such as proline. Data regarding

proline contents (Figure 42) showed that inoculation with rhizobacterial strains both in the

presence and absence of L-TRP significantly decreased the proline contents in maize plants

grown under salt stressed soil conditions.

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Figure 41: Effect of rhizobacterial inoculation (with and without L-TRP) on total chlorophyll contents (SPAD values) under salt affected

field conditions. Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average of three

replicates. Control (Uninoculated), M4 (Pseudomonas fluorescens), M7 (Serratia proteamaculans)

118

Figure 42: Effect of rhizobacterial inoculation (with and without L-TRP) on proline contents of maize under salt affected field conditions.



119

In the year I experiment reduction in proline contents ranged from 23to 29% over the

control when rhizobacterial strains were tested in the absence of L-TRP. While along with L-

TRP application reduction in proline contents by these strains ranged from 22 to 34% over

their respective un-inoculated controls. Minimum proline contents were recorded by the co-

inoculation with M4xM7 both in the absence and presence of L-TRP application.

In the year II experiment the maximum reduction (31 and 34%) was recorded by

M4xM7 over respective un-inoculated control in the absence and presence of L-TRP

application, respectively. Sole inoculation with M4 and M7 also significantly reduced the

proline contents both in the presence and absence of L-TRP application. Just an application of

L-TRP also decreased the proline contents but this decrease was non significant as compared

to control.

4.5.10 Crude protein contents in grains

The results regarding crude protein contents in grains (Figure 43) indicate that

rhizobacterial inoculation significantly improved the protein contents in the presence and

absence of L-TRP application. In the year I trial, the increase in protein contents due to

inoculation with M4 and M7 along with L-TRP application was 9 and 8% over respective

control, respectively. In the 2nd year trial these stain showed an enhancement in protein

concentration by 11 and 10% over respective un-inoculated control. These strains applied in

the absence of L-TRP also significantly improved the protein contents.

The combined application of these strains i.e. M4xM7 gave more promising results

than their sole application. An increase up to 15% in the first year experiment and 16% in the

second year experiment over respective un-inoculated control was recorded by M4xM7 in the

presence of L-TRP application. Combined inoculation in the absence of L-TRP application

also significantly improved the protein contents. Alone application of L-TRP also improved

the protein concentration in both years.

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Figure 43: Effect of rhizobacterial inoculation (with and without L-TRP) on crude protein contents in grains of maize under salt affected

field conditions. Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average of three


121

4.5.11 Phosphorous concentration in grains

The results regarding phosphorous concentration showed that inoculation/co-

inoculation significantly improved the P contents in grains of maize grown under salt affected

field conditions (Figure 44).

In the absence of L-TRP application, sole inoculation with M4 and M7 caused up to

16 and 11% increase in phosphorous concentration over respective control in year I and up to

19 and 14% in year II as compared with their respective control. Along with L-TRP

application these strains caused up to 17 and 21% increase in P contents over their respective

control in year I and up to 17 and 19% increase in year II experiment.

In the absence of L-TRP application co-inoculation with M4xM7 caused up to 21%

increase in phosphorous concentration in first year experiment and up to 25% increase over

its respective uninoculated control in second year trial. While along with L-TRP application

M4xM7 caused an increase of 24% in the 1st year and 24% increase in the 2nd year over its

respective uninoculated control. Improvement in phosphorous concentration was also noted

by just application of L-TRP over uninoculated control.

4.5.12 K:Na in leaves

Data in Figure 45 highlighted that inoculation/coinoculation significantly improved K:Na in

both year experiments.

In year I, maximum increase in K:Na in the presence of L-TRP was recorded when

M4 and M7 (M4xM7) were combined in combination. The increase was 410% over the

respective uninoculated control. M4 and M7 in single inoculation along with L-TRP gave

415% and 399% increase over their respective uninoculated controls.

Inoculation/coinoculation in the absence of L-TRP also significantly improved the K/Na. Non

significant improvement as compared with control was also noted by the sole application of

L-TRP application.

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Figure 44: Effect of rhizobacterial inoculation (with and without L-TRP) on phosphorous concentration iin grains of maize under salt

affected field conditions. Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average

of three replicates. Control (Uninoculated), M4 (Pseudomonas fluorescens), M7 (Serratia proteamaculans)

123

Figure 45: Effect of rhizobacterial inoculation (with and without L-TRP) on K+/Na+ of maize under salt affected field conditions. Bars



124

In the second year experiment M4xM7 in the absence and presence of L-TRP showed

up to 361 and 511% increase in K:Na over their respective un-inoculated control. M4 and M7

applied as single inoculation along with L-TRP showed up to 4188 and 402% increase over

their respective control. These strains in the absence of L-TRP also significantly improved the

K/Na. sole application of L-TRP as a treatment also positively improved the potassium ratio.

4.5.13 Total phenol contents

The results regarding total phenol contents are shown in Figure 46. It is evident from

the data that phenolic contents improved by inoculation/coinoculation. The improvement in

total phenolic contents in presence of L-TRP ranged from 36 to56% in first year and from 29

to36% in second year as compared with respective un-inoculated control. In the absence of L-

TRP maximum improvement in total phenolic contents up to 32% in the first year and 25% in

the second year, over respective un-inoculated controls was recorded by MxM7. Sole

inoculation with M4 and M7 in the absence of L-TRP also improved the total phenolic

contents in both years. The application of L-TRP alone was also found effective in improving

the total phenolic contents. Overall inoculation/ coinoculation along with L-TRP performed

better than without L-TRP.

4.5.14 Total soluble sugars

The data in the Figure 47 shows the effect of inoculation/coinoculation on total

soluble sugars of maize grown salt affected field conditions.

In the year I sole inoculation with M4 and M7 showed 41 and 45% increase in total

soluble sugars in the absence of L-TRP and 35 and 39% increase in total soluble sugars as

compared to their respective uninoculated controls. Maximum increase (50%) in total soluble

sugars was recorded by M4xM7 in the absence and presence of L-TRP, respectively.

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Figure 46: Effect of rhizobacterial inoculation (with and without L-TRP) on total phenol content of maize under salt affected field

conditions. Bars showing similar letters do not differ statistically at p ≤ 0.05, Duncan’s Multiple Range test. Data is average of three


126

Figure 47: Effect of rhizobacterial inoculation (with and without L-TRP) on total soluble sugars of maize under salt affected field



127

In the second year, M4xM7 increase in the total soluble sugars up to 32 and 25% over

the respective uninoculated control in the absence and presence of L-TRP, respectively. Sole

inoculation with M4 and M7 also significantly improved the total soluble sugars both in the

presence and absence of L-TRP. Non significant increase in total soluble sugars was also

observed with the sole application of L-TRP.


The results of the Effect of rhizobacterial inoculation on electrolyte leakage are shown

in Figure 48. In the year I experiment, inoculation/co-inoculation significantly reduced the

electrolyte leakage both in the presence and absence of L-TRP. The combined application of

both strains was found more effective than their sole inoculation. Along with L-TRP

application the maximum reduction (35%) in electrolyte leakage was recorded by M4xM7.

Sole inoculation with M4 and M7 also reduced the electrolyte leakage up to 32 and 30%

respectively, as compared to uninoculated control. Without L-TRP M4xM7 showed

maximum reduction in electrolyte leakage up to 31% followed by sole inoculation with M4

and M7, which showed a 27 and 27% reduction in electrolyte leakage as compared to un-

inoculated control.

In second year trial, coinoculation showed maximum reduction in electrolyte leakage

both in the presence and absence of L-TRP. This increase was up to 33% without L-TRP and

37% when co-inoculation was tested in the presence of L-TRP. Single inoculation also

reduced the electrolyte leakage significantly. Strain M4 was found more effective than M7

and caused 29 and 33% decrease in electrolyte leakage in the presence and absence of L-TRP

respectively, as compared with uninoculated control.

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Figure 48: Effect of rhizobacterial inoculation (with and without L-TRP) on electrolyte leakage of maize under salt affected field



129

4.5.16 SOD

The results of the effect of inoculation/coinoculation on the SOD values are demonstrated in

Figure 49. It is clear from the results that the rhizobacterial inoculation/co-inoculation

significantly reduced the SOD values both in the presence and absence of L-TRP.

In the 1st year trial, M4xM7 caused a maximum reduction in SOD values up to 64 and

72% in the absence and presence of L-TRP respectively, as compared with respective

uninoculated control. Single inoculation with M4 and M7 also caused significant reduction in

SOD values up to 71 and 69% in the presence of L-TRP and up to 58 and 54% in the absence

of L-TRP respectively, over their respective un-inoculated control.

In the 2nd year trial, co-inoculation with M4xM7 resulted in a maximum decrease of

up to 65% and 75% in the absence and presence of L-TRP respectively over its respective un-

inoculated control. In the absence of L-TRP single inoculation with M4 and M7 caused 45

and 52% decrease in SOD values over their respective uninoculated control. Along with L-

TRP application these strains caused 55 and 61% decrease in SOD values.

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Figure 49: Effect of rhizobacterial inoculation (with and without L-TRP) on SOD activity of maize under salt affected field conditions. Bars



131

4.6 Identification and characterization of selected strains

Both selected strains were characterized (Table 9) for different biochemical

characters. The results of these tests are summarized below.

It is evident from the results that both of these strains were positive in oxidase

activity, catalase activity, chitinase activity and phosphate solubilization. Both strains were

also positive for ACC-deaminase activity. The results of root colonization showed that both

stains colonized the roots but maximum root colonization (6.11 × 106 cfu g-1) was observed

by the strain M4 follwed by the strain M7. These strains also possesed the exopoly-saccharide

production ability.

The selected strains were identified by BIOLOG® as Pseudomonas fluorescens (M4)

and Serratia proteamaculans (M7).

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Table 9: Characterization of selected rhizobacterial strains

Strains Percent

match to

Biolog

ACC-

deaminase

Activity

Phosphate

solubilization

Chitinase

activity

Oxidase

activity

Catalase

activity

Exopoly-

saccharide

production

Root

colonization

(cfu g-1)

M4 68% +ve +ve +ve +ve +ve +ve 6.11 × 107

M7 59% +ve +ve +ve +ve +ve +ve 5.32 × 106

M4, Pseudomonas fluorescens; M7, Serratia proteamaculans

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CHAPTER-5

DISCUSSION

In the present study we isolated a number of rhizobacterial strains from the rhizosphere of

maize plants growing under salt-affected soil conditions. These strains were tested for their

ability to tolerate salinity stress by using osmoadaptation assay. Out of these strains, 19 most

tolerant strains were selected and their auxin production ability in the presence and absence of

L-TRP was measured. Eleven strains which tolerated salinity and produced auxin were

selected to test their ability of growth promotion of maize seedlings under salt stressed axenic

conditions. After this experiment the two most efficient strains (M4 and M7) were selected

for further evaluation in pot and field trials to improve the growth and yield of maize under

salt stressed conditions.

5.1 Salt tolerance of rhizobacterial strains

In this study the isolated rhizobacterial strains were subjected to osmoadaptation

assay. Results of this assay revealed that salinity adversely affected the growth of

rhizobacterial strains. At normal conditions, the growth of strains was optimum but the

growth of rhizobacterial strains varied under salinity stress. The growth of some strains was

improved even at higher salinity levels. This might be due to the ability of these

rhizobacterial strains to tolerate saline conditions. Such variability among bacterial strains to

tolerate salinity has been reported by Loret et al. (1995). Mansah et al. (2006) and Sgroy et al.

(2009) have also reported the variable growth rates of bacteria isolated from the rhizosphere

of salt tolerant plants.

Salinity adversely affects the survival and growth of the bacteria (Tate, 1995; Troman and

Weaver, 2006). The growth of bacteria is inhibited or reduced under saline conditions

(Troman and Weaver, 2006). However, some bacteria are able to withstand salinity stress.

Kaseem et al. (1985) reported that rhizobia had better ability to tolerate higher salinity than

their host plant. The ability of these bacterial strains to tolerate higher levels of salinity may

be due to changes in morphology and metabolism, which makes them able to cope and adopt

saline conditions. Bacterial species vary in their ability to withstand salinity (Bernard et al.,

134

1986; Elsheikh and Wood, 1990). The strains isolated from salt-affected conditions are more

able to tolerate salinity stress than those isolated from normal soil conditions (Hua et al.,

1982). Salt tolerant bacteria adapt to saline conditions by using different strategies. They

expend energy to exclude salt from their cytoplasm to avoid protein aggregation (‘salting

out’). In order to survive the high salinities, these bacteria employ two differing strategies to

prevent desiccation through osmotic movement of water out of their cytoplasm. Both

strategies work by increasing the internal osmolarity of the cell. In the first strategy organic

compounds are accumulated in the cytoplasm. These osmoprotectants are known as

compatible solutes. These can be synthesised or accumulated from the environment The most

common compatible solutes are neutral or zwitterionic and include amino

acids, sugars, polyols, betaines and ectoines, as well as derivatives of some of these

compounds. The second, more radical, adaptation involves the selective influx

of potassium (K+) ions into the cytoplasm (Santos and da Costa, 2002). The bacterial cells

also respond to salinity stress by changing their morphology. The cell ultrastructure and

morphology of salt tolerant strain of cowpea Rhizobium was severely affected under salt

stress. The cells appeared as spiral or filament-like structures, and the cell size greatly

expanded. The cell envelope was distorted, and the homogeneous cytoplasm was disrupted

(Zahran et al., 1992). It has been reported (Busse et al., 1989) that cells of a strain of R.

meliloti appeared with irregular morphology at potentials below -0.5 MPa. Strains of rhizobia

from different species modified their morphology under salt stress, and rhizobia with altered

morphology have been isolated from salt-affected soils in Egypt (Zahran et al., 1992). High

osmotic stress modified the synthesis pattern of extracellular and capsular polysaccharides

of R. leguminosarum (Breedveld, et al., 1991.). The colonies of R. meliloti grown in the

presence salt stress show a decrease in mucoidy and lower level of exopolysaccharides

production (Lloret et al., 1998). The synthesis pattern in SDS-PAGE of lipopolysaccharides

(LPS) from various species of bacteria (Zahran, 1992; Zahran et al., 1994.) was modified by

salt.

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5.2 Effect of rhizobacterial inoculation on growth of maize in axenic conditions

Abiotic stresses such as salinity cause reduction in the growth and development of

plants by impairing their physiological and biochemical mechanisms. These effects of

stresses can be minimized by the application of bacterial inoculants, which help the plants to

maintain their growth under stressed conditions by provoking natural mechanisms (Yang et

al., 2008; Vardharajula et al., 2011). Plant growth promoting rhizobacteria most commonly

improve the plant growth by increasing the root growth (Lucy et al., 2004) by producing

phytohormones. Plant hormones play a vital role in controlling the root growth. In the root

zone, auxin produced by the bacteria cause changes in root architecture. These changes result

in better root growth and increased root surface area, which ultimately result in better uptake

of nutrients and water leading to enhanced growth of whole plant (Somers et al., 2004;

Principe et al., 2007). On the other hand under stressful conditions plant produced ethylene

results in decreased root and shoots growth. But rhizobacteria containing ACC-deaminase

activity can degrade the precursor of ethylene (ACC), thus lower the internal levels of

ethylene in plants and rescue them from the stress (Mayak et al., 2004). In our growth room

study we observed that salinity caused a negative effect on the growth of maize seedlings. A

progressive decrease in root/shoot length and fresh/dry biomass of maize seedlings was

recorded with increasing salinity levels. However, inoculation with rhizobacterial strains

caused significant improvement in root/shoot growth of maize seedlings. This might be due

to their phytohormone production ability such as auxin and regulating the hormonal status of

plants and lowering the ethylene concentration. Elevated amounts of ethylene production and

alteration in hormonal (auxin) balance in plants have been documented by many researchers

(Mayak et al., 2004; Yue et al., 2007; Zahir et al., 2008). Many researchers have documented

the enhanced growth of different crops by microbial cultures mainly due to their

phytohormone production ability such as auxin (Glick, 1995; Noel et al., 1996; Okon and

Vanderleyden, 1997). The results of this study highlight the importance of screening auxin

producing PGPR for improving the growth and yield of maize under salt stressed conditions.

5.3 Effect of rhizobacterial inoculation on physiological parameters of maize

The ability of plants to cope salinity stress has been accessed on growth, yield,

biochemical and genetic basis. Other than these relative water contents, photosynthetic

136

activity, leaf gas exchange, electrolyte leakage are also good indicators of stresses (Burling et

al., 2013; Maccaferri et al., 2013). In our field and pot studies, it was found that salinity

adversely affected the transpiration rate, stomatal conductance and relative water contents.

The decrease in transpiration rate may be due to reduction in stomatal conductivity (Chedlia

et al., 2007). Plants close their stomata as an adaptive mechanism to withstand osmotic stress

and conserve water for later growth stages (Misra et al., 2002; Tardien, 2005; Hatami et al.,

2010). Due to this closure of stomata carbon dioxide intake is affected and photosynthesis

rate of plants subjected to salinity stress is decreased (Misra et al., 2002). Lawlor and Cornic

(2002) and Ben-Asher et al. (2006) also reported changes in stomatal conductivity and

decrease in photosynthetic activity. Increased concentration of Na+ causes reduction in carbon

dioxide assimilation (Chartzoulakis et al., 2002).

Salinity stress deprives plants from water and alongside cause ionic imbalance which

results in overall reduced growth of plants (Wyn Jones, 1981). The measurement of relative

water contents is an important parameter to assess stress tolerance in plants (Fisher et al.,

2000; Pereyra et al., 2006). In this study, we observed that inoculation /co-inoculation

application significantly enhanced the relative water contents. Our findings are also supported

by the results of other researchers (Nadeem et al., 2009; Naveed et al., 2014) who also found

improvement in relative water contents due to rhizobacterial inoculation. The reason of this

improvement in relative water contents may be due to longer roots caused by rhizobacterial

inoculation, which might enable the plant to absorb water from the deeper layers of soil

where concentration of salts is relatively low (Dodd et al., 2004; Hamida et al., 2004). This

increase in water contents may also be due to production of growth regulators such as auxins.

Cohen et al. (2008) also reported such improvements in relative water contents of leaves and

described it may be due to the bacterial production of plant growth regulators. Nadeem et al.

(2009) also conducted an experiment and reported such improvement in relative water

contents of maize leaves by using rhizobacterial strains under salinity stress. Osmotic stress

caused by salinity results in limited water supply which may lead to reduction in chlorophyll

contents (Paknejad et al., 2007). Higher chlorophyll contents are positively correlated with

increase in yield and under stress conditions elevated chlorophyll contents may result in

improvement in yield of plants.

137

When plants are subjected to osmotic stress, proline is accumulated in plants. Higher

concentration of such organic osmolytes in plants serves as a solute for osmotic adjustment

(Silverita et al., 2003). High concentration of proline is associated with stress conditions (Rai

et al., 2003). Extra energy is utilized to accumulate such osmolytes in addition to normal

metabolic costs. In our study, it was found that salinity stress caused a marked increase in

proline contents however, rhizobacterial inoculation /co-inoculation decreased the proline

contents. This decrease in proline contents might be due to the ameliorative effects of

rhizobacterial strains which helped the plants to withstand salinity stress and decreased the

proline contents. Similar results have been documented where bacterial inoculation was found

effective in lowering the proline concentration (Han and Lee, 2005; Nadeem et al., 2009).

In present study, we observed that salinity markedly decreased the chlorophyll

contents while rhizobacterial inoculation improved the chlorophyll contents. Our results are

also supported by studies of other researchers who found increase in chlorophyll contents due

to rhizobacterial inoculation (Nadeem et al., 2009). This increase in chlorophyll contents can

be attributed to increase in photosynthetic area of leaves due to inoculation/co- inoculation

with rhizobacterial strains which was otherwise decreased by salinity stress (Marcelis and

Van-Hooijdook, 1990). Ion toxicity caused by salinity stress also causes the death of older

leaves which results in the prevention of nutrient and hormonal supply to young leaves

(Munns, 1993; Tamman, 2003) which might lead to reduction in chlorophyll contents. Hamid

and Al-Komy (1998) have also reported such improvement in chlorophyll contents owing to

bacterial inoculation. The increased chlorophyll contents might have resulted in higher

photosynthetic rate and production of more photosynthates which ultimately resulted in

higher yields.

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5.4 Effect of rhizobacterial inoculation on nutritional balance in maize

Changes in nutrient uptake is the most common phenomenon which plants undergo in

different stresses such as salinity stress (Greenway and Munns, 1980). This results in lower

uptake of essential nutrients due to which plant suffer nutrient deficiencies which result in

poor growth. In our study we observed that salinity stress caused marked decrease in N and P

contents of maize plants. Phosphorous is essential macronutrient which plays a vital role in

normal growth and development of plants. Martenez and Lauchli (1994) reported that

phosphorous uptake by plants was inhibited under salinity stress. We found that

rhizobacterial inoculation significantly improved the mineral nutrient contents (nitrogen,

phosphorous and crude protein contents). This might be due to increased root biomass which

can explore more soil area and results in enhanced uptake of nutrients from larger soil

volume. Our results are in line with Nadeem et al. (2006), Yao (2004) who also reported

enhanced nutrient uptake by microbial inoculation under stressful conditions.

Similarly, Gaballah and Comaa (2005) found decrease in protein contents of faba

bean. However, we observed inoculation /co-inoculation to be very effective in reversing this

pattern and increase the concentration of inorganic nutrients such as N and P in grains and

leaves of maize plant. In our study, crude protein contents of maize grains were also

improved due to rhizobacterial inoculation /co-inoculation. Other scientists have also reported

such improvement in inorganic nutrients and crude protein contents due to rhizobacterial

inoculation/ co-inoculation (Hamidia et al., 2004). Mayak and co-workers (2004) also

reported increase in P contents in tomato plants grown under salt stress conditions. Under

drought conditions, increase in N and P contents of lettuce plant inoculated with Bacillus sp.

have documented by Vivas et al. (2003). Similarly, Han and Lee (2005) also reported

improvement in N, P and Ca concentrations in soya bean plants under salinity stress by

inoculation with PGPR. Dovvlacre and Okon (2007) have correlated this increased uptake of

N and P with root elongation caused by Azospirillum inoculation. It can be stated that

increase in the root area and provision of growth hormones such as auxins would have helped

the plant in taking more nutrients from the soil. It is reported in many studies that salinity

stress causes reduction in uptake and assimilation of essential nutrients in plants, thus their

139

supplementation is required to mitigate the harmful effects of salinity stress (Endris and

Mohammad, 2007; Khayyat at al., 2007; Heider and Jamshid, 2010).

5.5 Effect of rhizobacterial inoculation on ionic balance in plants

Salt tolerance of plants can be indicated by K+/ Na+ (Hamida et al., 2004). It was

found in our study that salinity stress caused the ionic imbalance in plants. It resulted in

higher intake of Na+ than K+. There are two phases of salinity stress in plants (Munns, 1993).

In first phase, salinity causes osmotic stress which results in decrease in water absorption

from soil (Mayak et al., 2004). In the second step, salinity induces ionic stress/toxicity.

Second phase of salinity stress is reported to be more injurious than the first phase as it results

in alteration in uptake, distribution and compartmentalization of ions in the plants. Potassium

acts as an osmoregulator and maintains water balance in plants under stress conditions.

Decreased K+ concentration results in drop in leaf water potential. This decrease in K+

concentration may result in reduction in stomatal conductivity and activity of ribulose

bisphosphate carboxylase which ultimately impairs the photosynthesis (Zhao et al., 2001;

Szepesi et al., 2008).

Salinity also disturbs the physiological activity relating to the ionic and osmolyte

accumulation such as proline (Steward et al., 1974). Ehasanpour and Amini (2003) reported

that the uptake and transport of phosphate within plants was highly sensitive to salinity stress.

Uptake of Na+ and K+ are disturbed due to salinity (Ashraf, 2004). Fortmere and Schubert

(1995) demonstrated that Na+ toxicity was the major cause of stress during the second phase

of salinity in maize. Similarly, Schubert and coworkers (2009) described that grain formation

and reduction in overall yield of maize under saline conditions was caused by osmotic stress

and Na+ toxicity.

In our pot and field studies, plants were exposed to long term salinity stress as they

were grown till maturity and it is highly likely that they have suffered both phases of salinity

stress. We observed higher Na+ concentration in our plants subjected to salinity stress and it

may be the one of the stresses. Higher uptake of Na+ compared to K+ has been reported

previously (Pervaiz et al., 2002; Ashraf, 2005). This increased uptake of Na+ results in

reduction of K+/Na+. Plants uptake Na+ and K+ through common channels thus higher Na+

140

concentration in soil decreased the K+ uptake due to ionic competition (Rains and Epstein,

1997). Similar findings of increased Na+ under salt stress conditions were reported by Saqib

(2000) and Nadeem et al. (2010).

Salinity stress causes not only the disturbances in mineral ion uptake but also their

transport within the plants is altered due to which their natural ratios in different plant parts

are distributed (Glenn et al., 1999). The transport of K+ and Ca2+ to the actively growing plant

parts is inhibited which affects the quality and quantity of produce. Roots of the plant are less

vulnerable to ion toxicity than shoot and leaves as the roots can maintain constant level of

ions by expelling out the extra Na+ and Cl- into soil or into shoot and leaves. So, higher

amounts of these deleterious ions are accumulated in shoots and leaves (Tester and

Davenport, 2003). Sodium causes metabolic toxicity by competing with K+ for binding sites

which is essential for different cellular functions. Potssium is involved in controlling the

activity of more than 50 enzymes and Na+ cannot serve as the alternative of the K+ to carry

out these functions. Thus, higher Na+/K+ cause disturbances in enzyme functions. Higher K+

concentrations are also required for the synthesis of different proteins. Thus, disturbances in

protein synthesis in plants under saline conditions may be caused by Na+ toxicity (Blaha et

al., 2000). Prat and Fathi-Ettai (1990) suggested that salinity stress causes changes in

morphology, ultra-structure and metabolic activities of plant species which ultimately result

in reduction in growth and yield of plants.

Ion homeostasis is the main phenomena which enables the plants to tolerate salinity

stress (Singh et al., 2011). Uptake of higher concentration of K+ as compared to Na+ is vital to

maintain higher K+/Na+ in cells (Kavitha et al., 2012). In our study, we found that

rhizobacterial inoculation /co-inoculation significantly improved the K+/Na+. The interaction

of plant roots with the rhizobacteria reduces the uptake of toxic elements and/or enhances the

uptake of essential nutrients (White, 2003). This might be due to lowered uptake of Na+ than

K+ due to longer roots caused by inoculation/coinoculation with rhizobacterial strains as

reported previously (Yue et al., 2007; Ahmad et al., 2012). Reduction in Na+ concentration

may be due to dilution effect as salt stressed plants inoculated with the rhizobacterial strains

have higher water contents than uninoculated plants. Such salt induced dilution effects have

been reported in different crops e.g. Physalis peviviana (Miranda et al., 2010), Spinacia

141

oleraces (Kaya et al., 2001) and maize (Collado et al., 2010). The increased K+ concentration

have also been correlated with better growth and yield under saline conditions by Giri et al.

(2007). Thus, it can be stated that this improvement in ionic balance caused by rhizobacterial

strains may be one of the reasons of better growth and yield of maize under salt stress.

Membrane permeability (MP) is a good indicator of salt stress and is judged by measuring

electrolyte leakage (Farkhondeh et al., 2012; Mansoor, 2012). In our study, we observed

marked increase in electrolyte leakage in plant subjected to salinity stress. We also observed

that a strong relationship existed between chlorophyll contents and electrolyte leakage. This

type of relationship has been documented in many studies (Chen et al., 1991; Kaya et al.,

2001a). It is likely that membrane integrity somehow is dependent upon chlorophyll contents,

because decrease in chlorophyll contents as caused by salinity stress induces senescence and

membrane become more permeable (Niu et al., 1995; De Araujo et al., 2006)

5.6 Antioxidant enzymes

Under salt stress situations plants accumulate higher levels of antioxidant enzymes

such as SOD, CAT, POD and APX as a defense mechanism to counter oxidative stress

caused by salinity (Munns and Tester, 2008; Ashraf, 2009; Kaye et al., 2011). In our studies

we also found marked increase in antioxidant enzymes under salinity stress. Our results are in

line with other researchers (Apel and Hirt, 2004: Ahmad et al., 2008; Kohler, 2008).

Rhizobacterial inoculation lowers the level of antioxidants (Shahbazi et al., 201; Baniaghil et

al., 2013). Along with production of antioxidant enzymes plants also accumulate some other

compounds such as phenolics and sugars which not only act as osmoregulators but also

scavenge reactive oxygen species (ROS) (Wang et al., 2003; Fernandez et al., 2012;

Theocharis et al., 2012). In our study, we found increase in content of sugars and phenolics

under salt stressed conditions. Our results are in line with Fernendez et al. (2012) who

reported higher sugar contents owing to bacterial inoculation under abiotic stress.

Sandhya et al. (2010) conducted a study to investigate the effect of five drought

tolerant PGPR strains on the growth and antioxidative activity of maize. The results of the

study revealed that the activity of antioxidant enzymes was lower in inoculated plants as

compared with the uninoculated plants. In barley plants the antioxidative activity was also

reduced. A study conducted by Omer et al. (2009) revealed that under salinity stress activity

142

of antioxidant enzymes such as catalase and peroxidase increase significantly in two

cultivars of barley. On the other hand when these plants were inoculated with Azospirillum

baselines, they showed lowering in the activity of such enzymes, which removed the

deleterious effects of salinity and enhanced the growth and yield of barley plants under salt

effected conditions.

5.7 Combined application of L-TRP and rhizobacterial strains

In soil tryptophan dependent biosynthesis of auxin significantly improves the growth and

development of plants (Sarwar et al., 1992). In our study, we observed that sole application of

L-TRP positively improved the maize growth under salt affected soil conditions implying that

native soil microflora was also active in synthesizing auxins from L-TRP or this might be due

to the uptake of L-TRP directly by plant’s root, which have resulted in improved growth

under saline conditions. Hussain et al., (1995) reported the synergistic effect of Rhizobium

inoculation and L-TRP in lentil in terms of 30% more yield than untreated control. The

exogenous provision of phytohormones as a result of microbial activity might affect plant’s

endogenous hormonal levels, either by supplementing the plants own suboptimal levels or by

interacting with the synthesis, translocation, or inactivation of existing hormone levels. Plants

themselves synthesize a diversity of growth-regulating phytohormones but they also respond

to exogenous applications of these plant growth regulators PGRs which, after being taken up

by the plant, may affect its growth directly as growth stimulants or indirectly by modifying

the rhizosphere (Khalid et al., 2006; Zahir et al., 2010). These results are supported by the

work of Datta et al. (1997), who exogenously applied phytohormones and recorded

improvement in wheat growth under salt affected conditions. They found that presoaking

wheat seeds with indole acetic acid mitigated the adverse effects of salinity. We found in our

study that separate application of L-TRP and PGPR strains caused positive effects on plant

growth and development under salt stressed conditions, but their combined application gave

better results regarding growth improvement. The results of Zahir et al. (2000) also support

our findings, which demonstrated that combined use of precursor (L-TRP) and inoculum

(Azotobacter) was better than their sole application. This improvement in growth and yield

can be attributed to the production of plant growth hormone (auxin) in the rhizosphere that

can optimize the internal hormonal level of plant or by increased uptake of essential nutrients

143

due to improved root growth mediated by the phytohormones produced by PGPR. From this

study, it can be suggested that combined application of L-TRP and PGPR strain (precursor-

inoculum interaction) can be very effective technique to induce salt stress tolerance in maize.

5.8 Mechanism used by PGPR for growth promotion in maize grown under salt stress

conditions

Soil salinity is one of the major limiting factors in crop growth and productivity all

over the world (Nemati et al., 2011). Different strategies are in practice to ameliorate the

adverse effects of salinity stress. Among many of the used strategies is the use of plant

growth promoting rhizobacteria (Kohler et al., 2009; Fernandez-Aunion et al., 2010). The

positive effects of such bacteria have been reported by many researchers on different crops

(Creus et al., 1997). In our study we found that both of the rhizozobacterial strains improved

the maize growth under saline conditions. This may be due to the presence of multiple traits

that these strains possessed. Both of the strains were able to coloninize the roots but Strain

M4 had the greater tendency of root colonization than M7 which may be the reason of better

performance of M4 as compared with strain M7 in single inoculation. The combined

application of both strains showed better performance which means that these strains had not

antagonistic effects on each other but developed a synergistic relationship. The enhacement

of maize growth might be due to multiple traits that both of these strains possessed.

Plant growth regulators such as auxins (IAA) are suggested to be involved in the

synthesis/activity of antioxidant enzymes and some of their isoforms also play role in the

metabolism of these growth regulators (Synkova et al., 2004; Tongetti et al., 2012). Reactive

oxygen species are produced at higher level under stress conditions and cause serious damage

to membranes. Despite posing a threat, they also activate a defense mechanism in plants

against stress (Pitzschke et al., 2006). Auxin (IAA) and reactive oxygen species (ROS) play a

critical role in salt stress tolerance. But the actual mechanism that how this cross-talk between

auxins and ROS activate salt tolerance is still to be under stood (Tognetti et al., 2012).

In our study, inoculation with rhizobacterial strains enhanced the growth and yield of

maize most probably due to the production of plant growth hormones such as indole acetic

acid (IAA). Glick (1995) suggested that hormone production is the key mechanism used by

144

the bacterial strains for improving the growth and yield of crop plants. Other researchers have

also documented such growth improvement in plants using the bacterial strains which had the

ability to produce auxins (Fallik et al., 1989; Xie et al., 1996; Dobbelaere et al., 2000; Pati et

al., 1995; Biswas et al., 2000b; Dazzo et al., 2000; Hussain et al., 2009). It is documented that

salinity causes progressively decline in plant hormones (Prakash and Parathapasenan, 1990;

Nilsen and Orcutt, 1996) thus it can be interpreted that reduction in growth and yield caused

by salinity may be due to hormonal imbalance. This imbalance in hormonal level can be

corrected by the exogenous application of hormones (Balki and Padole, 1982; Gulnaz et al.,

1999; Sastry and Shekhawa, 2001; Afzal et al., 2005; Akbari et al., 2007) or by using plant

growth promoting rhizobacteria capable of producing auxins.

Auxin is involved in the transcription of many genes which are known as primary

auxin response genes. For different crops these genes have been identified (Hagen and

Guilfoyle, 2002). Increased production of auxins (IAA) is correlated with root proliferation

which enables the plants to take more water from large soil area (lambrecht et al., 2000;

Steenhoudt and Vanderleyden, 2000). IAA positively affects the root growth directly or

indirectly by lowering the ethylene levels (Dimkpa et al., 2009; Lugtenberg and Kamilova,

2009). Junghans et al. (2006) has also correlated the higher contents of free auxins in

Arabidopsis with root proliferation under salt stress. Bianco and Defez (2009) used IAA

producing rhizobacteria and found that harmful effects of salinity were ameliorated in

Medicago plants.

Plant growth promoting rhizobacteria can affect the growth of plants through the

production of plant growth hormones such as auxins (IAA). These bacteria interact with

plants through the production of auxins (Malhothra and Srivaatava, 2006; Anjum et al.,

2011). In our study, we screened the PGPR on the basis of their auxin production ability and

found that most of the strains were able to produce auxins and this production was even

increased in the presence of L-TRP. Ramiz et al. (1993) have also reported that rhizobacterial

strains produced auxins with varying degrees. Similar to our findings other researchers

(Khalid et al., 2004, 2006; De and Basu, 2007) have reported the auxin production in

rhizobacterial strains.

145

It is highly likely that along with auxin production ability of these strains other

characters of these strains might have helped the plants to tolerate the adverse effects of

salinity. In our laboratory study, we found that all the strains possessed the ACC-deaminase

activity. ACC-deaminase activity in rhizobacterial strains have also been reported by other

researchers (Glick, 2005; Shahroon et al., 2006; Belimov et al., 2009; Nadeem et al., 2009).

Under salinity stress conditions ethylene concentration increased in plants which has

inhibitory effect on plant growth (Ables, 1992). The bacterial strains possessing the ACC-

deaminase activity can lower the ethylene concentration by causing hydrolysis of ACC,

which act as precursor of ethylene (Mayak et al., 2004; Cheng et al., 2007) thus decreasing

the adverse effects of salinity on plants. Many other researchers have reported such

reductions in ethylene by the use of PGPR containing ACC-deaminase activity (Glick, 1998;

Indragandhi et al., 2008; Zahir et al., 2011).

We observed that both the selected strains varied in their growth promotion ability, which

might be due to presence of different other growth promoting characters such as root

colonization, phosphate solubilazation and chitinase activity along with their ability to

produce auxins. These strains possessed phosphate solubilization ability which might have

helped the plants to get extra nutrients. Chitinase activity of these strains might have helped

the plants against their pathogens thus resulting in better growth. Our results are also

supported by the work of Nadeem et al. (2007) who reported that rhizobacterial strains varied

in their ability to improve the plant growth. Salt tolerance in plants also depends on restricted

or controlled uptake of Na+ and continued uptake of K+ (Jeschke and Wolf, 1988). In our

study microbial strains also produced exopolysaccharides. These exopolysaccharides can bind

Na+ (Geddie and Sutherland, 1993), thus decrease the uptake of Na+ in plant and help them in

alleviating salt stress (Ashraf et al., 2004).

Conclusions and future directions

Soil salinity is worldwide problem that poses a serious threat to agricultural production. With

severe and sudden climatic changes this problem is expected to spread at a fast pace. Various

physical, chemical and biological approaches are being used to hammer this problem. But

most of these techniques seem either very expensive or very time consuming. Since near past,

146

soil bacteria have been employed to induce salt stress tolerance in plants. Among such

bacteria are the ones that are capable of producing a hormone, auxin. Many reports document

that endogenous level of plant hormones such as auxin is disturbed due to soil salinization. It

is also well documented that auxin controls the various aspects of plant’s growth and

development. So alteration in hormonal balance is considered one of the main reasons of

growth reduction in plants during salinity stress. The auxin producing rhizobacteria can

supplement the auxin to plants and recover the hormonal alterations caused by salinity. The

results of the present study signify the effectiveness of auxin producing rhizobacteria for

improving the growth and yield of maize in salt affected soils.

However, further research is needed to make the use of this approach more effective in terms

of plant growth enhancement. The future research should be planned giving special emphasis

on these particular aspects,

Population of auxin producing rhizobacteria can be increased by seed or soil

inoculation.

Genetic identification of these strains is needed, so that these strains can be

genetically manipulated for getting better performance

In next phase endophytic auxin producing bacteria be selected to protect the bacterial

population from environmental constraints.

A deep insight into the interaction of these bacteria with the plants can be helpful to

develop statistical models which will help in managing the salt affected soils.

These auxin producing rhizobacteria can be used by seed/fertilizer/pesticide

companies to develop bio-inoculants for maize production in salt affected soils.

147

CHAPTER-6

SUMMARY

Maize (Zea mays) is important cereal crop of the world. Under arid and semiarid

conditions the production of maize is restricted due to number of biotic and abiotic stresses.

Soil salinity is one of the major abiotic stresses that hamper the productivity of the crops

throughout the world especially arid and semiarid regions like those in Pakistan. Soil salinity

adversely affects the growth of plants due to many factors including hormonal imbalance.

Plant growth promoting rhizobacteria have the ability to produce these plant growth

hormones. Inoculation with such bacterial strains can supply adequate amount of plant

growth hormones thus mitigate the adverse effects of salinity. A series of experiments were

carried out to evaluate the beneficial effects of auxin producing PGPR on growth and

performance of maize under salt affected soil conditions.

These experiments and their results are summarized below:

A number of rhizobacterial strains were isolated from the rhizosphere of the maize

grown under salt affected fields.

The isolated strains were subjected to osmoadaptation assay to test their ability to

withstand the salinity stress. The rhizobacterial strains were grown at four salinity

levels i.e. control, 10, 20 and 30 dS m-1 and the optical density was measured. The

results showed that all the strains varied in their ability to withstand salinity.

Twenty selected strains were accessed for their auxin production ability. All the

strains produced varied amounts of indole acetic acid (IAA) in the presence and

absence of L-TRP.

The rhizobacterial strains producing auxin were screened for their ability to promote

growth of maize seedlings under axenic conditions. The results indicated that salinity

treatment significantly reduced the growth of maize seedlings but inoculation with

auxin producing PGPR improved the growth of maize seedlings. These strains varied

in their growth promotion ability.

148

Two most effective strains producing auxin were selected for pot and field

experiments to evaluate their potential for improving the growth and yield of maize in

the presence and absence of L-TRP under salt affected soil conditions. The results of

these studies are summarized below

a) The growth and yield of maize was adversely affected by the salinity stress under pot

and field experiments.

b) The rhizobacterial inoculation alone and in combination significantly improved the

growth and yield of maize under salt affected soil conditions both in the presence and

absence of L-TRP. The results of combined application of bacterial strains along with

L-TRP application were more pronounced than sole application of strains.

c) Physiological parameters like carbon dioxide assimilation, stomatal conductance,

relative water contents, transpiration rate and photosynthetic rate etc. were decreased

by salinity treatment while inoculation with rhizobacterial strains significantly

improved these parameters in the presence and absence of L-TRP in both pot and field

experiments.

d) Salinity stress caused ionic imbalance in the plants and decreased the K+/Na+. The

rhizobacterial inoculation increased the K+ concentration and decreased the Na+ thus

improved the K+ to Na+ ratio in plants.

e) Concentration of osmolytes such as proline and soluble sugars increased under salinity

stress. However, inoculation with auxin producing rhizobacteria decreased the

concentration of these osmolytes in plants.

f) Mineral nutrient concentration was adversely by the salinity as the concentration of N,

P and K decreased under salinity stress. But the concentration of mineral nutrients was

significantly improved by the rhizobacterial inoculation. The concentration of N, P, K

and protein contents was increased due to inoculation thus improving the quality of

produce along with an increased grain yield of maize.

g) Antioxidant activity of the plants was increased under salinity stress, however,

rhizobacterial inoculation significantly lowered the activity of such enzymes showing

their ability to mitigate salinity stress.

149

h) The strains varied in their ability to tolerate the salinity stress. The strains showing

more growth at higher salinity levels also performed better for improving growth and

yield of maize under salinity stress.

i) The strains having more root colonization ability and auxin production ability along

with other characteristics also performed better in improving the growth and yield of

maize under saline conditions.

j) Combined application of auxin producing rhizobacterial strains along with L-TRP can

be an economic option for improving the growth and yield of maize under salt


On overall basis combined application of rhizobacterial strains producing auxin

showed better results than their sole inoculation. The results of the inoculation were

more pronounced when it was carried out along with L-TRP application. Such

bacteria could be very effective as inoculants to improve the growth and yield of

maize under salt affected soil conditions. These results of these experiments support

our hypothesis that auxin producing rhizobacteria can improve maize growth in saline

conditions.

150

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2002-ag-1672 (m. asif iqbal) ph.d. thesis...

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